Use of rutin and rapamycin in the preparation of a drug for synergistically inhibiting tumors with chemotherapeutic agents

Abstract

The present invention provides the use of rutin and rapamycin in the preparation of a drug for synergistically inhibiting tumors with chemotherapeutic agents. The present invention reveals that rutin can specifically target and inhibit the senescence-associated secretory phenotype (SASP), which can act synergistically with rapamycin. After rutin and rapamycin are combined with chemotherapeutic drugs, a significant synergistic effect is presented, which can promote the inhibition of tumors, and the promotion effect is unexpected.

Description

Technical Field

[0001] The present invention belongs to the field of pharmacy, and more specifically, the present invention relates to the use of rutin and rapamycin in the preparation of a drug for synergistically inhibiting tumors with chemotherapeutic agents. Background Art

[0002] Cell senescence refers to a relatively stable and usually irreversible state of cell cycle arrest in eukaryotic cells. In this state, proliferating cells become tolerant to growth-promoting stimuli, usually caused by stress signals such as DNA damage. The expression level of inflammatory cytokines in senescent cells increases significantly, and this phenomenon is called the Senescence-Associated Secretory Phenotype (SASP). Senescent cells can promote the carcinogenesis or increase the malignancy of adjacent precancerous cells by secreting extracellular matrix proteins, inflammation-related factors, and cancer cell growth factors, and these proteins are called SASP factors.

[0003] Senescent cells mainly participate in various physiological and pathological processes of the body through three pathways: (1) The gradual accumulation of changes in gene expression and morphology of senescent cells can affect the functions of corresponding tissues; (2) Senescent cells limit the regenerative potential of stem cells and undifferentiated progenitor cells, resulting in a decline in cell regeneration ability; (3) Senescent cells not only show growth cycle arrest, but also release a large number of cytokines, chemokines, growth factors, and proteases through autocrine and paracrine pathways, affecting the microenvironment of adjacent cells and tissues, leading to and accelerating aging and related diseases.

[0004] Stimuli such as DNA damage, telomere dysfunction, oncogene activation, and oxidative stress can all induce cells to exhibit SASP, and its mechanism is closely related to transcriptional cascades, autocrine loops, and persistent DNA damage responses. However, overexpressing or inhibiting the classical senescence pathways p53 and p16 INK4A / Rb cannot affect the expression of SASP, indicating that although cell cycle arrest and SASP in senescent cells often occur synergistically, their regulatory pathways do not completely overlap. It is reported that the DNA damage response increases the secretion of SASP factors IL-6 and IL-8 by activating ataxia telangiectasia mutated gene, Nijmegen breakage syndrome protein 1, and checkpoint kinase 2. The DNA damage response (DDR) is immediately activated after cell damage, and it takes about 1 week or even longer for mature SASP to appear in senescent cells. Moreover, a transient DNA damage response cannot induce cell senescence or SASP, indicating that there are other mechanisms in addition to the DNA damage response that jointly induce SASP.

[0005] Studies have shown that DDR, p38MAPK, and mTOR signals, as upstream drivers, and NF-κB and c / EBPβ, as downstream transcription factors, are all involved in the regulation of the senescence-associated secretory phenotype (SASP) in senescent cells. The activities of the transcription factors NF-κB and c / EBPβ increase during cellular senescence and are involved in regulating the expression of cytokines that mediate cellular stress and inflammatory signals. When cells senesce, the phosphorylated NF-κB / RelA subunit translocates into the nucleus, binds to the SASP promoter genes, and regulates the expression of SASP factors. Therefore, NF-κB is commonly referred to as the master regulator of SASP. The level of zinc finger transcription factor 4 (GATA4) is relatively high in senescent cells in the livers and kidneys of mice and in the brains of the elderly. GATA4 can affect the expression of SASP-related genes such as IL-6, IL-8, and CXCL1 by regulating the activity of NF-κB in senescent cells. p38MAPK is a member of the serine / threonine protein kinase family and is an important signal transduction molecule. Activating or blocking p38MAPK is sufficient to affect the formation of SASP in senescent cells. p38MAPK is activated a few days after the onset of the senescence program and indirectly activates NF-κB by activating mitogen- and stress-activated protein kinases - MSK1 and MSK2, causing the accumulation of p65 and p50 in the nucleus, which is consistent with the early development process of SASP. Senescent cells do not directly secrete the pro-inflammatory factor IL-1α, but a large amount of IL-1α is distributed on the surface of senescent cells. Together with NF-κB, they form a positive feed-forward loop to promote the coding transcription of inflammatory factors and establish and maintain SASP. mTOR promotes the secretion of SASP factors by regulating the level of IL-1α. Rapamycin does not affect the level of IL-1α mRNA but significantly reduces the expression of IL-1α protein on the surface of senescent cells. mTOR can also regulate the downstream signal of p38MAPK, MAPKAPK2, to affect the secretion of SASP factors. During cellular senescence, MAPKAPK2 phosphorylates the RNA-binding protein ZFP36L1, thereby limiting its ability to degrade the transcripts of SASP factors. The transcription factor c / EBPβ is associated with oncogene-induced cellular senescence. During senescence, c / EBPβ is recruited to the IL-6 promoter and directly promotes the transcription of SASP factors. c / EBPβ is also an important component of the IL-6 positive feed-forward autocrine loop, which can activate the inflammatory network of SASP and is an important regulator of the early spread of SASP. HMGB2 targets c / EBPβ to regulate SASP and promotes the expression of SASP genes by inhibiting the spread of heterochromatin. During cellular senescence, a large amount of HMGB2 binds to chromatin, eliminating the silencing effect of senescence-associated heterochromatic foci (SAHF) on SASP genes, resulting in increased expression of IL-8, IL-6, etc.

[0006] Epigenetic alterations affect aging by influencing DNA damage repair, telomere length, and metabolic pathways, or by activating the expression of senescence-associated genes and miRNAs. Multiple lines of evidence suggest that changes in chromatin state are closely related to the control of cellular senescence. Cells can sense different senescence stimuli, which activate signaling pathways that drive changes in chromatin state. However, the pathways by which senescence signals cause such changes remain largely unknown. Therefore, revealing the regulatory mechanisms underlying cellular senescence and the development of its specific phenotypes from an epigenetic perspective, and thereby identifying key molecules and their signaling pathways with targeted value, is an emerging direction in aging biology and geriatrics in the future, and there is an urgent need to conduct in-depth exploration to provide important scientific basis and potential intervention measures for clinical medicine.

[0007] Although an increasing number of experiments support that targeting cellular senescence can treat multiple senescence-related diseases such as tumors simultaneously, rigorous human clinical trials are still needed to help people better evaluate the benefits and risks of anti-aging drugs. Although various known SASP inhibitors internationally can significantly attenuate SASP, they do not essentially kill senescent cells. To pharmacologically reduce the burden of senescent cells, scientists are developing small molecules, peptides, and antibodies of this nature, namely "senolytics", to selectively eliminate senescent cells.

[0008] In addition to "senolytics" drugs, the field also focuses on "senomorphics" drugs. "Senolytics" mainly exert their effects by eliminating senescent cells, while the function of "senomorphics" is achieved by modulating the biological properties of senescent cells rather than eliminating them.

[0009] In summary, the field still needs to explore more drugs that affect SASP, with the aim of providing more approaches for the prevention and treatment of cellular senescence and tumors. Summary of the Invention

[0010] The object of the present invention is to provide the use of rutin and rapamycin in the preparation of a drug for synergistically inhibiting tumors with chemotherapeutic agents.

[0011] In a first aspect of the present invention, there is provided the use of rutin and rapamycin or their derivatives for use in combination with a chemotherapeutic drug to prepare a composition for specifically targeting and inhibiting the senescence-associated secretory phenotype (SASP) and inhibiting tumors and / or reversing tumor drug resistance; wherein, the chemotherapeutic drug is a chemotherapeutic drug that induces the senescence-associated secretory phenotype after administration, including mitoxantrone or bleomycin; the derivatives include pharmaceutically acceptable salts, esters, isomers, solvates, or prodrugs of rutin and rapamycin.

[0012] In one or more embodiments, the tumor suppression includes suppressing the tumor itself, as well as suppressing the migration and invasion of the tumor.

[0013] In one or more embodiments, the senescence-associated secretory phenotype is the senescence-associated secretory phenotype caused by DNA damage.

[0014] In one or more embodiments, the DNA damage is the DNA damage caused by chemotherapeutic drugs.

[0015] In one or more embodiments, in the composition, the rutin or rapamycin or their derivatives are also used for: inhibiting the expression of the broad-spectrum senescence-associated secretory phenotype; preferably, inhibiting the expression of the broad-spectrum senescence-associated secretory phenotype without affecting cell senescence; interfering with the interaction between ATM and HIF1α and TRAF6, and inhibiting the acute stress-related phenotype (ASAP); eliminating the malignancy conferred on cancer cells by senescent stromal cells in a paracrine manner; increasing the apoptosis rate of tumor cells; and / or, inhibiting the components of the senescence-associated secretory phenotype, such as IL8, IL6, IL1a, IL1b, CXCL3, MMP3, GM-CSF.

[0016] In one or more embodiments, the tumors include: prostate cancer, breast cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, bladder cancer, skin cancer, kidney cancer, esophageal cancer, cholangiocarcinoma, brain cancer.

[0017] In one or more embodiments, the chemotherapeutic drug is mitoxantrone; in the composition (or when used in combination), the weight ratio of mitoxantrone, rutin, and rapamycin is 1:20-80:20-80; preferably, the weight ratio of mitoxantrone, rutin, and rapamycin is 1:30-70:30-70; more preferably, the weight ratio of mitoxantrone, rutin, and rapamycin is 1:40-60 (such as ratios of 1:45:45, 1:50:50, 1:55:55, etc.).

[0018] In another aspect of the present invention, there is provided a pharmaceutical composition or kit for specifically targeting and suppressing the senescence-associated secretory phenotype and suppressing tumors and / or reversing tumor drug resistance, including: rutin and rapamycin or their derivatives, and a chemotherapeutic drug; wherein, the chemotherapeutic drug is a chemotherapeutic drug that induces the senescence-associated secretory phenotype after administration, including mitoxantrone or bleomycin; the derivatives include pharmaceutically acceptable salts, esters, isomers, solvates or prodrugs of rutin and rapamycin.

[0019] In one or more embodiments, when mitoxantrone is combined with rutin and rapamycin, the weight ratio of mitoxantrone, rutin, and rapamycin is 1:20 to 80:20 to 80; preferably, the weight ratio of mitoxantrone, rutin, and rapamycin is 1:30 to 70:30 to 70; more preferably, the weight ratio of mitoxantrone, rutin, and rapamycin is 1:40 to 60 (such as ratios of 1:45:45, 1:50:50, 1:55:55, etc.).

[0020] In another aspect of the present invention, there is provided a method for preparing a pharmaceutical composition or a kit for inhibiting tumors and / or reversing tumor drug resistance, comprising: mixing rutin and rapamycin or their derivatives with a chemotherapeutic drug; or placing rutin and rapamycin or their derivatives and a chemotherapeutic drug in the same kit; wherein, the chemotherapeutic drug is a chemotherapeutic drug that induces a senescence-associated secretory phenotype after administration, including mitoxantrone or bleomycin; preferably, when mitoxantrone is mixed or combined with rutin and rapamycin, the weight ratio of mitoxantrone, rutin, and rapamycin is 1:20 to 80:20 to 80; preferably, the weight ratio of mitoxantrone, rutin, and rapamycin is 1:30 to 70:30 to 70; more preferably, the weight ratio of mitoxantrone, rutin, and rapamycin is 1:40 to 60 (such as ratios of 1:45:45, 1:50:50, 1:55:55, etc.); the derivatives include pharmaceutically acceptable salts, esters, isomers, solvates, or prodrugs of rutin and rapamycin.

[0021] In one or more embodiments, rutin and rapamycin or their derivatives are mixed with a chemotherapeutic drug and divided into unit dosage forms according to the treatment course.

[0022] In another aspect of the present invention, there is provided the use of rutin and rapamycin or their derivatives for preparing a composition that specifically targets and inhibits the senescence-associated secretory phenotype; preferably, the rutin and rapamycin or their derivatives interfere with the interaction between ATM and HIF1α and TRAF6, inhibit the acute stress-related phenotype; eliminate the malignancy conferred by senescent stromal cells to cancer cells in a paracrine manner; and / or increase the apoptosis rate of tumor cells; the derivatives include pharmaceutically acceptable salts, esters, isomers, solvates, or prodrugs of rutin and rapamycin.

[0023] In another aspect of the present invention, there is provided a method for screening potential substances that promote the targeted inhibition of the senescence-associated secretory phenotype by rutin and rapamycin (or their derivatives) and inhibit tumors and / or reverse tumor drug resistance, the method comprising:

[0024] (1) Providing a tumor microenvironment system, which system includes tumor cells (preferably may also include stromal cells);

[0025] (2) Treat the system of (1) with chemotherapeutic drugs to induce a senescence-associated secretory phenotype in the tumor microenvironment; wherein, the chemotherapeutic drug is a chemotherapeutic drug that induces a senescence-associated secretory phenotype (SASP) after administration, including mitoxantrone or bleomycin; before, during, or after inducing the senescence-associated secretory phenotype in the tumor microenvironment, treat with rutin and rapamycin; and

[0026] (3) Add the candidate substance to the system of (2) and observe its effect on the tumor microenvironment system. If the candidate substance can statistically promote (significantly promote, such as promoting by 10%, 20%, 30%, 50% or more or higher) the inhibition of the senescence-associated secretory phenotype and the inhibition of tumors and / or reversal of tumor drug resistance by rutin and rapamycin, then the candidate substance is a potential substance that can be used in combination with rutin and rapamycin to inhibit tumors.

[0027] In one or more embodiments, the apoptosis situation or the senescence-associated secretory phenotype situation is evaluated by observing the caspase 3 cleavage activity or the expression of SASP factors. Preferably, the SASP factors include, but are not limited to: IL6, IL8, IL1a, IL1b, CXCL3, MMP3, GM-CSF.

[0028] In one or more embodiments, it is evaluated by observing the interaction of ATM with HIF1α and TRAF6 (inhibiting the acute stress-related phenotype). If the ability of rutin and rapamycin to interfere with the interaction of ATM with HIF1α and TRAF6 (inhibiting the acute stress-related phenotype) is promoted (significantly promoted, such as promoting by 10%, 20%, 30%, 50% or more or higher), then the candidate substance is a potential substance that can be used in combination with rutin and rapamycin to inhibit tumors.

[0029] In one or more embodiments, it is evaluated by observing the situation of the malignancy conferred by senescent stromal cells on cancer cells in a paracrine manner. If the candidate substance can generate senescent stromal cells and its ability to confer malignancy on cancer cells in a paracrine manner, then the candidate substance is a potential substance that can be used in combination with rutin and rapamycin to inhibit tumors.

[0030] In another aspect of the present invention, a method for screening potential substances that inhibit the senescence-associated secretory phenotype is provided. The method includes: (1) providing a stromal cell system and inducing a senescence-associated secretory phenotype in the system; before, during, or after the senescence-associated secretory phenotype, treat with rutin and rapamycin; (2) add the candidate substance to the system of (1) and observe its effect on the stromal cell system. If it can specifically promote the inhibitory effect of rutin and rapamycin on the senescence-associated secretory phenotype, then the candidate substance is a potential substance that can be used in combination with rutin and rapamycin to inhibit the senescence-associated secretory phenotype.

[0031] In one or more embodiments, a control group is further included to clearly distinguish the differences between the tumor microenvironment system in the test group and the control group, or the differences between rutin targeting and inhibiting the senescence-associated secretory phenotype and inhibiting tumors and / or reversing tumor drug resistance and the control group.

[0032] In one or more embodiments, the candidate substances include (but are not limited to): small molecule compounds designed specifically or present in a broad-spectrum compound library / biomolecule library, mixtures (such as plant extracts), biological macromolecules, signal pathway regulatory reagents, etc.

[0033] Other aspects of the present invention will be apparent to those skilled in the art from the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 . General experimental flow chart and technical route overview for large-scale screening of the natural product (NMA) library to obtain drugs or raw materials with anti-aging potential.

[0035] Figure 2 . Screening of various components of the natural library using human-derived stromal cells PSC27 under in vitro conditions to analyze the effects of specific drugs on the survival rates of proliferating cells and senescent cells. Blue dots within the pink rectangular area represent drugs that meet the characteristics of senolytics.

[0036] Figure 3 . Results of the survival counts of proliferating cells and senescent cells after treatment of PSC27 cells with 18 components of the NMA library. CTRL, proliferating cells. SEN, senescent cells. PCC1, procyanidin C1, used as a positive control for senolytics.

[0037] Figure 4 . Results of the survival counts of proliferating cells and senescent cells after treatment of PSC27 cells with 19 components of the NMA library (the final concentration of each component is 3 μg / ml respectively. If different concentrations are recommended, mark them in the figure, and the same applies hereinafter). CTRL, proliferating cells. SEN, senescent cells.

[0038] Figure 5 . Analysis of the expression levels of the SASP marker factor IL8 (interleukin 8) in proliferating cells and senescent cells after treatment of PSC27 cells with 19 components of the NMA library (the final concentration of each component is 1 μg / ml respectively). All data are normalized results compared to the CTRL group. ^, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0039] Figure 6. After treating PSC27 cells with 18 components of the NMA library (the final concentration of each component is 1 μg / ml), the expression levels of the SASP marker factor IL8 in proliferating cells and senescent cells were analyzed. All data are the normalized results compared to the CTRL group. ^, P > 0.05; *, P < 0.05; **, P < 0.01.

[0040] Figure 7 . The molecular formula of the natural plant chemical rutin.

[0041] Figure 8 . High-resolution mass spectra of the total ion chromatogram (TIC, upper) and the base peak chromatogram (BPC, lower) obtained after HPLC-ESI-QTOF-MS analysis.

[0042] Figure 9 . Results after SA-β-Gal staining on days 7 - 10 after treating proliferative human stromal cells PSC27 (at an early passage of around p10) with the chemotherapeutic drug bleomycin (BLEO) at a concentration of 50 μg / ml in vitro. Left panel, representative pictures; right panel, statistical results. CTRL, control cells; BLEO, cells after bleomycin treatment. DMSO, solvent; Rutin, rutin. ^, P > 0.05; ****, P < 0.0001.

[0043] Figure 10 . Results after BrdU staining of PSC27 cells after treatment with the chemotherapeutic drug bleomycin (BLEO) (50 μg / ml). Left panel, representative pictures; right panel, statistical data. CTRL, control cells; BLEO, cells after bleomycin treatment. DMSO, solvent; Rutin, rutin. ^, P > 0.05; ***, P < 0.001.

[0044] Figure 11 . Fluorescence quantitative PCR (qRT-PCR) was used to detect and analyze the relative expression levels of typical SASP factors in senescent cells induced by BLEO and treated with rutin at different concentrations (20 - 100 μM). All data are the normalized results compared to the CTRL group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0045] Figure 12 . Bioinformatics analysis of the transcriptional expression differences of PSC27 cells under three conditions and shown as a heat map. CTRL, control cells; BLEO, cells after bleomycin treatment; B / R, senescent cells after rutin treatment. Red stars, SASP component protein factors.

[0046] Figure 13. The results of GSEA analysis showed that the expression of SASP marker factors was significantly upregulated in senescent cells induced by BLEO, but decreased significantly after rutin treatment. SASP signature, SASP molecular tag.

[0047] Figure 14 . Bioinformatics analysis of the differential expression of the whole transcriptome of PSC27 cells in the senescent state and the rutin-treated senescent state was shown in a heatmap. Note that 3733 and 886 genes were downregulated or upregulated respectively (P<0.01, Foldchange>4).

[0048] Figure 15 . KEGG pathway analysis of the representative functions of 100 molecules significantly downregulated by rutin in senescent cells in molecular function.

[0049] Figure 16 . KEGG pathway analysis of the representative components of 100 molecules significantly downregulated by rutin in senescent cells in cellular component.

[0050] Figure 17 . KEGG pathway analysis of the representative processes of 100 molecules significantly downregulated by rutin in senescent cells in biological process.

[0051] Figure 18 . Immunoblot analysis of the DDR signaling pathway and SASP expression in PSC27 cells under the action of BLEO and / or rutin. GAPDH, protein loading control.

[0052] Figure 19 . Immunoprecipitation (mediated by TAK1 antibody) and blotting analysis of protein interactions in PSC27 cells under the action of BLEO and / or rutin. Input, total protein lysate. GAPDH, protein control.

[0053] Figure 20 . Immunoprecipitation (mediated by ATM antibody) and blotting analysis of protein interactions in PSC27 cells under the action of BLEO and / or rutin. Input, total protein lysate. GAPDH, protein control.

[0054] Figure 21 . Immunoblot analysis of the cell lysates of PSC27 cells under the action of BLEO and / or rutin after nuclear-cytoplasmic separation. C, cytoplasmic; N, nuclear. Lamin A / C, nuclear protein control. GAPDH, cytoplasmic protein control.

[0055] Figure 22. Fluorescent quantitative PCR (qRT-PCR) was used to detect and analyze the relative expression levels of a group of typical SASP molecules in senescent cells induced by BLEO, as well as after treatment with PX-478 or C25-140. All data were normalized results compared to the CTRL group. ^, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0056] Figure 23 . The fluorescence reagent DCFH-DA was used to detect the changes in the production of reactive oxygen species (ROS) at the PSC27 cell level under different conditions. On the left, representative pictures, scale bar, 20μm; on the right, statistical analysis.

[0057] Figure 24 . Detection of the proliferation ability of prostate cancer cells under in vitro culture conditions. The conditioned media of PSC27 cells under various conditions were collected and used to culture PC3, DU145, M12, and LNCaP, respectively. **, P<0.01; ***, P<0.001. Scale bar, 100μm. Rapamycin, rapamycin. RR, Rutin / Rapamycin.

[0058] Figure 25 . Measurement of the migration ability of prostate cancer cells under in vitro culture conditions. The conditioned media of PSC27 cells under various conditions were collected and used to culture PC3, DU145, M12, and LNCaP, respectively. **, P<0.01; ***, P<0.001; ****, P<0.0001. Scale bar, 100μm. RR, Rutin / Rapamycin.

[0059] Figure 26 . Determination of the invasion ability of prostate cancer cells under in vitro culture conditions. The conditioned media of PSC27 cells under various conditions were collected and used to culture PC3, DU145, M12, and LNCaP, respectively. **, P<0.01; ***, P<0.001; ****, P<0.0001. Scale bar, 100μm. RR, Rutin / Rapamycin.

[0060] Figure 27. Determination of the drug resistance potential of prostate cancer cells under in vitro culture conditions. Conditioned media collected from PSC27 cells under various conditions were used to culture PC3, DU145, M12, and LNCaP, respectively. *, P < 0.01; ***, P < 0.001; ****, P < 0.0001. IC50, half-inhibitory concentration. RR, Rutin / Rapamycin.

[0061] Figure 28 . Dose-response curve (nonlinear regression / curve fitting). Based on the culture of PC3 cells that were either naturally aged or BLEO-induced senescent from PSC27 with chemotherapeutic drugs, followed by treatment with a wide range of concentrations of rutin.

[0062] Figure 29 . After the CTRL group and the BLEO-damaged group of PSC27 cells were mixed with PC3 in vitro, they were transplanted into the subcutaneous tissue of mice as reconstructed tissues to form xenografts. At the end of the 8th week, the tumors were dissected and obtained, and the volumes of the tumors under each group condition were detected and compared. ^, P > 0.05; ***, P < 0.001; ****, P < 0.0001.

[0063] Figure 30 . Schematic diagram of the drug administration method for preclinical trial mice. Human-derived stromal cells PSC27 were mixed with cancer cells PC3 in vitro (1:4) and then transplanted into the subcutaneous of mice to form xenografts. After treatment with multiple treatment cycles under single-drug or combined drug administration conditions, the mice were finally sacrificed, and the changes in the molecular expression levels and other indicators of their tumor tissues were analyzed pathologically.

[0064] Figure 31 . Schematic diagram of the drug administration time and method for preclinical trial mice. Each 2 weeks was a drug administration cycle, and MIT (mitoxantrone) was intraperitoneally administered to mice on the first day of the 3rd / 5th / 7th week. Intraperitoneal rutin administration to mice started on the first day of the 5th week, once a week. After the 8-week treatment course ended, the mice were dissected and subjected to pathological identification and expression analysis.

[0065] Figure 32 . Statistical analysis based on the terminal volume of tumors. The chemotherapeutic drug MIT alone or together with the anti-aging drug rutin was used to administer to mice, and after the end of the 8th week, the tumor sizes of each group were compared and analyzed. On the left, statistical analysis. On the right, representative tumor pictures of each group.

[0066] Figure 33. Fluorescent quantitative PCR (qRT-PCR) was used to detect and analyze the changes in the transcript expression levels of a group of typical SASP factors in the tumor-bearing sites of mice among different groups. The data of each group were normalized against the sample with the lowest expression value within the group. The factors included IL6, IL8, AREG, and IL1a.

[0067] Figure 34 . Fluorescent quantitative PCR (qRT-PCR) was used to detect and analyze the changes in the transcript expression levels of another group of typical SASP factors in the tumor-bearing sites of mice among different groups. The data of each group were normalized against the sample with the lowest expression level within the group. The factors included MMP1, MMP3, ANGPTL4, and SPINK1.

[0068] Figure 35 . Fluorescent quantitative PCR (qRT-PCR) was used to detect and analyze the changes in the transcript expression levels of a group of cell senescence-specific biomarkers in the tumor-bearing sites of mice among different groups. The data of each group were normalized against the sample with the lowest expression level within the group. The biomarkers included p16 INK4a and p21 CIP1 .

[0069] Figure 36 . Comparison of cell senescence in the lesions of PC3 / PSC27 tumor-bearing animals in preclinical trials. Representative pictures after SA-β-Gal staining. Left, representative pictures. Right, statistical analysis. Scale bar, 100 μm.

[0070] Figure 37 . Statistical analysis of the terminal volume of systemic tumors. The chemotherapeutic drug MIT was administered to mice alone or in combination with rutin and the anti-aging drug Rapamycin. After 8 weeks, the tumor sizes of each group were compared and analyzed. The 6 groups on the left were PC3 cells only; the 6 groups on the right were formed by co-transplanting PC3 cells and PSC27 cells to form a reconstructed tissue. ^, P>0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0071] Figure 38 . Statistical analysis of the terminal volume of systemic tumors. The chemotherapeutic drug MIT was administered to mice alone or in combination with rutin and the traditional anti-aging drug Vitamin C. After 8 weeks, the tumor sizes of each group were compared and analyzed. The 6 groups on the left were PC3 cells only; the 6 groups on the right were formed by co-transplanting PC3 cells and PSC27 cells to form a reconstructed tissue. ^, P>0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0072] Figure 39. Statistical analysis of the terminal volume of systemic tumors. The chemotherapeutic drug VCR was used alone or in combination with rutin and the traditional anti-aging drug Vitamin C to administer drugs to mice. After the end of the 8th week, the tumor sizes of each group were compared and analyzed. The 6 groups on the left, PC3 cells only; the 6 groups on the right, PC3 cells and PSC27 cells were used to form a reconstructed tissue and transplanted. ^, P>0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. VCR, vincristine.

[0073] Figure 40 . After specifically separating cancer cells in the lesions on the sections using the LCM technique, the proportions of DNA damage and apoptosis (Caspase 3 cleaved) in each group of mice were analyzed. ^, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001. RR, Rutin / Rapamycin.

[0074] Figure 41 . The serum protein content of typical SASP factors in the circulating state in mice was detected using ELISA. On the left, AREG. On the right, EREG. RR, Rutin / Rapamycin.

[0075] Figure 42 . Statistical analysis of the terminal volume of systemic tumors (reconstructed tissue formed by breast cancer cells MDA-MB-231 and breast stromal cells HBF1203). The chemotherapeutic drug MIT was used alone or in combination with rutin and the anti-aging drug Rapamycin to administer drugs to mice. After the end of the 8th week, the tumor sizes of each group were compared and analyzed. The 6 groups on the left, MDA-MB-2313 cells only; the 6 groups on the right, MDA-MB-231 cells and HBF1203 cells were used to form a reconstructed tissue and transplanted. ^, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. DOX, doxorubicin.

[0076] Figure 43.Systematic tumor terminal volume statistical analysis (reconstructed tissue formed by breast cancer cells MDA-MB-231 and breast stromal cells HBF1203). Chemotherapeutic drug MIT alone or in combination with rutin and anti-aging drug Rapamycin was used to administer drugs to mice, and the tumor sizes of each group were compared and analyzed after the end of the 8th week. 6 groups on the left, MDA-MB-231 cells only; 6 groups on the right, reconstructed tissue formed by MDA-MB-231 cells and HBF1203 cells was transplanted. ^, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. VIN, vinblastine.

[0077] Figure 44 .Comparative analysis of the body weight data of PCa mice at the end of the treatment course under various different drug administration treatment conditions in preclinical trials. ^, P>0.05. RR, Rutin / Rapamycin.

[0078] Figure 45 .Comparative analysis of the serological data of PCa mice at the end of the treatment course under the above different drug administration treatment conditions. Creatinine, urine (kidney index), ALP and ALT (liver index) data were compared in parallel. ^, P>0.05. RR, Rutin / Rapamycin.

[0079] Figure 46 .Comparative analysis of the body weight data of immunocompetent mice (C57BL / 6J) at the end of the treatment course under various different drug administration treatment conditions. ^, P>0.05. RR, Rutin / Rapamycin.

[0080] Figure 47 .Comparative analysis of the serological data of immunocompetent mice (C57BL / 6J) at the end of the treatment course under the above different drug administration treatment conditions. Creatinine, urine (kidney index), ALP and ALT (liver index) data were compared in parallel. ^, P>0.05. RR, Rutin / Rapamycin.

[0081] Figure 48 .Comparative analysis of the counts of major types of blood cells of immunocompetent mice (C57BL / 6J) at the end of the treatment course under different drug administration treatment conditions in preclinical trials. The number of WBC, lymphocyte and neutrophil per unit volume was compared in parallel. ^, P>0.05. RR, Rutin / Rapamycin. Detailed implementation mode

[0082] After extensive and in-depth research, the present inventor has revealed that rutin can specifically target and inhibit the senescence-associated secretory phenotype (SASP), and it can act synergistically with rapamycin. After rutin and rapamycin are combined with chemotherapeutic drugs, significant synergistic effects are presented, which can promote tumor inhibition, and the promotion effect is unexpected.

[0083] In the present invention, the "individual", "subject" or "patient" refers to a mammal, particularly a human.

[0084] In the present invention, some typical "SASP factors" include IL6, IL8, AREG, IL1α, MMP1, MMP3, ANGPTL4 and SPINK1, etc.

[0085] In this article, rutin and rapamycin can also be used to reduce the drug resistance of patients to cancer therapies. The cancer therapies include chemotherapy or radiation therapy; chemotherapy such as MIT therapy, and radiation therapy such as ionizing radiation, mainly including α, β, γ and X-ray as well as proton and neutron beam therapy, etc.

[0086] The term "administer" or "administration" used in the present invention means providing the compound or pharmaceutical composition of the present invention to an object suffering from a disease or disorder to be treated or prevented or at risk thereof.

[0087] In the present invention, the term "comprising" or "including" means that various components can be applied together in the mixture or composition of the present invention. The terms "consisting essentially of..." and "consisting of..." are included in the term "comprising".

[0088] Rutin / Rapamycin

[0089] After preliminary in-depth screening and research, the present inventor has found that rutin can inhibit the expression of broad-spectrum SASP, and it inhibits SASP components such as IL6, IL8, IL1a, IL1b, CXCL3, MMP3 and GM-CSF, and has significant application potential in inhibiting the gene expression closely related to the pro-inflammatory response and secretory activity of senescent cells. At the same time, rutin inhibits the acute stress-related phenotype (ASAP) by interfering with the interaction between ATM and HIF1α and TRAF6. The research also shows that rutin can eliminate the malignancy conferred by senescent stromal cells to cancer cells in a paracrine manner.

[0090] Furthermore, the present inventor has found that rutin and rapamycin can act synergistically. When they are combined with chemotherapeutic drugs, they can effectively play a beneficial complementary role in targeting the lesion, achieving a surprising synergistic effect.

[0091] The structural formula of the rutin is as follows:

[0092]

[0093] The molecular formula of rapamycin is C51H79NO13, and its structural formula is as follows:

[0094]

[0095] In the present invention, the "compound" (including rutin, rapamycin, its salts or prodrugs, etc.) can be a compound in a pure form, or a compound with a purity greater than 85% (preferably greater than 90%, such as 95%, 98%, 99%).

[0096] Those skilled in the art should understand that after learning the structure of the compounds of the present invention, the compounds of the present invention can be obtained by various methods well-known in the art using well-known raw materials, such as chemical synthesis or extraction from organisms, and these methods are all included in the present invention. In addition, rutin or rapamycin can also be a commercial product.

[0097] The present invention also includes pharmaceutically acceptable salts, esters, isomers, solvates or prodrugs of rutin or rapamycin, as long as they also have the same or substantially the same functions as the compounds of rutin or rapamycin. In the present invention, the "pharmaceutically acceptable" components are substances that are applicable to humans and / or animals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, substances with a reasonable benefit / risk ratio. The "pharmaceutically acceptable salts" can be acid salts and base salts of rutin or rapamycin.

[0098] The "pharmaceutically acceptable acid salts" refer to salts that can maintain the biological activity and properties of the free base, and such salts will not exhibit undesirable biological activity or other changes. Such salts can be composed of inorganic acids, such as but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and similar acids. Such salts can also be composed of organic acids, such as but not limited to acetic acid, dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphorsulfonic acid, capric acid, caproic acid, octanoic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfonic acid, etc.

[0099] "Pharmaceutically acceptable basic salts" refer to salts that can maintain the biological activity and properties of the free acid, and such salts do not exhibit undesirable biological activity or other changes. These salts are prepared by adding inorganic bases or organic bases to the free acid. Salts obtained from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts and similar salts. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts and magnesium salts. Salts obtained from organic bases include, but are not limited to, primary, secondary, tertiary ammonium salts, substituted amines including naturally substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, danol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, harmine, choline, betaine, phenethylbenzylamine, N,N'-dibenzylethylenediamine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamide resins and similar structures. Preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0100] The compounds disclosed in this patent may exist in the form of solvates (such as hydrates), including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates and similar structures. In the present invention, prodrugs of rutin or rapamycin are also included, and the "prodrug" refers to a compound that, when administered by an appropriate method, is metabolized or undergoes a chemical reaction in the body of the subject to be converted into the required rutin or rapamycin.

[0101] In the present invention, isomers of rutin or rapamycin are also included. Since the compounds have one or more asymmetric centers, these compounds can exist as racemic mixtures, individual enantiomers, individual diastereoisomers, mixtures of diastereoisomers, cis or trans isomers.

[0102] Those skilled in the art should understand that after learning the structure of the compounds of the present invention, the compounds of the present invention can be obtained by a variety of methods well known in the art using known raw materials, such as chemical synthesis or extraction from organisms (such as animals or plants) or methods of modification based on extraction, and these methods are all included in the present invention. The compounds of the present invention can be synthesized using known methods; the synthesized compounds can be further purified by methods such as column chromatography and high performance liquid chromatography. In addition, the compounds of the present invention can also be obtained by commercial purchase.

[0103] Combined use of rutin and rapamycin with chemotherapeutic drugs

[0104] As described above, the present inventors have found that the combined use of rutin and rapamycin with specific chemotherapeutic drugs can effectively play a beneficial complementary role targeting diseases, achieving a surprising synergistic effect. Preferably, the chemotherapeutic drug is a chemotherapeutic drug that induces a senescence-associated secretory phenotype after administration, including mitoxantrone or bleomycin; the derivatives include pharmaceutically acceptable salts, esters, isomers, solvates or prodrugs of rutin or rapamycin.

[0105] In the screening of drugs for inhibiting SASP expression, the present inventors have found that a few NMA components exhibit significant senomorphics potential, and among them rutin is prominent. Further screening has found that the synergistic effect of rutin and rapamycin is even more prominent.

[0106] Currently, rapamycin or its derivatives are mainly used as immunosuppressive drugs in the prior art. It acts by binding to the corresponding immunophilin RMBP to inhibit the G0 and G1 phases of the cell cycle and block the entry of G1 into the S phase, and its effects are: ① inhibiting the proliferation of T and B cells; ② inhibiting the lymphocyte proliferation induced by IL-1, IL-2, IL-6 and IFN-γ; ③ inhibiting the production of IgG and donor-specific antibodies (cytotoxic antibodies); ④ inhibiting the proliferation of monocytes. The present inventors have also used it in the research of tumors in previous work. However, in this field, it has also been found that it has an opposite effect on tumors. Some studies have found that it promotes cancer, for example, it is considered to promote cancer by activating Akt (activating Akt in a variety of cancer cells through IGF-1R, mTORC2, deteriorating gliomas, etc.); some studies have also considered that it worsens the conditions of pancreatic cancer and liver cancer; some studies have also considered that rapamycin promotes cancer through the MAPK / ERK pathway, promotes cancer through PDGFRβ / MAPK, promotes cancer through MAPK / Mnk / eIF4E, etc. Therefore, there are great controversies in previous studies on the effect of rapamycin on tumors.

[0107] Rutin can inhibit the expression of broad-spectrum SASP without affecting cell senescence itself. Rutin has a significant inhibitory effect on the expression of typical SASP components, including IL6, IL8, IL1a, IL1b, CXCL3, MMP3 and GM-CSF. The analysis of the RNA-seq data set and the analysis of the transcriptome expression pattern of PSC27 show that, surprisingly, most SASP factors are indeed downregulated by rutin, although some genes not directly related to the expression state of cell senescence or SASP have also changed. The GSEA output result graph largely confirms the effect of rutin on the expression of senescent cells, indicating a significant specific inhibition of SASP. Therefore, rutin and rapamycin have significant application potential in inhibiting the expression of genes closely related to the pro-inflammatory response and secretory activity of senescent cells.

[0108] Rutin inhibits ASAP by interfering with the interaction between ATM and HIF1α and TRAF6. The potential target of rutin is downstream of ATM but upstream of TAK1, p38MAPK and other regulatory factors. Such a target would functionally involve the acute response that induces cellular senescence. Data from p-ATM-mediated IP and corresponding immunoblot analysis indicate that ATM and TRAF6 can interact, but rutin significantly weakens this interaction. More importantly, there is an interaction between ATM and HIF1α, which is also interfered with by rutin. By regulating the interaction between ATM and its key targets HIF1α and TRAF6 with the natural agent rutin, the expression of SASP can be inhibited while maintaining cellular senescence. Although rutin does not alter the production of ROS in proliferating cells, it significantly inhibits the ability of senescent cells to produce ROS, thus being basically consistent with its ability to scavenge free radicals.

[0109] Rutin can eliminate the malignancy conferred by senescent stromal cells to cancer cells in a paracrine manner. The inventors investigated the effect of rutin on the behavior of human prostate cancer (PCa) cells. After treatment with the CM of senescent PSC27 cells, a significant increase in the proliferation of several PCa cell lines, PC3, DU145, M12 and LCaP, was observed, accompanied by enhanced migration and invasion. However, after treating cancer cells with rutin, these acquired functions (migration and invasion) almost completely disappeared. After exposure to the CM derived from senescent stromal cells, the drug-resistant viability of cancer cells increased significantly, but after adding rutin, the drug resistance decreased by approximately 80%. Therefore, rutin can significantly deprive senescent stromal cells of the drug resistance they confer on cancer cells. When rutin is used in combination with rapamycin, a more significant decrease in the malignant activity of cancer cells can be further achieved based on the inhibitory effect produced by rutin.

[0110] Rutin combined with chemotherapy can improve the efficacy of anticancer treatment. Although MIT alone caused tumor shrinkage in tumors containing only PC3 cells, the administration of senomorphics did not show a significant effect; even when these drugs were used in combination with MIT, no further benefit was brought, indicating that PC3 tumor growth is independent of SASP at the tissue level, especially in the absence of stromal cells. Notably, when PC3 cells were combined with their stromal cells to form tissue reconstructions, the inventors observed a significant increase in tumor volume (p < 0.0001), further verifying the tumor-promoting effect of stromal cells in vivo. When animals carrying PC3 / PSC27 tumors were exposed to MIT, the tumor volume decreased significantly (35.5%, p < 0.001). After the dual treatment of rapamycin or rutin combined with MIT, the tumors showed further shrinkage, and this shrinkage in volume was extremely significant. Among them, the dual treatment of rapamycin and MIT caused the tumor volume to shrink further by 34.1% compared with MIT alone (p < 0.01); while the dual treatment of rutin and MIT caused the tumor volume to shrink further by up to 48.9% compared with MIT alone (p < 0.0001). This shows that the combination of rutin and MIT has a more excellent effect than the combination of rapamycin and MIT. And when rapamycin and rutin were jointly used for treating tumors with the chemotherapy drug MIT, the intervention achieved the best effect, and the tumor volume shrank further on the basis of MIT monotherapy (68.3%, p < 0.0001), which was unexpected. No such effect was observed when rutin was used in combination with some other drugs such as the antioxidant vitamin C. Rutin and rapamycin do not achieve this effect when combined with all chemotherapy drugs. For example, no benefit was observed when vinblastine (VIN) was used as a chemotherapy drug.

[0111] MIT itself can cause significant DNA damage and apoptosis in cancer cells. In PC3 / PSC27 xenografts, treatment with rutin alone neither caused a typical DDR nor enhanced cell death, indicating that the response of these tumors was limited when animals were only exposed to rutin. After rutin was used in combination with MIT, the apoptosis rate increased significantly. When rutin and rapamycin were simultaneously combined with MIT, the indices of DNA damage and apoptosis could be further increased on the basis of the combination of rutin and MIT, and in this case, the clearance of cancer cells in the lesion reached the best effect.

[0112] When rutin was used together with MIT, the autoproteolysis of Caspase 3 was enhanced. MIT-mediated chemotherapy led to an increase in the levels of AREG and EREG proteins in the circulating blood of animals, and this pattern was basically reversed in the case of using rutin. Once rutin was combined with rapamycin, it could further reduce the protein levels of AREG and ERE in the blood of mice on the basis of rutin alone used with MIT.

[0113] The research results of multiple types of tumors also show that the intervention effect of the SASP-targeted strategy on tumor drug resistance is not limited to specific cancer types, but may also be applicable to a wider range of various solid malignant tumors.

[0114] Based on the above new findings of the present inventors, the present invention provides a use of rutin and rapamycin for preparing a composition for specifically targeting and inhibiting the senescence-associated secretory phenotype (SASP) and inhibiting tumors and / or reversing tumor drug resistance.

[0115] As used in the present invention, unless otherwise specified, the "tumor" refers to a tumor that generates a senescence-associated secretory phenotype in the tumor microenvironment after treatment with genotoxic drugs, and / or a tumor that develops drug resistance after treatment with genotoxic drugs. For example, it includes: prostate cancer, breast cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, bladder cancer, skin cancer, kidney cancer, esophageal cancer, cholangiocarcinoma, brain cancer.

[0116] As used in the present invention, unless otherwise specified, the "chemotherapy drug" refers to a chemotherapy drug that induces a senescence-associated secretory phenotype (SASP) in the tumor microenvironment after administration. Preferably, it is mitoxantrone or bleomycin.

[0117] In some embodiments of the present invention, the "senescence-associated secretory phenotype" is the senescence-associated secretory phenotype that occurs under DNA damage; preferably, the DNA damage is DNA damage caused by chemotherapy drugs; more preferably, the chemotherapy drugs include genotoxic drugs.

[0118] Drug screening

[0119] After learning about the close correlation between rutin and rapamycin and the tumor microenvironment or SASP and their working mechanisms, drugs with further optimized inhibitory effects can be screened based on this feature. Drugs that truly act on senescent cells in the tumor microenvironment and are useful for inhibiting tumors, reversing tumor drug resistance, or inhibiting / delaying the senescence-associated secretory phenotype can be found from the substances. Or one or more substances that can be combined with rutin and rapamycin to exert a synergistic effect can be found from the substances.

[0120] Therefore, the present invention provides a method for screening potential substances that promote the inhibition of tumors by chemotherapeutic drugs, and the method includes: (1) providing a tumor microenvironment system, which includes tumor cells and stromal cells; (2) treating the system of (1) with chemotherapeutic drugs to induce the senescence-associated secretory phenotype in the tumor microenvironment; (3) adding a candidate substance to the system of (2) and observing its effect on the tumor microenvironment system. If it can specifically target and inhibit the senescence-associated secretory phenotype and / or promote the growth of stromal cells (non-senescent cells), that is, increase the proliferation rate of stromal cells, then it is a potential substance that promotes the inhibition of tumors by chemotherapeutic drugs. In step (2), it also includes: treating with rutin and rapamycin before, during, or after inducing the senescence-associated secretory phenotype in the tumor microenvironment; in step (3), it also includes: if the candidate substance can statistically promote rutin and rapamycin to clear senescent cells in the tumor microenvironment and / or promote the growth of stromal cells, then the candidate substance is a potential substance that can be used in combination with rutin and rapamycin to inhibit tumors.

[0121] The present invention also provides a method for screening potential substances that inhibit the senescence-associated secretory phenotype, and the method includes: (1) providing a stromal cell system and inducing the senescence-associated secretory phenotype in the system; treating with rutin and rapamycin before, during, or after the senescence-associated secretory phenotype; (2) adding a candidate substance to the system of (1) and observing its effect on the stromal cell system. If it can specifically promote the inhibitory effect of rutin and rapamycin on the senescence-associated secretory phenotype, then the candidate substance is a potential substance that can be used in combination with rutin and rapamycin to inhibit the senescence-associated secretory phenotype.

[0122] In a preferred embodiment of the present invention, when performing the screening, in order to more easily observe the changes in the corresponding indicators in the test group, a control group can also be set up, and the control group can be a system that does not add the candidate substance but has the same other conditions as the test group.

[0123] As a preferred embodiment of the present invention, the method further includes: performing further cell experiments and / or animal tests on the obtained potential substances to further select and determine substances that are truly useful for inhibiting tumors, reversing tumor drug resistance, or inhibiting / delaying the senescence-associated secretory phenotype.

[0124] On the other hand, the present invention also provides potential substances that inhibit tumors, reverse tumor drug resistance, or inhibit / delay the senescence-associated secretory phenotype obtained by using the screening method. These preliminarily screened substances can form a screening library, so that people can finally screen out truly useful drugs from them.

[0125] Drug combination

[0126] The present invention provides a pharmaceutical composition, which contains an effective amount (such as 0.00001 - 50 wt%; preferably 0.0001 - 20 wt%; more preferably, 0.001 - 10 wt%) of the above-mentioned rutin and rapamycin, a chemotherapeutic drug (such as 0.000001 - 20 wt%; preferably 0.00001 - 10 wt%; more preferably, 0.0001 - 2 wt%), and a pharmaceutically acceptable carrier. In addition, it should be understood that for the convenience of clinical administration or according to the requirements of the clinical treatment plan, the mixing of the above-mentioned rutin, rapamycin and chemotherapeutic drug is not necessary, and they can also be independently placed in separate containers, placed in a kit or medicine box, and used in combination when needed. The chemotherapeutic drug is a chemotherapeutic drug that induces the senescence-associated secretory phenotype after administration, preferably including mitoxantrone or bleomycin.

[0127] As used herein, the term "effective amount" refers to an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0128] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents. This term refers to such pharmaceutical carriers: they are not necessarily the active ingredients themselves and have no excessive toxicity after administration. Suitable carriers are well-known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the composition may contain liquids, such as water, saline, buffers. In addition, auxiliary substances may also be present in these carriers, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, etc. The carrier may also contain a cell transfection reagent. Forms of the drug suitable for injection include: sterile aqueous solutions or dispersions and sterile powders (for the temporary preparation of sterile injection solutions or dispersions). In all cases, these forms must be sterile and must be fluid to facilitate the expulsion of the fluid by a syringe. They must be stable under the manufacturing and storage conditions and must be able to prevent the contamination effects of microorganisms (such as bacteria and fungi).

[0129] As used herein, the term "comprising" or "including" includes "containing", "consisting essentially of", and "consisting of". The term "consisting essentially of" means that in the composition, in addition to containing the main active ingredients (such as rutin, rapamycin and chemotherapeutic drugs), it may also contain a small amount of minor ingredients and / or impurities that do not affect the active ingredients. For example, it may contain a sweetening agent to improve the taste, an antioxidant to prevent oxidation, and other commonly used additives in the art.

[0130] It should be understood that after learning the uses of rutin and rapamycin and their working mechanisms in the tumor microenvironment or SASP environment, various methods well-known in the art can be used to administer rutin, rapamycin, and / or chemotherapeutic drugs to mammals or humans. These methods are all encompassed by the present invention.

[0131] The dosage forms of the composition of the present invention can be diverse, as long as they can effectively deliver the active ingredients to the mammalian body. For example, they can be selected from: injections, tablets, capsules, powders, granules, syrups, solutions, suspensions, tinctures, oral liquids, or aerosols.

[0132] The effective amounts of rutin and rapamycin of the present invention can vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art based on various factors (such as through clinical trials). Such factors include, but are not limited to: the pharmacokinetic parameters of rutin and rapamycin such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's weight, the patient's immune status, the route of administration, etc.

[0133] In specific embodiments of the present invention, some dosing regimens for animals such as mice are given. Converting the dosing doses for animals such as mice to dosing doses applicable to humans is easily done by those skilled in the art. For example, it can be calculated according to the Meeh-Rubner formula: Meeh-Rubner formula: A = k × (W 2 / 3 ) / 10,000. In the formula, A is the body surface area, calculated in m 2 ; W is the body weight, calculated in g; K is a constant, which varies with the animal species. Generally speaking, for mice and rats it is 9.1, for guinea pigs it is 9.8, for rabbits it is 10.1, for cats it is 9.9, for dogs it is 11.2, for monkeys it is 11.8, and for humans it is 10.6. It should be understood that depending on the drug and the clinical situation, the conversion of the dosing dose can vary according to the evaluation of an experienced pharmacist.

[0134] The pharmaceutical composition of the present invention can also be formulated in the form of unit dosage forms for convenient and regular and quantitative medication.

[0135] As used herein, the terms "unit dosage form" and "unit form" refer to the dosage form prepared from the composition of the present invention for convenient administration, which is the dosage form required for single administration, including but not limited to various solid forms (such as tablets) and liquid forms. The unit dosage form contains the composition of the present invention in an amount suitable for single, daily, or unit-time administration.

[0136] In some preferred embodiments of the present invention, the composition is in unit dosage form. When the composition is prepared in unit dosage form, one dose of the composition in unit dosage form is taken every few days or weeks.

[0137] The present invention also provides a kit containing the above-mentioned pharmaceutical composition or directly containing rutin, rapamycin and / or chemotherapeutic drugs as described above. In addition, the kit may further include an instruction manual for explaining the usage method of the drugs in the kit.

[0138] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, such as those described in "Molecular Cloning: A Laboratory Manual", Third Edition, edited by J. Sambrook et al., Science Press, or according to the conditions recommended by the manufacturer.

[0139] Materials and Methods

[0140] 1. Cell culture

[0141] (1) Cell line maintenance

[0142] The normal human prostate primary stromal cell line PSC27 (obtained from Fred Hutchinson Cancer Research Center, USA) was cultured in an incubator at 37 °C and 5% CO2, and proliferated and passaged in PSCC complete culture medium (Gibco, USA, or the same product of Thermo Fisher).

[0143] (2) Cell cryopreservation and resuscitation

[0144] a. Cell cryopreservation

[0145] Cells in the logarithmic growth phase were collected with 0.25% trypsin, centrifuged at 1000 rpm for 2 min, the supernatant was discarded, and the cells were resuspended in freshly prepared cryopreservation solution. The cells were aliquoted into labeled sterile cryopreservation tubes. Then, after gradient cooling, they were finally transferred to liquid nitrogen for long-term storage.

[0146] b. Cell resuscitation

[0147] The cells cryopreserved in liquid nitrogen were taken out and immediately placed in a 37 °C water bath to rapidly thaw. 2 ml of cell culture medium was directly added to make the cells evenly suspended. After the cells adhered to the wall, the culture medium was replaced with a new one.

[0148] (3) In vitro experimental treatment

[0149] To cause cell damage, when the PSC27 cells grew to 80% (abbreviated as PSC27-CTRL), 50 μg / ml bleomycin (BLEO) was added to the culture medium. After 12 hours of drug treatment, the cells were simply washed 3 times with PBS and left in the culture medium for 7 - 10 days, and then subsequent experiments were carried out.

[0150] 2. Screening of natural product library

[0151] Pharmacodynamic analysis was performed on a natural product library (NMA) (Shyuanye Biotechnology) with a large number of various components, mostly medicinal plant extracts (including substances from individual animal sources) and having anti-aging potential. Various products were diluted to 96-well plates at certain concentration gradients, with a density of 5000 cells per well. DMEM was used as the culture medium, and the working concentration of natural products (or compounds) was generally controlled at 1 μM - 1 mM. 3 - 7 days after drug treatment, cell proliferation was measured using the CCK-8 Cell Counting Kit (based on the WST-8 principle, Vazyme), and cell apoptosis activity was determined using the Caspase 3 / 7 activity kit (Promega).

[0152] The initially identified candidate drugs were further screened for 30 days. The drugs entering the second-round candidate range were diluted into 6-well plates, with 20,000 cells per well. The culture medium and candidate drugs were changed every other day. To determine the effects of each drug on cell phenotype and viability, etc., the project conducted confirmatory analysis according to different concentrations of the drugs.

[0153] 3. Immunoblotting and immunofluorescence detection

[0154] Cell lysate-derived proteins were separated using NuPAGE 4 - 12% Bis-Tris gel and transferred onto nitrocellulose membranes (Life Technologies). The blots were blocked with 5% non-fat milk at room temperature for 1 h, incubated overnight at 4°C with the required primary antibody at the concentration specified by the manufacturer's protocol, then incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz) at room temperature for 1 h. Membrane blot signal detection was carried out using an enhanced chemiluminescence (ECL) detection reagent (Millipore) according to the manufacturer's protocol, and an ImageQuant LAS 400 Phospho-Imager (GE Healthcare) was used. As a standard protein marker, the inventors used the PageRuler Plus Prestained Protein Ladder (no. 26619) provided by Thermo Fisher Scientific.

[0155] For immunofluorescence staining, target cells were pre-seeded on coverslips for at least 24 h after culturing in a petri dish. After a brief wash, the cells were fixed with 4% paraformaldehyde in PBS for 8 min and blocked with 5% normal goat serum (NGS, ThermoFisher) for 30 min. Mouse monoclonal antibody anti-phospho-Histone H2A.X (Ser139) (clone JBW301, Millipore) and mouse monoclonal antibody anti-BrdU (Cat#347580, BD Biosciences), and the secondary antibody Alexa 488 (or 594)-conjugated F(ab’)2 were added sequentially to the slides coated with fixed cells. The nuclei were counterstained with 2 μg / ml of 4’,6-diamidino-2-phenylindole (DAPI). The most representative image was selected from three observation fields for data analysis and result presentation. An FV1000 laser scanning confocal microscope (Olympus) was used to obtain confocal fluorescence images of the cells.

[0156] 4. Whole transcriptome sequencing analysis (RNA-sequencing)

[0157] Whole transcriptome sequencing was performed on the human primary prostate stromal cell line PSC27 under different treatment conditions. Total RNA samples were obtained from the stromal cells. Their integrity was verified by Bioanalyzer 2100 (Agilent). The RNA was sequenced using Illumina HiSeq X10, and gene expression levels were quantified by the software package rsem (https: / / deweylab.github.io / rsem / ). Briefly, rRNA in the RNA samples was removed using the RiboMinus Eukaryote kit (Qiagen, Valencia, CA, USA); and strand-specific RNA-seq libraries were constructed using TruSeq Stranded Total RNA preparation kits (Illumina, San Diego, CA, USA) according to the manufacturer's instructions before deep sequencing.

[0158] Paired-end transcriptomic reads were mapped to the reference genome (GRCh38 / hg38), and reference annotation was performed using the Bowtie tool from Gencode v27. Duplicate reads were identified using the picard tools (1.98) script to mark duplicates (https: / / github.com / broadinstitute / picard), and only non-duplicate reads were retained. Reference splice junctions were provided by the reference transcriptome (Ensembl build 73). FPKM values were calculated using Cufflinks, and differential gene expression was called using the maximum likelihood estimation function of Cufflinks. Genes with significantly changed expression were defined by a false discovery rate (FDR)-corrected P value < 0.05, and only ensembl genes 73 with status "Known" and biotype "coding" were used for downstream analysis.

[0159] Next, Reads were trimmed using Trim Galore (v0.3.0) (http: / / www.bioinformatics.babraham.ac.uk / projects / trim_galore / ), and quality assessment was performed using FastQC (v0.10.0) (http: / / www.bioinformatics.bbsrc.ac.uk / projects / fastqc / ). Subsequently, the DAVID bioinformatics platform (https: / / david.ncifcrf.gov / ) and the Ingenuity Pathways Analysis (IPA) program (http: / / www.ingenuity.com / index.html) were utilized. Preliminary analysis of the raw data was performed on the free online platform of the Majorbio I-Sanger Cloud Platform (www.i-sanger.com), and the raw data was deposited in the NCBI Gene Expression Omnibus (GEO) database with the accession code GSE156448.

[0160] 5. Protein-protein interaction network analysis

[0161] Protein-protein interaction (PPI) analysis was performed using STRING 3.0. Specific proteins meeting the criteria were imported into the online analysis software (http: / / www.networkanalyst.ca), and a minimum interaction network was selected for further hub and module analysis.

[0162] 6. Gene Set Enrichment Analysis (GSEA)

[0163] Based on the data obtained from the preliminary RNA-seq analysis, for each significantly differentially expressed gene, the genes were ranked using the "wald statistics" obtained from DESeq2, and GSEA was performed on these ranked lists of all the curated gene sets available in MSigDB (http: / / software.broadinstitute.org / gsea / msigdb). DESeq2 independent filtering was based on the mean of the normalized read counts to filter out genes with very low expression levels. The SASP and GSEA signatures were as described in the inventors' previous published literature (Zhang et al., 2018a).

[0164] 7. Quantitative PCR (RT-PCR) for measuring gene expression

[0165] (1) Extraction of total cellular RNA

[0166] It was extracted with Trizol reagent. After quantifying the RNA with a spectrophotometer, a small amount of total RNA was taken for 1% agarose gel electrophoresis to check the RNA status and quality.

[0167] (2) Reverse transcription reaction

[0168] (3) Real-time quantitative PCR reaction

[0169] After the reaction was completed, the amplification of each gene was viewed by software analysis, and the corresponding threshold cycle number was exported. The 2-ΔΔCt method was used to calculate the relative expression level of each gene. The peaks and waveforms of the melting curve were analyzed to determine whether the amplified product obtained was a specific single target fragment.

[0170] 8. SA-β-Gal staining

[0171] Senescence-associated β-galactosidase (SA-β-Gal) staining was performed according to the previously reported protocol (Debacq-Chainiaux et al., 2009). Briefly, cells in culture dishes were washed with PBS and fixed at room temperature. Cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde for 3 min. Then, SA-β-Gal was stained with freshly prepared staining solution overnight at 37 °C. Images were taken the next day and the percentage of positive cells per unit area was calculated.

[0172] 9. Clonal expansion assay

[0173] Single-cell clonal expansion assay. Briefly, cells were plated in gelatin-coated 12-well plates at a density of 2000 cells / well. The number of cell clones was calculated after crystal violet staining.

[0174] 10. Drug-induced senescent cell apoptosis

[0175] PSC27 cells were plated in 96-well dishes and induced to senesce under treatment with 50 μg / ml BLEO. Rutin and PCC1 were added at concentrations of 100 μM and 50 μM, respectively. The cell culture medium was supplemented with Incucyte Nuclight Rapid Red reagent (Essen Bioscience) and Incucyte C-3 / 7 apoptosis reagent (Essen Bioscience). Representative fields were photographed.

[0176] 11. Mouse xenograft inoculation and preclinical treatment trials

[0177] All experiments involving mice were conducted in strict accordance with the relevant regulations for laboratory animals. Immunodeficient mice (NOD-SCID mice, ICR) aged 6 - 8 weeks (body weight approximately 25 g) were used for the animal experiments related to this invention. Stromal cells PSC27 and epithelial cells PC3 were mixed at a pre-determined ratio of 1:4, and each graft contained 1.25×10 6 cells for tissue reconstruction. Xenografts were implanted subcutaneously into mice, and the animals were euthanized at the end of the 8th week after the transplantation surgery. The tumor volume was calculated according to the following formula: V = (π / 6) x ((l + w) / 2) 3 (V, volume; l, length; w, width).

[0178] In preclinical treatment trials, mice transplanted subcutaneously were fed a standard experimental diet. After 2 weeks, the chemotherapeutic drug mitoxantrone (MIT, at a dose of 0.2 mg / kg) and / or rutin (500 μl, at a dose of 10 mg / kg), rapamycin (500 μl, at a dose of 10 mg / kg) were administered intraperitoneally. The time points were as follows: all three drugs were administered to the mice on the first day of weeks 3, 5, and 7. The entire treatment course consisted of 3 cycles of drug administration, with each cycle lasting 2 weeks. After the treatment course ended, the mouse tumors were collected for volume measurement and histological analysis. Each mouse received a cumulative dose of 0.6 mg / kg body weight of MIT, 30 mg / kg body weight of rutin, and 30 mg / kg body weight of rapamycin. To induce the expression of SASP factors systemically under chemotherapy, MIT was administered to the mice via intravenous infusion according to the above steps and sequence, but the dose was reduced to 0.1 mg / kg body weight per administration (the cumulative dose of MIT received throughout the treatment course was 0.3 mg / kg body weight) to reduce drug-related toxicity. The chemotherapy trial ended at the end of the 8th week. After the mice were sacrificed, they were immediately dissected, and their transplanted tumors were collected and used for pathological system analysis. For mice bearing breast cancer tumors, the preclinical procedures were the same as above, and the chemotherapeutic drugs doxorubicin (DOX, at a dose of 1.0 mg / kg) and / or rutin (500 μl, at a dose of 10 mg / kg), rapamycin (500 μl, at a dose of 10 mg / kg) were administered intraperitoneally. For alternative chemotherapeutic drugs, vincristine or vinblastine (at a dose of 1.0 mg / kg) was administered intraperitoneally.

[0179] 12. Biostatistical methods

[0180] In this invention, all in vitro experiments involving cell proliferation rate, survival rate, SA-β-Gal staining, etc., and in vivo experiments on mouse transplanted tumors and preclinical drug treatments were repeated more than 3 times, and the data were presented in the form of mean ± standard error. Statistical analysis was based on the original data and was calculated by one-way analysis of variance (ANOVA) or a two-tailed Student’s t-test, and results with P < 0.05 were considered to have significant differences.

[0181] The correlations between factors were examined using Pearson’s correlation coefficients. When mice were obtained and grouped in cages across several cohorts, survival analysis was performed using the Cox proportional hazard model. This model took the sex and age of the treatment as fixed effects, and the cohort and initial cage assignment as random effects. Since, in the study, some mice were moved from their initial cages to minimize stress from single-cage housing, the inventors also performed an analysis without cage effects. The results of these two analyses did not differ greatly in directionality or statistical significance, enhancing confidence in the inventors' results. Survival analysis was performed using statistical software R (version 3.4.1; library ‘coxme’). In most experiments and result evaluations, the researchers were blind to the assignments. The inventors assigned mice to experimental groups using baseline body weight (to achieve similar body weights between groups), and thus randomization was only performed within groups matched for body weight. All replicates in the present invention were from different samples, with each sample from a different experimental animal.

[0182] Example 1: Drug screening shows that rutin is a potential anti-aging small molecule drug

[0183] To identify new compounds that can effectively target senescent cells, the inventors conducted unbiased drug screening using a library consisting of a collection of many natural ingredients (NMAs), most of which are phytochemicals. For this purpose, the inventors selected a human primary prostate stromal cell line, PSC27, as a cell model. PSC27 consists mainly of fibroblasts, but also contains a small number of non-fibroblast cells such as smooth muscle cells and endothelial cells. This cell line forms a typical SASP when exposed to stressors such as genotoxic chemotherapy and ionizing radiation. To induce senescence, cells were treated with a pre-optimized and established sub-lethal dose of bleomycin (BLEO) (50 μg / ml), and an increase in positive staining for senescence-associated β-galactosidase (SA-β-GAL), a decrease in BrdU incorporation, and an enhanced DNA damage response (DDR) were observed. A screening strategy was established to compare the effects of the individual drugs tested on the survival and expression profiles of senescent cells ( Figure 1 ).

[0184] The inventors first determined the efficacy of these drug components in the NMA library on senescent PSC27 and explored their potential as an experimental cell model for large-scale drug screening. The inventors' preliminary data indicated that many compounds were able to alter the biological activity of senescent cells (SEN), but not affect proliferating cells ( Figures 2 - 4)。This property means that PSC27 is an ideal and qualified model for subsequent experimental exploration because it allows selective targeting of senescent cell populations rather than growing control cell populations, largely ensuring the feasibility of further in-depth research using this human-derived matrix line. In a large-scale screening of the NMA library, the inventors did not even identify a single senolytic, which is considered to have the potential to selectively kill senescent cells in culture, such as the positive control procyanidin C1( Figure 2 、 3 ), indicating a severe scarcity of natural anti-aging drugs in nature and the difficulty of expanding the substance reserve of this specific class of anti-aging drugs in reality.

[0185] However, the inventors observed that a few NMA components exhibited significant senomorphics potential, which is indeed worthy of further study( Figure 5 、 6 ). Among them, rutin is particularly prominent( Figure 5 、 7 ).

[0186] Among the senolytic agents that have a significant effect on downregulating the expression of a marker factor of SASP, namely interleukin 8 (IL8), the inventors selected rutin for in-depth analysis( Figure 7 、 8 ). As a flavonoid extracted from wild plants, rutin has a wide range of biological activities, and its effects are mainly focused on the protection against hyperglycemia, kidney disease, neuropathy, and cardiovascular diseases. However, its medical significance, including regulating the activity of senescent cells, especially SASP expression, and related therapeutic capabilities and functional mechanisms, still remains an unknown and largely blank field to date.

[0187] Example 2: Rutin can inhibit the expression of broad-spectrum SASP without affecting cell senescence itself

[0188] As mentioned above, rutin inhibits the expression of the SASP marker factor IL8, but it is still unclear whether it affects cell senescence and SASP in a broad sense, or the vast majority of other SASP factors. To answer these questions, the inventors conducted in vitro tests and found that SA-β-GAL staining and BrdU incorporation remained basically unchanged regardless of cell proliferation or senescence, with the latter induced by bleomycin (BLEO), a genotoxic drug commonly used in clinical oncology for cancer patients( Figure 9 、 10 ).

[0189] Among the cultures with different concentrations used by the inventors, rutin at 100 μM produced the best inhibitory effect on suppressing the expression of typical SASP components, including IL6, IL8, IL1a, IL1b, CXCL3, MMP3, and GM-CSF( Figure 11 ).

[0190] In addition, analysis of the RNA-seq dataset, analyzing the transcriptome expression pattern of PSC27 showed that, surprisingly, most SASP factors were indeed downregulated by rutin, although some genes not directly related to the expression status of cellular senescence or SASP were also altered( Figure 12 ).

[0191] As supporting evidence, the GSEA output result graph largely confirmed the effect of rutin on the expression of senescent cells, indicating significant specific inhibition of SASP( Figure 13 ).

[0192] Further bioinformatics analysis showed that 4619 transcripts were significantly modified in rutin-exposed senescent PSC27 cells under culture conditions (based on a 2-fold change in log2, p < 0.01) (3733 downregulated, 886 upregulated)( Figure 14 ).

[0193] After mapping the transcripts to the Gene Ontology (GO) database containing HPRD, Entrez gene, and UniPROT accession identifiers, the inventors noticed that the most prevalent molecular functions (top 100 representatives) of the upregulated genes were cytokine activity, metallopeptidase activity, structural molecule activity, and receptor activity( Figure 15 ).

[0194] For the proteins encoded by these genes, the most typical cellular components in biological activity are those transported to the extracellular space, followed by those located in the cytoplasm and nucleus, although many products do belong to the subclasses of soluble components, extracellular space, extracellular region, and exosomes( Figure 16 ). In addition, the most prominent biological processes associated with the upregulated genes are intercellular communication, inflammatory response, cell growth and / or maintenance, regulation of base, nucleoside, nucleotide, and nucleic acid metabolism( Figure 17 ).

[0195] In summary, the inventors' data indicate that rutin has significant application potential in inhibiting the gene expression closely related to the pro-inflammatory response and secretory activity of senescent cells.

[0196] Example 3: Rutin inhibits ASAP by interfering with the interaction between ATM and HIF1α and TRAF6

[0197] Next, the inventors explored the molecular mechanisms underlying the impact of rutin on the development of cell senescence-related phenotypes, particularly SASP. To evaluate the potential role of rutin in intracellular DDR signaling, an event that permits genomic DNA damage to activate downstream inflammatory responses.

[0198] The inventors first collected total lysates of PSC27 cells before and after treatment with rutin in proliferating or senescent cells. Immunoblotting results showed that ATM, one of the central regulators of DDR signaling, was significantly activated upon BLEO-induced senescence ( Figure 18 ).

[0199] In response to intrinsic or environmental stimuli, proliferating cells tend to first exhibit acute stress-related phenotypes (ASAP), including processes involving ATM activation and nuclear-to-cytoplasmic translocation, TRAF6-mediated monoubiquitination, and TAK1 phosphorylation, which can occur during the stress damage process observable within 2 - 3 days after cell exposure. As an important component of the cell response induced by genotoxic stress and a key regulator of ASAP, in the experiment, the cytoplasmic kinase TAK1 was significantly phosphorylated, a change that functionally primed it to subsequently participate in a double feed-forward mechanism to coordinate the development of SASP.

[0200] The inventors further noted that the activation of the TAK1 downstream target p38MAPK, the PI3K / AKT / mTOR signaling axis (an intermediary of sustained SASP signaling, represented by mTOR and AKT phosphorylation), and the upregulation of the ASAP marker molecule IL8 (also a marker of SASP in most cell lines) occurred in an acute manner after BLEO treatment ( Figure 18 ).

[0201] However, when rutin was present, these significant changes were generally reduced, except that ATM phosphorylation was largely unaffected, indicating that the potential target of rutin was downstream of ATM but upstream of TAK1, p38MAPK, and other regulators, and such a target would functionally involve inducing the acute response of cell senescence ( Figure 18 )).

[0202] During this process, there was a direct interaction link between activated TRAF6 and TAK1, a phenomenon that occurred shortly after DNA damage but was inhibited by the TAK1 inhibitor 5Z-7-oxozeaenol. However, it was unclear whether rutin disrupted this process. The inventors chose to perform phosphorylation TAK1 (p-TAK1)-mediated IP and subsequent immunoblot analysis and found that both the activation of TAK1 and the TRAF6-TAK1 interaction were significantly inhibited by rutin ( Figure 19)。These data confirm that the direct targets of rutin should extend beyond the interaction between TRAF6 and TAK1.

[0203] To identify the mechanism of action of rutin in the rapid process, where early responses ultimately lead to SASP formation as a chronic phenomenon, the inventors performed a genome-wide localization of molecules that are likely to interact with ATM. Bioinformatics analysis revealed 279 unique ATM interactors and 379 unique TRAF6 interactors in human cells, with 22 ATM and TRAF6 interacting molecules shared in the final output. Further research excluded the need to explore the vast majority of molecules that can interact with ATM and TRAF6, and hypoxia-inducible factor 1α (HIF1α) is a candidate molecule worthy of further investigation.

[0204] Data from p-ATM-mediated IP and corresponding immunoblot analysis indicate that ATM and TRAF6 can interact, but rutin undoubtedly significantly weakens this interaction. More importantly, there is an interaction between ATM and HIF1α, which is also disrupted by rutin ( Figure 20 ).

[0205] As further evidence, the inventors observed a significant cytoplasmic-nuclear translocation of the two major subunits, p65 and p50, of the NF-κB transcription complex during cellular senescence, although this trend was largely eliminated in the presence of rutin ( Figure 21 ). Notably, HIF1α exhibited a nuclear translocation similar to that of p65 and p50 ( Figure 21 ), suggesting that this factor is involved in regulating the expression of genes associated with senescence and / or senescence-related phenotypes, particularly SASP. Like the transcription complex NF-κB, HIF1α is a key transcription factor during the adaptive response, coordinating the transcription of many genes involved in angiogenesis, erythropoiesis, glycolytic metabolism, and inflammation, while its significance in senescence remains under-explored. HIF1α, as a central factor that transmits the damage signal from ATM to the nucleus, is responsible for the upregulation of a series of factors crucial for the maintenance of senescence and the development of SASP.

[0206] To confirm this speculation, the inventors evaluated the expression of a subset of genes encoding SASP factors or senescence-specific markers. Data showed that both the selective HIF-1α inhibitor PX-478 and C25-140, a small molecule compound that reduces TRAF6-mediated ubiquitin chain formation, significantly decreased the expression of typical SASP factors detected in the experiment ( Figure 22 ). However, p16 INK4A and p21 CIP1The expression of [seems to be unaffected. By regulating the interaction between ATM and its key targets HIF1α and TRAF6 with the natural preparation rutin, the expression of SASP can be inhibited while maintaining cellular senescence, and this unique feature is largely consistent with the criteria of the senescent morphology.

[0207] In the genotoxic damage caused by BLEO, PSC27 cells showed a significantly elevated ROS level ( Figure 23 ). Although rutin did not alter the production of ROS in proliferating cells, it significantly inhibited the ability of senescent cells to produce ROS, thus being basically consistent with its ability to scavenge free radicals.

[0208] Example 4. Rutin can eliminate the malignancy conferred by senescent stromal cells to cancer cells in a paracrine manner

[0209] After treatment with the CM of senescent PSC27 cells, the inventors observed a significant increase in the proliferation of several PCa cell lines, PC3, DU145, M12, and LCaP (p < 0.01) ( Figure 24 ), accompanied by enhanced migration and invasion ( Figure 25 , Figure 26 ). However, after treating cancer cells with rutin, these gain-of-function effects were significantly reduced ( Figures 24 - 26 ). The final concentration of rutin in the experiment was 100 μM. To further improve the inhibitory effect, the inventors further conducted drug screening. After analyzing a large number of candidate drugs, they finally focused on rapamycin and found that its combination with rutin had an obvious synergistic effect. When rutin was used in combination with rapamycin, it could further cause a more significant decrease in the malignant activity of cancer cells based on the inhibitory effect produced by rutin ( Figures 24 - 26 ).

[0210] Some factors of SASP, such as WNT16B, SFRP2, SPINK1, and AREG, can confer significant drug resistance to cancer cells. However, it is still uncertain whether the resistance-promoting ability of SASP can be blocked by rutin. The inventors found that after exposure to the CM derived from senescent stromal cells, the drug-resistant viability of cancer cells increased significantly, but after adding rutin (final concentration of 100 μM), the drug resistance decreased by approximately 80% ( Figure 27 ).

[0211] Although PSC27-BLEO CM increased the viability of PC3 exposed to 0.1 - 1.0 μM chemotherapeutic drug MIT, and MIT was close to the serum concentration of patients in the clinical background under this experimental condition, rutin (final concentration of 100 μM) could significantly inhibit the cancer cell resistance conferred by the CM of senescent stromal cells ( Figure 28)。Furthermore, surprisingly, when rapamycin is used in combination with rutin, better effects can be produced, and even the acquired drug resistance characteristics of cancer cells are basically restored to the state of the control group (i.e., the primary PSC27 cell group) (according to Figure 28 it can be seen that its curve basically coincides with the curve of the PSC group), which is quite unexpected.

[0212] Therefore, rutin can significantly deprive senescent stromal cells of the drug resistance they endow cancer cells with, and when rapamycin is used in combination with rutin, there is a synergistic effect, that is, this deprivation ability is further enhanced, providing a basis for developing new treatment regimens to improve the anti-cancer effect.

[0213] Example 5. Rutin combined with chemotherapy can improve the efficacy of anti-cancer treatment in preclinical trials

[0214] In view of the effects of rutin on the cancer cell expression profile and in vitro phenotype, the inventors next asked about the therapeutic effects that rutin might show under in vivo conditions. To address this issue, the inventors generated tissue reconstructions by mixing PSC27 cells and PC3 cells at a pre-optimized ratio (1:4) before subcutaneous implantation into the posterior abdomen of severe combined immunodeficiency (SCID) experimental mice. At the end of an 8-week period, the tumor size of the animals was measured. Compared with the tumors generated by PC3 and primary naive PSC27 cells (PSC27S Naive ), the xenografts composed of PC3 and PSC27 SEN showed a significant increase ( Figure 29 ). However, the tissue reconstructions generated from pre-treated PSC27 SEN cells in vitro showed a significantly reduced tumor volume (p<0.0001), which is very similar to the effectiveness of rapamycin in treating tumors, that is, a single in vitro targeted drug treatment of human senescent cells can produce long-term in vivo consequences.

[0215] To closely mimic the clinical situation under chemotherapy conditions, the inventors designed a preclinical protocol containing genotoxic drugs and / or rutin ( Figure 30 , Figure 31 ). Two weeks after human cells were implanted into the mice, stable uptake of the tumor by the host animals was usually observed, and a single dose of MIT or placebo was given on the first day of the 3rd, 5th, and 7th weeks until the end of the 8-week protocol ( Figure 31 ). Compared with the placebo, MIT treatment led to a significant reduction in tumor size, verifying the effectiveness of MIT as a genotoxic drug ( Figure 32 ). It is worth noting that typical SASP factors including IL6, IL8, AREG, IL1α, MMP1, MMP3, ANGPTL4, and SPINK1 were generally significantly up-regulated, and at the same time, a group of typical senescence markers including p16INK4A and p21 CIP1 and SA-β-GAL were both elevated, indicating the development of in vivo senescence and SASP in response to MIT treatment ( Figure 33 , Figure 34 , Figure 35 ). Interestingly, MIT treatment resulted in the expression of certain SASP factors such as MMP3 that were commonly observed in stromal and cancer cell populations, and typical senescence markers including p16 INK4A and p21 CIP1 , indicating that chemotherapy induced overall in vivo senescence, although SASP seemed to exhibit different development trends between these two cell populations ( Figure 34 , Figure 35 ). The SA-β-GAL staining results confirmed a considerable degree of tissue-level senescence induced by MIT, in sharp contrast to rutin; while rutin seemed neither to promote nor inhibit senescence ( Figure 36 ), a feature that was basically consistent with the inventors' in vitro observations.

[0216] The inventors next asked whether technically inhibiting the development of broad-spectrum SASP caused by therapeutic injury could further enhance the tumor's response to treatment. To this end, rapamycin, a very well-established anti-aging drug, especially in targeting the development of SASP, was used starting from the first preclinical treatment; at the same time, the inventors also used rutin as a parallel administration to compare the intervention effects of the two. Although MIT itself led to tumor shrinkage in tumors containing only PC3 cells (p < 0.001), the administration of senomorphics did not show a significant effect (p > 0.05) ( Figure 37 ).

[0217] Notably, even when these drugs were used in combination with MIT, no further benefits were brought (p > 0.05), indicating that PC3 tumor growth was independent of tissue-level SASP, especially in the absence of stromal cells. Strikingly, when PC3 cells were combined with their stromal cells to form tissue recombinants, the inventors observed a significant increase in tumor volume (p < 0.0001), further verifying the tumor-promoting effect of stromal cells in vivo ( Figure 37 ). However, when animals bearing PC3 / PSC27 tumors were exposed to MIT, the tumor volume decreased significantly (35.5%, p < 0.001). After the dual treatment of rapamycin or rutin in combination with MIT, the tumor showed a further reduction, and this reduction in volume was extremely significant. Among them, the dual treatment of rapamycin and MIT led to a further 34.1% reduction in tumor volume compared to single-use MIT (p < 0.01); while the dual treatment of rutin and MIT led to a further 48.9% reduction in tumor volume compared to single-use MIT (p < 0.0001) ( Figure 37) This result indicates that the combination of rutin and MIT has relatively better effects than the combination of rapamycin and MIT.

[0218] It should be noted that when rapamycin and rutin are jointly used for treating tumors with the chemotherapy drug MIT, the intervention achieves the best effect, and the tumor volume is further reduced on the basis of MIT monotherapy (68.3%, p < 0.0001)( Figure 37 ), which is unexpected.

[0219] However, when the inventor used vitamin C, a natural antioxidant and also a SASP inhibitor, to replace rapamycin and combine with rutin and MIT for treating tumors, these intervention effects could not be replicated, and the tumor volume increased instead, basically returning to the effect of MIT monotherapy( Figure 38 ). Therefore, even though they are all commonly used and effective SASP inhibitors in the field of aging, very different effects can occur when rutin is combined with different small molecule compounds and jointly intervenes in tumors with chemotherapy drugs. Among them, the synergistic effect caused by the combined use of rutin and rapamycin provides new options and ideas for future anti-cancer treatment.

[0220] On this basis, the inventor studied whether the treatment effects of other chemotherapy drugs would be significantly improved when combined with rutin and / or rapamycin. The results showed that this was not the case, and the anti-tumor treatments of multiple chemotherapy drugs did not benefit from the combined use of rutin and / or rapamycin, such as vincristine (VCR)( Figure 39 ).

[0221] To reveal the internal mechanism of SASP-induced cancer drug resistance, the inventor chose to obtain and dissect tumors from animals on the 7th day after the start of treatment, which is a time point before the development of drug-resistant clones. Compared with the placebo, MIT itself can cause significant DNA damage and apoptosis in cancer cells( Figure 40 ). In PC3 / PSC27 xenografts, the treatment with rutin alone neither causes typical DDR nor enhances cell death, indicating that the response of these tumors is very limited when animals are only exposed to rutin.

[0222] After the combined use of rutin and MIT, the apoptosis rate is further increased, suggesting synergistic cytotoxicity when combined with MIT. The in vivo apoptosis pattern is basically consistent with the tumor regression pattern after different drug treatments. The IHC staining results based on the degree of Caspase3 self-cleavage show that when rutin is used together with MIT, the apoptosis index increases significantly( Figure 40)。When rutin is used in combination with rapamycin and MIT simultaneously, it can further increase the indices of DNA damage and apoptosis on the basis of the combination of rutin and MIT. Figure 40 ) This indicates that the elimination of cancer cells in the lesion can achieve the best effect in this case.

[0223] ELISA data show that MIT-mediated chemotherapy leads to an increase in the levels of AREG and EREG proteins in the circulating blood of animals, and this pattern is significantly reversed when rutin is used. Figure 41 ) When rutin is combined with rapamycin, it can further reduce the protein levels of AREG and EREG in the blood of mice on the basis of rutin used alone with MIT. Figure 41 )

[0224] In view of the significant efficacy of combination therapy in cancer treatment, the inventors further expanded the research on this type of drug and tumor intervention. The progression and treatment outcomes of breast cancer were confirmed by generating xenografts composed of MDA-MB-231 (malignant breast cancer cells) and HBF1203 (mammary stromal cells), which is a combination of human breast-derived cells previously used by the inventors for cancer research. Similarly, the MDA-MB-231 / HBF1203 tumors basically replicated the preclinical experimental results of PCa under the intervention of specific chemotherapeutic drugs such as doxorubicin (DOX). Figure 42 ) However, not all chemotherapeutic drug interventions can achieve this effect. For example, vinblastine (VIN) does not have the characteristics of drug combination therapy. Figure 43 )

[0225] The results show that the SASP targeting strategy for tumor drug resistance intervention is not limited to specific cancer types and may be applicable to a wider range of various solid malignancies, but its actual applicability is only applicable to certain chemotherapeutic drugs. Generally speaking, the underlying mechanism determining this difference in intervention effects requires further in-depth analysis in subsequent studies.

[0226] As an important link before modern drugs move towards translational medicine and enter clinical practice, the inventors then detected the safety of rutin, a new type of senomorphics drug, and rapamycin when used in combination with traditional chemotherapeutic agents for anti-cancer. The results showed that there were no significant fluctuations in the physiological data of the experimental mice, including daily body weight, liver and kidney toxicity indicators (such as creatinine, urea, ALP, ALT, etc.) and blood components (such as globulin, white blood cells, lymphocytes, and platelets). Figures 44 - 48 )

[0227] These results indicate that rutin is a very effective and safe natural small molecule senomorphics drug, and rapamycin and rutin can exert a synergistic effect, greatly promoting the effect of chemotherapeutic drugs, and thus having a very promising application prospect in clinical treatment in the future.

[0228] Example 6. Analysis of the effects of various drug combinations on anti-tumor

[0229] In the previous extensive screening studies of the present inventors, the effects of various drug combinations on anti-tumor were compared. Some drug combinations did not show significantly better effects after being used in combination, while some drug combinations even led to a decrease in the tumor inhibitory effect after being used in combination.

[0230] The compound combinations that led to a decrease in the tumor inhibitory effect after drug combination and the results of tumor inhibition determination are listed in Table 1. The drug administration method in the experiment was the same as that in the aforementioned pre-clinical treatment trial, and the components without the dosage marked in the table were used in accordance with the dosage given in the aforementioned pre-clinical treatment trial.

[0231] Table 1

[0232]

[0233] Therefore, the combinations of the vast majority of drugs did not achieve an improvement in the pharmaceutical sense. However, after extensive screening and combination studies, the present invention obtained a combination of rutin, rapamycin and mitoxantrone with a synergistic effect.

[0234] The above-described embodiments merely represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims. At the same time, all the documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference.

Claims

1. Use of rutin and rapamycin or their derivatives for preparing a pharmaceutical composition for use in combination with a chemotherapeutic drug to inhibit tumors and / or reverse tumor drug resistance; wherein, the chemotherapeutic drug is mitoxantrone, a chemotherapeutic drug that induces a senescence-associated secretory phenotype after administration; the derivatives are pharmaceutically acceptable salts of rutin and rapamycin; in the pharmaceutical composition, the weight ratio of mitoxantrone, rutin, and rapamycin is 1: 30-70: 30-70; the tumor is prostate cancer or breast cancer.

2. The use according to claim 1, characterized in that, the senescence-associated secretory phenotype is a senescence-associated secretory phenotype caused by DNA damage.

3. The use according to claim 2, characterized in that, the DNA damage is DNA damage caused by a chemotherapeutic drug.

4. The use according to claim 1, characterized in that, in the pharmaceutical composition, the rutin or rapamycin or their derivatives are further used for: inhibiting the expression of a broad-spectrum senescence-associated secretory phenotype, which inhibits the expression of the broad-spectrum senescence-associated secretory phenotype without affecting cell senescence; interfering with the interaction between ATM and HIF1α and TRAF6, and inhibiting an acute stress-related phenotype; eliminating the malignancy conferred on cancer cells by senescent stromal cells in a paracrine manner; increasing the apoptosis rate of tumor cells; and / or inhibiting the components IL8, IL6, IL1a, IL1b, CXCL3, MMP3, GM-CSF of the senescence-associated secretory phenotype.

5. The use according to claim 1, characterized in that, in the pharmaceutical composition, the weight ratio of mitoxantrone, rutin, and rapamycin is 1: 40-60: 40-60.

6. Use of rutin and rapamycin or their derivatives and a chemotherapeutic drug in the preparation of a kit for inhibiting tumors and / or reversing tumor drug resistance; wherein, the chemotherapeutic drug is mitoxantrone, a chemotherapeutic drug that induces a senescence-associated secretory phenotype after administration; wherein, the weight ratio of mitoxantrone, rutin, and rapamycin is 1: 30-70: 30-70; the derivatives include pharmaceutically acceptable salts of rutin and rapamycin; the tumor is prostate cancer or breast cancer.

7. The use according to claim 6, characterized in that, the weight ratio of mitoxantrone, rutin, and rapamycin is 1: 40-60: 40-60.

8. A method for screening potential substances that promote the inhibition of tumors and / or reversal of tumor drug resistance by rutin and rapamycin, the method comprises: (1) providing a tumor microenvironment system, which system comprises tumor cells; (2) treating the system of (1) with a chemotherapeutic drug to induce a senescence-associated secretory phenotype in the tumor microenvironment; wherein, the chemotherapeutic drug is mitoxantrone, a chemotherapeutic drug that induces a senescence-associated secretory phenotype after administration; before, during, or after inducing the senescence-associated secretory phenotype in the tumor microenvironment, treating with rutin and rapamycin; and (3) Add the candidate substance to the system of (2), and observe its effect on the tumor microenvironment system. If the candidate substance can statistically promote the inhibition of senescence-associated secretory phenotype and tumor by rutin and rapamycin and / or reverse tumor drug resistance, then the candidate substance is a potential substance that can be used in combination with rutin and rapamycin to inhibit tumors; Wherein: The development of apoptosis or senescence-associated secretory phenotype is evaluated by observing the caspase 3 cleavage activity or the expression of SASP factors, and the SASP factors include: IL6, IL8, IL1a, IL1b, CXCL3, MMP3, GM-CSF; It is evaluated by observing the interaction between ATM and HIF1α and TRAF6. If the ability of rutin and rapamycin to interfere with the interaction between ATM and HIF1α and TRAF6 is promoted, then the candidate substance is a potential substance that can be used in combination with rutin and rapamycin to inhibit tumors; It is evaluated by observing the malignancy of cancer cells conferred by senescent stromal cells in a paracrine manner. If the candidate substance can produce senescent stromal cells and endow them with the ability to confer malignancy on cancer cells in a paracrine manner, then the candidate substance is a potential substance that can be used in combination with rutin and rapamycin to inhibit tumors; The tumor is prostate cancer or breast cancer.

9. The method according to claim 8, characterized in that The method further includes setting a control group to clearly distinguish the differences between the tumor microenvironment system in the test group and the control group, or the differences between the rutin-targeted inhibition of senescence-associated secretory phenotype and tumor and / or reversal of tumor drug resistance and the control group.

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