Application of fangchinoline in preparation of products for inducing aging of tumor cells

Abstract

The invention discloses an application of fangchinoline alkali in preparation of a product for inducing aging of tumor cells. The invention provides a novel application of fangchinoline alkali in induction of tumor cell senescence, which is beneficial to deep understanding of biological behaviors of tumor cells. By using the fangchinoline alkali as the tumor cell aging inducer, the aging process and the biological mechanism of tumor cells can be effectively simulated in an in-vitro or in-vivo model, so that a powerful tool is provided for biological research.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical medicines, and in particular to the application of fangchinoline in the preparation of products for inducing tumor cell senescence. Background Art

[0002] Tumor cells have the potential to proliferate indefinitely, so anti-tumor strategies targeting their growth regulation have attracted much attention in recent years. Among them, inducing tumor cell senescence is an important therapeutic approach. Cell senescence is a stable and irreversible state of cell cycle arrest that can exert anti-tumor effects by inhibiting cell proliferation. Combining the induction of tumor cell senescence with the removal of these senescent cells has formed a new dual treatment strategy, especially by using senescent cell removers (senolytics) to enhance the anti-tumor effect.

[0003] Currently, clinical trials are evaluating the effectiveness of this combination treatment strategy, especially on the basis of traditional chemotherapy drugs such as etoposide. Although these drugs can effectively induce tumor cell senescence, they can also cause DNA damage, leading to serious side effects, especially in normal cells. In order to reduce this toxic side effect, inhibitors targeting PARP such as olaparib have been used in some tumor patients, especially those with BRCA1 / 2 or PALB2 mutations. However, due to the low proportion of patients with these gene mutations, the scope of application of PARP inhibitors is relatively limited.

[0004] Therefore, there is an urgent need to provide a tumor cell senescence agent that can induce tumor cells into a senescent state while avoiding damage to normal cells to the greatest extent, thereby improving the patient's treatment experience and enhancing the anti-tumor efficacy. Summary of the invention

[0005] In view of the defects in the prior art, the present invention proposes the use of Fangchinoline in the preparation of products that induce tumor cell senescence. The present application has found through a large amount of experimental data that Fangchinoline (Fangchinoline), an extract of the traditional Chinese medicine powder Fangji, can be used as a tumor cell senescence inducer, effectively inducing senescence in a variety of tumor cells, and has important research value in the preparation of research tools for inducing tumor cell senescence and different applications in tumor treatment drugs.

[0006] The present invention provides the use of fangchinoline in preparing a product for inducing tumor cell senescence. The chemical substance registration number (CAS Registry Number) of fangchinoline is 436-77-1, and the chemical structure is as follows:

[0007]

[0008] In some embodiments, the product is a tumor cell growth retardant or a tumor cell senescence agent.

[0009] In some embodiments, the product is a pharmaceutical.

[0010] In some embodiments, the medicament comprises fangchinoline and a pharmaceutically acceptable carrier.

[0011] In some embodiments, the fangchinoline is the only active ingredient.

[0012] In some embodiments, the drug further comprises cardiac glycosides, preferably ouabain.

[0013] In some embodiments, the tumor cell is any one of colorectal cancer cells, pancreatic cancer cells, liver cancer cells, bladder cancer cells, breast cancer cells, cervical cancer cells, prostate cancer cells, glioma cells, melanoma cells, nasopharyngeal cancer cells, lung cancer cells and gastric cancer cells; preferably, it is a melanoma cell, a breast cancer cell or a colorectal cancer cell.

[0014] The present invention also provides an anti-tumor drug, which comprises fangchinoline and cardiac glycosides.

[0015] In some embodiments, the cardiac glycoside is ouabain.

[0016] In some embodiments, the dosage form of the anti-tumor drug is any one of tablets, capsules, powders, granules, and injections.

[0017] In some embodiments, the tumor is any one of colorectal cancer, pancreatic cancer, liver cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, nasopharyngeal carcinoma, lung cancer and gastric cancer; preferably melanoma, breast cancer or colorectal cancer.

[0018] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0019] 1. This application provides a new use of fangchinoline in inducing tumor cell senescence, which helps to gain a deeper understanding of the biological behavior of tumor cells. By using fangchinoline as an inducer, the aging process and biological mechanism of tumor cells can be effectively simulated in in vitro or in vivo models, thereby providing a powerful tool for biological research.

[0020] 2. Fangchinoline can be used for drug screening to evaluate the efficacy of potential anticancer drugs through its induction effect on tumor cell senescence.

[0021] 3. By utilizing the unique effects of fangchinoline, we can further explore the regulatory mechanism of cell aging, thus providing a scientific basis for the development of new tumor treatment strategies.

[0022] 4. Compared with traditional tumor cell senescence inducers, fangchinoline has low toxicity to normal cells and will not cause DNA damage to normal cells such as HCMEC / D3 and 293T.

[0023] 5. This application shows a stronger effect of promoting tumor cell apoptosis by combining fangchinoline and the cardiac glycoside drug ouabain compared to the use of either alone, which is expected to provide new ideas for tumor treatment, reduce tumor cell survival, delay tumor progression, and thereby improve the clinical efficacy of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 These are the results of inducing tumor cells to enter G0 / 1 phase cycle arrest after treatment with fangchinoline in Example 1 of the present invention; Figure A is the flow cytometry result of Mel-167 cells; Figure B is the statistical result of Figure A; Figure C is the flow cytometry result of SW620 cells; Figure D is the statistical result of Figure C; Figure E is the flow cytometry result of HCC1806 cells; Figure F is the statistical result of Figure E; data are expressed as mean ± SEM, n = 3, p < 0.05 is considered significant.

[0026] Figure 2 Figure 1 is the effect of fangchinoline on the expression of aging markers induced by Example 1 of the present invention; Figure A is the Western blot result of Mel-167; Figure B is the Western blot result of the malignant melanoma cell line A375; Figure C is the Western blot result of SW620; Figure D is the Western blot result of HCC1806; Figure E is the result of PCR detection of the relative mRNA expression of Ki-67, LaminB1, and CDKN1A in the Mel-167 cell line; Figure F is the protein expression result of p-H2A.X after fangchinoline treatment of normal cells HCMEC / D3 and 293T cells; Fan, fangchinoline; data are expressed as mean ± SEM, n = 3, p < 0.05 is significant.

[0027] Figure 3Figure 1 is the SA-β-Gal staining result of Example 1 of the present invention; Figure A is the staining result of IGR37 melanoma cells; Figure B is the staining result of A375 melanoma cells; Figure C is the staining result of Pem22 melanoma cells; Figure D is the staining result of Sw620 colorectal cancer cells.

[0028] Figure 4 It is the transcriptome sequencing result of Example 1 of the present invention; Figure A is the p53 signaling pathway of tumor cells; Figure B is the expression of Senmayo (aging-related gene set); Figure C is the heat map of SASP-related genes.

[0029] Figure 5 The results of real-time quantitative PCR detection in Example 1 of the present invention; Figure A shows the mRNA expression results of multiple SASP-related genes, CXCL8, IGFBP3, CCL3, CCL20, JUN, ICAM1, and GDF15.

[0030] Figure 6 The in vitro experiment of Example 2 of the present invention verifies the effect of fangchinoline on tumor cell proliferation; Figures A to C are growth curves of different tumor cells, Mel-167, PEM22, and A375 are malignant melanoma cell lines, HCC1806, 4T1, and Brx142 are breast cancer cell lines, and SW620, MC38, and CT26 are colorectal cancer cell lines; Figure D is the IC50 (half inhibition rate) of fangchinoline on Mel-167 cells; Figure E is a density and state diagram of cells after Mel-167 cells are treated with fangchinoline; Figure F is the cell proliferation result of non-tumor cells after 72 hours of treatment with fangchinoline, HUVEC is human umbilical vein endothelial cell; Fan, fangchinoline; data are expressed as mean ± SEM, n = 3, p < 0.05 is significant.

[0031] Figure 7 Figure 2 is the tumor proliferation result of fangchinoline combined with ouabain in Example 2 of the present invention; Figure A is the cell viability result; Figure B is the protein detection result of apoptosis marker.

[0032] Figure 8 This is an in vivo experiment to verify the effect of fangchinoline on tumor cell proliferation in Example 3 of the present invention; Figure A is a metastasis tumor growth curve drawn by statistical analysis of the in vivo imaging data at each time point; Figure B is the results of liver metastasis and lung metastasis; Figure C is the weight change results of mice after medication. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0034] The present invention discloses a new use of fangchinoline in inducing tumor cell senescence. By using different concentrations of fangchinoline to treat a variety of tumor cells, biochemistry and high-throughput RNA sequencing methods are used to clarify that it can induce the senescence of melanoma, breast cancer, and colorectal cancer cells, and exert anti-tumor growth and metastasis effects. Fangchinoline can exert anti-tumor growth and metastasis effects without relying on BRCA1 / 2 or PALB2 mutations, is not limited by specific gene mutations, and can be applied to a wider range of patient groups, especially patients without BRCA1 / 2 or PALB2 mutations, broadening its scope of application. By using fangchinoline combined with the cardiac glycoside drug ouabain for sequential therapy to treat tumor cells, tumor cell senescence is further promoted, and senescent cells are effectively induced to enter the apoptosis program, thereby achieving the effect of accelerating the clearance of tumor cells.

[0035] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used are all commercially available unless otherwise specified.

[0036] Preparation of fangchinoline solution: Fangchinoline powder was dissolved in DMSO to prepare a 50 mM stock solution, which was stored in a -20 degree refrigerator away from light.

[0037] Example 1 Fangchinoline induces tumor cell senescence

[0038] Melanoma cells, breast cancer cells HCC1806 and colorectal cancer cells SW620 were cultured in vitro and treated with different concentrations of fangchinoline for 72 hours. Flow cytometry, Western blot, SA-β-Gal staining, RNA-seq and RT-qPCR were used to study the effect of fangchinoline on tumor cell senescence.

[0039] (1) Flow cytometry to detect changes in tumor cell cycle

[0040] After treatment with different concentrations (0, 3, 6, 9, 12 μM) of fangchinoline, the cells were digested with trypsin, washed once with 1× PBS (phosphate buffered saline), resuspended and counted, and anhydrous ethanol was added to a final concentration of 75% ethanol. The samples were placed at -20°C for 2 hours and then stained.

[0041] The components of the staining solution include: 0.1% Nonidet P-40 (ethylphenyl polyethylene glycol NP-40) in PBS, RNaseA (Takara, #2158, 10 mg / mL, final concentration 10 μg / mL, 1:1000), and PI (life technologies #P3566, propidium iodide, 1 mg / mL, final concentration 1:500).

[0042] Cell cycle analysis was performed using a BD FACS Aria III flow cytometer, and the test results were analyzed using ModFit LT5.0 software, and data statistics were performed using GraphPad Prism.

[0043] The results are as follows Figure 1 As shown, after treatment with fangchinoline, the number of G0 / 1 phase cells in melanoma cells Mel-167, breast cancer cells HCC1806 and colorectal cancer cells SW620 cells increased, and the number of S phase cells in the replication phase decreased, indicating that fangchinoline can effectively induce G0 / 1 phase arrest in tumor cells.

[0044] (2) Western blot detection of aging markers

[0045] The changes of senescence markers Ki-67, p-p53, p53, p21, p-RB and LaminB1 were detected by Western blot. The steps are as follows:

[0046] The cells were lysed using RIPA cell lysis buffer (Biyuntian Biotechnology, #P0013B), the total protein of the cells was extracted, and the protein concentration was determined using the BCA protein quantification kit (Thermo fisher, #23227). After the protein concentration was obtained, 30 μg of each sample was taken for Western blot sample preparation. Polyacrylamide gel (SDS-PAGE) gel (Biyuntian Biotechnology, #P0012A) was used for protein electrophoresis, and the electrophoresis conditions were 120V, 60min. The protein separated by electrophoresis was transferred from the gel to a PVDF membrane; then it was blocked with 5% skim milk powder, incubated with the corresponding primary antibody, and incubated with HRP-labeled secondary antibody; protein expression was detected using a chemiluminescence instrument.

[0047] The results are as follows Figure 2 As shown in the figure, after treatment with 2 μM fangchinoline, the expression of p21 protein in Mel-167, HCC1806 and SW620 cells was significantly increased, and the protein expression of Ki-67, Lamin B1 and phosphorylated RB was significantly decreased compared with the control group ( Figure 2 A~D), Figure 2 In E, Ki-67 and LaminB1 mRNA expression levels were significantly decreased, and CDKN1A

[0048] The mRNA expression of (p21) was significantly increased, indicating that treatment with a certain concentration of fangchinoline can promote the expression of tumor cell senescence-related proteins, thereby promoting tumor cell senescence.

[0049] (3) SA-β-Gal staining to assess the number of senescent cells

[0050] Melanoma cell lines Mel-167, PEM22, A375, breast cancer cell lines HCC1806, 4T1, Brx142, and colorectal cancer cell lines SW620, MC38, and CT26 were treated with different concentrations of fangchinoline for 6 days, and then fixed with the fixative solution of the SA-β-Gal staining kit (Biosharp, #BL133A). The staining solution was prepared according to the instructions of the kit, and the fixed cells were stained overnight; the stained cells were photographed under a microscope and the SA-β-Gal positive cells were counted using Image J software, and the cells with positive β-galactosidase staining were senescent cells.

[0051] Figure 3 The results showed that after treatment with different concentrations of fangchinoline, the staining area in melanoma cells IGR37, A375, Pem22 and colorectal cancer cells SW620 gradually increased, and the number of senescent tumor cells increased, indicating that fangchinoline can effectively increase the number of SA-β-Gal-positive tumor cells and promote tumor cell senescence.

[0052] (4) RNA-seq detection of cell transcriptome

[0053] After the cell lines were cultured in vitro and treated with different concentrations of fangchinoline for 72 hours, total RNA was extracted using the NucleoZol RNA extraction kit (Macherey-Nagel, #740404.200); the RNA-seq library construction kit (VAHTS Universal V10 RNA-seq Library Prep Kit #NR606-01) was used; the constructed library was sent to Genentech for next-generation sequencing. Gene set enrichment analysis (GSEA) was performed.

[0054] Figure 4 The results showed that after treatment with fangchinoline, the p53 signaling pathway in tumor cells was activated ( Figure 4 A), Senmayo (aging-related gene set) up-regulated ( Figure 4 B) The mRNA expression of many SASP-related genes increased after drug treatment. Fangchinoline can effectively activate the gene expression of the p53 signaling pathway gene set and the Senmayo SASP gene set.

[0055] (5) RT-qPCR detection of mRNA expression of SASP-related genes

[0056] Total cellular RNA was extracted using the NucleoZol RNA extraction kit (Macherey-Nagel, #740404.200); the RNA was reverse transcribed using a reverse transcription kit (Acori Biotechnology, #AG11728) to obtain cDNA; and then CXCL8, IGFBP3, CCL3, CCL20, JUN, ICAM1, and GDF15 were quantitatively detected using a qPCR kit (Acori Biotechnology, #AG11718).

[0057] Figure 5 The results showed that after melanoma cells were treated with fangchinoline, the mRNA expression of SASP-related genes was significantly increased, which was consistent with the analysis results of RNA-seq, and could induce tumor cell senescence.

[0058] Example 2 Fangchinoline induces anti-tumor growth and metastasis of melanoma cells in vitro

[0059] This example tests whether fangchinoline can exert anti-tumor growth and metastasis effects in vitro after inducing tumor cell senescence.

[0060] (1) Inhibition of tumor cell growth after treatment with fangchinoline

[0061] Each tumor cell and normal cell were seeded in a 96-well plate at a density of 4000 cells / well and treated with different concentrations of fangchinoline for 72 hours. The Luminescent Cell Viability Assay Kit (Promega, #G7570) was used to measure the total viability of cells and calculate the relative growth rate of cells.

[0062] The results are as follows Figure 6 As shown in the data, after being treated with different fangchinoline, the cell viability of each tumor cell decreased with the increase of concentration, indicating that fangchinoline can effectively inhibit the in vitro growth of melanoma cells, breast cancer cells, and colorectal cancer cells, but has extremely low toxicity to normal cells such as vascular endothelial cells (HCMEC-1, HCMEC / D3, HUVEC) and astrocytes, and will not cause DNA damage to normal cells HCMEC / D3 and 293T.

[0063] (2) Effect of fangchinoline combined with ouabain on cell proliferation

[0064] The cells were seeded in a 96-well plate at a density of 7000 cells / well and treated with fangchinoline for 48 hours and then with different concentrations of ouabain for 24 hours. The total viability of cells was determined using the Luminescent Cell Viability Assay Kit (Promega, #G7570). Western blot was used to detect the changes in apoptosis markers of tumor cells when fangchinoline was combined with ouabain.

[0065] The results are as follows Figure 7 As shown, the use of fangchinoline alone only caused cell senescence but did not induce apoptosis. After treatment with fangchinoline combined with ouabain, the apoptosis indicators PARP, Caspase9, Caspase8, and Caspase3 decreased, while the apoptosis-activated spliceosomes c-PARP and c-Caspase9 increased, indicating that fangchinoline combined with ouabain can cause melanoma apoptosis and thus exert a stronger inhibitory effect on tumor cell proliferation.

[0066] Example 3 Fangchinoline induces anti-tumor growth and metastasis of melanoma cells in vivo

[0067] Evaluation of the inhibitory effect of fangchinoline on melanoma metastasis in mice:

[0068] NSG mice were purchased from Guangzhou Nanmo Company and raised in the Experimental Animal Center of Southern University of Science and Technology. 500,000 melanoma cells were injected into NSG mice by tail vein injection. Five days later, the mice were intraperitoneally injected with fangchinoline as solvent, 40% PEG300+50% saline+5% DMSO+5% Tween80, and the dosage was 10 mg / kg / day. The growth of metastases was detected every 5 to 7 days using a live imaging device, and the growth curve of metastases was statistically analyzed.

[0069] Figure 8 The results showed that compared with the control group, after in vivo injection of fangchinoline, the total fluorescence intensity decreased, the fluorescence intensity of liver metastasis and lung metastasis was significantly reduced, and the tumor's metastatic ability was weakened, indicating that fangchinoline treatment can effectively inhibit the metastasis of melanoma.

[0070] In summary, the present application discloses a new use of fangchinoline in inducing tumor cell senescence, which can exert significant anti-tumor growth and metastasis efficacy after inducing tumor cell senescence. In addition, fangchinoline can be used in combination with the cardiac glycoside drug ouabain to further enhance fangchinoline-induced tumor cell senescence, and effectively induce senescent cells to enter the apoptosis program, thereby accelerating the elimination of tumor cells.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of fangchinoline in the preparation of products for inducing tumor cell senescence.

2. The use according to claim 1, characterized in that: The product is a tumor cell growth retardant or a tumor cell aging preparation.

3. The use according to claim 1, characterized in that: The product described is a drug.

4. The use according to claim 3, characterized in that: The medicine comprises fangchinoline and a pharmaceutically acceptable carrier.

5. The use according to claim 4, characterized in that: The fangchinoline is the only active ingredient.

6. The use according to claim 4, characterized in that: The drugs also include cardiac glycosides.

7. An anti-tumor drug, characterized in that: The anti-tumor drugs include fangchinoline and cardiac glycosides.

8. The anti-tumor drug according to claim 7, characterized in that: The cardiac glycoside substance is ouabain.

9. The anti-tumor drug according to claim 7, characterized in that: The dosage form of the anti-tumor drug is any one of tablets, capsules, powders, granules and injections.

10. The antitumor drug according to any one of claims 7 to 9, characterized in that: The tumor is any one of colorectal cancer, pancreatic cancer, liver cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, nasopharyngeal carcinoma, lung cancer and gastric cancer.

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