Application of arginine deprivation in delaying aging and combined with chemotherapy drugs in treating tumors

Abstract

The present invention provides an application of arginine deprivation in delaying cell senescence and treating tumors. The present invention alleviates cell senescence by depriving arginine, thereby inhibiting the synthesis and release of SASP in senescent cells in the tumor microenvironment, thereby achieving a certain degree of improvement in the efficacy of tumor chemotherapy.

Description

Technical Field

[0001] The present invention relates to adjuvant chemotherapy for tumors, and in particular to the application of arginine deprivation in conjunction with genotoxic chemotherapy drugs in treating tumors. Background Art

[0002] Cancer, a malignant disease that threatens human health, has long been a hot topic in biomedical research regarding its diagnosis, treatment, and recovery. In 1989, British surgeon Stephen Paget first proposed the concept of the tumor microenvironment (TME). His "seed and soil" theory has profoundly influenced the understanding of tumor cell metastasis and recurrence. Decades of research and discovery in cancer have led to the definition of tumors as heterogeneous organs with a complexity far exceeding that of healthy tissues. The TME, fundamental to tumor development, metastasis, metabolism, and survival, is primarily composed of heterogeneous tumor cells, in addition to other tissues and cells that are compromised by tumor cells. The TME of solid tumors primarily comprises a variety of tissues and cells, including the extracellular matrix, carcinoma-associated fibroblasts (CAFs), immune cells, neuroendocrine cells, pericytes, smooth muscle, endothelial cells, and blood vessels. The TME of hematologic malignancies, on the other hand, is primarily composed of bone marrow stromal cells, endothelial cells, monocytes, macrophages, osteoblasts, osteoclasts, natural killer cells, and various T and B cells.

[0003] In 1961, Hayflick and Moorhead first discovered that human fibroblasts cultured in vitro initially have a strong proliferative capacity. However, after 30 generations of culture, the cells permanently lose this ability to proliferate, a phenomenon known as cellular senescence. Since its discovery, two competing interpretations have been offered regarding its role in living organisms. One view holds that cellular senescence is an important protective mechanism. Cells harboring oncogene mutations have the capacity for indefinite proliferation, and their abnormal division and accumulation can lead to tumor development, where cellular senescence can play an anti-tumor role. The other view holds that cellular senescence is detrimental to the organism. Excessive accumulation of senescent cells can impair tissue regeneration and repair, thereby contributing to the development of age-related diseases. The senescence-associated secretory phenotype (SASP), as the primary characteristic and executor of the functions of senescent cells, plays a crucial role in human health.

[0004] Chemotherapeutic drugs are a classic approach in clinical cancer treatment. They damage tumor cell genomic DNA and induce apoptosis, achieving the goal of tumor treatment. However, off-target chemotherapy drugs can damage benign tissues and cells within the tumor microenvironment, including various stromal cells, immune cells, and endothelial cells. These damaged cells exhibit characteristics typical of senescent cells, and through the SASP, senescent cells can reshape the tumor microenvironment, thereby reducing the therapeutic efficacy of chemotherapy and leading to disease progression. SASP factors are the primary executors of senescent cell function, and different SASP factors may exert distinct effects on different cell types. For example, some SASP factors (such as IL-6, IL-8, and TNF-α) can enhance the senescent phenotype and induce senescence in normal cells through autocrine and paracrine pathways; however, certain angiogenic factors and growth factors can promote angiogenesis in the tumor microenvironment. Therefore, off-target drug-induced cellular senescence and SASP factor release are influenced by multiple factors, including drug dose, disease progression, and signaling pathways. Studies have reported that senescent cells, through the SASP, can promote tumor cell proliferation, invasion, migration, angiogenesis, and drug resistance, among other malignant phenotypes. Therefore, therapy-induced senescence (TIS) is an urgent problem to be solved in clinical cancer treatment. Currently, chemotherapy-assisted therapies mainly include: senescent cell clearance drugs (Senolytics), immune-mediated senescent cell clearance and senescent cell SASP inhibitors (Senomorphics).

[0005] Arginine, a key metabolite in the human body, is inextricably linked to numerous human functions and diseases, including aging, tumors, and immunity. Studies have reported that arginine can delay cellular aging, thereby improving human health (Weili Zhong et al., Diabetes Research and Clinical Practice 89 (2010) 38–45). Arginine also plays a crucial role in the activation of immune system macrophages. When macrophages differentiate into M1 / M2 macrophages, their gene expression of iNOS and ARG1 is significantly upregulated, indicating enhanced cellular arginine metabolism. Finally, arginine plays a crucial role as a pharmaceutical additive in wound healing, and various therapeutic strategies to promote wound healing incorporate arginine as an additive to achieve enhanced therapeutic effects.

[0006] Reports in the field suggest that food restriction, intermittent fasting, dietary control, and dietary regulation can, to some extent, prevent Alzheimer's disease, control weight, and promote neuroregeneration. However, these are all general dietary manipulations, and the impact of arginine deprivation on disease is still unclear. Summary of the Invention

[0007] The present invention provides the use of a substance capable of causing arginine deprivation in the preparation of a reagent for delaying cell senescence, reducing lysosomal β-galactosidase activity in senescent cells, reducing DNA damage response in senescent cells, and reducing SASP gene expression in senescent cells.

[0008] In one or more embodiments, substances that can cause arginine deprivation include: arginine-free or low-arginine culture medium, arginine-free or low-arginine food, agents that increase arginine consumption, and agents that reduce arginine absorption or transport.

[0009] In one or more embodiments, the low arginine medium or low arginine food contains less than 400 μM arginine, such as 350 μM or less, 300 μM or less, 250 μM or less, 200 μM or less, 150 μM or less, 100 μM or less, 50 μM or less, or 20 μM or less arginine. Preferably, the culture medium is DMEM medium.

[0010] In one or more embodiments, the low-arginine food does not contain arginine-rich foods, such as nuts or seafood.

[0011] In one or more embodiments, the agent that increases arginine consumption or the agent that decreases arginine uptake or transport is an arginine transport inhibitor, such as an inhibitor of the gene SLC7A1.

[0012] In one or more embodiments, the arginine is L-arginine.

[0013] In one or more embodiments, the senescent cells comprise PSC27 cells or NIH3T3 cells.

[0014] The present invention also provides a method for delaying cell senescence in vitro, reducing the activity of lysosomal β-galactosidase in senescent cells, reducing the DNA damage response of senescent cells, or reducing the expression of SASP factors in senescent cells, comprising depriving the cells of arginine.

[0015] In one or more embodiments, the arginine deprivation comprises one or more steps selected from the group consisting of:

[0016] (1) Cultivate cells using medium without or low in arginine,

[0017] (2) Reduce the absorption of arginine by cells.

[0018] In one or more embodiments, the senescent cells comprise PSC27 cells or NIH3T3 cells.

[0019] The present invention also provides the use of a substance that can cause arginine deprivation in the preparation of a reagent that inhibits the malignant proliferation of tumor cells, reduces the malignant invasion or migration of tumor cells, reduces the drug resistance of tumor cells, or enhances the killing effect of chemotherapy drugs on tumor cells by alleviating the senescence of non-tumor cells in the tumor microenvironment or the expression of their SASP factors.

[0020] In one or more embodiments, the chemotherapeutic drug is a genotoxic chemotherapeutic drug.

[0021] In one or more embodiments, the chemotherapy drug comprises bleomycin, satraplatin, mitoxantrone, or doxorubicin.

[0022] In one or more embodiments, substances that can cause arginine deprivation include: arginine-free or low-arginine culture medium, arginine-free or low-arginine food, agents that increase arginine consumption, and agents that reduce arginine absorption.

[0023] In one or more embodiments, the low arginine medium or low arginine food contains less than 400 μM arginine, such as 350 μM or less, 300 μM or less, 250 μM or less, 200 μM or less, 150 μM or less, 100 μM or less, 50 μM or less, or 20 μM or less arginine. Preferably, the culture medium is DMEM medium.

[0024] In one or more embodiments, the agent that increases arginine consumption or the agent that decreases arginine uptake is an arginine transport inhibitor, such as an inhibitor of the gene SLC7A1.

[0025] In one or more embodiments, the arginine is L-arginine.

[0026] In one or more embodiments, the tumor cell is a prostate tumor cell or a melanoma cell. Preferably, the tumor cell comprises PC-3, DU145, RM-1, or B16-F10.

[0027] The present invention also provides an in vitro method for inhibiting the malignant proliferation of tumor cells, reducing the malignant invasion or migration of tumor cells, reducing the drug resistance of tumor cells, or enhancing the killing effect of chemotherapy drugs on tumor cells by alleviating the senescence of non-tumor cells in the tumor microenvironment or the expression of their SASP factors, comprising depriving the cells of arginine.

[0028] In one or more embodiments, the chemotherapeutic drug is a genotoxic chemotherapeutic drug.

[0029] In one or more embodiments, the chemotherapy drug comprises bleomycin, satraplatin, mitoxantrone, or doxorubicin.

[0030] In one or more embodiments, the arginine deprivation comprises one or more steps selected from the group consisting of:

[0031] (1) Cultivate cells using medium without or low in arginine,

[0032] (2) Reduce the absorption of arginine by cells.

[0033] In one or more embodiments, the tumor cell is a prostate tumor cell or a melanoma cell. Preferably, the tumor cell comprises PC-3, DU145, RM-1, or B16-F10.

[0034] The present invention also provides a use of a substance capable of causing arginine deprivation and a chemotherapy drug in preparing a reagent for treating tumors.

[0035] In one or more embodiments, the chemotherapeutic drug is a genotoxic chemotherapeutic drug.

[0036] In one or more embodiments, the chemotherapy drug comprises bleomycin, satraplatin, mitoxantrone, or doxorubicin.

[0037] In one or more embodiments, substances that can cause arginine deprivation include: arginine-free or low-arginine culture medium, arginine-free or low-arginine food, agents that increase arginine consumption, and agents that reduce arginine absorption.

[0038] In one or more embodiments, the low arginine medium or low arginine food contains less than 400 μM arginine, such as 350 μM or less, 300 μM or less, 250 μM or less, 200 μM or less, 150 μM or less, 100 μM or less, 50 μM or less, or 20 μM or less arginine. Preferably, the culture medium is DMEM medium.

[0039] In one or more embodiments, the agent that increases arginine consumption or the agent that decreases arginine uptake is an arginine transport inhibitor, such as an inhibitor of the gene SLC7A1.

[0040] In one or more embodiments, the arginine is L-arginine.

[0041] In one or more embodiments, the tumor is a prostate tumor cell or a melanoma cell.Preferably, the tumor is a tumor containing PC-3, DU145, RM-1 or B16-F10 tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Lysosomal β-galactosidase staining of senescent cell lines from human prostate senescent cells (PSC27) and mouse senescent cells (NIH3T3).

[0043] Figure 2 DNA damage response assay in human prostate senescent cells (PSC27) and mouse senescent cells (NIH3T3).

[0044] Figure 3 To detect the DNA synthesis ability of human prostate senescent cells (PSC27) and mouse senescent cells (NIH3T3).

[0045] Figure 4 Detection of senescence-related gene expression in human prostate senescent cells (PSC27) and mouse senescent cells (NIH3T3).

[0046] Figure 5 Detection of arginine levels in human prostate senescent cells (PSC27) and mouse senescent cells (NIH3T3).

[0047] Figure 6 Lysosomal β-galactosidase staining of human prostate senescent cells (PSC27) and mouse senescent cells (NIH3T3) in the presence or absence of arginine.

[0048] Figure 7 DNA damage response was assessed in human prostate senescent cells (PSC27) and mouse senescent cells (NIH3T3) in the presence or absence of arginine.

[0049] Figure 8 The expression of senescence-related genes in human prostate senescent cells (PSC27) and mouse senescent cells (NIH3T3) was detected in the presence or absence of arginine.

[0050] Figure 9 Different concentrations of arginine were added to human prostate senescent cells (PSC27) and mouse senescent cells (NIH3T3) in the presence of arginine to detect the effect of arginine metabolism on the occurrence and development of SASP in senescent cells.

[0051] Figure 10 Different concentrations of arginine were added to human prostate senescent cells (PSC27) and mouse senescent cells (NIH3T3) in the absence of arginine to detect the effect of arginine metabolism on the occurrence and development of SASP in senescent cells.

[0052] Figure 11 The nuclear diameter of human prostate senescent cells (PSC27) and mouse senescent cells (NIH3T3) was measured in the presence or absence of arginine.

[0053] Figure 12Cell volume was measured in human prostate senescent cells (PSC27) and mouse senescent cells (NIH3T3) in the presence or absence of arginine.

[0054] Figure 13 To detect the malignant proliferation ability of human tumor cells.

[0055] Figure 14 To detect the malignant proliferation ability of mouse tumor cells.

[0056] Figure 15 To detect the migration and invasion ability of human tumor cells.

[0057] Figure 16 To detect the migration and invasion ability of mouse tumor cells.

[0058] Figure 17 Detection of drug resistance of human tumor cells.

[0059] Figure 18 Detection of drug resistance of human and mouse tumor cells.

[0060] Figure 19 To test the therapeutic ability of arginine deprivation in synergistic effect with chemotherapy drugs on tumors in tumor-bearing mice. DETAILED DESCRIPTION

[0061] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a preferred technical solution.

[0062] As used herein, "treatment" and "treating" refer to any action that provides benefits to patients suffering from tumors, including improving the condition by alleviating or inhibiting at least one symptom and delaying disease progression. The "subject" or "patient" involved in the treatment, treatment, and diagnosis described herein is an animal suffering from a tumor, typically a mammal, such as a rat (e.g., mouse), rabbit, or human.

[0063] The inventors discovered that arginine is a key metabolite of senescent cells. Depriving senescent cells of arginine can alleviate cellular senescence and inhibit the development and progression of the SASP in senescent cells, thereby reducing the ability of tumor cells to proliferate malignantly. Therefore, arginine deprivation can be used as an adjunct therapy for tumor chemotherapy.

[0064] Arginine and its detection

[0065] Arginine is an important metabolite in the human body and can be detected by microplate reader, flow cytometer, etc. These detection methods and steps are known in the art. In an exemplary embodiment, the cellular arginine level is detected using a high-performance genetically encoded arginine fluorescent probe (see CN202010098995.5).

[0066] An exemplary arginine detection method is as follows:

[0067] Microplate reader detection: Stable cell lines were established. 24 hours after infection, the culture medium was aspirated and the cells were washed with preheated PBS and then cultured with 4 mL of complete culture medium. After 48 hours of culture, the cells were passaged, and positive cells were screened by adding an appropriate concentration of puromycin. The stable cell lines were digested, neutralized, resuspended, and counted, then inoculated onto microplates. After cells adhered, drug-induced cell senescence was used before detection. The culture medium was aspirated, and the samples were washed with preheated HBSS glucose buffer. After adding HBSS glucose buffer, the samples were subjected to microplate detection. The fluorescence values ​​of the samples were read using a microplate reader.

[0068] Flow cytometer detection: The cells were seeded in a well plate and returned to the incubator for culture. After the cells adhered, drugs were used to induce cell senescence and maintain them to a fully senescent state. The cells were washed, digested, and neutralized, and the suspension was transferred to a centrifuge tube and centrifuged. The supernatant was aspirated and the cells were resuspended in PBS solution containing 4% serum and transferred to a 96-well plate for use. The samples were detected using the FITC channel and KO525 channel of the flow cytometer, and the FITC / KO525 fluorescence ratio was calculated.

[0069] Herein, the sample for detecting arginine is a sample derived from body fluid, preferably a sample derived from blood, such as serum, plasma, or whole blood.

[0070] Senescent cells

[0071] According to the disclosures in this application, benign cells in the tumor microenvironment undergo senescence after stimulation with genotoxic chemotherapy drugs and release multiple SASP factors, which can significantly enhance the proliferation and migration of tumor cells. Depriving senescent cells of arginine can inhibit the development of the SASP in senescent cells.

[0072] As used herein, the term "genotoxic chemotherapy drug" is a chemotherapy drug that affects nucleic acids and changes their function. The drug may bind directly to DNA or may cause DNA damage by affecting enzymes that replicate DNA. The therapeutic purpose of using genotoxic chemotherapy drugs is to induce DNA damage in cancer cells, but the selectivity of the drug's action is based on rapidly dividing cells, such as stem cells in the bone marrow, which are usually killed along with cancer cells. Therefore, the drug has an effect on both normal cells and cancer cells. In an exemplary embodiment, the genotoxic chemotherapy drug is selected from one or more of bleomycin (BLEO), satraplatin (SAT), mitoxantrone (MIT) and doxorubicin (DOX).

[0073] As used herein, the term "cellular senescence" refers to a phenomenon in which cells initially have a strong proliferative capacity but permanently lose this proliferative capacity over time. This phenomenon is called cellular senescence. Senescent cells as described herein can be determined according to standards known to those skilled in the art, using conventional methods in the art, such as observing senescence-associated β-galactosidase (SA-β-Gal) staining, activation of DNA damage foci, and development of the SASP.

[0074] As used herein, the term "senescence-associated secretory phenotype (SASP) factors" refers to a series of inflammatory cytokines, chemokines, growth factors and proteases secreted by cells after cellular senescence, such as IL-1, IL-6, IL-8, IL-1β, IL-33 and TNF-amRNA.

[0075] As used herein, the term "arginine deprivation" refers to a state in which a cell or organism lacks arginine. Arginine deprivation can be achieved in a variety of ways, including but not limited to culturing cells without arginine, reducing or eliminating arginine intake in the diet (e.g., reducing arginine-rich foods), reducing the use of arginine drugs, reducing the intake of arginine as an additive food, increasing the consumption of arginine in the environment or in the body through reagents, and reducing the absorption of arginine by cells or organisms. In some embodiments, arginine deprivation of cells can be achieved by culturing cells in an arginine-free medium, and arginine deprivation of mice can be achieved by feeding them an arginine-free mouse diet. In some embodiments, arginine deprivation is the administration of drugs that increase arginine consumption and / or reduce arginine absorption, such as arginine transport inhibitors, such as inhibitors of the gene SLC7A1, including but not limited to ADI-PE20, rhArg-PEG, PEG-BCT-100, and cGCN2.

[0076] Here, arginine deprivation in subjects receiving genotoxic chemotherapy can inhibit the development of the SASP in senescent cells and reduce the invasion and migration of malignant tumor cells. In an exemplary embodiment, supernatant (CM) of senescent cells can be collected and used in tumor malignancy characterization experiments, such as testing the malignant proliferation, invasion, migration, and drug resistance of tumor cells.

[0077] As used herein, the term "subject" or "patient" is a mammal, such as a mouse, rabbit, human, etc.

[0078] Uses and treatments

[0079] Also provided herein is the use of arginine deprivation as described in any embodiment herein in synergistically with a genotoxic chemotherapy drug for treating tumors. Also provided herein is the use of arginine deprivation as described in any embodiment herein in delaying cellular senescence. Also provided herein is the use of arginine deprivation as described in any embodiment herein in synergistically with a genotoxic chemotherapy drug for preparing a preparation for treating tumors.

[0080] In some embodiments, the present invention also provides an arginine-free composition as described in any embodiment herein for use in cooperating with genotoxic chemotherapy drugs to treat tumors, for use in delaying cell senescence, and for use in cooperating with genotoxic chemotherapy drugs to prepare a preparation for treating tumors.

[0081] The present invention also provides a method for treating tumors by arginine deprivation in conjunction with genotoxic chemotherapy drugs, wherein the method comprises treating the tumor under the premise of depriving cells of arginine.

[0082] Herein, the dosage and frequency of administration can be determined by medical staff based on the specific condition, the patient's age and gender, etc. Generally, for the treatment of a specific disease, a therapeutically effective amount refers to a dose sufficient to improve or alleviate in some way the symptoms associated with the disease. Such a dose can be administered as a single dose, or it can be administered according to an effective treatment regimen. The dosage may cure the disease, but administration is usually to improve the symptoms of the disease. Repeated administration is generally required to achieve the desired symptom improvement. In certain embodiments, the frequency of administration can be multiple times a day, twice a day, every two days, every three days, every four days, every five days or every six days, or once every two weeks, or once a month.

[0083] The present invention has the following beneficial effects:

[0084] 1. Depriving senescent cells of arginine can alleviate cell senescence, which is mainly manifested by reduced lysosomal β-galactosidase activity in senescent cells, weakened DNA damage response, weakened nuclear diffusion, and reduced aging-induced cell volume increase and SASP synthesis and release.

[0085] 2. Depriving senescent cells in the tumor microenvironment of arginine reduces the malignant phenotype of tumor cells, which mainly includes reducing the malignant proliferation, invasion, migration, epithelial-mesenchymal transition and drug resistance of tumor cells caused by off-target chemotherapy drugs.

[0086] 3. Arginine deprivation combined with chemotherapy drugs can enhance the therapeutic effect of tumors.

[0087] The compounds of the present invention can be obtained from commercial sources or synthesized according to or with reference to disclosed methods. Other aspects of the present invention will be apparent to those skilled in the art from the disclosure herein. The present invention will be further illustrated below by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. Unless otherwise indicated, the methods and reagents used in the examples are conventional methods and reagents in the art.

[0088] Example 1 Construction and detection of cell senescence model

[0089] 1.1 Experimental Materials and Reagents

[0090] PSC27 human prostate fibroblasts and NIH3T3 mouse embryonic fibroblasts were provided by the Chinese Academy of Sciences. The incubators and safety cabinets required for cell culture were produced by ThermoFIsh. The culture medium and culture dishes required for cell culture were produced by Gibco. The chemotherapy drugs that induce cell senescence were produced by MCE. The Sa-β-Gal staining kit was provided by Beyotime.

[0091] 1.2 Experimental steps

[0092] 1.2.1 Genotoxic chemotherapy drugs induce cellular senescence

[0093] 1) Inoculate cells into the well plates required for the experiment and return the cells to the incubator for culture;

[0094] 2) After the cell confluence reaches 80%, treat the cells with chemotherapy drugs for 12 hours and then change the medium. The drug concentrations used for PSC27 cells are: bleomycin (BLEO, 50μg / mL), satraplatin (SAT, 10nM), mitoxantrone (MIT, 50nM), and doxorubicin (DOX, 2.5μM). The drug concentrations used for NIH3T3 cells are: bleomycin (BLEO, 50μg / mL), satraplatin (SAT, 20nM), mitoxantrone (MIT, 2μM), and doxorubicin (DOX, 5μM);

[0095] 3) Continue culturing the cells for 10 days and then proceed with research after the cells have fully aged.

[0096] 4) Senescent cells in the arginine deprivation group were cultured in an arginine-free medium during drug treatment; samples requiring arginine were supplemented with L-arginine to a concentration of 400 μM in the culture medium.

[0097] 1.2.2 SA-β-Gal staining of senescent cells

[0098] 1) A certain number of cells were seeded into a 6-well plate and then induced to undergo senescence using drugs. When the cells reached a fully senescent state, the cells were tested.

[0099] 2) Remove cells from the incubator, discard the culture medium, and wash the cells with preheated PBS;

[0100] 3) Aspirate and discard PBS, add 1 mL of staining and fixing solution, and let stand at room temperature for 15 minutes;

[0101] 4) While cells are being fixed, prepare the staining mixture according to the manufacturer's instructions.

[0102] 5) After the cells are fixed, aspirate and discard the staining fixative and wash the sample three times with PBS, each time for 3 minutes;

[0103] 6) Add the prepared staining solution mixture to the cleaned sample and place it in a CO2-free 37°C incubator for incubation;

[0104] 7) Most samples require staining for 12–16 hours. After staining, wash with PBS. Add an appropriate amount of PBS and store in a refrigerator at 4°C for subsequent imaging and microscopic examination.

[0105] 1.2.3 Detection of DNA replication capacity of senescent cells

[0106] 1) Disinfect the coverslips with 75% alcohol and wash with PBS, then place them in a 6-well plate for later use.

[0107] 2) After cell digestion, count the cells and seed them into a 6-well plate containing a coverslip;

[0108] 3) After the cells have adhered, dilute 10 μL of 1 mM BrdU solution with 1 mL of complete culture medium and incubate the cells with medium containing BrdU.

[0109] 4) After culturing the cells for 2-3 days, the culture medium was aspirated and the cells were washed with preheated PBS. After washing, 4% paraformaldehyde was added and allowed to stand at room temperature for 15 minutes to fix the cell samples.

[0110] 5) After fixation, aspirate and discard the 4% paraformaldehyde and wash three times with PBS. Add 1.5M HCl and incubate at room temperature for 30 minutes. Aspirate and discard the HCl and wash three times with PBS, each for 5 minutes.

[0111] 6) Neutralize the sample by adding 0.1 M sodium tetraborate solution and incubating at room temperature for 20 minutes. After incubation, wash three times with PBS, each time for 5 minutes.

[0112] 7) Add 1% Triton X-100 in PBST and incubate the sample at room temperature for 10 minutes to permeabilize the membrane. After incubation, wash the sample three times with PBS, each time for 5 minutes.

[0113] 8) Prepare 5% BSA blocking solution with PBST, incubate the sample with the blocking solution, and let it stand at room temperature for 1 hour;

[0114] 9) After blocking, aspirate and discard the blocking solution, and wash three times with PBS, each time for 5 minutes;

[0115] 10) Dilute the primary antibody 1:100 with blocking solution, add 50 μL of antibody dilution to each slide and incubate at room temperature for 1 hour;

[0116] 11) After incubation, wash the samples three times with PBS for 5 minutes each. Dilute the secondary immunofluorescence antibody 1:100 with blocking solution. Then, add 50 μL of antibody dilution solution to each sample and incubate at room temperature for 1 hour.

[0117] 12) After incubation, the samples were washed three times with PBS, each time for 5 minutes;

[0118] 13) Dilute DAPI nuclear dye 1:1000 with blocking solution, add 50 μL of dilution to each sample and incubate for 30 minutes;

[0119] 14) After incubation, wash the sample three times with PBS for 5 minutes each. Add 30 μL of anti-fluorescence quencher to the slide and place the coverslip sample-side up on the slide. Seal the sample with nail polish on all sides to prevent drying. Store the sealed sample at 4°C in the dark for subsequent photography and microscopic examination.

[0120] 1.2.4 Detection of DNA damage foci in senescent cells

[0121] 1) Disinfect the coverslips with 75% alcohol and wash with PBS, then place them in a 6-well plate for later use.

[0122] 2) After cell digestion, count the cells and seed them into a 6-well plate containing a coverslip;

[0123] 3) Induce cell senescence with drugs after the cell confluence reaches 80%;

[0124] 4) After the cells are completely senescent, the culture medium is discarded and the cells are washed with preheated PBS. After washing, 4% paraformaldehyde is added and allowed to stand at room temperature for 15 minutes to fix the cell samples;

[0125] 5) After fixation, discard the 4% paraformaldehyde and wash three times with PBS, each time for 5 minutes;

[0126] 6) Add PBST containing 1% Triton X-100 to the sample and incubate at room temperature for 10 minutes to permeabilize the membrane. Wash the membrane three times with PBS, each for 5 minutes.

[0127] 7) Prepare 5% BSA blocking solution with PBST and incubate the sample with the blocking solution at room temperature for 1 hour;

[0128] 8) After blocking, discard the blocking solution and wash three times with PBS, each time for 5 minutes;

[0129] 9) Dilute the primary antibody 1:100 with blocking solution, add 50 μL of antibody dilution to each slide and incubate at room temperature for 1 hour;

[0130] 10) After incubation, wash the samples three times with PBS for 5 minutes each. Dilute the secondary immunofluorescence antibody 1:100 in blocking buffer. Then, add 50 μL of antibody dilution buffer to each sample and incubate at room temperature for 1 hour.

[0131] 11) After incubation, the samples were washed three times with PBS, each time for 5 minutes;

[0132] 12) Dilute DAPI nuclear dye 1:1000 with blocking solution and add 50 μL of the dilution to each sample for incubation for 30 minutes.

[0133] 13) After incubation, wash the sample three times with PBS for 5 minutes each. Add 30 μL of anti-fluorescence quencher to the slide and place the coverslip sample-side up on the slide. Seal the sample with nail polish on all sides to prevent drying. Store the sealed sample at 4°C in the dark for subsequent photography and microscopic examination.

[0134] 1.2.5 Detection of DNA damage foci in senescent cells

[0135] 1) Disinfect the coverslips with 75% alcohol and wash with PBS, then place them in a 6-well plate for later use.

[0136] 2) After cell digestion, count the cells and seed them into a 6-well plate containing a coverslip;

[0137] 3) Induce cell senescence with drugs after the cell confluence reaches 80%;

[0138] 4) After the cells are completely senescent, the culture medium is discarded and the cells are washed with preheated PBS. After washing, 4% paraformaldehyde is added and allowed to stand at room temperature for 15 minutes to fix the cell samples;

[0139] 5) After fixation, discard the 4% paraformaldehyde and wash three times with PBS, each time for 5 minutes;

[0140] 6) Add PBST containing 1% Triton X-100 to the sample and incubate at room temperature for 10 minutes to permeabilize the membrane. Wash the membrane three times with PBS, each for 5 minutes.

[0141] 7) Prepare 5% BSA blocking solution with PBST and incubate the sample with the blocking solution at room temperature for 1 hour;

[0142] 8) After blocking, discard the blocking solution and wash three times with PBS, each time for 5 minutes;

[0143] 9) Dilute the primary antibody 1:100 with blocking solution, add 50 μL of antibody dilution to each slide and incubate at room temperature for 1 hour;

[0144] 10) After incubation, wash the samples three times with PBS for 5 minutes each. Dilute the secondary immunofluorescence antibody 1:100 in blocking buffer. Then, add 50 μL of antibody dilution buffer to each sample and incubate at room temperature for 1 hour.

[0145] 11) After incubation, the samples were washed three times with PBS, each time for 5 minutes;

[0146] 12) Dilute DAPI nuclear dye 1:1000 with blocking solution and add 50 μL of the dilution to each sample for incubation for 30 minutes.

[0147] 13) After incubation, wash the sample three times with PBS for 5 minutes each. Add 30 μL of anti-fluorescence quencher to the slide and place the coverslip sample-side up on the slide. Seal the sample with nail polish on all sides to prevent drying. Store the sealed sample at 4°C in the dark for subsequent photography and microscopic examination.

[0148] 1.2.6 Detection of SASP factors in senescent cells

[0149] 1.2.6.1 Total RNA extraction from cells

[0150] 1) Take the sample to be tested out of the incubator and discard the culture medium;

[0151] 2) Add 1 mL of MagZol to the culture dish, pipette the sample with a nuclease-free pipette tip, and transfer it to a 1.5 mL centrifuge tube;

[0152] 3) After the sample is completely dissolved, add 200 μL of chloroform to a 1.5 mL centrifuge tube, shake to mix, and let stand at room temperature for 5 minutes;

[0153] 4) Place the tube in a pre-cooled refrigerated centrifuge and centrifuge at 4°C, 12,000 rpm for 15 minutes. After centrifugation, transfer the upper clear layer to a new 1.5 mL centrifuge tube.

[0154] 5) Add an equal volume of isopropanol to a 1.5 mL centrifuge tube, incubate at room temperature for 10 minutes, and then centrifuge at 12,000 rpm for 10 minutes at 4°C to precipitate RNA.

[0155] 6) Aspirate the liquid in the centrifuge tube, add 1 mL of pre-chilled 75% ethanol, and centrifuge at 7,500 rpm at 4°C for 5 minutes.

[0156] 7) Aspirate the liquid in the centrifuge tube and centrifuge at 12,000 rpm at 4°C for 5 min to completely remove any residual liquid.

[0157] 8) Add 30 μL of nuclease-free water to the centrifuge tube to dissolve the precipitate, label the sample name and concentration, and store in a -80°C freezer.

[0158] 1.2.6.2cDNA synthesis

[0159] This experiment uses the Tiangen Bio-One-Step RNA Reverse Transcription Kit. The specific experimental steps are as follows:

[0160] 1) To ensure the accuracy of subsequent qPCR experimental data, the extracted total cell RNA needs to be quantified;

[0161] 2) Add 1 μg of total cellular RNA to a PCR tube and add nuclease-free water to make up the volume to 8 μL;

[0162] 3) Add 2 μL of reverse transcriptase mix and place the sample in a PCR instrument for reverse transcription. The reverse transcription protocol is: amplification at 42°C for 30 minutes, incubation at 95°C for 3 minutes to inactivate the enzyme, and storage at 16°C.

[0163] 4) After reverse transcription, dilute the cDNA sample with 90 μL of nuclease-free water. After dilution, use it for qPCR detection or store it in a -20°C refrigerator.

[0164] 1.2.6.3 Fluorescence quantitative PCR

[0165] Fluorescence quantitative PCR experiments were performed using Quanshijin Top Green qPCR SuperMix and Roche's Light Cycler 480 real-time PCR instrument were used. Primers were synthesized by BGI Biotechnology; primer sequences are provided in the experimental materials. Diluted template, primers, and SYBR were added to a 384-well microplate according to the system outlined in Table 1. The assay procedure is shown in Table 2.

[0166] Table 1 List of fluorescent quantitative PCR primers

[0167]

[0168]

[0169] Table 2 Fluorescence quantitative PCR system

[0170]

[0171] Table 3 Fluorescence quantitative PCR program

[0172]

[0173] 1.2.7 Detection of arginine content in living cells

[0174] 1.2.7.1 Construction of genetically encoded arginine fluorescent probe cell lines

[0175] The present invention uses a high-performance genetically encoded arginine fluorescent probe (see CN202010098995.5) to detect cellular arginine levels.

[0176] 1) Thaw 293T cells and passage them when they resume growth and reach 80-90% confluence;

[0177] 2) Transfection experiments can be performed after the cells reach 80% confluence after passage;

[0178] 3) Using a 10 cm dish as an example, prepare the transfection mixture according to the system given in Table 2.2.7.

[0179] 4) After the prepared mixed solution A and mixed solution B are allowed to stand at room temperature for 5 minutes, the two parts of the liquid are mixed and allowed to stand at room temperature for another 20 minutes;

[0180] Table 4 Cell transfection system

[0181]

[0182] 5) The 293T cells to be transfected are replaced with fresh culture medium containing 5% serum and the mixture is added, and the cells are returned to the incubator for culture;

[0183] 6) After 12 hours of cell culture, discard the transfection medium, add pre-warmed complete culture medium, and return the cells to the incubator for culture;

[0184] 7) Collect viral supernatants after 24, 48, and 72 hours of incubation and freeze in a -80°C refrigerator for later use;

[0185] 8) The viral supernatant was centrifuged at 3,000 rpm / min for 10 min to pellet residual cell debris and added to the culture dish to infect cells as needed;

[0186] 9) Different cell lines have different sensitivities to viruses; therefore, the infection time and viral load need to be optimized for different cell lines.

[0187] 10) 48 hours after virus infection, the cells can be assayed for fluorescent probe expression using a microscope, flow cytometer, or microplate reader. When the cell confluence reaches 80%, the cells are passaged. Since the target plasmid carries a puromycin resistance gene, an appropriate concentration of puromycin is added to screen the cells to ensure a high positive rate of cell lines.

[0188] 1.2.7.2 ELISA assay for arginine content in living cells

[0189] 1) Digest, neutralize, resuspend, and count the cell line stably expressing the arginine probe. 15,000 cells are seeded into a 96-well microtiter plate. After the cells adhere, induce cellular senescence with a drug before testing.

[0190] 5) Aspirate the culture medium and wash the sample three times with prewarmed HBSS glucose buffer. Add 100 μL of HBSS glucose buffer to the sample and perform enzyme-linked immunosorbent assay. Read the fluorescence values ​​of the sample at the Ex / Em = 420 / 532 nm channel and the Ex / Em = 485 / 528 nm channel using a microplate reader, and calculate the 485 nm / 420 nm ratio.

[0191] 1.2.7.3 Flow cytometric detection of arginine content in living cells

[0192] 1) 40,000 cells stably expressing the arginine probe were seeded in a 12-well plate and returned to the incubator for culture;

[0193] 2) After the cells attach, chemotherapy drugs are used to induce cell senescence and maintain the cells in a fully senescent state;

[0194] 3) After the cells are completely senescent, wash, digest, and neutralize the cells, transfer the entire suspension to a 1.5 mL centrifuge tube, and centrifuge at 1,000 rpm for 5 minutes;

[0195] 4) Discard the supernatant, resuspend the cells in 150 μL of PBS solution containing 4% serum, and transfer to a 96-well plate for later use;

[0196] 5) Detect the sample using the FITC channel and KO525 channel of the flow cytometer, and calculate the FITC / KO525 fluorescence ratio.

[0197] 1.3 Experimental Results

[0198] 1.3.1 Genotoxic chemotherapy drugs induce enhanced lysosomal β-galactosidase activity in senescent cells

[0199] Figure 1 The results of cell Sa-β-Gal staining are shown. The experimental results show that the cells were positively stained with Sa-β-Gal after treatment with genotoxic chemotherapy drugs, indicating that genotoxic chemotherapy drugs can induce cell senescence and enhance the activity of lysosomal β-galactosidase in senescent cells.

[0200] 1.3.2 Enhanced DNA damage response in senescent cells

[0201] Figure 2 The results of γH2AX immunofluorescence analysis show that cellular DNA damage response is enhanced after treatment with genotoxic chemotherapy drugs. γH2AX, a typical molecular marker of DNA damage, is significantly increased in number and brightness in senescent cells, suggesting that genotoxic chemotherapy drugs induce cellular senescence by triggering a strong DNA damage response.

[0202] 1.3.3 Decreased DNA replication capacity in aging cells

[0203] Figure 3 The results of a BrdU immunofluorescence assay are shown. BrdU, a nucleoside analog, participates in DNA replication alongside thymidine during cell division, thereby labeling genomic DNA. The results indicate that cells treated with genotoxic chemotherapy drugs exhibit negative BrdU immunofluorescence staining, indicating a loss of DNA replication and proliferation, and that genotoxic chemotherapy drugs can induce cellular senescence.

[0204] 1.3.4 Enhanced SASP in Senescent Cells

[0205] Figure 4 The results of cell SASP factor gene expression are shown. The experimental results show that the SASP gene expression of senescent cells treated with genotoxic chemotherapy drugs is significantly upregulated, indicating that the drugs successfully induce cell senescence and that senescent cells can release a large number of SASP factors.

[0206] 1.3.5 Arginine content in senescent cells is significantly downregulated

[0207] Figure 5 The results of the cell cytoplasmic arginine content are shown. The experimental results show that the cytoplasmic arginine content of senescent cells treated with genotoxic chemotherapy drugs is significantly downregulated, indicating that the arginine metabolism of senescent cells is enhanced and arginine is an important metabolite of senescent cells.

[0208] Example 2 Effects of Arginine Deprivation on Senescent Cells

[0209] 2.1 Experimental Materials and Reagents

[0210] The arginine-free DMEM culture medium required in this example was produced by Dalian Meilun Biotechnology Co., Ltd., and the remaining experimental materials and reagents were the same as those in Example 1.

[0211] 2.2 Experimental methods

[0212] Preparation of arginine-deprived senescent cell samples. Cells were cultured in arginine-free DMEM medium while senescence was induced with genotoxic agents. For samples requiring arginine, L-arginine powder was added to a final concentration of 400 μM (the L-arginine content in normal culture medium). Samples were cultured in arginine-free medium and assayed 10 days after treatment.

[0213] 2.3 Experimental Results

[0214] 2.3.1 Arginine deprivation reduces lysosomal β-galactosidase activity in aging cells

[0215] Figure 6 The results of Sa-β-Gal staining of senescent cells after arginine deprivation are shown. The experimental results show that arginine deprivation can reduce Sa-β-Gal staining of senescent cells and reduce the activity of lysosomal β-galactosidase.

[0216] 2.3.2 Arginine deprivation reduces DNA damage response in senescent cells

[0217] Figure 7 The experimental results of γH2AX immunofluorescence of DNA damage foci in senescent cells after arginine deprivation are shown. The experimental results show that the number and activation degree of DNA damage foci in senescent cells decreased significantly after arginine deprivation.

[0218] 2.3.3 Arginine deprivation reduces the SASP in senescent cells

[0219] Figure 8 The gene expression levels of SASP factors in senescent cells after arginine deprivation were shown. The experimental results showed that the expression of SASP genes in senescent cells was significantly downregulated after arginine deprivation.

[0220] 2.3.4 Excessive arginine does not promote the expression of SASP factors in senescent cells

[0221] Figure 9 The results show that the addition of different concentrations of arginine to cells in the presence of arginine affects the occurrence and development of SASP in senescent cells. The experimental results indicate that the arginine metabolism of senescent cells has a limit, and the additional addition of arginine will not promote the occurrence of SASP in senescent cells.

[0222] 2.3.4 Arginine metabolism affects the SASP of senescent cells

[0223] Figure 10 The results show that under the premise of arginine deprivation, the addition of different concentrations of arginine affects the occurrence and development of SASP in senescent cells. The experimental results show that arginine deprivation can inhibit the occurrence and development of SASP in senescent cells, and the addition of arginine can promote the expression of SASP in senescent cells and the SASP expression level increases with the increase of arginine concentration.

[0224] 2.3.5 Arginine deprivation reduces nuclear proliferation in senescent cells

[0225] Figure 11 The statistical data of the nuclear diameter of senescent cells are shown, and the experimental results show that the nuclear diameter of senescent cells is significantly reduced after arginine deprivation.

[0226] 2.3.6 Arginine deprivation reduces cell volume in senescent cells

[0227] Figure 12 The statistical data of the cell volume of senescent cells are shown, and the experimental results show that the volume of senescent cells is significantly reduced after deprivation of arginine.

[0228] In summary, this example found that arginine is an important metabolite of senescent cells, and depriving senescent cells of arginine can alleviate cell senescence.

[0229] Example 3 Arginine deprivation and tumor chemotherapy

[0230] 3.1 Experimental Materials and Reagents

[0231] The crystal violet staining solution required for this example was produced by Yisheng Biotechnology Co., Ltd., the cell apoptosis detection kit was produced by Shichengjin Biotechnology Co., Ltd., the Transwell culture dish was produced by Jiete Co., Ltd., the BALB / c nude mice were provided by Jicui Pharmaceutical Co., Ltd., and the mouse bedding and feed were all produced by Xietong Biological Co., Ltd.

[0232] 3.2 Experimental methods

[0233] 3.2.1 Collection of cell-conditioned medium

[0234] 1) When the confluence of normal cells reaches 80% or senescent cells reach complete senescence, wash the cells with preheated PBS;

[0235] 2) Add serum-free medium and culture the cells for 3 days;

[0236] 3) After three days, the supernatant was collected, centrifuged at 3,000 rpm for 5 min to pellet cell debris, and stored at -80°C until use;

[0237] 4) After the cells are cultured for 3 days with complete medium, CM can be collected again.

[0238] 3.2.2 Tumor cell clone formation experiment

[0239] 1) 10,000 tumor cells (human prostate tumor cells PC-3 and DU145, mouse prostate tumor cells RM-1, mouse skin melanoma cells B16-F10) were seeded in a 12-well plate and duplicate samples were prepared;

[0240] 2) 24 hours after tumor cells were inoculated and allowed to adhere, corresponding CM was added and cultured for 3 days;

[0241] 3) One sample was cultured with CM for 3 days, the culture medium was aspirated, and the tumor cells were washed with PBS;

[0242] 4) Add 4% paraformaldehyde to fix the tumor cells at room temperature for 15 minutes;

[0243] 5) Aspirate and discard 4% paraformaldehyde and wash the tumor cells with PBS three times, 5 minutes each time;

[0244] 6) Add crystal violet solution and incubate at room temperature for 30 minutes;

[0245] 7) Recover the crystal violet solution and wash the sample with clean water until no purple solution flows out;

[0246] 8) After the sample is dried, store it at room temperature for subsequent microscopic examination or photography;

[0247] 9) For another sample, the culture medium was aspirated and the tumor cells were washed with PBS;

[0248] 10) Add 200 μL of trypsin to each well to digest the cells, and then add 500 μL of complete culture medium to neutralize them;

[0249] 11) Use a pipette to pipette the cells to ensure uniform cell distribution, then discard 30 μL of the cell suspension and mix it with an equal volume of 0.2% trypan blue solution, and count the cells using a cell counter.

[0250] 3.2.3 Tumor cell scratch assay

[0251] 1) Tumor cells were seeded in 6-well plates at a cell density of 70%;

[0252] 2) After the cells have attached, use a blue pipette tip and a ruler to draw a straight line in the middle of each well, keeping the pipette tip vertical and not tilted.

[0253] 3) Gently tap the side of the 6-well plate to aspirate and discard the culture medium and floating cells;

[0254] 4) Add CM to 6-well plates as needed to culture tumor cells;

[0255] 5) Because different tumor cells have different growth rates and migration abilities, tumor cells were observed every 12 hours and the experiment was stopped after the wound healed;

[0256] 6) Aspirate the culture medium and wash the cells with PBS;

[0257] 7) Add 4% paraformaldehyde to fix the cells for 15 minutes at room temperature;

[0258] 8) Aspirate and discard 4% paraformaldehyde and wash the cells with PBS three times, 5 minutes each time;

[0259] 9) The samples were stored in PBS for subsequent observation and photography.

[0260] 3.2.4 Tumor cell migration assay

[0261] 1) Remove the tumor cells from the incubator, discard the culture medium, and wash the cells with preheated PBS;

[0262] 2) Digest the tumor cells with trypsin and neutralize with complete culture medium. Transfer all the liquid to a 15 mL centrifuge tube and centrifuge.

[0263] 3) After resuspending the cells, count the cells using a cell counter and seed 100,000 cells in a Transwell cell culture chamber;

[0264] 4) Add the desired CM to the well plate, place the Transwell chamber seeded with tumor cells into the well plate, and culture with CM for 24 hours to induce cell migration;

[0265] 5) Aspirate the culture medium and wash the cells with preheated PBS;

[0266] 6) Aspirate and discard PBS, and fix the tumor cells on the surface of the Transwell chamber with 4% paraformaldehyde;

[0267] 7) Aspirate and discard the 4% paraformaldehyde and wash the sample with PBS. Gently wipe the cells in the Transwell chamber with a cotton swab to remove tumor cells that have not migrated.

[0268] 8) Wash the Transwell chamber three times with PBS, 5 minutes each time;

[0269] 9) Permeabilize the sample with 1% Triton X-100 in PBST and let stand at room temperature for 10 minutes;

[0270] 10) Aspirate and discard PBST, and wash the sample three times with PBS, each time for 5 minutes;

[0271] 11) Dilute the nuclear dye 1:1000 with 5% BSA in PBST. Place 100 μL of the dilution on the surface of the parafilm and incubate the migrating cells with the dye dilution in the dark for 10 minutes.

[0272] 12) Wash the sample three times with PBS for 5 minutes each time and take the image immediately after washing to avoid fluorescence quenching.

[0273] 3.2.5 Tumor cell invasion assay

[0274] 1) The experimental steps are basically the same as those for the tumor cell migration assay, except that the Transwell chamber needs to be pretreated;

[0275] 2) Melt the Matrigel on ice and pre-cool the 1.5 mL centrifuge tubes and pipette tips required for the experiment in a -20°C refrigerator.

[0276] 3) After the Matrigel has melted and the experimental equipment has frozen, dilute the Matrigel with serum-free medium at a ratio of 1:3, and transfer 30 μL of the dilution into the Transwell chamber;

[0277] 4) Place the Transwell chamber containing Matrigel in an incubator and incubate for 3 hours. After the Matrigel solidifies, inoculate cells for invasion assay.

[0278] 5) Add the desired CM to the well plate, place the Transwell chamber seeded with tumor cells into the well plate, and culture with CM for 48 hours to induce cell migration;

[0279] 6) Aspirate the culture medium and wash the cells with preheated PBS;

[0280] 7) Aspirate and discard PBS, and fix the tumor cells on the surface of the Transwell chamber with 4% paraformaldehyde;

[0281] 8) Aspirate and discard the 4% paraformaldehyde and wash the sample with PBS. Gently wipe the cells in the Transwell chamber with a cotton swab to remove tumor cells that have not migrated.

[0282] 9) Wash the Transwell chamber three times with PBS, 5 minutes each time;

[0283] 10) Permeabilize the sample with 1% Triton X-100 in PBST and let stand at room temperature for 10 minutes;

[0284] 11) Aspirate and discard PBST, and wash the sample three times with PBS, each time for 5 minutes;

[0285] 12) Dilute the nuclear dye 1:1000 with 5% BSA in PBST. Place 100 μL of the dilution on the surface of the parafilm and incubate the migrating cells with the dye dilution in the dark for 10 minutes.

[0286] 13) Wash the sample three times with PBS for 5 minutes each time and take the image immediately after washing to avoid fluorescence quenching.

[0287] 3.2.6 Tumor cell drug resistance experiment

[0288] The apoptosis detection kit used in this experiment was purchased from Quanshijin Company.

[0289] 1) Tumor cells were digested, neutralized, resuspended, and counted, and 20,000 tumor cells were seeded into 12-well plates for culture;

[0290] 2) After the cells adhered and resumed growth, apoptosis of tumor cells was induced according to the half-inhibitory concentration (IC50) of MIT on tumor cells, and CM was added to treat the cells at the same time;

[0291] 3) After 24 hours of drug treatment, apoptotic cells in the supernatant were collected and adherent cells in the culture dish were collected by trypsin digestion;

[0292] 4) Wash the cells twice with pre-chilled PBS and collect the cells by centrifugation at 1,000 rpm and 4°C for 5 min;

[0293] 5) Remove PBS and resuspend cells in 100 μL of pre-chilled 1× Annexin V binding buffer;

[0294] 6) Add 5 μL of Annexin V-FITC and 5 μL of PI, gently pipette around the cells to mix, and incubate at room temperature in the dark for 15 minutes;

[0295] 7) Add 400 μL of pre-chilled 1× Annexin V binding buffer and gently pipette the cells to mix. Place the sample on ice and wait for flow cytometry analysis.

[0296] 3.2.7 Mouse tumor-bearing experimental procedures

[0297] 1) Digestion, neutralization, resuspending, and counting of mouse RM-1 tumor cells;

[0298] 2) Digestion, neutralization, resuspending, and counting of mouse NIH3T3 fibroblasts;

[0299] 3) Evenly mix fibroblasts and tumor cells at a ratio of 1:4 and count them;

[0300] 4) Each tumor requires 6.25^10 6 cells (5^10 6 RM-1 cells and 1.25^10 6 NIH3T3 cells), the mixed cells were resuspended in 200 μL PBS and inoculated into the groin of nude mice;

[0301] 5) After the mice were raised for 2 weeks and the tumor mass was established, the chemotherapy drug mitoxantrone was injected intraperitoneally at a concentration of 0.4 mg / kg.

[0302] 6) Mice in the arginine deprivation group received chemotherapy drugs and were fed an arginine-free diet;

[0303] 7) Mice need to receive five drug treatments, once a week;

[0304] 8) After the mice were treated, they were euthanized with anesthesia. The tumors in the mice were removed and the expression of V = (π / 6) × ((l + w) / 2) was calculated according to the literature (Anand N, Murthy S, Amann G, et al. Protein elongation factor eef1a2 is aputative oncogene in ovarian cancer. Nat Genet. 2002, 31(3): 301-305). 3 Calculate tumor volume.

[0305] 3.3 Experimental Results

[0306] 3.3.1 Effect of arginine deprivation in tumor microenvironmental senescent cells on tumor cell proliferation

[0307] Figure 13 and Figure 14 The experimental results of crystal violet staining and cell statistics of tumor cells are shown. The experimental results indicate that senescent cells in the tumor microenvironment can promote the malignant proliferation of tumor cells by releasing SASP factors. By depriving arginine, the occurrence and development of SASP in senescent cells can be inhibited, thereby reducing the ability of tumor cells to proliferate malignantly.

[0308] 3.3.2 Effects of arginine deprivation in tumor microenvironmental senescent cells on tumor cell invasion and migration

[0309] Figure 15 and Figure 16The data show the statistical data of tumor cell invasion and migration. The experimental results show that benign cells in the tumor microenvironment undergo senescence and release a variety of SASP factors after stimulation by chemotherapy drugs. SASP factors can significantly enhance the ability of tumor cells to proliferate and migrate. Depriving senescent cells of arginine can inhibit the occurrence and development of SASP in senescent cells, thereby reducing the invasion and migration of malignant tumor cells. After cell senescence, a large amount of arginine is consumed. Senescent cells cultured in arginine-free medium have no arginine intake for a long time (10 days), and their arginine metabolism is restricted to a certain extent, which leads to the inhibition of their SASP and a significant reduction in the release of SASP factors in their supernatant. Tumor cells are cultured with the supernatant of senescent cells. Due to the reduction of SASP factors, the ability of SASP to promote tumor cell proliferation is reduced.

[0310] 3.3.3 Effect of arginine deprivation in tumor microenvironmental senescent cells on tumor cell drug resistance

[0311] Figure 17 and Figure 18 The flow cytometry results and statistical data from a tumor cell drug resistance experiment show that benign cells in the tumor microenvironment undergo senescence after chemotherapy and release multiple SASP factors, which can significantly enhance the drug resistance of tumor cells. Depriving senescent cells of arginine can inhibit the development of the SASP in senescent cells, thereby reducing the drug resistance of tumor cells.

[0312] 3.3.4 In vivo study of arginine nutritional intervention on tumor chemotherapy

[0313] Figure 19 The study demonstrated the in vivo effects of arginine deprivation on tumor chemotherapy. Experiments revealed that tumor volume in tumor-bearing mice significantly decreased after receiving chemotherapy drugs. Furthermore, feeding the treated mice an arginine-free diet further enhanced the therapeutic efficacy of the chemotherapy drugs against the tumors, suggesting that arginine deprivation could serve as an adjuvant therapy for tumor chemotherapy, thereby enhancing clinical tumor treatment outcomes.

Claims

1. Use of arginine-free DMEM medium in the preparation of a reagent for delaying cell senescence caused by genotoxic chemotherapy drugs, wherein the cells are non-tumor cells, and the genotoxic chemotherapy drugs are selected from one or more of the following: bleomycin, satraplatin, mitoxantrone and doxorubicin.

2. A method for non-therapeutic in vitro delaying of cell senescence induced by genotoxic chemotherapy drugs, non-therapeutic in vitro reduction of lysosomal β-galactosidase activity in senescent cells induced by genotoxic chemotherapy drugs, non-therapeutic in vitro reduction of DNA damage response in senescent cells induced by genotoxic chemotherapy drugs, or non-therapeutic in vitro reduction of SASP gene expression in senescent cells induced by genotoxic chemotherapy drugs, comprising the step of arginine deprivation of the cells, wherein the arginine deprivation comprises culturing the cells in arginine-free DMEM medium, wherein the cells are non-tumor cells, and the genotoxic chemotherapy drugs are selected from one or more of the following: bleomycin, satraplatin, mitoxantrone, and doxorubicin.

3. An in vitro non-therapeutic method for enhancing the killing effect of mitoxantrone on tumor cells, comprising the step of depriving the cells of arginine, wherein the arginine deprivation comprises culturing the cells in arginine-free DMEM medium, wherein the tumor cells are prostate tumor cells RM-1.

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