CN115317613A - Targets and applications related to chemoresistance in colorectal cancer - Google Patents

Abstract

The invention discloses a target spot related to drug resistance of a colorectal cancer chemotherapeutic drug and application thereof. The invention discovers for the first time that: thiol oxidative stress may play an important role in 5-Fu drug-resistant CRC reversal, and TGM2 can be degraded through S-glutathione dependence, 5-Fu-induced apoptosis of 5-Fu drug-resistant CRC cells is enhanced, and thus the treatment effect of 5-Fu is enhanced. Changes in the expression level of TMG2 protein in CRC cells affect the treatment sensitivity to 5-Fu, and TGM2 is associated with drug resistance and poor prognosis of CRC patients to 5-Fu. TGM2 is likely to be a potential therapeutic target and may be regulated by thiol oxidative stress through S-glutathionylation by C193. Compared with 5-Fu single medicine, the combination of 5-Fu and 2-APPA can obviously improve the treatment effect of colorectal cancer.

Description

Targets and applications related to chemotherapeutic drug resistance in colorectal cancer

technical field

The invention belongs to the fields of biomedicine and pharmacy, and specifically relates to targets and applications related to colorectal cancer chemotherapeutic drug resistance.

Background technique

Colorectal cancer (CRC) is one of the most common cancers in the world. According to the 2018 global cancer statistics, CRC ranks first in cancer-related death and third in incidence. Currently, the first-line treatment for patients with colorectal cancer is surgery combined with chemotherapy drugs. As a basic drug in colorectal cancer chemotherapy, 5-Fu inhibits the biological activity of thymine synthase, prevents DNA synthesis in colorectal cancer cells, and further interferes with tumor growth and metastasis. However, long-term use of 5-Fu in patients with colorectal cancer tends to cause tumor insensitivity to 5-Fu. According to a large number of literature reports, one of the most important reasons for the failure of chemotherapy using 5-Fu as the base drug is that colorectal cancer patients are resistant to 5-Fu. Of particular concern is that colorectal cancer patients who were previously sensitive to 5-Fu are more likely to show resistance to 5-Fu when their tumors recur. The effective rate of 5-Fu-based chemotherapy in patients with advanced colorectal cancer is only 10-15%, and more than 80% of patients with advanced colorectal cancer eventually relapse, although they initially responded to chemotherapy. Drug resistance to 5-Fu has been regarded as one of the main obstacles to chemotherapy for advanced colorectal cancer, therefore, there is an urgent need to identify therapeutic targets and develop new treatment strategies to improve 5-Fu-based therapy in colorectal cancer patients. response to chemotherapy.

Studies have shown that the mechanisms of CRC's resistance to 5-Fu are: the function of transmembrane transporters is enhanced, resulting in a decrease in intracellular drug concentration; the activity of thymidine synthase (TS) is increased, which antagonizes the effect of 5-Fu on inhibiting TS; The overexpression of dehydrogenase (DPD) accelerates the catabolism of 5-FU; the function of inhibitor of apoptosis protein family (IAPs) is enhanced, which reduces drug-induced apoptosis; phosphatidylinositol 3 kinase/protein kinase B (PI3K-Akt ) signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, etc. are abnormally activated; heat shock protein 27 (HSP27) is highly expressed, and its protective effect on tumor cell damage is enhanced.

Molecules that play a key role in the above process are often biomarkers related to tumor prognosis, and are also important targets for reversing drug resistance and improving chemotherapy effects. Due to the many causes and mechanisms of chemotherapy resistance in colorectal cancer, reversing chemotherapy resistance is full of challenges. It is still necessary to develop new and effective therapeutic targets to combat the endless emergence of drug resistance. A large number of scientific research and clinical trials are needed to overcome difficulties. Therefore, it is of great significance for the intervention and treatment of colorectal cancer to actively carry out research on the mechanism of colorectal cancer drug resistance and find new key molecules and therapeutic targets.

Contents of the invention

In view of the 5-Fu drug resistance in the treatment of colorectal cancer, it is urgent to discover new factors regulating drug resistance in colorectal cancer and to clarify the molecular mechanism of drug resistance in colorectal cancer, so as to provide new targets and targets for clinical treatment of colorectal cancer. Strategy. In view of this, the purpose of the present invention is to find the key target for improving the sensitivity of 5-Fu drug-resistant colorectal cancer to 5-Fu.

In order to achieve the above purpose, the present invention conducts related research on the drug resistance mechanism of colorectal cancer.

The present invention uses the small molecule compound 2-APPA (CAS No. 1133387-90-2, structural formula as follows)

Taking drug-resistant colorectal cancer cells CT8/5Fu and HCT15/5Fu as examples, it was found that:

① After adding 2-AAPA to treat HCT8/5Fu and HCT15/5Fu cells, the GSH:GSSG ratio in the cells was down-regulated (2-AAPA acts as a GR inhibitor), and thiol oxidative stress was up-regulated (2-AAPA acts as a thiol Oxidative stress inducer), TGM2 mRNA expression and TGM2 protein level down-regulation (2-AAPA plays the role of inhibiting TGM2 mRNA expression and down-regulating TGM2 protein level), TGM2 S-glutathionylation significantly increased (2-AAPA plays a role in inducing the S-glutathionylation of TGM2, and C193 is the main site of S-glutathionylation modification on TGM2), HCT8/5Fu and HCT15/5Fu cells are sensitive to 5-Fu Resistance reversal and increased sensitivity.

②The combined treatment of 2-AAPA and 5-Fu significantly enhanced the apoptosis induction of HCT8/5Fu and HCT15/5Fu cells, and it was achieved by inducing the cleavage of apoptosis proteins capase3 and PARP. In the HCT15/5Fu xenograft tumor model, after 28 days of drug action, compared with the 5-Fu single drug group, the tumor volume of the combined 2-AAPA and 5-Fu group was significantly smaller, and the tumor cell proliferation was significantly inhibited, 5-Fu-induced apoptosis was significantly enhanced, and TGM2 protein level was significantly down-regulated. Moreover, the tumor-bearing mice tolerated the drug well, and no symptoms such as weight loss occurred. Description: 2-AAPA can significantly reverse the 5-Fu resistance of 5-Fu drug-resistant CRC cells in vitro and in vivo, and increase the sensitivity of 5-Fu drug-resistant CRC cells to 5-Fu. The combination of 2-AAPA and 5-Fu can be used to treat 5-Fu resistant colorectal cancer.

③ Compared with HCT8 and HCT15 cells (non-drug-resistant group), the TGM2 of HCT8/5Fu and HCT15/5Fu cells (drug-resistant group) increased significantly. 2-AAPA alone or in combination with 5-Fu can significantly reduce the mRNA expression of TGM2 in HCT8/5Fu or HCT15/5Fu cells. The combination of 2-AAPA and 5-Fu can significantly reduce the protein level of TGM2 in HCT8/5Fu or HCT15/5Fu cells. Moreover, 2-AAPA decreased the protein level of TGM2 in HCT8/5Fu and HCT15/5Fu cells in a time- and dose-dependent manner, and co-treatment with 2-AAPA and MG132 significantly restored the protein level of TGM2. Description: 2-AAPA-induced thiol oxidative stress, increased apoptosis, and downregulation of TGM2 are achieved through proteasomal degradation of TGM2 protein, and more specifically, through the S-glutathionylation-dependent proteasomal degradation pathway Completed.

Based on the above-mentioned research results, the present invention proposes the following technical solutions:

The first aspect of the present invention provides the application of substances that inhibit the expression of TGM2 gene or down-regulate the level of TGM2 protein, which is at least one of the following (1) to (3):

(1) Preparation of medicines for improving the sensitivity of drug-resistant colorectal cancer cells to 5-fluorouracil;

(2) Preparation of drugs for reducing or reversing drug resistance of colorectal cancer cells to 5-fluorouracil;

(3) Preparation of medicines for increasing the apoptosis of drug-resistant colorectal cancer cells.

Preferably, the substance that inhibits the expression of TGM2 gene or down-regulates the level of TGM2 protein includes: TGM2 inhibitors of TGM2 knockdown lentiviral particles or small molecule compounds.

The second aspect of the present invention provides the application of substances that induce S-glutathionylation of TGM2 protein, which is at least one of the following (1) to (3):

(1) Preparation of medicines for improving the sensitivity of drug-resistant colorectal cancer cells to 5-fluorouracil;

(2) Preparation of drugs for reducing or reversing drug resistance of colorectal cancer cells to 5-fluorouracil;

(3) Preparation of medicines for increasing the apoptosis of drug-resistant colorectal cancer cells.

Preferably, the substance that induces S-glutathionylation of TGM2 protein is a substance that induces S-glutathionylation of C193 site of TGM2 protein.

The third aspect of the present invention provides the application of GR inhibitors, which is at least one of the following (1)-(3):

(1) Preparation of medicines for improving the sensitivity of drug-resistant colorectal cancer cells to 5-fluorouracil;

(2) Preparation of drugs for reducing or reversing drug resistance of colorectal cancer cells to 5-fluorouracil;

(3) Preparation of medicines for increasing the apoptosis of drug-resistant colorectal cancer cells.

The fourth aspect of the present invention provides the application of a thiol oxidative stress inducer, which is at least one of the following (1)-(3):

(1) Preparation of medicines for improving the sensitivity of drug-resistant colorectal cancer cells to 5-fluorouracil;

(2) Preparation of drugs for reducing or reversing drug resistance of colorectal cancer cells to 5-fluorouracil;

(3) Preparation of medicines for increasing the apoptosis of drug-resistant colorectal cancer cells.

The fifth aspect of the present invention provides a drug for treating colorectal cancer, including: 5-fluorouracil and at least one of the following 5-fluorouracil resistance reversal agents:

(X1) A substance that inhibits the expression of TGM2 gene or down-regulates the level of TGM2 protein;

(X2) a substance that induces S-glutathionylation of the TGM2 protein;

(X3) GR inhibitors;

(X4) Thiol oxidative stress inducer.

Preferably, the substance that inhibits the expression of TGM2 gene or down-regulates the level of TGM2 protein includes: TGM2 inhibitors of TGM2 knockdown lentiviral particles or small molecule compounds.

The sixth aspect of the present invention provides the application of TGM2 detection reagent in the preparation of at least one of the following diagnostic reagents:

(Y1) Diagnostic reagents for 5-fluorouracil resistance in colorectal cancer;

(Y2) A diagnostic reagent for the prognosis of colorectal cancer patients.

Preferably, the TGM2 detection reagent includes a TGM2 mRNA detection reagent or a TGM2 protein detection reagent.

Preferably, the TGM2 detection reagents include the reagents required to detect TGM2 content by common detection methods such as immunoblotting, immunohistochemistry, enzyme-linked immunosorbent assay, RT-qPCR detection, and immunofluorescence detection.

Preferably, the TGM2 detection reagent is a reagent for real-time quantitative PCR detection of TGM2, and the PCR primers for TGM2 are as follows:

Forward primer (forward): 5'-TGGGTGGAGTCGTGGATG-3';

Reverse primer (reverse): 5'-GGGAACGGTTGATGGATTT-3'.

In the present invention, the drug-resistant colorectal cancer cells are colorectal cancers that highly express TGM2.

In the present invention, the drug-resistant colorectal cancer cells are HCT8/5Fu or HCT15/5Fu cells.

In the present invention, the drug resistance of the drug-resistant colorectal cancer cells is 5-fluorouracil drug resistance.

Compared with the prior art, the present invention has the following beneficial technical effects:

The present invention finds for the first time that GR-mediated thiol oxidative stress may promote 5-Fu-induced apoptosis of drug-resistant CRC cells by down-regulating TGM2 protein levels, thereby enhancing the therapeutic effect of 5-Fu. Thiol oxidative stress may play an important role in the reversal of 5-Fu-resistant CRC.

The present invention found for the first time that the change of TMG2 protein expression in CRC cells affects its chemosensitivity to 5-Fu, TGM2 may serve as a potential therapeutic target, and can be oxidized by thiols through S-glutathionylation of C193 Regulated. Thiol oxidative stress can enhance 5-Fu-induced apoptosis in 5-Fu-resistant CRC cells through S-glutathionylation-dependent degradation of TGM2. TGM2 is associated with resistance to 5-Fu and poor prognosis in CRC patients.

This study proposed a new treatment plan for colorectal cancer for the first time: 5-Fu combined with 2-APPA.

Description of drawings

Figure 1A shows the in vitro cytotoxicity control of parental cell HCT8 and drug-resistant cell HCT8/5Fu treated with different concentrations of 5-Fu. Figure 1B shows the in vitro cytotoxicity control of parental cell HCT15 and drug-resistant cell HCT15/5Fu treated with different concentrations of 5-Fu.

Figure 2A shows the control of reactive oxygen species levels in parental HCT8 and HCT15 cells, 5-Fu-resistant HCT8/5Fu and HCT15/5Fu cells. Figure 2B shows the control chart of GSH/GSSG ratio in HCT8/5Fu cells treated with different groups. Figure 2C shows the comparison chart of GSH/GSSG ratio in HCT15/5Fu cells treated with different groups. Figure 2D shows the intracellular ROS levels of HCT8/5Fu and HCT15/5Fu treated with different groups. Figure 2E shows the intracellular ROS levels of HCT8/5Fu and HCT15/5Fu treated with N-acetyl-L-cysteine (NAC) in different groups.

Figure 3A and Figure 3B are the results of flow cytometry detection of HCT8/5Fu cell apoptosis in different groups and the corresponding quantification histograms of apoptosis, respectively. Figure 3D and Figure 3C are the results of flow cytometry detection of HCT15/5Fu cell apoptosis in different groups and the corresponding quantification histograms of apoptosis, respectively. Figure 3E and Figure 3F are the results of apoptosis of HCT8/5Fu cells treated with Z-VAD-FMK in different groups and the corresponding quantification histograms of apoptosis. Figure 3H and Figure 3G are the results of apoptosis of HCT15/5Fu cells treated with Z-VAD-FMK in different groups and the corresponding quantification histograms of apoptosis. Figure 3I is the Western blot analysis results of apoptosis-related proteins in HCT8/5Fu cells treated with different groups. Figure 3J shows the results of Western blot analysis of apoptosis-related proteins in HCT15/5Fu cells treated with different groups.

Figure 4A shows the results of RT-PCR analysis of HCT8/5Fu cells treated in groups. Figure 4B shows the results of RT-PCR analysis of HCT15/5Fu cells treated in groups. Figure 4C shows the results of Western blot analysis of TGM2 protein in HCT8/5Fu and HCT15/5Fu cells treated in groups.

Figure 5A shows the results of Western blot analysis of TGM2 protein levels in HCT8-vector cells and HCT8-TGM2 cells, HCT15-vector cells and HCT15-TGM2 cells. Figure 5B shows the results of Western blot analysis of TGM2 protein levels in HCT8/5Fu-vector cells and HCT8/5Fu-TGM2 cells, HCT15/5Fu-vector cells and HCT15/5Fu-TGM2 cells. Figure 5C shows the results of Western blot analysis of TGM2 protein levels in HCT8/5Fu-Scramble cells and HCT8/5Fu-shTGM2 cells, HCT15/5Fu-Scramble cells and HCT15/5Fu-shTGM2 cells.

Figure 6A shows the in vitro cytotoxicity control of HCT8-vector cells and HCT8-TGM2 cells treated with different concentrations of 5-Fu. Figure 6B shows the in vitro cytotoxicity control of HCT15-vector cells and HCT15-TGM2 cells treated with different concentrations of 5-Fu. Figure 6C shows the in vitro cytotoxicity control of HCT8/5Fu-Scramble cells and HCT8/5Fu-shTGM2 cells treated with different concentrations of 5-Fu. Figure 6D shows the in vitro cytotoxicity control of HCT15/5Fu-Scramble cells and HCT15/5Fu-shTGM2 cells treated with different concentrations of 5-Fu.

Fig. 7A shows the control chart of the apoptosis results of HCT15/5Fu-vector cells and HCT15/5Fu-TGM2 cells treated in different groups. Figure 7B presents the histogram of apoptosis quantification in Figure 7A. Fig. 7D shows the control chart of the apoptosis results of HCT15/5Fu-Scramble cells and HCT15/5Fu-shTGM2 cells treated in different groups. Figure 7C presents the histogram of apoptosis quantification in Figure 7D.

Figure 8A shows the results of Western blot analysis of the protein level of TGM2 in HCT8/5Fu and HCT15/5Fu cells treated with different doses of 2-AAPA. Figure 8B shows the results of Western blot analysis of TGM2 protein levels in HCT8/5Fu and HCT15/5Fu cells treated with the same dose of 2-AAPA for different periods of time. Figure 8C shows the results of Western blot analysis of the protein levels of TGM2 in HCT8/5Fu and HCT15/5Fu cells treated in different groups for 6 hours.

Figure 9B shows the mass spectrum of the S-glutathionylated peptide containing Cys193 detected in the Flag-TGM2 protein. Figure 9C shows the results of immunoblot analysis of S-glutathionylation in HCT15/5Fu cells transfected with WT-Flag-TGM2 or Flag-TGM2-C193S plasmids with or without 2-AAPA treatment. FIG. 9D is a quantification histogram of apoptosis results of HCT15/5Fu cells transfected with WT-Flag-TGM2 or Flag-TGM2-C193S treated by different groups.

Fig. 10A is a picture of tumors in nude mouse xenograft tumor models of HCT15/5Fu cells treated with different groups of drugs for 28 days. Fig. 10B is the change data of tumor volume measured during the tumor-bearing period of nude mice treated by different groups. FIG. 10C is a graph comparing the body weight of tumor-bearing mice treated by different groups. Figure 10D and Figure 10E show representative IHC-TGM2 images and quantification of staining, respectively, in tumor samples from four groups. Figure 10F and Figure 10G show representative IHC-Ki67 images and quantification of staining in tumor samples from four groups, respectively. Figure 10H and Figure 10I show representative TUNEL images and fluorescence quantification in four grouped tumor samples, respectively. Among them, compared with the control group, * indicates P<0.05, ** indicates P<0.01; compared with the 5-Fu group, # indicates P<0.05, ### indicates P<0.001.

Fig. 11 is a disease-specific survival curve between TGM2 gene expression level and colorectal cancer patients.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and specific examples. It should be understood that these examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. Those who do not indicate specific techniques or conditions in the following examples shall be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased in the market. The following data are the mean ± standard deviation of three independent experiments. In each figure, *P<0.05, **P<0.01, ***P<0.001. In the examples, the concentration unit adopts the customary writing method in the field: when M, mM or μM, they represent mol/L, mmol/L or μmol/L, respectively.

1. Material description:

2-AAPA (CAS No. 1133387-90-2) can be purchased from Sigma, and can also be purified from p-phenylene diisothiocyanate and N-acetyl-L-cysteine. For specific preparation, please refer to the following Literature: T.Seefeldt, Y.Zhao, W.Chen, A.S.Raza, L.Carlson, J.Herman, A.Stoebner, S.Hanson, R.Foll, X.Guan, Characterization of a novel dithiocarbamate glutathione reductase inhibitor and its use as a tool to modulate intracellular glutathione. The Journal of Biological Chemistry 284, 2729-2737 (2009).

5-Fu was purchased from Sigma. Pan-caspase inhibitor (Z-VAD-FMK) was purchased from Sigma. Proteasome inhibitor MG132 was purchased from Selleck Company. FBS, DMEM culture medium, and trypsin were purchased from Gibco Company, and antibodies (primary antibody and secondary antibody) were purchased from Proteinteck Company. DMEM complete culture solution is DMEM culture solution containing 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. Control activated lentiviral particles, control knockdown lentiviral particles, TGM2 overexpressed lentiviral particles, and TGM2 knockdown lentiviral particles were purchased from Santa Cruz.

Human colorectal cancer cell lines HCT8 and HCT15 were purchased from ATCC Cell Bank. The plasmid of Flag-TGM2 and the plasmid of Flag-TGM2-C193S were constructed by Vigene Bioscience.

BALB/c nude mice, 6 weeks old, female, were purchased from Shanghai Slack Experimental Animal Co., Ltd., experimental animal license number: SCXK (Shanghai) 2017-0005, and all experimental animals were kept in an SPF grade animal room.

Construction of drug-resistant cell lines HCT8/5Fu and HCT15/5Fu

Passage HCT8 cells to 24-well plates, inoculate 20,000 cells per well, place them in a 5% CO ₂ incubator, and culture them in complete DMEM medium for 24 hours at 37°C; then add 20 μM of 5 -Fu, continue to cultivate for 72h; then, change the fresh DMEM complete culture medium and continue to cultivate for 24h, then add 30μM 5-Fu in the DMEM complete culture medium, continue to cultivate for 72h; adjust the 5-Fu concentration in the DMEM complete culture medium (according to The following order is increasing: 20 μM → 30 μM → 40 μM → 50 μM → 60 μM → 70 μM → 80 μM → 90 μM → final concentration 100 μM), repeat the above culture process, and obtain 5-Fu resistant HCT8/5Fu cell lines.

Passage HCT15 cells to 24-well plate, inoculate 20,000 cells per well, place in 5% CO ₂ incubator, and culture in DMEM complete medium for 24 hours at 37°C; then add 2 μM 5 -Fu, continue to cultivate for 72h; then, change the fresh DMEM complete culture medium and continue to cultivate for 24h, then add 3 μ M 5-Fu in the DMEM complete culture medium, continue to cultivate for 72h; adjust the 5-Fu concentration in the DMEM complete culture medium (according to Increment in this order: 2μM→3μM→4μM→5μM→6μM→7μM→8μM→9μM→final concentration 10μM), repeat the above culture process to obtain 5-Fu resistant HCT15/5Fu cell line.

The routine culture of HCT8/5Fu and HCT15/5Fu drug-resistant cell lines adopts DMEM complete medium, and maintains with 100μM and 10μM 5-Fu respectively to ensure the stability of their drug resistance.

2. Method description

2.1 MTT test

The cells in the logarithmic growth phase were digested with 0.25% trypsin and blown into a cell suspension, and the cells were seeded in a 96-well cell culture plate with a final volume of 200 μL per well (the number of cells was 5000/well). Move the 96-well plate into a 37°C, 5% CO ₂ saturated humidity incubator for 24 hours to adhere to the wall, discard the DMEM complete culture solution, and replace it with DMEM complete culture solution containing different concentrations of drugs for cultivation. Set up 4 duplicate wells for each concentration, and place in a 37°C, 5% CO ₂ saturated humidity incubator to continue culturing for 48 hours. MTT solution was added to each well and incubated at 37 °C for 4 h. Detect the respective absorbance, calculate the cell viability of each group, and draw the curve. _IC50 values were calculated using GraphPad Prism software. The drug resistance index (RI) was calculated according to the following formula: RI=IC ₅₀ of 5-Fu drug-resistant CRC cells/IC ₅₀ of non-drug-resistant CRC parental cells.

2.2 LC-MS detection of intracellular GSH and GSSG

Collect cells by trypsinization, lyse the cells with 3% 5-sulfosalicylic acid solution, centrifuge at 16000×g for 10 min at 4°C to remove protein precipitates, dilute the supernatant with 0.2% formic acid solution, and use ThermoFisher Q-Exactive Combined system analysis of GSH and GSSG concentration. Internal reference: p-aminobenzoic acid. Liquid phase column: Thermo Scientific HypersilGOLD C18 column (2.1×100 mm, 1.9 μm). Mobile phase A [0.2% (v/v) formic acid in water]; mobile phase B [0.2% (v/v) formic acid in acetonitrile]: 4min 0%-30%, 6min 30%-80%, 8min 80%. Flow rate: 0.2 mL/min. The mass spectrometry ESI source was set to positive ion SIM mode. The m/z values of GSH, GSSG and internal reference were: 307.08381(M+H) ⁺ , m/z 612.15196(M+H) ⁺ and m/z 138.05550(M+H) ⁺ , respectively.

2.3 CellRox green fluorescent probe detects reactive oxygen species in cells

Cells were seeded in 6-well plates. After adhering to the wall, the cells were treated with different grouping drugs, and then cultured with 5 μM CellRox Green reagent in complete DMEM medium at 37°C for 30 min. Cells were harvested by trypsinization and washed three times with PBS. Intracellular green fluorescence intensity was then analyzed by flow cytometry. Data analysis was performed by FlowJo software.

2.4 Apoptosis experiment

Take the cells in the logarithmic growth phase, digest them into a single cell suspension, inoculate 2× ¹⁰⁵ cells/well in a 6-well cell culture plate, and culture in an incubator at 37°C and 5% CO ₂ saturated humidity for 24 hours to adhere to the wall Afterwards, grouping treatment was carried out: DMEM complete culture solution containing different grouping drugs was added respectively, and the culture was continued for 48 hours in an incubator at 37° C. and 5% CO ₂ saturated humidity, and the cells were collected. Cell apoptosis was detected by flow cytometry according to the Annexin V FITC/PI kit of BD Company.

2.5 Western blotting

Add cell lysate to lyse in ice bath for 30 min, centrifuge at 16000×g, 4°C for 30 min, collect supernatant, determine protein content, and separate protein by SDS-PAGE. After electrophoresis, the protein was transferred to PVDF membrane. After blocking with 5% skimmed milk powder, the primary antibody (apoptosis-related protein, TGM2 protein) was blocked overnight, and then blocked with horseradish peroxidase-labeled secondary antibody for 2 hours. Aminobenzidine (DAB) solution was used for color development and scanning analysis.

2.6 Lentiviral Particle Transfection to Construct TGM2 Overexpression Cells and TGM2 Knockdown Cells

Take the cells in the logarithmic growth phase, digest them into a single cell suspension, inoculate 5×10 ⁵ cells/well in a 12-well cell culture plate, and culture in an incubator at 37°C and 5% CO ₂ saturated humidity for 24 hours to adhere to the wall . The next day, cells were cultured in DMEM complete medium containing 5 μg/mL Polybrene, and 5 μL of various lentiviruses (TGM2 overexpressed lentiviral particles, control activated lentiviral particles, TGM2 knockdown lentiviral particles) were added to each well of cells. lentiviral particles, control knockdown lentiviral particles). On the third day, fresh DMEM complete culture medium was replaced. On the fourth day, the cells were subcultured and cells successfully transfected with lentivirus were screened with puromycin to construct: TGM2 overexpression cells HCT8-TGM2 and HCT15-TGM2, HCT8/5Fu-TGM2 and HCT15/5Fu-TGM2, and corresponding vector cells HCT8-Vector, HCT15-Vector, HCT8/5Fu-Vector, HCT15/5Fu-Vector; TGM2 knockdown cells HCT8/5Fu-shTGM2 and HCT15/5Fu-shTGM2, and corresponding The carrier cells HCT8/5Fu-Scramble and HCT15/5Fu-Scramble.

2.7 Real-time quantitative PCR to determine the mRNA expression level of TGM2

Cells were collected and total RNA was extracted with RNA rapid purification kit (Shanghai Yishan Biotechnology Co., Ltd.). cDNA was synthesized by all-in-one RT kit (Shanghai Yishan Biotechnology Co., Ltd.). Real-time quantitative PCR was completed by ABI 7300 PCR instrument. The PCR primers of TGM2 are as follows: forward: 5'-TGGGTGGAGTCGTGGATG-3'; reverse: 5'-GGGAACGGTTGATGGATTT-3'.

2.8 Immunoprecipitation detection of protein S-glutathionylation on Flag-TGM2 and its mass spectrometry analysis

Cells were collected and lysed with IP buffer containing protease inhibitors (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40 (V/V), 2 mM EDTA) at 4°C for 1 h. After the cell lysate was centrifuged at 16000×g for 10 min, the supernatant was collected and the protein concentration was determined. Take 800 μg cell lysate and 2 μg Flag antibody and incubate overnight at 4°C. Protein S-glutathionylation levels were analyzed by immunoblotting with an anti-glutathione antibody. Flag-TGM2 protein was captured from cell lysate with protein A/G agarose, digested with trypsin overnight at 37°C, and analyzed for S-glutathionylation sites by LC-MS/MS Data were analyzed by Proteome Discoverer 1.4 software (ThermoFisher).

Embodiment 1 adopts MTT method to measure the in vitro cytotoxicity of 5-Fu to each cell line

The method of 2.1 was used to conduct MTT test on the parental cells HCT8, HCT15, drug-resistant cells HCT8/5Fu, HCT15/5Fu, respectively. The culture solution containing different concentrations of drugs was DMEM complete culture solution containing different concentrations of 5-Fu, and the concentration gradient of 5-Fu was set as: 0, 7.8 μM, 15.6 μM, 31.3 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, 1000 μM. Figure 1A shows the in vitro cytotoxicity control of different concentrations of 5-Fu on parental cells HCT8 and drug-resistant cells HCT8/5Fu, and Figure 1B shows the effects of different concentrations of 5-Fu on parental cells HCT15 and drug-resistant cells HCT15 In vitro cytotoxicity control of /5Fu. It can be seen that under different concentrations of treatment, the drug-resistant cells showed better cell activity than the corresponding parental cells. At the same time, calculate the IC ₅₀ values of 5-Fu on HCT8, HCT15 cells, HCT8/5Fu, and HCT15/5Fu cells, as described in Table 1 and Table 2 below. It can be seen that compared with HCT8 and HCT15 cells, the IC ₅₀ values of 5-Fu on HCT8/5Fu and HCT15/5Fu cells were significantly increased, respectively. The RI values of HCT8/5Fu and HCT15/5Fu cells were 21.1 and 15.5, respectively, indicating that HCT8/5Fu and HCT15/5Fu cells were resistant to 5-Fu.

Adopt the method of 2.1 to carry out MTT test to drug-resistant cell HCT8/5Fu, the culture solution that contains different concentrations of drugs is the DMEM complete culture solution that contains the 2-APPA of 5-Fu of different concentrations and 40 μ M; Adopt the method of 2.1 to drug resistance Sex cells HCT15/5Fu were tested for MTT. The culture solution containing different concentrations of drugs was DMEM complete culture solution containing different concentrations of 5-Fu and 45 μM 2-APPA. The concentration gradient of 5-Fu was still set as: 0, 7.8 μM, 15.6 μM, 31.3 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, 1000 μM. The IC ₅₀ values of 5-Fu on HCT8/5Fu and HCT15/5Fu cells at the specified 2-AAPA doses were calculated according to the results of MTT test. At the same time, compared with the IC ₅₀ value of 5-Fu on HCT8/5Fu and HCT15/5Fu cells without adding 2-AAPA, and calculated the reversal fold. Reversal factor (RF) = IC ₅₀ of 5-Fu on cells without 2-AAPA/IC ₅₀ of 5-Fu on cells with 2-AAPA. The details are shown in Table 1 and Table 2 below, where RI is the drug resistance index; RF is the reversal factor.

Table 1. Determination of IC ₅₀ values of HCT8/5Fu cells to 5-Fu

Table 2. Determination of IC ₅₀ values of HCT15/5Fu cells to 5-Fu

It can be seen from Table 1 that under the action of 40 μM 2-AAPA, the sensitivity of HCT8/5Fu cells to 5-Fu increased, and the RF was 2.2 times. That is, 2-AAPA can reverse the drug resistance of HCT8/5Fu to 5-Fu. It can be seen from Table 2 that under the action of 45 μM 2-AAPA, the sensitivity of HCT15/5Fu cells to 5-Fu was increased, and the RF was 3.7 times. That is, 2-AAPA can reverse the drug resistance of HCT15/5Fu cells to 5-Fu.

Example 2 Intracellular thiol oxidative stress and reactive oxygen species levels

The methods of 2.2 and 2.3 were used to detect the quantification of GSH and GSSG and the generation level of ROS in HCT8, HCT8/5Fu, HCT15 and HCT15/5Fu cells. LC-MS results showed that the GSH concentrations in HCT8, HCT8/5Fu, HCT15 and HCT15/5Fu cells were 13.53±0.77, 15.55±0.25, 11.42±0.34 and 14.71±1.97 (unit: nmol/mg protein); HCT8, HCT8 The concentration of GSSG in /5Fu, HCT15 and HCT15/5Fu cells were 0.79±0.13, 1.39±0.33, 0.34±0.05 and 0.49±0.04 (unit: nmol/mg protein), respectively. From this, the intracellular GSH:GSSG ratio, a key indicator indicative of intracellular thiol oxidative stress, was calculated. The GSH:GSSG ratios in HCT8, HCT8/5Fu, HCT15 and HCT15/5Fu cells were: 17.50±2.79, 11.21±0.12, 33.57±1.8, 29.71±1.88, respectively. It can be seen that the GSH:GSSG ratio is significantly lower in drug-resistant cells than in parental cells. The results of evaluating the production level of intracellular ROS using CellRox Green reagent are shown in Figure 2A. As can be seen from Figure 2A, compared with the parental HCT8 and HCT15 cells, the production of reactive oxygen species in HCT8/5Fu and HCT15/5Fu drug-resistant cells was significantly increased. These results indicated that thiol oxidative stress was significantly increased in HCT8/5Fu and HCT15/5Fu resistant cells.

HCT8/5Fu cells were randomly grouped into groups: ①Ctrl control group (DMEM complete culture solution without drugs); ②2-AAPA single drug group: DMEM complete culture solution containing 2-AAPA at a concentration of 40 μM; ③5-Fu single drug group : DMEM complete culture solution containing 400 μM 5-Fu; ④Combination combination group: DMEM complete culture solution containing 40 μM 2-AAPA and 400 μM 5-Fu, and different cultures were set up for each group of cells Time: 2h, 4h, 8h and 24h. LC-MS was used to detect intracellular GSH and GSSG in each group and calculate the ratio of GSH/GSSG. For each group of cells treated for 2 h, the cells were stained with CellRox Green reagent, and the ROS levels were analyzed by flow cytometry. The results showed that in HCT8/5Fu cells, 2-AAPA alone or in combination could significantly reduce the ratio of GSH:GSSG (as shown in Figure 2B), and the combination of 2-AAPA and 5-Fu significantly increased the generation of ROS (as shown in Figure 2D Show).

HCT15/5Fu cells were randomly divided into groups: ①Ctrl control group (DMEM complete culture solution without drugs); ②2-AAPA single drug group: DMEM complete culture solution containing 45 μM 2-AAPA; ③5-Fu single drug group : DMEM complete culture solution containing 5-Fu at a concentration of 100 μM; ④Combination group: DMEM complete culture solution containing 2-AAPA at a concentration of 45 μM and 5-Fu at a concentration of 100 μM, and different cultures were set up for each group of cells Time: 2h, 4h, 8h and 24h. LC-MS was used to detect intracellular GSH and GSSG in each group and calculate the ratio of GSH/GSSG. For each group of cells treated for 2 h, the cells were stained with CellRox Green reagent, and the ROS levels were analyzed by flow cytometry. It was found that in HCT15/5Fu cells, 2-AAPA alone or in combination could significantly reduce the GSH:GSSG ratio (as shown in Figure 2C), and the combination of 2-AAPA and 5-Fu significantly increased ROS generation (as shown in Figure 2D ).

The HCT8/5Fu cells were randomly divided into groups as follows: ①Ctrl control group (DMEM complete culture solution without drugs); ②NAC group: DMEM complete culture solution containing 5 mM NAC treated for 1 h; ③Containing 40 μM 2-AAPA and 400μM 5-Fu DMEM complete culture solution was treated for 2h; ④Combination combination group: first treated with DMEM complete culture solution containing 5mM NAC for 1h, and then treated with 2-AAPA with a concentration of 40μM and 400μM NAC 5-Fu DMEM complete culture solution treatment 2h. HCT15/5Fu cells were randomly divided into groups as follows: ①Ctrl control group (DMEM complete culture solution without drugs); ②NAC group: DMEM complete culture solution containing 5 mM NAC treated for 1 h; ③Containing 45 μM 2-AAPA and The DMEM complete culture solution with a concentration of 100 μM 5-Fu was treated for 2 h ④Combination combination group: first treated with DMEM complete culture solution containing 5 mM NAC for 1 h, and then treated with 2-AAPA with a concentration of 45 μM and 5-Fu with a concentration of 100 μM Fu was treated with DMEM complete culture solution for 2h. Cells in each group were stained with CellRox-Green reagent, and ROS levels were analyzed by flow cytometry, and the results are shown in Figure 2E. Findings: Antioxidant NAC pretreatment can neutralize ROS induced by the combination of 2-AAPA and 5-Fu in HCT8/5Fu and HCT15/5Fu cells. This indicated that NAC pretreatment significantly reduced the thiol oxidative stress induced by the combination of 2-AAPA and 5-Fu in HCT8/5Fu and HCT15/5Fu cells.

These results indicated that 2-AAPA induced thiol oxidative stress in HCT8/5Fu and HCT15/5Fu cells, and the combination of 2-AAPA and 5-Fu induced reactive oxygen species production in HCT8/5Fu and HCT15/5Fu cells.

Combining Example 1 and Example 2, it can be concluded that: 2-AAPA induces HCT8/5Fu and HCT15/5Fu cells to produce thiol oxidative stress and reactive oxygen species in combination with 5-Fu through up-regulating thiol oxidative stress, and increases High thiol oxidative stress significantly enhanced the sensitivity of 5-Fu-resistant CRC cells to 5-Fu treatment, increasing the sensitivity of HCT8/5Fu and HCT15/5Fu cells to 5-Fu by 2.2-fold and 3.7-fold, respectively .

Example 3 Cell Apoptosis Experiment

Apoptosis experiments were performed on drug-resistant cells HCT8/5Fu in the following groups: ①Ctrl control group (DMEM complete culture solution without drugs); ②2-AAPA single-drug group: DMEM complete culture solution containing 40 μM 2-AAPA ; ③ 5-Fu single drug group: DMEM complete culture solution containing 400 μM 5-Fu; ④ Combination combination group: DMEM complete culture solution containing 40 μM 2-AAPA and 400 μM 5-Fu. The culture time is 48h. The results of HCT8/5Fu cell apoptosis detected by flow cytometry are shown in Figure 3A, the corresponding apoptosis quantification histogram is shown in Figure 3B, and the Western blot analysis chart of apoptosis-related proteins is shown in Figure 3I. It can be seen that compared with the control group, 2-AAPA single-drug group and 5-Fu single-drug group, the apoptosis of the combination group was significantly increased, and the difference was statistically significant (P<0.001) (see Figure 3B). The apoptosis-related proteins capase3 and PARP were significantly reduced, and the cleaved capase3 and PARP were significantly increased (see Figure 3I). These results indicated that the combination of 2-AAPA and 5-Fu significantly enhanced the induction of apoptosis in HCT8/5Fu cells, and it was achieved by inducing the cleavage of capase3 and PARP in HCT8/5Fu cells.

Apoptosis experiments were performed on drug-resistant cells HCT15/5Fu in the following groups: ①Ctrl control group (DMEM complete culture solution without drugs); ②2-AAPA single drug group: DMEM complete culture solution containing 45 μM 2-AAPA Added; ③5-Fu single drug group: DMEM complete culture solution containing 100 μM 5-Fu; ④Combination combination group (DMEM complete culture solution containing 45 μM 2-AAPA and 100 μM 5-Fu). The culture time is 48h. The results of HCT15/5Fu cell apoptosis detected by flow cytometry are shown in Figure 3D, the corresponding apoptosis quantification histogram is shown in Figure 3C, and the Western blot analysis chart of apoptosis-related proteins is shown in Figure 3J. It can be seen that compared with the control group, 2-AAPA single-drug group and 5-Fu single-drug group, the apoptosis in the combination group was significantly increased, and the difference was statistically significant (P<0.01) (see Figure 3C). Apoptosis-related proteins capase3 and PARP were significantly reduced, and cleaved capase3 and PARP were significantly increased (see Figure 3J). These results indicated that the combination of 2-AAPA and 5-Fu significantly enhanced the induction of apoptosis in HCT15/5Fu cells, and it was achieved by inducing the cleavage of capase3 and PARP in HCT15/5Fu cells.

This shows that: 2-AAPA enhances the sensitivity of HCT8/5Fu and HCT15/5Fu cells to 5-Fu, and it is realized by enhancing the apoptosis induction of drug-resistant cells in combination with 5-Fu.

Apoptosis experiments were performed on drug-resistant cells HCT8/5Fu in the following groups: ①Ctrl control group: DMEM complete culture medium without drugs; ②Z-VAD group: treated with DMEM complete culture medium containing 10 μM Z-VAD-FMK for 1 h ;③First combination group: treated with DMEM complete culture solution containing 40μM 2-AAPA and 400μM 5-Fu for 48h ④Second combination group: firstly treated with DMEM complete culture solution containing 10μM Z-VAD-FMK After pretreatment for 1 hour, they were treated with DMEM complete culture solution containing 2-AAPA at a concentration of 40 μM and 5-Fu at a concentration of 400 μM for 48 hours. The results of HCT8/5Fu cell apoptosis detected by flow cytometry are shown in Figure 3E, and the corresponding histogram of apoptosis quantification is shown in Figure 3F. It can be seen from the figure that compared with the first combination group, the apoptosis of HCT8/5Fu cells in the second combination group was significantly decreased, and the difference was statistically significant (P<0.05). Description: Z-VAD-FMK blocked the apoptosis of HCT8/5Fu cells induced by combined treatment of 2-AAPA and 5-Fu.

Apoptosis experiments were performed on drug-resistant cells HCT15/5Fu in the following groups: ①Ctrl control group: DMEM complete culture medium without drugs; ②Z-VAD group: treated with DMEM complete culture medium containing 10 μM Z-VAD-FMK for 1 h ;③The first combination group: treated with DMEM complete culture solution containing 45 μM 2-AAPA and 100 μM 5-Fu for 48 hours ④The second combination group: first treated with DMEM complete culture solution containing 10 μM Z-VAD-FMK After pretreatment for 1 hour, they were treated with DMEM complete culture solution containing 2-AAPA at a concentration of 45 μM and 5-Fu at a concentration of 100 μM for 48 hours. The results of HCT15/5Fu cell apoptosis detected by flow cytometry are shown in Figure 3H, and the corresponding apoptosis quantification histogram is shown in Figure 3G. It can be seen from the figure that compared with the first combination group, the apoptosis of HCT15/5Fu cells in the second combination group was significantly decreased, and the difference was statistically different (P<0.01). Description: Z-VAD-FMK blocked the apoptosis of HCT15/5Fu cells induced by combined treatment of 2-AAPA and 5-Fu.

Embodiment 4 differential gene

RNA sequencing was performed on HCT8, HCT15, HCT8/5Fu, and HCT15/5Fu cells, and it was found that there were several differentially expressed genes between the drug-resistant group (HCT8/5Fu and HCT15/5Fu) and the non-drug-resistant group (HCT8 and HCT15 cells) . Among them, the TGM2 (transglutaminase 2) gene belongs to the up-regulated gene. That is, TGM2 in HCT8/5Fu and HCT15/5Fu cells (drug-resistant group) was significantly increased compared with HCT8 and HCT15 cells (non-drug-resistant group).

The drug-resistant cells HCT8/5Fu were treated in the following groups for 24 hours: ①Ctrl control group (DMEM complete culture medium without drugs); ②2-AAPA single-drug group: DMEM complete culture medium containing 40 μM 2-AAPA; ③5 -Fu single drug group: DMEM complete culture solution containing 400 μM 5-Fu; ④Combination combination group: DMEM complete culture solution containing 40 μM 2-AAPA and 400 μM 5-Fu. The drug-resistant cells HCT15/5Fu were treated in the following groups for 24 hours: ①Ctrl control group (DMEM complete culture solution without drugs); ②2-AAPA single drug group: DMEM complete culture solution containing 45 μM 2-AAPA; ③5 -Fu single drug group: DMEM complete culture solution containing 100 μM 5-Fu; ④Combination combination group: DMEM complete culture solution containing 45 μM 2-AAPA and 100 μM 5-Fu. RT-PCR analysis was performed on each group of the two cells, and the results are shown in Figure 4A and Figure 4B. RT-PCR results showed: compared with the control group (1.00), the mRNA of TGM2 in the HCT8/5Fu and HCT15/5Fu cells of the 2-AAPA monotherapy group were 0.52±0.01 and 0.60±0.05 respectively; and TGM2 mRNA in HCT15/5Fu cells were 0.36±0.02 and 0.27±0.11, respectively. It can be seen that 2-AAPA used alone or in combination with 5-Fu can significantly reduce the mRNA expression of TGM2 in HCT8/5Fu or HCT15/5Fu cells. The treatment time of each group was set to 48h, and the TGM2 protein level in each cell was analyzed by Western blot. The results are shown in Figure 4C, which also showed that the combination of 2-AAPA and 5-Fu can significantly reduce the level of HCT8/5Fu or HCT15/5Fu cells. The protein level of TGM2 in.

Example 5 Construction of TGM2 Overexpression Cells and Knockdown Cells

The TGM2 protein levels in Vector vector cells, overexpression cells, Scramble vector cells and knockdown cells were analyzed by Western blot. The results showed that compared with vector cells HCT8-Vector and HCT15-Vector, TGM2 in HCT8-TGM2 and HCT15-TGM2 cells Protein levels were significantly increased (Figure 5A). Compared with the vector cells HCT8/5Fu-Vector and HCT15/5Fu-Vector, the TGM2 protein levels were significantly increased in HCT8/5Fu-TGM2 and HCT15/5Fu-TGM2 cells (Figure 5B). Compared with the vector cells HCT8/5Fu-Scramble and HCT15/5Fu-Scramble, the TGM2 protein levels were significantly decreased in HCT8/5Fu-shTGM2 and HCT15/5Fu-shTGM2 cells (Fig. 5C).

Example 6 Drug resistance experiments of TGM2 overexpression cells and TGM2 knockdown cells

MTT tests were performed on the vector cells HCT8-Vector and HCT15-Vector, and the overexpression cells HCT8-TGM2 and HCT15-TGM2, respectively, using the method in 2.1. The culture solution containing different concentrations of drugs was DMEM complete culture solution containing different concentrations of 5-Fu, and the concentration gradient of 5-Fu was set as: 0, 7.8 μM, 15.6 μM, 31.3 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, 1000 μM. Figure 6A shows the control of in vitro cytotoxicity of different concentrations of 5-Fu on HCT8-Vector and HCT8-TGM2, and Figure 6B shows the control of in vitro cytotoxicity of different concentrations of 5-Fu on HCT15-Vector and HCT15-TGM2. The results showed that: at different concentrations of 5-Fu, compared with HCT8-Vector and HCT15-Vector vector cells, HCT8-TGM2 and HCT15-TGM2 overexpression cells showed better cell viability, indicating that HCT8-TGM2 and HCT15- TGM2 overexpression cells are resistant to 5-Fu. This means that the stable overexpression of TGM2 significantly induces the drug resistance of HCT8-TGM2 and HCT15-TGM2 cells to 5-Fu.

MTT tests were carried out on the carrier cells HCT8/5Fu-Scramble and HCT15/5Fu-Scramble, knockdown cells HCT8/5Fu-shTGM2 and HCT15/5Fu-shTGM2, respectively, using the method in 2.1. The culture solution containing different concentrations of drugs was DMEM complete culture solution containing different concentrations of 5-Fu, and the concentration gradient of 5-Fu was set as: 0, 7.8 μM, 15.6 μM, 31.3 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, 1000 μM. Figure 6C shows the in vitro cytotoxicity control of different concentrations of 5-Fu on HCT8/5Fu-Scramble and HCT8/5Fu-shTGM2, and Figure 6D shows the effect of different concentrations of 5-Fu on HCT15/5Fu-Scramble and HCT15/5Fu - In vitro cytotoxicity control of shTGM2. The results showed that: at different concentrations of 5-Fu, compared with the carrier cells HCT8/5Fu-Scramble and HCT15/5Fu-Scramble, HCT8/5Fu-shTGM2 and HCT15/5Fu-shTGM2 cells (shTGM2) showed lower cell activity, indicating that HCT8/5Fu-shTGM2 and HCT15/5Fu-shTGM2 cells are less resistant to 5-Fu. This means that knockdown of TGM2 significantly reduces the drug resistance of HCT8/5Fu-shTGM2 and HCT15/5Fu-shTGM2 cells to 5-Fu. That is, stable knockdown of TGM2 can reverse the resistance of drug-resistant cells to 5-Fu.

Example 7 Apoptosis Experiment of TGM2 Overexpression Cells and TGM2 Stable Knockdown Cells

Carrier cells HCT15/5Fu-Vector and overexpression cells HCT15/5Fu-TGM2 were subjected to multiple groups of apoptosis experiments: ①Ctrl control group (DMEM complete culture medium without drugs); ②2-AAPA single drug group: containing concentration of 45 μM 2-AAPA in DMEM complete culture solution; ③ 5-Fu single drug group: DMEM complete culture solution containing 100 μM 5-Fu; ④ Combination combination group (containing 45 μM 2-AAPA and 100 μM 5 -Fu's DMEM complete medium). The processing time is 48h. Analysis was performed by flow cytometry. The apoptosis results and their quantification histograms are shown in Figure 7A and Figure 7B. It can be seen that in the group using 2-AAPA alone or in combination, the apoptosis of HCT15/5Fu-TGM2 cells was significantly reduced compared with the vector cell HCT15/5Fu-Vector, which means that the stable overexpression of TGM2 reduced the HCT15/5Fu-Vector 2-AAPA-induced apoptosis in TGM2 cells. This illustrates the role of TGM2 in 2-AAPA-induced apoptosis of colorectal cancer cells: 2-AAPA induces apoptosis by reducing TGM2 protein levels, and stable overexpression of TGM2 will reduce 2-AAPA-induced apoptosis in CRC cells. That is, 2-AAPA targets TGM2 and increases the sensitivity of HCT15/5Fu cells to 5-Fu by inducing apoptosis.

Carrier cells HCT15/5Fu-Scramble and knockdown cells HCT15/5Fu-shTGM2 were subjected to multiple groups of apoptosis experiments: ①Ctrl control group (DMEM complete culture medium without drugs); ②2-AAPA single drug group: containing concentration of 45 μM 2-AAPA DMEM complete culture solution; ③ 5-Fu single drug group: DMEM complete culture solution containing 100 μM 5-Fu; ④ Combination combination group: 45 μM 2-AAPA and 100 μM 5 -Fu's DMEM complete culture solution. The processing time is 48h. The apoptosis results and their quantification histograms are shown in Figures 7D and 7C. In the 2-AAPA single drug group, the apoptosis of HCT15/5Fu-shTGM2 cells was significantly lower than that of the carrier cell HCT15/5Fu-Scramble; in the 5-Fu single drug group, the apoptosis of HCT15/5Fu-shTGM2 cells was significantly lower than that of The carrier cell HCT15/5Fu-Scramble had a significant increase; in the combination group of 5-Fu and 2-AAPA, the apoptosis of HCT15/5Fu-shTGM2 cells was significantly increased compared with the carrier cell HCT15/5Fu-Scramble. This means that the knockdown of TGM2 reduces the target of 2-AAPA and reduces the apoptosis induced by it. At the same time, the apoptosis induced by 5-Fu increases, which means that TGM2 is related to the drug resistance of 5-Fu.

Taken together, these results suggest that high expression of TGM2 induces drug resistance to 5-Fu in colorectal cancer cells.

Example 8 Effect of 2-AAPA on TGM2 protein level in drug-resistant cells

HCT8/5Fu and HCT15/5Fu cells were each treated with 2-AAPA at different doses (0 μM, 40 μM, 60 μM, 80 μM) for 48 hours, and then the protein level of TGM2 in the cells was analyzed by Western blot, the results are shown in Figure 8A; HCT8/5Fu and HCT15/5Fu cells were each treated with 80 μM 2-AAPA for different times (0h, 2h, 4h and 6h), and then the protein level of TGM2 in the cells was analyzed by Western blot, the results are shown in Figure 8B. It was found that 2-AAPA decreased the protein level of TGM2 in HCT8/5Fu and HCT15/5Fu cells in a time- and dose-dependent manner. Description: 2-AAPA can regulate/reduce the protein level of TGM2 in drug-resistant cells.

HCT8/5Fu and HCT15/5Fu cells were treated in different groups for 6 hours: ① control group (DMEM complete culture solution without drugs); ② 2-AAPA single drug group: DMEM complete culture solution containing 2-AAPA at a concentration of 80 μM ; ③ proteasome inhibitor MG132 single drug, DMEM complete culture solution containing MG132 with a concentration of 10 μM; ④ combination group (DMEM complete culture solution containing 2-AAPA with a concentration of 80 μM and MG132 with a concentration of 10 μM), and then passed Western The protein level of TGM2 was detected by blot. The results are shown in Figure 8C. Findings: 2-AAPA and MG132 co-treatment significantly restored TGM2 protein levels compared to 2-AAPA treatment. These results indicated that 2-AAPA could down-regulate the protein level of TGM2 in drug-resistant cells, which was achieved by proteasome degradation of TGM2 protein.

Example 9 S-glutathionylation of TGM2 protein

The Flag-TGM2 plasmid was transfected into HCT8/5Fu and HCT15/5Fu cells with Lipofectamine 3000 kit, and then the transfected cells were treated with DMEM complete culture solution containing 60 μM 2-AAPA for 4 hours. At the same time, the transfected cells were treated with drug-free DMEM complete culture solution for 4 hours as a control group.

The treated cells were collected, and the protein S-glutathionylation level of Flag-TGM2 transfected cells after different treatments was analyzed by Western blotting in 2.7. The results are shown in Figure 9A. It can be seen that, compared with the control group, The S-glutathionylation of TGM2 was significantly increased in the two cell lines (HCT8/5Fu and HCT15/5Fu) treated with 2-AAPA, indicating that TGM2 is the substrate of S-glutathionylation by 2-AAPA.

LC-MS/MS results of Flag-TGM2 protein captured from HCT15/5Fu cell lysates showed: S-glutathionylation of Cys193 was not observed in the control group; in 2-AAPA-treated HCT15/5Fu samples Only one S-glutathionylated peptide containing Cys193 was detected in , and its mass spectrum is shown in Figure 9B. Specifically, the precursor ion of the peptide NIPWNFGQFEDGILDICLLDVNPK containing S-glutathionylated Cys193 was detected at m/z 1097.87952 [(m+3H) ³⁺ ] at a retention time of 80.24 min, and a series of fragment ions were also observed (See Table 3). S-glutathionylation of Cys193 was further confirmed by the detection of a y11 ion (ICLILLDVNPK) containing one molecule of glutathione (305.06816 Da) at m/z 514.66040 [(m+3H) ³⁺ ]. That is, C193 is the main site of S-glutathionylation modification on TGM2.

Table 3. NIPWNFGQFEDGILDIC*LILLDVNPK peptide fragment ions containing S-glutathionylated Cys193.

The TGM2-C193S mutant can be constructed by mutating Cys193 of Flag-TGM2 to serine (Ser). Use the Lipofectamine 3000 kit to transfect the plasmids of Flag-TGM2 and Flag-TGM2-C193S into HCT15/5Fu cells respectively to obtain HCT15/5Fu cells transfected with the wild type of Flag-TGM2 and transfected with Flag-TGM2-C193S Mutant HCT15/5Fu cells. The transfected cells were treated with DMEM complete medium containing 60 μM 2-AAPA for 4 h. At the same time, the transfected cells were treated with drug-free DMEM complete culture solution for 4 hours as a control group.

The results of Western blot analysis are shown in Fig. 9C. It can be seen that when the cells were treated with 2-AAPA, the S-glutathionylation of C193S-Flag-TGM2 was significantly reduced compared with WT-Flag-TGM2. This indicates that TGM2-C193 is the regulatory target of 2-AAPA. Combined with the co-treatment results of MG132 and 2-AAPA, it can be concluded that: 2-AAPA down-regulates the level of TGM2 protein, which is completed through the S-glutathionylation-dependent proteasomal degradation pathway.

Flow cytometry analysis of the apoptosis of HCT15/5Fu cells transfected with WT-Flag-TGM2 or Flag-TGM2-C193S in the following groups: ①Ctrl control group (DMEM complete culture medium without drugs); ②2-AAPA Single drug group: DMEM complete culture medium containing 2-AAPA at a concentration of 45 μM; ③5-Fu single drug group: DMEM complete culture medium containing 100 μM 5-Fu; ④Combination combination group (containing 2-AAPA at a concentration of 45 μM AAPA and 5-Fu at a concentration of 100 μM in DMEM complete medium). The processing time is 48h. Analysis was performed by flow cytometry. The quantification histogram of apoptosis results is shown in Fig. 9D. It was found that the apoptosis induced by 2-AAPA single drug group or combination group in HCT15/5Fu cells transfected with Flag-TGM2-C193S was significantly less than that in HCT15/5Fu cells transfected with wild-type Flag-TGM2; In the drug group, HCT15/5Fu cells transfected with Flag-TGM2-C193S induced more apoptosis, indicating that they were more sensitive to 5-Fu. This result suggests that TGM2-C193 is a regulatory target of 2-AAPA-induced apoptosis.

Example 10 Construction and analysis of xenograft tumor model of HCT15/5Fu cells

The HCT15/5Fu xenograft tumor model was established on BALB/c nude mice, and the therapeutic effects of 2-AAPA single drug, 5-Fu single drug and 2-AAPA and 5-Fu combined drug on HCT15/5Fu xenograft tumor were evaluated. The details are as follows: Inject HCT15/5Fu cells into nude mice (5×10 ⁶ cells/mouse). When the tumor grows to about 100 mm ³ , the tumor-bearing mice are randomly divided into groups, and administered daily according to the following groups: ① Physiological Saline group (20% PEG200 normal saline solution) ② 2-AAPA single drug group (35 mg/kg body weight) ③ 5-Fu single drug group (25 mg/kg body weight) ④ 2-AAPA (35 mg/kg body weight) + 5-Fu (25 mg /kg body weight) combined group. The tumor volume was measured 2-3 times a week, the mice were weighed, and the data were recorded. The formula for calculating tumor volume (V) is: V=1/2×a×b ² where a and b represent length and width, respectively.

Figure 10A is a picture of tumors in tumor-bearing mice grown for 28 days. Fig. 10B is the change data of tumor volume measured during the tumor-bearing period of nude mice. Figure 10A and Figure 10B show that after 28 days of drug action, the tumor volume of the combined drug group of 2-AAPA and 5-Fu was much smaller than that of the 5-Fu single drug group. This shows that the combination of 2-AAPA and 5-Fu significantly increases the tumor inhibition rate. Figure 10C shows the comparison of the body weights of the mice in the above four groups, and the results show that there is no significant difference in the body weights of the nude mice in each group. It shows that the tumor-bearing mice can well tolerate the above drugs, and no symptoms such as weight loss occur.

In addition, using immunohistochemical kits, immunohistochemical staining analysis was performed on TGM2 of tumors in tumor-bearing mice treated with drugs for 28 days in different groups. The proliferation index Ki-67 and TUNEL-positive tumor cells in tumor-bearing mice treated with drugs for 28 days in different groups were analyzed by Servicebio. Figure 10D, Figure 10F, and Figure 10H respectively show representative TGM2 images, IHC-Ki67 images, and TUNEL images in the tumor samples of the four groups, and Figure 10E, Figure 10G, and Figure 10I are images of Figure 10D, The results of quantitative processing of staining in Figure 10F and Figure 10H. It can be seen from each figure that in the transplanted tumor tissue, compared with the control group, the 2-AAPA single drug group and the 5-Fu single drug group did not significantly down-regulate the TGM2 protein level and Ki67, but the TGM2 protein level and Ki67 in the combined group were significantly down-regulated, which shows that It shows that the 2-AAPA single drug group and the 5-Fu single drug group did not significantly inhibit the proliferation of tumor cells, but the combination of 2-AAPA and 5-Fu significantly inhibited the proliferation of tumor cells. At the same time, in the transplanted tumor tissue, compared with the normal saline group, neither the 2-AAPA single drug group nor the 5-Fu single drug group significantly induced tumor cell apoptosis, while the tumor samples of the 2-AAPA and 5-Fu combined drug group The number of TUNEL-positive tumor cells significantly increased, confirming that 2-AAPA can significantly enhance 5-Fu-induced apoptosis. It was demonstrated that thiol oxidative stress caused by the combination of 2-AAPA and 5-Fu could significantly enhance 5-Fu-induced apoptosis in vivo, and increase the therapeutic sensitivity of HCT15/5Fu cell xenograft tumor model to 5-Fu. This suggests that, in vivo, thiol oxidative stress can induce apoptosis by downregulating TGM2 and increase the sensitivity to 5-Fu in a tumor-bearing nude mouse model of 5-Fu-resistant CRC cells. 2-AAPA can increase the sensitivity of 5-Fu resistant CRC cells to 5-Fu in animals. This means that 2-AAPA can significantly reverse the 5-Fu resistance of 5-Fu resistant CRC cells both in vitro and in vivo.

Example 11 TGM2 protein expression level in colorectal cancer patient sample

Disease-specific survival curves derived from TGM2 gene expression levels in colorectal cancer patients were generated according to UCSC xena (https://xena.ucsc.edu/). TCGA colorectal cancer (COAD) was selected as the database and TGM2 as the target gene, and then the Kaplan-Meier diagram of disease-specific survival was generated, as shown in Figure 11. It was found that colorectal cancer patients with higher levels of TGM2 in their tumors had a shorter disease-specific survival time.

At the same time, taking 42 colorectal cancer patients as objects, the expression levels of TGM2 in 42 pairs of CRC tissues and adjacent normal tissues were detected by immunohistochemistry (IHC). The staining scores showed that the expression levels of TGM2 protein in tumor tissues were significantly higher than Normal tissue (P<0.0001). These results suggest that TGM2 can be used as a candidate marker for the prognosis of CRC patients and has functional relevance.

These results suggest that TGM2 is associated with poor prognosis in colorectal cancer patients.

It can be seen that the object of the present invention has been fully and effectively achieved. The method and principle of the present invention have been shown and described in the embodiments, and the implementation can be modified arbitrarily without departing from the principle. Therefore, the present invention includes all modified embodiments based on the spirit and scope of the claims.

Claims (10)

1. The substance for inhibiting the TGM2 gene expression level or reducing the TGM2 protein level is at least one of the following substances (1) to (3):

(1) Preparing a medicament for increasing the sensitivity of drug-resistant colorectal cancer cells to 5-fluorouracil;

(2) Preparing a medicine for reducing or reversing the drug resistance of colon drug-resistant intestinal cancer cells to 5-fluorouracil;

(3) Preparing a medicine for increasing drug-resistant colorectal cancer cell apoptosis.

2. The use of claim 1, wherein the substance that inhibits the expression of TGM2 gene or down-regulates the level of TGM2 protein comprises: TGM2 knockdown of a TGM2 inhibitor of a lentiviral particle or a small molecule compound.

3. The substance inducing S-glutathionylation of TGM2 protein is used in at least one of the following (1) to (3):

(1) Preparing a medicament for increasing the sensitivity of drug-resistant colorectal cancer cells to 5-fluorouracil;

(2) Preparing a medicine for reducing or reversing the drug resistance of colon drug-resistant intestinal cancer cells to 5-fluorouracil;

(3) Preparing a medicine for increasing drug-resistant colorectal cancer cell apoptosis.

4. The use of claim 1, wherein the substance inducing S-glutathionylation of TGM2 protein is a substance inducing S-glutathionylation at C193 site of TGM2 protein.

The use of a GR inhibitor is at least one of the following (1) to (3):

(1) Preparing a medicament for increasing the sensitivity of drug-resistant colorectal cancer cells to 5-fluorouracil;

(2) Preparing a medicament for reducing or reversing the drug resistance of colon cancer cells to 5-fluorouracil;

(3) Preparing a medicine for increasing drug-resistant colorectal cancer cell apoptosis.

6. The application of the mercaptan oxidative stress inducer is at least one of the following (1) to (3):

(1) Preparing a medicament for increasing the sensitivity of drug-resistant colorectal cancer cells to 5-fluorouracil;

(2) Preparing a medicine for reducing or reversing the drug resistance of colon drug-resistant intestinal cancer cells to 5-fluorouracil;

(3) Preparing a medicine for increasing drug-resistant colorectal cancer cell apoptosis.

7. A medicament for treating colorectal cancer, comprising: 5-fluorouracil and a 5-fluorouracil resistance reversal agent of at least one of:

(X1) a substance that inhibits the expression level of TGM2 gene or down-regulates the level of TGM2 protein;

(X2) a substance inducing S-glutathionylation of TGM2 protein;

(X3) GR inhibitors;

(X4) thiol oxidative stress inducer.

8. The medicament for treating colorectal cancer according to claim 7, wherein the substance inhibiting the expression level of TGM2 gene or down-regulating the level of TGM2 protein is TGM2 inhibitor of TGM2 knockdown lentivirus particles or small molecule compounds.

Use of TGM2 detection reagent for the preparation of at least one of the following diagnostic reagents:

(Y1) a diagnostic reagent for 5-fluorouracil resistance of colorectal cancer;

(Y2) a diagnostic reagent for the prognosis of a patient with colorectal cancer.

10. The use of claim 8, wherein the TGM2 detection reagent comprises a TGM2mRNA detection reagent or a TGM2 protein detection reagent.

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