| BACKGROUNDColon cancer is one of the most commonly diagnosed cancers,its incidence and mortality are growing in our country.Many patients are at an advanced stage at the time of diagnosis.Chemotherapy is a commonly used strategy for colon cancer treatment,as many patients have regional or distant spread at the time of diagnosis.But the strong side effects of chemotherapy drugs and chemotherapy resistance seriously affect the treatment effect and prognosis of colon cancer patients.Therefore,clinical treatment of colon cancer is still a great challenge,and searching for novel drugs for colon cancer treatment with high selectivity and low toxicity has become a recent focus of attention.Traditional Chinese medicine is an important source of anti-cancer drugs.Tetramethylpyrazine is an alkaloid monomer extracted from ligusticum chuanxiong.Recent studies have demonstrated that tetramethylpyrazine has potent inhibitory effects on a number of types of tumors,such as liver cancer,gastric carcinoma,prostate cancer,cervical cancer and breast cancer.However,there are few reports on whether tetramethylpyrazine has the effect on inhibiting the growth of colon cancer cells and its specific mechanism.OBJECTIVESTo detect the antitumor effect of tetramethylpyrazine on colon cancer and explore the antitumor mechanism preliminarily.To analyze the killing effect of tetramethylpyrazine on different colon cancer cell lines,detect the regulatory effect of tetramethylpyrazine on the cell proliferation and cell apoptosis of colon cancer cells and explore the regulatory mechanism of tetramethylpyrazine on cell apoptosis pathway of colon cells by changing mitochondrial reactive oxygen metabolism.In addition,the antitumor effect of tetramethylpyrazine was tested in colon cancer tumor-bearing mice.MOTHODSThe crystal violet staining method was used to observe the cell viability of tetramethylpyrazine on colon cancer cells.The Cell Counting Kit-8(CCK-8)method was used to detect the cell proliferation.Calculate the concentration of TMP that resulted in 50%inhibition(IC50)of cell proliferation through the concentration-dependent and time-dependent effects of tetramethylpyrazine.Flow cytometry was used to detect the cell apoptosis rate and cell cycle distribution.Annexin-V/PI double staining method was used to detect the type and proportion of cell apoptosis.DCFH-DA probe was used to detect intracellular reactive oxygen species.Active oxygen inhibitor(NAC)and Caspase inhibitor(Z-VAD-FMK)combined with tetramethylpyrazine treated with colon cancer cells were used to observe the effect of clearing reactive oxygen species and blocking apoptosis-related target proteins on the apoptosis of colon cancer cells.A colon cancer nude mouse model was used to analysis the reactive oxygen species content by detecting the malondialdehyde.The Caspase 3 and Caspase 9 activity detection kit was used to detect the Caspase 3 and Caspase 9 protein content.RESULTS1.Tetramethylpyrazine suppressed the cell viability in a dose-dependent manner on six colon cancer cell lines,especially in SW480 and HCT116 cells.The IC50 values of SW480 and HCT116 cells were the lowest among all cell lines.2.With increasing tetramethylpyrazine concentration and prolonged exposure time,the number of cells can be seen under the microscope gradually decreased.SW480 and HCT116 cell morphology became distorted,round,and fragmented after tetramethylpyrazine treatment,especially when the concentration of ligustrazine reached 600 ?g/ml.The proliferation and viability of SW480 and HCT116 cells gradually decreased with increasing tetramethylpyrazine concentration and prolonged exposure time.3.With increasing tetramethylpyrazine concentration,the proportion of cells in S phase gradually decreases.Compared with the control group,a significant decrease in the percentage of S phase was detected in cells treated with tetramethylpyrazineat 600?g/ml.4.With increasing tetramethylpyrazine concentration,the rate of apoptosis increased in a concentration-dependent manner.Compared with the control group,SW480 and HCT116 cells treated with tetramethylpyrazine showed a marked increase in apoptosis rate,especially when the concentration of tetramethylpyrazine reached 600 ?g/ml.The SW480 cells treated with tetramethylpyrazine showed increased cell numbers in early apoptosis(annexin+/pi-,lower right quadrant),whereas the HCT116 cells showed late apoptosis(annexin+/pi+,upper right quadrant).5.Compared with the DMSO control group,the number of HCT116 and SW480 cells in the tetramethylpyrazine group was significantly reduced,and the cell morphology became distorted,round,and fragmented.Compared with the tetramethylpyrazine+NAC and tetramethylpyrazine+Z-VAD-FMK groups,the number of cells was significantly increased.The apoptosis rate of TMP group was significantly higher than that of DMSO group.The apoptosis rate of tetramethylpyrazine group was significantly higher than that of DMSO control group.The apoptosis rate of tetramethylpyrazine+NAC and tetramethylpyrazine+Z-VAD-FMK group was significantly lower than the tetramethylpyrazine group.The cell survival rate of the NAC group was higher than the Z-VAD-FMK group.6.Compared with the DMSO control group,the content of active oxygen in the tetramethylpyrazine group was significantly increased.Compared with the tetramethylpyrazine treated group,the content of active oxygen in the tetramethylpyrazine+NAC group was significantly decreased.The content of active oxygen in the tetramethylpyrazine+Z-VAD-FMK group and the tetramethylpyrazine treated group did not change significantly in SW480 colon cancer cells.In HCT116 colon cancer cells,the content of reactive oxygen species decreased in the tetramethylpyrazine+Z-VAD-FMK group compared with the tetramethylpyrazine treated group.7.Compared with the control group,Caspase 3,9 and PARP protein in the tetramethylpyrazine group had obvious shear activation,and the protein content was significantly increased.Compared with tetramethylpyrazine group,the activation of Caspase 3,9 and PARP can be significantly inhibited by Z-VAD-FMK and NAC.Compared with tetramethylpyrazine+Z-VAD-FMK group,the protein expression of 3,9 and PARP reduced and was significantly inhibited in tetramethylpyrazine+NAC group.8.In animal experiments,the growth of transplanted tumors was significantly inhibited in the high-concentration tetramethylpyrazine group in a dose-dependent manner.The weight distribution of the transplanted tumor is:1.62±0.48 g(drug concentration 0 mg/kg),0.92±0.21 g(drug concentration 50 mg/kg)and 0.58±0.19 g(drug concentration 100 mg/kg),there are significant differences in a dose-dependent manner.9.The content of malondialdehyde and Caspase3,9 protein in the transplanted tumors of the animal model of the high-concentration tetramethylpyrazine group(100 mg/kg)was significantly higher than the low-concentration group(50 mg/kg)and the control group(0 mg/kg)).The differences are significant in a dose-dependent manner.CONCLUSIONS1.Tetramethylpyrazine can significantly inhibit the cell proliferation and promote cell apoptosis of colon cancer cells in a time and concentration dependent manner.2.Tetramethylpyrazine can block colon cancer cells in G1 phase,and then inhibit the synthesis of S phase,thereby inhibiting cell proliferation.3.The cell apoptosis of colon cancer cells induced by tetramethylpyrazine is closely related to the production of large amounts of reactive oxygen species(ROS)to activate the caspase-dependent apoptosis pathway,and ROS inhibitors can effectively inhibit the cell apoptosis induced by tetramethylpyrazine.4.Increasing the expression of ROS in colon cancer cells induced by tetramethylpyrazine plays a regulatory effect in inducing apoptosis in the upstream of caspase-dependent apoptosis pathway.5.Tetramethylpyrazine can significantly inhibit the proliferation of colon cancer xenograft tumors in nude mice,and increase the expression of ROS,Caspase 3 and Caspase 9 in the xenograft tumors of nude mice and induce the apoptosis of tumor cells. |
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