| Hepatocellular carcinoma(HCC)is a common and highly malignant tumor.Therefore,there is an urgent need to explore new drugs for hepatocellular carcinoma treatment.Plants have been used as a resource for anticancer drugs,and many current anticancer drugs from plants have been reported.Trichosanthes kirilowii is a herbaceous plant widely distributed throughout China.Many biologically active ingredients have been extracted from Trichosanthes kirilowii,such as terpenoids,sterols,flavonoids,nitrogenous compounds,and wood lipoproteins.Numerous studies have shown that these compounds have a variety of pharmacological activities,including anti-myocardial ischemia,anti-hypoxia,anti-platelet aggregation,sputum,anti-inflammatory,cytotoxic and antioxidant effects.TKP is a low-toxicity antitumor activity protein extracted from the fruit of Trichosanthes kirilowii.It is a serine protease.Studies have shown that TKP has significant anti-colorectal cancer activity,and TKP can induce apoptosis in colorectal cancer cells through the PI3 K / AKT-mediated mitochondrial pathway.However,the effect of TKP on liver cancer has not been studied.The purpose of this study is to research the effect of TKP on HCC cells and further clarify its molecular mechanism,which will provide theoretical basis and experimental data for the future treatment of HCC.The research includes the following parts:The first part: The effects of TKP on the proliferation and aerobic glycolysis of HCC cells.The HCC cells Bel-7402,Hep G2 and normal liver cells HL7702 were treated with TKP at concentrations of 0,2.5,5,10,20,and 40 ?g/m L for 24 h,respectively,and then the survival rate of cells was measured by MTT method.MTT results revealed that TKP significantly inhibited the survival of Bel-7402 and Hep G2 cells,But it has no obvious effect on the survival of normal liver cells HL7702.Cell clone formation experiments was applied to further verify the inhibitoryeffect of TKP on the proliferation of HCC cells.The Bel-7402 and Hep G2 cells were treated with TKP at a concentration of 0 and 2.5 ?g/m L,and then stained with crystal violet at a concentration of 5% after 7 days.The results showed that TKP could significantly inhibit the colony formation ability of Bel-7402 and Hep G2 cells.The Bel-7402 and Hep G2 cells were treated with TKP at concentrations of 0,2.5,5,and 10 ?g/m L,respectively,for 24 h.After collecting the culture solution,the glucose content and lactic acid content in the culture solution were measured with glucose kit and lactic acid kit.The results showed that the amount of glucose in the culture medium was increased significantly,while the yield of lactic acid was decreased significantly.In addition,we also used real-time PCR to detect m RNA levels of key proteins in aerobic glycolysis,such as lactate dehydrogenase LDHA,pyruvate dehydrogenase kinase PDK,and glucose transporter 1 GLUT1.The results showed that the m RNA levels of LDHA,PDK,and GLUT1 were significantly decreased after TKP treatment.These results indicated that TKP can significantly inhibit the proliferation and aerobic glycolysis of HCC cells.Part ?: TKP inhibits aerobic glycolysis of HCC cells by regulating PKM2.The Bel-7402 and Hep G2 cells were treated with TKP at concentrations of 0,2.5,5,and 10 ?g/m L for 24 h,respectively.Western blot was used to detect the effect of TKP on pyruvate kinase PKM2 in Bel-7402 and Hep G2 cells.The results showed that TKP significantly inhibited the total PKM2 protein level in a dose-dependent manner,and that after treatment with TKP,PKM2 protein was significantly reduced in the nucleus and cytoplasm.In addition,we also tested the effect of TKP on pyruvate kinase activity in HCC cells.The results showed that TKP significantly inhibited the enzyme activity of PKM2.These results indicated that TKP significantly inhibits PKM2 expression,nuclear translocation,and pyruvate kinase activity.To further confirm the key role of PKM2 in TKP-induced aerobic glycolysis inhibition,weoverexpressed PKM2 in Bel-7402 and Hep G2 cells.After examining the glucose and lactic acid contents in the culture medium,it was found that the decrease in glucose and lactic acid production due to TKP treatment was significantly reversed.In addition,in cells overexpressing PKM2,the expression levels of GLUT1,PDK and LDHA were significantly increased.These results demonstrated that TKP regulates PKM2 and thereby inhibits aerobic glycolysis of HCC cells.Part ?: TKP reduces PKM2 expression,cell proliferation and aerobic glycolysis by inhibiting the ?-catenin/c-Myc/hn RNPA1 pathway.The Bel-7402 and Hep G2 cells were treated with TKP at concentrations of 0,2.5,5,and 10 ?g/m L,respectively,for 24 h.The m RNA levels of?-catenin,c-Myc,and hn RNPA1 were detected by real-time quantitative PCR.It was shown that the m RNA levels of ?-catenin,c-Myc and hn RNPA1 were significantly down-regulated after TKP treatment.After the expression of ?-catenin was up-regulated with Li Cl,the m RNA levels of ?-catenin,c-Myc and hn RNPA1 and the protein levels of PKM2 were detected by real-time quantitative PCR and western blotting.The results showed that after adding Li Cl,the m RNA levels of ?-catenin,c-Myc and hn RNPA1 were significantly up-regulated,and the inhibitory effects of TKP on PKM2 expression and nuclear translocation were significantly reversed.At the same time,the decrease in glucose consumption and lactic acid production caused by TKP were also reversed after Li Cl treatment,and m RNA expression levels of key proteins for aerobic glycolysis such as GLUT1,PDK and LDHA also were increased significantly.MTT results showed that co-treatment with Li Cl and TKP could reduce the inhibitory effect of TKP on the proliferation of HCC cells.These results indicated that TKP regulates PKM2 by blocking the?-catenin/c-Myc/hn RNPA1 pathway,thereby inhibiting HCC cell proliferation and aerobic glycolysis.In summary,TKP significantly inhibits the proliferation of HCC cells by blocking PKM2-mediated aerobic glycolysis.Further researchshows that TKP inhibits PKM2 expression by inhibiting the?-catenin/c-Myc/hn RNPA1 pathway. |
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