Anti-Hepatocellular Carcinoma Cells Activity And Preliminary Mechanisms Of Action Of Novel Bradykinin Receptor Inhibitors

With the continuous changes of living environment and food structure,the number of cancer patients has increased year by year,among which hepatocellular carcinoma has a high incidence rate and a poor treatment outcome.Due to the complexity of hepatocellular carcinoma and the multiplicity of etiological factors,the treatment methods are still limited to traditional chemotherapy and surgery,and the prognosis is poor.Bradykinin is a kind of linear non-apeptide widely existing in human body,which is highly expressed in inflammatory regions and tumor microenvironment.Various studies have shown that bradykinin can promote the proliferation,migration and invasion of various cancers such as stomach,breast,lung and prostate cancer.Bradykinin acts in many types of cancer by binding to its specific receptors,bradykinin 1 receptor and bradykinin 2 receptor.Studies have shown that selectively blocking the bradykinin receptors can inhibit the development of liver cancer.Therefore,screening for novel bradykinin receptor inhibitors to inhibit the promoting effect of bradykinin on primary liver cancer could provide new drugs for future treatment of liver cancer.This study includes the following three aspects: in the first part,the bradykinin receptor inhibitors J051-71 and J051-105 were synthesized by silica gel column chromatography and purified by expression.The second part mainly studied the inhibitory effects and mechanisms of action of the two bradykinin receptor inhibitors on the liver cancer cell lines HepG2 and BEL-7402.The third part studied the effect of bradykinin receptor inhibitors on the apoptosis of hepatocellular carcinoma cells and the related mechanism.We successfully synthesized the pure bradykinin receptor inhibitors J051-71 and J051-105 through the mature and optimized method of organic chemical synthesis developed by previous students in our laboratory and the time-of-flight mass spectrometer test.In this study,we investigated the possible role of bradykinin receptor inhibitors in the proliferation and apoptosis of hepatocellular carcinoma cells.The research data showed that bradykinin receptor inhibitors J051-71 and J051-105 inhibited the proliferation of liver cancer cells HepG2 and BEL-7402.MTT colorimetry experiments and colony formation assay showed that J051-71 and J051-105 had certain inhibitory effects on the proliferation of hepatocellular carcinoma cells,but such inhibitory effects were not correlated with cell cycle.Western blot was used to verify that bradykinin receptor inhibitors reduced the expression of bradykinin B1 receptor in liver cancer cells,thereby reducing the protein expression of p-ERK 1/2 and down-regulating the activation of ERK 1/2 signaling pathway.In addition,J051-71 and J051-105 reduced p-ERK 1/2 activation in bradykinin-mediated HCC cells.This also confirmed that bradykinin receptor inhibitors inhibited the activation of ERK signaling pathway by inhibiting the expression of B1 R protein,and finally inhibited the proliferation process of liver cancer cells.Bradykinin receptor inhibitors J051-71 and J051-105 also induced and promoted the apoptosis of the two liver cancer cells HepG2 and BEL-7402.Flow cytometry showed that the apoptosis rate of HCC cells increased with the concentration of bradykinin receptor inhibitors.JC-1 mitochondrial membrane potential detection method revealed the down-regulation of mitochondrial membrane potential in liver cancer cells in a dose-and time-dependent manner.Western blot also confirmed that bradykinin receptor inhibitors increased the levels of cleaved PARP and activated caspase 3 in a dose-and time-dependent manner,thus confirming the induction of apoptosis.In summary,this study proves that bradykinin receptor inhibitors J051-71 and J051-105 can down-regulate the activation of ERK signaling pathway by binding to B1 R and reducing the expression level of B1 R,thus inhibiting the proliferation of liver cancer cells.J051-71 and J051-105 can also trigger the activation of the apoptosis mechanism in HCC cells,resulting in the spontaneous death of HCC cells.Our discovery of novel bradykinin receptor inhibitors may provide promising new approaches for the treatment of primary liver cancer.