Antitumor Effect And Mechanism Of 7-oxostaurosporine

Pancreatic cancer,known as the king of cancer,is a highly malignant digestive tract tumor.The total 5-year survival rate is only 9.3%.Targeted molecular therapy has become a research focus in cancer therapy.However,the pathogenesis of pancreatic cancer is complex,and there is no effective targeted inhibitor for clinical use.Finding new diagnosis and treatment targets is the focus of pancreatic cancer research.In our previous studies,we isolated and identified a new compound 7-oxostaurosporine?33C?with significant antitumor activity from marine microorganisms.33c is a kind of staurosporine,and staurosporine is a good PKC Inhibitor.Up to now,there is no report about its antitumor effect.The effect and mechanism of 33c in vitro and in vivo were studied.The results showed 33C can significantly induce apoptosis of human pancreatic cancer cells by inhibiting the PKC-NF?B signaling pathway,has good antitumor effect in vivo,and has less toxic and side effects.Simultaneously activated PKC?and P65 are highly expressed in the tumor tissue of patients with pancreatic cancer,in the cytoplasm and nucleus,and are negatively related to the patient's prognosis.The main research contents and results are as follows:1.7-oxostaurosporine?33C?was extracted from solid fermentation products of Streptomyces sp.NB-A13 by silica gel column chromatography,gel column chromatography and high performance liquid chromatography.The structures were identified by NMR.2.Cell Counting Kit-8 kit was used to detect tumor cells survival rate at different concentrations of 33C for 24 hours.CCK8 test results showed that at a treatment concentration of 3.2?M,the inhibition rate on pancreatic cancer and lung cancer cell lines is more than 90%,and when the 33C treatment concentration is reduced,the inhibition rate on tumor cells also follows.Decreased,showing a certain concentration-dependent relationship.The IC₅₀ of 33C for each tumor cell was calculated to be within the range of 18-3.2?M.3.Establish Bx PC-3 transplanted tumor model and pancreatic cancer PDX subcutaneous tumor mouse model,and investigate the tumor suppressive effect of 33C in vivo.Record the tumor volume change of each group,calculate the tumor volume and draw the tumor volume growth curve.During the administration period,the tumor volume of the nude mice in each group kept increasing,but compared with the control group,the tumor growth rate in the Napabucasin group and the 33C group was significantly slower,in which the 33C group suppressed Bx PC-3 transplantation tumors and pancreatic cancer PDX growth.4.By observing the general condition of the mice,the mice are in good mental state.Compared with the negative control group,the weight of the mice in each administration group slightly decreased,but there was no significant difference.To observe the toxic and side effects of 33C on mice,perform HE staining organs.The results showed that the heart,liver,spleen,lung,and kidney of nude mice in each group were observed by HE staining.No significant pathological changes were found in the main tissues and organs of group 33C?6mg/kg?.5.Bx PC-3,PANC-1 and MIA paca-2 cells were treated with 33C at different concentrations,and the apoptotic rate of the cells was measured by flow cytometry.The results showed that after treating cells with different concentrations of 33C?0,0.25?M,0.5?M,1?M?for 24 hours,the results of flow cytometry showed that the apoptosis rate of pancreatic cancer cells cells increased with the increase of drug concentration.At 1?M,pancreatic cancer cells induced significant apoptosis.6.WB immunoblot was used to detect the effects of 33C on the protein expression and phosphorylation levels of various PKC isoforms in human pancreatic cancer cells and test the effects of 33C at different concentrations on the protein expression and phosphorylation level of PKC?.The results showed that PKC??T538?subtype phosphorylation level was reduced in 0.25?M 33C-treated cells,when cells were treated with 0.5?M 33C,PKC??T538?subtype phosphorylation level was significantly reduced and PKC??T505?subtype phosphorylation level was decreased.The levels of PKC??T538?and PKC??T505?subtype phosphorylation in 1?M 33C-treated cells decreased significantly.However,the phosphorylation levels of other PKC isoforms in Bx PC-3 cells treated were not changed.7.HTRF in vitro kinase assay was used to study the effect of 33C on PKC?and PKC?activity,and the IC₅₀ values of 33C on PKC?and PKC?kinase were calculated.HTRF test results show that 33C inhibits PKC?(IC₅₀=10.94 n M)and PKC?(IC₅₀=3.262 n M)is significantly better than Sotrastaurin inhibits PKC?(IC₅₀=198.6 n M)and PKC?(IC₅₀=33.85 n M).8.si RNA silenced PKC?in pancreatic cancer Bxpc-3 and As PC-1.WB immunoblot was used to detect the effect of 33C on protein expression and phosphorylation level of NF-?B.The results showed that p-p65 phosphorylation was inhibited when si RNA silenced PKC?expression in pancreatic cancer Bxpc-3 and As PC-1,and the p-p65 phosphorylation has not changed after PKC?was silenced.9.Use tissue chip technology to study the expression of protein kinases PKC?and NF-?B in pancreatic cancer and its clinical significance,and to study its correlation with the prognosis of patients with pancreatic cancer.Activated PKC?and P65 are highly expressed in the tumor tissues,pancreatic cytoplasm and nucleus of patients with pancreatic cancer,and arenegativelycorrelated with the patient's prognosis.