Molecular Mechanism Of Tanshinone Analogues Against Prostate Cancer Based On Data Processing

Objective:To explore the molecular mechanism of tanshinone analogues inducing apoptosis in prostate cancer cells.Methods:The gene expression profile GSE69223 of prostate cancer samples was downloaded from the GEO database.In R language,the key genes between prostate cancer tissues and adjacent tissues were screened by setting threshold?log2?Fold Change??>2 and pvalue<0.05,and compared with those of prostate cancer cells treated with and without tanshinone analogue TB1.Molecular docking technology was used to screen target proteins which could be directly combined with tanshinone analogues TB1.The effects of different concentrations of tanshinone analogues TB1 on proliferation of prostate cancer cells were detected by colony forming assay.MTT assay was used to determine the effects of TB1 on proliferation of prostate cancer cells.The apoptosis of prostate cancer cells was investigated by Hoechst staining method.The apoptosis and distribution of prostate cancer cells treated with tanshinone analogues TB1 were analyzed by flow cytometry.The effects of tanshinone analogues TB1 on the migration of prostate cancer cells were investigated by scratch test.The apoptosis of prostate cancer cells treated with tanshinone analogues TB1 was analyzed by flow cytometry.Meanwhile,the effects of tanshinone analogues TB1 on apoptosis and cycle arrest related genes and related proteins in prostate cancer cells were investigated by PCR and Western blot experiments.Results:The standardized results of the chip showed that all the samples in the data set could be included in the analysis area.The results of the key genes data mining obtained 41 up-regulated genes and 101 down regulated genes compared with the adjacent tissue samples,and there was no overlap between the TB1 gene and the control group.It was suggested that tanshinone analogues TB1 may have cytotoxicity to normal prostate cells.The parameters of molecular docking were the largest,and the minimum?G of ligand protein system was the smallest,that was probably the target of direct binding of TB1 to tanshinone analogues,So P53 protein is the most suitable target for normal prostate cells.The results of clone formation experiments show that with the increase of TB1 concentration of tanshinone analogues,the colony formation of prostate cancer cells gradually decreased,indicating that tanshinone analogues TB1 inhibited the proliferation of prostate cancer cells.MTT assay results showed that tanshinone analogues TB1 had a proliferation inhibition effect on prostate cancer cells PC-3M-IE8 and PC-3M-2B4,and had a time dependence on PC-3M-IE8?P<0.05?.The IC₅₀value was obtained by fitting the curve with SPSS analysis software.It was found that at 12h,tanshinone analogue TB1 had significantly lower IC₅₀value than that of the positive cisplatin IC₅₀value.In Hoechst 33258 apoptosis staining assay,the result showed that with the increase of the concentration of tanshinone analogue TB1,the prostate cancer cells showed significant nuclear shrinkage and chromosome shrinkage,and dense staining appeared at high concentration.After the action of tanshinone analogue TB1,prostate cancer cells appeared early and late apoptosis and S phase arrest.Random scratch test showed that tanshinone analogues TB1 had no effect on the migration ability of prostate cancer cells.PCR and Western blot showed that the expression of P53 protein in prostate cancer cells increased after tanshinone analog TB1 treatment.It further increased the expression of downstream protein P21 protein and further combined P21 with Cyclin A--CDK2 complex,resulting in the cell cycle failing to proceed normally,thereby blocking the S phase,leading to apoptosis of prostate cancer cells.On the other hand,tanshinone analogues TB1 induced apoptosis through P53 mediated apoptosis,and the expression level of P53 protein increased under tanshinone analog TB1,the expression of downstream protein Bax was further regulated.The mitochondrial membrane potential was detected by flow cytometry,the results showed that the membrane potential of prostate cancer cells treated with tanshinone analogues TB1 decreased.WB detection showed that the expression of Bax protein increased and the expression of Bcl-2 protein decreased.The mitochondrial membrane potential decreased,and the cytochrome C in mitochondria was released into the cytoplasm.The Caspase cascade results in Caspase-3 degradation of intracellular substrates,resulting in apoptosis.Tanshinone analog TB1 also activates the death receptor pathway,leading to Caspase-8 activation,which further activates Caspase-3 and ultimately leads to apoptosis.Conclusion:This study showed that tanshinone analogue TB1 has a certain inhibitory effect on prostate cancer cells,which can block prostate cancer cell cycle at s stage and induce apoptosis,and can also induce apoptosis through mitochondrial pathway and death receptor pathway.But it also has cytotoxicity to normal cells.In the later period,we can reduce its cytotoxicity to normal cells by combining small molecule carriers and microbubbles.This study can provide some reference for the development of anti prostate cancer drugs.