Anticancer Activity Study Of Actinomycin V And Its Underlying Mechanisms

Cancer is considered as the leading cause of human death.Relevant statistics show that because of the large population size,close to 22%of global new cancer cases and nearly 27%of global cancer deaths occur in China,and the proportion is still rising.Therefore,the discovery or treatment of cancer has become a major direction for Chinese scientists.The traditional strategies include surgery,chemotherapy and radiotherapy.Among them,chemotherapy is still the most important and effective method in clinical trials,and natural medicine plays an essential role in chemotherapy.One of the important ways to discover leading drugs and new drugs is to find compounds with high physiological activity from natural products.Actinomycin is a class of chromopeptide antibiotics isolated from microorganisms.Actinomycin D is the most studied and has been approved as an anti-cancer drug for clinical treatment,especially for children with nephroblastoma,which has an ideal effect with an overall recovery rate of 80%.Inadequately,its used in clinic is severely limited by the hepatotoxicity.Actinomycin V is an analog of actinomycin D,in which L-4-proline is replaced by L-4-ketoproline.Studies have shown that actinomycin V has a better inhibitory effect on a variety of tumor cells than actinomycin D,while the underlying mechanism has not been elucidated.In this present study,we aim to investigate the anti-tumor effects of actinomycin V and also the mechanism under this action.MTT colorimetric assays were performed to measure the cytotoxic effects of actinomycin V and actinomycin D on several cancer and normal cell lines.Results showed that both the cytotoxicity and selectively index of actinomycin V are better than actinomycin D,indicating a more ideal anti-tumor effect of actinomycin V.Therefore,we selected human non-small cell lung cancer cells that are most sensitive to the treatment of actinomycin V for further research.Flow cytometry analysis showed that actinomycin V significantly induced apoptosis on A549 cells with little induction effect on NCI-H1299 cells.DAPI staining and Western blotting further confirmed that actinomycin V treatment decreased the expression level of Bcl-2 protein and increased the apoptotic body formation,nuclear condensation,and also the protein level of Bax Meanwhile,results from flow cytometry analysis revealed that actinomycin V treatment also induced G2/M phase arrest in A549 cells but showed no obvious cycle arrest effect on NCI-H1299 cells.Western Blotting found that treatment with actinomycin V up-regulated the expression of phosphor-Cdc2 and down-regulated the expression of M-phase promoting factors including Cyclin B1,Cdc2 and Cdc25A,which indicated that actinomycin V treatment arrested cells in G2 phase without entering M phase.The difference in the effect of actinomycin V on two types of human non-small cell lung cancer cells has drawn our attention.We noticed that NCI-H1299 is p53-deficient while A549 cells with wild-type p53.In this connection,our further studies aimed to investigate the role of p53 protein on actinomycin V-induced apoptosis and G2/M phase arrest on A549 cells.Results from western blotting and rt-PCR revealed that actinomycin V treatment increased the protein and also mRNA levels of p53,p21 wafl/Cio1 and Bax.Furthermore,pre-treatment with p53 inhibitor Pifithrin-α(PFT-α)significantly reduced the effects of actinomycin V on cell cycle arrest and apoptosis,and subsequently improved cells survival.When cells were pre-treated with 5 μmol/L PFT-α for 1 h,the proportion of apoptotic cells decreased from 29.56%to 14.38%and G2/M phase arrested cells decreased from 33.53%to 15.37%after 1 nmol/L actinomycin V treatment for 24 h.These results confirmed that p53 involved in actinomycin V-induced apoptosis and G2/M phase arrest on A549 cells.In conclusion,actinomycin V inhibits the proliferation of A549 cells through inducing p53 expression and triggering p53-dependent cellular responses,including apoptosis and cell cycle arrest,which is a promising anti-tumor lead compound and deserve further investigation.