Investigation On The Anti-tumor Activity And Mechanism Of Novel Podophyllotoxin Derivatives

Cancer,as a kind of incurable disease,has seriously affected human life.It is mainly characterized by the ability of cells to grow rapidly and spread to the adjacent areas.Because of its complex pathogenesis and intractable characteristics,cancer is still largely treated by surgery.But postoperative metastasis and recurrence cannot be completely avoided,among that drug assistance is essential in the treatment process.Natural products play an important role in the field of drug research and the development of new anticancer drugs from natural products is one of the hot topics in cancer treatment.Podophyllotoxin is a natural lignan.It can interfere with tubulin synthesis and lead to apoptosis of cancer cells.Structurally,it contains four continuous chiral centers and four nearly planar fused rings.Podophyllotoxin and its β-configurated congener,epipodophyllotoxin,showed significant anticancer effects via inhibition of polymerization of tubulin and topoisomerase II respectively,leading to cell cycle arrest and suppression of the formation of the mitotic-spindles microtubules.Other biological activities including insecticidal,antifungal,antiviral,anti-inflammatory,immunosuppression,and so on,have been proved to be possessed by podophyllotoxin and its derivatives.Nevertheless,the clinical application of podophyllotoxin is severely hampered by its poor water solubility and high toxicity to normal cells.These limitations and the multiple pharmaceutical activities of podophyllotoxin inspired dozens of successful structural modification applying diversified strategies.Etoposide and teniposide are two glycosyl podophyllotoxin derivatives that have already been used for cancer therapy,revealing their potency as putative anticancer drugs.Structural activity relationship revealed that C-4 position modification was tolerable for its potency.Therefore,other C-4 non-sugar substituted derivatives has become the focus of medicinal chemists.In this study,47 podophyllotoxin derivatives modified by C-4 position were chose as the research objects.And HL-7702,A549,He La,MCF-7 and PC-3 were selected as test cell lines by MTT assay.The results showed that piperazine podophyllotoxin derivatives with α-configurated exhibited better inhibitory effect on the growth of cancer cells.And most of the derivatives also showed certain inhibitory effects on different cancer cells.Piperazine podophyllotoxin derivative 9 had the most significant inhibitory effect on the four tumor cell lines.In particular,it showed the strongest inhibitory activity on MCF-7.Subsequently,the proliferation of four cancer cells treated with compound 9 was studied.Colony formation experiments showed that compound 9 significantly inhibited the colony formation ability of MCF-7 cells and affected cell proliferation.To elucidate the antitumor mechanism of compound 9 on MCF-7 cells,mitochondrial membrane potential was measured.The results showed that the mitochondrial membrane potential of MCF-7 cells decreased.And,this change may be related to apoptosis.Subsequently,Hoechst 33342/PI double-stained MCF-7 cells were used.Under fluorescence microscope,it is observed that compound 9 induced apoptosis and necrosis in different degrees.Furthermore,apoptosis and necrotic pathways were blocked using 20 μM Z-VAD-FMK and 10 μM Necrostin-1.The results showed that both inhibitors could reduce the antitumor effect induced by compound 9.In addition,the mechanism of compound 9 on apoptosis of MCF-7 cells was elucidated by the changes of Bcl-2/Bax,caspase-3 and caspase-8.The results showed that Bcl-2/Bax,Caspase-3 and Caspase-8 were involved in the regulation of compound 9 on apoptosis of MCF-7.Since it is well known that podophyllotoxin depolymerizes the tubulin network,microtubules of MCF-7 cells treated with compound 9 were fluorescently stained.The results showed that compound 9 could also depolymerize the tubulin network of MCF-7.To further elucidate the binding mechanism of the compound 9,molecular docking calculation of compound 9 into the colchicine binding pocket of tubulin was investigated.Molecular docking results showed that compound 9depolymerized microtubules by occupying the colchicine site of tubulin.In conclusion,most of the 47 podophyllotoxin derivatives showed certain cytotoxicity to cancer cells.Especially,compound 9 had the most significant effect on MCF-7.Mechanism studies suggested that it may inhibit MCF-7 cells growth by inducing mitochondrial caspase apoptosis,depolymerizing cell microtubules,and occupying colchicine action sites.Therefore,these compounds can be further developed as novel anticancer drugs and might provide a new potential chemotherapy candidate for the treatment of breast cancer.