In Vitro Anti-cancer Activities And Mechanism Studies Of Podophyllotoxin

Podophyllotoxin (PPT), a natural compound extracted from the podophyllum plants, is known to have important antiviral and antineoplastic properties. In recent years, many works have shown that the podophyllotoxin is a very important anticancer drug precursor. More attention has been paid to the anti-cancer activities of its derivatives. In this study, the inhibition of cell growth by PPT, its derivatives picropodophyllotoxin (PPP) and4-demethyl-podophyllotoxin (4DPG) were investigated. Our data showed that PPT, PPP and4DPG have different effects on the inhibition of cell growth to human normal liver QSG7701cells, hepatoma SMMU7721cells, and the cervical carcinoma HeLa cells. Among the three drugs, the IC50of PPP is500nM; followed by4DPG48nM, which is more effective than PPP; the inhibition of tumor cell growth of PPT is most significant and the IC50is the lowest and24nM. Then, more cell lines were chosen to test the cytotoxicity and the antitumor activity of PPT and4DPG, such as the normal mouse fibroblast NIH/3T3and human leukemia Jurkat cells, pleomorphic glioblastoma T98G cells and glioma SH-SY5Y cells. The results showed that PPT and its derivatives4DPG both have broad antitumor activities. Since the inhibition of PPT is the most effective to HeLa cells, the cellular and molecular mechanisms of growth inhibition of PPP in HeLa cells were explored. When treated cells with24nM PPT for24h and48h, respectively, the nucleus turned to become shrinking and finally formed apoptotic bodies, and the distribution of intracellular cytoskeleton also changed by fluorescence microscopy. Moreover, cell apoptosis, mitochondrial membrane potential and cell cycle were detected by flow cytometry (FCM). We found that HeLa cells became apoptotic, that the mitochondrial membrane potential was collapsed at24h, and that the cell cycle was obviously arrested at the G2/M phase. In addition, by using proteomic techniques, seven proteins were found to be significantly regulated by podophyllotoxin compared to the untreated control. They were calreticulin (CRT), protein disulfide isomerase A3(PDIA3), hepatoma-derived growth factor (HDGF), Reticulocalbin1(RCN1), Nucleophosmin/B23(NPM), Triosephosphate isomerase1(TPI1), Superoxide dismutase1(SOD1). Among them, CRT, PDIA3, HDGF and RCN1were downregulated and three others were upregulated. To further validate the results, real time fluorescence quantitative PCR was used to detected the alteration in gene expression of CRT, NPM, Bcl-2, Bax and Bcl-XL; and western blotting with specific antibodies were done. Our results showed that the transcription levels and the corresponding protein levels were consistent, that is, CRT (CRT) and Bcl-2(Bcl-2) were downregulated, while NPM(NPM) and Bax (Bax) were upregulated after the treatment of podophyllotoxin. In summary, we believe that podophyllotoxin inhibit cell growth via several cell signaling pathways. For another, due to the cytotoxicity of PPT in normal cells, we expect to construct an effective tumor targeted drug delivery system. An experimental approach was established to detect and separate of PPT and its carrier by high performance liquid chromatography. The carrier was connected to PPT with disulfide bonds, which would link to a tumor-targeted motif. We have proved that the disulfide bond between PPT and its carrier would break by2mM glutathione (GSH). The preliminary data lay the foundation for tumor targeted drug delivery system.