XWL-1-48, A Novel Oral Derivative Of Podophyllotoxin, Arrests Tumor Growth In Vitro And Vivo And GL331Induces Growth Arrest In HepG2Cells In Vitro

Tumor is a major threat to human health and the chemotherapy has been successful in the treatment of tumor. The use of oral anticancer drugs has shown a steady increase. Most patients prefer anticancer oral therapy to intravenous treatment primarily for the convenience of a home-based therapy. A better patient compliance, drug tolerability, convenience and possible better efficacy for oral therapy as compared to intravenous emerge as the major reasons to use oral anticancer agents among oncologists. Topoisomerases (Topos) are a well-validated target for anticancer therapy. Podophyllotoxin is the traditional DNA topoisomerase Ⅱ (Topo Ⅱ) inhibitor which shows high antitumor activity, but it cannot be used as antitumor agent because of its serious side effects clinically. Within the nucleus, DNA Topo Ⅱ solves the topological problems of DNA via a DNA breakage reunion mechanism to facilitate replication, transcription, recombination, chromosome condensation and decondensation. Etoposide and teniposide are semisynthetic derivatives of the natural podophyllotoxin and inhibit activity of Topo Ⅱ and are currently used clinically against various cancers, including small-cell lung cancer, testicular carcinoma, lymphoma, and Kaposi’s sarcoma. However, They still have several limitations such as poor water solubility, metabolic inactivation and development of drug resistance. So researchers are trying to find other new derivatives of podophyllotoxin that could overcome such deficiencies. XWL-1-48is a novel congener of VP16and is more watersoluble than VP16for oral administration. This study mainly focued on the anti-tumor activeities of XWL-1-48in vitro and in vivo and explored the underlying mechanisms. In addition, GL331is a novel podophyllotoxin deriveative which has ever entered phase Ⅱ clinical trial. The present work further investigated the molecular mechanism of another podophyllotoxin derivative, GL331, to induce cell cycle arrest and apoptosis in HepG2cells. This study is comprised of two parts, that is, part I XWL-1-48, a novel oral podophyllotoxin derivative, arrests tumor growth in vitro and in vivo; part Ⅱ:GL331induces growth arrest in HepG2cells in vitro. Part I:XWL-1-48, a novel oral podophyllotoxin derivative, arrests tumor growth in vitro and in vivoThe present study mainly studied the anti-tumor effects of XWL-1-48in vitro and in vivo and explored the underlying mechanisms. kDNA-decatenation reaction indicated that XWL-1-48could inhibit the Topo Ⅱα activity in an concentration-dependent manner; Furthermore, it induced cytotoxicity in different kinds of solid tumor cells and the IC50value ranged from0.34μM to2.11μM, which was more potent than its congener GL331, expecially in HepG2and MCF-7cells. Meanwhile, XWL-1-48exerted it cytotoxicity on the former two cell lines in a time-dependent way, which was more potent than GL331and VP16. In addition, it exhibited preliminary anti-MDR activity in KBV cells. XWL-1-48could also supperss the colony forming ability in HepG2cells and MCF-7cells. These results indicated that XWL-1-48could significantly inhibit the tumor growth in vitro. In vivo experiment, XWL-1-48exerted its antitumor activity in a dose-dependent way in H22bearing mice model. The inhibitory effect of XWL-1-48at8mg/kg is almost the same as VP16at26mg/kg. XWL-1-48also dose-dependently arrested the growth of HepG2and MCF-7xenografts in nude mouse. The inhibitory effect of XWL-1-48at4mg/kg was also almost the same as its congener VP16at23mg/kg. These results demonstrated that XWL-1-48exhibited excellent anti-tumor activity in vivo and it was more potent than VP16, with comparable toxicity. The present work also demonstrated that treatment with XWL-1-48resulted in S arrest in HepG2and MCF-7cells. And at the same time, it could induce typical apoptotic characters as indicated by morphologic changes which were further confirmed by PI-AnnexinV flow cytometric analysis. The up-regulated phosphorylation of H2AX and ATM in situ indicated that XWL-1-48could cause DNA damage in HepG2cells. Furthermore, it could activate the DNA damage signaling pathway ATM-P53-P21and ATM-Chk2-Cdc25A, down-regulated the levels of Cyclin A and p-Cdk2(Thr160), up-regulated the level of p-Cdk2(Thr14), all of which finally led to the inactivation of Cyclin A/Cdk2complex. XWL-1-48-induced DNA damage response could be reversed by ATM siRNA except for Cyclin A, followed by a partially disruption of S arrest, which indicated that XWL-1-48-induced S arrest was an ATM-dependent characteristic to some extent. XWL-1-48induced apoptosis through down-regulating apoptotic protein Bcl-2, up-regulating the Bax/Bcl-2radio and pro-apoptotic protein FasL or Fas (in HepG2cells and MCF-7cells, respectively), and finally, XWL-1-48could activate the Caspase-3enzyme. Meanwhile this study indicated that XWL-1-48-induced S arrest and apoptosis were not p53-dependent, which was proved by p53siRNA treatment. The study also found that XWL-1-48could downregulate the Mdm2level in a dose-dependent manner and this effect was not p53nor ATM-dependent. Furthermore, XWL-1-48could decrease the levels of p-Akt (Ser473), p-Mdm2(Serl66) and Mdm2, which indicated that it could supperss the activation of the P13K-Akt-Mdm2survival pathway to decrease Mdm2.Taken together, these data prove that XWL-1-48is a novel oral derivative of podophyllotoxin which exhibit poten anti-tumor activity in vitro and in vivo. It is also indicated that XWL-1-48is more effective while no more side effects, as compared to VP16. Through inhibiting Topo Ⅱ activity, DNA damage is caused and DNA damage response is triggered by XWL-1-48, which further induces the S arrest and apoptosis. At the same time, XWL-1-48down-regulates the Mdm2level by suppressing the activation of PI3K-Akt-Mdm2survival pathway. All of these results contributed to the anti-tumor effects of XWL-1-48. Part Ⅱ:GL331induces growth arrest in HepG2cells in vitroGL331, a topoisomerase Ⅱ inhibitor, has been found to trigger DNA damage response (DDR) to induce cell cycle arrest and apoptosis in various tumor cells. However, the underlying mechanism is not yet fully understood in HepG2cells. This study investigated the molecular mechanism involved in the GL331-induced cell cycle arrest via DDR in HepG2cells. As a result, GL331could induce S arrest and up-regulate the phosphorylation of the histone H2AX variant (y-H2AX). Ataxia telangiectasia mutated protein kinase (ATM) was activated by GL331through its autophosphorylation at Serl981, which led to the activation of DNA damage signaling pathways including p53/p21and Chk2/Cdc25A cascades. The DNA damage cascades triggered by GL331finally induced the inactivation of Cyclin A/Cdk2complexes to some extent. These phenomena could be reversed by ATM siRNA, followed by a partial disruption of S arrest. The present results suggested that the S arrest induced by GL331via DDR was in an ATM-dependent manner to some degree. GL331induced apoptosis through down-regulating apoptotic protein Bcl-2, up-regulating the Bax/Bcl-2radio and pro-apoptotic protein FasL, and finally activating the Caspase-3enzyme. Taken together, these data exhibit an important role of DDR triggered by GL331to induce S arrest and apoptosis and provide a new support for GL331to function as an anti-cancer drug.