ABSTRACT
Deoxypodophyllotoxin (DPT), a naturally occurring microtubule destabilizer, inhibits tubulin polymerization and causes cell cycle arrest at G2/M phase in tumor cells. However, the anti-tumor effect and specific mechanism of DPT in non-small cell lung cancer (NSCLC) are still poorly understood. In this study, we determined the anti-tumor effect and potential mechanism of DPT in the NSCLC cell line, NCI-H460 (H460). First, we demonstrated that DPT significantly inhibits the proliferation of H460 cells in vitro and the growth of H460 xenografts in vivo. In further studies, DPT triggered necroptosis in H460 cells with the following characteristics: (I) necrotic cell death morphology; (II) autophagy; (III) loss of plasma membrane integrity; (IV) loss of mitochondria membrane potential; (V) elevation of reactive oxygen species levels; and (VI) specific inhibition of necroptosis via a small molecule, necrostatin-1. This study also revealed that DPT has a similar effect towards the drug-sensitive cancer cell line, H460, and the drug-resistant cell line, H460/Bcl-xL. To our knowledge, this is the first report to document the induction of necroptosis by a microtubule-targeting agent to circumvent cancer drug resistance, thereby providing a new potential choice for clinical cancer therapy, especially drug-resistant cancer therapy.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Cell Death/drug effects , Cell Proliferation/drug effects , Podophyllotoxin/analogs & derivatives , Animals , Antineoplastic Agents, Phytogenic/administration & dosage , Autophagy/drug effects , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Culture Techniques , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm , Drugs, Chinese Herbal , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Membrane Potential, Mitochondrial/drug effects , Mice , Mice, Nude , Necrosis/pathology , Podophyllotoxin/administration & dosage , Podophyllotoxin/pharmacology , Reactive Oxygen Species/metabolism , Xenograft Model Antitumor AssaysABSTRACT
A functioning vascular supply is essential for solid tumor growth and metastases, which means that blood vessels are an ideal target for antitumor drug discovery. Targeting tumor vasculature involves two main approaches, anti-angiogenesis and vascular disruption. The anti-angiogenic and vascular disrupting activities of deoxypodophyllotoxin (DPT), a natural microtubule destabilizer, were examined with several in vitro, ex vivo and/or in vivo models. First, we demonstrated that DPT significantly inhibits the proliferation, migration and tube formation of endothelial cells and inhibits angiogenesis in rat aortic ring and chick chorioallantoic membrane assays. In further studies, DPT induced cytoskeleton reorganization in endothelial cells, which likely contributed to the anti-angiogenic effect at non-cytotoxic concentrations. DPT treatment at higher concentrations for longer time induced the cell cycle arrest, which may contributes to its anti-proliferation effect and anti-angiogenic activity. And DPT dramatically inducted the expression of cyclin B1 and p21 (WAF1/CIP1). Meanwhile, DPT disrupted capillary-like networks in vitro and newly formed vessels from rat aortic rings. Endothelial cell contraction associated with an increase in F-actin via the Rho/Rho kinase pathway likely contributed to the vascular disrupting activity. Taken together, our results provided the initial evidence that DPT exerts potent anti-angiogenic and vascular disrupting effects. This study also provides important insight into the mechanism of action of promising new anticancer drugs with both anti-angiogenic and vascular disrupting activities.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Blood Vessels/drug effects , Blood Vessels/pathology , Neovascularization, Pathologic/drug therapy , Podophyllotoxin/analogs & derivatives , Amides/pharmacology , Animals , Cell Cycle/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Chickens , Chorioallantoic Membrane/blood supply , Chorioallantoic Membrane/drug effects , Cyclin B1/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Drugs, Chinese Herbal , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/pathology , Humans , In Vitro Techniques , Male , Mice, Nude , Microtubules/drug effects , Microtubules/metabolism , Neovascularization, Pathologic/pathology , Neovascularization, Physiologic/drug effects , Paclitaxel/pharmacology , Podophyllotoxin/pharmacology , Podophyllotoxin/therapeutic use , Pyridines/pharmacology , Rats , Rats, Sprague-Dawley , Xenograft Model Antitumor AssaysABSTRACT
The objective of this study was to investigate the apoptosis-inducing effect of an oxidative analogue of gambogic acid (GA) on the human hepatocellular carcinoma cell line HepG2 and explore the related molecular mechanisms. HepG2 cells were treated with the analogue of GA and the growth inhibition was analysed by MTT assay. The morphological changes in cells were observed under an inverted light microscope and a fluorescence microscope. In addition, both the cell-cycle arrest and the apoptosis rate were detected by flow cytometry. Western blot was used to evaluate the alteration of protein expression. The viability of HepG2 cells was markedly inhibited in a concentration-dependent manner and obvious morphological changes were confirmed, including condensed chromatin and reduced volume. Increased percentage of apoptotic cells was displayed and altered expression level of several apoptosis-associated proteins, P53, Bcl-2, Bax and pro-caspase-3, was obtained. The newly synthesized analogue of GA exhibited potential anticancer activity, induced remarkable apoptosis in HepG2 cells, probably through the intrinsic mitochondrial pathway, and promised to be a new candidate for future cancer therapy.
