riccardin-d and Lung-Neoplasms

Riccardin D is a macrocyclic bisbibenzyl compound extracted from liverwort plant Dumortiera hirsuta. Our previous study showed that riccardin D induced apoptosis of human leukemia cells by targeting DNA topoisomerase II (topo II). Riccardin D has been considered as a novel DNA topo II inhibitor and potential chemotherapeutic agent for treatment of cancers. In this study, we evaluated the inhibitory effects of riccardin D on growth of human non-small cell lung cancer (NSCLC) both in vitro and in vivo. Riccardin D effectively inhibited the proliferation of NSCLC cells as estimated by the MTT assay. Further examination showed that the ability of invasion and migration of NSCLC cells was suppressed on exposure to riccardin D as estimated by the assays of scratch and transwell chamber. The anticancer activity of riccardin D was verified in mice bearing human NSCLC H460 xenografts. Riccardin D injection produced a 44.5% inhibition of cancer growth without apparent signs of toxicity to animals. Further, riccardin D induced apoptosis of NSCLC cells as evidenced by the increases of cells with externalization of phosphatidylserine and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive in H460 xenografts. The analysis of apoptotic proteins showed that riccardin D activated the caspases cascade signaling pathway as demonstrated by the increases of cleaved caspase-3 and cleaved PARP in NSCLC cells in vitro and in H460 xenografts in mice. The pBR322 DNA relaxation assay indicated that riccardin D inhibited the activity of DNA topo II in H460 and A549 cells, suggesting the mechanism of riccardin D in induction of NSCLC apoptosis. In addition, we studied the activity and expression of matrix metalloproteinases (MMPs) in NSCLC cells. The activities of MMP-2 and MMP-9 in supernatants of NSCLC cells were suppressed on exposure to riccardin D as estimated by gelatin zymography assay. The inhibitory effects of riccardin D on expressions of MMP-2 and MMP-9 were verified in H460 xenografts in mice and the decreases of vascular endothelial growth factor (VEGF) and Erk1/2 might associate with the inhibition of MMPs and NSCLC growth. Together, our results suggest that riccardin D has a high inhibitory effect on human NSCLC growth through induction of apoptosis.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; DNA Topoisomerases, Type II; Female; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phenyl Ethers; Stilbenes; Topoisomerase II Inhibitors; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Riccardin D is a novel macrocyclic bisbibenzyl compound extracted from Chinese liverwort plant Dumortiera hirsuta. Our previous studies showed that riccardin D is a DNA topo II inhibitor and has therapeutic potential for treatment of cancers. In this combined in vitro and in vivo study, we examined the inhibitory effects of riccardin D on tumor angiogenesis and the subsequent effect of anticancer activity was evaluated. Incubation with riccardin D weakly inhibited the proliferation of human umbilical vascular endothelial cells (HUVEC) as estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The scratch wound experiment showed that riccardin D effectively decreased the motility and migration of HUVEC cells. Riccardin D inhibited the formation of capillary tube as demonstrated by decrease of branch points formed by HUVEC cells on 3-D Matrigel. We examined the levels of angiogenic factors including vascular endothelial growth factor (VEGF), VEGF receptor 2, epidermal growth factor receptor (EGF receptor), and matrix metalloproteinase (MMPs) in HUVEC cells. The expressions of VEGF, phospho-VEGF receptor 2, EGF receptor and MMP-2 were significantly reduced by riccardin D as estimated by Western blot assay and real-time quantitative PCR analysis. The decrease of VEGF was also detected in riccardin D-treated human lung cancer H460 cells. The anticancer activity of riccardin D was then evaluated in a mouse model in which riccardin D delayed the growth of H460 xenografts without obvious toxicity to animals after three weeks injection. To evaluate the role of antiangiogenesis of riccardin D in mice, CD34 immunohistochemical staining was employed to analyze the mean vascular density in H460 xenograft tissues. The number of blood vessels was significantly decreased after riccardin D treatment. These results suggest that riccardin D display the inhibitory effect on growth of human lung carcinoma cells and that the inhibition of angiogenesis may involve in anticancer activity of riccardin D.

Topics: Angiogenesis Inhibitors; Animals; Blood Vessels; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Human Umbilical Vein Endothelial Cells; Humans; Lung Neoplasms; Macrocyclic Compounds; Mice; Neovascularization, Pathologic; Phenyl Ethers; Stilbenes; Xenograft Model Antitumor Assays