Incarvine C suppresses proliferation and vasculogenic mimicry of hepatocellular carcinoma cells via targeting ROCK inhibition.
BACKGROUND
Studies have described vasculogenic mimicry (VM) as an alternative circulatory system to blood vessels in multiple malignant tumor types, including hepatocellular carcinoma (HCC). In the current study, we aimed to seek novel and more efficient treatment strategies by targeting VM and explore the underlying mechanisms in HCC cells.


METHODS
Cell counting kit-8 (CCK-8) assay and colony survival assay were performed to explore the inhibitory effect of incarvine C (IVC) on human cancer cell proliferation. Flow cytometry was performed to analyze the cell cycle distribution after DNA staining and cell apoptosis by the Annexin V-PE and 7-AAD assay. The effect of IVC on Rho-associated, coiled-coil-containing protein kinase (ROCK) was determined by western blotting and stress fiber formation assay. The inhibitory role of IVC on MHCC97H cell VM formation was determined by formation of tubular network structures on Matrigel in vitro, real time-qPCR, confocal microscopy and western blotting techniques.


RESULTS
We explored an anti-metastatic HCC agent, IVC, derived from traditional Chinese medicinal herbs, and found that IVC dose-dependently inhibited the growth of MHCC97H cells. IVC induced MHCC97H cell cycle arrest at G1 transition, which was associated with cyclin-dependent kinase 2 (CDK-2)/cyclin-E1 degradation and p21/p53 up-regulation. In addition, IVC induced apoptotic death of MHCC97H cells. Furthermore, IVC strongly suppressed the phosphorylation of the ROCK substrate myosin phosphatase target subunit-1 (MYPT-1) and ROCK-mediated actin fiber formation. Finally, IVC inhibited cell-dominant tube formation in vitro, which was accompanied with the down-regulation of VM-key factors as detected by real time-qPCR and immunofluorescence.


CONCLUSIONS
Taken together, the effective inhibitory effect of IVC on MHCC97H cell proliferation and neovascularization was associated with ROCK inhibition, suggesting that IVC may be a new potential drug candidate for the treatment of HCC.
Department of Clinical Pharmacy, Shanghai General Hospital, Shanghai Jiao Tong University School of medicine, No. 100 Haining Road, Shanghai, 200080, P. R. China. grissomzhang@hotmail.com.
Ji-Gang
JG
Zhang
Department of Clinical Pharmacy, Shanghai General Hospital, Shanghai Jiao Tong University School of medicine, No. 100 Haining Road, Shanghai, 200080, P. R. China. rainbowdan0513@hotmail.com.
Dan-Dan
DD
Zhang
Department of Clinical Pharmacy, Shanghai General Hospital, Shanghai Jiao Tong University School of medicine, No. 100 Haining Road, Shanghai, 200080, P. R. China. wuxinkaoyan007@163.com.
Xin
X
Wu
Department of Clinical Pharmacy, Shanghai General Hospital, Shanghai Jiao Tong University School of medicine, No. 100 Haining Road, Shanghai, 200080, P. R. China. yzwyy0506@126.com.
Yu-Zhu
YZ
Wang
Department of Clinical Pharmacy, Shanghai General Hospital, Shanghai Jiao Tong University School of medicine, No. 100 Haining Road, Shanghai, 200080, P. R. China. gsy15815@163.com.
Sheng-Ying
SY
Gu
Department of Clinical Pharmacy, Shanghai General Hospital, Shanghai Jiao Tong University School of medicine, No. 100 Haining Road, Shanghai, 200080, P. R. China. zghlxqx@163.com.
Guan-Hua
GH
Zhu
Department of Clinical Pharmacy, Shanghai General Hospital, Shanghai Jiao Tong University School of medicine, No. 100 Haining Road, Shanghai, 200080, P. R. China. lixiaoyulxb@sina.com.
Xiao-Yu
XY
Li
Department of Clinical Pharmacy, Shanghai General Hospital, Shanghai Jiao Tong University School of medicine, No. 100 Haining Road, Shanghai, 200080, P. R. China. liqin0626@hotmail.com.
Qin
Q
Li
Department of Clinical Pharmacy, Shanghai General Hospital, Shanghai Jiao Tong University School of medicine, No. 100 Haining Road, Shanghai, 200080, P. R. China. gaolinliu@aliyun.com.
Gao-Lin
GL
Liu
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D053961:Azabicyclo Compounds / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D003373:Coumaric Acids / Q000494:pharmacology*; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D009389:Neovascularization, Pathologic / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D000972:Antineoplastic Agents, Phytogenic; D053961:Azabicyclo Compounds; D003373:Coumaric Acids; D047428:Protein Kinase Inhibitors; C000609003:incarvine C; D054460:rho-Associated Kinases