Vincristine enhances amoeboid-like motility via GEF-H1/RhoA/ROCK/Myosin light chain signaling in MKN45 cells.
BACKGROUND
Anti-cancer drugs are widely used in cancer treatment frequently combined with surgical therapy and/or radiation therapy. Although surgery and radiation have been suggested to facilitate invasion and metastasis of tumor cells in some cases, there is so far little information about the effect of anti-cancer drugs on cellular invasive ability and metastasis. In this study, using four different anti-cancer drugs (vincristine, paclitaxel, cisplatin and etoposide), we examined whether these drugs influence the invasive ability of tumor cells.


METHODS
Human gastric adenocarcinoma MKN45 cells were used to evaluate the effect of anti-cancer drugs. After drug treatment, cellular invasive ability was assessed using the Matrigel invasion chamber. Cytoskeletal changes after treatment were examined microscopically with F-actin staining. In addition, we monitored cellular motility in 3D matrigel environment by time-lapse microscopic analysis. The drug-induced activation of RhoA and ROCK was evaluated by pull-down assay and Western blotting using an antibody against phosphorylated myosin light chain (MLC), respectively. Where necessary, a ROCK inhibitor Y27632 and siRNA for guanine nucleotide exchange factor-H1 (GEF-H1) were applied.


RESULTS
Among all drugs tested, only vincristine stimulated the invasive ability of MKN45 cells. Microscopic analysis revealed that vincristine induced the formation of non-apoptotic membrane blebs and amoeboid-like motility. Vincristine significantly enhanced RhoA activity and MLC phosphorylation, suggesting the involvement of RhoA/ROCK pathway in the vincristine-induced cytoskeletal reorganization and cellular invasion. Furthermore, we found that Y27632 as well as the siRNA for GEF-H1, a RhoA-specific activator, attenuated MLC phosphorylation, the formation of membrane blebs and the invasive ability after vincristine treatment.


CONCLUSIONS
These results indicate that vincristine activates GEF-H1/RhoA/ROCK/MLC signaling, thereby promoting amoeboid-like motility and the invasive ability of MKN45 cells.
Department of Environmental Veterinary Sciences, Laboratory of Radiation Biology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Masato
M
Eitaki
Tohru
T
Yamamori
Shunsuke
S
Meike
Hironobu
H
Yasui
Osamu
O
Inanami
D000199:Actins / Q000378:metabolism; D000230:Adenocarcinoma / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects*; D002945:Cisplatin / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D005047:Etoposide / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D018994:Myosin Light Chains / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D017239:Paclitaxel / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D064067:Rho Guanine Nucleotide Exchange Factors; D013274:Stomach Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D014750:Vincristine / Q000494:pharmacology*; D054460:rho-Associated Kinases / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
C577356:ARHGEF2 protein, human; D000199:Actins; D000970:Antineoplastic Agents; D020662:Guanine Nucleotide Exchange Factors; D018994:Myosin Light Chains; D064067:Rho Guanine Nucleotide Exchange Factors; C066737:RHOA protein, human; D014750:Vincristine; D005047:Etoposide; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; D017239:Paclitaxel; D002945:Cisplatin