Determination of possible relation between RhoC and extracellular proteolysis in terms of invasion and metastasis in colon cancer cells

Introduction: Most of deaths derived from colorectal cancer are due to metastasis rather than primary tumor; therefore colorectal cancer is the fourth leading cause of cancer death after lung, prostate and breast cancers. It is known that matrix metalloproteinases (MMPs) which are primarily responsible for degradation of extracellular matrix and locate in the extracellular area contribute carcinogenesis and metastasis of colorectal cancer. In addition RhoC, a member of small GTPases family, is involved in cell migration by regulating cytoskeletal components and highly correlated with metastasis. Aim: The aim of this research is to investigate the possible relation between RhoC that regulates the cytoskeletal components and MMP that is responsible for extracellular proteolysis in terms of invasion and metastasis in primary and metastatic colon cancer cells. Material and Methods: At first, RhoC gene expression was silenced with siRNA transfection in primary (HT-29, HCT-116 and SW480) and metastatic (SW620 and LoVo) colon cancer cells. After this transfection ROCK-I, mDia, MMP-2, MMP-7, MMP-9, MMP-14, TIMP-1 and TIMP-2 mRNA expression levels were investigated with Real Time PCR; the protein expression levels of MMP-2, MMP-9, MMP-7, MMP-14 and TIMP-2 were determined with Western Blot. In addition, secretion and activity levels of MMP-2 and MMP-9 were detected with Gelatin Zymography. After RhoC silencing, migration and invasion capability of colon cancer cells were evaluated with Cell Migration and Matrigel Invasion techniques. Results: The knockdown of RhoC resulted in decreased MMP-9 mRNA expression levels and significantly increased TIMP-2 mRNA and protein expression levels in HT-29 cells. In HCT116 cells, MMP-9 mRNA expression levels were significantly decreased and TIMP-1 mRNA expression levels were increased. Furthermore, RhoC inhibition declined proMMP-2, proMMP-9 and active MMP-9 activities in HT-29 cells and also proMMP-2 activity in SW480 cells. In addition, cell migration was inhibited in RhoC silenced HT-29, SW480, SW620 ve LoVo cell lines and cell invasion was decreased in RhoC silenced HT-29, HCT116, SW480 SW620 and LoVo cells. Conclusion: In conclusion, we predict that our results may contribute for developing new anti-metastatic therapeutic agents that target both RhoC signalling and MMP activity to prevent colorectal cancer metastasis.