Effect Of Rho GTPase In The Inhibition Of Tumor Metastasis By ω-3 Polyunsaturated Fatty Acid

Cancer is the most life-threatening disease to humankind and tumor metastasis is one of the major factors on death due to cancer.The mechanism of tumor metastasis is abnormally complex and influenced by many factors.Besides the interactions between tumor and microenvironment and the integrated regulation by the human body,the biological characteristics(adhesion ability,invasion ability,migration ability)are found to be closely related with tumor metastasis.And all of the biological characteristics are closely related with the cytoskeleton mainly composed of the microfilaments and microtubules.Cytoskeleton reorganization plays a key role in the processes including pseudopod formation,adhesion of cell and the extracellular matrix,formation of focal adhesion complex,invasion into the extracellular matrix,distant migration in the blood or lymph vessel and growing in target organs or tissues,and all of these processes are mainly regulated by Rho GTPase. Rho GTPase belongs to the Ras superfamily with the molecular mass of about 20-25 Kd.Rho GTPase family proteins all have the ability to bind GTP and develop the activity of GTP enzymes. RhoA,Rac1 and Cdc42 are the three main Rho GTPase studied currently.Rho GTPase is the key regulator of cytoskeleton and can induce the organization of the microfilaments by actins and the microtubules by microtubulins.The regulation of cytoskeleton by Rho GTPase plays an important role in the adhesion,invasion and metastasis of tumor cells.As a metastasis-related gene of cancer,the overexpression of Rho GTPase exists in the highly metastatic tumor cells and the metastatic sites,which is closely related with tumor metastasis.Rho GTPase is the key molecule of several signal transduction pathways and the inhibition of the activity of Rho GTPase or the blockage of such pathways mediated by Rho GTPase can effectively attenuate the invasion and migration of tumor cells.ω-3 Polyunsaturated fatty acid is a group of very important nutrients with multiple biological functions,mainly including eicosapentaenoic acid(EPA) and docosahexaenoic acid(DHA) which have much influence on the carcinogenesis and tumor progression. Recently research have found that the increase intake ofω-3 PUFA of patients with chemotherapy can effectively attenuate tumor metastasis,make the metastatic sites diminished or disappeared and can also increase the sensitivity of tumor cells for chemotherapeutics but do not increase the drug toxicity.Sinceω-3 PUFA has some effects on tumor metastasis and the molecular mechanisms are still unclear,research on the effect ofω-3 PUFA on tumor metastasis and exploration of the molecular mechanism ofω-3 PUFA on tumor metastasis are important for the prevention and therapy of cancer.In this paper,MTT assay was firstly used to determine the effects ofω-3 PUFA(EPA or DHA) on the proliferation of PC-3 cells and the cell cycle analysis by flow cytometry was used to observe the influence ofω-3 PUFA on the cell cycle distribution.Adhesion assay,invasion assay and migration assay were conducted to explore the effects ofω-3 PUFA on tumor metastasis and RT-PCR,western blot and immunocytochemistry assay were used to detect the influence ofω-3 PUFA on the expression of the mRNA and protein of Rho GTPase.Moreover,immunofluorescence cytochemistry technology was used to observe the change of the microfilaments and microtubules in the metastasis of PC-3 cells treated byω-3 PUFA.According to the research above,we could observe the effect ofω-3 PUFA on the metastasis of PC-3 cells and explore the influence ofω-3 PUFA on the expression of Rho GTPase and the cytoskeleton reorganization.Finally we could demonstrate the role of Rho GTPase in the inhibition of tumor metastasis byω-3 PUFA.The results and conclusions are summarized as follows:1. MTT assay found that both EPA and DHA could inhibit the growth of PC-3 cells in a dose and time-dependent manner.Analysis of the cell cycle distribution of PC-3 cells treated by EPA or DHA of 60μmol/L demonstrated the significant increase at G0/G1 phase and decrease at the S+G2/M phase,which suggested that the cell cycle was arrested at the G0/G1 phase.2. Adhesion assay,invasion assay and migration assay have found that the adhesion ability of PC-3 cells treated withω-3 PUFA of 60μmol/L was significantly decreased at each time points compared to the control group and the number of invasion cells and migration cells from the inner of millicell chambers were significantly lesser than the control group,which demonstrated the significant inhibition of the adhesion,invasion and migration of PC-3 cells treated withω-3 PUFA. 3. RT-PCR assay and western blot assay have revealed that the expression of RhoA,Rac2,Cdc42 mRNA and Rac1,Rac2,Cdc42 protein all significantly suppressed byω-3 PUFA of 60μmol/L and immunocytochemistry assay found that the expression and distribution of RhoA in PC-3 cells was decreased.All of the results demonstrated thatω-3 PUFA can significantly inhibit the expression of Rho GTPase.4. Immunofluorescence assay has found the significant influence ofω-3 PUFA on the cytoskeleton reorganization.In the control group,the microbubules principally located in the cell membrane and the cytoplasm near the membrane with bunched or reticular arrangement and the microfilaments distributed in the cytosplam reticularly.Afer treated withω-3 PUFA of 60μmol/L,we found that the microbubules got shorter and the bunched structure collapsed or even disappeared. And the network structure of microfilaments also got dissolved or collapsed.All of the results demonstrated that the changes of the structure and distribution of the microfilaments and microtubules byω-3 PUFA.In conclusion,ω-3 PUFA not only could significantly inhibit the the ability of the proliferation of PC-3 cells and block the cell cycle,but also could inhibit the ability of adhesion,invasion and migration of PC-3 cells.Moreover,the expression of Rho GTPase and the structure of cytoskeleton could both been influenced byω-3 PUFA.In a word,the inhibitive effect ofω-3 PUFA on tumor metastasis may be mediated by the down-regulated expression and activity of Rho GTPase and the inhibitive effect of cytoskeleton reorganization regulated by Rho GTPase.