Effect Of ω-3 Polyunsaturated Fatty Acids On The Growth Of Colon Cancer Cells

Objective:There are growing data showing that the polyunsaturated fatty acids(PUFAs)prevent the pathogenesis and growth of cancers. Through the research of ω-3 PUFAs on colorectal cancer cell proliferation inhibition and mechanism, we plan to provide a theoretical basis for clinical application of ω-3 PUFAs in the prevention and treatment of colorectal cancer. YAP, an oncoprotein, is a core protein in Hippo pathway.GPCR120 is a membrane protein, with high expression in colon cancer cells.The mechanism of PUFA suppressing proliferation and promoting apoptosis of colon cancer cells, may provide a new way for the research on the occurrence and development of colon cancer.Methods:1.The proliferation effect of polyunsaturated fatty acid DHA and EPA on colon cancer cells was measured by MTT, and the apoptosis effect was measured by flow cytometry.2.The expression of YAP and the level of p-YAP in HT-29 and LOVO cells under the treatment of different concentrations of DHA and EPA were detected by Western Blot.3.The translocation between nucleus and cytoplasma of YAP in HT-29 and LOVO cells under DHA and EPA treatment were observed by confocal microscopy.4.Real-time PCR analysis was used to detect the mRNA level of YAP in HT-29 and LOVO cells with or without transfection of siRNAs of YAP before treatment of DHA and EPA.5.Knockdown of LATS and MST with si RNA, then perform Western Blot to detect the effect of ω-3 polyunsaturated fatty acids on YAP expression.6.The expression of YAP and p-YAP in HT-29 and LOVO cells treated with DHA and EPA or pretreated with siRNAs of LATS or MST was measured by Western Blot.7.The identification of membrane protein GPCR40 and GPCR120 of human normal colon tissue or colon cancer tissue were confirmed by immunohistochemistry.8.Knockdown of the membrane protein GPCR40 and GPCR120 with siRNA,and detect the protein level of P-YAP and P-LATS in HT-29 and LOVO cells under the treatment of DHA and EPA by Western Blot.9.The expression of p-LATS and p-YAP in HT-29 and LOVO cells treated with DHA and EPA after transfection with dnGαs or vehicle was detected by Western Blot.10.The expression of p-LATS and p-YAP in HT-29 and LOVO cells with or without pretreatment of the PKA inhibitor H-89 under DHA and EPA treatment was measured by Western Blot.Results:MTT assay showed that ω-3 PUFAs inhibit the proliferation of HT-29 and LOVO cells, in a time- and dose-dependent manner. Flow cytometry results showed that the ω-3 PUFAs induce colon cancer cells apoptosis.When HT-29 and LOVO cells were treated with different concentrations of ω-3PUFAs the protein level of phosphorylated YAP(p-YAP) was increased in a manner of time and concentration dependence. Confocal microscopy experiment revealed that YAP aggregated in cytoplasm obviously, with a reduction in nuclei, after 75μmol/Lω-3 PUFAs treatment. Quantitative real-time PCR analysis showed that the target genes of YAP, including pro-proliferation and anti- apoptosis genes, were decreased in mRNA level in HT-29 and LOVO cells, when treated with 75μmol/L ω-3 PUFAs.In addition, knockdown of YAP abolished this decrease tendency of these target genes. These results revealed that the down-regulated expression of these pro-proliferation and anti-apoptosis genes induced by ω-3 PUFAs are mediated by YAP. Western Blot has indicated that the expression of phosphorylated LATS(p-LATS) are obviously increased in HT-29 and Lovo cells after ω-3 PUFAs treatment in a time- and dose-dependent manner. After specific siRNAs were utilized to knockdown MST1 or LATS1, p-YAP protein levels declined dramatically. Finally,subsequent treatment of ω-3 PUFAs could not induce p-YAP due to knockdown of upstream factors such as MST1 or LATS1.We examined GPCR40 and GPCR120 expressions in paraffin-embedded CRC tissues of patients using immunohistochemistry(IHC) staining. Both of the two GPCRs were expressed in cancerous tissues. The effect of GPCR40 and GPCR120 knockdown on YAP and LATS phosphorylation in colonic cancer cell lines were measured by western blot. The results indicated that the loss of GPCR40 or GPCR120 function through siRNA could largely block ω-3 PUFAs-induced increas of YAP and LATS phosphorylation.HT-29 and LOVO cells transfected with dnGαs showed the decline of pLats and pYAP levels, even if DHA or EPA could not reverse this decreased tendency. We pretreated HT-29 and LOVO cells with the PKA inhibitor H-89 and Western blotting assays demonstrated that pLats and pYAP greatly reduced, whether DHA or EPA could not induce them rising again.Conclusions:1. DHA and EPA suppressed proliferation and induced apoptosis in colon cancer cells in a time- and dose-dependent manner.2. DHA and EPA induced phosphorylation and cytoplasmic translocation of YAP protein in HT-29 å'Œ LOVO cells.3.ω-3 PUFAs decreased mRNA level of the target genes of YAP, including pro-proliferation and anti- apoptosis genes, in HT-29 and LOVO cells4. Phosphorylation of YAP induced by DHA or EPA was through the Hippo pathway, thus regulating proliferation and apoptosis in colon cancer cells.5. GPCR120, GPCR40, Gαs and PKA were involved in mediating ω-3PUFAs-induced p-YAP. GPCR40/GPCR120 functions as a specific receptor for DHA and EPA. GPCRs that mainly activate the stimulatory subunit of the trimeric G protein Gαs signaling could stimulate Lats kinase activity, therefore increasing YAP/TAZ phosphorylation.6. Gs–PKA signalling pathway was upstream of the canonical Hippo pathway and was dispensable for ω-3 PUFAs-induced YAP phosphorylation.