The Study Of The Inhibiting Effect Of EPA On Theproliferationofhuman Cholangiocarcinoma Cell Line FRH-0201

The unsaturated fatty acids whose carbon chains contain more than one double valence bond per molecule are called polyunsaturated fatty acids (PUFA). The studies in and outside of China have demonstrated that PUFA can kill tumor cells in vitro. PUFA can be classified into two groups, omega-3(ω-3) and omega-6(ω-6), according to the location of their first double bond with respect to the terminal methyl group. Omega-3polyunsaturated fatty acids (ω-3PUFA) are considered essential fatty acids for human body. Omega-3PUFA also is an essential component of membrane phosphatides and involves in the adjustment of intracellular metabolism. It has been proved through in vivo and in vitro pharmacological experiments that ω-3PUFA can inhibit the growth, invasion and metastasis of tumors, enhance the therapeutic efficacy of certain anti-cancer drugs and prolong the survival time of tumor-bearing hosts. We have discovered in previous studies that docosahexaenoic acid (DHA) could significantly inhibit the proliferation of human gastric cancer cell line AGS. Eicosapentenoic acid (EPA) is another member of ω-3PUFA family, and a lot of in vivo and in vitro studies showed that it has the inhibiting effect on multiple tumors. The present study adopted cholangiocarcinoma cell line FRH-0201which can well represent all groups of primary lesion cells of cholangiocarcinoma as an experimental model. The purpose was to study the inhibiting effect of EPA on the proliferation of the cell line in vitro and its mechanism.1、The impact of EPA on the proliferation of human cholangiocarcinoma cell line FRH-0201The results from the MTT method indicated that EPA could significantly inhibit the proliferation of FRH-0201cell line when the cells were exposed to EPA for48hours, with the highest inhibiting rate of89.92±0.42%and IC50value of32.20±1.70μg/ml. The cells in each treatment group showed marked morphological changes when observed under an inverted microscope:the cells were shrinking, there were more intercellular granules, and the number of dead cells was increased. EPA showed no significant inhibiting effect on the proliferation of normal mouse fibroblast cell line L929, and there was no significant difference (P>0.05) in OD values between the treatment groups of various EPA concentrations, and between each treatment group and the control group.Trypan blue exclusion method was used to determine cell viability and growth curves were generated for all the groups. It was found that EPA of each concentration could inhibit the proliferation of FRH-0201cell line when the cells were exposed to EPA for less than96hours and it showed a time-dependent effect. The analysis with flow cytometry demonstrated that when FRH-0201cells were exposed to EPA of32μg/ml,50μg/ml and70p.g/ml for48hours each, the percentage of cells in G0/G1phase was increased while the percentage of cells in S and G2/M phases was significantly decreased with the increase of EPA concentration. Therefore, the cells were remaining in G0/G1phase of the cell cycle.2. Apoptotic effect of EPA in vitro on human cholangiocarcinoma cell line FRH-0201It was discovered with acridine orange staining under a fluorescence microscope that the DNA in the cell nuclei of FRH-0201cell line in the control group showed even fluorescent yellow-green color, while the substances in the cell nuclei or cytoplasm of FRH-0201cell line in the treatment groups showed dense fluorescent yellow-green color and even strongly stained yellow-green debris, which demonstrated classic morphological changes of apoptosis.A transmission electron microscope was used to observe the cellular ultra-structure of FRH-0201. It was discovered that after the cells were exposed to EPA of40μg/ml for24hours, vacuoles were formed in the cytoplasm and condensed chromosomes gathered near the nuclear membranes, which formed masses of high electron density close to the membranes. These also demonstrated classic morphological changes of apoptosis.Analysis of apoptosis was performed by Annexin V/PI double staining assay. The results showed that when the cells were exposed to EPA of50μg/ml and70μg/ml for48hours, the apoptosis rates were37.3%and62.2%respectively, which were significantly higher than the one in the control group (1.3%). And when the cells were exposed to EPA of50μg/ml and70μg/ml for48hours each, the percentages of late-stage apoptotic cells and necrotic cells were7.0%and7.5%respectively. 3, The primary study on the mechanism of FRH-0201apoptosis induced by EPA in vitroFlow cytometry was used to assess mitochondrial transmembrane potential (△ψm) changes after JC-1staining of the cells. The result indicated that FRH-0201mitochondrial△ψm was significantly decreased with the increase of EPA dose (P<0.05).After FRH-0201cells were exposed to the EPA of each concentration for48hours, immunohistochemistry was used to detect the expression of cytochrome c, and the result showed that there were brown granules in the cytoplasm of the cells with the protein expression. The result of semiquantitative analysis with computer indicated that the density of brown granules (p<0.05) and the percentage of positive cells (p<0.001) in the treatment groups were both significantly higher than that in the control group.Western blotting was used to assess the effect of EPA on the expressions of caspase-3and caspase-9in FRH-0201cells. The results demonstrated that compared to the control group, the expressions of caspase-3and caspase-9were significantly enhanced after the cells were exposed to the EPA of each concentration for24hours.4. The effect of EPA on SOD and MDA in FRH-0201cell lineA full-automatic biochemical analyzer was used to detect the contents of superoxide dismutase (SOD, wavelength550nm) and malondialdehyde (MDA, wavelength532nm) in FRH-0201cells. The results demonstrated that the activity of SOD in FRH-0201cells was lowered to a significant extent (p<0.05) or extremely significant extent (p<0.001), and the content of MDA in FRH-0201cells was increased to an extremely significant extent (p<0.01; p<0.001) after the cells were exposed to the EPA of each concentration. 5. ConclusionEPA can significantly inhibit the proliferation of FRH-0201cells cultured in vitro, while it has no remarkable inhibiting effect on the proliferation of normal mouse fibroblast cell line, L929. It can make FRH-0201cells remain in G0/G1phase, impacting the cell cycle. It can also lower mitochondrial transmembrane potential,induce the release of cytochrome c and activate caspase-9, triggering the activation of caspase-3cascade, which will result in the apoptosis of FRH-0201cells.