| Objective:To investigate the effects of levocarnitine(L-CNT)and 5-fluorouracil(5-Fu)on the apoptosis of MGC803 cells,and the underlying mechanisms were studied.Methods:1.Determination of the concentration of L-CNT and 5-FuThe MTT assay was used to analyze the different concentrations of L-CNT(0.08,0.8,8,16 mmol·L-1)and 5-Fu(0.019,0.19,1.9,19,95 mmol·L-1)on the proliferation of MGC803 cells at different time,the concentration of L-CNT and 5-Fu and the time of action were selected according to the MTT assay results.2.Effects of L-CNT on 5-Fu-induced apoptosis of MGC803 cellsMGC803 cells were divided into negative group(control),L-CNT group,5-Fu group and L-CNT+5-Fu group in vitro.Then the morphological changes of the cells were observed by inverted microscope.The apoptosis rate of cells was detected by Annexin V-FITC/PI double staining.3.The underlying mechanisms of L-CNT on the 5-Fu-induced apoptosis of MGC803 cellsReal-time PCR was taken to detect the ability of MGC803 cells to respond to L-CNT following analysis of the expression of carnitine metabolic enzymes CPT1 A,CPT1B,CRAT and OCTN2.Cell cycle was analyzed by flow cytometry.Western blot was applied to detect the expression of proteins including Bcl-2,Bax,ANT1 and cleaved-PARP which are related to apoptosis,cyclin D1 and p21 which are related to cell cycle.Results:1.Determination of the concentration of L-CNT and 5-FuMTT assay showed that in the concentration range of 0.08-0.8 mmol·L-1,the cell proliferation rate increased with the the increasing concentrations of L-CNT,and the proliferation rate decreased in the concentration range of 8-16 mmol·L-1.MGC803 cells showed different degrees of dose-dependent inhibition to 5-Fu using the MTT assay.The IC20 value of 5-Fu for 24 h was 0.01 mmol·L-1,IC50 value was 9.01 mmol·L-1.The IC20 value of 5-Fu for 48 h was 0.006 mmol·L-1,IC50 value was 0.81 mmol·L-1.With the combination treatment of 0.01 mmol· L-1 5-Fu and L-CNT at different concentrations for 24 h,the cell proliferation rates decreased with the increase of L-CNT concentration.According to the MTT results,the concentrations of L-CNT and 5-Fu were 0.8 mmol ·L-1 and 0.01 mmol·L-1,respectively,and the action time was 24 h.2.L-CNT enhances the apoptosis induced by 5-Fu on MGC803 cellsCompared with control group,L-CNT(0.8 mmol·L-1)group cells tightly linked and cells membrane were sharp.There was no significant changes in the proportion of cell apoptosis in the L-CNT(0.8 mmol·L-1)group(P>0.05).The proliferation rate of MGC803 cells was decreased significantly after 5-Fu(0.01 mmol·L-1)with or without L-CNT(0.8 mmol·L-1)treatment(F=188.234,P<0.001).Cells were characterized by shrinkage and cell membrane was blebbing.The apoptosis rates of cells were markedly increased(F=88.219,P<0.001).Compared with 5-Fu(0.01 mmol·L-1)group,the proliferation rates of MGC803 cells were decreased from 66.60±3.22 % to 60.00±1.77 %,the apoptosis rates were increased from 13.47±1.92 % to 19.60±1.06 % after the combination treatment of 5-Fu(0.01 mmol·L-1)with L-CNT(0.8 mmol·L-1)(P<0.001).3.L-CNT regulates cell cycle to enhance the apoptosis induced by 5-Fu on MGC803 cellsCompared with control group,the expression of CPT1 A,CPT1B and OCTN2 genes in L-CNT(0.8 mmol·L-1)group were significantly higher(P<0.05),CRAT expression was increased slightly(P>0.05).The G0/G1 phase proportion of cells was decreased from 23.69±1.10 % to 11.35±1.33 %(P<0.001).The S phase proportion of cells was increased from 65.30±1.35 % to 73.79±0.68 %(P<0.001).And the G2/M phase proportion of cells was increased from 11.01±2.35 % to 14.86±1.89 % slightly(P>0.05).The expression of apoptosis proteins Bcl-2,Bax and cleaved-PARP were slightly increased(P>0.05).The ratio of Bcl-2/Bax was slightly increased from 0.80±0.07 to 0.93±0.11(P>0.05).The expression of ANT1 and cyclin D1 proteins were markedly increased(P<0.01,P<0.01).The expression of p21 protein was not markedly changed after L-CNT treatment.In 5-Fu(0.01 mmol·L-1)group,the expression of CPT1 A,CPT1B and OCTN2 genes were significantly lower(P<0.05),CRAT expression was increased slightly(P>0.05).The G0/G1 phase proportion of cells was reduced,the S phase proportion of cells was increased and the G2/M phase proportion of cells was decreased after 5-Fu(0.01 mmol·L-1)with or without L-CNT(0.8 mmol·L-1)treatment.At the same time,the expression of pro-apoptotic proteins Bax,ANT1 and cleaved-PARP were significantly increased(F=30.210,P<0.01;F=26.878,P<0.01;F=670.722,P<0.001),and the expression of anti-apoptotic protein Bcl-2 was decreased(F=19.255,P<0.01).Compared with 5-Fu(0.01 mmol·L-1)group,the expression of CPT1 A was increased from 0.17±0.08 to 1.27±0.14 significantly(P<0.001),and the CPT1 B from 0.16±0.04 to 1.98±0.38(P<0.01),as well as the expression of CRAT and OCTN2 was increased slightly(P>0.05)after treated with the combination of L-CNT(0.8 mmol·L-1)and 5-Fu(0.01 mmol·L-1).The G0/G1 phase proportion of cells was decreased from 73.61±1.80 % to72.95±0.91 % slightly.The S phase proportion of cells was increased from 20.11±0.92 % to 27.05±0.91 %(P<0.01).And the G2/M phase proportion of cells was decreased from 6.27±0.95 % to 0.03±0.01 %(P<0.05).The expression of Bcl-2,Bax,cleaved-PARP and cyclin D1 proteins were all significantly increased(P<0.05,P<0.01,P<0.01,P<0.001),and the expression of p21 protein was not markedly changed.Conclusions:1.0.8 mmol·L-1 L-CNT could not induce apoptosis in MGC803 cells,and the percentage of cells in S phase was increased mainly by upgulating cyclin D1 protein expression.2.0.01 mmol·L-1 5-Fu induced MGC803 cell apoptosis by blocking cell cycle S phase and opening mitochondrial membrane permeability.3.0.8 mmol·L-1 L-CNT could enhance the apoptosis induction of MGC803 cells by 0.01 mmol·L-1 5-Fu.The mechanisms may be involved in regulating the cell cycle and influencing the expression of Bcl-2,Bax and ANT1 proteins meditated by mitochondrial apoptotic pathway. |
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