The Effect Of Curcumin Combined With L-FP On Gastric Cancer MGC-803 Cells And Its Mechanism

Objective:Curcumin(CUR),a fat soluble phenolic pigment, is the main active component of turmeric,which has the function of anti-tumor,anti-inflammation,suppressor mutation and so on.L-FP,the project of chemotherapy,consists of low-dose cisplatin(DDP) combination with5-fluorouracil(5-FU),which is commonly used to treat Gastric Cancer(GC). In order to provide theoretical and experimental basis for combining application of curcumin and L-FP on treatment of gastric cancer in clinical,this paper aims to investigate the mechanism and effects of curcumin combination with L-FP on growth, proliferation, migration and apoptosis of human gastric cancer cell line MGC-803 in vitro.Methods:By the research methods of cell biology and molecular biology,observe the effects of curcumin and / or different doses of L-FP on growth,clone,migration, apoptosis,cell cycle and apoptosis-related regulatory protein Caspase-3,Caspase-8,Bax and bcl-2 of human gastric cancer MGC-803 cell in vitro. Main observation indexes and methods are as follows:1. MTT Assay : Measure OD with Microplate Reader, and calculate the growth inhibition rate when human gastric cancer MGC-803 cells on96-well plates are treated with curcumin and/or different doses of L-FP for 24,48 and 72 hours.2. Colony formation assay:The MGC-803 cells on 6-well plates are incubated in different concentration of curcumin and/or L-FP for 24 h.Curcumin and/or L-FP containing medium is then removed,and the cells are incubated for an additional 10 d in complete medium.The inhibitory rate was calculated after counting colonies by staining with Giemsa.3. Transwell migration assay: Calculate the migration rate by measuring with MTT assay when human gastric cancer MGC-803 cells on Transwell are treated with curcumin and/or different doses of L-FP for 10 hours.4. AO/EB fluorescent staining: when the MGC-803 cells on 6-well plates are treated with or without curcumin and/or L-FP at different concentrations for 24 hours, photograph and observe of cell morphology by using a fluorescence microscope after staining by AO/EB, and then calculate the apoptotic percentage.5. Flow cytometry analysis:The apoptosis is analyzed by flow cytometry using Annexin V-FITC and PI double staining and the cell cycle is analyzed by using PI single staining when the MGC-803 cells on 6-well plates are treated with or without curcumin and/or L-FP at different concentrations for 24 hours.6. Colorimetric method based on enzyme standard instrument: After the MGC-803 cells on 6-well plates are treated with or without curcumin and/or L-FP at different concentrations for 24 hours,The activity of Caspase-3 and Caspase-8 are measured by Caspase-3 and Caspase-8activity assay kits according to manufacturer's protocol.And then calculate the relative Caspase-3 and Caspase-8 activity.7. Western blot method: The total protein is extracted and the expression of apoptosis-elated regulatory protein Bax and Bcl-2 is detected by Western blot method after the MGC-803 cells on 6-well plates are treated with or without curcumin and/or L-FP at different concentrations for 24 hours.Results:1. Effect of curcumin and/or L-FP on the growth of MGC-803 cells:After incubated with curcumin or/and L-FP at different concentrations for 24h, 48 h and 72 h, compared to controls, all experiment groups exerted significantly obvious anti-growth effects(*P<0.05 or **P<0.01 or ***P<0.001);After treated by drugs for 24 h and 72 h,there is no significant difference on cell growth speed between MD L-FP group and curcumin combined LD L-FP group or between HD L-FP group and curcumin combined MD L-FP group.Moreover,the cell growth speed of combined group is significantly lower than L-FP single group after 48h(*P<0.05).2. Impact of curcumin and/or L-FP on the colony formation ability of MGC-803 cells: Compared to controls, the colony formation ability of all experiment groups significantly decreased(*P<0.05); The inhibitory rate of combined group is significantly higher than that of curcumin or L-FP single group(*P<0.05).3. Impact of curcumin and/or L-FP on the migration ability of MGC-803cells:A statistically significant lower number of cells migrated through the transwell filter when curcumin and/or L-FP were added in the migration chamber compared to the control group without curcumin and FP(*P<0.05 or**P<0.01);The migration rate of combined group is no significant difference from that of L-FP single group(*P<0.05).4. Effect of curcumin and/or L-FP on the cell cycle of MGC-803 cells: There was change in S phase arrest in response to treatment with curcumin and/or L-FP compared with control group(*P<0.05),while combined groups arrested the more cells in S phase than curcumin or L-FP single group(*P<0.05).5. Effect of curcumin and/or L-FP on the apoptosis of MGC-803 cells:Compared to controls,all experiment groups exerted significantly obvious induction of apoptotic effects(*P<0.05), The apoptotic percentage of combined group is significantly higher than curcumin or L-FP single group,And there is no significant difference on apoptotic percentage between flow cytometry and AO/EB(P>0.05).6. Effect of curcumin and/or L-FP on the apoptosis-elated regulatory proteins in MGC-803 cells:The experiment groups with curcumin and/or L-FP significantly decreased the expression of Bcl-2 and increased the expression of Bax compared to the control group without curcumin and L-FP; and the relative Caspase-3 and Caspase-8 activity of experiment groups with curcumin and/or L-FP significantly elevated compared to control group without curcumin and L-FP(*P<0.05);The combined groups were lower of the relative Bcl-2 protein expression or higher of the relative Bax protein expression than L-FP single groups(*P<0.05),besides the relative Bax protein expression of CUR+MD L-FP group and HD L-FP group had no significant difference(P>0.05). The relative Caspase-3 and Caspase-8 activity of CUR+MD L-FP group were higher than HD L-FP group(*P<0.05 or**P<0.01),while that of CUR+LD L-FP group and MD L-FP group had no significant difference(P>0.05).Conclusions:1. Curcumin can enhance anti-growth effect of L-FP on MGC-803 cells.2. Curcumin can enhance inhibition of proliferation and colony formation and migration ability of L-FP's on MGC-803 cells.3. Curcumin can enhance cell cycle arrest at S phase effect of L-FP on MGC-803 cells.4.Curcumin can enhance apoptosis effect of L-FP on MGC-803 cells;There is no significant difference on apoptotic percentage between flow cytometry and AO/EB.5. This suggested that curcumin enhanced the induction of apoptotic effect of FP in MGC-803 cells by inhibition of Bcl-2 and the promotion of Bax,followed by elevating the activation of Caspase-3 and Caspase-8.