| Objective:(1)To observe the effects of metformin combined with daunorubicin on the apoptosis and proliferation of human colon cancer cell line(SW480) and investigate its possible mechanism.(2)To study the inhibitory effect of metformin combined with erythromycin on tumor inhibition of human colon cancer cell line(SW480) in nude mice.Methods:(1) Human colon cancer cells(SW480)were cultured in vitro.Different concentrations of metformin(2.5,5,10,20,40,80mmol·L-1) by the MTT assay,daunorubicin(0.025,0.05,0.1,0.2,0.4,0.8 ?mol·L-1) and different concentrations of metformin(2.5,5,10,20,40,80 mmol·L-1) and daunorubicin(0.2 ?mol·L-1) at different times(24 h,48 h,72 h) on the proliferation of SW480 colon cancer cells, and to determine the concentration and time of the drug.The experiment was divided into four groups: blank group, metformin group(20 mmol·L-1),erythromycin group(0.2 ?mol·L-1), metformin combined with erythromycin group(20mmol·L-1+0.2 ?mol·L-1).After 24 h drug detect the corresponding indicators:(1)MTT assay detected the cell survival;(2) Colony forming assay for detection of colony formation in cells;(3) Propidium iodide staining(PI) and Annexin V/PI staining were used to detected the cells apoptosis rate;(4)Western blot detected the expression of Bcl-2 and Bax protein;(5)Using RT-PCR to detect the expression of Bcl-2ã€Bax and m RNA protein;(2)Using human colon cancer SW480 cells in nude mice and establish a model of transplanted tumor in animal experiments: select 5-6weeks of healthy female nude mice.When the cells were grown to logarithmic phase,adjusting the cell concentration of 1×107 /m L,cell suspension 0.2 m L per mice injected under the left armpit.The 40 mice were randomly,this mice were divided into four groups:model(NS), metformin(100 mg·kg-1), erythromycin(0.1 mg·kg-1),metformin combined with erythromycin group(100 mg·kg-1+0.1mg·kg-1), The dose was given for each 0.2 m L,Once every 3 days,delivery after 6 cycles,the mice were killed after detection of the corresponding index.(1)Measuring the size of the tumor size, drawing the curve of the tumor;(2) Weigh the tumor mass and calculate the tumor inhibition rate;(3)HE staining was used to observe the metastasis and invasion of tumor;(4) TUNEL assay was used to detect apoptosis;Result:(1)The results of vitro cell experiment:(1) MTT assay showed that different concentrations of metformin(2.5,5,10,20,40,80 mmol·L-1), daunorubicin(0.025,0.05,0.1,0.2,0.4,0.8mmol·L-1) action in SW480 cells 24 h, 48 h, 72 h after can reduce cell viability. Two combined on cell growth inhibition rate was increased, and the inhibition in a dose and time dependent manner.After treatment with metformin(20 mmol·L-1), the survival rates of 24 h,48h and72 h were 66.35%, 50.37% and 42.02%, respectively.With 0.2 ? mol·L-1 daunorubicin treated SW480 cells 24,48,72 h later, cell survival rates were 63.12%,48.13%,45.24%.The combination of metformin and daunorubicin,the survival rates of 24 h, 48 h and 72 h were 60.63%, 45.29% and 32.16%,respectively. Its significantly lower than each of the single use group(P<0.05).(2)Colony clone showed that,after crystal violet staining after cloning is displayed at the reduction purple spots,while after the cells were treated with metformin and daunorubicin monotherapy and combination treatment group compared with the control group,the number of colony-forming colonies were the difference was statistically significant(P<0.05), further observation after the two combined, the combination group compared with daunorubicin alone group,spots colony formation is reduced even more significantly,the difference was statistically significant(P<0.05).(3)Single staining(PI) staining and Annexin V/PI double staining by flow cytometry showed:the spontaneous apoptosis in SW480 cells is very low, after medication,metformin and daunorubicin alone could increase cell apoptosis rate after both combined acting on the cell apoptosis was significantly increased compared with blank group differences were statistically significant(P<0.05), compared with daunorubicin alone group, the combination group apoptosis rate was increased,the difference They were significant(P<0.05).(4) Western blot and RT-PCR showed that,Bcl-2 protein and m RNA expression compared with the control group, the expression of each group decreased, the difference was significant(P<0.05);compared with daunorubicin alone group,the United treatment group was significantlyreduced,the difference was significant(P<0.05);the expression of Bax protein and m RNA,compared with the control group,all other expression were increased,the difference was significant(P<0.05);and daunorubicin ADM alone group, the combination group was significantly increased expression levels,the difference was significant(P<0.05).(2)The results of animal experiments :(1)Tumor volume of the combined group, with growth alone group, the slow growth, the volume is reduced,the difference was significant(P<0.05).Wet weight and tumor inhibitory rate was calculated.The results showed that compared with the model group,daunorubicin group and the combination group had significantly reduced tumor weight,the difference was statistically significant(P<0.05),compared with daunorubicin group,combined group tumor weight reduction more significantly,the difference was statistically significant(P<0.05).(2) Hematoxylin and eosin(HE) staining results showed that the most abundant blood vessels in the outer layer of the tumor,the combination of metformin combined with other groups, compared with other groups,the normal cells of the region less, and more necrotic areas.(3)TUNEL detection of apoptotic cells, the microscopic display result show the combined group is apoptosis mostobvious.Conclusion:(1)Metformin combined with erythromycin on human colon cancer SW480 cell proliferation inhibition and can promote apoptosis, the mechanism may be related to the promotion of Bax protein expression, inhibition of Bcl-2 protein.(2)Metformin combined with erythromycin on SW480 colon carcinoma in nude mice have anti-tumor growth effect, which may be related to the promotion of apoptosis. |
