| Objective:Atractylodes,a Compositae plant,which comes from a dry root of Atractylodes.It is mainly produced in Zhejiang,Anhui,Hubei and Hunan.Its taste is sweet,bitter and warm.Atractylodes has multiple functions such as invigorating the spleen and invigorating qi,drying dampness and diuresis,antiperspiration and placenta.Atractylenolide III,a main component of Atractylenolide,has significant anti-inflammation and anti-tumor effects.It remains unclear whether Atractylenolide III can inhibit the growth of HCT-116 cells and induce cell apoptosis.Therefore,this study takes HCT-116 cells as the research object,observes the effect of Atractylolide III on the growth of HCT-116 cells,and further explores cell apoptosis induced by Atractylolide III and its possible anti-tumor molecular mechanism to provides some experimental evidence that Atractylolide III is applied for the tumor treatment in particular Colon cancer.Methods:(1)Atractylolide III antitumor activity in vitro screening: CCK-8 method was used to detect the effects of atractylode III at different concentrations on the cell growth inhibition of A549,HePG2,PC12,Panc-28 and HCT-116 cells.(2)The screening of intervention concentration and action time of Atractylode III: Some certain concentrations gradient of Atractylolide III(6.25,12.5,25,50,100,200 ?mol/L)were used to treat HCT-116 cells for 12 h,24 h and 48 h,respectively,and the effect of growth inhibition of HCT-116 cells was detected by CCK-8 assay to screen the suitable treatment concentreation and time.(3)CCK-8 assay was used to detect the anti-tumor effect of Atractylolide III on HCT-116 cells in vitro: The HCT-116 cells were divided into negative control group,positive control group(5-FU,0.5 ?g/mL),Atractylolide III groups(50,100,200 ?mol/L).Firstly,the HCT-116 cells were regularly cultured.The cells of negative control group were not treated,the cells of positive control group were treated with 0.5 ?g/mL 5-FU for 24 h,the Atractylolide III groups was respectively treated with 50,100 and 200 ?mol/L Atractylolide III for 24 h.Finally,the cell growth inhibition of HCT-116 cells was detected by CCK-8 assay;(4)Hoechst 33258 staining was used to detect the apoptosis of HCT-116 cells: The cells were divided into negative control group,positive control group(5-FU,0.5 ?g/mL)and Atractylolide III groups(50,100,200 ?mol/L).The HCT-116 cells were cultured in six-wells plate.The cells of negative control group were not treated.The cells of positive control group were treated with 0.5 ?g/mL 5-FU for 24 h.The cells of Atractylolide III groups were respectively treated with 50,100 and 200 ?mol/L Atractylolide III for 24 h.The apoptotic ratio of HCT-116 cells was analyzed according to the proportion of cell nucleus staining with Brillant blue.(5)Annexin V-FITC flow cytometry was used to analyze the effect of Atractylolide III on the apoptotic rate of HCT-116 cells: the HCT-116 cells were divided into negative control group,positive control group(5-FU,0.5 ?g/mL)and Atractylolide III groups(50,100,200 ?mol/L).The HCT-116 cells were cultured in six-wells plate under regular method.The cells of negative control group were treated with nothing.The cells of positive control group was treated with 0.5 ?g/mL 5-FU for 24 h.The atractylolide III groups were respectively treated with 50,100 and 200 ?mol/L Atractylolide III for 24 h.All cells were performed in accordance to kit instruction and final apoptotic cells in different groups were severally examined by flow cytometry.The apoptotic cell rate was used to investigate the effect of Atractylolide III with various concentration in HCT-116 cells;(6)RT-PCR assay was used to determine the expression levels of apoptosis-related genes Bax,Bcl-2,caspase 3 and caspase 9 in HCT-116 cells: After treatement with 5-FU or Atractylolide III,the HCT-116 cells were divided into negative control group,positive control group(5-FU,0.5 ?g/mL)and Atractylolide III groups(50,100,200 ?mol/L).The HCT-116 cells were cultured in six-wells plate under regular method.The cells of negative control group was treated with nothing.The cells of positive control group was treated with 0.5 ?g/mL 5-FU for 24 h.The Atractylolide III groups were respectively treated with 50,100,200 ?mol/L Atractylolide III for 24 h.The cells were performed in accordance to kits instruction and final several apoptotic gene levels were analyzed by Bio-rad Real-Time PCR Systems.(7)Western blot was used to detect the expression of apoptosis-related proteins in HCT-116 cells: After treatement with 5-FU or Atractylolide III,the HCT-116 cells were divided into negative control group,positive control group(5-FU,0.5 ?g/mL)and Atractylolide III groups(50,100,200 ?mol/L).The HCT-116 cells were cultured in six-wells plate under regular method.The cells of negative control group was treated with nothing.The cells of positive control group was treated with 0.5 ?g/mL 5-FU for 24 h.The Atractylolide III groups were respectively treated with 50,100 and 200 ?mol/L atractylolide III for 24 h.All proteins of HCT-116 cells were extracted,electrophoresis,transmembrane,incubation of first antibodies and secondary antibodies according to the instruction of reagents kits.Finally,the expression of apoptosis-related proteins on PVDF membrane was detected by ECL chemiluminescence assay to exposure in Bio-rad ChemiDoc? XRS+ System and gray value of protein bands were analyzed by Image J.Results:(1)Screening of antitumor activity in vitro: The results showed that Atractylolide III of different concentrations(25,50,100,200 ?mol/L)have no significant inhibitory effect on the cell growth of HePG2,Panc-28 and A549 cells compared with the control group.Atractylolide III(100,200 ?mol/L)was applied to interfer with PC12 tumor cells.It can inhibit slightly the cell growth,but there was no significant difference compared to negative control group.The growth inhibitory ratio of HCT-116 cells can be markredly elevated to 16.71±1.38%,17.33±1.08%,34.55±3.14%,73.98±2.95%,respectively,after the intervention of Atractylolide III 25,50,100 and 200 ?mol/L,respectively.There was a significant difference compared to negative control group.(2)The Screening of treatment concentration and time of Atractylolide III: The cell growth inhibitory ratio of HCT-116 cells treated with Atractylolide III(6.25,12.5,25,50,100 ?mol/L)for 12 h appeared no significant difference when compared with the control group.The cell viability of HCT-116 cells treated with Atractylolide III 200 ?mol/L for 12 h was slightly increased but no significant difference compared with that of control group(p > 0.05).And the cell growth of HCT-116 cells treated with Atractylolide III 6.25,12.5 and 25 ?mol/L for 24 h also showed no significant change.The cell growth inhibitory ratio of HCT-116 cells treated with Atractylolide III 50,100 and 200 ?mol/L for 24 h were markedly increased and showed significant difference when compared with control group(p < 0.05).The cell growth inhibitory ratio of HCT-116 cells treated with Atractylolide III 6.25,12.5,25,50,100 and 200 ?mol/L for 48 h was significantly elevated than that of the control group(p < 0.05),and showed a certain concentration dependence.These results suggested convincingly that the cells were treated with 50,100 and 200 ?mol/L Atractylolide III for 24 h in following experiments.(3)The determination of antitumor activity of Atractylolide III in HCT-116 cells in vitro.The results from CCK-8 assay showed that the cell growth inhibitory ratio of HCT-116 cells treated with 5-FU and Atractylolide III at different concentrations(50,100,200 ?mol/L)for 24 h was increased significantly when compared with the negative control group(p < 0.05).(4)Hoechst 33258 staining was used to detect the effect of Atractylolide III on apoptosis of HCT-116 cells.The results suggested that HCT-116 cells were treated with 5-FU(0.5 ?g/mL)and Atractylolide III(50,100,200 ?mol/L)for 24 hours.The percentage of cells nuclei with bright blue increased significantly to suggest that apoptotic cells was increased markerdly in a concentration-dependent manner.And there was a significant difference compared to negative control group(p < 0.05).(5)Annexin V-FITC flow cytometry was used to analyze the effect of Atractylolide III on the apoptotic rate of HCT-116 cells.The results showed that the apoptotic rate of HCT-116 cells treated with 5-FU(0.5 ?g/mL)and Atractylolide III(50,100,200 ?mol/L)for 24 hours was significantly increased than that of cells in the negative control group.The difference was statistically significant(p < 0.05).(6)RT-PCR was used to determine the expression of apoptosis-related genes in HCT-116 cells treated with Atractylolide III: The results showed that the levels of apoptotic genes Bax,caspase-9 and caspase-3 were markerdly elevated in HCT-116 cells of positive control group(5-FU,0.5 ?g/mL)and Atractylolide III concentration groups(50,100,200 ?mol/L)were treated for 24 h.However,the expression level of Bcl-2 gene was significantly decreased compared with the negative control group(p < 0.05).(7)Western blot was used to detect the expression of apoptosis-related proteins Bax,Bcl-2,caspase-9 and caspase-3 in HCT-116 cells treated with atractylolide III: The results showed that the levels of pro-apoptotic proteins Bax,caspase-9 and caspase-3 in HCT-116 cells of positive control group(5-FU,0.5 ?g/mL)and atractylolide III groups(50,100,200 ?mol/L)treated for 24 h were significantly increased,but the level of Bcl-2 was significantly decreased,which compared with the negative control group,the difference was statistically significant(p < 0.05).Conclusion:(1)Atractylode III has no significant growth inhibitory effect for tumor cells HePG2,Panc-28,A549 and PC12.However,Atractylode III has significant inhibitory effect on the growth of human colon cancer HCT-116 cells in vitro,the optimal intervention concentration was 50,100,200 ?mol/L,and the optimal treatment time was 24 h.(2)Atractylolide III can significantly inhibit the cell growth and promote cell apoptosis in HCT-116 cells,it shows a certain concentration dependence.(3)Atractylolide III may promote the expression of pro-apoptotic related genes Bax,caspase-9,caspase-3 and proteins Bax,caspase-9,cleaved caspase-3,but inhibit the expression of anti-apoptotic related gene/ protein Bcl-2 by regulating the Bax/Bcl-2 apoptotic signaling pathway,thus promoting the development of apoptosis of HCT-116 cells. |
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