| Objective: To study the biological function and molecular mechanism of zoledronate(ZOL) on HCT116 cells.Methods:1)CCK-8 assay was used to detect inhibitory effect of ZOL on HCT116 cell proliferation. HCT116 cells were incubated with different concentrations of ZOL for 72 h, cell viability was measured and IC50 was calculated. 2) Colony-forming unit assays were used to detect the effect of ZOL on the colony formation ability of HCT116 cells. 3) Flow cytometry analysis was used to detect the apoptosis of HCT116 cells after treated with 25μmol/L or 50μmol/L ZOL for 48 h. 4) HCT116 cells were treated with 25μmol/L or 50μmol/L ZOL for 24 h, 48 h or 72 h. After cells were stained with JC-1, the mitochondrial membrane potential dye, for 15 min in dark, fluorescence intensity and fluorescence positive ratio were measured with a fluorescence microscopy and a flow cytometry. 5) Western blotting analyzed the distribution of cytochrome C in mitochondria and cytoplasm and the activation level of Caspase-3 in cytoplasm after HCT116 cells were treated with ZOL at the concentration of 25μmol/L or 50μmol/L; 6) An xenografts nude mice model was established with HCT116 cells. Tumor growth, weight and tissue morphology were observed after administration of ZOL.Results: 1) ZOL inhibited the proliferation of HCT116 cells in a dose dependent manner, and IC50 was 26.79μmol/L. 2) The number of colonies in ZOL treatment group at IC50 dose was significantly decreased when compared to control. 3)After treated with 25μmol/L or 50μmol/L ZOL for 48 h, the apoptosis ratio of HCT116 cells was significantly increased(11.6%±0.5%, 25μM; 49.2%±3.4%, 50μM; p=0.0028). 4) After HCT116 cells were treated with 25μmol/L or 50μmol/L ZOL for 24 h, 48 h or 72 h, fluorescence microscopy images showed the red fluorescence intensity of JC-1 significantly decreased, and flow cytometry analysis showed that the proportion of JC-1 positive cells(in red) decreased. The ratio were 96.49%±2.1%, 86.13%±3.2% and 74.23%±5.3% separately(p=0.004) after 25μmol/L ZOL treated for 24 h, 48 h or 72 h, while the ratio were 55.21% ± 5.9%, 66.65% ± 2.3%, 38.71% ± 4.3% separately after 50μmol/L ZOL treated for 24 h, 48 h or 72 h. 5) Western blot analysis showed that after HCT116 cells were treated with ZOL at the concentration of 25μmol/L or 50μmol/L, cytochrome C decreased in mitochondria but increased in cytoplasm while the activated Caspase-3 in cytoplasm increased, which indicated that cytochrome C was released from mitochondria into cytoplasm and led to the activation of caspase 3. 6) In vivo experiment results showed that ZOL significantly slowed tumor growth. The last measure of tumor volume was 2289.4 ± 363.8mm3 in the control group, while 926.42 ± 238.3mm3 in 2mg/kg ZOL group. Tumor tissue was stripped at the end of the test. Tumor weight was 1.65 ± 0.31 g in 2mg/kg ZOL group, which was significantly smaller than that in control group, 2.53 ± 0.33 g, with an inhibition rate of 34.8%. Histologic images displayed more apoptosis bodies in sections of 2mg/kg ZOL-treated tumors.Conclusion: ZOL could inhibit cell proliferation and colony formation of HCT116 cells. ZOL decreased mitochondrial transmembrane potential, led to increased permeability and resulted in cytochrome C release from mitochondria to the cytoplasm, and activated caspase-3, which finally resulted in cell apoptosis. Animal experiments showed that ZOL could inhibit tumor growth in vivo and induce tumor apoptosis. This study suggested that ZOL could be developed as a potential agent for treating CRC. |
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