The Effects Of ABL-N On Proliferation And Apoptosis Of Colorectal Cancer Cell Line CT₂₆

Colorectal cancer is a common digestive tract cancer. At present, colorectal cancer was treated mainly by operation radical resection, at the same time with complementary treatment of chemo-radiation to prevent relapsing and metastasis. In previous studies for Inula Britannica L, it has been found that acetylbritannilactone (ABL), a sesquiterpene lactone compound, has influence on cell cycle of tumor cells, but the stability and solubility of ABL are poor. ABL-N, a derivative of ABL, was used and colorectal cancer cell line CT₂₆ was selected and treated with different concentrations of ABL-N in vitro in this experiment to study the effects of ABL-N on proliferation and apoptosis of CT₂₆ cells.Objective:(1) To observe the effect of ABL-N on cell proliferation of CT₂₆ cells.(2) To explore the influence of ABL-N on cell cycle of CT₂₆ cells.(3) To explore the influence of ABL-N on apoptosis of CT₂₆ cells and further discuss the possible mechanisms of ABL-N-induced apoptosis.Methods:(1) MTT assay was used to detect the effect of ABL-N on proliferation of CT₂₆ cells. The cells were treated with different concentrations of ABL-N for 24 h and inhibitory rate was calculated according to the average OD values in the test groups and control group.(2) The plat colony formation assay was used to further test the effect of ABL-N on proliferation of CT₂₆ cells. The cells in test groups were treated with different concentrations of ABL-N for 24 h, and then cultured in fresh medium for 9 d. The cells in control group were cultured in solvent medium for the same time. The cells were stained, the number of clones in each group was counted and colony formation rate was calculated. (3) Flow cytometry was used to detect the cell cycle and apoptosis rate of CT₂₆ cells in control and test groups. CT₂₆ cells in the test group were treated with ABL-N for 24 h, which in the control group were cultured in solvent medium for 24 h. The cell cycle and apoptosis rate of CT₂₆ cells were determined by flow cytometry.(4) Transmission electron microscope was used to detect morphological changes of apoptosis of colorectal cancer cell line CT₂₆ treated with ABL-N. CT₂₆ cells in the test group were treated with ABL-N for 24 h, which were cultured in solvent medium for 24 h in the control group. The morphological changes of apoptosis of CT₂₆ cells were detected by transmission electron microscope.(5) Immunofluorescence staining and laser confocal scanning microscope were used to detect expression of Caspase-3 and Caspase-9 in CT₂₆ cells treated with ABL-N. CT₂₆ cells in the test groups were treated with different concentrations of ABL-N for 24 h, which were cultured in solvent medium for 24 h in the control group. The cells were fixed for 15 min with 4% paraformaldehyde, blocked for 30 min, incubated with primary antibody overnight in 4℃, and incubated with secondary antibody for 2 h at room temperature. The fluorescence intensity of expression of Caspase-3 and Caspase-9 was determined by laser confocal scanning microscope.Results:(1) MTT results: The inhibitory rates were 1.6%, 16.9%, 53.4% and 92.1% respectively in ABL-N 5, 10, 20 and 40mg/L groups. OD value in ABL-N 5mg/L group was lower than that in control group, but there was no statistically significant difference between the two groups (P>0.05). ABL-N 10, 20 and 40mg/L could significantly inhibit the proliferation of CT₂₆ cells (P<0.05 or P<0.01). ABL-N inhibited proliferation of colorectal cancer cell line CT₂₆ in a concentration-dependent manner.(2) Flat colony formation assay results: The colony formation rates of CT₂₆ cells in groups treated with ABL-N 5, 10 and 20mg/L were (44.7±3.8)%, (28.6±2.5)% and (4.6±1.4)%, respectively. There was no colony formation in the group treated with ABL-N 40mg/L. Compared with the control group (53.4±1.9)%, ABL-N 5mg/L could significantly inhibit the colony formation of CT₂₆ cells (P<0.05), and ABL-N 10, 20 and 40mg/L significantly inhibited colony formation (P<0.01). The inhibitory effect of ABL-N on colony formation of CT₂₆ cells was also concentration-dependent.(3) Flow cytometry results: The percentage of CT₂₆ cells in G₁ phase was 87.5% and 67.9% respectively in ABL-N (20mg/L, 24 h) group and control group. ABL-N treatment (20mg/L, 24 h) decreased the percentage of CT₂₆ cells in S phase from 29.6% to 11.8%. The apoptosis rate was 55.8% in the group treated with ABL-N 20mg/L, which was 4.14% in control group. The results indicated that ABL-N induced cell cycle arrest of CT₂₆, inhibited cell growth and induced apoptosis.(4) Morphological features of apoptosis were observed by transmission electron microscopy. The features of apoptosis could be observed such as higher electron density, vacuoles formation, and less mitochondria and rough endoplasmic reticulum in the cytoplasm, twist, crimple and rupture of nuclear membrane, nuclear karyopyknosis, and the characteristic half-moon shape of condensed chromatin in the CT₂₆ cells treated with ABL-N 20mg/L for 24 h.(5) The results of expression of apoptosis-related genes Caspase-3 and Caspase-9 by immunofluorescence staining and laser confocal scanning microscope: The expression of Caspase-3 and Caspase-9 in the cytoplasm of CT₂₆ cells was significantly higher in the groups treated with ABL-N 20 and 40 mg/L than that in the control group (P<0.01), and the expression of Caspase-3 and Caspase-9 was higher in ABL-N 40mg/L group than that in ABL-N 20mg/L group (P<0.01), which indicated that ABL-N induced expression of apoptosis-related genes Caspase-3 and Caspase-9 in the cytoplasm of CT₂₆ cells in a concentration-dependent manner.Conclusion:(1) ABL-N inhibited proliferation of CT₂₆ cells in a concentration-dependent manner. (2) ABL-N induced cell cycle arrest of CT₂₆ cells, and the cell cycle arrested in G₁ phase.(3) ABL-N induced apoptosis of CT₂₆ cells, which might be related to increasing expression of Caspase-3 and Caspase-9.