Effect Of ICA On The Expression Of AKT、p-AKT And NF-κB In PI3K/AKT Signaling Pathway In ACC-M Cell Line

Objective: Research the PI3 K inhibitor LY294002, ICA and their combined application of ACC-M cells, changes in the expression of AKT, p-AKT, NF-κB in PI3K/AKT signaling pathway. Preliminary study on the mechanism of ICA in salivary adenoid cystic carcinoma. Methods: The experiments were divided into negative control group, positive control group(LY294002 IC50 group), ICA IC50 group, ICA IC20+LY294002 IC10 group and ICA IC40+LY294002 IC10 group. 1. With different concentrations of ICA, LY294002 role ACC-M cells for 24 h, 48 h, 72 h, MTT method was used to determination of cell inhibition rate, find best time of drug action and calculate the action time of inhibitory concentration(IC10 IC20 IC25 IC30 IC40 IC50) screened of the drug concentration in combination group. 2. The morphological changes of the cells were observed by optical microscope. 3. Flow cytometry was used to detect the apoptosis of the cells in each group. 4. To detect the expression of AKT, p-AKT, NF-κB protein in each group by Western-blot. Results: 1. Different concentrations of ICA(25μg/ml, 50μg/ml, 100μg/ml, 200μg/ml, 400μg/ml) role of ACC-M cells for 24 h, 48 h, 72 h, found ICA has significantly effect inhibited the proliferation of ACC-M cells(P<0.05) and is concentration and time dependent. Different concentrations of LY294002(6.25μmol/l, 12.5μmol/l, 25μmol/l, 50μmol/l, 100μmol/l) role of ACC-M cells for 24 h, 48 h, 72 h, found that LY294002 on ACC-M cells has significantly inhibited the proliferation(P<0.05), with the same concentration and time dependence. It was found that the optimal action time of ICA and LY294002 was 48 h, the calculated ICA and LY294002 IC50 were 123.67μg/ml(185.2μmol/l) and 30.88μmol/l respectively, and the matching concentration of IC50 in the combined group(ICA IC40+LY294002 IC10) was detected. 2. Compared with the negative control group, reduce the cell density of the LY294002 IC50 group, ICA IC50 group, ICA IC20+LY294002 IC10 group, ICA IC40+LY294002 IC10 group, the gap is bigger, cells gradually by polygons become fusiform cells detached from the wall, and a portion of the cells floating in the culture medium showed that the apoptosis of state. 3. Cell apoptosis rate was significantly increased in the ICA IC40+LY294002 IC10 group than in the ICA IC50 group, LY294002 IC50 group and ICA IC20+LY294002 IC10 group(P<0.05). The apoptosis rate of ICA IC20+LY294002 IC10 group was higher than that of the ICA IC50 group, but lower than that of the LY294002 IC50 group. 4. Compared with the control group and LY294002 IC50 group, ICA IC50 group and ICA IC20+LY294002 IC10 group, ICA IC40+LY294002 IC10 group in p-AKT protein expression was significantly decreased, and ICA IC20+LY294002 IC10 group, ICA IC40+LY294002 IC10 group p-AKT expression was lower than that of LY294002 IC50 and ICA IC50 two single drug group(P < 0.05). LY294002 IC50 group in AKT, NF-κB protein expression and compared to the control group was not statistically significant(P > 0.05), and ICA IC50 group and ICA IC20+LY294002 IC10 group, ICA IC40+LY294002 IC10 group of AKT, NF-κB protein expression compared with the control group compared to slightly decreased(P < 0.05), and ICA IC20+ LY294002 IC10 group, ICA IC40+LY294002 IC10 group of AKT and NF-κB expression below ICA IC50 group. Conclusion: ICA and LY294002 were able to inhibit the proliferation of ACC-M cells, when ICA and LY294002 were used in combination than either drug alone can effectively inhibit the growth of ACC-M cells in vitro and its mechanism of action may act by inhibiting the expression of PI3K/AKT signal pathway and AKT, p-AKT, NF-κB protein.