Effect Of Cinnamaldehyde On Biological Behavior Of Human Colorectal Cancer Cells And Inducing Apoptosis Via Inhibition Of PI3K/Akt Signaling Pathway

Objective:To investigate the relationship between effects of cinnamaldehyde on biological behavior of human colorectal cancer cells and inducing apoptosis via inhibition of PI3K/Akt signaling pathway in vitro.Method:①After treated with 0,20,40,60,80 μg/ml of CA for 12,24 and 48h, the effect of different concentration on inhibition rate of the cells were observed via the MTT assay. ② Treated with different concentrations of CA (0,20,40,80μg/ml) for 24 h, cell Transwell assay and Cell matrix adhesion assay were used to measure the effect of adherence ability of cinnamaldehyde on human colorectal cancer cells, nvasion and adhesion-related genes (E-cadherin,MMP-2,MMP-9) were detected by western blot analysis.③Evaluated the effects of CA on CRC cells by using Annexin V-FITC and observed the nuclear morphological changes of cells by using Hoechst 33258 staining; Aoptosis-related genes (Bcl-2,Bax,PARP,cleaved-PARP) were detected by western blot analysis.④ To further explore the effect of cinnamaldehyde on PI3K/Akt pathway, Cells were treated with 40μg/ml CA along, the expression of PI3K, p-PI3K, Akt and p-Akt was detected by western blotting analysis. To clear the blocking effect and blocking site, Cells were pretreated with 10 ng/ml IGF-I for the 2 h, and then treated with 40μg/ml CA. Further, cells were pretreated with IGF-I for 2 h, followed by exposure to LY294002 for 24 h as a positive control. The expression of PI3K, p-PI3K, Akt and p-Akt was detected by western blotting analysis. ⑤ o confirm whether the apoptosis of CRC cells by CA was mainly regulated through the PI3/AKt pathway, IGF-1 and LY294002 were used to evaluate the underlying mechanism. Cells were pretreated with 10 ng/ml IGF-I for the 2 h, and then treated with 40μg/ml CA.Further; cells were pretreated with IGF-I for 2 h, followed by exposure to LY294002 for 24 h as a positive control. The expression levels of Bcl-2, Bax, cleaved-PARP and PARP were detected by western blotting analysis.Result:Three kinds of colorectal cancer cells with various stages of differentiation invasion (HCT116, LoVo, SW480) were treated with CA with 0,10,20,40,60,80 μg/ml of CA for 12,24 and 48h, the effect of different concentration on inhibition rate of the cells were observed via the MTT assay. Inhibition rates of the cells were significantly higher compared with the control group at the same time points, and the inhibition of CA presented an approximate dose-and time-dependent manner.24 h were found to be optimal administration time for the next work. The IC50 values of CA inhibition of HCT116, LoVo and SW480 growth at 24 h were 30.7ng/ml, 30.6μg/ml and 35.69μg/ml, respectively. After treated with different concentrations of CA(0,20, 40,80μg/ml) for 24 h, the number of the cells invaded across 8 μm diameter pores to the lower chamber markedly decreased compared with control group, indicating that CA could suppress the invasion of CRC presented a dose-dependent manner.Meanwhile, similar to the results of transwell invasion assay, the adhesion rates of cells treated with CA at all time points were lower than those of control group. Further data demonstrated that the inhibitory rate of adhesion increased as concentrations and time rose up. Consequently, CA could dramatically inhibit the adhesion of CRC cells to fibronectin and the inhibition rates presented a dose-and time-dependent manner. Western blot found that CA significantly reduced the expression of MMP-2 and MMP-9 in a concentration-dependent manner, the expression of E-cadherin was up-regulated as the dose of CA increased which may be responsible for the reduction of cell invasion and adhesion observed in CRC cells.By using Hoechst 33258 staining the apoptotic cells were exhibited highly fluorescent condensed chromatin. As to the treated cells, we observed small, fragmented, and condensed nuclei with typical apoptotic morphology as in contrast with normal symmetrical, blue nuclei, we evaluated the effects of CA on CRC cells by using Annexin V-FITC.The results indicated that both early and late apoptotic rates of the cells could increase by CA, and the apoptotic rates of 20,40,80μg/ml of CA were significantly higher than the control group. Western blot result revealed that the Bax, cleaved-PARP expression was obviously increased, whereas the PARP, Bcl-2 expression was decreased, leading to an up-regulation in the ratio of Bax/Bcl-2. This might be one of the molecular mechanisms in which CA induced apoptosis in CRC cells. Further experiments found that moderate doses of cinnamaldehyde can significantly inhibited the PI3K/Akt pathway, Western blotting analysis indicates that CA inhibited IGF-1-induced the expression of p-AKT protein. The total p-PI3K level also inhibited although the total PI3K, total Akt level in each experimental group did not obviously change. To confirm whether the apoptosis of CRC cells by CA was mainly regulated through the PI3K/Akt pathway, IGF-1 and LY294002 were used to evaluate the underlying mechanism. The results indicated that IGF-1-induced anti-apoptotic was inhibited by LY294002 and CA.Conclusion:Cinnamaldehyde can effectively inhibit the proliferation of human colon cancer cells. Cinnamaldehyde had inhibitory effects on the invasion and metastasis, CA significantly reduced the expression of MMP-2 and MMP-9, the expression of E-cadherin was up-regulated which may be responsible for the reduction of cell invasion and adhesion observed in CRC cells. CA induces apoptosis of human colorectal cancer cells. The mechanism involved is Bax, cleaved-PARP expression was increased, whereas the PARP, Bcl-2 expression was decreased.On the other hand, CA could inhibit PI3K/Akt signaling pathway through down-regulate p-PI3K and p-AKT. CA could down regulate PI3K/Akt dependent transcriptional activity, resulting the regulation of apoptosis-related genes expression.