| Objective:To research the effect of baicalein on the cytotoxicity of cisplatin and the mechanism in the process in human lung cancer cells A549/CDDP.Methods:1. MTT assay was used to detect cell viability.2. Annexin-V/PI double staining was used to assess cell apoptosis.3. The ability of invasion and migration in A549/CDDP cells were observed by transwell assay.4. Western blot assay was used to explore the expression of E-cadherin, Vimentin,p-Akt, Akt, p65, IκBα, c-IAP1, c-IAP2, survivin and Bcl-x L.5. Si RNA transfection was used to knockdown NF-κB p65 in A549/CDDP cells,downregulation the activity of NF-κB.6. A549/CDDP xenograft model was used to observe the inhibition of A549/CDDP proliferation by baicalein or CDDPin vivo.7. Statistical analysis between groups was performed by t-test with Sigma Plot16.0 software.Results:1.Baicalein enhanced the cytotoxicity of CDDP in A549/CDDP cells by inducing apoptosis Baicalein inhibited proliferation of A549 /CDDP cells until 200μM baicalein was used. 100 μM baicalein significantly enhanced the cytotoxicity of CDDP in A549/CDDP cells, and baicalein significantly enhanced the apoptosis which was induced by CDDP.2.Baicalein increased the chemosensitivity of CDDP in A549/CDDP in vivo.The xenograft model was established, our results found that the combination of baicalein with CDDP synergistically significantly reduced the tumor growth compared with CDDP group or baicalein group.3. A549/CDDP cells undergo EMT.Compared with the A549 cells, A549/CDDP cells showed the morphological changes, decreased E-cadherin, increased vimentin, and increased migratory and invasive behavior. These results demonstrated that the A549/CDDP cells undergo EMT.4. Baicalein reversed CDDP-induced EMT in A549/CDDP cells.A549/CDDP cells were treated with baicalein, our results found that baicalein significantly increased invasion and migration activity, and enhanced the expression of E-cadherin, while decreased the expression of vimentin, the results showed that baicalein reversed EMT in A549/CDDP cells.5. Baicalein reversed EMT through the inhibition of PI3K/Akt pathway in A549/CDDP cells The A549/CDDP cells demonstrated increased p-Akt compared to A549 cells.The expression of p-Akt was inhibited when A549/CDDP cells were treated with LY294002, which blocked the PI3K/Akt pathway. LY294002 also significantly enhanced the expression of E-cadherin, decreased the expression of Vimentin,down-regulated the invasion and migration activity, and sensitized A549/CDDP cells to CDDP. Baicalein obviously decreased the expression of p-Akt, these results demonstrated that These results indicate that baicalein reversed EMT and increased the sensitivity of CDDP via PI3K/Akt pathway.6. Baicalein reversed EMT and increased the cytotoxicity of CDDP via the inhibition of PI3K/Akt/NF-κB pathway The redults showed that the A549/CDDP cells demonstrated enhanced expression of p65, decreased expression of IκBa compared with the parental A549 cells. P65 si RNA was used to knockdown the expression of p65, we foundthat p65 si RNA enhanced E-cadherin, decreased vimentin, and reduced the invasion and migration of A549/CDDP cells. MTT assay demonstrated that p65 si RNA sensitized A549/CDDP cells to CDDP. These results suggest that EMT can be reversed by suppressing the activity of NF-κB. Moreover, the result of western blot showed thatbaicalein significantly enhanced the IκBa levels and decreased p65 expression, while LY294002 also inhibited NF-κB, which blocked the PI3K/Akt pathway. So we concluded that baicalein reversed EMT and increased the cytotoxicity of CDDP via the inhibition of PI3K/Akt/NF-κB pathway.7. Baicalein inhibited NF-κB- mediated target proteins.Baicalein significantly down-regulated the expression of NF-κB-activated antiapoptotic genes such as sc-IAP1, c-IAP2, survivin and Bcl-x L.Conclusion:Baicalein can reverses the resistance of A549 cells to cisplatin in vivo and in vitro by the down-regulation of the PI3K/Akt/NF-κB-mediated EMT and NF-κB-activated antiapoptotic genes. |
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