Cx43 Reverses The Resistance Of A549 Lung Adenocarcinoma Cells To Cisplatin By Inhibiting EMT

Objective: To determine the effect of Cx43 in promoting lung cancer progression and CDDP resistance by regulating EMT in vitro.Methods: 1. MTT assay was used to examine cell viability. Cells were treated with increasing concentrations of CDDP(20-320 μM) for 48 h, and the cell viability was assessed by MTT assay, then calculate resistance index(RI). 2. Western blotting assay was used to explore the expressions of E-cadherin, Vimentin, Snail, Slug and Cx43. 3. Overexpression of Cx43 and inhibition of Cx43 expression by si RNA transfection. 4. We measured the migratory activity of the cells by wound-healing assays. 5. Matrigel invasion assay was used to compare invasive activity of the cells. 6. Statistical analysis. Statistical analysis between groups was performed using an unpaired Student’s t-test with Sigma Plot 10.0 software.Results: 1. The determination of resistant strains. The IC50 of A549/CDDP cell was 311.2 μM and IC50 of A549 cell was 53.6 μM, the A549/CDDP cell line demonstrated a 5.8-fold higher resis-tance to CDDP than the A549 cell line. 2. Acquired resistance of A549/CDDP cells to CDDP induces the cells to undergo EMT. We observed the morphological and the expression of EMT markers changes in A549/CDDP cells.These results demonstrated that the acquired resistance to CDDP induced A549/CDDP cells to undergo EMT. 3. A549/CDDP cells display increased potential for migration and invasion. Cells that have undergone EMT display increased migratory and invasive behaviors. Thus, in the following experiment, we measured the invasive and migratory activity of the cells by Transwell and wound-healing assays. We found that the A549/CDDP cells displayed increased potential for migration and invasion. 4. A549/CDDP cells show decreased expression of Cx43. Western blotting was used to detect the expression of Cx43 in A549 and A549/CDDP cells.Bar graphs derived from the densitometric scanning of the blots, significantly different from A549 cells. 5. Overexpression of Cx43 reverses EMT in A549/CDDP cells. We overexpressed Cx43 by transfection of pc DNA-Cx43 in A549/CDDP cells. Western blotting was used to confirm this effect.Results shows that the pc DNA- transfected cells exhibited an elongated fibroblast- like morphology, whereas Cx43-A549/CDDP cells, which had a high Cx43 expression level, displayed epithelial morphology. Moreover, compared with the pc DNA-transfected cells, the expression level of EMT markers also demonstrate that upregulation of Cx43 by pc DNA-Cx43 converted EMT to mesenchymal-epithelial transition(MET) in the A549/CDDP cells. 6. Knockdown of Cx43 expression induces EMT in A549 cells. We downregulated Cx43 expression in A549 cells which were sensitive to CDDP using si RNA. Compared with the NC control cells, the cells displayed a spindle-like fibroblastic phenotype, and the expression of EMT markers showed that A549 cells underwent EMT. These results suggest that Cx43 is involved in the regulation of CDDP-induced EMT in human lung cancer cells. 7. Cx43 regulates invasive and migratory properties of cells. To further investigate the effect of Cx43 on EMT in human lung cancer, we investigated the invasive and migratory properties of the cells. The results revealed that when A549/CDDP cells were transfected with Cx43, the capability of these cells to migrate and invade were obviously reduced relative to the pc DNA-transfected cells. In contrast, A549 cells transfected with Cx43 si RNA showed significant enhancement in their invasive and migratory properties. These results provide further evidence that Cx43 is involved in the regulation of CDDP-induced EMT in human lung cancer cells. 8. Cx43 regulates CDDP-induced cytoxicity in human adeno-carcinoma cells. To observe the effect of Cx43 on the cytotoxic effect of CDDP, we manipulated Cx43 expression in two ways: overexpression of Cx43 by transfection of pc DNA-Cx43 in A549/CDDP cells and knockdown of Cx43 expression with si RNA-Cx43 in A549 cells. The results showed that compared with the pc DNA-transfected cells, overexpression of Cx43 in the A549/CDDP cell line significantly reversed resistance of the cells to CDDP. Conversely, knockdown of Cx43 expression with si RNA-Cx43 resulted in insensitivity of A549 cells to CDDP. These results suggest that downregulation of Cx43 which induces EMT may underlie the resistance of A549 cells to CDDP.Conclusion: Cx43 plays a critical role in promoting lung cancer progression and CDDP resistance by regulating EMT.