Knockdown of EpCAM enhances the chemosensitivity of breast cancer cells to 5-fluorouracil by downregulating the antiapoptotic factor Bcl-2

Abstract

Resistance to fluoropyrimidine-based chemotherapy is the main reason for the failure of cancer treatment, and drug resistance is associated with an inability of tumor cells to undergo apoptosis in response to treatment. Alterations in the expression of epithelial cell adhesion molecule (EpCAM) affect the sensitivity or resistance of tumor cells to anticancer treatment and the activity of intracellular signaling pathways. However, the role of EpCAM in the induction of apoptosis in breast cancer cells remains unclear. Here, we investigated the effect of EpCAM gene knockdown on chemosensitivity to 5-fluorouracil (5-FU) in MCF-7 cells and explored the underlying mechanisms. Our results showed that knockdown of EpCAM promoted apoptosis, inhibited cell proliferation and caused cell-cycle arrest. EpCAM knockdown enhanced the cytotoxic effect of 5-FU, promoting apoptosis by downregulating the expression of the anti-apoptotic protein Bcl-2 and upregulating the expression of the pro-apoptotic proteins Bax, and caspase3 via the ERK1/2 and JNK MAPK signaling pathways in MCF-7 cells. These results indicate that knockdown of EpCAM may have a tumor suppressor effect and suggest EpCAM as a potential target for the treatment of breast cancer.

Figure 1. Effect of si-EpCAM and/or 5-FU treatment on cell viability in vitro.

MCF-7 cells were treated with 5-FU (7.5 µg/ml and 20 µg/ml) and/or si-EpCAM. A. MCF-7 cells were treated with si-EpCAM, the expression of EpCAM was detected. A negative siRNA control and Lipofectamin 2000 were served as control. B. Cell viability was detected with the CCK-8 assay. Cell viability was expressed as a percentage of control cells (MCF-7 cells). B. The results are presented as the inhibitory ratio of MCF-7 cells. *P<0.05.

Figure 2. Effect of EpCAM silencing and/or 5-FU treatment on the morphology of MCF-7 cells.

A. Morphologic changes of MCF-7 cells treated with the indicated concentration of si-EpCAM and 5-FU alone or together for 48 h. Magnification: 100×. B. MCF-7 cells were treated with the indicated concentrations of si-EpCAM and 5-FU alone or together for 48 h, then stained with DAPI (400×). Condensed and fragmented nuclei in cells are indicated by arrowheads.

Figure 3. Effect of EpCAM silencing and/or 5-FU treatment on apoptosis in MCF-7 cells.

A: Apoptosis was examined using annexin V-FITC/PI staining and flow cytometry analysis. A representative flow cytometric analysis of apoptosis in MCF-7 cells is shown. The fluorescence intensity of annexinV/FITC is plotted on the x-axis, and PI is plotted on the y-axis. FITC−/PI−, FITC+/PI−, FITC+/PI+, FITC−/PI+ was regarded as living, early apoptotic, late apoptotic and necrotic cells, respectively. B: The percentage of apoptotic cells was examined by annexin V-FITC/PI staining and flow cytometry analysis. Results are presented as mean±SD of three separate experiments. *P<0.05 versus 5-FU.

Figure 4. Effect of si-EpCAM and/or 5-FU treatment on cell cycle distribution in MCF-7 cells.

Cells were incubated with si-EpCAM and 5-FU alone or in combination at the indicated concentrations for 48 h, and cell cycle distribution was evaluated using PI staining and flow cytometry. One representative flow cytometric analysis of cell cycle distribution is shown.

Figure 5. Effect of si-EpCAM and/or 5-FU treatment on apoptosis-related factors in MCF-7 cells.

(A) MCF-7 cells were treated with 7.5 µg/ml and 20 µg/ml 5-FU for 48 h. Cells were harvested and analyzed by western blotting with antibodies against Bcl-2, Bax and caspase3. (B) MCF-7 cells were treated with si-EpCAM and/or 5-FU (7.5 µg/ml) for 48 h, and the expression of Bcl-2, Bax and caspase 3 was determined by immunoblotting.

Figure 6. Involvement of the ERK1/2 and JNK signaling pathways in si-EpCAM and 5-FU induced apoptosis.

(A) Effect of si-EpCAM and/or 5-FU treatment on the ERK and JNK signalingpathways.MCF-7 cells were treated with si-EpCAM and/or 5-FU (7.5 µg/ml) for 48 h, and the expression of pJNK and pERK1/2 was determined by immunoblotting. (B) The schematic illustration summarizes the effect of si-EpCAM on apoptosis in MCF-7 cells through activation of ERK and JNK signaling pathways induced by 5-FU.