Mechanisms of Gefitinib-mediated reversal of tamoxifen resistance in MCF-7 breast cancer cells by inducing ERα re-expression

Abstract

Estrogen receptor (ER)-positive breast cancer patients may turn ER-negative and develop acquired drug resistance, which compromises the efficacy of endocrine therapy. By investigating the phenomenon that gefitinib can re-sensitise tamoxifen (TAM)-resistant MCF-7 breast cancer cells (MCF-7/TAM) to TAM, the present study verified that gefitinib could reverse the acquired drug resistance in endocrine therapy and further explored the underlying mechanism.ERα-negative MCF-7/TAM cells were established. Upon treating the cells with gefitinib, the mRNA and protein levels of ERα and ERβ, as well as the expression of molecules involved in the MAPK pathway, were examined using the RT-PCR and immunocytochemistry. The RT-PCR results showed that the mRNA levels of ERα and ERβ in MCF-7/TAM cells were up-regulated following gefitinib treatment; specifically, ERα was re-expressed, and ERβ expression was up-regulated. The expression of molecules involved in the MAPK pathway, including RAS, MEK1/2, and p-ERK1/2, in MCF-7/TAM cells was significantly up-regulated, compared with MCF-7 cells. After the gefitinib treatment, the expression levels of MEK1/2 and p-ERK1/2 were significantly down-regulated. ERα loss is the primary cause for TAM resistance. Gefitinib reverses TAM resistance primarily by up-regulating the ERα mRNA level and inducing the re-expression of ERα. The MAPK pathway plays a key role in ERα re-expression.

Figure 1. Dose curve and time course of the effect of Gefitinib on the proliferation of MCF-7 and MCF-7/TAM cells.

(A): MCF-7 and MCF-7/TAM cells were treated with different doses of Gefitinib. The absorbance values determined at 492 nm in an ELISA analyser demonstrated that Gefitinib at different concentration gradients exhibited differing degrees of inhibition on cell proliferation. (B): The absorbance values of MCF-7/TAM cells treated with different concentrations of Gefitinib for 24 h or 48 h. Effects on MCF-7 and MCF-7/TAM cell proliferation in different time and different doses of Gefitinib for detection of CCK-8. (C): the effect of different doses of Gefitinib to inhibit the proliferation of M0 and M/T group, there showed inhibitory concentrations of Gefitinib in two kinds of cells for the proliferation. (D): different concentrations of Gefitinib changes in the range of 24 h, 48 h absorbance in group M/T cells. The experiments consisted of the following groups: the MCF-7 group (M0), the MCF-7/TAM group (M/T), and the MCF-7/TAM-Gefitinib (10 μg/ml) group (G10). All the experiments were repeated three times.

Figure 2. Gefitinib enhances the sensitivity of MCF-7/TAM cells to TAM.

The inhibitory effects of 10 μg/mL Gefitinib, 1.5 μmol/L TAM, or 10 μg/mL Gefitinib plus 1.5 μmol/L TAM on MCF-7/TAM cell proliferation were evaluated. The data revealed that treatment with Gefitinib re-sensitisedMCF-7/TAM cells to TAM and inhibited cell proliferation. All the experiments were repeated three times.* denotes p < 0.05.

Figure 3. RT-PCR results for the ERα and ERβ mRNA levels in MCF-7/TAM cells.

(A): RT-PCR result for the ERα mRNA level. (B): Histogram of the ERα mRNA level. (C): RT-PCR result for the ERβ mRNA level. (D): Histogram of the ERβ mRNA level. All the experiments were repeated three times. * denotes p < 0.05.

Figure 4. Immunocytochemical staining for ERα and ERβ expression in cells.

(A): Sections 1a–1c represent ERα expression in the M0, M/T, and G10 groups, respectively. Sections 2a–2c represent ERβ expression in the M0, M/T, and G10 groups, respectively. ERα expression in cells of the M/T group was rarely observed. ERα was re-expressed in cells of the G10 group after treatment with 10 μg/mL Gefitinibfor 48 h. The ERβ expression in cells of the M/T group was also slightly down-regulated. These data were obtained from 3 independent experiments (DAB staining, ×400). (B): The expression of ERα in M/T group were significantly reduced than M0 group, with10 μg/ml Gefitini treatment for 48 h, the change of ERβ protein expression level is not obvious. All the experiments were repeated three times. *denotes p < 0.05.

Figure 5. Western blot results for the expression of molecules in the MAPK pathway.

(A): The expression of Ras, MEK1/2, and p-ERK1/2 in the M/T group was significantly enhanced compared with the expression levels detected in the M0 group. Treatment with 10 μg/mL Gefitinib for 48 h significantly reduced the expression of Ras, MEK1/2, and p-ERK1/2 compared with the expression levels detected in the M/T group. (B): Histograms of protein expression for Ras, MEK1/2, and p-ERK1/2. All the experiments were repeated three times.*denotes p < 0.05.