ER alpha negative breast cancer cells restore response to endocrine therapy by combination treatment with both HDAC inhibitor and DNMT inhibitor

Abstract

Purpose: Estrogen receptor alpha (ER alpha) mediates the growth stimulation of estrogen in breast cancer cells and is a useful predictive factor for response to endocrine therapy. It is reported that ER alpha was induced in ER alpha negative breast cancer cells by both DNA methyltransferase-1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (AZA) and histone deacetylase (HDAC) inhibitor trichostatin A (TSA). However, whether the breast cancer cells with induced ER alpha restore response to endocrine therapy requires to be further researched.

Patients and methods: Reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to explore the change in the mRNA of ER alpha, PR and pS2 in the ER alpha negative breast cancer cells MDA-MB-435 treated with two chemicals (AZA + TSA). Water-soluble tetrazolium salt-8 (WST-8) method was used to study the proliferation rate of the breast cancer cells. Flow cytometer (FCW) was used to analyze the distribution of cell cycle of these breast cancer cells. Some xenograft models in nude mice were used to further study the results we found in vitro.

Results: In this study we observed that the mRNA of ER alpha, PR and pS2 in the ER alpha negative breast cancer cells MDA-MB-435 was re-expressed by treatment with AZA + TSA. The proliferation assay analysis showed AZA + TSA suppressed the proliferation of MDA-MB-435 cells, which were further suppressed by addition of 4-OH Tamoxifen (4-OHT). On the contrary, the proliferation of cells treated with 4-OHT alone showed no difference compared with the vehicle control. Cell cycle analysis showed AZA + TSA treated cells showed S phase arrest, which was partially attenuated by addition of estradiol (E2); furthermore, the effect of E2 on stimulation of cell cycle could be reversed by 4-OHT in the treated cells with induced ER alpha. In vivo experiment xenograft volume of MDA-MB-435 cells treated with AZA + TSA was smaller than that of the control (P < 0.01), and the xenograft of AZA + TSA treated cells was further suppressed by ovariectomy (P < 0.01).

Conclusions: Our data indicate that DNMT1 inhibitor AZA and HDAC inhibitor TSA play important roles in restoring sensitivity of the ER alpha negative breast cancer cells to endocrine therapy in vitro and in vivo.

Fig. 1

mRNA expression of ERα, PR and pS2 in MDA-MB-435 cells with different treatment and in MCF-7 cells using RT-PCR. a MDA-MB-435 cells were treated as follows: 1 vehicle control; 2 2.5 μmol/L AZA for 96 h, 100 ng/mL TSA added only during the last 12 h; 3 5.0 μmol/L AZA for 96 h, 100 ng/mL TSA added only during the last 12 h. MCF-7 cells were as positive control. b The quantification was obtained by densitometry analysis using NIH Image

Fig. 2

Growth inhibition effects of indicated chemicals on MDA-MB-435 cells using the proliferation assay (WST-8 method). Means and standard errors (SE) of the four groups were shown in form of columns and error bars, respectively. a Compared with vehicle control and tamoxifen treatment, AZA + TSA inhibited MDA-MB-435 cells growth and further inhibition was observed by addition of tamoxifen. b Growth of MDA-MB-435 treated with AZA and TSA at indicated concentrations with or without 4-OHT. The concentration of AZA was increased gradually

Fig. 3

The effects of indicated chemicals on cell cycle distribution in MDA-MB-435 cells for indicated hours. a Flow cytometric histograms of MDA-MB-435 cells treated with indicated chemicals. b Representative quantitation of the distribution of the stages in these experimental cells. Means and standard errors (SE) of the three groups were shown in form of columns and error bars, respectively

Fig. 4

Xenograft of MDA-MB-435 cells with or without AZA + TSA treatment in female nude mice with or without ovarian ablation (OA). a The xenografts of the treated cells in three groups: MDA-MB-435 in the nude mice without OA; MDA-MB-435 treated with 2.5 μmol/L AZA and 100 ng/μL TSA in the nude mice without OA; MDA-MB-435 treated with 2.5 μmol/L AZA and 100 ng/μL TSA in the nude mice which had underwent OA. b Representative quantitation of the volume of these xenografts. The error bars represent the standard errors