Altered methylation of glucosylceramide synthase promoter regulates its expression and associates with acquired multidrug resistance in invasive ductal breast cancer

Jiannan Liu,Ying Liu,Daoping Zhang,Xiangshuo Kong,Yi Su,Xiaodan Wang,Aina Liu,Dong You,Leilei Yuan,Ping Sun,Xiaofang Zhang

doi:10.18632/oncotarget.9337

Abstract

Overexpression of glucosylceramide synthase (GCS) increases multidrug resistance (MDR) in many cancer cells. However, its mechanism is unknown. The aim of the present study is to detect the association of methylation at the GCS gene promoter with its expression and MDR in invasive ductal breast cancer. 40 cases GCS-positive and 40 cases GCS-negative primary breast carcinoma samples, three drug-sensitive breast cancer cell lines and one multidrug-resistant breast cancer cell line were used. Immunohistochemistry, methylation-specific PCR (MSP), quantitative real-time (qPCR), westernblot and cytotoxicity assay techniques were employed. Thwe results revealed that there was a statistically negative correlation between GCS CpG islands methylation and GCS phenotype in patients with breast cancer. GCS CpG islands methylation was negatively associated with high ER, meanwhile positively with high HER-2 status. Similar results were obtained from the analysis of breast cancer cell lines. Treatment with the demethylating agent 5-aza-2′-deoxycytidine (5-Aza-dc) changed the GCS promoter methylation pattern in three sensitive cells and also caused increased drug resistance of them. These results suggested that the changes of DNA methylation status of the GCS promoter correlates with multidrug resistance in breast cancer.

Highlights

IntroductionThe leading cause of death among women in most countries worldwide, is rapidly increasing in China [1, 2]
Breast cancer, the leading cause of death among women in most countries worldwide, is rapidly increasing in China [1, 2]
Treatment with the demethylating agent 5-aza-2′-deoxycytidine (5-Aza-dc) changed the glucosylceramide synthase (GCS) promoter methylation pattern in three sensitive cells and caused increased drug resistance of them. These results suggested that the changes of DNA methylation status of the GCS promoter correlates with multidrug resistance in breast cancer

Summary

Introduction

The leading cause of death among women in most countries worldwide, is rapidly increasing in China [1, 2]. Multidrug resistance (MDR) remains to be a serious obstacle in breast cancer treatment [3, 4]. Accumulating evidence has indicated the important role of glucosylceramide synthase (GCS) in MDR [5, 6]. GCS is a transmembrane protein encoded by the UGCG gene in humans. It can transfer UDP-glucose to ceramide to form glucosylceramide, and allow cells to escape from ceramide-induced cellular apoptosis [7, 8]. Liu et al introduced GCS cDNA into MCF-7 cells, which increased

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