Targeting glucosylceramide synthase downregulates expression of the multidrug resistance gene MDR1 and sensitizes breast carcinoma cells to anticancer drugs

Abstract

Drug resistance in breast cancer remains a major cause for the failure of chemotherapy. Glucosylceramide synthase (GCS) plays an important role in multidrug resistance (MDR) in breast cancer. P-glycoprotein (P-gp) also confers a cross-resistance of many unrelated drugs. In this study, we studied the MDR effect and potential mechanisms of breast cancer after constructing permanent breast cancer cell lines with GCS knockout by using recombinant vectors targeting GCS (pSUPER-GCSshRNAs). The GCSshRNA stably transfected cells were successfully established and significant lower levels of GCS mRNA and protein expression were confirmed. In in vitro experiments, the GCSshRNA stably transfected cells showed a significantly reduced level of MDR1 and P-gp expression and decreased drug efflux ability. Reduced level of GCS expression conveyed a significant reversal of drug resistance by MTT assay and increased caspase-3 activity. In in vivo experiments by using nude mice with xenograft tumors, a significant inhibition of tumor growth was observed after comparing with the control group. Furthermore, enhanced response of chemotherapy was acquired by reduced expression of GCS as well as MDR1 in vivo. In conclusion, GCSshRNA could efficiently suppress GCS and MDR1 expression in vitro and in vivo and these findings may be used as one of the methods to reverse MDR in breast cancer.

Ceramide Glycosylation Catalyzed by Glucosylceramide Synthase and Cancer Drug Resistance

“…Oligonucleotides (20-mer) that target the open reading frame of GCS (ORF 18–37), as in a phosphorothioate DNA (Liu et al, 2004; Liu et al, 2008), or a 2′- O -methyl RNA with phosphorothioate DNA (mixed backbone oligonucleotide, MBO-asGCS) (Liu et al, 2010b; Patwardhan et al, 2009), sensitize drug cytotoxicity in resistant human NCI/ADR-RES, A2780AD, KB-A1, SW620/AD, and murine EMT6/AR1 cancer cells. RNA interference by treatment of siRNA duplex (Gouaze et al, 2005) or vector-mediated transfection of short hairpin DNA (pSUPER-GCSshRNA) (Liu et al, 2010a; Sun et al, 2010b; Sun et al, 2006; Zhang et al, 2009; Zhang et al, 2011) sensitizes drug-resistant human breast cancer cells and leukemia cells; however, these siRNA agents have not yet been tested in vivo . MBO-asGCS directly administered in animal models has relatively higher uptake by tumors than other tissues, and less nonspecific toxicity (Patwardhan et al, 2009).…”

Therapeutic potential of targeting ceramide/glucosylceramide pathway in cancer

“…Furthermore, these oligonucleotides decrease the size of the tumors by increasing the levels of C18:ceramide and apoptosis mediated by caspases [67]. In breast cancer, in vivo and in vitro suppression of GCS by GCS siRNA also decreases P-gp expression and the tumor size; therefore, it reverses multidrug resistance [68]. Transfection of adriamycin-resistant MCF-7/ADM human breast cancer cells with both GCS and MDR1 siRNAs results in significant, and more importantly, efficient reversal of multidrug resistance [69].…”

C6-Ceramide and targeted inhibition of acid ceramidase induce synergistic decreases in breast cancer cell growth

“…Targeting these enzymes blocks the tumor cell's ability to metabolize ceramide to less toxic and/or pro-survival intermediates. Reported antitumor effects resulting from targeted inhibition of ceramide metabolizing enzymes include induction of apoptosis [16,17], growth inhibition [17][18][19], and increased sensitivity to radiation [20] and chemotherapeutics [21,22]. Use of exogenous, cell-permeable ceramide analogs, C6-and C8-cer, has also demonstrated promising anti-cancer activity in preclinical studies [23][24][25][26][27].…”