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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.