| Ceramide, the central molecule of sphingolipids, mediates apoptotic responses in cancer cells by regulating expression of genes by phosphorylation, acetylation or methylation of proteins that serve as regulators of transcription factors and pre-mRNA alternative splicing Ceramide is converted to higher glycosphingolipids which promote cellular processes like proliferation, tumor metastasis, stem cells generation and drug resistance. Drug resistance in cancers blocks the cellular actions of chemotherapy. Glucosylceramide synthase (GCS), a key enzyme catalyzing ceramide glycosylation, is overexpressed in various drug-resistant cancer cell lines and in metastatic tumors. To determine whether GCS is a target for the reversal of drug resistance, we evaluated effects of antisense approach against GCS in drug-resistant models in vitro and in vivo. MBO-asGCS (mixed backbone anti-GCS oligonucleotide; 50 nM)) significantly increased doxorubicin sensitivity by 83-fold in drug-resistant human ovarian cancer NCI/ADR-RES cells and by 43-fold in drug-resistant murine breast cancer EMT6/AR1 cells. In the in vivo studies, administration of MBO-asGCS (1 mg/kg/3-day, intratumoral injection for 42 days) inhibited tumor growth more than 64% and increased doxorubicin-sensitivity by 58%, compared to MBO-scrambled (SC) alone or with doxorubicin (2 mg/kg/week, i.p. injection for 42 days). Further assessments of quantitative PCR, Western blot, ceramide glycosylation, caspases and apoptosis indicated that the effects of MBO-asGCS rely on the suppression of endogenous GCS expression and the enhancement of ceramide-induced apoptosis. Also, it is reported that ceramide induces p53-mediated apoptosis. Mutation of tumor suppressor p53 is frequently detected in many different types of cancers. Restoration of p53 that is to increase the expression of wild-type p53 and to eliminate mutant expression can substantially increase the efficacy of chemotherapy and stop malignant progression. We, for the first time, report that GCS suppression restores p53 dependent-apoptosis in p53 mutant drug-resistant cancer cells. A lose of a 21 base-pair in the mRNA of exon-5 can result in the accumulation of mutant p53 and cells insensitiveness to induced- apoptosis in NCI/ADR-RES cells. We found that treatment of MBO-asGCS increased the levels of wild-type (wt) p53 mRNA and protein in dose-dependent manner (50-200 nM). In response to doxorubicin-induced DNA damage, phosphorylated p53 (Ser 15) was significantly increased, that in turn increased the protein levels of p21 and Box, and apoptosis (52% vs. 3%) after MBO-asGCS treatment as compared with controls. Silencing of GCS using MBO-asGCS (1 mg/kg) restored wt p53 expression and substantially sensitized NCI/ADR-RES tumors to doxorubicin-induced apoptosis in vivo. Increasing endogenous ceramide by inhibition of its glycosylation using PDMP or enhanced ceramide production using sphingomylinase promoted wt p53 expression at the levels of mRNA, protein and its phosphorylation; decreasing ceramide by inhibition of ceramide synthesis using fumonisin (FBI) repressed wt p53 expression. Further mechanic studies indicated that increased ceramide modulated the function of PP1-dependent SRp30a and thus regulated alternative pre-mRNA splicing. Present study indicates that cancer cells marked with mutant p53 phenotype are capable of expressing functional p53; ceramide regulates expression of wt form of p53 at post-transcription processing. |
