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Glucosylceramide synthase upregulates MDR1 expression in the regulation of cancer drug resistance through cSrc and beta-catenin signaling.

Liu YY, Gupta V, Patwardhan GA, Bhinge K, Zhao Y, Bao J, Mehendale H, Cabot MC, Li YT, Jazwinski SM - Mol. Cancer (2010)

Bottom Line: The expression of P-glycoprotein and the function of its drug efflux of tumors were decreased by 4 and 8 times after MBO-asGCS treatment, even though this treatment did not have a significant effect on P-glycoprotein in normal small intestine.GSL profiling, silencing of globotriaosylceramide synthase and assessment of signaling pathway indicated that GCS transfection significantly increased globo series GSLs (globotriaosylceramide Gb3, globotetraosylceramide Gb4) on GSL-enriched microdomain (GEM), activated cSrc kinase, decreased beta-catenin phosphorylation, and increased nuclear beta-catenin.Conversely, MBO-asGCS treatments decreased globo series GSLs (Gb3, Gb4), cSrc kinase and nuclear beta-catenin, and suppressed MDR-1 expression in dose-dependent pattern.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Basic Pharmaceutical Sciences, University of Louisiana at Monroe, Monroe, Louisiana 71209, USA. yliu@ulm.edu

ABSTRACT

Background: Drug resistance is the outcome of multiple-gene interactions in cancer cells under stress of anticancer agents. MDR1 overexpression is most commonly detected in drug-resistant cancers and accompanied with other gene alterations including enhanced glucosylceramide synthase (GCS). MDR1 encodes for P-glycoprotein that extrudes anticancer drugs. Polymorphisms of MDR1 disrupt the effects of P-glycoprotein antagonists and limit the success of drug resistance reversal in clinical trials. GCS converts ceramide to glucosylceramide, reducing the impact of ceramide-induced apoptosis and increasing glycosphingolipid (GSL) synthesis. Understanding the molecular mechanisms underlying MDR1 overexpression and how it interacts with GCS may find effective approaches to reverse drug resistance.

Results: MDR1 and GCS were coincidently overexpressed in drug-resistant breast, ovary, cervical and colon cancer cells; silencing GCS using a novel mixed-backbone oligonucleotide (MBO-asGCS) sensitized these four drug-resistant cell lines to doxorubicin. This sensitization was correlated with the decreased MDR1 expression and the increased doxorubicin accumulation. Doxorubicin treatment induced GCS and MDR1 expression in tumors, but MBO-asGCS treatment eliminated "in-vivo" growth of drug-resistant tumor (NCI/ADR-RES). MBO-asGCS suppressed the expression of MDR1 with GCS and sensitized NCI/ADR-RES tumor to doxorubicin. The expression of P-glycoprotein and the function of its drug efflux of tumors were decreased by 4 and 8 times after MBO-asGCS treatment, even though this treatment did not have a significant effect on P-glycoprotein in normal small intestine. GCS transient transfection induced MDR1 overexpression and increased P-glycoprotein efflux in dose-dependent fashion in OVCAR-8 cancer cells. GSL profiling, silencing of globotriaosylceramide synthase and assessment of signaling pathway indicated that GCS transfection significantly increased globo series GSLs (globotriaosylceramide Gb3, globotetraosylceramide Gb4) on GSL-enriched microdomain (GEM), activated cSrc kinase, decreased beta-catenin phosphorylation, and increased nuclear beta-catenin. These consequently increased MDR1 promoter activation and its expression. Conversely, MBO-asGCS treatments decreased globo series GSLs (Gb3, Gb4), cSrc kinase and nuclear beta-catenin, and suppressed MDR-1 expression in dose-dependent pattern.

Conclusion: This study demonstrates, for the first time, that GCS upregulates MDR1 expression modulating drug resistance of cancer. GSLs, in particular globo series GSLs mediate gene expression of MDR1 through cSrc and beta-catenin signaling pathway.

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Silencing GCS Represses MDR1 Expression by Decreasing cSrc/β-Catenin Signaling. After MBO-asGCS treatments (0, 50, 100, 200 nM), drug resistant NCI/ADR-RES cells were cultured in 10% FBS RPMI-1640 medium for 7 days. The NCI/ADR-RES cells were incubated with verapamil (10 μg, 2 hr) in 5% FBS RPMI-1640 medium to inhibit P-gp function. (A) Western blots. Equal amounts of total cellular proteins or nuclear proteins (50 μg/lane) were resolved by 4-20% gradient SDS-PAGE and immunoblotted with indicated primary antibodies. GD3 syn, GD3 synthase; Gb3 syn, Gb3 synthase; p-cSrc, phosphorylated cSrc; p-FAK, phosphorylated FAK; p-β-catenin, phosphorylated β-catenin. (B) MDR1 expression. MDR1 promoter activity (top panel) and P-gp protein (bottom panel) were assessed as described in Methods, after 7 days of MBO-asGCS treatments. *, p < 0.001 compared with vehicle. (C) Paclitaxel accumulation and efflux. Cells were incubated with Flutax-2 (0.5 μM) in medium at 37°C for 2 hr to measure paclitaxel accumulation (top panel). After washing with ice-cold PBS, cells were incubated with fresh medium for an additional 2 hr to measure paclitaxel efflux (bottom panel). *, p < 0.001 compared with vehicle treatment.
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Figure 6: Silencing GCS Represses MDR1 Expression by Decreasing cSrc/β-Catenin Signaling. After MBO-asGCS treatments (0, 50, 100, 200 nM), drug resistant NCI/ADR-RES cells were cultured in 10% FBS RPMI-1640 medium for 7 days. The NCI/ADR-RES cells were incubated with verapamil (10 μg, 2 hr) in 5% FBS RPMI-1640 medium to inhibit P-gp function. (A) Western blots. Equal amounts of total cellular proteins or nuclear proteins (50 μg/lane) were resolved by 4-20% gradient SDS-PAGE and immunoblotted with indicated primary antibodies. GD3 syn, GD3 synthase; Gb3 syn, Gb3 synthase; p-cSrc, phosphorylated cSrc; p-FAK, phosphorylated FAK; p-β-catenin, phosphorylated β-catenin. (B) MDR1 expression. MDR1 promoter activity (top panel) and P-gp protein (bottom panel) were assessed as described in Methods, after 7 days of MBO-asGCS treatments. *, p < 0.001 compared with vehicle. (C) Paclitaxel accumulation and efflux. Cells were incubated with Flutax-2 (0.5 μM) in medium at 37°C for 2 hr to measure paclitaxel accumulation (top panel). After washing with ice-cold PBS, cells were incubated with fresh medium for an additional 2 hr to measure paclitaxel efflux (bottom panel). *, p < 0.001 compared with vehicle treatment.

Mentions: We silenced GCS with MBO-asGCS in NCI/ADR-RES cells that overexpressed GCS and MDR1, to verify the mechanism underlying GCS modulation of MDR1. After one week of MBO-asGCS treatments, GCS protein levels, but neither GD3 synthase nor Gb3 synthase, were decreased to 30%, 5% and 2% in cells treated with increasing concentrations of MBO-asGCS (50, 100, 200 nM), as compared with vehicle control, respectively (Figure 6A). The levels of the phosphorylated cSrc and FAK proteins, but not the total cSrc, were correspondingly decreased with GCS protein levels. Interestingly, nuclear β-catenin was decreased to approximately 10%, whereas phosphorylated β-catenin was increased as the levels of GCS, p-cSrc, and p-FAK were decreased in these cells after MBO-asGCS treatments. P-gp protein levels were decreased to 85%, 30% and 25% with decreases in MDR1 promoter activity of cells treated with increasing concentrations of MBO-asGCS (50, 100, 200 nM) (Figure 6B). Sequentially, we found that cellular accumulation of paclitaxel was elevated by 2.5-fold, 11-fold and 22-fold in cells treated with MBO-asGCS, compared to vehicle control, respectively (Figure 6C). Conversely, cell efflux of paclitaxel was reduced to 89%, 48%, and 31% in cell after these treatments. As anticipated, verapamil treatment (10 μM) inhibited P-gp function, as effectively as MBO-asGCS (50 nM) in the cellular accumulation and efflux of paclitaxel in NCI/ADR-RES cells (Figure 6C).


Glucosylceramide synthase upregulates MDR1 expression in the regulation of cancer drug resistance through cSrc and beta-catenin signaling.

Liu YY, Gupta V, Patwardhan GA, Bhinge K, Zhao Y, Bao J, Mehendale H, Cabot MC, Li YT, Jazwinski SM - Mol. Cancer (2010)

Silencing GCS Represses MDR1 Expression by Decreasing cSrc/β-Catenin Signaling. After MBO-asGCS treatments (0, 50, 100, 200 nM), drug resistant NCI/ADR-RES cells were cultured in 10% FBS RPMI-1640 medium for 7 days. The NCI/ADR-RES cells were incubated with verapamil (10 μg, 2 hr) in 5% FBS RPMI-1640 medium to inhibit P-gp function. (A) Western blots. Equal amounts of total cellular proteins or nuclear proteins (50 μg/lane) were resolved by 4-20% gradient SDS-PAGE and immunoblotted with indicated primary antibodies. GD3 syn, GD3 synthase; Gb3 syn, Gb3 synthase; p-cSrc, phosphorylated cSrc; p-FAK, phosphorylated FAK; p-β-catenin, phosphorylated β-catenin. (B) MDR1 expression. MDR1 promoter activity (top panel) and P-gp protein (bottom panel) were assessed as described in Methods, after 7 days of MBO-asGCS treatments. *, p < 0.001 compared with vehicle. (C) Paclitaxel accumulation and efflux. Cells were incubated with Flutax-2 (0.5 μM) in medium at 37°C for 2 hr to measure paclitaxel accumulation (top panel). After washing with ice-cold PBS, cells were incubated with fresh medium for an additional 2 hr to measure paclitaxel efflux (bottom panel). *, p < 0.001 compared with vehicle treatment.
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Figure 6: Silencing GCS Represses MDR1 Expression by Decreasing cSrc/β-Catenin Signaling. After MBO-asGCS treatments (0, 50, 100, 200 nM), drug resistant NCI/ADR-RES cells were cultured in 10% FBS RPMI-1640 medium for 7 days. The NCI/ADR-RES cells were incubated with verapamil (10 μg, 2 hr) in 5% FBS RPMI-1640 medium to inhibit P-gp function. (A) Western blots. Equal amounts of total cellular proteins or nuclear proteins (50 μg/lane) were resolved by 4-20% gradient SDS-PAGE and immunoblotted with indicated primary antibodies. GD3 syn, GD3 synthase; Gb3 syn, Gb3 synthase; p-cSrc, phosphorylated cSrc; p-FAK, phosphorylated FAK; p-β-catenin, phosphorylated β-catenin. (B) MDR1 expression. MDR1 promoter activity (top panel) and P-gp protein (bottom panel) were assessed as described in Methods, after 7 days of MBO-asGCS treatments. *, p < 0.001 compared with vehicle. (C) Paclitaxel accumulation and efflux. Cells were incubated with Flutax-2 (0.5 μM) in medium at 37°C for 2 hr to measure paclitaxel accumulation (top panel). After washing with ice-cold PBS, cells were incubated with fresh medium for an additional 2 hr to measure paclitaxel efflux (bottom panel). *, p < 0.001 compared with vehicle treatment.
Mentions: We silenced GCS with MBO-asGCS in NCI/ADR-RES cells that overexpressed GCS and MDR1, to verify the mechanism underlying GCS modulation of MDR1. After one week of MBO-asGCS treatments, GCS protein levels, but neither GD3 synthase nor Gb3 synthase, were decreased to 30%, 5% and 2% in cells treated with increasing concentrations of MBO-asGCS (50, 100, 200 nM), as compared with vehicle control, respectively (Figure 6A). The levels of the phosphorylated cSrc and FAK proteins, but not the total cSrc, were correspondingly decreased with GCS protein levels. Interestingly, nuclear β-catenin was decreased to approximately 10%, whereas phosphorylated β-catenin was increased as the levels of GCS, p-cSrc, and p-FAK were decreased in these cells after MBO-asGCS treatments. P-gp protein levels were decreased to 85%, 30% and 25% with decreases in MDR1 promoter activity of cells treated with increasing concentrations of MBO-asGCS (50, 100, 200 nM) (Figure 6B). Sequentially, we found that cellular accumulation of paclitaxel was elevated by 2.5-fold, 11-fold and 22-fold in cells treated with MBO-asGCS, compared to vehicle control, respectively (Figure 6C). Conversely, cell efflux of paclitaxel was reduced to 89%, 48%, and 31% in cell after these treatments. As anticipated, verapamil treatment (10 μM) inhibited P-gp function, as effectively as MBO-asGCS (50 nM) in the cellular accumulation and efflux of paclitaxel in NCI/ADR-RES cells (Figure 6C).

Bottom Line: The expression of P-glycoprotein and the function of its drug efflux of tumors were decreased by 4 and 8 times after MBO-asGCS treatment, even though this treatment did not have a significant effect on P-glycoprotein in normal small intestine.GSL profiling, silencing of globotriaosylceramide synthase and assessment of signaling pathway indicated that GCS transfection significantly increased globo series GSLs (globotriaosylceramide Gb3, globotetraosylceramide Gb4) on GSL-enriched microdomain (GEM), activated cSrc kinase, decreased beta-catenin phosphorylation, and increased nuclear beta-catenin.Conversely, MBO-asGCS treatments decreased globo series GSLs (Gb3, Gb4), cSrc kinase and nuclear beta-catenin, and suppressed MDR-1 expression in dose-dependent pattern.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Basic Pharmaceutical Sciences, University of Louisiana at Monroe, Monroe, Louisiana 71209, USA. yliu@ulm.edu

ABSTRACT

Background: Drug resistance is the outcome of multiple-gene interactions in cancer cells under stress of anticancer agents. MDR1 overexpression is most commonly detected in drug-resistant cancers and accompanied with other gene alterations including enhanced glucosylceramide synthase (GCS). MDR1 encodes for P-glycoprotein that extrudes anticancer drugs. Polymorphisms of MDR1 disrupt the effects of P-glycoprotein antagonists and limit the success of drug resistance reversal in clinical trials. GCS converts ceramide to glucosylceramide, reducing the impact of ceramide-induced apoptosis and increasing glycosphingolipid (GSL) synthesis. Understanding the molecular mechanisms underlying MDR1 overexpression and how it interacts with GCS may find effective approaches to reverse drug resistance.

Results: MDR1 and GCS were coincidently overexpressed in drug-resistant breast, ovary, cervical and colon cancer cells; silencing GCS using a novel mixed-backbone oligonucleotide (MBO-asGCS) sensitized these four drug-resistant cell lines to doxorubicin. This sensitization was correlated with the decreased MDR1 expression and the increased doxorubicin accumulation. Doxorubicin treatment induced GCS and MDR1 expression in tumors, but MBO-asGCS treatment eliminated "in-vivo" growth of drug-resistant tumor (NCI/ADR-RES). MBO-asGCS suppressed the expression of MDR1 with GCS and sensitized NCI/ADR-RES tumor to doxorubicin. The expression of P-glycoprotein and the function of its drug efflux of tumors were decreased by 4 and 8 times after MBO-asGCS treatment, even though this treatment did not have a significant effect on P-glycoprotein in normal small intestine. GCS transient transfection induced MDR1 overexpression and increased P-glycoprotein efflux in dose-dependent fashion in OVCAR-8 cancer cells. GSL profiling, silencing of globotriaosylceramide synthase and assessment of signaling pathway indicated that GCS transfection significantly increased globo series GSLs (globotriaosylceramide Gb3, globotetraosylceramide Gb4) on GSL-enriched microdomain (GEM), activated cSrc kinase, decreased beta-catenin phosphorylation, and increased nuclear beta-catenin. These consequently increased MDR1 promoter activation and its expression. Conversely, MBO-asGCS treatments decreased globo series GSLs (Gb3, Gb4), cSrc kinase and nuclear beta-catenin, and suppressed MDR-1 expression in dose-dependent pattern.

Conclusion: This study demonstrates, for the first time, that GCS upregulates MDR1 expression modulating drug resistance of cancer. GSLs, in particular globo series GSLs mediate gene expression of MDR1 through cSrc and beta-catenin signaling pathway.

Show MeSH
Related in: MedlinePlus