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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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Effects of GCS on P-gp in NCI/ADR-RES Transfectants. (A) GCS and P-gp proteins detected by Western blot. Detergent-soluble protein (50 μg/lane) from NCI/ADR-RES (ADR-RES), NCI-ADR-RES/GCS (GCS) and NCI/ADR-RES/asGCS (asGCS) cells was immunoblotted with anti-GCS or anti-P-gp antibody. GAPDH was used as loading control. (B) Ceramide glycosylation catalyzed by GCS. Cells were incubated with NBD C6-Cer (100 nM) in 1% BSA RPMI-1640 medium, at 37°C for 2 hr. C6-Cer and C6-GlcCer were identified on chromatograms with commercial standard (St.) and measured using spectrophotometry. *, p < 0.001 compared to ADR-RES cells. (C) Immunostaining of GCS and P-gp. Cells were incubated with anti-human GCS (green) and anti-P-gp (red) following addition of Alexa 488- and Alexa 667-conjugated secondary antibodies. DAPI in mounting solution was used for nucleus counterstaining (blue). Ctrl, NCI/ADR-RES cells were incubated with the secondary antibodies alone, as specificity control; Fluo, merged fluorescence microphotograph (× 200). (D) Doxorubicin accumulation. After 1 hr incubation with doxorubicin (0.1 mg/ml), cellular doxorubicin was documented by fluorescence microscopy (× 200) and analyzed by HPLC, following methanol extraction. Doxorubicin amount was normalized to 100,000 cells. *, p < 0.001.
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Figure 1: Effects of GCS on P-gp in NCI/ADR-RES Transfectants. (A) GCS and P-gp proteins detected by Western blot. Detergent-soluble protein (50 μg/lane) from NCI/ADR-RES (ADR-RES), NCI-ADR-RES/GCS (GCS) and NCI/ADR-RES/asGCS (asGCS) cells was immunoblotted with anti-GCS or anti-P-gp antibody. GAPDH was used as loading control. (B) Ceramide glycosylation catalyzed by GCS. Cells were incubated with NBD C6-Cer (100 nM) in 1% BSA RPMI-1640 medium, at 37°C for 2 hr. C6-Cer and C6-GlcCer were identified on chromatograms with commercial standard (St.) and measured using spectrophotometry. *, p < 0.001 compared to ADR-RES cells. (C) Immunostaining of GCS and P-gp. Cells were incubated with anti-human GCS (green) and anti-P-gp (red) following addition of Alexa 488- and Alexa 667-conjugated secondary antibodies. DAPI in mounting solution was used for nucleus counterstaining (blue). Ctrl, NCI/ADR-RES cells were incubated with the secondary antibodies alone, as specificity control; Fluo, merged fluorescence microphotograph (× 200). (D) Doxorubicin accumulation. After 1 hr incubation with doxorubicin (0.1 mg/ml), cellular doxorubicin was documented by fluorescence microscopy (× 200) and analyzed by HPLC, following methanol extraction. Doxorubicin amount was normalized to 100,000 cells. *, p < 0.001.

Mentions: We observed the role of GCS in the regulation of MDR1 expression in NCI/ADR-RES and its GCS transfectants. GCS protein levels were increased in NCI/ADR-RES/GCS cells and significantly decreased in NCI/ADR-RES/asGCS cells (Figure 1A). Consistent with these, GCS enzyme activity was decreased to 52% (0.9 vs. 1.7 GC/Cer) in NCI/ADR-RES/asGCS cells, whereas the activity was increased to 110% (1.9 vs. 1.7 GC/Cer) in NCI/ADR-RES/GCS, as compared with parental NCI/ADR-RES cells (Figure 1A,B). Coordinately, P-gp was significantly decreased to 40% in NCI/ADR-RES/asGCS cells, as detected by Western blotting and immunostaining (Figure 1A,C). HPLC assays indicated that cellular accumulation of doxorubicin was increased by 3-fold in NCI/ADR-RES/asGCS cells (2.54 ± 0.15 vs. 0.80 ± 0.51 ng/105 cells) as compared with NCI/ADR-RES cells (Figure 1D). Substantial intracellular accumulation of doxorubicin was also observed in NCI/ADR-RES/asGCS cells under fluorescence microscopy (Figure 1D).


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)

Effects of GCS on P-gp in NCI/ADR-RES Transfectants. (A) GCS and P-gp proteins detected by Western blot. Detergent-soluble protein (50 μg/lane) from NCI/ADR-RES (ADR-RES), NCI-ADR-RES/GCS (GCS) and NCI/ADR-RES/asGCS (asGCS) cells was immunoblotted with anti-GCS or anti-P-gp antibody. GAPDH was used as loading control. (B) Ceramide glycosylation catalyzed by GCS. Cells were incubated with NBD C6-Cer (100 nM) in 1% BSA RPMI-1640 medium, at 37°C for 2 hr. C6-Cer and C6-GlcCer were identified on chromatograms with commercial standard (St.) and measured using spectrophotometry. *, p < 0.001 compared to ADR-RES cells. (C) Immunostaining of GCS and P-gp. Cells were incubated with anti-human GCS (green) and anti-P-gp (red) following addition of Alexa 488- and Alexa 667-conjugated secondary antibodies. DAPI in mounting solution was used for nucleus counterstaining (blue). Ctrl, NCI/ADR-RES cells were incubated with the secondary antibodies alone, as specificity control; Fluo, merged fluorescence microphotograph (× 200). (D) Doxorubicin accumulation. After 1 hr incubation with doxorubicin (0.1 mg/ml), cellular doxorubicin was documented by fluorescence microscopy (× 200) and analyzed by HPLC, following methanol extraction. Doxorubicin amount was normalized to 100,000 cells. *, p < 0.001.
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Figure 1: Effects of GCS on P-gp in NCI/ADR-RES Transfectants. (A) GCS and P-gp proteins detected by Western blot. Detergent-soluble protein (50 μg/lane) from NCI/ADR-RES (ADR-RES), NCI-ADR-RES/GCS (GCS) and NCI/ADR-RES/asGCS (asGCS) cells was immunoblotted with anti-GCS or anti-P-gp antibody. GAPDH was used as loading control. (B) Ceramide glycosylation catalyzed by GCS. Cells were incubated with NBD C6-Cer (100 nM) in 1% BSA RPMI-1640 medium, at 37°C for 2 hr. C6-Cer and C6-GlcCer were identified on chromatograms with commercial standard (St.) and measured using spectrophotometry. *, p < 0.001 compared to ADR-RES cells. (C) Immunostaining of GCS and P-gp. Cells were incubated with anti-human GCS (green) and anti-P-gp (red) following addition of Alexa 488- and Alexa 667-conjugated secondary antibodies. DAPI in mounting solution was used for nucleus counterstaining (blue). Ctrl, NCI/ADR-RES cells were incubated with the secondary antibodies alone, as specificity control; Fluo, merged fluorescence microphotograph (× 200). (D) Doxorubicin accumulation. After 1 hr incubation with doxorubicin (0.1 mg/ml), cellular doxorubicin was documented by fluorescence microscopy (× 200) and analyzed by HPLC, following methanol extraction. Doxorubicin amount was normalized to 100,000 cells. *, p < 0.001.
Mentions: We observed the role of GCS in the regulation of MDR1 expression in NCI/ADR-RES and its GCS transfectants. GCS protein levels were increased in NCI/ADR-RES/GCS cells and significantly decreased in NCI/ADR-RES/asGCS cells (Figure 1A). Consistent with these, GCS enzyme activity was decreased to 52% (0.9 vs. 1.7 GC/Cer) in NCI/ADR-RES/asGCS cells, whereas the activity was increased to 110% (1.9 vs. 1.7 GC/Cer) in NCI/ADR-RES/GCS, as compared with parental NCI/ADR-RES cells (Figure 1A,B). Coordinately, P-gp was significantly decreased to 40% in NCI/ADR-RES/asGCS cells, as detected by Western blotting and immunostaining (Figure 1A,C). HPLC assays indicated that cellular accumulation of doxorubicin was increased by 3-fold in NCI/ADR-RES/asGCS cells (2.54 ± 0.15 vs. 0.80 ± 0.51 ng/105 cells) as compared with NCI/ADR-RES cells (Figure 1D). Substantial intracellular accumulation of doxorubicin was also observed in NCI/ADR-RES/asGCS cells under fluorescence microscopy (Figure 1D).

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