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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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Gb3 Synthesis and β‐Catenin Recruitment Are Involved in MDR1 Transactivation. To silence Gb3 synthase, cells were transfected with siRNA-Gb3S (100 nM) or control siRNA (siRNA-SC) twice and grown in 10% FBS RPMI-1640 medium for 7 days. (A) MDR1 promoter activity. *, P < 0.001 compared with siRNA-SC. (B) Western blot. Gb3 syn, Gb3 synthase; p-cSrc, phosphorylated cSrc. (C) Cellular efflux. *, p < 0.001 compared with siRNA-SC. (D) Immunostaining. Cells were incubated with anti-human Gb3 synthase (red) and anti-P-gp (green) following addition of Alexa 667- and Alexa 488-conjugated secondary antibodies. DAPI in mounting solution was used for nucleus counterstaining (blue). Fluo., merged fluorescence microphotograph (x 200). (E) β-catenin/Tcf4 on P-gp expression. NCI/ADR-RES cells were exposed to FH535, β-catenin/Tcf4 inhibitor in 5% FBS medium for 24 hr.
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Figure 8: Gb3 Synthesis and β‐Catenin Recruitment Are Involved in MDR1 Transactivation. To silence Gb3 synthase, cells were transfected with siRNA-Gb3S (100 nM) or control siRNA (siRNA-SC) twice and grown in 10% FBS RPMI-1640 medium for 7 days. (A) MDR1 promoter activity. *, P < 0.001 compared with siRNA-SC. (B) Western blot. Gb3 syn, Gb3 synthase; p-cSrc, phosphorylated cSrc. (C) Cellular efflux. *, p < 0.001 compared with siRNA-SC. (D) Immunostaining. Cells were incubated with anti-human Gb3 synthase (red) and anti-P-gp (green) following addition of Alexa 667- and Alexa 488-conjugated secondary antibodies. DAPI in mounting solution was used for nucleus counterstaining (blue). Fluo., merged fluorescence microphotograph (x 200). (E) β-catenin/Tcf4 on P-gp expression. NCI/ADR-RES cells were exposed to FH535, β-catenin/Tcf4 inhibitor in 5% FBS medium for 24 hr.

Mentions: In order to characterize the role of GSLs in MDR1 expression, we selectively silenced the enzyme responsible for the synthesis of globo series GSLs. Cells were transfected with siRNA against Gb3 synthase to block globo series GSL production. As shown in Figure 8A, silencing Gb3 synthase significantly decreased MDR1 promoter activity, particularly in NCI/ADR-RES/GCS cells (p < 0.001, compared with NCI/ADR-RES cells). Consistently, silencing of Gb3 synthase considerably decreased p-cSrc, β-catenin and P-gp protein levels and efflux in both cell lines, as detected in Western blot (Figure 8B), cellular efflux (Figure 8C) and immunostaining (Figure 8D). We further treated NCI/ADR-RES cells with FH535, inhibiting β-catenin recruitment to T-cell factor (Tcf) [45]. We found that FH535 (20 μM) decreased P-gp protein to 25% of control (Figure 8E); however, it did not affect either Gb3 synthase or p-cSrc in Western blotting.


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)

Gb3 Synthesis and β‐Catenin Recruitment Are Involved in MDR1 Transactivation. To silence Gb3 synthase, cells were transfected with siRNA-Gb3S (100 nM) or control siRNA (siRNA-SC) twice and grown in 10% FBS RPMI-1640 medium for 7 days. (A) MDR1 promoter activity. *, P < 0.001 compared with siRNA-SC. (B) Western blot. Gb3 syn, Gb3 synthase; p-cSrc, phosphorylated cSrc. (C) Cellular efflux. *, p < 0.001 compared with siRNA-SC. (D) Immunostaining. Cells were incubated with anti-human Gb3 synthase (red) and anti-P-gp (green) following addition of Alexa 667- and Alexa 488-conjugated secondary antibodies. DAPI in mounting solution was used for nucleus counterstaining (blue). Fluo., merged fluorescence microphotograph (x 200). (E) β-catenin/Tcf4 on P-gp expression. NCI/ADR-RES cells were exposed to FH535, β-catenin/Tcf4 inhibitor in 5% FBS medium for 24 hr.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2903501&req=5

Figure 8: Gb3 Synthesis and β‐Catenin Recruitment Are Involved in MDR1 Transactivation. To silence Gb3 synthase, cells were transfected with siRNA-Gb3S (100 nM) or control siRNA (siRNA-SC) twice and grown in 10% FBS RPMI-1640 medium for 7 days. (A) MDR1 promoter activity. *, P < 0.001 compared with siRNA-SC. (B) Western blot. Gb3 syn, Gb3 synthase; p-cSrc, phosphorylated cSrc. (C) Cellular efflux. *, p < 0.001 compared with siRNA-SC. (D) Immunostaining. Cells were incubated with anti-human Gb3 synthase (red) and anti-P-gp (green) following addition of Alexa 667- and Alexa 488-conjugated secondary antibodies. DAPI in mounting solution was used for nucleus counterstaining (blue). Fluo., merged fluorescence microphotograph (x 200). (E) β-catenin/Tcf4 on P-gp expression. NCI/ADR-RES cells were exposed to FH535, β-catenin/Tcf4 inhibitor in 5% FBS medium for 24 hr.
Mentions: In order to characterize the role of GSLs in MDR1 expression, we selectively silenced the enzyme responsible for the synthesis of globo series GSLs. Cells were transfected with siRNA against Gb3 synthase to block globo series GSL production. As shown in Figure 8A, silencing Gb3 synthase significantly decreased MDR1 promoter activity, particularly in NCI/ADR-RES/GCS cells (p < 0.001, compared with NCI/ADR-RES cells). Consistently, silencing of Gb3 synthase considerably decreased p-cSrc, β-catenin and P-gp protein levels and efflux in both cell lines, as detected in Western blot (Figure 8B), cellular efflux (Figure 8C) and immunostaining (Figure 8D). We further treated NCI/ADR-RES cells with FH535, inhibiting β-catenin recruitment to T-cell factor (Tcf) [45]. We found that FH535 (20 μM) decreased P-gp protein to 25% of control (Figure 8E); however, it did not affect either Gb3 synthase or p-cSrc in Western blotting.

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