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

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.

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.

Figure 2

Silencing of GCS by MBO-asGCS Represses MDR1 Expression and Sensitizes Drug-Resistant Cancer Cells. (A) P-gp and GCS in drug-resistant cancer cells. Cells were cultured in growth medium for 24 hr, and then treated with vehicle or MBO-asGCS (50 nM) for an additional 48 hr. Equal amounts of protein (50 μg/lane) were resolved by 4-20% gradient SDS-PAGE and immunoblotted with anti-GCS and anti-P-gp antibodies. GAPDH was used as endpoint control, and GCS/GAPDH or P-gp/GAPDH represents optical densities of the bands. -, vehicle (Lipofectamine 2000); +, MBO-asGCS (50 nM). *, p < 0.001 compared with vehicle treatment. (B) Cell response to doxorubicin. After pretreatment of MBO-asGCS (50 nM) or vehicle, cells were incubated with 5% FBS medium at the presence of doxorubicin for an additional 72 hr. *, p < 0.001 compared with vehicle.

Figure 3

Silencing of GCS by MBO-asGCS Represses MDR1 Expression and Reverses Tumor Resistance to Doxorubicin in vivo. (A) Tumor growth. Tumors generated from NCI/ADR-RES cells (~3 mm in diameter, 10 mice/group) were treated with MBO-asGCS (1 mg/kg every 3 days) or doxorubicin (2 mg/kg/week) and combination thereof. Data represent the mean ± SE; *, p < 0.001 compared to saline group (open squares); **, p < 0.001 compared to doxorubicin treatment (solid squares). (B) Western blots of tumor tissues. After treatments, extracted tumor proteins (100 μg/μl, three samples per group) were resolved by 4-20% SDS-PAGE and immunoblotted with anti-GCS or anti-P-gp antibodies, respectively. (C) Immunostaining. After retrieval, antigens on tissue sections (5 μm) were recognized by anti-GCS (green) and anti-P-gp (red) antibodies with fluorescence conjugated secondary antibodies. Microphotographs of merged fluorescence (Fluo.) with H&E staining (H&E) were originally magnified by 200.

Figure 4

Effects of GCS Silencing on P-gp Regulated Drug Accumulation and Efflux in Tumors. NCI/ADR-RES tumors were treated with MBO-asGCS (1 mg/kg/3 days, 3 mice/group) or saline for 7 days. (A) Doxorubicin accumulation. After 4 hr and 24 hr peritoneal administration of doxorubicin (1 mg/kg), serum and tumor tissues were collected and prepared for HPLC assays. Doxorubicin levels were represented per μl of serum or per mg of tumor tissue. *, p < 0.001 compared with serum of saline treatment at 24 hr; **, P < 0.001 compared with saline treatments. (B) Paclitaxel accumulation. After two administrations of MBO-asGCS, tissue suspensions (25 mg/reaction) were incubated with Flutax-2 (1 μM) in medium containing collagenase IV, immediately following mincing. Accumulation of paclitaxel was measured after 2 hr incubation. *, p < 0.001 compared with saline treatment of tumors. (C) Paclitaxel efflux. After accumulation described in (B), tissues were incubated with fresh medium for an additional 2 hr to measure paclitaxel efflux. *, p < 0.001 compared with saline treatment of tumors.

Figure 5

GCS Upregulates MDR1 Expression through Enhanced cSrc/β-Catenin Signaling. After a series of transient GCS transfection (0, 2.0, 4.0, 8.0 μg of pcDNA 3.1-GCS plasmid DNA in 100-mm dish), OVCAR-8 cells were cultured in 10% FBS RPMI-1640 medium for 7 days. OVCAR-8 cells transfected with pcDNA 3.1-GCS (8 μg) were then treated with 10 μM PP2 for 24 hr (+PP2). (A) Western blots. Equal amounts of detergent-soluble total cellular proteins or nuclear proteins (50 μg/lane) were resolved on 4-20% gradient SDS-PAGE and immunoblotted with indicated primary antibodies. 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 GCS transient transfection in OVCAR-8 cells. *, p < 0.001 compared with mock transfection; **, p < 0.001 compared with vehicle treatment in cells transfected with GCS (8 μg DNA). (C) Paclitaxel accumulation and efflux. Cells were incubated with Flutax‐2 (0.5 μM) in medium at 37°C. Accumulation of paclitaxel (top panel) was measured after 2 hr incubation. After washing with ice-cold PBS, cells were re-incubated with fresh medium for an additional 2 hr to measure efflux (bottom panel). *, p < 0.001 compared with the mock transfection. **, p < 0.001 compared with vehicle treatment in cells transfected with GCS (8 μg DNA).

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.

Figure 7

Globo Series GSLs Mediate MDR1 Transactivation. (A) Glycosphingolipids. Cells were cultured in 10% FBS RPMI-1640 medium and harvested by trypsin-EDTA. Extracted lipids (5 μl aliquot of 100 μl) were resolve by TLC and GSLs were visualized by spraying with diphenylamine-aniline phosphoric acid reagent. GlcCer, glucosylceramide. (B) Gb3, a receptor of verocytotoxin on GCS transfectants. Cells were incubated with increasing concentrations of verocytotoxin in 5% FBS RPMI-1640 medium for 72 hr. *, p < 0.001 compared to ADR-RES. (C) GEM GSLs. GEMs of cells were prepared with gradient sucrose and extracted lipids (100 μg of GEM protein) were applied to HPTLC plates. (D) cSrc phosphorylation in GEMs. Equal amounts of GEM protein (50 μg/lane) were resolved by 4-12% gradient SDS-PAGE and immunoblotted with antibodies. p-cSRc/cSrc represents optical densities of the bands; *, p < 0.001 compared with mock.

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.

Figure 9

GSL Synthesis and MDR1 Expression. GCS, glucosylceramide synthase; GlcCer, glucosylceramide; Tcf4, T-cell factor 4; FAK, focal adhesion kinase; cSrc, proto-oncogene (Schmidt-Ruppin A-2); Gb3, globotriaosylceramide; Gb5, globopentaosylceramide; MSGb5, monosyl-Gb5.