Glucosylceramide synthase upregulates MDR1 expression in the regulation of cancer drug resistance through cSrc and β-catenin signaling
© Liu et al. 2010
Received: 11 December 2009
Accepted: 11 June 2010
Published: 11 June 2010
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.
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 β-catenin phosphorylation, and increased nuclear β-catenin. These consequently increased MDR1 promoter activation and its expression. Conversely, MBO-asGCS treatments decreased globo series GSLs (Gb3, Gb4), cSrc kinase and nuclear β-catenin, and suppressed MDR-1 expression in dose-dependent pattern.
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 β-catenin signaling pathway.
Chemotherapy is the principal treatment option for patients with late stage cancers. Despite considerable advances in drug discovery, metastatic solid malignancies remain incurable, due to their poor response to most of the conventional antineoplastic agents. Acquired drug resistance of cancer cells severely limits the success of chemotherapy, particular in solid tumors [1, 2]. The ABCB1 transporter, known as P-glycoprotein (P-gp) is encoded by human multidrug resistance 1 gene (MDR1) and is an important mediator of drug resistance [2, 3]. Like other membrane transport proteins in ABC (ATP binding cassette) family, P-gp is found in various cellular membranes of organisms from bacteria to mammals. P-gp plays roles in the absorption, distribution, and excretion of pharmacological compounds in normal tissues [4, 5]. However, overexpression of MDR1 in tumors results in increase of P-gp and active effluxing of a variety of natural product anticancer agents from cells [2, 6]. The polymorphism of MDR1, particularly the 'silent' polymorphism, blocks the effects of currently available P-gp antagonists and thus limits the success of these agents in clinical trials [7–10].
Drug resistance is the outcome of multiple-gene interactions in cancer cells under the stress of antineoplastic agents. Several drug-resistant markers including Bcl-2, mutant p53, and glucosylceramide synthase (GCS) are overexpressed in drug-resistant cancers [5, 11–13]. However, little is known about the molecular mechanism underlying MDR1 overexpression and how it interacts with other genes to impart drug-resistance. Recently, an emerging body of evidence indicates a curious association of multidrug resistance with ceramide glycosylation [13–18]. GCS (UDP-glucose:ceramide glucosyltransferase, UGCG) transfers a glucose residue from UDP-glucose to ceramide and produces glucosylceramide [19, 20]. This first step in glycosphingolipid (GSL) synthesis tightly regulates the production of all upstream GSLs . Ceramide, a lipid second messenger, induces growth arrest or apoptosis in cancer cells; this induced-apoptosis is in part responsible for the therapeutic efficiency of antineoplastic regimens including anthracyclines, taxanes, and vinca alkaloids and radiation therapy [15, 22–25]. Overexpression of GCS can result in drug resistance, as introduction of GCS confers cell resistance to doxorubicin, daunorubicin, and tumor necrosis factor-α [16, 26, 27]. GCS is overexpressed in many MDR cancer cell lines [17, 28], and in leukemia, breast cancer, and renal cell cancer [29–31]. Interestingly, GCS is coincidently overexpressed with MDR1 in drug-resistant cells [28, 32] and in leukemia cells from patients who have poor-response to chemotherapy [31, 33]. We have studied the effects of ceramide glycosylation on MDR1 and found that GCS upregulates MDR1 expression through activation of cSrc and β-catenin signaling.
Silencing GCS represses MDR1 expression and sensitizes cancer cells to chemotherapeutic agents
Silencing GCS represses MDR1 expression and restores tumor sensitivity to doxorubicin
GCS upregulates MDR1 expression through cSrc kinase and β-catenin signaling
Silencing GCS represses MDR1 transactivation via inhibition of cSrc and β-catenin signaling
Globo series GSLs modulate MDR1 expression
Furthermore, we examined whether GCS alter Gb3 concentration and cSrc kinase in GEMs. As shown in Figure 7C and 7D, silencing of GCS by MBO-asGCS (100 nM) significantly decreased Gb3 level, and p-cSrc to 32% in GEMs of NCI/ADR-RES cells. On the contrary, GCS transfection significantly increased Gb3 and doubled p-cSrc in GEMs of OVCAR-8 cells. Alterations of Gb3 and cSrc kinase in GEMs following GCS gene manipulations significantly changed P-gp expression levels as well.
GCS is a key enzyme for ceramide glycosylation and GSL synthesis. This study demonstrates that GCS upregulates MDR1 expression and modulates drug resistance of cancer. It reveals that GSLs, in particular globo series GSLs mediate gene expression through cSrc and β-catenin signaling.
Previous works indicate that GCS and MDR1 are co-overexpressed in drug-resistant leukemia  and in drug-resistant cancer cells including human ovarian cancer (NCI/ADR-RES), cervical cancer (KB-V1), leukemia (HL-60/VCR), melanoma (MeWo Eto) and colon cancer (SW620/AD) [13, 28]. However, it is not clear how GCS or MDR1 affects each other to promote drug-resistance. Suppressing GCS with siRNA or a GCS inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), down-regulates the expression and function of P-gp in human breast cancer cells ; however, inhibition of GCS by other types of GCS inhibitors (N-butyl-deoxygalactonojirimycin, OGB-1; N-nonyl-deoxygalactonojirimycin, OGB2) did not appear to have any effect on P-gp functional activity in chronic lymphocytic leukemia cells, even though OGB-1 and OGB2 sensitized these cells . P-pg has been proposed as a Golgi glucosylceramide flippase that enhances neutral GSL synthesis, since transfection of MDR1 increases globo series GSLs, and inhibition of P-gp with cyclosporine A decreases neutral GSL biosynthesis in cells [32, 44, 47, 48]. To characterize the role of GCS in MDR1-GCS co-overexpression, we tested P-gp expression after GCS gene silencing in several different types of cancer cells and in tumors. We have found that silencing of the GCS down-regulates P-gp expression, inhibits its efflux activity, and consequently sensitizes MDR cells including NCI/ADR-RES, A2780-AD, KB-A1, SW620/AD and EMT6/AR1 (Figure 1, 2). Furthermore, suppressing GCS with MBO-asGCS substantially decreases P-gp protein, enhances the accumulation of doxorubicin or paclitaxel, and sensitizes tumors to chemotherapy (Figure 3, 4). We have reported that doxorubicin upregulates GCS expression and results in drug resistance in cells . Herein it has been found that doxorubicin treatment up-regulates GCS expression and importantly, P-gp expression in tumors (Dox vs. saline, Figure 3B); MBO-asGCS simultaneously suppresses GCS and MDR1 overexpression (MBO-asGCS vs. saline, Figure 3B), even under doxorubicin challenge (MBO-asGCS + Dox vs. Dox, Figure 3B). Taken together, these results demonstrate that GCS has a regulatory role in MDR1 expression and genesis of drug resistance. Inhibition of GCS appears to be an efficient approach not only to prevent the formation of drug resistance during the course of cancer chemotherapy, but also to reverse drug resistance of cancers.
It has taken time to understand how GSLs generated by GCS modulate gene expression. By introducing GCS into OVCAR-8 cells that express low levels of GCS and P-gp, we have found that GCS consequently upregulates MDR1 expression and enhances P-gp efflux through cSrc and β-catenin signaling. Inhibition of Src kinase by PP2 further indicates that GSLs in cell membrane may mediate the phosphorylation of cSrc and of β-catenin that decreases β-catenin levels in the nucleus (Figure 5). This finding has been confirmed by selective silencing of GCS (not Gb3 synthase or GD3 synthase) using MBO-asGCS, in NCI/ADR-RES cells that over express GCS and P-gp (Figure 6). The promoter of the human MDR1 contains multiple Tcf4/LEF (T-cell factor 4/lymphoid enhancer factor) binding motifs, CTTTGA/TA/T [49, 50]. It has been demonstrated that MDR1 is a direct target gene of the β-catenin/Tcf4 transcriptional complex, and activation of β-catenin increases P-gp expression [51–53]. It has been reported that active cSrc elevates the levels of β-catenin, and inhibition of cSrc decreases the binding of β-catenin to the promoters of β-catenin/Tcf4 complex targets such as cyclin D1 and c-Myc [54, 55]. In present study, inhibitions of cSrc kinase by PP2 and β-catenin/Tcf4 recruitment by FH535 sequentially prevent MDR1 transactivation (Figure 5, 6, 8). These data strongly support the model that GCS enhances cSrc signaling and β-catenin, and transactivates MDR1 expression.
This study demonstrates, for the first time, that GCS upregulates MDR1 expression and modulates drug resistance of cancer. GSLs, in particular of globo series GSLs mediate gene expression of MDR1 through cSrc and β-catenin signaling.
Drug-resistant NCI/ADR-RES human ovarian cancer cells (designed as MCF-7-AdrR previously) [63, 64] were kindly provided by Dr. Kenneth Cowan (UNMC Eppley Cancer Center, Omaha, NE) and Dr. Merrill Goldsmith (National Cancer Institute, Bethesda, MD, USA). The ovarian carcinoma cells A2780-AD, which is resistant to doxorubicin , was kindly provided by Dr. Thomas C. Hamilton (Fox Chase Cancer Center, Philadelphia, PA). Doxorubicin-selected KB-A1 cells  were from Dr. Michael M. Gottesman (National Cancer Institute, Bethesda, MD). Drug resistant SW620/Ad colon cancer cells  were kindly provided by Drs. Susan Bates and Antonio Fojo (National Cancer Institute, Bethesda, MD). Drug-resistant murine EMT6/AR1 breast carcinoma cells [68, 69] were kindly provided by Dr. Ian Tannock (Ontario Cancer Institute, Toronto, ON, Canada). The OVCAR-8 human ovarian carcinoma cells were provided by Dr. M. Hollingshead of Division of Cancer Treatment and Diagnosis Tumor Repository at National Cancer Institute (Frederick, MD). NCI/ADR-RES, KB-A1 and SW620Ad cells were maintained in RPMI-1640 medium containing 10% (v/v) FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 584 mg/liter L-glutamine. A2780-AD cells were cultured in RPMI-1640 medium containing 100 nM doxorubicin in addition to the above components. EMT6/AR1 cells were maintained in Dulbecco's modified eagle medium (DMEM) containing 1 μg/ml of doxorubicin for 2 days/week in addition to the above components. Cells were cultured in a humidified incubator with 95% air and 5% CO2 at 37°C. Doxorubicin hydrochloride was purchased from Sigma (St. Luis, MO). NCI/ADR-RES cells transfected with human GCS gene (NCI/ADR-RES/GCS) and GCS antisense (NCI/ADR-RES/asGCS) were cultured in RPMI 1640 containing the above components and G418 (400 μg/mL) [13, 70].
Mixed-backbone oligonucleotide and inhibitors
A mixed-backbone oligonucleotide, designed to target the ORF 18-37 of human GCS [34, 71], was verified and designated as MBO-asGCS . MBO-asGCS were 20-mer phosphorothioate DNAs, except that four bases at the 5' end and the 3' end were replaced by 2'-O-methyl RNA. MBO-asGCS was synthesized and purified by reverse-phase HPLC and desalting (Integrated DNA Technologies, Inc., Coralville, IA). The MBO-asGCS was introduced into cells with Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) in Opti-MEM I reduced-serum medium (Invitrogen). To repress MDR1 expression, cells were transfected with MBO-asGCS (100 nM) twice and grown in 10% FBS RPMI-1640 medium for 7 days. To inhibit P-gp function, NCI/ADR-RES cells were exposed to verapamil (10 μM) in 5% RPMI-1640 at 37°C for 2 hr, before the analysis of accumulation and efflux. Verapamil hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO).
To silence Gb3 synthase, NCI/ADR-RES and NCI/ADR-RES/GCS cells were transfected with siRNA targeting human Gb3 synthase (siRNA-Gb3S 100 nM) or scrambled control siRNA (siRNA-SC 100 nM) twice and grown in 10% FBS RPMI-1640 medium for 7 days. The siRNA targeting human Gb3 synthase and control siRNA-A were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). β-1,3-Gal-TL siRNA (sc-62006) was designed to knockdown human β-1,3-galactosyltransferase (GeneID: 145173). Control siRNA-A was consists of a scrambled sequence that will not lead to the specific degradation of Gb3. siRNAs (100 nM) were introduced into these cells with Lipofectamine 2000.
A Src kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (PP2) [37, 38] was purchased from Enzo Life Sciences (Plymouth Meeting, PA). An effective β-catenin/Tcf inhibitor, FH535  was purchased from Sigma-Aldrich (St. Louis, MO). OVCAR-8/GCS cells were incubated with PP2 (10 μM) in 5% RPMI-1640 medium for 24 hr. NCI-ADR-RES cells were exposed to FH535 (1 to 20 μM) in 5% RPMI-1640 medium for 24 hr.
Western blotting analysis
Western blotting was conducted as described previously [13, 17]. After treatments, cells or tissue homogenates were lysed using NP40 cell lysis buffer (Biosource, Camarillo, CA, US) to extract the total cellular protein for Western blot. The nuclear proteins were extracted as described previously . Briefly, cells were suspended in 100 μl of Tween-20 lysis buffer (25 mM Tris/Hepes, pH 8.0, 250 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5% Tween-20), and kept on ice for 15 min. The nuclei were pelleted at 6000 g for 5 min at 4°C, and then resuspended in 100 μl of the lysis buffer containing 500 mM NaCl and incubated on ice for additional 15 min. After incubation, the samples were mixed with 100 μl of the lysis buffer (without NaCl). The supernants were collected for Western blotting following a spin-down at 10,000 g for 15 min. Equal amounts of these proteins (50 μg/lane) were resolved using 4-20% gradient SDS-PAGE (Invitrogen). The transferred blot was blocked with 5% fat-free milk in PBS and immuno-blotted with primary antibodies (anti-GCS goat IgG, anti-P-pg mouse, cSrc, phosphorylated cSrc, phosphorylated FAK, β-catenin, phosphorylated β-catenin) at 4°C, overnight. The antigen-antibody in blots was detected by using a second antibody-conjugated HRP and enzyme-linked chemiluminescence plus substrate (GE Healthcare). GAPDH or β-tubulin was used as loading control for total proteins or nuclear proteins.
Cells (10,000 cells/chamber) were grown in 4-chamber slides with 10% FBS culture medium for 48 hr. After methanol fixation, cells were blocked and then incubated with anti-GCS serum and anti-P-gp antibody (1:100) in block solution (Vector Laboratories, Burlingame, CA), overnight at 4°C. GCS antibody and P-gp antibody on cells were recognized by Alexa Fluor®488 goat anti-rabbit IgG and Alexa Fluor 667 goat anti-mouse IgG (Invitrogen). Cell nuclei were counterstained with DAPI (4', 6 diamidino-2-phenylindole) in mounting solution (Vector Laboratories). The slides were observed using a Nikon TE-2000 phase contrast microscope, and the images were captured by a Retiga 2300™ monochrome digital camera using IPLab™ image analysis program (Scanalytics Inc., Rockville, MD).
Cell viability assay
Cell viability was analyzed by quantitation of ATP, an indicator of active cells using CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI), as described previously . Briefly, cells (4,000 cells/well) were grown in 96-well plates with 10% FBS RPMI-1640 medium for 24 hr. MBO-asGCS (50 nM) was introduced into cells by Lipofectamine 2000 (vehicle) in Opti-MEM reduced-serum medium, for 4 hr. Cells were then incubated with increasing concentrations of agents in 5% FBS medium for another 72 hr. Cell viability was determined by the measurement of luminescent ATP using a Synergy HT microplate reader (BioTek, Winnooski, VT. USA), following incubation with CellTiter-Glo reagent (Promega, Madison, WI, USA).
Verocytotoxin was kindly provided by Dr. Clifford A. Lingwood (University of Toronto and Hospital for Sick Children, Toronto, Canada). After 24 hr growth in 96-well plates, cells were incubated with verocytotoxin in 5% FBS RPMI-1640 medium for an additional 72 hr.
Cellular ceramide glycosylation assay
Cells were grown 24 hr in 35-mm dishes (1 × 106 cells/dish) in 10% FBS RPMI-1640 medium and MBO-asGCS (50 nM) was introduced as described above. After 12 hr growth in 10% RPMI-1640 medium, cells were switched to 1% bovine serum albumin (fatty acid free) medium containing 50 μM NBD C6-ceramide complexed to BSA (Invitrogen). After 2 hr incubation at 37°C, lipids were extracted, and resolved on partisil high performance TLC plates with fluorescent indicator in a solvent system containing chloroform/methanol/3.5 N ammonium hydroxide (85:15:1, v/v/v), as described previously [17, 73]. NBD C6-glucosylceramide and NBD C6-ceramide were identified using AlphaImager HP imaging system (Alpha Innotech, San Leandro, CA) and quantitated on a Synergy HT multi-detection microplate reader (BioTek). For quantitation, calibration curves were established after TLC separation of NBD C6-ceramide (Invitrogen) and NBD C6-glucosylceramide (N-hexanol-NBD-glucosylceramide; Matreya, Pleasant Gap, PA).
Cells were cultured in 10% FBS RPMI-1640 medium and harvested by trypsin-EDTA. Approximately 400 mg of pelleted cells was lyophilized and extracted twice with 4 ml of chloroform/methanol (2/1, v/v). The two extracts were combined, evaporated to dryness and subjected to saponification by suspending the residue in 1 ml of 0.5 N NaOH. After incubation at 55°C for 1 hr, the mixture was neutralized with glacial acetic acid, evaporated to dryness, suspended in 1 ml of water, exhaustively dialyzed against water and lyophilized. The lyophilized powder was dissolved in 100 μl chloroform/methanol (2/1) and a 5-μl aliquot was spotted on a TLC plate (Merck, Darmstadt, Germany). The plate was developed in chloroform/methanol/12 mM MgCl2 (50/40/10, v/v/v), and GSLs were visualized by spraying the plate with diphenylamine-aniline phosphoric acid reagent as described previously .
GSLs on GEMs were prepared and analyzed in NCI/ADR-RES/asGCS, OVCAR-8/GCS and each mock-transfected cell lines, as described previously [58, 75] with modification. Briefly, cells (1 × 107) were harvested, suspended in 1 ml of lysis buffer containing 1% Triton X-100 (TX-100), and 75 units of Aprotinin in TNEV solution (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM NaVO4), homogenized and incubated on ice for 20 min. Cell lysates were centrifuged for 5 min at 1300 g to remove nuclei and large cellular debris. The supernatant collected (700 μl) was mixed with equal volume (700 μl) of 85% sucrose (wt/vol) in TNEV solution. The diluted Triton X-100 lysates were overlaid with 30% (6 ml) and 5% (3.3 ml) of sucrose TNEV solution in SW41 centrifuge tube. The samples were centrifuged for 18 h at 200,000 g at 4°C. White bands located at ~5-7% sucrose were collected as GEM fraction and its protein content was determined using BCA Protein Assay Kit. The lipids were extracted with chlofrom/methanol/water (1:1:1, v/v/v) from 200 μg of GEM protein. Extracted lipids were resuspended in choloform-methanol (1:1, v/v) and applied to partisil HPTLC plates. Lipids were resolved using the solvent system of chloroform/methanol/water (65:25:4 v/v/v). Acid alcohol (90% methanol/5% sulfuric acid, 5% acetic acid; Sigma-Aldrich) was used for the chemical detection of glycosphingolipids. Neurtral glycospingolipids qualmix and ceramide trihexosides (Gb3) were purchased from Matreya (Pleasant Gap, PA) and used as standards in TLC.
High-pressure liquid chromatography (HPLC) analysis of doxorubicin
The concentrations of doxorubicin in cells, serum and tumors were analyzed, as described previously with minor modifications [76, 77]. Cells (2.5 × 105 cells/well) were grown in 6-well plates with 10% FBS RPMI-1640 medium. After 24 hr, cells were shifted to medium containing doxorubicin (100 μM) for 2 hr incubation, at 37°C. Following ice-cold PBS rinsing, cellular doxorubicin was extracted using 3 ml of methanol. For tumor samples, ~80 mg of tissue was homogenized in 200 μl of ice-cold methanol. After centrifugation (7,000 g, 10 min), the supernatant of samples was injected into the HPLC system with an auto-sampler. Doxorubicin was resolved on a Pecosphere C18 reversed-phase column with mobile phase of 50 mM sodium phosphate buffer (pH 2.0):acetonitrile:1-propanol (65:25:2; v/v/v; flow rate of 0.8 ml/min). Doxorubicin was detected with the use of a scanning fluorescence detector at λexcitation 480 nm and λemission 550 nm. The retention time was approximately 7 minutes for doxorubicin. Standard curves were linear within the range of 1 ng/ml to 100 ng/ml (equal to 0.002 ~ 0.17 μM). Samples containing high doxorubicin concentrations were diluted as needed.
For the analysis of doxorubicin in serum, proteins were precipitated with 10% trichloroacetic acid. The supernatant obtained after centrifugation (7,000 g, 10 min) was used for HPLC assay.
Paclitaxel accumulation and efflux
The measurements were performed as described previously [78, 79]. After treatments or transfection, cells were grown in 10% FBS RPMI-1640 medium for 24 hr and then shifted to 5% FBS RPMI-1640 medium containing Fluotax-2 (Oregon green 488 paclitaxel, 0.5 μM) and incubated at 37°C for 2 hr. After ice-cold wash and trypsinization, accumulation of paclitaxel was measured. For efflux, at the end of the 2 hr incubation, fresh media was added following wash and re-incubated at 37°C for an additional 2 hr. Fluorescent paclitaxel was measured at λexcitation 485 nm and λemission 529 nm using a Synergy HT microplate reader. Cellular accumulation of paclitaxel was normalized to cell number and paclitaxel added (total intensity). The efflux was normalized against accumulated paclitaxel in cells. Flutax-2 (Oregon green 488 paclitaxel) was purchased from Invitrogen.
After two MBO-asGCS administrations (1 mg/kg every 3-days, ip, 3 mice/group), the small intestine (ileum) and tumors were resected. Tissues (25 mg/reaction) were incubated with fluorescent paclitaxel (1.0 μM) in 200 μl of 1% BSA RPMI-1640 medium containing collagenase IV, immediately following mincing. Accumulation of paclitaxel was measured after 2 hr incubation and 3 times of washes with ice-cold PBS. For efflux, samples were incubated with fresh medium for an additional 2 hr following accumulation and washed 3 times with ice-cold PBS.
Drug-resistant tumor models and treatments
Drug-resistant NCI/ADR-RES tumors were established by using the methods described previously [35, 80]. Athymic nude mice (Foxn1 nu /Foxn1 + , 4-5 weeks, female) were purchased from Harlan (Indianapolis, IN) and maintained in the Vivarium, University of Louisiana at Monroe, according to the approved protocol. Cultured cells after 3 to 5 passages were washed with and resuspended in serum-free RPMI-1640 medium. A suspension of NCI/ADR-RES cells (1 × 106 cells in 20 μl per mouse) was injected into the left flank of the mouse. The mice were monitored by measuring tumor growth, body weight and clinical observation. Tumor-bearing mice were randomly divided into multiple treatment and control groups (ten mice per group). MBOs, dissolved in RPMI 1640 medium were given at the dose of 1 mg/kg, twice per week, at the tumor site. The control group received medium only. In combination therapy, doxorubicin was given by intraperitoneal injection at 2 mg/kg once a week with medium or MBOs for 42 days, respectively.
Tumors were removed, fixed and maintained in paraffin blocks. Microsections from each tumor (5 μm) were H&E stained and identified by pathologist (Dr. J. Bao). For immunostaining, antigens were retrieved in steaming sodium citrate buffer (10 mM, 0.05% Tween-20, pH 6.0, 10 min). After blocking with 2% block solution (Vector Laboratories, Burlingame, CA), the slides were incubated with primary antibodies (1:100) at 4°C, overnight.
MDR1 promoter assay
The human MDR1 promoter reporter, pMDR1  was kindly provided by Dr. Kathleen W. Scotto (University of Medicine and Dentistry of New Jersey, New Brunswick, NJ). MDR1 promoter (sequence from -1202 to +118) drives luciferase expression from pGL2B. After treatments or transfection, cells (2.5 × 105 cells/well) were placed into 6-well plates with 10% FBS RPMI-1640 medium. After 24 hr culture, pMDR1 plasmid (4 μg/well) and pGL4 renilla luciferase reporter driven by thymidine kinase promoter (pGL4-hRluc/TK; 4 μg/well) were introduced into cells with Lipofectamine 2000 and cells were cultured in 10% FBS medium for additional 48 hr. Cell lysates were incubated with Dual-luciferase reporter assay system reagents (Promega). The intensities of firefly luciferase (MDR1 promoter activity) and renilla luciferase (TK promoter activity) were measured using a Synergy HT multidetection microplate reader. MDR1 promoter activity was normalized to protein and TK promoter.
All data represent the mean ± SD. Experiments in triplicate were repeated 2 or 3 times in cell models. Student's t test was used to compare mean values, using a Prism 4 program (GraphPad software, San Diego, CA).
This work was supported by United States Public Health Service/NIH grant P20 RR16456 from the NCRR (Y.Y.L, S.M.J), Department of Defense Breast Cancer Research Program DAMD17-01-1-0536 (Y.Y.L.). We thank Dr. Clifford A. Lingwood (University of Toronto and Hospital for Sick Children, Toronto, Canada) for providing verocytotoxin and Dr. Kathleen W. Scotto (University of Medicine and Dentistry of New Jersey, New Brunswick, NJ) for providing human MDR1 promoter reporter.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.