Lipid raft modulation by Rp1 reverses multidrug resistance via inactivating MDR-1 and Src inhibition

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Lipid raft modulation by Rp1 reverses multidrug resistance via inactivating MDR-1 and Src inhibition Un-Jung Yun a, Ji-Hye Lee a, Kyung Hee Koo a, Sang-Kyu Ye b, Soo-Youl Kim c, Chang-Hun Lee c,**, Yong-Nyun Kim a,* a

Comparative Biomedicine Research Branch, Division of Cancer Biology, Research Institute, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do, 410-769, Republic of Korea Department of Pharmacology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-779, Republic of Korea c Cancer Cell and Molecular Biology Branch, Division of Cancer Biology, Research Institute, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do, 410-769, Republic of Korea b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 7 December 2012 Accepted 27 February 2013 Available online xxx

Multidrug resistance (MDR) is a major obstacle to effective cancer therapy. The membrane transporter MDR-1 (P-gp, ABCB1), a member of the ATP-binding cassette (ABC) transporter family, effluxes anticancer drugs from cancer cells. Increased activity of MDR-1 is known to be the main mechanism for multidrug resistance. MDR-1 is known to be localized in the cholesterol- and sphingolipid-enriched plasma membrane microdomains, known as lipid rafts. Disruption of lipid rafts by cholesterol depletion alters lipid raft functions, indicating that cholesterol is critical for raft function. Because ginsenosides are structurally similar to cholesterol, in this study, we investigated the effect of Rp1, a novel ginsenoside derivative, on drug resistance using drug-sensitive OVCAR-8 and drug-resistant NCI/ADR-RES and DXR cells. Rp1 treatment resulted in an accumulation of doxorubicin or rhodamine 123 by decreasing MDR-1 activity in doxorubicin-resistant cells. Rp1 synergistically induced cell death with actinomycin D in DXR cells. Rp1 appeared to redistribute lipid rafts and MDR-1 protein. Moreover, Rp1 reversed resistance to actinomycin D by decreasing MDR-1 protein levels and Src phosphorylation with modulation of lipid rafts. Addition of cholesterol attenuated Rp1-induced raft aggregation and MDR-1 redistribution. Rp1 and actinomycin D reduced Src activity, and overexpression of active Src decreased the synergistic effect of Rp1 with actinomycin D. Rp1-induced drug sensitization was also observed with several anti-cancer drugs, including doxorubicin. These data suggest that lipid raft-modulating agents can be used to inhibit MDR-1 activity and thus overcome drug resistance. ß 2013 Elsevier Inc. All rights reserved.

Keywords: MDR-1 Lipid rafts Ginsenosides Src Actinomycin D

1. Introduction Chemotherapeutics represent the most effective cancer treatment; however, cancer cells can become resistant to various drugs [1]. The development of multidrug resistance is a major cause of failure of cancer chemotherapy. Various factors may cause multidrug resistance, including impaired drug delivery to tumor cells and genetic or epigenetic alterations that affect drug sensitivity in the cancer cells [1]. More than all, multidrug resistance is caused by increased expression of ATP-binding cassette (ABC) transporter family members, which efflux anti-

* Corresponding author. Tel.: +82 31 920 2415; fax: +82 31 920 2468. ** Corresponding author. Tel.: +82 31 920 2201; fax: +82 31 920 2006. E-mail addresses: [email protected] (U.-J. Yun), [email protected] (J.-H. Lee), [email protected] (K.H. Koo), [email protected] (S.-K. Ye), [email protected] (S.-Y. Kim), [email protected] (C.-H. Lee), [email protected] (Y.-N. Kim).

cancer drugs from cells [1,2]. ABC transporters, including MDR-1 (ABCB1, P-gp), MRP-1 (ABCC1), and BCRP (ABCG2) are expressed in many human cancers and involved in metastasis of cancers as well as multidrug resistance [1,3,4]. MDR-1 is the most well-known ABC transporter that expels a broad range of chemicals, including anticancer drugs, out of cells by utilizing ATP hydrolysis. MDR-1 is implicated in both intrinsic and acquired drug resistance and is upregulated in a number of human tumors [1,5]. MDR-1 is a transmembrane protein that localizes to the membrane microdomains known as lipid rafts. Lipid rafts are cholesterol- and sphingolipid-enriched plasma membrane microdomains. Caveolae are a subclass of lipid rafts that are distinguished from other lipid rafts by the presence of caveolin-1 [6,7]. Cholesterol is a critical lipid component for maintenance of intact structure and function of lipid rafts; cholesterol depletion results in disruption of lipid rafts and thus alterations in cellular functions [8,9]. Because many different signaling molecules are found in the lipid rafts, these microdomains are considered platforms in the organization of signaling

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Please cite this article in press as: Yun U-J, et al. Lipid raft modulation by Rp1 reverses multidrug resistance via inactivating MDR-1 and Src inhibition. Biochem Pharmacol (2013), http://dx.doi.org/10.1016/j.bcp.2013.02.025

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molecules into functional complexes. Lipid rafts are associated with growth factor receptor signaling, Src and Akt activation, and cytoskeleton reorganization, which are linked to cell transformation and cancer progression [10–13]. In addition, studies have reported that lipid rafts are associated with drug resistance and MDR-1 function [14–16], indicating that lipid raft modulation may cause the loss of multidrug resistance. Ginsenosides, the pharmacologically active components of Panax ginseng, are known to have anti-cancer effects [17,18]. Rg3, one of the active ginsenosides, has been shown to have antitumor activity in various cancers [18]. In addition, Rg3 reduces membrane fluidity and modulates MDR-1 activity in mice implanted with adriamycin-resistant P388 murine leukemia cells [19]. Another ginsenoside, Rh2, attenuates adriamycin resistance by inhibiting MDR-1 activity in adriamycin-resistant MCF-7/Adr cells [20]. Combined with cisplatin treatment, Rh2 significantly inhibits tumor growth and prolongs survival of mice [21]. However, the molecular mechanisms whereby ginsenosides impact on drug resistance have not been elucidated. Ginsenosides are structurally similar to cholesterol. Therefore, we hypothesized in a previous study that ginsenosides can alter lipid rafts and thus influence cellular functions such as cell survival. Rh2 induces apoptosis of A431 human epidermoid carcinoma cells by modulating lipid raft-associated signaling pathways, including Akt activation [7]. Rp1, a novel ginsenoside derivative, is known to have anti-metastatic and anti-proliferative potential by inducing G1 arrest and apoptosis [22,23]. In this study, we demonstrate that Rp1 can overcome multidrug resistance by modulating lipid rafts and downregulating MDR-1 in a Src-dependent manner. 2. Materials and methods 2.1. Materials Ginsenoside Rp1, Rg3, and Rh2 were gifted from Ambo institute (Seoul, Korea) and dissolved in dimethylsulfoxide (DMSO, Sigma– Aldrich, St. Louis, MO). Doxorubicin, paclitaxel, and actinomycin D, rhodamine 123, and verapamil were purchased from Sigma– Aldrich Corporation (St. Louis, MO). The Src inhibitor PP2 was purchased from Calbiochem (San Diego, CA). Alexa Fluor 555- and 488-conjugated cholera toxin subunit B (CTxB) was from Molecular Probes by Life technologies (Eugene, OR). Anti-MDR-1 (sc13131), anti-HA (sc-805), anti-c-Src (sc-18), anti-Caveolin-1 (sc894), normal rabbit IgG (sc-2027), normal mouse IgG (sc-2025), HRP-conjugated rabbit IgG (sc-2004), and HRP-conjugated mouse IgG (sc-2005) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-H2AX (Ser139) was obtained from Millipore Corporation (Bedford, MA). Anti-H2AX (7631), antiphospho-Src (Y416, 2101) and anti-PARP (9542) were purchased from Cell signaling technology (Beverly, MA). Anti-Calnexin (610523) was purchased from BD Bioscience (San Diego, CA). Anti-b-actin was obtained from Sigma–Aldrich Corporation (St. Louis, MO).

Co., Grand Island, NY) at 37 8C in a humidified atmosphere containing 5% CO2. DXR cells were grown under selective pressure of 1 mM doxorubicin. The cells were allowed to adhere overnight and grown to approximately 70% confluence and were then serumstarved for 4 h using RPMI containing 0.1% bovine serum albumin (BSA, USB Corp., Cleveland, OH) prior to treatment. Cells were treated with indicated concentrations of reagents in the RPMI containing 0.1% BSA [7]. 2.3. Cell proliferation assay The cells were plated in 96-well cell culture plate, followed by treatment. The effects of the treatments on cell growth were determined with celltiter 96 aqueous nonradioactive cell proliferation assay kit (MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme-thoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, Promega, Madison, WI) as described in the manufacturer’s instruction. Absorbance was measured at 490 nm with a powerwave HT spectrophotometer (Biotek instruments, Winooski, VT). Each experiment was performed in triplicate. 2.4. Immunoblot analysis Cells were lysed with 2 x sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) lysis buffer (20 mM Tris, pH 8.0, 2% SDS, 2 mM dithiothreitol (DTT), 1 mM Na3VO4, 2 mM ethylenediaminetetraacetic acid (EDTA), 20% glycerol) and sonicated [7,24]. Protein concentration of each sample was determined using a Micro-BCA protein assay reagent (Pierce Chemical Co., Rockford, IL) as described by the manufacturer, and equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in TBS-T (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.1% Tween 20) containing 5% non-fat dried milk. The membranes were then incubated with the primary antibody at 4 8C overnight, washed three times with TBS-T, incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or goat anti-rabbit IgG secondary antibodies for 1 h at room temperature, and then washed with TBS-T three times. The immune complexes were visualized using the enhanced chemiluminescence method. Densitometry on immunoblotting images were performed using Image J software (NIH, Bethesda, MD). bactin was used as a loading control for densitometry measurements of immunoblotting. Data are summarized as histograms of mean  S.D. of three independent experiments. 2.5. Annexin V/PI staining For detection of apoptotic cells, cells were harvested and incubated for 10 min at room temperature with fluorescein isothiocyanate (FITC)-conjugated annexin V reagent and PI in binding buffer as described by the manufacturer using FITC annexin V apoptosis detection kit I (BD Biosciences, San Jose, CA), and then analyzed by flow cytometry. The Data were analyzed with Cell Quest Software (BD Bioscience, San Jose, CA).

2.2. Cell culture 2.6. Construct, siRNA and transfection The human ovarian cancer cell line, OVCAR-8 (NCI, MTA Number: 2702-09) is a kind gift from Dr. Soo-Youl Kim (National cancer center, Goyang, Korea) and high dose doxorubicin resistant cell (DXR) derived from NCI/ADR-RES was obtained from Dr. Kyung-Chae Jeong (National cancer center, Goyang, Korea). OVCAR-8 and DXR cells were grown in RPMI with L-glutamine (Hyclone, Logan, UT) supplemented with 10% FBS (Hyclone, Logan, UT), 100 units/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ ml amphotericin B (Antibiotics-Antimycotic, Gibco Laboratories

Constitutive active Src construct was kindly provided by Dr. Jung Weon Lee (Seoul National University, Seoul, Korea). Cells were transiently transfected with active Src plasmid or vector for control, respectively using metapectene pro (Biontex laboratories, Martinsried, Germany) as described by the manufacturer. Small interfering RNA (siRNA) for knock-down of negative control (SN1003), MDR-1 (1000410) and Src (100544) were obtained from Bioneer corporation (Daejun, Korea). Cells were transfected with

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siRNA duplexes using metapectene pro as described by the manufacturer. 2.7. Immunofluorescence and confocal microscopy imaging Cells were attached to glass cover slips and fixed with 2% paraformaldehyde in PBS (Phosphate buffered saline), rinsed with PBS. Cells fixed with paraformaldehyde were then permeablized with 0.2% Triton X-100 in PBS for 5 min. Cells fixed were blocked in PBS containing 10% FBS or 3% BSA for 1 h at room temperature. For staining, cells were incubated with anti-primary antibodies or non-specific IgG overnight at 4 8C, washed in PBS and then exposed to Alexa 488- or 568-conjugated secondary IgG, and alexa 488-or 555-conjugated CTxB for GM-1 staining for 1 h at room temperature, respectively. The glass cover slips were washed in PBS and mounted on glass slides. For confocal fluorescence microscopy, cells were examined using Carl Zeiss fluorescence microscopy LSM Meta 510 CLSM (Carl Zeiss, Jena, Germany). 2.8. Rhodamine 123 and doxorubicin efflux assay To investigate the activity of the MDR-1, cellular efflux assay was performed as previously described [25]. Rhodamine 123 was added at 1 h before drug-release and cells were incubated 1 more hour to the indicated time. Cells were washed and incubated with rhodamine 123- and drug-free medium for 1 more hour to measure efflux. For doxorubicin efflux analysis, cells were treated for 4 h with 30 mM doxorubicin alone or in combination with Rp1, verapamil. And then, cells were washed with doxorubicin free medium and incubated for 1 more hour such as rhodamine 123 efflux analysis. Following harvest, cells were washed with PBS and analyzed by flow cytometry. 2.9. Isolation of detergent insoluble membrane Isolation of detergent insoluble membrane was carried out using Triton X-100 as detergent. Cells were washed with cold PBS and treated with Triton X-100 buffer (25 mM HEPES pH 7.4, 2 mM MnCl2, 1 mM PMSF, 10 mM Na3VO4, 0.15% Triton X-100, 1 mg/ml leupeptin, 1 mg/ml aprotinin) at 4 8C for 10 min. Supernatant was harvested (Soluble fraction), and then cells on dish were lysed with 2 x SDS- PAGE lysis buffer described previously and sonicated (Insoluble fraction). 2.10. Purification of lipid rafts Cells were washed with cold PBS and solubilized in 1 ml lysis buffer (75 mM Nacl, 500 m; EDTA, 12.5 mM HEPES, pH 6.5, 1 mM PMSF, protease inhibitor cocktail) containing triton X-100. The cells were prepared as previously described [7], including homogenization with douce tissue grinder, gradient centrifugation with sucrose. 1 ml 80% sucrose (dissolved in 15 mM NaCl, 25 mM HEPES, pH 6.5) was mixed with 1 ml cell lysate homogenized. It was overlaid with 6 ml 30% sucrose followed by 3 ml 5% sucrose. The gradient was centrifuged with a SW41 rotor at 4 C for 20 h at 40,000 r.p.m. (Beckman Coulter Optima LE-80K Ultracentrifuge, Palo Alto, CA). Gradient fractions of 1 ml each were harvested from the top. Equal volumes of fractions were subjected to immunoblot analysis. 2.11. RNA extraction and RT-PCR RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) and cDNA was synthesized using Maxime RT premix (25081, Intron Biotechnology, Seoul, Korea). Equal amounts of cDNA were used for PCR using Accupower PCR premix (K-2016, Bioneer, Daejun,

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Korea). PCR products were analyzed on agarose gels. The primers used were as follows: MDR-1, 5’-GTT ACT CTT AGC AAT TGT ACC CAT CAT-3’ and 5’-CAA AGA CAA CAG CTG AAA ATA CTA ACA-3’; bactin, 5’-ACA ACG GCT CCG GCA TGT GC-3’ and 5’- CGG TTG GCC TTG GGG TTC AG-3’. 2.12. Data analysis All data points represented the mean value of experiments in triplicates. Statistical significance was determined by Student’s ttest, with P < 0.05 taken to show significant differences between means. 3. Results 3.1. Rp1 redistributes MDR-1 and lipid rafts and enhances drug accumulation in multidrug-resistant cells. To investigate the role of lipid rafts in drug resistance, we used the OVCAR-8 human ovarian carcinoma cell line and NCI/ADR-RES, which is a doxorubicin-resistant cell line selected from OVCAR-8. We further selected NCI/ADR-RES with doxorubicin up to 50 mM and designated these cells the DXR cell line. First, we evaluated drug resistance with anticancer drugs, including actinomycin D and paclitaxel, as well as doxorubicin, by MTS proliferation assay. Compared with OVCAR-8 cells, DXR cells showed resistance to these three anti-cancer drugs (Fig. 1A–C). These drugs are substrates of ABC transporters, including MDR-1 [1,14]. We assessed MDR-1 expression and found that it was expressed in the DXR cells but not in the OVCAR-8 cells (Fig. 1D). When cells were fractionated with Triton X-100, most of the MDR-1 protein was found in the insoluble fraction, where caveolin-1, a marker for lipid rafts, was enriched (Fig. 1E). We further fractionated cells to purify lipid raft fractions and observed that MDR-1 was present in the lipid raft fractions as verified by caveolin-1 and Src enrichment (Fig. 1F). In addition, MDR-1 was co-localized with GM-1, a marker of lipid rafts, in immunofluorescence analysis (Fig. 1G). These results indicate that multidrug-resistant DXR cells express high levels of MDR-1 protein in lipid rafts. We have previously demonstrated that lipid raft disruption with MbCD, a cholesterol-depleting agent, or with Rh2, a ginsenoside, can lead to apoptosis via altering lipid raft-associated signaling [7,8]. Therefore, we hypothesized that lipid raft alteration could enhance drug sensitivity by regulating MDR-1 activity. To explore this possibility, we employed ginsenosdes including Rh2 and Rg3, and a ginsenoside derivative, Rp1, that are structurally similar to cholesterol (Fig. 2A). 5 mM of Rg3, Rh2, or Rp1 alone appeared not to affect cell viability but these ginsenosides decreased cell viability when they were combined with actinomycin D. Among them Rp1 was the most effectively synergistic for actinomycininduced cell growth inhibition (Fig. 2B). As shown in Fig. 2C, 5 mM Rp1 did not affect cell growth, but 10 mM Rp1 inhibited cell growth in both drug-sensitive OVCAR-8 cells and drug-resistant DXR cells. We used a non-cytotoxic dose of Rp1, 5 mM, to investigate its effect on drug sensitivity. To test whether Rp1 affected the distribution of MDR-1 and lipid rafts, cells were stained for MDR-1 and GM-1 after Rp1 treatment. As shown in Fig. 2D, Rp1 altered the distribution of MDR-1 protein and lipid rafts from dispersed to aggregated phenomena without affecting their co-localization. One of the main functions of MDR-1 is to efflux drugs, thereby lowering intracellular drug accumulation [26]. To test whether the altered distribution of MDR-1 and lipid rafts influences MDR-1 activity, we measured intracellular accumulation of MDR-1 substrates rhodamine 123 and doxorubicin. Verapamil, a well-known MDR-1 inhibitor, increased intracellular rhodamine123 levels compared to control; however, Rp1 increased intracellular rhodamine 123

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Fig. 1. Multidrug resistance and MDR-1 association with lipid rafts in OVCAR-8 and DXR cells. Serum-starved OVCAR-8 and DXR cells were treated with the indicated concentration of doxorubicin (DOX) (A), actinomycin D (ActD) (B), or paclitaxel (PTX) (C) for 24 h, and cell growth inhibition was assessed by MTS assay. Data are presented as the averages of triplicate measurements, with error bars representing standard deviations (*, P < 0.05). (D) OVCAR-8 and DXR cells were lysed, and equal amount of cell lysates were analyzed for MDR-1 protein expression by immunoblot analysis using anti-MDR-1 antibody. b-actin was used as a loading control. MDR-1 expression was determined by densitometry analysis of three independent immunoblots and data are summarized as histograms of mean with standard deviations (p < 0.05, * versus OVCAR-8). (E) DXR cells were fractionated into Triton X-100-soluble (S) and -insoluble (IS) fractions as described in Section 2. Then, equal volumes of samples were subjected to immunoblot analysis using anti-MDR-1 and anti-caveolin-1 antibodies. Expression of MDR-1 and caveolin-1 was determined by densitometry analysis of three independent immunoblots (p < 0.05, * versus soluble fraction). (F) DXR cells were homogenized in a lysis buffer containing 1% Triton X-100, as described in Section 2. After sucrose gradient centrifugation, fractions containing lipid rafts (caveolin-1-enriched fractions) were collected and subjected to immunoblot analysis. Expression of MDR-1, cSrc, and caveolin-1 was determined by densitometry analysis of three independent immunoblots (p < 0.05, * versus Fraction #2). (G) DXR cells were permeabilized, stained with CTxB-Alexa 555 for GM-1, a lipid raft marker, and anti-MDR-1 antibody. Nuclei were stained with Hoechst 33342 (H33342). Magnification: 40  (zoom = 2  ). Scale bar = 10 mm. These experiments were performed twice on separate samples with comparable results.

accumulation more than verapamil (Fig. 2E). Similarly, doxorubicin accumulation was more pronounced in Rp1-treated cells than in verapamil-treated cells (Fig. 2F). These data indicate that Rp1 modulates the distribution of MDR-1 protein and lipid rafts and attenuates MDR-1 activity without apparent cytotoxicity. 3.2. Rp1 increases sensitivity to actinomycin D by attenuating MDR-1 expression. Because Rp1 redistributed MDR-1 and enhanced rhodamine 123 and doxorubicin accumulation, we tested whether Rp1 can sensitize cancer cells to various chemotherapeutic agents, including doxorubicin, actinomycin D, or cisplatin. Rp1 appeared to enhance cell growth inhibition synergistically with doxorubicin or actinomycin D, but not with cisplatin that is not a substrate of MDR-1 (Fig. 3A, 3B, and 3C). This sensitization effect of Rp1 was more enhanced when DXR cells were treated with actinomycin D than with doxorubicin. To investigate whether co-treatment of cells with Rp1 and actinomycin D results in apoptosis, we performed flow cytometric analysis of annexin V- and PI-stained cells. Actinomycin D induced more cell death when combined with

Rp1 (Fig. 3D). Consistent with these apoptosis results, caspase-3 activation assessed by cleavage of PARP, a caspase-3 substrate, was increased in the cells co-treated with Rp1 and actinomycin D than in cells treated with these agents independently (Fig. 3E). However, cisplatin-induced cleavage of PARP is not further increased by Rp1 (Fig. 3F). These results suggest that Rp1 can sensitize DXR cells to anti-cancer drugs, including doxorubicin and actinomycin D. Actinomycin D, a DNA intercalating agent, is known to induce histone g-H2AX foci [27]. We used this phenomenon as an indicator of DNA double-strand breaks caused by actinomycin D. As shown in the immunostaining data, there was little g-H2AX foci formation in the DXR cells treated with a relatively low concentration of actinomycin D (1 mM). However, distinct gH2AX foci were formed (Fig. 4A) and g-H2AX levels were augmented biphasically with time by co-treatment with Rp1 and actinomycin D although H2AX levels remained unchanged (Fig. 4B), indicating that Rp1 enhances DNA damage induced by actinomycin D. To investigate whether this synergistic effect is associated with MDR-1 localization, cells were stained for MDR-1 and GM-1. Interestingly, co-treatment of DXR with Rp1 and actinomycin D remarkably reduced MDR-1 staining and caused

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Fig. 2. Effect of Rp1 on MDR-1 activity and MDR-1 localization. (A) Schematic representation of cholesterol and Rp1. (B) Synergistic effects of Rp1, Rg3, and Rh2 on actinomycin D-induced cell growth inhibition. Serum-starved DXR cells were treated with either 5 mM Rp1, 5 mM Rg3, or 5 mM Rh2 alone or co-treated with 1 mM actinomycin D for 24 h. Cell growth inhibition was assessed by MTS assay. Data are presented as the averages of triplicate measurements, with error bars representing standard deviations (*, P < 0.05). (C) Serum-starved OVCAR-8 and DXR cells were treated with the indicated concentrations of Rp1 for 24 h, and cell growth inhibition was assessed by MTS assay. Data are presented as the averages of triplicate measurements, with error bars representing standard deviations (P < 0.05, * versus untreated OVCAR-8 cells, ** versus untreated DXR cells). (D) Serum-starved DXR cells were treated with 5 mM Rp1 for 16 h, permeabilized, stained with CTxB-Alexa 555 and anti-MDR-1 antibody, and analyzed by confocal microscopy. Isotype antibody was used as a negative control for MDR-1 staining. Magnification: 40  (zoom = 2  ). Scale bar = 10 mm. (E) Serum-starved DXR cells were treated with 5 mM Rp1 or 10 mM verapamil (VPL), an MDR-1 inhibitor, for 4 h, and MDR-1 activity was monitored by rhodamine 123 efflux assay as described in Section 2. Verapamil was used as a positive control. Following harvest, cells were washed and subjected to flow cytometric analysis. (F) Serum-starved cells were treated with 30 mM doxorubicin alone or in combination with 5 mM Rp1 or 10 mM verapamil for 4 h. Cells were then washed with doxorubicin-free medium and treated as described in (E). These experiments were performed with similar results separately.

lipid raft aggregation (Fig. 4C), which was observed before (Fig. 2D). To examine if the combination of Rp1 and actinomycin D can regulate MDR-1 protein levels, we performed immunoblotting analysis. As shown in Fig. 4D, although actinomycin D or Rp1 alone appeared to increase MDR-1 protein levels, these two together decreased MDR-1 protein levels without affecting MDR-1 mRNA levels. To further test whether this co-treatment affects MDR-1 distribution, we fractionated cells into detergent-soluble and -insoluble fractions. We found that co-treatment with actinomycin D and Rp1 resulted in shifts of both MDR-1 and caveolin-1 from insoluble to soluble fractions, whereas calnexin, an endoplasmic reticulum marker that has been used as a marker of the soluble fraction, remained unchanged (Fig. 4E). These data reveal that Rp1 potentiates sensitivity to actinomycin D by downregulating and/or redistributing MDR-1 protein from lipid rafts. 3.3. Cholesterol supplementation reverses the Rp1-dependent increase of sensitivity to actinomycin D. Previously, we demonstrated that cholesterol depletion induces cell death through lipid raft disruption and internalization, which

are reversed by cholesterol supplementation [8]. Because Rp1 altered the distribution of MDR-1 and lipid rafts, we tested whether cholesterol affects Rp1-mediated drug sensitivity. Intriguingly, as shown in Fig. 5A, co-treatment with Rp1 and actinomycin D led to morphological alterations, which were attenuated by cholesterol addition. Consistent with this observation, cholesterol decreased cell death induced by Rp1 and actinomycin D together (Fig. 5B). Consequently, cholesterol attenuated caspase-3 activity assessed by PARP cleavage and MDR-1 downregulation induced by Rp1 and actinomycin D (Fig. 5C). Next, we tested whether MDR-1 recovery by cholesterol is associated with MDR-1 activity with an rhodamine 123 efflux assay. As expected, rhodamine123 accumulation was highest in cells co-treated with Rp1 and actinomycin D. However, this elevated rhodamine 123 accumulation was decreased by cholesterol addition (Fig. 5D). Accordingly, cholesterol treatment decreased g-H2AX protein levels (Fig. 5E) and g-H2AX foci formation induced by Rp1 and actinomycin D (Fig. 5F). To test whether cholesterol addition changes the distribution of MDR-1 and lipid rafts, we observed their localization by immunofluorescence. MDR-1 and GM-1 were visualized as aggregates in Rp1- and

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Fig. 3. Synergistic effects of Rp1 on anti-cancer drug-induced apoptosis. (A, B, and C) Serum-starved DXR cells were co-treated with 5 mM Rp1 and either with the indicated concentrations of doxorubicin (A), actinomycin D (B), or cisplatin (C) for 24 h. Cell growth inhibition was examined by MTS assay. Data are presented as the averages of triplicate measurements, with error bars representing standard deviations (*, P < 0.05). (D) Serum-starved DXR cells were treated with 1 mM actinomycin D, 5 mM Rp1, or actinomycin D and Rp1 together for 16 h. Apoptosis was measured by flow cytometry of annexin V- and PI-stained cells. (E and F) Serum-starved DXR cells were treated with the indicated concentrations of actinomycin D and Rp1 (E) or cisplatin and Rp1 (F). Equal amounts of cell lysates from each treatment were subjected to immunoblot analysis using anti-PARP and anti-b-actin antibodies. Cleaved PARP was determined by densitometry and data are summarized as histograms of mean  S.D. of three independent experiments. (*, p < 0.05; NS, no significant difference). These experiments were observed in independent experiments.

actinomycin D-treated cells but were dispersed in cholesterolsupplemented cells (Fig. 5G). To further investigate the role of cholesterol and/or lipid rafts in drug resistance, we depleted cholesterol from cells using MbCD and simvastatin, a cholesterol synthesis inhibitor. Combining actinomycin D and MbCD or simvastatin treatments synergistically inhibited cell growth (Fig. 6A). Low doses of MbCD (1 mM) and simvastatin (5 mM) alone did not deform cell morphology; however, co-treatment with these agents and actinomycin D increased cell death as determined by cell morphology (Fig. 6B). In addition, there were increases in g-H2AX foci formation when cells were co-treated with cholesterol-depleting agents (Fig. 6C). MbCD and actinomycin D co-treatment did not decrease MDR-1 expression but did increase PARP cleavage. Simvastatin increased PARP cleavage and inhibited actinomycin D-induced MDR-1 upregulation (Fig. 6D and E). These results indicate that Rp1 and actinomycin D exerts their synergistic effect through lipid raft modulation and MDR-1 downregulation, which can be reversed by cholesterol, a critical component of lipid rafts. 3.4. Rp1 reverses resistance to actinomycin D by reducing Src activation. Because Src family kinase activity has been associated with drug resistance, we examined whether changes in Src activity are linked to drug sensitivity induced by Rp1. Actinomycin D itself reduced Src activity, and co-treatment with Rp1 abolished Src activity with a decrease in total levels of Src, although Rp1 alone did not affect Src activity (Fig. 7A). This was also true in cells

co-treated with cholesterol-depleting agents and actinomycin D (Fig. 7A). Cell growth was significantly decreased by combining a Src inhibitor, PP2, with actinomycin D (Fig. 7B). PP2 alone did not change MDR-1 levels but decreased MDR-1 levels increased by actinomycin D when combined with actinomycin D. Consequently, PARP cleavage was increased in cells co-treated with PP2 and actinomycin D, compared with cells treated with PP2 or actinomycin D alone (Fig. 7C). Rhodamine123 accumulation was also increased by PP2 and actinomycin D co-treatment (Fig. 7D). To further investigate Src involvement in drug resistance, we knockdowned either Src or MDR-1 using their specific siRNAs as shown in the Fig. 7E. Src knock-down decreased MDR-1 levels in the actinomycin D-treated cells. Consequently, actinomycin D-induced PARP cleavage was augmented in the Src knock-downed cells and in the MDR-1 knock-downed cells (Fig. 7E). Next, we forced expression of HA-tagged active Src to determine the importance of Src activity in drug resistance. Consistent with the results above, Rp1 and actinomycin D reduced Src phosphorylation in control vector-transfected cells. However, Src phosphorylation remained high even with Rp1 and actinomycin D co-treatment in active Src-expressing cells (Fig. 8A). Rp1 and actinomycin D also reduced cell viability in the vector-transfected cells, and active Src expression considerably attenuated this phenomenon (Fig. 8B). In addition, g-H2AX foci formation was evident in untransfected cells treated with Rp1 and actinomycin D but was abolished in active Src-expressing cells (green) (Fig. 8C). Furthermore, MDR-1 staining in untransfected cells was decreased by Rp1 and actinomycin D treatment but remained elevated in active Src-expressing cells (Fig. 8D). All these data indicate that Rp1 exerts its synergistic

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Fig. 4. Effect of Rp1 on MDR-1 levels and actinomycin D-induced DNA damage. (A) Serum-starved DXR cells were treated with 1 mM actinomycin D, 5 mM Rp1, or both 1 mM actinomycin D and 5 mM Rp1 for 16 h. After treatment, the cells were fixed, permeabilized, and subjected to immunofluorescence analysis using anti-g-H2AX antibody. Nuclei were stained with Hoechst 33342. Magnification: 40  (zoom = 2  ). Scale bar = 10 mm. (B) Serum-starved DXR cells were treated as in (A) for the indicated times, and cell lysates were subjected to immunoblot analysis using anti-g-H2AX, anti-H2AX and anti-b-actin antibodies. Levels of g-H2AX normalized by H2AX are summarized as histograms of mean  S.D. of three independent experiments. (p < 0.05, * versus actinomycin D only at each time point). (C) Serum-starved DXR cells were treated as described in (A) and stained for MDR-1 and GM-1 using anti-MDR-1 antibody and CTxB-Alexa 555. Nuclei were stained with Hoechst 33342. Magnification: 40  (zoom = 2  ). Scale bar = 10 mm. (D) Serum-starved DXR cells were treated with the indicated concentrations of actinomycin D and 5 mM Rp1. Protein extracts from each treatment were subjected to immunoblot analysis using anti-MDR-1 and anti-b-actin antibodies, and mRNA expression was analyzed by RT-PCR. b-actin was used as a loading control. MDR-1 levels were determined by densitometry and data are summarized as histograms of mean  S.D. of three independent experiments. (p < 0.05, * versus actinomycin D only). (E) DXR cells were treated with 1 mM actinomycin D, 5 mM Rp1, or both 1 mM actinomycin D and 5 mM Rp1 for 24 h. After treatment, DXR cells were fractionated into Triton X-100-soluble (S) and insoluble (IS) fractions, and then equal volumes of fractions were subjected to immunoblot analysis. Calnexin and caveolin-1 were used as markers for soluble and insoluble fractions, respectively. Expression of MDR-1 and caveolin-1 was determined by densitometry analysis of three independent immunoblots (p < 0.05, * versus actinomycin D only). Similar results were observed in independent experiments.

effect with anti-cancer drugs by altering lipid raft distribution and thus downregulating drug resistance-related proteins, such as MDR-1 and Src. 4. Discussion Multidrug resistance is a major cause of treatment failure in cancer, and overcoming multidrug resistance is a prerequisite for effective chemotherapy. In the present study, we demonstrated that ginsenoside Rp1, a semi-synthesized derivative of a ginseng saponin, can overcome drug resistance by inhibiting MDR-1 activity. We showed that Rp1, structurally similar to cholesterol, can modulate cholesterol-enriched membrane microdomains, lipid rafts, where MDR-1 is present, resulting in a shift of MDR1 from lipid rafts to non-raft fractions. Combining Rp1 and actinomycin D synergistically induced cell death by downregulating MDR-1 protein levels and activity through Src inactivation. Cholesterol is a critical lipid for the structure and function of lipid rafts, which function as a platform for signaling molecules [11,28]. MDR-1 is a lipid raft-associated protein, and altering total cellular cholesterol can modulate MDR-1 activity. For example, cholesterol depletion using MbCD induces the accumulation of

intracellular doxorubicin and increases the cytotoxic effect of doxorubicin in both drug-sensitive and multidrug-resistant HL-60 cells [29]. In addition, cholesterol depletion shifts MDR-1 from raft fractions to non-raft fractions [30,31] and thus increases rhodamine 123 accumulation, which is reversed by cholesterol supplementation in L-MDR cells overexpressing MDR-1 [31]. These studies indicate that cholesterol content and lipid raft integrity are important for MDR-1 activity. Ginsenosides may act as lipid raft modulators because they are structurally very similar to cholesterol and the ginsenosides Rh1, Rh2, Rg3, and Rg5 are known to have anti-tumor effects [32]. They exert their anti-tumor effects by regulating multiple signaling pathways, such as death receptor induction, caspase activation, angiogenesis inhibition, and autophagy induction [33]. In addition, our previous study demonstrated that Rh2 changes lipid raft distribution and induces lipid raft internalization and Akt inactivation, which are responsible for Rh2-mediated apoptosis in the human epidermoid carcinoma cell line, A431 [7]. This indicates that the anti-tumor effects of ginsenoside Rh2 are associated with changes in lipid raft-associated molecular events. We also demonstrate in the present study that Rp1 can alter lipid rafts and thus impair MDR-1 activity, leading to drug sensitivity.

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Fig. 5. Effect of cholesterol addition on increased actinomycin D sensitivity by Rp1. (A) Serum-starved DXR cells were treated with 1 mM actinomycin D, 5 mM Rp1, or both 1 mM actinomycin D and 5 mM Rp1. At 2 h after treatment, 0.1 mM cholesterol was added to actinomycin D and Rp1-treated cells. Images were taken using phase contrast microscopy. Magnification: 5  . Scale bar = 100 mm. (B) DXR cells were treated as described in (A), and cell growth inhibition was measured by MTS assay. Data are presented as the averages of triplicate measurements, with error bars representing standard deviations (*, P < 0.05). (C) DXR cells were treated as described in (A), and equal amounts of cell lysates were subjected to immunoblot analysis using anti-MDR-1, anti-PARP, and anti-b-actin antibodies. Cleaved PARP and MDR-1 were determined by densitometry and data are summarized as histograms of mean  S.D. of three independent experiments (p < 0.05, * versus actinomycin D only, ** versus actinomycin D plus Rp1). (D) DXR cells were treated as in (A), and rhodamine123 accumulation was monitored as described in Section 2. (E) DXR cells were treated as described in (A), and cellular proteins were subjected to immunoblot analysis using anti-g-H2AX and anti-b-actin antibodies. g-H2AX was determined by densitometry and data are summarized as histograms of mean  S.D. of three independent experiments (*, p < 0.05). (F and G) DXR cells treated as in (A) were fixed, stained with anti-g-H2AX antibody or isotype IgG antibody (F), or stained with anti-MDR-1 antibody and CTxB-Alexa 555 (G), and analyzed by confocal microscopy. Hoechst 33342 was used as a nuclear marker. Magnification: 40  (zoom = 2  ). Scale bar = 10 mm. These experiments were performed independently, with similar results.

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Fig. 6. Effect of lipid raft modulation on sensitivity to actinomycin D. (A) Serum-starved DXR cells were treated with 1 mM actinomycin D in the presence of the indicated concentrations of MbCD or simvastatin for 24 h, and cell growth inhibition was assessed by MTS assay. Data are presented as the averages of triplicate measurements, with error bars representing standard deviations (*, P < 0.05). (B) Serum-starved DXR cells were treated with 1 mM actinomycin D, 1 mM MbCD, 5 mM simvastatin, 1 mM actinomycin D plus 1 mM MbCD, or 1 mM actinomycin D plus 5 mM simvastatin for 24 h before the cells were analyzed by microscopy. Magnification: 5  . Scale bar = 100 mm. (C) Serum-starved DXR cells were treated as in (B) for 16 h and then fixed. Cells were permeabilized, stained with CTxB-Alexa 488 and anti-g-H2AX antibody, and analyzed by confocal microscopy. Nuclei were stained with Hoechst 33342. Magnification: 40  (zoom = 2  ). Scale bar = 10 mm. (D and E) DXR cells were treated as in (B), and samples from each treatment were subjected to immunoblot analysis using anti-MDR-1 and anti-PARP antibodies. Cleaved PARP was determined by densitometry and data are summarized as histograms of mean  S.D. of three independent experiments. (p < 0.05, * versus actinomycin D only). Similar results were observed in three independent experiments.

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Fig. 7. Association of Src activity with drug resistance. (A) Serum-starved DXR cells were treated with the indicated reagents for 24 h. Cell lysates were subjected to immunoblot analysis using anti-p-Src, anti-c-Src, and anti-b-actin antibodies. p-Src was determined by densitometry and data are summarized as histograms of mean  S.D. of three independent experiments (p < 0.05, * versus actinomycin D only, ** versus actinomycin D plus Rp1). (B) DXR cells were treated with actinomycin D or actinomycin D plus various concentrations of PP2 for 24 h, and cell growth inhibition was assessed by MTS assay. Data are presented as the averages of triplicate measurements, with error bars representing standard deviations (*, P < 0.05). (C) DXR cells were treated as in (B), and cell lysates were subjected to immunoblot analysis using MDR-1-, p-Src-, c-Src-, PARP-, and bactin-specific antibodies. MDR-1 and cleaved PARP were determined by densitometry and data are summarized as histograms of mean  S.D. of three independent experiments (p < 0.05, * versus actinomycin D only). (D) DXR cells were treated with 1 mM actinomycin D, 10 mM PP2, or both actinomycin D and PP2. Rhodamine 123 accumulation was monitored as described in Section 2. (E) DXR cells were transfected with si-RNAs for negative control (NC), MDR-1, or Src for 24 h, followed by actinomycin D treatment for 24 h. Cell lysates were subjected to immunoblot analysis using anti-MDR-1, anti-c-Src, anti-PARP, and anti-b-actin antibodies. Cleaved PARP was determined by densitometry and data are summarized as histograms of mean  S.D. of three independent experiments (*, p < 0.05). These experiments were performed twice with similar results.

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Fig. 8. Effect of Src activation on Rp1-induced drug sensitivity. (A) DXR cells were transfected with empty vector or HA-tagged, active Src plasmid for 20 h and cells were serum-starved for 4 h. Cells were then treated with 1 mM actinomycin D, 5 mM Rp1, or both 1 mM actinomycin D and 5 mM Rp1 for 24 h. Equal amounts of protein extracts were subjected to immunoblot analysis using anti-HA, anti-p-Src, and anti-b-actin antibodies. Active Src expression (HA-tag) and Src activity (p-Src) were determined by densitometry and data are summarized as histograms of mean  S.D. of three independent experiments (p < 0.05, * versus vector). (B) Serum-starved DXR cells treated as in (A) were analyzed by MTS assay for cell growth inhibition. Data are presented as the averages of triplicate measurements, with error bars representing standard deviations (*, P < 0.05). (C) DXR cells were transfected with active Src plasmid and treated with 1 mM actinomycin D plus 5 mM Rp1 for 12 h. Cells were stained with anti-HA and anti-g-H2AX antibodies and then visualized by confocal microscopy. Magnification: 40  (zoom = 2  ). Scale bar = 10 mm. (D) DXR cells treated as in (C) were stained with anti-HA and anti-MDR-1 antibodies, then examined by confocal microscopy. Magnification: 40  (zoom = 2  ). Scale bar = 10 mm. Similar results were obtained in three separate experiments.

Rp1 was semi-synthesized from crude ginsenosides (e.g., Rg5 and Rk1) and it showed improved stability and anti-tumor effects in vitro and in vivo [22,23]. Here, we tested whether Rp1 modulates lipid rafts and thus alters MDR-1 activity in multidrug-resistant DXR cells. A remarkable finding in our investigation is that Rp1 induces lipid raft clustering and decreases MDR-1 activity, thus sensitizing drug-resistant cells to anticancer drugs. Rp1 caused the redistribution of both lipid rafts and MDR-1, leading to intracellular accumulation of rhodamine 123 and doxorubicin (Fig. 2), indicating that Rp1 alters not only MDR-1 distribution but also inhibits MDR-1 activity. Accordingly, the combination of Rp1 with anti-cancer drugs resulted in enhanced apoptosis. More interestingly, Rp1 more effectively induced drug sensitivity than Rg3 or Rh2 (Fig. 2B), which are known to reverse drug resistance [19,34].

The NCI/ADR-RES cell line expresses high levels of ABCB1 (MDR-1) mRNA and almost no ABCB4 and ABCB5 mRNA [35]. NCI/ ADR-RES cells are doxorubicin-resistant cells established from the OVCAR-8 ovarian cancer cell line [36]. DXR cells were derived from the NCI/ADR-RES cell line by further selection with up to 50 mM of doxorubicin. DXR cells highly express MDR-1 (Fig. 1D) with very low levels of MRP and BCRP proteins (data not shown). Studies have shown that acquisition of drug resistance is accompanied by upregulation of lipids and proteins that constitute lipid rafts and caveolar membranes [37]. Lipid raft levels appeared to be slightly higher in DXR than in OVCAR-8 cells with little changes in caveolin-1 levels (data not shown). Although DXR cells are resistant to anti-cancer drugs (Fig. 1), Rp1 itself induced apoptosis of both OVCAR8 and DXR cells to a similar degree at the concentration of 10 mM (Fig. 2C), indicating that Rp1 is not an

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MDR-1 substrate. Although 5 mM Rp1 affected lipid raft distribution (Fig. 2), it fails to induce much apoptosis. Therefore, we used 5 mM Rp1 for the co-treatment experiments. Actinomycin D causes DNA damage and induces histone gH2AX foci formation [27], which is a marker of DNA damage [38] and pharmaco-dynamic biomarker to monitor the efficacy of anticancer drug [39]. Although neither 1 mM actinomycin D alone nor 5 mM Rp1 alone induced g-H2AX foci formation, the two together caused g-H2AX foci formation and an increase in g-H2AX protein levels. The synergistic effect between Rp1 and actinomycin D appeared to be due to decreased MDR-1 activity, as evaluated by intracellular rhodamine 123 retention. Synergistic effect of Rp1 is not limited to actinomycin D because Rp1 enhanced doxorubicinand paclitaxel-induced cell growth inhibition (Fig. 3A and data not shown). We propose that Rp1 causes lipid raft modulation. Rp1 might compete with cholesterol for lipid raft incorporation because its structure is similar to that of cholesterol, thereby altering lipid raft feature. Lipid rafts are liquid-ordered domains of the membrane with decreased fluidity and membrane cholesterol content influences membrane fluidity [40]. By competing with cholesterol, Rp1 might alter membrane fluidity, which inactivates MDR-1 activity. Alternatively, the altered localization of MDR-1 could contribute to Rp1-induced MDR-1 inactivation because Rp1 caused translocation of MDR-1 out of lipid rafts (Fig. 4). MDR-1 shift from lipid rafts to non-raft fractions has been correlated with MDR-1 inhibition induced by MbCD, rituximab, the anti-CD20 antibody, and an anti-CD19 antibody [25,41,42]. These data indicate that MDR-1 localization, at least in the intact lipid rafts, is important for MDR-1 function and thus MDR-1 mislocalization can lead to MDR1 inactivation and drug sensitivity. Interestingly, Rp1 and actinomycin D together downregulated MDR-1 protein levels without a decrease in MDR-1 RNA levels (Fig. 4). However, cholesterol addition recovered MDR-1 levels, distribution, and activity, and thus attenuated cell death induced by Rp1 and actinomycin D together (Fig. 5). Previously, we demonstrated that MbCD, a lipid raft-disrupting agent causes lipid raft internalization, which is reversed by cholesterol [8]. MDR-1 down-regulation is probably due to Rp1-induced lipid raft trafficking. It is possible that Rp1 alters lipid rafts and causes their internalization. Internalized lipid rafts might fuse to lysosome unless cholesterol is supplemented. We are currently investigating this possibility. Src is one of the best-studied targets for cancer therapy [43]. Acylated Src family kinases are localized to lipid rafts, and lipid raft-specific knockdown of Src inhibits cell adhesion and cell cycle in breast cancer cells [44]. Numerous studies have demonstrated that Src overexpression and Src activity are associated with resistance to chemotherapeutics [45,46]. Upregulation of MRP-1 in the lipid rafts mirrors that of glucosylceramide during multidrug resistance acquisition [47]. Glucosylceramide synthase was also shown to upregulate MDR-1 expression through Src and b-catenin signaling pathways [48]. In addition, Src overexpression is responsible for nilotinib resistance in chronic myeloid leukemia cell lines [49], and a Src inhibitor, sacratinib, reverses MDR-1mediated drug resistance in MCF-7/Adr cells [50], indicating that elevated Src activity contributes to multidrug resistance. Interestingly, MDR-1 expression itself can contribute to drug resistance via suppression of caspase activation in the absence of ATP-dependent drug efflux [51], indicating that MDR-1 expression levels are important for drug resistance. Here, we showed that although actinomycin D alone reduced Src activity, co-treatment with Rp1 almost abolished Src activity (Fig. 7) and that overexpression of active Src attenuated Rp1-mediated drug sensitivity (Fig. 8). When DXR cells were treated with actinomycin D and Rp1 together, MDR-1 protein levels were downregulated, whereas MDR-1 was slightly increased when treated with either agent alone (Fig. 4).

Moreover, overexpression of active Src maintained MDR-1 levels when DXR cells were treated with actinomycin D and Rp1 together (Fig. 8) and knock-down of Src by si-RNA decreased MDR-1 expression in the actinomycin D-treated cells, and thus sensitized cells to actinomycin D (Fig. 7). Therefore, inactivation of Src could be responsible for Rp1-mediated drug sensitivity by regulating MDR-1 levels. We cannot rule out the possibility that Rp1 might directly inhibit MDR-1 activity. Because multidrug resistance is due to both drug efflux by the overexpression of ABC transporters, such as MDR-1, and the molecular complexity of cancer, combination therapy is becoming more important to maximize anti-tumor effects and to decrease side effects [52,53]. In this regard, we demonstrated that combination of Rp1 with actinomycin D at low doses synergistically induces cell death through lipid raft modulation, MDR-1 downregulation, and Src inactivation. Our findings indicate that combination therapy with Rp1 may be a useful strategy to overcome chemotherapy resistance.

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Please cite this article in press as: Yun U-J, et al. Lipid raft modulation by Rp1 reverses multidrug resistance via inactivating MDR-1 and Src inhibition. Biochem Pharmacol (2013), http://dx.doi.org/10.1016/j.bcp.2013.02.025