RESEARCH PAPER Cancer Biology & Therapy 16:1, 149--159; January 2015; © 2015 Taylor & Francis Group, LLC Wnt pathway activation and ABCB1 expression account for attenuation of Proteasome inhibitor-mediated apoptosis in multidrug- resistant cancer cells Kowit Yu Chong1,2,3,*, Chih-Jung Hsu1, Tsai-Hsien Hung2, Han-Shu Hu1, Tsung-Teng Huang4, Tzu-Hao Wang5, Chihuei Wang6, Chuan-Mu Chen7,8,9, Kong Bung Choo10, and Ching-Ping Tseng1,2,3 1Department of Medical Biotechnology and Laboratory Science; College of Medicine; Chang Gung University; Tao-Yuan, Taiwan, Republic of China; 2Graduate Institute of Biomed- ical Sciences; Division of Biotechnology; College of Medicine; Chang Gung University; Tao-Yuan, Taiwan, Republic of China; 3Molecular Medicine Research Center; College of Medicine; Chang Gung University; Tao-Yuan, Taiwan, Republic of China; 4Center for Molecular and Clinical Immunology; College of Medicine; Chang Gung University; Tao-Yuan, Taiwan, Republic of China; 5Department of Obstetrics and Gynecology; Chang-Gung Memorial Hospital; Lin-Kou Medical Center; Tao-Yuan, Taiwan, Republic of China; 6Depart- ment of Medical Biotechnology; Kaohsiung Medical University; Kaohsiung, Taiwan, Republic of China; 7Department of Life Sciences; National Chung Hsing University; Taichung, Taiwan, Republic of China; 8Agricultural Biotechnology Center; National Chung Hsing University; Taichung, Taiwan, Republic of China; 9Rong-Hsing Translational Medicine Center; National Chung Hsing University; Taichung, Taiwan, Republic of China; 10Department of Preclinical Sciences; Faculty of Medicine and Health Sciences and Centre for Stem Cell Research; Universiti Tunku Abdul Rahman; Selangor, Malaysia Keywords: apoptosis, cancer, cell cycle, multiple drug resistance, proteasome inhibitor, Wnt Multiple drug resistance (MDR) is a major obstacle to attenuating the effectiveness of chemotherapy to many human malignancies. Proteasome inhibition induces apoptosis in a variety of cancer cells and is recognized as a novel anticancer therapy approach. Despite its success, some multiple myeloma patients are resistant or become refractory to ongoing treatment by bortezomib suggesting that chemoresistant cancer cells may have developed a novel mechanism directed against the proteasome inhibitor. The present study aimed to investigate potential mechanism(s) of attenuation in a MDR cell line, MES-SA/Dx5. We found that compared to the parental human uterus sarcoma cell line MES-SA cells, MES-SA/Dx5 cells highly expressed the ABCB1 was more resistant to MG132 and bortezomib, escaping the proteasome inhibitor-induced apoptosis pathway. The resistance was reversed by co-treatment of MG132 and the ABCB1 inhibitor verapamil. The data indicated that ABCB1 might play a role in the efflux of MG132 from the MES-SA/ Dx5 cells to reduce MG132-induced apoptosis. Furthermore, the canonical Wnt pathway was found activated only in the MES-SA/Dx5 cells through active b-catenin and related transactivation activities. Western blot analysis demonstrated that Wnt-targeting genes, including c-Myc and cyclin D1, were upregulated and were relevant in inhibiting the expression of p21 in MES-SA/Dx5 cells. On the other hand, MES-SA cells expressed high levels of p21 and downregulated cyclin D1 and caused cell cycle arrest. Together, our study demonstrated the existence and participation of ABCB1 and the Wnt pathway in an MDR cell line that attenuated proteasome inhibitor-induced apoptosis. Introduction involvement of some unknown alternative mechanisms of drug resistance in cancer cells which seemed to be adverse to the phar- Multidrug resistance (MDR) of cancer cells has become a bar- macological properties of the ABCB1 inhibitors tested.2 An inno- rier to clinical applications of anticancer agents.1 P-glycoprotein vative approach of proteasome inhibition capable of inducing 1 (permeability glycoprotein, abbreviated as P-gp or Pgp), also apoptosis in multiple myeloma cells has been reported.3 The pro- known as multidrug resistance protein 1 (MDR1) or ATP-bind- teasome inhibitor, bortezomib (VelcadeÒ, formerly known as PS- ing cassette sub-family B member 1 (ABCB1) or cluster of differ- 341), inhibited the NFkB pathway leading to decreased cell pro- entiation 243 (CD243), is a glycoprotein that in humans is liferation and induction of apoptosis. Bortezomib obtained FDA encoded by the ABCB1 gene. Since ABCB1 is highly related to approval in 2005 for clinical treatment against relapsed multiple MDR in cancer cells, ABCB1 inhibitors have been tested in clini- myeloma and mantle cell lymphoma.4,5 Despite successes with cal trials in attempts to circumvent MDR. Unfortunately, such this drug, some patients develop resistant or become refractory to attempts have failed in the phase III trials because of possible ongoing treatment; clinical responses to bortezomib in other *Correspondence to: Kowit Yu Chong; Email: [email protected] Submitted: 08/25/2014; Revised: 10/29/2014; Accepted: 11/09/2014 http://dx.doi.org/10.4161/15384047.2014.987093 www.taylorandfrancis.com Cancer Biology & Therapy 149 hematologic malignancies and solid tumors remain low.6,7 To Results date, several mechanisms of resistance to proteasome inhibitors have been reported, including upregulation of heat shock pro- Cytotoxic effects of proteasome inhibitors on multidrug- teins,8 increased activity of the aggresome pathway,9 mutations resistant cancer cells or overexpression of the b5 unit of the proteasome catalytic To understand the damage caused by proteasome inhibi- unit10 and overexpression of ABCB1.11,12 tors in multidrug-resistant cancer cells, different concentra- Combined use of an ABCB1 inhibitor and bortezomib was tions of proteasome inhibitors, MG132 and bortezomib, shown to increase anticancer cytotoxicity in Ewing’s family were used to treat MES-SA and MES-SA/Dx5 cancer cells. tumors cells.11 Furthermore, siRNA knockdown of ABCB1 Results obtained in MTT assays (Fig. 1A and 1B)andlive/ expression in K562 cells rendered treated cells to be more sensi- dead viability assay (Fig. 1C and 1D) were consistent. The tive to bortezomib cytotoxicity; based on this observation, borte- survival rate of both cell lines declined with increasing drug zomib has been considered as a possible ABCB1 substrate.12,13 concentrations, and the half maximal inhibitory concentra- On the other hand, some studies have indicated that treating tions (IC50) of the 2 cell lines were significantly different. m multidrug-resistant cancer cells with a proteasome inhibitor can The IC50 of MG132 treatment was 0.2 MinMES-SAand m alleviate the effects of ABCB1 protein translation and posttrans- 2 M in MES-SA/Dx5 cells (Fig. 1A). The IC50 of bortezo- lational modifications thereby facilitating the accumulation of mib treatment was 6 nM in MES-SA cells and 300 nM in immature ABCB1, and consequently reducing signs of multidrug MES-SA/Dx5 cells (Fig. 1B), respectively. Thus, these results resistance.12,14,15 indicated that compared to data on the MES-SA cells, multi- It has also been shown that human DLD1 colon cancer cells drug-resistant MES-SA/Dx5 cells seemed to possess a higher induce ABCB1 gene expression through T-cell Factor 4 and the tolerance against proteasome inhibitors. Furthermore, the b-catenin transcription factor complex, causing ABCB1 expres- MES-SA/Dx5, which overexpressed ABCB1 but not ABCC1 sion levels to increase.16 Furthermore, studies have indicated that and ABCG2, were selected from MES-SA cell lines by doxo- a T-cell Factor 4 and b-catenin transcription factor complex rubicin treatment.23,24,25 Western blot analysis showed that bind at a site located at nucleotides -261 to -1813 upstream of ABCB1 expression occurred only in MES-SA/Dx5 cells, and the promoter of the ABCB1 gene.17 Thus, ABCB1 is a known there was significant accumulation of the ABCB1 protein in downstream target gene of the Wnt pathway. Furthermore, T- coincidence with the elevation of MG132 concentration cell Factor 4 assists b-catenin translocation from the cytosol to (Fig. 1E). Our data, thus, indicate that a direct association the nucleus to participate in the activation of downstream target of multidrug resistance in MES-SA/Dx5 cells with ABCB1 genes such as c-Myc, cyclin D1, MMPs.18 Liang et al. proposed function to reduce the cytotoxic effects of proteasome that the proto-oncogene c-Myc could inhibit p21 expression, inhibitors. facilitating cell cycle progression.19 Another study demonstrated To further verify the correlation between doxorubicin and that MG132 caused glioma cancer cells to remain in the G2 ABCB1, both cell types were treated with doxorubicin in the phase.20 Furthermore, p53 and p21 co-expression caused the cell presence of an ABCB1 inhibitor, verapamil. MES-SA cells cycle in cancer cells to remain at the G2 phase, preventing the showed similar survival rates regardless of the presence of verapa- cells from entering mitosis and, thus, arresting cell growth.21,22 mil (Fig. 2A). As for the MES-SA/Dx5 cells, following verapamil Together, these findings suggest that chemoresistant cancer cells treatment, the survival rate was reduced to 60% (Fig. 2B) indi- may have developed a Wnt pathway-related mechanism directed cating that the ABCB1 inhibitor increased anticancer drug cyto- against the proteasome inhibitor-induced apoptosis. toxicity on MES-SA/Dx5 most likely by inhibiting the Hence, the multidrug-resistant cell line, MES-SA/Dx5, estab- extracellular efflux and exclusion of doxorubicin by ABCB1. We lished from a poorly differentiated human uterine sarcoma cell further investigated correlation between the proteasome inhibitor line, MES-SA cells, grown in the presence of doxorubicin and and ABCB1 by treating both cells with MG132 in the presence were overexpressing ABCB1 but not ABCC1 and of verapamil. Results showed that following 60-hour treatment ABCG2,23,24,25 was used in this study. Two proteasome inhibi- with MG132 and in the presence of verapamil, the survival rate tors, namely MG132 and Bortezomib, were used to investigate of the treated MES-SA cells decreased to 30% (Fig. 2C), and possible mechanism(s) of drug resistance developed in the multi- that of MES-SA/Dx5 to 80% (Fig. 2D).