RESEARCH PAPER Biology & Therapy 16:1, 149--159; January 2015; © 2015 Taylor & Francis Group, LLC Wnt pathway activation and ABCB1 expression account for attenuation of inhibitor-mediated 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 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 , 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 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 . 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 through T-cell Factor 4 and the tolerance against proteasome inhibitors. Furthermore, the b-catenin 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 -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- 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, and p21 co-expression caused the cell presence of an ABCB1 inhibitor, verapamil. MES-SA cells cycle in cancer cells to remain at the , 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). Compared to MES-SA drug-resistant cancer cells. We found that Wnt-targeting genes, cells, the survival rate of MES-SA/Dx5 cells was more greatly including c-Myc and cyclin D1, were upregulated and were rele- affected by verapamil. Furthermore, the DNA contents treated vant in inhibiting the expression of p21 in MES-SA/Dx5 cells. cells were analyzed with propidium iodine dye. sub-G1 apoptotic Moreover, we also found that the canonical Wnt pathway was population of MES-SA cells increased to 3% (Fig. 2E). and that only activated in ABCB1-overexpressed MES-SA/Dx5 cells of MES-SA/Dx5 to 15% (Fig. 2F). Compared to MES-SA cells, through active b-catenin and its related transactivation activities the sub-G1 apoptotic population of MES-SA/Dx5 cells was sig- regulating the cell cycle. Together, our study demonstrated the nificantly affected by verapamil. These results verified that MES- existence and involvement of ABCB1 and that the Wnt pathway SA/Dx5 cells relied on ABCB1 to express multidrug resistance to in a MDR cell line would attenuate proteasome inhibitor- reduce the cytotoxic effects induced by proteasome inhibitors induced apoptosis. (Fig. 1E).

150 Cancer Biology & Therapy Volume 16 Issue 1 Involvement of the apoptosis pathway of proteasome inhibitor in triggering cell death of multidrug- resistant cancer cells To evaluate possible involvement of the apoptosis pathway in proteasome inhibitor-triggered cell death, the MES-SA and MES-SA/DX5 cancer cells were treated with respective IC50 concentrations of MG132, and the expression of the transcription factor p53, apoptosis enzyme 3 and 7 and the DNA repair enzyme, poly ADP-ribose polymerase (PARP), were analyzed. Western blot results showed that p53 protein expression levels increased along with elevated MG132 concentrations (Fig. 3A). An increase in p53 expression levels possibly indi- cates activation of the family apoptosis enzymes. Among such enzymes, caspase 3 performs a pivotal function in apoptosis signal transduc- tion.26 Caspase-Glo 3/7 assay was used to measure the activities of caspases 3 and 7 in both cell types. Results showed that in both MES-SA and MES-SA/Dx5 cell lines, activities of caspases 3 and 7 increased with increasing MG132 concentrations (Fig. 3C). Activation of the intracellu- lar caspase 3 usually involves loss of activity of certain enzymes, such as 27 DNA repair enzyme PARP. Results Figure 1. Multidrug-resistant MES-SA/Dx5 cancer cells is more resistant to proteasome inhibitors showed that when MG132 concentra- MG132- and bortezomib-induced cytotoxicity. Cells were incubated with the indicated concentra- tion was increased, the levels of intact tion of (A, C) MG132 or (B, D) bortezomib for 60 h. At the end of the drug treatment, cells were har- PARP declined gradually vested to measure viability using (A, B) the MTT assay, and (C, D) the live/dead viability assay. (E) Expression of ABCB1 of both cell lines after MG132 treatment at the indicated drug concentrations while the amounts of cleaved PARPs incubated for 60 h. ABCB1 was identified by Western blot analysis. The surviving fractions are expressed increased (Fig. 3B). Thus, the same as the mean § SD from 3 independent experiments. **P < 0 .05 indicates the differences between the apoptotic pathway of MG132 contrib- MG132- or bortezomib-treated cells and relative to the respective untreated controls. uted to in triggering cell death in both cancer cell lines and the effects are independent on multidrug resistance. cytosol to the nucleus to participate in the activation of the downstream target gene such as c-Myc, cyclin D1 and MMPs.18 Involvement of the Wnt pathway in regulating drug Results showed no notable difference in T-cell Factor 4 expres- resistance in MDR cancer cells against proteasome inhibitor sion levels in the 2 cell lines; most T-cell Factor 4 protein was Are there some unknown mechanisms that exist in ABCB1- located in the cytosol and not in the nucleus (Fig. 4B). We next overexpressed MDR MES-SA/Dx5 cells to attenuate proteasome evaluated whether T-cell Factor 4 and the b-catenin transcription inhibitors-induced apoptosis? Since ABCB1 is a known down- factor complex could regulate transcription of c-Myc and cyclin stream target gene of the Wnt pathway, we hypothesized that D1. The Wnt reporter gene system included TOPflash which MES-SA/Dx5 cells upregulated the expression of ABCB1 down- contained a wild-type b-catenin/ factor-binding elements, stream target genes through the Wnt pathway. Western blot whereas FOPflash contained a mutated b-catenin/T cell factor- results showed that b-catenin expression levels increased substan- binding elements for T-cell Factor 4 and the b-catenin transcrip- tially following proteasome inhibition in MES-SA/Dx5 com- tion factor complex. In MES-SA cells, of either the pared to MES-SA cells, especially in the nuclear fraction TOPflash or FOPflash reporter vector both resulted in relatively (Fig. 4A). T-cell Factor 4 assists b-catenin translocation from the low T-cell Factor 4 activities (Fig. 4C) due to low b-catenin

www.taylorandfrancis.com Cancer Biology & Therapy 151 Figure 3. Proteasome inhibitor-induced apoptosis through increased expression of p53, caspase 3/7 and cleaved PARP in a dose-dependent manner. (A-B) Expression of p53 (A) and PARP (B)in MES-SA and MES-SA/D£5 cells after MG132 treatment for 60 h exam- ined by Western blotting. (C) Caspase 3/7 activities of MES-SA and MES- Figure 2. ABCB1 inhibitor resensitizes the multidrug-resistant MES- SA/D£5 cells were detected by the Caspase-Glo 3/7 assay after 60 h SA/Dx5 cancer cells to proteasome inhibitor-induced cytotoxicity. MG132 treatment at the indicated dosages. The Caspase 3/7 activities (A-B) MES-SA (A) and MES-SA/Dx5 cells (B) were incubated with the indi- were detected by luminescence and were proportional to its intensity cated concentrations of doxorubicin and 2 mM verapamil for 60 h. (C-D) normalized by cell viability. Data are expressed as relative luciferase MES-SA (C) and MES-SA/Dx5 cells (D) were incubated with the indicated activity (RLU) as the mean § SD from 3 independent experiments. concentration of MG132 and 2 mM verapamil for 60 h. At the end of *P < 0 .05 and **P < 0 .01 indicate the differences between the MG132- treatment with MG132 or doxorubicin in the presence of the verapamil, treated cells and the respective untreated controls. cells were harvested for measurement of viability using the MTT assay. (E-F) MES-SA (E) and MES-SA/Dx5 cells (F) were incubated with the indi- m cated concentration of MG132 and 2 M verapamil for 48 h. At the end following TOPflash transfection was 150 times higher than the of treatment with MG132 in the presence of the verapamil, cells were subjected to cell cycle analysis by FACS after propidium iodide staining, intensity in the similarly-transfected MES-SA cells. These results as described in Materials and methods. The sub-G1 population repre- indicated that T-cell Factor 4 and the b-catenin transcription fac- sents apoptotic as well as dead cells. The surviving fractions are tor complex in the nucleus of MES-SA/Dx5 cells possessed the expressed as the mean § SD from 3 independent experiments. *P < 0 ability to regulate the transcription of b-catenin/Tcf-4 down- P < .05 and ** 0 .01 indicate the differences between the ABCB1 inhibi- stream target genes. Transfection of mutant b-catenin-expressing tor-treated cells and the respective untreated controls. plasmids into MES-SA/Dx5 cells also resulted in an elevation in luminescence intensity (Fig. 4C). MG132 treatment of TOP- expression in this cell line. On the other hand, in MES-SA/Dx5 flash-transfected MES-SA/Dx5 cells also led to increases in lumi- cells, transfection of the FOPflash negative control group also nescence intensity. MES-SA/Dx5 cells transfected with mutant resulted in low luminescence; however, luminescence intensity b-catenin and subsequently treated with MG132 resulted in the

152 Cancer Biology & Therapy Volume 16 Issue 1 highest luminescence intensity (Fig. 4C). Thus, the results revealed that besides upregulating b-catenin expression in the nucleus, the transcrip- tional functions of T-cell Factor 4 and the b-catenin transcription factor com- plex were also upregulated in MES-SA/ Dx5 cells to facilitate the activation of ABCB1 downstream target genes. To confirm the regulatory of the expression of ABCB1 downstream tar- get genes through the b-catenin, we deliver the indicated concentration of CTNNB1 shRNA to MES-SA/Dx5 cell, the TOP reporter assay indicated that Tcf transctivation activity were sig- nificant reduced in dose-dependent manner of CTNNB1 knockdown MES-SA/Dx5 cell (Fig. 4D). Tran- scriptional control of ABCB1 by the b-catenin/TCF response elements in its promoter site had been proved in the previous study.28 We further to evaluate the ABCB1 expression level and function in CTNNB1 knockdown MES-SA/Dx5 cell. The ABCB1-Luc reporter assay data showed that ABCB1 promoter transactivation activ- ity was reduced in dose-dependent manner of CTNNB1 knockdown MES-SA/Dx5 cell (Fig. 4D). Thus, the results revealed that besides upregulat- ing b-catenin expression in the Figure 4. The Wnt pathway is activated in multidrug-resistant MES-SA/Dx5 cancer cells through nucleus, the transcriptional functions active b-catenin and its related transactivation activities against proteasome inhibitor. (A-B) b of T-cell Factor 4 and the b-catenin Expression of the Wnt pathway ligand -catenin (A) and the transcription factor Tcf-4 (B) of nuclear and cytoplasmic fractions in MES-SA and MES-SA/D£5 cell lines after bortezomib treatment for 60 h transcription factor complex were also examined by Western blotting. (C) b-catenin/TCF transcription activities of MES-SA and MES-SA/D£5 upregulated in MES-SA/Dx5 cells to cells treated with MG132. The b-catenin/TCF transcription activities of the MG132-treated cells were facilitate the activation of ABCB1 detected by the TOP/FOP assay. The TOP-Flash or FOP-Flash reporter-transfected MES-SA and MES-SA/ downstream target genes. D£5 cells were incubated with different concentrations of MG132 for 60 h. MES-SA and MES-SA/D£5 cells transfected with the mutant-type b-catenin were used as controls. At the end of the treatment, cell lysates were harvested and were assayed for luciferase activities. The b-catenin/TCF transcription Involvement of cell cycle in activities were represented as a ratio of firefly/renilla. (D)The b-catenin/TCF transcription activities or regulating drug resistance in MDR ABCB1 transactivation activity of the CTNNB1 shRNA transfected MES-SA/Dx5 cells was detected by cancer cells against proteasome TOP assay or ABCB1 promoter–Luciferase reporter assay, respectively. Data are expressed as relative inhibitor luciferase activity (RLU) as the mean § SD from 3 independent experiments. *P < 0 .05 and **P < 0 To investigate whether MES-SA/ .01 indicate the differences between the MG132-treated cells or CTNNB1 shRNA transfected cells and the respective untreated controls. Dx5 cells enhanced resistance against proteasome inhibitors through the Wnt pathway-targeted downstream genes, such as c-Myc and cyclin D1, western blot analysis was performed lines; however, in higher treatment concentrations of MG132, the to assay the levels of these 2 proteins in MES-SA and MES-SA/ cyclin D1 expression level in MES-SA cells reduced rapidly, possi- Dx5 cells. The results showed higher expression levels in both c- bly due to the function of other regulating factors in the cancer Myc and cyclin D1 in these cells (Fig. 5A). Following MG132 cells. To explain the considerable reduction of cyclin D1 levels in treatment, c-Myc remained highly expressed in MES-SA/Dx5 MES-SA cells in high MG132 concentrations, we further exam- cells and displayed no effects to the cells even with increased drug ined the expression level of the p21 protein, a cell cycle regulator concentrations. The cyclin D1 expression levels increased responsible for cyclin D1 regulation. Western blot results showed markedly with increasing MG132 concentrations in the 2 cell that following an increase in MG132 concentration, p21

www.taylorandfrancis.com Cancer Biology & Therapy 153 Figure 5. Cell cycle-regulated resistance against proteasome inhibitor in the mul- tidrug-resistant MES-SA/Dx5 cancer cells (A) Expression of Wnt-targeting genes, c- Myc, cyclin D1 and a cell cycle regulator, p21, in MES-SA and MES-SA/Dx5 cells after MG132 treatment for 60 h examined by Western blotting. (B) Expression of ABCB1, b-catenin, c-Myc, cyclin D1 and p21 in CTNNB1 shRNA transient transfected MES- SA/Dx5 cell examined by Western blotting. (C) Cells were incubated with the indicated concentrations of MG132 for 60 h. At the end of the treatment, cells were harvested for measurement of viability using the MTT assay and cell cycle distribution using Flow cytometry/PI staining. (D) Cells were trans- fected with the indicated concentrations of CTNNB1-shRNA plasmid for 24 h, and incu- bated with the indicated concentrations of MG132 for 60 h. At the end of the treat- ment, cells were harvested for measure- ment for of viability by using (D) the MTT assay and (E) the colony formation assay. The colony formation was scored after 10 d incubation. Data represent mean § SD of 3 independent experiments. *P < 0 .05 and **P < 0 .01 indicate differences between the MG132-treated cells or CTNNB1 shRNA transfected cells and the respective untreated controls.

deliver the CTNNB1 shRNA vector to MES-SA/Dx5 cell. The Western blot data indicated that ABCB1, b-catenin, c-Myc and cyclin D1 were significant decreased in dose-dependent manner of CTNNB1 knockdown MES-SA/Dx5 cell (Fig. 5B). Following MG132 treatment, MES- SA cells expressed a large amount of the cell cycle regulator protein p21, which could subsequently regulate cell cycle distribution of the cell population.21 As for MES-SA/Dx5 cells expressing the Wnt pathway protein, whether MG132 affected cell cycle distribution was next investigated by PI staining. Results showed that following an increase in the MG132 concentration, cell cycle distribution in MES-SA cells showed a expression levels in MES-SA cells markedly increased (Fig. 5A). considerable increase in the G2 phase The results clearly revealed that after cell injury induced by from 12% to 64%, and a reduction of the from 31% to MG132, MES-SA cells expressed high levels of p21 leading to 7%. However, following MG132 treatment, the cell cycle distri- inhibition of cyclin D1 expression. In contrast, the c-Myc expres- bution in MES-SA/Dx5 cells displayed similar results: increase in sion level was also high, causing p21 expression to become consid- the G2 phase from 20% to 63% and reduction in the S phase erably inhibited in MES-SA/Dx5 cells. Furthermore, to confirm from 38% to 22% (Fig. 5C). Thus, even after the damage caused the regulatory of the expression of Wnt pathway-targeted down- by MG132, MES-SA/Dx5 cells could still sustain normal cell stream genes in MDR cancer cells through the b-catenin, we cycle progression and increase cancer cell proliferation.

154 Cancer Biology & Therapy Volume 16 Issue 1 Possible inhibition of b-catenin leading to resensitizition of complex as well as ABCB1 expression in MES-SA/Dx5 cells. MG132 toxicity in drug-resistant MES-SA/Dx5 cells was examined. Regardless of MG132 treatment, b-catenin expression increased About 40% decreased cell viability was observed when MES-SA/ markedly in the nucleus of MES-SA/Dx5 cancer cells relative to Dx5 cells were treated with indicated concentration of MG132 data obtained with MES-SA cells (Fig. 4). Western blot results in MTT assays (Fig. 5D) and colony formation assays (Fig. 5E), showed b-catenin bands with higher MW in the cytosol than in the viability and colony formation ability significantly decreased the nucleus, possibly due to phosphorylation of b-catenin in the in a dose-dependent manner of MG132 treated CTNNB1- cytosol. Smaller protein bands (marked with asterisks in Fig. 4A) shRNA transfected MES-SA/Dx5 cells (Fig. 5D-E). Our data, are possibly b-catenin products of caspase reactions. Similar therefore, support the proposition that expression of the Wnt b-catenin smaller-fragment proteins have also been previously pathway contribute significantly to the mitigation of the cyto- reported in a study using MG132 treatment to activate caspases toxic damages caused in the MES-SA/Dx5 cancer cells. in hepatocellular carcinoma cells.33 Treatment of MES-SA/Dx5 cancer cells with the proteasome inhibitor MG132 induced upregulated transcriptional activities Discussion of T-cell Factor 4 and b-catenin transcription factor complex on downstream target genes (Fig. 5). ABCB1 is a known down- Several studies have reported using proteasome inhibitors as stream target gene of the Wnt pathway. Hence, data reported in ABCB1 substrates in their studies.11,12,15,29-32 The drugs this study have led us to propose that MG132 activation of the MG132 and bortezomib applied in this study both contain ben- Wnt pathway possibly increases ABCB1 expression. This propo- zene rings and have molecular weights of approximately 400 Da, sition is consistent with the Western blot results indicating ele- which are fitting as ABCB1 substrates. We noted that doxorubi- vated ABCB1 expression in conjunction with MG132 cin resistance-selected MES-SA/Dx5 exhibit 10-fold and 25-fold concentration (Fig. 1E). Moreover, this result also explains why cross-resistance to the proteasome inhibitors MG132 and borte- MG132 treatment did not inhibit ABCB1 expression. zomib, respectively. Our studies was consistent with original Treatment with a proteasome inhibitor not only causes apopto- studies by Sharma et al, 1992 showed that peptide aldehyde sis in cancer cells, but also results in cell-cycle arrest. The relation- structures like MG132 are bona fide substrates for ABCB1,29 but ship between cell cycle progression and apoptosis is a boronated peptide compound like bortezomib has relative poor complementary.34 In the apoptosis experiments using 2 cancer cell ABCB1 substrate affinity resulting in moderate resistance levels lines reported here yielded similar results showing increases in p53 (approx. 5-fold). Besides these differences it should also be taken expression in response to increases in the concentration of the pro- into account that MG132 target multiple catalytic proteasome teasome inhibitor (Fig. 3). Aside from inducing the activation of subunits,29 whereas bortezomib has preferential binding to the cell apoptosis proteins, p53 also indirectly regulates cell cycle pro- b5 unit of the proteasome catalytic unit.10 Moreover, these drugs gression.21,22 This is primarily achieved through the activation of could also efflux to outside of the . It has been the cell-cycles regulating p21 protein, thus, achieving the observed shown that siRNA knockdown of ABCB1 expression in K562 effects in cell cycle regulation. The p21 protein is an inhibitor of cells is more sensitive to bortezomib cytotoxicity and considered cyclin dependent kinase (CDK), and is arrested in the cell cycle bortezomib has been considered as a possible ABCB1 substrate.13 via CDK-cyclin inhibition sparing time for damaged DNA to Combined use of an ABCB1 inhibitor and bortezomib increases repair in the nucleus before continuing the cell cycle processes. anticancer cytotoxicity on Ewing’s family tumors cells13 and it is Studies have indicated that MG132 treatment of MES-SA assumed that bortezomib is a possible ABCB1 substrate cells caused accumulation of p21 and inhibition of intracellular drug.12,13 The published results are essentially similar with our cyclin D1 (Fig. 5A). Cyclin D1 is a cell cycle protein that forms study in the weakened cytotoxicity of proteasome inhibitors due a complex with CDK4 to regulate cell cycle progression from the to the presence of ABCB1 (Fig. 1). Subsequent to adding an G1 phase to the S phase. MES-SA/Dx5 cancer cells, however, ABCB1 inhibitor to MES-SA/Dx5 cells, cell survival rates were did not express p21 (Fig. 5A), mainly due to activation of the tar- reduced, though not as markedly as in the MES-SA cancer cells get gene c-Myc downstream of the Wnt pathway. Liang J et al. (Fig. 2). Therefore, MG132 and doxorubicin possible act via dif- (2003) proposed that the anticancer gene c-Myc could inhibit ferent mechanisms in cells. Alternatively, the combined applica- p21 expression, facilitating cell cycle progression.19 However, the tion of MG132 and an ABCB1 inhibitor results in drug function of p21 in cell cycle regulation following its accumula- interference due to their concerted action. tion remains to be investigated. Following proteasome dysfunction in cancer cells, human Previous studies demonstrated that MG132 caused glioma DLD1 colon cancer cells were shown to induce ABCB1 gene cancer cells to stay in the G2 phase.20 Furthermore, p53 and p21 expression through T-cell Factor 4 and b-catenin transcription co-expression caused the cell cycle in cancer cells to remain at the factor complex, causing increases in the ABCB1 expression lev- G2 phase, preventing them from performing mitosis and, thus, els.16 Furthermore, studies have indicated that a T-cell Factor 4 arresting cell growth.21,22 The results of our study (Fig. 5C) are and b-catenin transcription factor complex binding site is located consistent with the abovementioned conclusions indicating that, upstream of the ABCB1 gene promoter.17 Our study, therefore, in MES-SA cells, the S phase was notably reduced while the G2 targeted T-cell Factor 4 and b-catenin transcription factor phase increased. MES-SA/Dx5 cells, following the same

www.taylorandfrancis.com Cancer Biology & Therapy 155 treatment with MG132, displayed a gradual decrease in the S phase but the decrease was more substantial in the G2 phase (Fig. 5C) due to Wnt pathway activation of c-Myc, consequently inhibiting p21 expression. These results confirm that activated Wnt pathways in MES-SA/Dx5 cells render the cells resistant to the MG132 effects on the cell cycle enabling normal progression of the cell cycle and cancer cell proliferation. Following MG132 treatment, p21 in MES-SA cells accumu- lated considerably, causing a substantial increase in the G2 phase and a marked reduction in the S phase (Fig. 5C). This indicates that cells remaining in the G2 phase could not proceed into the M phase to perform mitosis. MES-SA/Dx5 cells lacked p21 expression, and while there was an increase in the G2 phase, S phase reduction was relatively limited (Fig. 5C). Thus, even after Figure 6. (A)proposed mechanism of attenuated Proteasome Inhibi- the damage caused by MG132, MES-SA/Dx5 cells could still sus- tor-induced cytotoxicity in the multidrug-resistant MES-SA/Dx5 can- tain normal cell cycle progression and increase cell proliferation. cer cells Since MES-SA/Dx5 and the parental MES-SA cancer cells both Taken together, Wnt pathway expression contributes positively to trigger similar apoptosis pathway after MG132 or bortezomib treatment, the mitigation of the cytotoxic damages caused by MG132 in the MES-SA/Dx5 cells efflux MG132 or bortezomib out of the cell via ABCB1 MES-SA/Dx5 cancer cells. expression, resulting in reduced MG132 or bortezomib cytotoxicity. MES- SA/Dx5 cells then increase the expression of the cancer-cell growth fac- Results from our study have indicated that MG132 treat- tors cyclin D1 and c-Myc through the Wnt pathway to subsequently ment caused an increase in the p53 expression levels, inhibit p21 regulation on the cell cycle. These events render MES-SA/Dx5 increased Caspase 3/7 activities and elevated amounts of cells capable of resisting cell cycle suspension in the G2 phase caused by fragmented PARP level in MES-SA cancer cells (Fig. 3C). MG132 or bortezomib, permitting the cancer cells to proliferate normally Furthermore, p53 regulation on the expression of the down- and consequently alleviating proteasome Inhibitor cytotoxicity. stream p21 gene results in cell-cycle arrest in the G2 phase leading to cessation of MES-SA cell growth in a proposed mechanism depicted in Figure 6. Though MES-SA/Dx5 and the parent MES-SA cancer cells both resulted in similar Western blot analysis apoptosis pathway after MG132 treatment, MES-SA/Dx5 Total cellular proteins were isolated from cell lines by the cells were able to transport MG132 out of the cell via PRO-PREPTM Protein Extraction Solution (INtRon, 17081). ABCB1 expression, resulting in reduced bortezomib cyto- Nuclear and cytoplasmic proteins were isolated from cell lines toxicity. MES-SA/Dx5 cells then increased the expression of using the NE-PER Nuclear and Cytoplasmic Extraction Reagents the cancer cell growth factors cyclin D1 and c-Myc through (Pierce, 78835). Western blot was performed as described previ- the Wnt pathway, subsequently inhibiting p21 regulation ously.35 Briefly, approximately 40 mg of total proteins was on the cell cycle. This rendered MES-SA/Dx5 cells capable loaded onto each lane and the proteins were separated in SDS- of resisting cell cycle suspension in G2 phase caused by PAGE gels. After electrophoresis, the resolved proteins were MG132 (Fig. 5C), permitting the cells to populate nor- transferred to PVDF membrane (EMD Millipore, IPVH00010) mally, and also alleviating MG132 cytotoxicity. In conclu- and subsequently stained with Ponceau S (Sigma-Aldrich, sion, MES-SA/Dx5 cancer cells resisted cytotoxicity of P7170) to confirm complete protein transfer. The membranes proteasome inhibitors through the functions of ABCB1 and were blocked with 5% nonfat dry milk in PBS-T (0.1% Tween- the Wnt pathway. 20 (Sigma-Aldrich, P1379) in PBS, pH7.4) for 1 hour and probed overnight with the following antisera at appropriate dilu- tions: 1:1000 dilution of anti-ABCB1 (Santa Cruz, sc-13131), Materials and Methods 1:500 dilution of anti-p53 (Invitrogen, 081129), 1:500 dilution of anti-PARP (Cell Signaling, 9542), 1:500 dilution of anti- Cell culture Lamin B1 (Santa Cruz, sc-56145), 1:500 dilution of anti-Tcf 4 The multiple drug-resistant cell line MES-SA/Dx5 (ATCC, (Santa Cruz, sc-13027), 1:500 dilution of anti-c-Myc (Santa CRL-1977) was established from the human sarcoma cell line Cruz sc-70469), 1:500 dilution of anti-cyclin D1 (Santa Cruz, MES-SA (ATCC, CRL-1976) in the presence of increasing sc-8396) 1:500 dilution of anti-p21 (Cell Signaling, 2946), a doxorubicin concentrations.23 All cell lines were continuously 1:1,000 dilution of the anti-b-catenin (Invitrogen, 138400) and maintained in McCoy’s 5A medium (Invitrogen, 16600) supple- 1:10,000 dilution of an anti-actin (Novus, NN600501) antise- mented with 10% fetal bovine serum (Invitrogen, 10099) and rum in PBS–T. The identification of each protein was achieved 1% Antimycotics (Invitrogen, 15240) under 95% air and 5% with the Western Lighting Plus Reagent (Perkin Elmer,  CO2 at 37 C. The MES-SA/Dx5 cell line was maintained in the NEL103E001EA) using an appropriate alkaline phosphatase- continuous presence of 1.7 mM doxorubicin (Sigma-Aldrich, conjugated secondary . The level of each protein in the D1515). protein gel blot analysis was detected by the LAS-3000

156 Cancer Biology & Therapy Volume 16 Issue 1 chemiluminescence detection device (FUJIFILM). To adjust for Colony formation assay loading differences, the optical density of each protein was nor- The cells were plated at a density of 5 £ 105 cells/well in a 6- malized to that of the b-actin band. well plate and grown for 24 h. After CTNNB1 shRNA transfec- tion, the cells were grown in the replaced fresh medium for 24 h. The cells were then treated with the indicated concentration of shRNA MG132 for 48 h. The cells were then replated at a density of The CTNNB1 shRNA clone (TRCN0000003845; 1000 cells/10 cm2 dish and grown for 7 to 8 d until discrete colo- GCTTGGAATGAGACTGCTGAT) targeted at the human nies were visualized. After washing with PBS, the colonies were b CTNNB1 transcript for catenin, and the control scramble stained with 0.5% crystal violet (Sigma-Aldrich, C3886) and shRNA clone (CCTAAGGTTAAGTCGCCCTCGCTC) in a counted. After cell counting, cell number was estimated by dis- TRC001-based shRNA pLKO.1 vector was purchased from the solving the crystal violet in 70% ethanol (4 ml/well) and then National RNAi Core Facility (Academia Sinica). optical density (OD) values was measured at 562 were used as previously reported.38 In each group, cells transfected with the control scramble shRNA were set at 100%. Results (mean § Transfection and luciferase assays standard error) represent data from triplicate wells. MES-SA or MES-SA/Dx5 cells were plated at a density of 1 £ 105 cells/well in a 24-well plate. For Tcf transactivation 36 activity assay as described previously, cells were co-transfected Caspase 3/7 assays with 800 ng TOP-Flash, FOP-Flash reporter (Addgene) or the MES-SA and MES-SA/Dx5 cells were plated at a density of 37 mutated b-catenin overexpresssion plasmid, p-cat T41AS45A. approximately 8 £ 103 cells/well in a 96-well plate. The cells and 1 ng phRL-TK (Promega, E2261) premixed with Lipofect- were then treated with the indicated dosages of bortezomib for amine 2000 (Invitrogen, 11668027) for 6 h. The indicated con- 60 h. At the end of the drug treatment, 100 ml Caspase-Glo 3/7 centration of bortezomib (Millennium Pharmaceuticals) was Reagent (Promega, G8091) was added to the treated cells in each then added to the transfected cells and incubated for 60 h. For well. After 30 min of incubation at room temperature, the rela- ABCB1 transactivation activity assay, cells were co-transfected tive luminescence unit (RLU) was measured by GLOMAX 20/ with indicated concentration of CTNNB1 shRNA DNA, 20 Luminometer (Promega) and as an indication of Caspase 3/7 800 ng ABCB1 reporter (Addgene), and 1 ng phRL-TK (Prom- apoptotic activity. ega, E2261). At the end of the incubation, cells were lysed by Passive Lysis Buffer and were assayed by the Dual-Luciferase Reporter Assay System (Promega, E1910). The luminescence Live/Dead viability assay intensity was measured by GLOMAX 20/20 Luminometer In preparation of flow cytometry, the cells were then treated (Promega). The firefly luciferase activities were adjusted for back- with the indicated concentration of various drugs in different sets ground luminescence and normalized to renilla luciferase activi- of experiments. The concentrations of verapamil (Sigma-Aldrich, ties to adjust for transfection efficiencies. The firefly luciferase V4629) used were as previously reported.23 After 48 h of drug units were divided by the renilla units to obtain the relative lucif- incubation, the cells were collected at the indicated time points. erase units (RLU). The experiments were performed in tripli- The cells were then stained with Live-DyeTM, a cell-permeable cates, and data shown were obtained from 3 independent green fluorescent dye (Ex/Em D 488/518 nm) and propidium experiments. iodine, a cell non-permeable red fluorescent dye (Ex/Em D 488/ 615), by using the Live-Dead cell staining Kit (Biovision, K501) following the manufacturer’s protocol. Stained live and dead cells Cell viability MTT assay was assessed with the FACS Calibur flow cytometer and Cell- MES-SA or MES-SA/Dx5 cells were plated at an approximate Quest Pro software (Becton Dickinson) as previously described.35 density of 1 £ 105 cells/well in a 24-well plate. The cells were then treated with the indicated concentration of various drugs in different sets of experiments. The concentrations of verapamil Cell cycle analysis by FLOW cytometry (Sigma-Aldrich, V4629) were used as previously reported.23 The cell cycle was performed as previously described.39 Brief, After 60 h of drug incubation, the medium was removed and MES-SA and MES-SA/Dx5 cells were treated with MG132 for phosphate buffered saline (PBS) was used to wash the cells. Two 60 h following which the cells were washed with PBS and fixed hundreds microliter of Thiazolyl Blue Tetrazolium (Sigma- in 4% paraformaldehyde. Samples were treated with 10 mg/ml Aldrich, M2128) was added to each well and was incubated with RNase and Triton X-100 at 37C for 1 h. The cells were then the cells at 37C for 2 h. Subsequently, 400 ml DMSO was stained with 50 mg/ml propidium iodine (EMD Millipore, added to each well and incubation at 37C was continued for 537060). After 20 min of incubation at 4C in the dark, the cells 20 min. Absorbance of the mixture was read at 540 nm using a were analyzed using a FCM scan flow cytometer using the Cell- Microplate Reader (VersaMax). Cell viability (%) was calculated Quest software (Becton Dickinson). The percentage of cells in as the ratio of the surviving cells in each drug-treated experiment the apoptotic sub-G1 and G1, S-phase and G2-M phase were set to that of the control. calculated using the Modfit software (Verity Software House).

www.taylorandfrancis.com Cancer Biology & Therapy 157 Statistical analysis Funding The surviving fraction and the relative luminescence unit (RLU) This work was supported in part by the Ministry of Science § were measured in triplicate samples and were expressed as mean and Technology (Taiwan; NSC 95–2320-B-182–028-MY3, SD. The Student’s t-test or one way ANOVA was used for statistical NSC 99–2632-B-182–001-MY3, MOST 103–2320-B-182– < analysis. P 0.05 was considered as statistically significant. 021), Chang Gung Memorial Hospital Grant (CMRPD 34012, CMRPD 180133 and CMRPD 1B0473) and Ministry of Edu- cation (Taiwan; EMRPD1C0121). Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

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