OncoTargets and Therapy Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RESEARCH Synergistic effect of a retinoid X receptor-selective ligand bexarotene and docetaxel in prostate cancer This article was published in the following Dove Press journal: OncoTargets and Therapy Danyang Shen Purpose: To explore if bexarotene (BEX) synergistically enhances docetaxel (DTX) cyto- Huan Wang toxicity in castration-resistant prostate cancer cell lines. Qiming Zheng Materials and methods: MTT assay was used to measure the cytotoxic effect of DTX and Sheng Cheng BEX on castration-resistant prostate cancer (CRPC) cell proliferation and the combination Liwei Xu index (CI) values calculated to analyze the interaction between DTX and BEX. Flow cytometry and Western blot analysis identified the underlying mechanism for the synergistic Mingchao Wang effect of BEX and DTX. Gong H Li Results: When mitotic slippage happens, BEX can synergistically strengthen the anti- Li Q Xia proliferation of DTX in a way of significantly down-regulating cyclinB1 and CDK1 expres- Department of Urology, Sir Run Run sion, and then arresting cells in G2 phase. Shaw Hospital, Zhejiang University Conclusion: Results from this study showed that BEX-induced G2 arrest and DTX-induced School of Medicine, Hangzhou 310016, ’ mitotic arrest probably contributed to the synergistic effect of BEX and DTX. For personal use only. People s Republic of China Keywords: docetaxel, bexarotene, prostate cancer, combination therapy, cell cycle arrest Introduction Prostate cancer (PCa) is the most prevalent cancer in males and has become the third major cause of cancer-related death in men in the United States, with an estimated 161,360 new cases, and 16,730 deaths in 2017.1 Androgen deprivation therapy (ADT) is the cornerstone for treating metastatic prostate cancer. Nevertheless, a substantial proportion of patients who initially responded to OncoTargets and Therapy downloaded from https://www.dovepress.com/ by 54.70.40.11 on 20-Oct-2019 ADT would inevitably develop castration-resistant prostate cancer (CRPC),2 and warrant the development of other CRPC treatment approaches. In 2004, docetaxel (DTX) was approved by the US Food and Drug Administration as a first-line treatment for CRPC. However, dose-related DTX toxicities always limit its clinical use, and most CRPC patients will ultimately develop resistance to chemotherapy. Combination therapy of DTX with other agents has demon- strated its potential to reduce dose-related toxicities and maintain or even enhance efficacy.3 Retinoid X receptors (RXRs), a type of intracellular receptors of retinoids, serve Correspondence: Li Q Xia; Gong H Li Department of Urology, Sir Run Run as transcriptional factors via homodimerization or heterodimerization with multiple Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310016, People’s nuclear receptors, including retinoic acid receptors and peroxisome proliferator- Republic of China activated receptors. RXRs thus have a significant role in regulating tumor cell Tel +86 1 375 826 4531; 4 +86 1 358 870 8507 proliferation, differentiation, and apoptosis. Bexarotene (BEX) is a synthetic Fax +86 05 718 604 4817 retinoid analog that binds specifically and activates RXRs and approved for treating Email [email protected]; [email protected] cutaneous T-cell lymphoma (CTCL). Hypertriglyceridemia, hypercholesterolemia, submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 7877–7886 7877 DovePress © 2019 Shen et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work http://doi.org/10.2147/OTT.S209307 you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Powered by TCPDF (www.tcpdf.org) 1 / 1 Shen et al Dovepress and hypothyroidism, significant side-effects of BEX are Company (Jiangsu, China) and stored at 4 °C in the dark. manageable and reversible with corresponding therapy. BEX was from the Ligand Pharmaceuticals Inc. (San Considering the nonoverlapping adverse-effect profile of Diego, CA, USA.) and stored at −20 °C. BEX with most cytotoxic agents, it is used frequently in combination with most chemotherapy drugs. A phase I MTT assay trial of 15 patients identified a combination of 25 mg/m2/ Cells (6×103/well) were seeded in 96-well plates and allowed week DTX and 400 mg/m2/day BEX as the maximum to adhere overnight. Varying concentrations of DTX, BEX, dose in several solid tumors. Three of 10 non-small-cell and combination of drugs added to cells. After 24 and 48 h of lung carcinoma (NSCLC) patients and one angiosarcoma incubation, one-tenth volume of MTT (5 mg/mL, dissolved patient showed stable disease.5 Another two randomized in PBS buffer) was added to each well, and the plate incu- phase III trials showed that BEX was well tolerated in bated for another 2 h. The medium was removed, and 150 μL advanced NSCLC patients when added to the traditional of dimethyl sulfoxide (DMSO) added to dissolve the forma- chemotherapy (cisplatin/vinorelbine or carboplatin/pacli- zan crystals, and absorbance measured at 550 nm in a micro- fi taxel). Although BEX, in combination with chemotherapy, plate reader (Bio-Tek, VT, USA). The IC50 was de ned as failed to prolong survival in advanced NSCLC, a subgroup half-maximum inhibitory concentration, and all results cal- of patients who suffered severe hypertriglyceridemia in the culated as percentages of controls. BEX treatment group showed prolonged survival with BEX.6–8 Previously, Yen et al (2006) showed that low- Cell cycle dose BEX (1 µM) exposure reduced the spontaneous Analysis of cell cycle profile was by using the Cell Cycle mutation rate and thus effectively delayed the development Staining Kit (Multi Science, Hangzhou, China) following of resistance to chemotherapy drugs (paclitaxel, doxorubi- the manufacturer’s instructions. Cells were seeded in six- cin, and cisplatin) in PC3 cells. Moreover, the addition of well plates and treated with DTX (5 nΜ or 10 nΜ) and BEX (20 and 40 μM) alone or with a combination of drugs For personal use only. BEX desensitized the resistant cells to the cytotoxic agents.9–11 However, the potential role of BEX and DTX for 24 or 48 h. Cells were harvested, washed with PBS, μ combination regime in treating CRPC is not thoroughly and resuspended in 500 L DNA staining solution contain- μ addressed. ing 5 L permeabilization solution. Cells were incubated This study aimed to explore the ability of BEX to in the dark for 30 min, and cell cycle analysis performed enhance DTX therapeutic effects in CRPC. We showed using FACSCalibur (BD, NJ, USA). synergistic cytotoxicity of BEX and DTX in PC3 and DU145 CRPC cell lines. Following prolonged DTX- Apoptosis induced mitosis arrest, some prostate cancer cells “slip” Cell apoptosis was analyzed using the Cell Apoptosis Kit out of mitosis and this event can be related to drug (Multi Science, Hangzhou, China) following the manufac- 12,13 ’ OncoTargets and Therapy downloaded from https://www.dovepress.com/ by 54.70.40.11 on 20-Oct-2019 resistance. We revealed that when mitotic slippage turer s instructions. Cells were seeded in six-well plates and Μ Μ μ happens, BEX can synergistically strengthen the anti-pro- treated with DTX (5 n or 10 n ) and BEX (20 and 40 M) liferation of DTX in a way of significantly down-regulat- alone or with a combination of drugs for 24 and 48 h. Cells ing cyclinB1 and CDK1 expression, subsequently were then harvested, washed with PBS, and resuspended in μ μ μ arresting cells in G2 phase. 500 L binding buffer with 5 L Annexin V-FITC and 10 L PI. Cells were incubated in the dark for 5 min, and cell apoptosis analysis was performed using FACSCalibur (BD, Materials and methods NJ, USA). Cell lines and reagents The human PCa androgen-independent cell lines PC3 and Western blot DU145 were from the Cell Bank of Type Culture Collection Cells after treatment were washed with cold PBS and of Chinese Academy of Sciences. The cells were cultured in lysed on ice for 10 min in cell lysis buffer (Beyotime, RPMI-1640 media supplemented with 10% fetal bovine Shanghai, China) containing PMSF (FDbio-tech, serum (Cellmax, Peking, China) and penicillin-streptomy- Hangzhou, China) and protease inhibitor cocktail cin. The cells were incubated at 37 °C and 5% CO2 atmo- (Biotool, Huston, USA). Lysates collected and centrifuged sphere. DTX was obtained from Jiangsu Hengrui Medicine at 12,000× g for 20 min. Protein concentrations quantified 7878 submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 DovePress Powered by TCPDF (www.tcpdf.org) 1 / 1 Dovepress Shen et al by BCA Protein Assay Kit (Thermo Fisher, MA, USA), Results and 20 µg total protein separated on SDS/PAGE gels and Effect of BEX and DTX alone or in fl subsequently transferred to polyvinylidene di uoride combination on cell viability membranes. The membranes were blocked in 5% nonfat MTT assay was used to investigate the effects of DTX and milk at room temperature for 1 h. Afterward, the mem- BEX on PC3 and DU145 cell proliferation. We used branes were incubated overnight at 4 °C with primary sequential DTX and BEX doses singly or simultaneously antibodies for anti-cyclinD1, anti-cyclinE2, anti- to evaluate their antitumor efficacy and explore whether cyclinB1, anti-CDK1, anti–phospho-histone H3 (Cell they can act synergistically.