The Synergic Effect of Vincristine and Vorinostat in Leukemia in Vitro and In

Chao et al. Journal of Hematology & Oncology (2015) 8:82 DOI 10.1186/s13045-015-0176-7 JOURNAL OF HEMATOLOGY & ONCOLOGY RESEARCH ARTICLE Open Access The synergic effect of vincristine and vorinostat in leukemia in vitro and in vivo Min-Wu Chao1, Mei-Jung Lai2, Jing-Ping Liou3, Ya-Ling Chang1, Jing-Chi Wang4, Shiow-Lin Pan4*† and Che-Ming Teng1*† Abstract Background: Combination therapy is a key strategy for minimizing drug resistance, a common problem in cancer therapy. The microtubule-depolymerizing agent vincristine is widely used in the treatment of acute leukemia. In order to decrease toxicity and chemoresistance of vincristine, this study will investigate the effects of combination vincristine and vorinostat (suberoylanilide hydroxamic acid (SAHA)), a pan-histone deacetylase inhibitor, on human acute T cell lymphoblastic leukemia cells. Methods: Cell viability experiments were determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and cell cycle distributions as well as mitochondria membrane potential were analyzed by flow cytometry. In vitro tubulin polymerization assay was used to test tubulin assembly, and immunofluorescence analysis was performed to detect microtubule distribution and morphology. In vivo effect of the combination was evaluated by a MOLT-4 xenograft model. Statistical analysis was assessed by Bonferroni’s t test. Results: Cell viability showed that the combination of vincristine and SAHA exhibited greater cytotoxicity with an IC50 value of 0.88 nM, compared to each drug alone, 3.3 and 840 nM. This combination synergically induced G2/M arrest, followed by an increase in cell number at the sub-G1 phase and caspase activation. Moreover, the results of vincristine combined with an HDAC6 inhibitor (tubastatin A), which acetylated α-tubulin, were consistent with the effects of vincristine/SAHA co-treatment, thus suggesting that SAHA may alter microtubule dynamics through HDAC6 inhibition. Conclusion: These findings indicate that the combination of vincristine and SAHA on T cell leukemic cells resulted in a change in microtubule dynamics contributing to M phase arrest followed by induction of the apoptotic pathway. These data suggest that the combination effect of vincristine/SAHA could have an important preclinical basis for future clinical trial testing. Keywords: Vincristine, SAHA, HDAC6, Leukemia Background divide, microtubule-like reticular structures spread in Microtubules, dynamically structured polymers of α- the cytosol. As the cell enters the M phase, however, tubulin and β-tubulin, are required in many cellular the original reticular microtubules are remodeled and processes, including vesicle and organelle transporta- assembled into mitotic spindles, which connect with tion, cellular motility, cell shape maintenance, chromosomes and drive chromosome separation [2]. chromosome separation, and division [1]. Before cells All of the microtubule processes described above are crucial for microtubule polymerization (stabilization) and depolymerization (destabilization). * Correspondence: [email protected]; [email protected] †Equal contributors Drugs that inhibit microtubule dynamics have been 4The Ph.D. Program for Cancer Biology and Drug Discovery, College of used clinically in cancer treatment for more than Medical Science and Technology, Taipei Medical University, No. 250, 20 years [3]. These agents can be divided into two Wu-hsing St., Taipei 11031, Taiwan 1Pharmacological Institute, College of Medicine, National Taiwan University, major types: microtubule-destabilizing agents, such as No. 1, Jen-Ai Road, Sec. 1, Taipei, Taiwan vincristine, and microtubule-stabilizing agents, such as Full list of author information is available at the end of the article © 2015 Chao et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chao et al. Journal of Hematology & Oncology (2015) 8:82 Page 2 of 15 paclitaxel [4]. At lower concentrations, both types Results affect only microtubule dynamics, inducing abnormal Cytotoxic effects of vincristine and SAHA, alone and in mitotic spindle formation and causing cell arrest in combination, on human leukemic MOLT-4 cells theMphaseand,subsequently,cellapoptosis[5]. A 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium Microtubule-binding agents have played critical roles bromide (MTT) assay was performed to investigate in the history of chemotherapeutic drug development the cytotoxicity of the microtubule-destabilizing agent and remain a first choice in the treatment of solid tu- vincristine and the HDACi vorinostat (SAHA) on hu- mors and hematological malignancies. As with other man ALL MOLT-4 cells. We first tested the cytotoxic anticancer agents, however, the problems of drug re- effect of SAHA and vincristine alone and in combin- sistance, neuropathy, immunosuppression, and poor ation. As shown in Fig. 1a, there was no significant solubility must be addressed. The current trends in cytotoxicity at concentrations up to 500 nM of tubulin-binding agent development are to change the SAHA. However, SAHA had an IC50 of 840 nM for dosage to improve solubility or a combination of 48 h, when concentration reached the highest level these agents with other anticancer drugs to reduce (1000 nM). In addition, vincristine exhibited cytotox- toxicity and enhance activity [6]. icity against human leukemic MOLT-4 cells with an Histone deacetylase (HDAC) catalyzes the removal IC50 of 3.3 nM at 48 h (Fig. 1b). To determine of the acetyl group in histones and nonhistone pro- whether an interaction between SAHA and vincris- teins. HDACs have different biological functions due tine took place, the cytotoxic potency of a combin- to the highly divergent sequences outside the cata- ation assay was measured. Cells treated with 500 nM lytic domain [7]. HDAC6, which predominates in the SAHA and various concentrations of vincristine (0.3 cytoplasm [8], is responsible for tubulin deacetylation, to 3 nM) significantly inhibited cell survival com- which plays a key regulatory role in the stability of pared to each treatment alone (Fig. 1c). the dynamic microtubules [9–12]. HDAC overexpres- sion in many cancers has also been reported [13–15]. Thus, HDAC is believed to be a promising target for Effects of vincristine in combination with SAHA on human cancer therapy [16]. HDAC inhibitors (HDACis) T cell leukemic cell survival cause histone hyperacetylation, thus activating the si- To further explore the synergistic cytotoxic effects, lenced gene for normal cellular function (cell cycle, we determined the effects on cell cycle distribution. angiogenesis, immunoregulation, cell apoptosis) [17]. As compared with SAHA, treatment with vincristine In addition, HDACis have been found to target non- induced an increase in the G2/M phase of the cell histone proteins, including protein 53 (p53), E2F1, cycle. In particular, the combination of vincristine signal transducer and activator of transcription 1, nu- plus SAHA caused an almost complete arrest of cells clear factor-κβ, α-tubulin, heat shock protein 90, and in the G2/M phase following short-term treatment Ku 70 [18–20]. Suberoylanilide hydroxamic acid (24h)andasubsequentinductioninthesub-G1 (SAHA), a pan-HDACi, was the first HDACi ap- phase following long-term treatment (48 h) (Fig. 2a). proved by the FDA for cutaneous T cell lymphoma Figure 2b shows the statistical results. Next, the com- in 2006 [21–23]. Owing to their low toxicity and bination index (CI) method was used to evaluate the wide range of activity, HDACis are often combined synergistic combinations [25]. A CI value of >1.0, 1.0, with other anticancer drugs [24]. Most studies have and <1.0 indicates an antagonistic, additive, or syner- been performed using cell-based experiments [23], gistic interaction, respectively, between the drugs. In but many combination therapies are undergoing clin- the G2/M phase, the CI values of vincristine (0.3, 1, ical trials, often for hematological malignancies [17]. and 3 nM) combined with 500 nM SAHA were 1.63, The purpose of this study is to evaluate the com- 0.72, and 0.32, respectively, and the CI values in the bined effects of vincristine and SAHA on an acute T sub-G1 phase were 0.97, 0.77, and 0.28, respectively cell lymphoblastic leukemia (ALL) model. We deter- (Fig. 2c). And this synergistic combination effect also mined the mechanism underlying cell arrest in the was noted in the other T cell leukemic cell line, mitotic phase and subsequent apoptosis following CCRF-CEM (Fig. 2d), rather than in acute myeloid combination treatment. We found that vincristine and leukemic cells (Additional file 1: Figure S2). Moreover, SAHA have a synergic effect on microtubules through vincristine (1 or 3 nM) combined with various con- different mechanisms. Moreover, this combined influ- centrations of SAHA also shows synergistic effect ence was also observed in in vivo results. Conse- (Additional file 2: Figure S1). These data indicate that quently, we suggest that a vincristine and SAHA vincristine and SAHA synergistically induced cell ar- combination treatment could be used in the clinical rest in the G2/M phase and subsequently in the sub- setting. G1 phase. Chao et al. Journal of Hematology & Oncology (2015) 8:82 Page 3 of 15 Fig. 1 Cell viability effects of SAHA or vincristine alone and in combination on the MOLT-4 cell line. Cell viability was measured by MTT assay. a, b MOLT-4 cells were treated with various concentrations of SAHA and vincristine alone for 24 and 48 h, respectively. c The combination of SAHA (S)and vincristine (V) was compared to the effect of vincristine alone. Data are expressed as mean values ± SD of at least three separate determinations. *P < 0.05, ** P < 0.02, *** P <0.005 Chao et al. Journal of Hematology & Oncology (2015) 8:82 Page 4 of 15 Fig.

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