Toxicology in Vitro 28 (2014) 419–425

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Calcium channel blocker verapamil accelerates gambogic acid-induced cytotoxicity via enhancing proteasome inhibition and ROS generation Ningning Liu a,1, Hongbiao Huang a,1, Shouting Liu a, Xiaofen Li a, Changshan Yang a, Q. Ping Dou a,b, Jinbao Liu a,⇑ a

Protein Modification and Degradation Lab, Department of Pathophysiology, Guangzhou Medical University, Guangzhou, Guangdong 510182, People’s Republic of China The Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Departments of Oncology, Pharmacology and Pathology, School of Medicine, Wayne State University, Detroit, MI 48201-2013, USA b

a r t i c l e

i n f o

Article history: Received 4 August 2013 Accepted 18 December 2013 Available online 27 December 2013 Keywords: Verapamil Gambogic acid Proteasome Ubiquitin-proteasome system Apoptosis Cancer therapy CYP1A2

a b s t r a c t Verapamil (Ver), an inhibitor of the multidrug resistance gene product, has been proved to be a promising combination partner with other anti-cancer agents including proteasome inhibitor bortezomib. Gambogic acid (GA) has been approved for Phase II clinical trials in cancer therapy in China. We have most recently reported that GA is a potent proteasome inhibitor, with anticancer efficiency comparable to bortezomib but much less toxicity. In the current study we investigated whether Ver can enhance the cytotoxicity of GA. We report that (i) the combination of Ver and GA results in synergistic cytotoxic effect and cell death induction in HepG2 and K562 cancer cell lines; (ii) a combinational treatment with Ver and GA induces caspase activation, endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production; (iii) caspase inhibitor z-VAD blocks GA + Ver-induced apoptosis but not proteasome inhibition; (iv) cysteine-containing compound N-acetylcysteine (NAC) prevents GA + Ver-induced poly(ADP-ribose) polymerase cleavage and proteasome inhibition. These results demonstrate that Ver accelerates GAinduced cytotoxicity via enhancing proteasome inhibition and ROS production. These findings indicate that the natural product GA is a valuable candidate that can be used in combination with Ver, thus representing a compelling anticancer strategy. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Proteasome inhibition has been demonstrated as a novel therapeutic strategy in cancer therapy. Bortezomib (Velcade), a specific proteasome inhibitor, has been approved by the United States Food and Drug Administration (FDA) to treat multiple myeloma patients (Adams, 2004; Adams et al., 1999; Kane et al., 2006; Orlowski and Dees, 2003). Several second generation proteasome inhibitors are now under clinical trials for cancer therapy (Yang et al., 2009). Although bortezomib has achieved significant clinical benefit for

Abbreviations: GA, gambogic acid; Ver, verapamil; Ab, antibody; ER, endoplasmic reticulum; ROS, reactive oxygen species; z-VAD-FMK, carbobenzoxy-valylalanyl-aspartyl-(O-methyl)-fluoromethylketone; CT-like, chymotrypsin-like; DCFH-DA, 20 ,70 -dichlorfluorescein-diacetate; PARP, poly ADP-ribose polymerase; MDR, multidrug resistance; UPR, unfolded protein response; PI, propidium iodide; PVDF, polyvinylidene difluoride; HRP, horseradish peroxidase; NAC, Nacetylcysteine. ⇑ Corresponding author. Address: Department of Pathophysiology, Guangzhou Medical University, 195 Dongfengxi Road, Guangzhou, Guangdong 510182, People’s Republic of China. Tel.: +86 20 81340720; fax: +86 20 81340542. E-mail address: [email protected] (J. Liu). 1 Liu N. and Huang H. contributed equally to this work. 0887-2333/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.12.008

multiple myeloma in clinical trials, its effectiveness and administration have been limited by toxic side effect. To improve the efficacy of proteasome inhibition-based treatments, drugs augmenting the antitumor properties of bortezomib or other proteasome inhibitors and/or reducing their dose-dependent toxicities are required. Gambogic acid (GA) is a natural product isolated from gamboge, which has been used as a coloring agent and in traditional Chinese medicine for the treatment of various human conditions for hundreds of years (Auterhoff et al., 1962; Panthong et al., 2007). Recent studies have demonstrated that GA has a spectrum anticancer effect both in vitro and in vivo with low toxic side effects (Huang et al., 2011a; Liu et al., 2005; Yang et al., 2007; Yi et al., 2008). GA has been approved by the Chinese FDA for the treatment of solid cancers in Phase II clinical trials (Zhou and Wang, 2007). Even though several molecular targets of GA have been suggested, most of them might not be responsible for GA-induced cytotoxicity (Kasibhatla et al., 2005; Pandey et al., 2007; Rong et al., 2009; Wu et al., 2004; Xu et al., 2009; Yu et al., 2007). We have demonstrated that GA is able to selectively inhibit tumor proteasome activity, with potency comparable to bortezomib but lower toxicity. Compared with bortezomib, GA could produce tissue-specific

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proteasome inhibition and tumor-specific toxicity (Li et al., 2013; Shi et al., 2013). We have also demonstrated that GA could overcome hypoxia-induced myocardial hypertrophy via targeting the proteasome (Zhao et al., 2013). As an L-type calcium channel blocker, verapamil (Ver) has been used in clinics for many years. It was approved by the United States FDA in 1981 and has been used for the treatment of cardiac arrhythmias, hypertension, and, most recently, as a promising combination partner with bortezomib (Chen et al., 2012; Meister et al., 2010). Ver has also been reported as an inhibitor of the multidrug resistance (MDR) gene product in cancer therapy (Endicott and Ling, 1989). In the current study, we report that the combination of GA and Ver synergistically enhanced cytotoxicity and cell death in tumor cell cultures, which was associated with enhanced proteasome inhibition, caspase activation, generation of reactive oxygen species (ROS) and induction of endoplasmic reticulum (ER) stress.

incidence of cell death in the live culture condition, HepG2 and K562 cells were seeded into 12-well plates and PI was added directly to the cell culture medium, then the cells in the culture dish were kinetically imaged with an inverted fluorescence microscope equipped with a digital camera (Axio Obsever Z1, Zeiss). 2.4. Western blot analysis Western blot was performed as we described previously (Li et al., 2013). Briefly, an equal amount of total protein extracted from cultured cells were separated by 12% SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The blots were blocked with 5% milk for 1 h. Primary Abs and horseradish peroxidase (HRP)-conjugated secondary Abs were each incubated for 1 h. The bounded secondary antibodies were reacted to the ECL detection reagents and exposed to X-ray films (Kodak, Japan). 2.5. ROS production

2. Materials and methods 2.1. Materials GA and z-VAD-FMK [Carbobenzoxy-valyl-alanyl-aspartyl-(Omethyl)-fluoromethylketone] were purchased from BIOMOL International LP (Plymouth Meeting, PA). Ver was from National Institute for the Control Pharmaceutical and Biological Products (Beijing, China). Propidium iodide (PI)/Annexin V-FITC Apoptosis Detection Kit was from Keygen Company (Nanjing, China). Rabbit polyclonal antibodies (Abs) against nuclear PARP (poly (ADP-ribose) polymerase); rabbit monoclonal Abs against BIP (C50B12), caspase-3 (8G10) and Bcl-2 (50E3); mouse monoclonal Abs against C/EBP homologous protein (CHOP, L63F7), caspase-8 (1C12) and caspase-9 (C9) were from Cell Signaling (Beverly, MA). Rabbit polyclonal Abs against cleaved caspase-8 (Cleaved Asp384) was from Assay biotechnology Company, Inc. Rabbit polyclonal antibodies against cleaved caspase-9 p35 (D315) and cleaved caspase-3 p17 were from Bioworld Technology, Inc. Mouse monoclonal Abs against ubiquitin (P4D1) and Bax (B-9), rabbit polyclonal Ab against GAPDH (FL-335) and horseradish peroxidase (HRP)-labeled secondary Abs were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Enhanced chemiluminescence (ECL) reagents were from Amersham Biosciences (Piscataway, NJ). DCFH-DA (20 ,70 -dichlorfluorescein-diacetate) was from Beyotime Institute of Biotechnology (Jiangsu, China). 2.2. Cell viability assay Cell viability was detected by trypan blue exclusion assay. Briefly, HepG2 or K562 cancer cells (5  103 cells/well) were seeded in 96-well plate and treated with GA, Ver and the combination for 72 h, then the cells were detached by trypsinization and viable cells were determined by counting the viable cells with trypan blue staining. All the results are from at least three independent experiments. 2.3. Cell death assay Apoptosis assay was performed as we previously described (Huang et al., 2010). In brief, cultured HepG2 and K562 cells were harvested and washed with cold PBS and resuspended with the binding buffer, followed by Annexin V-FITC incubation for 15 min and PI staining for another 15 min at 4 °C in dark. The stained cells were analyzed with flow cytometry within 30 min. The morphological changes of cell death were performed as we reported (Huang et al., 2012). To monitor temporal changes in the

HepG2 or K562 cells were treated with GA and/or Ver for 24 h, and then the cells were incubated with the free serum medium with addition of 10 lM of DCFH-DA for 20 min at 37 °C. Then cells were collected for flow cytometry analysis. DCFH penetrates the cells and is in turn oxidized to DCF in the presence of ROS, and the DCF fluorescence intensity was determined by flow cytometric analysis. The fold changes of mean fluorescence intensities are shown in the diagram. Mean values and standard deviations were calculated from triplicates. 2.6. Cell-based chymotrypsin-like (CT-like) activity assay This was performed as we previously reported (Huang et al., 2011b). Cancer cells (4000 cells) were treated with either vehicle or GA, Ver and the combination for 6 h. The treated cells were incubated with the Promega Proteasome-Glo Cell-Based Assay Reagent (Promega Bioscience, Madison, WI) for 10 min. The CT-like proteasome activity was detected. Luminescence generated from each reaction was detected with luminescence microplate reader (Varioskan Flash 3001, Thermo Scientific, USA). Means and standard deviations are calculated from three independent experiments. 2.7. Statistical methods Mean ± SD are presented where applicable. Unpaired Student’s t-test or one way ANOVA is used where appropriate for determining statistic probabilities. P value less than 0.05 is considered significant. 3. Results 3.1. The combination of Ver and GA induced apoptosis in cancer cells in a synergistic manner Human hepatoma HepG2 and leukemia K562 cells were grown in RPMI 1640 supplemented with 10% FBS. The effect of GA or Ver alone and the combination on the cell viability was assessed by trypan blue exclusion assay. Treatment with increasing doses of either GA (0.3, 0.4, 0.5 lM) or Ver (20, 30, 40 lM) only slightly decreased cell viability in HepG2 cells after 72 h, while the combination of GA and Ver dramatically decreased the HepG2 cell viability (Fig. 1A). In K562 cells, GA plus Ver also yielded the similar synergistic effects, compared to each treatment alone (Fig. 1B). Next the induction of cell death by GA and Ver combination treatment was analyzed. As displayed in Fig. 1C, the combination of GA and Ver markedly increased Annexin V/propidium iodide

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Fig. 1. Combination of GA (gambogic acid) and Ver (verapamil) triggered in vitro synergistic cytotoxicity. (A, B) HepG2 cells or K562 cells were treated with GA, Ver or their combination for 72 h and cell viability was detected by Trypan blue assay. Mean ± SD (n = 3). DM: DMSO. P < 0.05, compared with GA or Ver treatment alone, respectively. (C) HepG2 or K562 cells were treated with either GA, Ver or their combination for 72 h. Cell death was detected by flow cytometry with Annexin V/propidium iodide (PI) staining. Typical flow images (upper) and summary of cell death (lower) were shown. Cell death includes early apoptotis in the lower-right quadrant, late apoptosis in the upper-right quadrant and necrosis in the upper-left quadrant. Mean ± SD (n = 3). *P < 0.05, versus non-combinational treatment. (D) HepG2 or K562 cells were treated as in (C) for 48 h, cell death was kinetically detected with direct PI staining in live cells. The representative merged images between red fluorescence and the phase-contrast were shown. Scale bar = 50 lm.

(PI)-positive cells in HepG2 and K562 cell lines. It was further found that GA plus Ver synergistically increased PI-positive cells by direct PI staining assay in live cells (Fig. 1D), indicating GA-induced cell death and morphological changes were highly enhanced by Ver. 3.2. Caspase activation is responsible for GA + Ver-induced apoptosis We reported that GA-induced cell- or tissue-specific proteasome inhibition is required for its mediated cytotoxicity (Li et al., 2013). It is well known that proteasome inhibition-induced cell death is associated with multiple pathways including caspase activation, ER stress and ROS production (Fribley et al., 2004; Miller et al., 2007; Simon et al., 2000). Hence, we investigated whether GA, Ver or their combination could affect these signaling pathways. We found that after the combination treatment of HepG2 or K562 cells, levels of procaspase-3, -8, and -9 dramatically decreased while their cleaved, active forms were increased greatly, accompanied by PARP cleavage, compared to each treatment alone (Fig. 2A), indicating the activation of caspases and the induction of apoptosis. In addition, Bcl-2 but not Bax was dramatically decreased with the combination treatment (Fig. 2A). Furthermore, GA or Ver alone had slight cytotoxicity and the combination induced 30% cell death after treatment for 36 h (in HepG2 cells) or 24 h (in K562 cells), while z-VAD, a pan-caspase blocker, almost completely blocked the induced cell death (Fig. 2B and C), confirming that

caspase activation is responsible for the cell death induced by GA and Vel combination. Therefore, the GA plus Ver treatment resulted in caspase activation followed by PARP cleavage and cellular apoptosis. We also found that the combined treatment with GA and Ver resulted in the activation of ER stress and the production of ROS in both HepG2 and K562 cancer cells, which have been found to be associated with caspase activation and proteasome inhibition (Fribley et al., 2004; Miller et al., 2007; Simon et al., 2000). While GA at 0.5 lM or Ver at 30 lM alone did not alter CHOP protein expression levels after 48 h treatment the combination of GA and Ver markedly increased CHOP protein expression; also, GA (0.5 lM) or Ver (30 lM) alone slightly decreased levels of BIP expression while their combination prevented the decrease in BIP protein expression (Fig. 2D). These results demonstrated that the combination of GA and Ver enhanced the ER stress. Furthermore, GA or Ver alone slightly induced levels of DCF fluorescence intensity while the combination treatment with GA and Ver for 24 h significantly increased DCF intensity (Fig. 2E and F), implying that the combination treatment enhances ROS production. 3.3. Caspase inactivation completely abrogates GA + Ver-induced apoptosis but not proteasome inhibition We have reported that GA indirectly inhibits proteasome function in the cells (Li et al., 2013). To study the mechanisms

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Fig. 2. Combinational treatment with GA and Ver induces caspase activation, ER stress and ROS production. (A) HepG2 cells were treated with GA and/or Ver as indicated for 36 h. Proteins extracts were subjected to Western blot by using antibodies against pro- or cleaved caspase-9, -8 and -3, Bax, Bcl2 and PARP. GAPDH was used as a loading control. (B, C) HepG2 cells or K562 cells were treated as in (A) in the absence or presence of a pan-caspase inhibitor z-VAD (20 lM) for 36 h (HepG2) or 24 h (K562), followed by cell death assay with Annexin V/PI staining by flow cytometry. Summary of cell death was shown in (B) for HepG2 cells and in (C) for K562 cells. Mean ± SD (n = 3). * P < 0.05, versus other treatment. (D) HepG2 cells were treated with GA, Ver and their combination for 48 h. Protein extracts were subjected to Western blot by using antibodies against CHOP and BIP. (E, F) HepG2 cells or K562 cells were treated with GA, Ver and their combination for 24 h. HepG2 cells were incubated with DCFH-DA (10 lM). DCF fluorescence was measured by flow cytometry. The merged flow images were shown in (E) and ROS fold changes were shown in (F). Mean ± SD (n = 3). *P < 0.05, versus other treatment. Scale bar = 100 lm.

via which Ver enhances the cytotoxic effect of GA, we first determined the proteasomal chymotrypsin-like (CT-like) activity in HepG2 and K562 cells by a cell-based CT-like activity assay. As shown in Fig. 3A, 0.5 lM of GA inhibited the proteasomal CT-like activity by 40%, and the combination of GA and 30 lM of Ver induced 52% inhibition after 6 h treatment. The combination of GA and Ver in K562 cells gave a similar result (Fig. 3B). Furthermore, GA or Ver alone produced a moderate accumulation of ubiquitinat-

ed proteins (Ub-prs, an indicator of proteasome inhibition), while their combination produced dramatic accumulation of Ub-prs in HepG2 and K562 cells after 24 h of treatment (Fig. 3C and D, lanes 4). To test the effect of caspase activation on GA + Ver-induced proteasome inhibition and PARP cleavage, HepG2 or K562 cells were exposed to GA, Ver alone or their combination for 24 h with or without a pan-caspase inhibitor z-VAD. The presence of z-VAD completely abrogated PARP cleavage (indicator of cell apoptosis)

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4. Discussion

Fig. 3. Caspase inhibitor reverses GA + Ver-induced apoptosis but not proteasome inhibition. (A, B) HepG2 cells or K562 were treated with GA (30 or 20 lM), Ver (0.5 or 0.3 lM) alone or the combination for 6 h, then CT-like activity was determined by using a cell-based CT-like assay kit. Mean ± SD (n = 3). *P < 0.05, versus each treatment alone. (C, D) HepG2 or K562 cells were treated as in (A, B) in the absence or presence of z-VAD (20 lM) for 24 h, ubiquitinated proteins and PARP were detected by Western blot. Typical Western images were shown and GAPDH was used as a loading control.

but only slightly affected the accumulation of Ub-prs induced by GA + Ver combination or GA alone (Fig. 3C and D, lanes 6 or 7).

3.4. N-acetylcysteine (NAC) reverses GA + Ver-induced PARP cleavage and proteasome inhibition To test the effect of cysteine-containing compound NAC on GA + Ver-induced proteasome inhibition and apoptosis, K562 or HepG2 cells were treated with GA, Ver alone or the combination for 24 h in the absence or presence of NAC (2 mM). It was found that NAC significantly inhibited GA + Ver-induced proteasome inhibition and PARP cleavage (Fig. 4A and B).

GA has been proven to be a promising anticancer agent due to its less toxic side effects compared with other anticancer drugs. To enhance GA’s selectivity for cancer cells, we further searched for agents that synergistically increase the antitumor effect of GA. Ver represented a promising candidate since it could reduce MDR1-mediated drug resistance in leukemia cells (Endicott and Ling, 1989). Our present study revealed that Ver enhanced the cytotoxic effect of GA leading to increased cell death in human hepatoma HepG2 and leukemia K562 cancer cells; GA + Ver-mediated cell death is dependent on proteasome inhibition, caspase activation, ER stress and ROS generation; blocking caspase activation by z-VAD and blocking proteasome inhibition/ROS generation by NAC inhibited GA + Ver-induced cell death. Our studies are consistent to the synergistic effects of Ver and bortezomib, a classical proteasome inhibitor (Chen et al., 2012; Meister et al., 2010). In cancer cells, both GA and Ver combination and bortezomib and Ver combination efficiently enhanced cytotoxicies as well as the caspase activation, production of ROS and UPR (unfolded protein response) induction. However, the involved mechanisms may be different. First, the GA + Ver combination, but not Bortezomib + Ver combination, could enhance Bip expression. Secondly, even though the two combinations both could enhance CHOP expression, GA + Ver, but not bortezomib + Ver could cause downregulation of Bcl-2. CHOP, which is associated with terminal UPR and apoptosis, was shown to downregulate Bcl-2 (McCullough et al., 2001). Although it is unclear what is responsible for the observed differences, we offer the below potential explanations: (i) each combination strategy may target different targets of the UPR pathway; (ii) unlike bortezomib, GA is not a specific proteasome inhibitor; (iii) the differences may be cell-selective, which need to be investigated in the future. Based on our results, one of the mechanisms for the synergistic effect of GA and Ver relied on caspase activation. GA and Ver enhanced caspase activation as shown by the induction of cleaved, active forms of caspases and the decreases of pro-caspases which were blocked by a pan-caspase inhibitor. In addition, it was found that the ratio of Bcl-2/Bax was significantly reduced after the combination treatment. Since overexpression of Bax has been shown to accelerate cell death, while overexpression of antiapoptotic proteins such as Bcl-2 represses the death function of Bax (Finucane et al., 1999; Li and Dou, 2000), therefore, the decreased ratio of Bcl-2/Bax might contribute to caspase activation and cell apoptosis. It is well known that ROS plays an important role in proteasome inhibition-induced cell death (Simon et al., 2000). Either GA or Ver alone induced a slight increase of ROS while the combination significantly increased the levels of ROS in cancer cells. Several pathways have been reported for ROS to induce cell death (Simon et al., 2000). Additionally, cysteine-containing compound NAC inhibited PARP cleavage, implying that ROS also contributes to GA + Ver-induced cell death. We have reported that GA yielded the similar gene expression (specifically proteasome-related ER stress gene) pattern to proteasome inhibitor bortezomib (Li et al., 2013). As shown in Fig. 2D, at the indicated doses, GA or Ver alone did not increase expression of proapoptotic protein CHOP and ER resident chaperone l-heavy chain binding protein (BiP) but the combination of GA and Ver produced high expression of these two proteins especially CHOP protein. Enhanced chaperone production is a hallmark of ER stress activation which contributes to proteasome inhibition-induced cell death (Fribley et al., 2004). This result demonstrated that ER stress is also associated with the combination-mediated cell death. Our above results indicate that caspase activation, ROS generation and ER stress activation all are involved in GA + Ver-induced cytotoxicity in the tested cancer cells.

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Fig. 4. NAC reverses GA + Ver-induced both PARP cleavage and proteasome inhibition. K562 or HepG2 cells were treated with either GA (0.3 or 0.5 lM), Ver (20 or 30 lM) alone or combination of the two agents as indicated in the absence or presence of NAC (2 mM) for 24 h. Ubiquitinated proteins and PARP cleavage were detected by Western blot. The typical Western images in K562 and HepG2 cells were shown in (A) and (B), respectively.

Next we investigated the relationship between proteasome inhibition and the mentioned pathways. By using cell-based CTlike activity assay and ubiquitinated protein detection by Western blotting, it was found that Ver not only enhanced accumulation of ubiquitinated proteins but also directly enhanced inhibition of the proteasomal activities by GA in HepG2 and K562 cells. It is known that proteasome inhibition by bortezomib or GA could induce ROS generation, ER stress and caspase activation (Fribley et al., 2004; Miller et al., 2007; Simon et al., 2000). To test what is responsible for the enhanced proteasome inhibition, the effect of caspase inhibitor was tested. We found that z-VAD could present the combination-induced cell death but only weakly suppresses proteasome inhibition, indicating that caspase activation is not the major factor interfering with proteasome function even though it has been reported that caspase activation could cleave 19S proteasome subunits thus inhibiting proteasome function (Sun et al., 2004). We found that MT1 is a metabolite of GA that might be responsible for GA-mediated proteasome inhibition (Li et al., 2013). Based on its structure, a cysteine-containing compound such as NAC could attack the epoxide MT1 to form a reduced form that would lose its proteasome-inhibitory activity. As expected, NAC dramatically reversed GA + Ver-induced not only cell apoptosis but also proteasome inhibition. Here we propose that the complete reverse by NAC of GA-mediated proteasome inhibition is due to interaction of NAC and MT1 of GA but not through scavengering ROS, as ROS itself like H2O2 (less than 100 lM) has a weak effect on ubiquitinated protein accumulation (data not shown). These results demonstrated that the enhanced proteasome inhibition is responsible for the combination-mediated changes. There are two possible mechanisms that might contribute to the synergistic proteasome inhibition of GA and Ver combination. First, Ver itself could inhibit proteasome peptidase activity and accumulate ubiquitinated proteins to some extent, as previously reported (Meister et al., 2010) and also observed in this study. Secondly, Ver possibly enhanced proteasome inhibition by blocking cellular CYP1A2 activity thus activating CYP2E1 for the active MT1 formation since Ver has been reported to be a strong CYP1A2 inhibitor (Fuhr et al., 1992). This is consistent to our previous report that CYP1A2 inhibition enhanced GA-induced proteasome inhibition and cell death (Li et al., 2013). Although the combination of GA and Ver has a strong synergistic effect on cytotoxicity in vitro, this effect should be confirmed in vivo in the future. Our findings suggest that Ver is a promising combination partner with GA in cancer therapy, thus providing a strategy in the future combinational clinical trial.

Author contributions JL and NL designed the experiments, analyzed the data and wrote the manuscript; NL, HH, SL, XL and CY performed the experiments or analyzed the data. QPD analyzed the data and edited the manuscript. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by the National High Technology Research and Development Program of China (2006AA02Z4B5), NSFC (81272451/H1609, 81070033/H0108), Key Project (10A057S) from Guangzhou Education Commission (to J.L.); by NSFC (81201719) and Foundation for Distinguished Young Talents in Higher Education of Guangdong (2012LYM_0109) (to H.H.). Appendix A. Supplementary material Transparency Document associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tiv. 2013.12.008. References Adams, J., 2004. The development of proteasome inhibitors as anticancer drugs. Cancer Cell 5, 417–421. Adams, J., Palombella, V.J., Sausville, E.A., Johnson, J., Destree, A., Lazarus, D.D., Maas, J., Pien, C.S., Prakash, S., Elliott, P.J., 1999. Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res. 59, 2615–2622. Auterhoff, H., Frauendorf, H., Liesenklas, W., Schwandt, C., 1962. The chief constituents of gamboge resins. 1. Chemistry of gamboge. Arch. Pharm. 295 (67), 833–846. Chen, Z., Romaguera, J., Wang, M., Fayad, L., Kwak, L.W., McCarty, N., 2012. Verapamil synergistically enhances cytotoxicity of bortezomib in mantle cell lymphoma via induction of reactive oxygen species production. Br. J. Haematol. 159, 243–246. Endicott, J.A., Ling, V., 1989. The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu. Rev. Biochem. 58, 137–171. Finucane, D.M., Bossy-Wetzel, E., Waterhouse, N.J., Cotter, T.G., Green, D.R., 1999. Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL. J. Biol. Chem. 274, 2225–2233. Fribley, A., Zeng, Q., Wang, C.Y., 2004. Proteasome inhibitor PS-341 induces apoptosis through induction of endoplasmic reticulum stress-reactive oxygen species in head and neck squamous cell carcinoma cells. Mol. Cell. Biol. 24, 9695–9704.

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