Pediatr Surg Int (2012) 28:149–159 DOI 10.1007/s00383-011-2988-z
ORIGINAL ARTICLE
Apoptosis sensitizers enhance cytotoxicity in hepatoblastoma cells Justus Lieber • Verena Ellerkamp • Julia Wenz • Bettina Kirchner • Steven W. Warmann • Jo¨rg Fuchs Sorin Armeanu-Ebinger
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Published online: 5 October 2011 Ó Springer-Verlag 2011
Abstract Purpose Drug resistance remains a major challenge for the treatment of high-risk hepatoblastoma (HB). To enhance effectiveness of chemotherapy we modulate apoptosis in HB cells in vitro. Methods Viability was monitored in HB cells (HuH6, HepT1) and fibroblasts in monolayer and spheroid cultures treated with ABT-737, obatoclax, HA14-1, and TW-37 and each in combination with CDDP, etoposide, irinotecan, paclitaxel, and DOXO in a MTT assay. Western blot analyses were performed to determine expressions of proand anti-apoptotic proteins. Results Obatoclax and ABT-737 led to a dose-dependent decrease of viability in HB cells at concentrations above 0.3 lM. TW-37 and HA14-1 were less effective. ABT-737 and obatoclax had additive effects when combined with
CDDP, etoposide, irinotecan, paclitaxel, or DOXO. This was also observed for fibroblast, however, for higher drug concentrations. In spheroid cultures, relative expression of Bcl-XL was increased, Bax was decreased, Mcl-1 was low, and Bcl-2 was not detected compared to 2D cultures, denoting an anti-apoptotic state in spheroids. Obatoclax and ABT-737 have overcome the resistance to CDDP. HuH6 cells have shown higher susceptability for apoptosis sensitizers than HepT1. Conclusion The data provide evidence that ABT-737 and obatoclax might improve treatment results in children with HB.
J. Lieber (&) V. Ellerkamp J. Wenz B. Kirchner S. W. Warmann J. Fuchs S. Armeanu-Ebinger Department of Pediatric Surgery and Pediatric Urology, University Children’s Hospital, Hoppe-Seyler-Strasse 1, 72076 Tu¨bingen, Germany e-mail:
[email protected]
Introduction
V. Ellerkamp e-mail:
[email protected] J. Wenz e-mail:
[email protected] B. Kirchner e-mail:
[email protected] S. W. Warmann e-mail:
[email protected] J. Fuchs e-mail:
[email protected] S. Armeanu-Ebinger e-mail:
[email protected]
Keywords BH3 mimetic drugs Apoptosis sensitizers Obatoclax ABT-737 HA14-1 TW-37 Hepatoblastoma Multidrug resistance
Treatment of primary malignancies might be limited by marked chemoresistance to currently available drugs in the case of some tumors initially, and in others during therapy [1]. Treatment results remain poor due to increased recurrence rates and tumor metastasisation in some cases. In children suffering from hepatoblastoma (HB), those with high-risk diseases (metastasized tumors, PRETEXT IV, and alpha-fetoprotein levels \100 lg/l) also present a poor 3-year survival of 69%, which is mainly contributed to multidrug resistance [2–7]. Preoperative chemotherapy should ideally lead to significant reduction of tumor burden to enable surgical respectability. In addition, the aim of chemotherapy is to eliminate free circulating tumor cells including intraoperative situations, in order to prevent tumor cell dissemination.
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In order to enhance effectiveness of chemotherapy, various mechanisms are currently under investigation, and those targeting apoptotic mechanisms constitute a promising alternative option [1]. Malignancies are characterized frequently by defects in apoptosis signaling. This renders the malignant cells resistant to endogenous and exogenous apoptotic stimuli, such as cytotoxic drugs. The defective apoptosis often results from overexpression of anti-apoptotic proteins in the Bcl protein family, such as Bcl-2 and Bcl-XL. Bcl-2 prevents cytochrome C release by sequestering pro-apoptotic BH3-only proteins such as tBid, Bad, Bax, and Bim [8]. The detailed knowledge of the structures of Bcl-2 and Bcl-XL has led to the identification of a number of short peptides and small organic molecules capable of inhibiting Bcl-2 and Bcl-XL function. These small molecules bind to the Bcl-2 homology domain 3 (BH3) binding groove of Bcl-2 and facilitate the activation of pro-apoptotic Bcl proteins increasing the sensitivity of cells for apoptosis. BH3 mimetic agents, also referred to as apoptosis sensitizers, hold considerable promise for enhancing the chemo-sensitivity of Bcl-2- and Bcl-XLoverexpressing cancers [9]. HB cells express high amounts of anti-apoptotic molecules encoded by genes of the Bcl family [10]. Therefore, inhibition of Bcl-2 function might enhance chemotherapy also in HB. Apoptosis-inducing effects of the small molecule ABT-737, as well as additive effects when administered in combination with cytotoxic drugs, have already been reported [11]. Herein, we describe the effects of various BH3 mimetic drugs, such as ABT-737, obatoclax, HA14-1, and TW-37, as single drugs and in combination with various chemotherapeutics commonly used in the HB treatment protocols, in order to improve results in high-risk HB.
Methods Drugs ABT-737 was kindly provided by Abbott (Abbott GmbH & Co, KG, Germany). For in vitro studies, ABT-737 was dissolved in DMSO at 1 mM and diluted with medium to seven final concentrations in the cell culture between 0.01 and 100 lM. Obatoclax, HA-14, and TW-37 were purchased from Absource Diagnostics GmbH (Munich, Germany), solubilized in DMSO and diluted with medium to final concentrations in the cell culture between 0.03 and 30 lM (obatoclax), 0.01 and 10 lM (HA-14), and 0.03 and 30 lM (TW-37). The highest DMSO concentration in cultures was 0.1 ll/ml. The cytotoxic agents cisplatin (CDDP, Neocorp AG, Weilheim, Germany), doxorubicin (DOXO, cell pharm GmbH, Hannover, Germany), paclitaxel (Neocorp AG,
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Weilheim, Germany), etoposide (Bristol-Myers Squibb GmbH & Co. KGaG, Munich, Germany), and irinotecan (Pfizer GmbH, Berlin, Germany) were commercially available as drug formulation. Cells and culture conditions The HB cell lines HepT1 [12] and HuH6 [13] were used for all experiments. Tumor cells were grown as monolayer in Dulbecco’s MEM medium (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum and 1% glutamine. The cells were grown at 37°C in a humidified atmosphere containing 5% carbon dioxide. All used cells were mycoplasma negative. For spheroid cultures, low attachment plates were used (Corning Inc., Corning, NY, USA) as previously described [14]. Fibroblasts were derived from human skin samples by tissue culture and grown as monolayer in the first three passages as described for tumor cells. Cell viability assay HB (HuH6, HepT1) cells and fibroblasts (10,000 cells/ 100 ll) were seeded out in 96-well plates (Becton-Dickinson GmbH, Heidelberg, Germany) and cultured as described above. At day 2, cytotoxic drugs (CDDP, DOXO, paclitaxel, etoposide, and irinotecan) were added to the cells at 7 different concentrations around IC50 [11]. BH3 mimetic drugs (ABT-737, obatoclax, HA14-1, and TW-37) were added to final concentrations between 0.01 and 30 lM. Experiments were repeated with fibroblasts and BH3 mimetic drugs alone, as well as BH3 mimetic drugs in combination with cytotoxic drugs, respectively. Drugs solutions were prepared shortly before administration. All assays were performed three times in triplicates. Cell viability was assessed by MTT [3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl-tetrazoliumbromide] assay (Sigma-Aldrich, Munich, Germany). 25 ll MTT (5 mg/ml) dissolved in PBS was added to each well. After incubation for 3 h, 100 ll/well lysis solution (10% SDS in acid water; Merck, Darmstadt, Germany) was added, and further incubated overnight in the dark at room temperature. Cell viability was assessed by measuring absorption at 570 nm using a Milena Kinetic Analyzer (DPC Bierman, Bad Nauheim, Germany). Percentages of cell viability were calculated by normalization between background of cultures without cells and untreated cultures as control. Dosedependent viability curves were computed by sigmoidal curves with variable slope to determine IC50. Western blot For Western blot analysis, HB cells were grown for 24 h as monolayer and spheroid cultures as described above. Cells
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were lysed in RIPA-buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS, and 1 mM EDTA) for 5 min on ice. Lysates were centrifuged at 14,0009g for 15 min at 4°C. Samples were mixed with Laemmli buffer (1:1) and heated at 95°C for 5 min. Gels were loaded with an equal volume (20 ll) of each specimen. Protein extracts were resolved by SDS-PAGE on 10% polyacrylamide gels and transferred to PVDF membranes. After incubation with blocking solution (5% non-fat dry milk in TBST: Tris-buffered saline containing 0.1% Tween;), membranes were probed overnight at 4°C with antibodies such as Mcl1, Bax, Bad, Bcl-2, and Bcl-XL, all from Santa Cruz Biotechnology Inc. (Heidelberg, Germany). Primary antibodies were diluted in blocking solution at a ratio of 1:2,000. GAPDH served as control for protein loading (1:200, sc-25778, Santa Cruz Biotechnology Inc., Heidelberg, Germany). Membranes were washed three times in TBST for 5 min and incubated with the corresponding HRP-labeled anti-mouse or antirabbit secondary antibody for 1 h at room temperature (GE Healthcare Europe GmbH, Freiburg, Germany). Visualization of antibody binding was carried out by chemiluminescence (Roti Lumin, Carl Roth, Karlsruhe, Germany). Densitometric analysis was done with AlphaDigiDoc software (Biozym Scientific, Oldendorf, Germany). Statistics Statistical analysis was carried out by one-way ANOVA on ranks test using GraphPad Prism 4.00 (GraphPad Software, San Diego, CA, USA, www.graphpad.com). Viability curves were fitted with a sigmoidal dose response function with variable slope. F test was used to compare curve parameters of treatment with and without BH3 mimetic alone. All numeric data are expressed as mean. Data plotted on graphs are mean and SD. Significance was assumed for all p \ 0.05.
Results Effects of BH3 mimetic drugs on HB cells Fibroblasts and HB cells were incubated with four different BH3 mimetic drugs (Fig. 1). Obatoclax and ABT-737 led to a decrease of viability in both HepT1 and HuH6 cells at concentrations above 0.3 and 1 lM, respectively. In contrast, fibroblasts did not show decrease of viability. Relative cell viability in cultures after treatment with obatoclax decreased dose-dependently for HB cell lines, although in HuH6 a drop of viability at lower concentration than in HepT1 was observed. IC50 for HuH6 was 0.5 lM and for HepT1 5 lM. HA14-1 showed no effect on HB cells at
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doses between 0.1 and 30 lM, but was toxic at 100 lM. TW-37 decreased cell viability not more than 50% at 10 lM. No effects were observed in fibroblasts after treatment with both BH3 mimetic drugs, HA14-1 and TW37 at the tested concentrations. Viability of HB cells after combination treatment with BH3 mimetic and cytostatic drugs BH3 mimetic drugs enhance the effect of various cytotoxic drugs in a combination treatment of tumor cell lines. Therefore, we measured the cell viability of HepT1 and HuH6 cells after co-incubation with five different cytotoxic agents at increasing concentrations and four BH3 mimetic drugs at three different concentrations. No additive effects were observed after combination treatment with HA14-1 and CDDP, etoposide, irinotecan, paclitaxel, and DOXO, respectively (Fig. 2). Similar results were obtained with TW-37 (data not shown). Treatment of HuH6 cells with obatoclax at three different concentrations (0.03, 0.1, and 0.3 lM) led to a dose-dependent decrease of relative cell viability when combined with any of the above mentioned cytostatic drugs, CDDP, etoposide, irinotecan, paclitaxel, and DOXO (Fig. 3). For example, treatment of HuH6 cells with 5.0 lg/ml CDDP decreased cell viability to 50%, whereas the combined therapy with 0.3 lM obatoclax further decreased cell viability to 20%. Additive effects were also seen with paclitaxel and DOXO and less considerably with etoposide and irinotecan. Similar effects were observed when treating HepT1 cells with a combination of obatoclax and CDDP. But HuH6 cells seemed to be more susceptible to CDDP as compared to HepT1, and preliminary data showed less cell viability reduction for combinations with 0.3 lM obatoclax in HepT1 cells. Consequently, HepT1 cells were incubated with 0.1, 0.3, and 1 lM obatoclax. Despite this, cell viability decreased to 25% in HuH6 cells when treated with 2.5 lg CDDP and with adding 0.3 lM obatoclax, whereas viability decreased only to 50% in HepT1 cells when treated with same doses of CDDP and 1 lM obatoclax. Similar results were obtained after treatment with ABT-737 [11]. Fibroblast cultures were used to estimate toxicity of treatment. In fibroblasts no reduction of cell viability was observed after treatment with CDDP up to 10.0 lg/ml (Fig. 4). After treatment with etoposide, we found toxic effects with doses exceeding 100 lg/ml resulting in complete reduction of cell viability. Similar toxic effects were observed after treatment with irinotecan [30 lg/ml and DOXO [0.3 lg/ml. Paclitaxel showed dose-dependent toxicity (IC50 = 3 lg/ml). After combined treatment with obatoclax (0.1, 0.3, and 1 lM) no additive effects occurred when adding CDDP, etoposide, and irinotecan. Combination treatment of fibroblasts with obatoclax and paclitaxel
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Fig. 1 Viability of HB cells and fibroblasts treated with BH3 mimetic drugs. HepT1, HuH6, and fibroblasts were incubated with obatoclax (a), ABT-737 (b), HA14-4 (c), and TW-37 (d) at different concentrations. Relative cell viability was determined 72 h later in a MTT assay
or DOXO, respectively, led to dose-dependent reduction of cell viability (Fig. 4). For example, at 0.3 lg/ml DOXO, viability of fibroblasts decreased from 75 to 20% when adding 1 lM obatoclax. The IC50 was 3 lg/ml when treating with paclitaxel alone, whereas the IC50 was 0.3 lg/ml when adding 1 lM obatoclax. Overcoming resistance to CDDP in spheroid cultures using obatoclax Imbalanced expression of pro-apoptotic (low) and antiapoptotic (high) proteins confers apoptosis resistance to tumor cells and contributes to resistance to cytostatic drugs. HuH6 and HepT1 cells show similar expression of Bcl-XL (anti-apoptotic) and Bax (pro-apoptotic) (Fig. 5). In 3D cultures the relative expression of Bcl-XL increases in HuH6 (0.76–1.20) and HepT1 (0.77–1.03), whereas Bax decreases in both cell lines, HuH6 (0.80–0.59) and HepT1 (0.93–0.79). Relative expression of Mcl-1 was low and Bcl-2 was not detected. These expression levels demonstrate an increased anti-apoptotic state in HB cells in 3D cultures compared to 2D cultures. Therefore, apoptosis sensitizer like BH3 mimetics may enhance drug activity in these cultures. Comparing the viability of HB cells after treatment with CDDP in 2D and 3D cultures, the main difference was the high sensibility of HB cells to low CDDP doses (0.3–5 lg/ml) in 2D cultures (Fig. 3), whereas in 3D cultures higher doses
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(5–10 lg/ml) were used to obtain same rates of cell viability reduction (Fig. 6). In both cultures, HuH6 cells were more susceptible to CDDP compared to HepT1 cells. This resistance has been overcome using BH3 mimetic drugs in combination with CDDP (Fig. 6). Adding obatoclax (0.3 lM) or ABT-737 (0.1 lM) led to a decrease of HB cell viability in 3D cultures when treated in combination with CDDP. CDDP was reduced eightfold obtaining same reduction of viability rates in HuH6 cells (Fig. 6a). Effects in HepT1 were less considerable reducing relative cell viability rates only 20% when adding BH3 mimetic drugs to CDDP therapy (Fig. 6b).
Discussion The use of multiple drugs with different mechanism or modes of action may increase the efficacy of the therapeutic effect, minimizing or slowing down the development of drug resistance and providing selective synergism against target versus host [15]. Rapid development of multiple drug resistance against current therapies is a major barrier in the treatment of cancer. Therefore, anticancer agents that can overcome acquired drug resistance in cancer cells are of great importance. In many cancers pro-survival proteins, such as Bcl-XL and Bcl-2, are up-regulated and contribute to resistance to chemotherapy, resulting in disease progression and metastasisation [16].
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Fig. 2 Viability of HB cells treated with a combination of HA14-1 and various cytotoxic drugs. HuH6 (a–e) and HepT1 (f–j) were incubated with HA14-4 at three different concentrations and CDDP (a, f), etoposide (b, g), irinotecan (d, i), paclitaxel (d, i), and DOXO (e, j) at seven different concentrations. Relative cell viability was determined 72 h later in a MTT assay
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154 Fig. 3 Viability of HB cells treated with a combination of obatoclax and various cytotoxic drugs. HuH6 (a–e) and HepT1 (f–j) were incubated with obatoclax at three different concentrations and CDDP (a, f), etoposide (b, g), irinotecan (d, i), paclitaxel (d, i), and DOXO (e, j) at seven different concentrations. Relative cell viability was determined 72 h later in a MTT assay
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Fig. 4 Viability of fibroblasts treated with obatoclax and various c cytotoxic drugs. Fibroblasts were incubated with obatoclax at three different concentrations and CDDP (a), etoposide (b), irinotecan (c), paclitaxel (d), and DOXO (e) at seven different concentrations. Relative cell viability was determined 72 h later in a MTT assay
Overexpression of Bcl-2 delays Bak conformational change, cytochrome C release, and apoptosis induced by cytotoxic drugs. In tumors with Bcl-2 overexpressionmediated chemoresistance, which includes childhood HB, apoptosis sensitizers binding to the Bcl-2 homology domain 3 (BH3) binding groove of Bcl-2 are considered to be a promising option. In this study, obatoclax and ABT737 induced apoptosis in HB cells and enhanced cytotoxic effects when combined with CDDP, etoposide, irinotecan, paclitaxel, and DOXO, of which some are commonly used in treatment protocols of HB. In contrast, HA14-1 and TW37, which also bind to Bcl-2 proteins and inhibit its function, had no pro-apoptotic effect on HB cells. Among BH3 mimetic compounds, HA14-1 is an organic compound originally discovered by computer modeling and the first small molecule which was predicted to bind to Bcl-2 with inhibitory effects [17]. It has been shown to induce apoptosis in various hematopoietic and solid tumors such as leukemias, lymphomas, breast, and ovarian carcinoma as well as neuroblastoma cells [17–20]. A synergism with a variety of anticancer agents to promote apoptosis is also described [21–25]. However, the response to HA14-1 was variable and described as partial in some cell lines, but inducing massive cell death in others. It is known, that expression of HA14-1 targets (Bcl-2 and Bcl-XL) did not correlate to these different responses; consequently, the potentiating effect of HA14-1 might be drug- and cell-type specific [18]. As the combination treatment in HB cell cultures did not result in additive effects, we assume that the targets of HA14-1 in HB cells were not regulated by cytotoxic drugs. Finally, HA14-1 is highly unstable and may rapidly decompose to inactive species in cell culture medium [26]. The same authors have observed that decomposition generates reactive oxygen species (ROS) resulting in potent pro-apoptotic activity, which makes interpretation of results in addition to effects of antagonizing anti-apoptotic Bcl-2 family proteins difficult. As HA14-1 did not show effects on HB cells in general, we yield no sense in repeating experiments using the stable analog sHA14-1 [27]. TW-37 also binds to the BH3 groove of Bcl-2 and competes with pro-apoptotic proteins (such as Bid, Bim, and Bad) preventing their heterodimerization with Bcl-2 and therefore allowing these proteins to induce apoptosis [28]. The anti-tumor action of TW-37 is assumed to be due to a combination of a pro-apoptotic effect on the tumor cells, as well as a specific anti-angiogenic effect [29].
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Fig. 5 Western blot analyses reveal increased anti-apoptotic state in spheroid HB cultures. HuH6 and HepT1 cells were grown for 24 h as monolayer and spheroid cultures. Relative expression levels of BclXL, Bax, and Mcl-1 are demonstrated
An anti-tumor effect has been described on HNSCC (head and neck small cell carcinoma), lymphomas, and pancreatic tumors. TW-37 also enhances cytotoxic effects when combined with CDDP, compared to treatment as a single drug [29–32]. In HB cells, effects of TW-37 as a single drug were moderate and comparable to above mentioned tumors from the literature. Surprisingly, we did not see additive effects after combination treatment with TW-37 and CDDP, as reported for HNSCC, in which CDDP is also a commonly used conventional chemotherapeutic drug in the treatment protocol [33]. Despite the fact that HNSCC and HB both express high levels of Bcl-2, the mechanism for the absence of additive effects in HB cells remains unclear. The IC50 for CDDP in HuH6 and HNSCC (UMFig. 6 Obatoclax has overcoming resistance to CDDP in spheroid cultures. HuH6 (a) and HepT1 (b) were incubated with and without ABT-737 or obatoclax and CDDP at seven different concentrations in spheroid cultures. Relative cell viability was determined 72 h later in a MTT assay
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SCC-1, UM-SCC-74A) was comparable, but doses of TW37 were threefold lower in HNSCC to significantly enhance effects of the combination treatment. In general, doses of TW-37 used were markedly higher in HuH6 and HepT1. Interestingly, TW-37 in the low- to mid-nanomolar range is accompanied by a marked accumulation of cells in the S phase of the cell cycle and without an equivalent increase in cell apoptosis in HNSCC [29]. This finding deserves further investigation in HB cells in order to improve understanding of TW-37 effects. ABT-737 inhibits the pro-survival function of Bcl-2, Bcl-XL, and Bcl-w, but exhibits low-affinity binding to the anti-apoptotic Mcl-1 and A1 proteins. Proteins of the Bcl family are overexpressed in HB tumors and Mcl is reduced, representing eligible conditions for high efficiency of ABT-737. In addition, HB cells expressed the pro-apoptotic Bak, which was described as a key molecule to sensitize tumor cells to ABT-737. However, this BH3 mimetic drug significantly potentiates the efficacy of established and novel chemotherapeutic drugs on HB cells, but singleagent activity is poor below doses of 1 lM. These results are consistent with those obtained in other solid tumors except SCLC reported in the literature [34–38]. In contrast, obatoclax has shown dose-dependent single-agent activity on HB cells at concentrations above 0.3 lM. Mechanistically, apoptosis induction by obatoclax can be preceded by liberation of Bak from Mcl-1, dissociation of Bim from Bcl-2 and Mcl-1 [39]. The additional binding on Mcl-1 proteins may enhance efficiency of obatoclax; however, a gene expression analysis revealed a twofold lower expression of Mcl-1 in native HB tissue and HuH6 cells than in normal liver tissue and a benefit of obatoclax was not expected [40, 41]. Furthermore, effects on HB cells of both BH3 mimetic drugs, ABT-737 and obatoclax, were more considerably in HuH6 compared to HepT1. The enhanced sensibility of
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HuH6 cells does not correlate with the relative expression of anti- and pro-apoptotic proteins, as Bcl-XL, Bax, and Mcl-1 are similar in both cell lines. In HepT1 cells higher concentrations of ABT-737 and obatoclax were used, but viability was further reduced in HuH6 only. We assume that a higher proliferation rate in HuH6 cells may explain the higher sensibility. It has been proposed that obatoclax can abolish cell growth independently of apoptosis by inducing a S–G2 cell cycle block [42]. This anti-proliferative effect could be separated from the pro-apoptotic effects of obatoclax in acute myeloid leukemia (AML) cell lines and primary AML samples, and it occurred in the absence of Bax/Bak/ Bim proteins, suggesting that this agent has multiple targets. The same para-apoptotic effect of obatoclax may contribute to the dose-dependent effects on proliferating fibroblasts in our study, as viability assays do not distinguish between cell death and block of mitosis. These Bcl2-independent targets of obatoclax may have clinical applicability, but mechanisms of these anti-proliferative effects on HB cells require further investigations in blood cells and in vivo. The combination therapy, including BH3 mimetic drugs, differently enhanced effects of cytotoxic drugs in HB cells, which was possibly due to their different mechanisms of action. The used cytotoxic drugs act as alkylating agents (CDDP), disturb RNA synthesis (DOXO), disturb spatial arrangement of DNA (irinotecan), inhibit organization of microtubules (paclitaxel) or interfere with DNA-repairing mechanisms (etoposide). Of these drugs, only CDDP and DOXO coopt mechanisms of apoptosis directly on the mitochondrial level inducing increased expression levels of P53, P21, and ROS (radical oxygen species) among others [43]. However, DOXO and also paclitaxel, which indirectly induce apoptosis, were found to be toxic in fibroblasts when combined with BH3 mimetic drugs, presuming that toxic effects may be enhanced and may have a narrow therapeutic window. In contrast, a combination with CDDP, etoposide and irinotecan will be better tolerated as revealed by fibroblast cultures. A combination of obatoclax with platin derivates and etoposide revealed increased transient adverse effects in some tumor patients; however, it was considered for a following phase II trial in small cell lung cancer [44]. CDDP is included in the treatment protocol of standard-risk HB achieving a response rate of 90%, enabling complete tumor resection in 95% and resulting in an overall survival of 95% (ClinicalTrials.gov number, NCT00003912.) [4]. In high-risk HB, the multidrug resistance (MDR) contributes to limited treatment results with only 78.7% achieving a partial response to chemotherapy and complete resection of liver tumors in 76.2% [2, 3, 5, 7]. Using CDDP in HB patients is still associated with adverse effects such as nephrotoxicity, ototoxicity, neurotoxicity,
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and myelosuppression [45]. In order to reduce these side effects, BH3 mimetic drugs are expected to enable doses reduction of cytotoxic drugs while maintaining their antitumor activity. After more than four cycles of CDDP 80% of the patients developed multidrug resistance [46]. In our model for multidrug-resistant HB, this resistance was overcome by BH3 mimetic drugs denoting the prominent role of anti-apotoptic mechanisms as the cause of resistance. This emphasizes that BH3 mimetics may also prevent or overcome resistance in HB.
Conclusion The data provide evidence that BH3 mimetic drugs might improve treatment results in patients with HB. Among the tested chemosensitizers ABT-737 and obatoclax might be used to reduce chemotherapeutic doses in HB. Modulating apoptosis pathways using these drugs in combination with established cytotoxic drugs may enhance resectability and reverse chemoresistance in HB. Acknowledgments The authors wish to acknowledge ABBOTT Laboratories for providing ABT-737.
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