The BH3 mimetic ABT-737 increases treatment efficiency of paclitaxel against hepatoblastoma

Abstract

Background: The primary goal of current chemotherapy in hepatoblastoma (HB) is reduction of tumour volume and vitality to enable complete surgical resection and reduce risk of recurrence or metastatic disease. Drug resistance remains a major challenge for HB treatment. In some malignancies inhibition of anti-apoptotic pathways using small BH3 mimetic molecules like ABT-737 shows synergistic effects in combination with cystotoxic agents in vitro. Now we analysed toxicology and synergistic effects of this approach in HB cells and HB xenografts.

Methods: Viability was monitored in HB cells (HUH6 and HepT1) and fibroblasts treated with paclitaxel, ABT-737 and a combination of both in a MTT assay. HUH6 xenotransplants in NOD/LtSz-scid IL2Rγnull mice (NSG) were treated accordingly. Tumour volume and body weight were monitored. Xenografted tumours were analysed by histology and immunohistochemistry (Ki-67 and TUNEL assay).

Results: ABT-737 reduced viability in HUH6 and HepT1 cells cultures at concentrations above 1 μM and also enhanced the cytotoxic effect of paclitaxel when used in combination. Thereby paclitaxel could be reduced tenfold to achieve similar reduction of viability of tumour cells. In contrast no toxicity in fibroblasts was observed at the same regiments. Subcutaneous HB (HUH6) treated with paclitaxel (12 mg/kg body weight, n = 7) led to delayed tumour growth in the beginning of the experiment. However, tumour volume was similar to controls (n = 5) at day 25. Combination treatment with paclitaxel and ABT-737 (100 mg/kg, n = 8) revealed significantly 10 fold lower relative tumour volumes compared to control and paclitaxel groups. Paclitaxel dependent toxicity was observed in this mice strain.

Conclusions: Our results demonstrate enhancement of chemotherapy by using modulators of apoptosis. Further analyses should include improved pharmacological formulations of paclitaxel and BH3 mimetics in order to reduce toxicological effects. Sensitising HB to apoptosis may also render resistant HB susceptible to established chemotherapy regimens.

Figure 1

Synergistic effect of paclitaxel and ABT-737 cultured cells. Fibroblasts (black square), HepT1 (black triangle) and HuH6 (black rhomb) cells were incubated with paclitaxel at seven different concentrations alone (A) and in combination with 0.3 μM ABT-737 (B). Relative cell viability was determined 72 hrs later in a MTT assay. No toxicity was observed in fibroblasts. For HB cells synergistic effects of combined treatment could be observed. Apoptosis was detected by Caspase 3 activity 24 h after combination treatment of HuH6 and HepT1 cells (C). Enhanced Caspase 3 activity was found in HuH6 cells incubated with paclitaxel and 0.3 μM ABT-737 when compared with paclitaxel alone.

Figure 2

Tumour growth after treatment with paclitaxel alone and in combination with ABT-737. (A) HuH6 derived s.c. tumours were treated with paclitaxel (white triangle), ABT-737 (black square), and with a combination of both (black triangle) at indicated time points. Untreated animals served as controls (white square). Data points indicate means and SD of tumour volumes at respective days. (B) After the first cycle of treatment (14 days) the combination of paclitaxel and ABT-737 revealed a significantly lower relative tumour growth compared to controls and paclitaxel treated tumours. Data represent the relative tumour volume of each animal. *p < 0.05 in a Student t-test.

Figure 3

Immunhistological analysis of HB tumours. Images show staining of Haematoxilin and Eosin (A-D), detection of Ki-67 (E-H) and Tunel assay (I-L). For each representative samples of controls (A, E, I), paclitaxel (B, F, J), ABT-737 (C, G, K) and combination (D, H, L) are provided. Combined treatment using paclitaxel and ABT-737 reveals high tissue damage and a low proliferation index. Multiple picnotic nuclei denote necrotic tissue destruction. Brown staining shows Ki-67 positive cells. Bright green fluorescences are apoptotic cells. Nuclear staining was done by Hematoxylin and DAPI, respectively.

Figure 4

Toxicity of paclitaxel in NSG mice. NSG mice were treated as indicated on 4 consecutive days. The body weight is shown as fraction for each individual mouse at day 5 and at the beginning of the experiment (A). Significant loss of 10% body weight was observed in the groups treated with paclitaxel (black circle) and paclitaxel/ABT-737 (black triangle) compared with control (black rhomb) animals (one way Anova, Dunnett's multiple test p < 0.01). HE staining of liver tissues revealed multiple picnotic cells after combination treatment (E), which were not found in the other 3 groups: control (B), paclitaxel (C) and ABT-737 (D).