nitrophenols and Hepatoblastoma

Pediatric hepatoblastoma (HB) is commonly treated by neoadjuvant chemotherapy and surgical tumor resection according to international multicenter trial protocols. Complete tumor resection is essential and survival rates up to 95% have now been achieved in those tumors classified as standard-risk HB. Drug resistance and occurrence of metastases remain the major challenges in the treatment of HB, especially in high-risk tumors. These conditions urgently require the development of alternative therapeutic strategies. One of those alternatives is the modulation of apoptosis in HB cells. HBs regularly overexpress anti-apoptotic proteins of the Bcl-family in comparison to healthy liver tissue. This fact may contribute to the development of chemoresistance of HB cells. Synthetic small inhibitory molecules with BH3-mimetic effects, such as ABT-737 and obatoclax, enhance the susceptibility of tumor cells to different cytotoxic drugs and thereby affect initiator proteins of the apoptosis cascade via the intrinsic pathway. Besides additive effects on HB cell viability when used in combination with cytotoxic drugs, BH3-mimetics also play a role in preventing metastasation by reducing adhesion and inhibiting cell migration abilities. Presumably, including additive BH3-mimetic drugs into existing therapeutic regimens in HB patients might allow dose reduction of established cytotoxic drugs and thereby associated immanent side effects, while maintaining the antitumor activity. Furthermore, reduction of tumor growth and inhibition of tumor cell dissemination may facilitate complete surgical tumor resection, which is mandatory in this tumor type resulting in improved survival rates in high-risk HB. Currently, there are phase I and phase II clinical trials in several cancer entities using this potential target. This paper reviews the available literature regarding the use of BH3-mimetic drugs as single agents or in combination with chemotherapy in various malignancies and focuses on results in HB cells.

Topics: Antibodies, Monoclonal; Apoptosis; Biphenyl Compounds; Child; Drug Resistance, Neoplasm; Hepatoblastoma; Humans; Indoles; Liver Neoplasms; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides; TNF-Related Apoptosis-Inducing Ligand

Drug resistance and metastasis remain major challenges in the treatment of high-risk hepatoblastoma (HB) and require the development of alternative therapeutic strategies. Modulation of apoptosis in HB cells enhances the sensitivity of these cells towards various drugs and has been discussed to enforce treatment. We investigated the impact of apoptosis sensitisers, BH3-mimetics, on the interaction between the host and HB to reduce tumour growth and dissemination while enhancing immunity. BH3-mimetics, such as obatoclax and ABT-737, enhanced the apoptosis-inducing effect of TRAIL and TNF-α resistant HB cells (HepT1 and HUH6). Tumour cell migration was inhibited by ABT-737 and more markedly by obatoclax. In an orthotopic model of HB, tumour uptake was reduced when the cells were pretreated with low concentrations of obatoclax. Only 1 of 7 mice developed HB in the liver, compared with an incidence of 0.8 in the control group. In summary, our study showed that apoptosis sensitisers had broader effects on HB cells than expected including migration and susceptibility to cytokines in addition to the known effects on drug sensitization. Sensitising HB to apoptosis may also allow resistant HB to be targeted by immune cells and prevent tumour cell dissemination.

Topics: Animals; Biomimetic Materials; Biphenyl Compounds; Cell Transformation, Neoplastic; Cells, Cultured; Disease Models, Animal; Drug Evaluation, Preclinical; Hepatoblastoma; Humans; Indoles; Liver Neoplasms; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Nude; Mice, Transgenic; Nitrophenols; Peptide Fragments; Piperazines; Proto-Oncogene Proteins; Pyrroles; Sulfonamides

Advanced stages of tumour and development of metastases are the two major problems in treating liver tumours such as hepatoblastoma (HB) and hepatocellular carcinoma (HCC), in paediatric patients. Modulation of apoptosis in HB cells enhances the sensitivity of these cells towards various drugs and has been discussed to enforce treatment. We analysed the effect of apoptosis modulators, BH3 mimetics, on mechanisms of dissemination such as adhesion or migration of HB and HCC cells. BH3 mimetics such as ABT-737 and obatoclax can reduce cell migration in a scratch assay as well as adhesion of HB and HCC cells to matrigel. Immunofluorescence staining of F-actin demonstrated that development of lamellipodia, which are important for migration, decreased. BH3 mimetics increase the level of activated caspases 3 and 7 in HUH6 cells. This results in the degradation of GTPase Cdc42, which can be determined by western blot analysis. A pan-caspase inhibitor can block the migration and degradation of Rho-GTPase. In summary, our study showed that BH3 mimetics not only enhance drug sensitivity but also may prevent metastasis by inhibiting HB and HCC cell motility.

Topics: Actins; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Caspase 7; Caspase Inhibitors; cdc42 GTP-Binding Protein; Cell Adhesion; Cell Movement; Cytoskeleton; Enzyme Activation; Fluorescent Antibody Technique; Hepatoblastoma; Humans; Indoles; Neoplasm Metastasis; Nitrophenols; Oligopeptides; Piperazines; Proteolysis; Pseudopodia; Pyrroles; Sulfonamides

The response of standard-risk hepatoblastoma (HB) to neoadjuvant cisplatin (CDDP) chemotherapy is excellent; however, in high-risk HB, drug resistance remains a major challenge. Alternative therapeutic strategies may consider combining cytotoxic drugs with apoptosis sensitizers as this has shown additive effects in various types of malignancies. Analysis of published expression databases have revealed an anti-apoptosis state in HB samples. Herein, we evaluated the synergistic effects of ABT-737 as a modulator of apoptosis in combination with CDDP in HB. To this end, clonogenic assays were performed with HepT1 and HUH6 HB cells to evaluate the synergistic effects of CDDP and ABT-737. Combination treatment with CDDP and ABT-737 reduced the clonogenicity of HB cells more than 5-fold compared to treatment with CDDP alone. Furthermore, the HUH6 mixed-type HB cells showed higher sensitivity to CDDP and combination treatment compared to the HepT1 embryonal-type cells. Subcutaneous HUH6 tumors in NOD/LtSz-scid IL2Rγnull mice were treated with CDDP (1.25 and 3 mg/kg body weight, n=6), ABT-737 (100 mg/kg, n=5) and the combination of both agents (n=5). Combined treatment led to a significantly reduced tumor growth compared to CDDP treatment alone (p<0.02). When using higher doses of CDDP (3 mg/kg) alone or in combination with ABT-737, dose-dependent toxicity was observed in this mouse strain. In conclusion, our results demonstrated the enhancement of chemotherapy efficacy by using modulators of apoptosis together with cytotoxic agents. Additive effects of ABT-737 may allow reduction in CDDP dosages with maintenance of antitumor activity. Sensitizing HB to apoptosis may also render resistant HB susceptible to established chemotherapy regimens.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Cell Line, Tumor; Cisplatin; Drug Resistance, Neoplasm; Gene Expression Profiling; Hepatoblastoma; Liver Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Nitrophenols; Piperazines; Sulfonamides; Weight Loss

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.. 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 pro- and anti-apoptotic proteins.. Obatoclax and ABT-737 led to a dose-dependent decrease of viability in HB cells at concentrations above 0.3 μM. 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.. The data provide evidence that ABT-737 and obatoclax might improve treatment results in children with HB.

Topics: Apoptosis; Benzamides; Benzopyrans; Biphenyl Compounds; Blotting, Western; Cell Survival; Drug Resistance, Neoplasm; Enzyme Inhibitors; Hepatoblastoma; Humans; Indoles; Liver Neoplasms; Nitriles; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides; Sulfones; Tumor Cells, Cultured

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.. 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).. 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.. 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.

Topics: Analysis of Variance; Animals; Antineoplastic Combined Chemotherapy Protocols; Biomimetic Materials; Biphenyl Compounds; Cell Growth Processes; Cell Line, Tumor; Drug Synergism; Hepatoblastoma; Humans; Immunohistochemistry; Liver Neoplasms; Mice; Mice, SCID; Nitrophenols; Paclitaxel; Piperazines; Sulfonamides; Xenograft Model Antitumor Assays

An increased expression of anti-apoptotic proteins is regularly found in malignant cells, contributing to their clonal expansion by conferring an improved survival ability. In Hepatoblastoma (HB) apoptosis regulation contributes to resistance and therapy failure, therefore we modulated apoptosis sensitivity of HB cells for an improved cytotoxic activity of commonly used drugs.. Apoptosis-related proteins were quantified in HB cells (HuH6 and HepT1) using protein assays. Interaction of ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xL, and Bcl-W with cytotoxic drugs was monitored in a proliferation assay. Apoptosis induction was measured by caspase-3 activity.. We found high levels of the anti-apoptotic protein Bcl-2 and Bcl-X as well as low levels of pro-apoptotic protein Bax and Bad in both HB cell lines. ABT-737 induced apoptosis in HuH6 and HepT1 cells at concentrations higher than 1 µM. ABT-737 also enhanced the cytotoxic effect of cisplatin (CDDP), doxorubicin (DOXO), etoposide and paclitaxel when used as combination therapy. HuH6 expressed slightly higher pro-apoptotic and lower anti-apoptotic protein levels than HepT1, which may explain the stronger enhancement of cytostatic drug effects in HuH6 cells when treated in combination with ABT-737.. The observed anti-apoptotic phenotype in HB cell lines may contribute to resistance to cytotoxic drugs used in the standard treatment protocol of HB. These pre-clinical results suggest that apoptosis sensitizers with BH-3 mimicry, such as ABT-737, should be further evaluated in preclinical models of HB.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Apoptosis; bcl-X Protein; Biphenyl Compounds; Blotting, Western; Cell Line, Tumor; Cisplatin; Doxorubicin; Drug Resistance, Neoplasm; Hepatoblastoma; Humans; Liver Neoplasms; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides