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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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).</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

BIM mediates synergistic killing of B-cell acute lymphoblastic leukemia cells by BCL-2 and MEK inhibitors

B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematological disease that kills ~50% of adult patients. With the exception of some BCR-ABL1(+) patients who benefit from tyrosine kinase inhibitors, there are no effective targeted therapies for adult B-ALL patients and chemotherapy remains first-line therapy despite adverse side effects and poor efficacy. We show that, although the MEK/ERK pathway is activated in B-ALL cells driven by different oncogenes, MEK inhibition does not suppress B-ALL cell growth. However, MEK inhibition synergized with BCL-2/BCL-XL family inhibitors to suppress proliferation and induce apoptosis in B-ALL cells. We show that this synergism is mediated by the pro-apoptotic factor BIM, which is dephosphorylated as a result of MEK inhibition, allowing it to bind to and neutralize MCL-1, thereby enhancing BCL-2/BCL-XL inhibitor-induced cell death. This cooperative effect is observed in B-ALL cells driven by a range of genetic abnormalities and therefore has significant therapeutic potential

Phosphorylation of Puma modulates its apoptotic function by regulating protein stability

Puma is a potent BH3-only protein that antagonises anti-apoptotic Bcl-2 proteins, promotes Bax/Bak activation and has an essential role in multiple apoptotic models. Puma expression is normally kept very low, but can be induced by several transcription factors including p53, p73, E2F1 and FOXO3a, whereby it can induce an apoptotic response. As Puma can to bind and inactivate all anti-apoptotic members of the Bcl-2 family, its activity must be tightly controlled. We report here, for the first time, evidence that Puma is subject to post-translational control through phosphorylation. We show that Puma is phosphorylated at multiple sites, with the major site of phosphorylation being serine 10. Replacing serine 10 with alanine causes reduced Puma turnover and enhanced cell death. Interestingly, Puma turnover occurs through the proteasome, and substitution of serine 10 causes elevated Puma levels independently of macroautophagy, Bcl-2 family member binding, caspase activity and apoptotic death. We conclude, therefore, that phosphorylation of Puma at serine 10 promotes Puma turnover, represses Puma's cell death potential and promotes cell survival. Owing to the highly pro-apoptotic nature of Puma, these studies highlight an important additional regulatory step in the determination of cellular life or death

Global gene disruption in human cells to assign genes to phenotypes

Insertional mutagenesis in a haploid background can disrupt gene function[superscript 1]. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones[superscript 1] enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites[superscript 2]. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT

Over-expression of Adenine Nucleotide Translocase 1 (ANT1) Induces Apoptosis and Tumor Regression in vivo

Background: Adenine nucleotide translocase (ANT) is located in the inner mitochondrial membrane and catalyzes the exchange of mitochondrial ATP for cytosolic ADP. ANT has been known to be a major component of the permeability transition pore complex of mitochondria and contributes to mitochondria-mediated apoptosis. Human ANT has four isoforms (ANT1, ANT2, ANT3, and ANT4), and the expression of the ANT isoforms is variable depending on the tissue and cell type, developmental stage, and proliferation status. Among the isoforms, ANT1 is highly expressed in terminally-differentiated tissues, but expressed in low levels in proliferating cells, such as cancer cells. In particular, over-expression of ANT1 induces apoptosis in cultured tumor cells. Methods: We applied an ANT1 gene transfer approach to induce apoptosis and to evaluate the anti-tumor effect of ANT1 in a nude mouse model. Results: We demonstrated that ANT1 transfection induced apoptosis of MDA-MB-231 cells, inactivated NF-κB activity, and increased Bax expression. ANT1-inducing apoptosis was accompanied by the disruption of mitochondrial membrane potential, cytochrome c release and the activation of caspases-9 and -3. Moreover, ANT1 transfection significantly suppressed tumor growth in vivo. Conclusion: Our results suggest that ANT1 transfection may be a useful therapeutic modality for the treatment of cancer

Obatoclax induces Atg7-dependent autophagy independent of beclin-1 and BAX/BAK

Direct pharmacological targeting of the anti-apoptotic B-cell lymphoma-2 (BCL-2) family is an attractive therapeutic strategy for treating cancer. Obatoclax is a pan-BCL-2 family inhibitor currently in clinical development. Here we show that, although obatoclax can induce mitochondrial apoptosis dependent on BCL-2 associated x protein/BCL-2 antagonist killer (BAX/BAK) consistent with its on-target pharmacodynamics, simultaneous silencing of both BAX and BAK did not abolish acute toxicity or loss of clonogenicity. This is despite complete inhibition of apoptosis. Obatoclax dramatically reduced viability without inducing loss of plasma membrane integrity. This was associated with rapid processing of light chain-3 (LC3) and reduction of S6 kinase phosphorylation, consistent with autophagy. Dramatic ultrastructural vacuolation, not typical of autophagy, was also induced. Silencing of beclin-1 failed to prevent LC3 processing, whereas knockout of autophagy-related (Atg)7 abolished LC3 processing but failed to prevent obatoclax-induced loss of clonogenicity or ultrastructural changes. siRNA silencing of Atg7 in BAX/BAK knockout mouse embryonic fibroblasts did not prevent obatoclax-induced loss of viability. Cells selected for obatoclax resistance evaded apoptosis independent of changes in BCL-2 family expression and displayed reduced LC3 processing. In summary, obatoclax exhibits BAX- and BAK-dependent and -independent mechanisms of toxicity and activation of autophagy. Mechanisms other than autophagy and apoptosis are blocked in obatoclax resistant cells and contribute significantly to obatoclax's anticancer efficacy

The Bcl-2 repertoire of mesothelioma spheroids underlies acquired apoptotic multicellular resistance

Three-dimensional (3D) cultures are a valuable platform to study acquired multicellular apoptotic resistance of cancer. We used spheroids of cell lines and actual tumor to study resistance to the proteasome inhibitor bortezomib in mesothelioma, a highly chemoresistant tumor. Spheroids from mesothelioma cell lines acquired resistance to bortezomib by failing to upregulate Noxa, a pro-apoptotic sensitizer BH3-only protein that acts by displacing Bim, a pro-apoptotic Bax/Bak-activator protein. Surprisingly, despite their resistance, spheroids also upregulated Bim and thereby acquired sensitivity to ABT-737, an inhibitor of anti-apoptotic Bcl-2 molecules. Analysis using BH3 profiling confirmed that spheroids acquired a dependence on anti-apoptotic Bcl-2 proteins and were ‘primed for death'. We then studied spheroids grown from actual mesothelioma. ABT-737 was active in spheroids grown from those tumors (5/7, ∼70%) with elevated levels of Bim. Using immunocytochemistry of tissue microarrays of 48 mesotheliomas, we found that most (33, 69%) expressed elevated Bim. In conclusion, mesothelioma cells in 3D alter the expression of Bcl-2 molecules, thereby acquiring both apoptotic resistance and sensitivity to Bcl-2 blockade. Mesothelioma tumors ex vivo also show sensitivity to Bcl-2 blockade that may depend on Bim, which is frequently elevated in mesothelioma. Therefore, mesothelioma, a highly resistant tumor, may have an intrinsic sensitivity to Bcl-2 blockade that can be exploited therapeutically

Withaferin a-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species

Withaferin A (WA), a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC). The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak. © 2011 Hahm et al

Synergistic Anticancer Effects of the 9.2.27PE Immunotoxin and ABT-737 in Melanoma

In cancer, combinations of drugs targeting different cellular functions is well accepted to improve tumor control. We studied the effects of a Pseudomonas exotoxin A (PE) - based immunotoxin, the 9.2.27PE, and the BH-3 mimetic compound ABT-737 in a panel of melanoma cell lines. The drug combination resulted in synergistic cytotoxicity, and the cell death observed was associated with apoptosis, as activation of caspase-3, inactivation of Poly (ADP-ribose) polymerase (PARP) and increased DNA fragmentation could be prevented by pre-treatment with caspase and cathepsin inhibitors. We further show that ABT-737 caused endoplasmic reticulum (ER) stress with increased GRP78 and phosphorylated eIF2α protein levels. Moreover, treatment with ABT-737 increased the intracellular calcium levels, an effect which was enhanced by 9.2.27PE, which as a single entity drug had minimal effect on calcium release from the ER. In addition, silencing of Mcl-1 by short hairpin RNA (shRNA) enhanced the intracellular calcium levels and cytotoxicity caused by ABT-737. Notably, the combination of 9.2.27PE and ABT-737 caused growth delay in a human melanoma xenograft mice model, supporting further investigations of this particular drug combination