Direct activation of the apoptosis machinery as a mechanism to target cancer cells

Apoptosis plays a pivotal role in the cytotoxic activity of most chemotherapeutic drugs, and defects in this pathway provide a basis for drug resistance in many cancers. Thus the ability to restore apoptosis by using small molecules could have important therapeutic implications. Using a cell-free assay to simultaneously target multiple components of the apoptosis pathway, we identified a class of compounds that activate caspases in a cytochrome c-dependent manner and induce apoptosis in whole cells. By reconstituting the apoptosis pathway with purified proteins, we determined that these compounds promote the protein–protein association of Apaf-1 into the functional apoptosome. These compounds exert cytostatic and cytotoxic effects on a variety of cancer cell lines while having little or no activity against the normal cell lines tested. These findings suggest that direct activation of the basic apoptosis machinery may be a viable mechanism to selectively target cancer

A combinatorial approach for selectively inducing programmed cell death in human pancreatic cancer cells

Pancreatic cancer is an extremely aggressive neoplasm whose incidence equals its death rate. Despite intensive analysis, the genetic changes that mediate pancreatic cancer development and effective therapies for diminishing the morbidity associated with this disease remain unresolved. Through subtraction hybridization, we have identified a gene associated with induction of irreversible growth arrest, cancer reversion, and terminal differentiation in human melanoma cells, melanoma differentiation associated gene-7 (mda-7). Ectopic expression of mda-7 when using a recombinant adenovirus, Ad.mda-7, results in growth suppression and apoptosis in a broad spectrum of human cancers with diverse genetic defects, without exerting deleterious effects in normal human epithelial or fibroblast cells. Despite the apparently ubiquitous antitumor effects of mda-7, pancreatic carcinoma cells are remarkably refractory to Ad.mda-7 induced growth suppression and apoptosis. In contrast, the combination of Ad.mda-7 with antisense phosphorothioate oligonucleotides, which target the K-ras oncogene (a gene that is mutated in 85 to 95% of pancreatic carcinomas), induces a dramatic suppression in growth and a decrease in cell viability by induction of apoptosis. In mutant K-ras pancreatic carcinoma cells, programmed cell death correlates with expression and an increase, respectively, in MDA-7 and BAX proteins and increases in the ratio of BAX to BCL-2 proteins. Moreover, transfection of mutant K-ras pancreatic carcinoma cells with an antisense K-ras expression vector and infection with Ad.mda-7 inhibits colony formation in vitro and tumorigenesis in vivo in nude mice. These intriguing observations demonstrate that a combinatorial approach, consisting of a cancer-specific apoptosis-inducing gene and an oncogene inactivation strategy, may provide the foundation for developing an effective therapy for pancreatic cancer

Heart-targeted overexpression of caspase3 in mice increases infarct size and depresses cardiac function

Up-regulation of proapoptotic genes has been reported in heart failure and myocardial infarction. To determine whether caspase genes can affect cardiac function, a transgenic mouse was generated. Cardiac tissue-specific overexpression of the proapoptotic gene Caspase3 was induced by using the rat promoter of α-myosin heavy chain, a model that may represent a unique tool for investigating new molecules and antiapoptotic therapeutic strategies. Cardiac-specific Caspase3 expression induced transient depression of cardiac function and abnormal nuclear and myofibrillar ultrastructural damage. When subjected to myocardial ischemia–reperfusion injury, Caspase3 transgenic mice showed increased infarct size and a pronounced susceptibility to die. In this report, we document an unexpected property of the proapoptotic gene caspase3 on cardiac contractility. Despite inducing ultrastructural damage, Caspase3 does not trigger a full apoptotic response in the cardiomyocyte. We also implicate Caspase3 in determining myocardial infarct size after ischemia–reperfusion injury, because its cardiomyocyte-specific overexpression increases infarct size

Intracellular antibody-caspase-mediated cell killing: An approach for application in cancer therapy

Antibodies have been expressed inside cells in an attempt to ablate the function of oncogene products. To make intracellular antibodies more generally applicable and effective in cancer therapy, we have devised a method in which programmed cell death or apoptosis can be triggered by specific antibody–antigen interaction. When intracellular antibodies are linked to caspase 3, the “executioner” in the apoptosis pathway, and bind to the target antigen, the caspase 3 moieties are self-activated and thereby induce cell killing. We have used this strategy in a model system with two pairs of intracellular antibodies and antigens. In vivo coexpression of an antibody-caspase 3 fusion with its antigenic target induced apoptosis that was specific for antibody, antigen, and active caspase 3. Moreover, the antibody-caspase 3 fusion protein was not toxic to cells in the absence of antigen. Therefore, intracellular antibody-mediated apoptosis should be useful as a specific therapeutic approach for the treatment of cancers, a situation where target cell killing is required

Increased in vivo apoptosis in cells lacking mitochondrial DNA gene expression

We have attempted to determine whether loss of mtDNA and respiratory chain function result in apoptosis in vivo. Apoptosis was studied in embryos with homozygous disruption of the mitochondrial transcription factor A gene (Tfam) and tissue-specific Tfam knockout animals with severe respiratory chain deficiency in the heart. We found massive apoptosis in Tfam knockout embryos at embryonic day (E) 9.5 and increased apoptosis in the heart of the tissue-specific Tfam knockouts. Furthermore, mtDNA-less (ρ(0)) cell lines were susceptible to apoptosis induced by different stimuli in vitro. The data presented here provide in vivo evidence that respiratory chain deficiency predisposes cells to apoptosis, contrary to previous assumptions based on in vitro studies of cultured cells. These results suggest that increased apoptosis is a pathogenic event in human mtDNA mutation disorders. The finding that respiratory chain deficiency is associated with increased in vivo apoptosis may have important therapeutic implications for human disease. Respiratory chain deficiency and cell loss and/or apoptosis have been associated with neurodegeneration, heart failure, diabetes mellitus, and aging. Furthermore, chemotherapy and radiation treatment of cancer are intended to induce apoptosis in tumor cells. It would therefore be of interest to determine whether manipulation of respiratory chain function can be used to inhibit or enhance apoptosis in these conditions