A novel inhibitory BAK antibody enables assessment of non-activated BAK in cancer cells.

BAX and BAK are pro-apoptotic members of the BCL2 family that are required to permeabilize the mitochondrial outer membrane. The proteins can adopt a non-activated monomeric conformation, or an activated conformation in which the exposed BH3 domain facilitates binding either to a prosurvival protein or to another activated BAK or BAX protein to promote pore formation. Certain cancer cells are proposed to have high levels of activated BAK sequestered by MCL1 or BCLXL, thus priming these cells to undergo apoptosis in response to BH3 mimetic compounds that target MCL1 or BCLXL. Here we report the first antibody, 14G6, that is specific for the non-activated BAK conformer. A crystal structure of 14G6 Fab bound to BAK revealed a binding site encompassing both the α1 helix and α5-α6 hinge regions of BAK, two sites involved in the unfolding of BAK during its activation. In mitochondrial experiments, 14G6 inhibited BAK unfolding triggered by three diverse BAK activators, supporting crucial roles for both α1 dissociation and separation of the core (α2-α5) and latch (α6-α9) regions in BAK activation. 14G6 bound the majority of BAK in several leukaemia cell lines, and binding decreased following treatment with BH3 mimetics, indicating only minor levels of constitutively activated BAK in those cells. In summary, 14G6 provides a new means of assessing BAK status in response to anti-cancer treatments.

Assessment of Betulinic Acid Cytotoxicity and Mitochondrial Metabolism Impairment in a Human Melanoma Cell Line.

Melanoma represents one of the most aggressive and drug resistant skin cancers with poor prognosis in its advanced stages. Despite the increasing number of targeted therapies, novel approaches are needed to counteract both therapeutic resistance and the side effects of classic therapy. Betulinic acid (BA) is a bioactive phytocompound that has been reported to induce apoptosis in several types of cancers including melanomas; however, its effects on mitochondrial bioenergetics are less investigated. The present study performed in A375 human melanoma cells was aimed to characterize the effects of BA on mitochondrial bioenergetics and cellular behavior. BA demonstrated a dose-dependent inhibitory effect in both mitochondrial respiration and glycolysis in A375 melanoma cells and at sub-toxic concentrations (10 µM) induced mitochondrial dysfunction by eliciting a decrease in the mitochondrial membrane potential and changes in mitochondria morphology and localization. In addition, BA triggered a dose-dependent cytotoxic effect characterized by apoptotic features: morphological alterations (nuclear fragmentation, apoptotic bodies) and the upregulation of pro-apoptotic markers mRNA expression (Bax, Bad and Bak). BA represents a viable therapeutic option via a complex modulatory effect on mitochondrial metabolism that might be useful in advanced melanoma or as reliable strategy to counteract resistance to standard therapy.

Immunohistochemical assessment of apoptosis-associated proteins: p53, Bcl-xL, Bax and Bak in gastric cancer cells in correlation with clinical and pathomorphological factors.

PURPOSE: The p53 protein as well as Bcl-2 family proteins such as Bax, Bak and Bcl-xL regulate apoptosis. The study objective was to analyze the expression of p53, Bak, Bcl-xL and Bax in gastric cancer and in healthy gastric mucosa. MATERIAL AND METHODS: The study group consisted of 66 patients with gastric cancer, treated surgically in II Department of General and Gastroenterological Surgery, Medical University of Bialystok. The expression of the studied proteins was assessed using the immunohistochemical method. RESULTS: Significant differences were found in the expressions of the studied proteins as compared to healthy gastric mucosa. The expressions of p53 and Bax were significantly higher (70% vs 13% and 50% vs 13%), whereas those of Bak and Bcl-xL significantly lower (18% vs 83% and 74% vs 97%) in cancer cells than in normal mucosa (p<0.001). Significant differences were also noted in the expressions of Bax and Bcl-xL in relation to histological type. In the intestinal type (Lauren I), the expressions of Bax and Bcl-xL were higher as compared to the diffuse type (Lauren II) (93% vs 43% and 91% vs 43%). Simultaneously, correlations were noted between changes in the expression of Bax vs Bcl-xL and Bak. High expression of Bax showed a positive correlation with reduced Bak and Bcl-xL (p<0.05). Moreover, positive expression of p53 caused poorer distant survival of patients (p<0.05). CONCLUSION: Our study concluded that disturbances in the expression of p53, Bax, Bcl-xL and Bak proteins are associated with their involvement in the process of carcinogenesis in the stomach. It is suggesting that they might appeared in the early phase of carcinogenesis.

In vivo and in vitro assessment of pathways involved in contrast media-induced renal cells apoptosis.

Contrast-induced nephropathy accounts for >10% of all causes of hospital-acquired renal failure, causes a prolonged in-hospital stay and represents a powerful predictor of poor early and late outcome. Mechanisms of contrast-induced nephropathy are not completely understood. In vitro data suggests that contrast media (CM) induces a direct toxic effect on renal tubular cells through the activation of the intrinsic apoptotic pathway. It is unclear whether this effect has a role in the clinical setting. In this work, we evaluated the effects of CM both in vivo and in vitro. By analyzing urine samples obtained from patients who experienced contrast-induced acute kidney injury (CI-AKI), we verified, by western blot and immunohistochemistry, that CM induces tubular renal cells apoptosis. Furthermore, in cultured cells, CM caused a dose-response increase in reactive oxygen species (ROS) production, which triggered Jun N-terminal kinases (JNK1/2) and p38 stress kinases marked activation and thus apoptosis. Inhibition of JNK1/2 and p38 by different approaches (i.e. pharmacological antagonists and transfection of kinase-death mutants of the upstream p38 and JNK kinases) prevented CM-induced apoptosis. Interestingly, N-acetylcysteine inhibited ROS production, and thus stress kinases and apoptosis activation. Therefore, we conclude that CM-induced tubular renal cells apoptosis represents a key mechanism of CI-AKI.