Abstract:
The seminal discovery that the BCL2 gene inhibits cell death rather than promotes cell proliferation gave the foundation for a nowadays widely appreciated theory that impaired tumor cell death is a decisive stage in a multi-step carcinogenesis. Recognition of the central role of mitochondria and Bcl-2 family members in the regulation and propagation of cell demise has, thus, recently uncovered novel targets for selective anti-cancer therapies. In this context, we have for the first time demonstrated monotherapeutic potential of a small molecule Bcl-2 antagonist, HA14-1, against follicular lymphoma (FL) cells with t(14;18) translocation and Bcl-2 overexpression. In agreement with others we have shown that cytotoxicity mediated by a small molecule Bcl-2 inhibitor HA14-1 in FL cells proceeds through rapid (4h) dissipation of the ∆Ψm, generation of ROS and caspase-dependent apoptosis. Moreover, we were first to prove the applicability of combinatorial treatment of HA14-1 and selected conventional chemotherapeutics (dexamethasone and doxorubicin) in FL cells. In the following work we investigated the quantitative relationship between ∆Ψm loss and caspase activation in HA14-1 treated cells. Employing both pharmacological inhibitor studies and novel state-of-the-art multiparametric flow cytometry assays, we revealed that following HA14-1 treatment caspase activation occurs solely as a consequence of mitochondrial breach. Additionally, we provided new evidence that HA14-1-evoked apoptosis appears to be at least partially PT-dependent. We also for the first time addressed the cell cycle specificity of HA14-1 action using multivariate flow cytometry approaches. As the interest in the role of ER and Golgi during induction/execution of apoptosis has been gaining momentum, they simultaneously attract growing appreciation in the development of novel anti-cancer therapies. Pertinent to the therapy of B-cell malignancies we report here for the first time the effects of an ER-Golgi transport inhibitor, Brefeldin A (BFA), alone and in combination with a small molecule Bcl-2 inhibitor HA14-1 or death receptor trigger, in the human FL cell lines bearing t(14;18) translocation. Of importance for future anti-cancer regimens, small molecule Bcl-2 antagonist, HA14-1 and agonistic anti-Fas mAb significantly enhanced BFA- mediated cytotoxicity and apoptosis, revealing novel and previously unexplored avenues to enhance ER-stress mediated cell killing in B-cell malignancies. Finally, as basic studies advance towards their ultimate translational goals there is a need for effective and rapid analytical methods allowing high-throughput detection of diverse cell demise modes. In this context patented SYTO® probes are gaining increasing interest as easy to use markers of apoptosis. Herein, applying the state-of-the-art multiparametric flow cytometry and multicolor cell imaging we identified for the first time different SYTO16 staining characteristics upon apoptotic and oncotic stimuli. We also tracked similarities and discrepancies between SYTO16 and ∆Ψm, sensitive probe, tertramethylrhodamine methyl ester (TMRM), demonstrating that stimulation with mitochondrial uncoupler FCCP and a small-molecule Bcl-2 inhibitor, HA14-1, induce distinct staining profiles with the decrease in TMRM fluorescence preceding the loss of SYTO16 fluorescence. To our knowledge this is the first report demonstrating such a distinct behavior of SYTO16 and TMRM and our data demonstrate that loss of SYTO16 is caspase-dependent, as is not a mere indicator of ∆ψm dissipation, postulated previously by some authors.