Abstract
Activation of initiator and effector caspases, mitochondrial changes involving a reduction in its membrane potential and release of cytochrome c (cyt c) into the cytosol, are characteristic features of apoptosis. These changes are associated with cell acidification in some models of apoptosis. The hierarchical relationship between these events has, however, not been deciphered. We have shown that somatostatin (SST), acting via the Src homology 2 bearing tyrosine phosphatase SHP-1, exerts cytotoxic action in MCF-7 cells, and triggers cell acidification and apoptosis. We investigated the temporal sequence of apoptotic events linking caspase activation, acidification, and mitochondrial dysfunction in this system and report here that (i) SHP-1-mediated caspase-8 activation is required for SST-induced decrease in pH(i). (ii) Effector caspases are induced only when there is concomitant acidification. (iii) Decrease in pH(i) is necessary to induce reduction in mitochondrial membrane potential, cyt c release and caspase-9 activation and (iv) depletion of ATP ablates SST-induced cyt c release and caspase-9 activation, but not its ability to induce effector caspases and apoptosis. These data reveal that SHP-1-/caspase-8-mediated acidification occurs at a site other than the mitochondrion and that SST-induced apoptosis is not dependent on disruption of mitochondrial function and caspase-9 activation.
Highlights
Apoptosis is a physiological process of cell death indispensable for the maintenance of multicellular organisms
We investigated the temporal sequence of apoptotic events linking caspase activation, acidification, and mitochondrial dysfunction in this system and report here that (i) SHP1-mediated caspase-8 activation is required for SST-induced decrease in pHi. (ii) Effector caspases are induced only when there is concomitant acidification. (iii) Decrease in pHi is necessary to induce reduction in mitochondrial membrane potential, cyt c release and caspase-9 activation and (iv) depletion of ATP ablates SST-induced cyt c release and caspase-9 activation, but not its ability to induce effector caspases and apoptosis
Caspase-9 can function as an initiator caspase when mitochondrial dysfunction is the primary event in apoptosis, whereas it serves to amplify the apoptotic signaling of other initiator caspases under conditions in which disruption of mitochondria is a late event (16 –19)
Summary
Apoptosis is a physiological process of cell death indispensable for the maintenance of multicellular organisms. Somatostatin (SST) receptor (SSTR)-mediated cytotoxic signaling triggers acidification and apoptosis in MCF-7 and T47D breast cancer cells: prevention of acidification by pH clamping inhibited its ability to induce apoptosis [33,34,35]. In the present study we undertook to delineate the temporal sequence of activation of different caspases in relation to cellular acidification and mitochondrial dysfunction during SST-induced apoptosis in MCF-7 cells. We present evidence demonstrating that caspase 8 activation is necessary for intracellular acidification to occur during SST-induced apoptosis and that the effector caspases are induced only as a consequence of the. Depletion of ATP prevented the activation of caspase-9 but only partially inhibited its ability to activate the terminal caspases and induce apoptosis These data suggest that SST-induced, acidification-dependent, apoptosis is not dependent on mitochondrial dysfunction
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