Resistance to caspase-8 and -9 fragments in a malignant pleural mesothelioma cell line with acquired cisplatin-resistance

Abstract

Apoptotic cysteine-aspartate proteases (caspases) are essential for the progression and execution of apoptosis, and detection of caspase fragmentation or activity is often used as markers of apoptosis. Cisplatin (cis-diamminedichloroplatinum (II)) is a chemotherapeutic drug that is clinically used for the treatment of solid tumours. We compared a cisplatin-resistant pleural malignant mesothelioma cell line (P31res1.2) with its parental cell line (P31) regarding the consequences of in vitro acquired cisplatin-resistance on basal and cisplatin-induced (equitoxic and equiapoptotic cisplatin concentrations) caspase-3, -8 and -9 fragmentation and proteolytic activity. Acquisition of cisplatin-resistance resulted in basal fragmentation of caspase-8 and -9 without a concomitant increase in proteolytic activity, and there was an increased basal caspase-3/7 activity. Similarly, cisplatin-resistant non-small-cell lung cancer cells, H1299res, had increased caspase-3 and -9 content compared with the parental H1299 cells. In P31 cells, cisplatin exposure resulted in caspase-9-mediated caspase-3/7 activation, but in P31res1.2 cells the cisplatin-induced caspase-3/7 activation occurred before caspase-8 or -9 activation. We therefore concluded that in vitro acquisition of cisplatin-resistance rendered P31res1.2 cells resistant to caspase-8 and caspase-9 fragments and that cisplatin-induced, initiator-caspase independent caspase-3/7 activation was necessary to overcome this resistance. Finally, the results demonstrated that detection of cleaved caspase fragments alone might be insufficient as a marker of caspase activity and ensuing apoptosis induction.

Figure 1

Effector caspase-3/7 activity and caspase-3 fragmentation in malignant pleural mesothelioma cells (P31) and a sub-line with in vitro acquired cisplatin-resistance (P31res1.2) determined in absence and presence of equitoxic and equiapoptotic concentrations of cisplatin, after exposure times as indicated. (a) Caspase-3/7 activity determined in a fluorometric assay using a fluorescent-tagged substrate, DEVD-AFC. ^**P<0.01, compared with control. Data presented as means±S.E.M. of three separate experiments. (b) Western blot of full-length and cleaved PARP as an endogenous indicator of caspase-3/7 activity. The membranes were stripped and reprobed for actin as a protein loading control. Image representative of at least three separate experiments. (c) Activation of caspase-3 determined by western blotting to detect full-length and cleaved versions of caspase-3. The membranes were stripped and reprobed for actin as a protein loading control. Image representative of at least three separate experiments. (d) P31 and P31res1.2 content of full-length caspase-3 and cleaved caspase-3 determined with a Proteome profiler array for human apoptosis-related proteins. Data presented as means±S.E.M. of duplicate analysis. (e) Detection of caspase-3 and caspase-7 by western blotting under control conditions in non-small cell lung cancer cells (H1299) and a sub-line with in vitro acquired cisplatin-resistance (H1299res)

Figure 2

Initiator caspase-8 and -9 fragmentation and activity in malignant pleural mesothelioma cells (P31) and a sub-line with in vitro acquired cisplatin-resistance (P31res1.2), determined in absence and presence of equitoxic and equiapoptotic concentrations of cisplatin, after exposure times as indicated. (a) Western blots of caspase-8 and -9 full-length proteins and cleaved fragments. The membranes were stripped and reprobed for actin as a protein loading control. Image representative of at least three separate experiments. (b) Caspase-8 and -9 activities determined in a fluorometric assay using fluorescent-tagged substrates, IETD-AFC for caspase-8 and LEHD-AFC for caspase-9. Basal activity was determined in control cells maintained without cisplatin and cisplatin-induced activity was determined in cells exposed to equitoxic concentrations of cisplatin as indicated. ^*P<0.05, ^**P<0.01, compared to control. Data presented as means±S.E.M. of three separate experiments. (c) Detection of caspase-9 by western blotting under control conditions in non-small cell lung cancer cells (H1299) and a sub-line with in vitro acquired cisplatin-resistance (H1299res)

Figure 3

Expression of inhibitor of apoptosis proteins and HtrA2 in in vitro acquired cisplatin-resistance. (a) P31 and P31res1.2 content of c-IAP1, c-IAP2, survivin, XIAP and HtrA2 determined with a Proteome profiler array for human apoptosis-related proteins. Data presented as means±S.E.M. of duplicate analysis. (b) Detection of the caspase-3 and -9 inhibitor XIAP and of the XIAP inhibitor HtrA2XIAP in malignant pleural mesothelioma cells (P31) and a sub-line with in vitro acquired cisplatin-resistance (P31res1.2), determined in absence and presence of equitoxic and equiapoptotic concentrations of cisplatin, after exposure times as indicated. Actin was used as protein loading control. Image representative of three separate experiments