Analysis of the cytochrome c-dependent apoptosis apparatus in cells from human pancreatic carcinoma

Abstract

Defects in the apoptotic system are likely to play a role in tumorigenesis. Pancreatic carcinoma cells are extremely resistant to apoptosis induction by chemotherapy suggesting that the apoptosis machinery is faulty. We investigated the integrity of the cytochrome c-dependent apoptotic apparatus in 10 human pancreatic carcinoma cell lines. Expression of Apaf-1, caspase-3, -6, -7, -8 and -9, Hsp-70 and XIAP was detected in all cell lines. The expression levels of Apaf-1 and caspase-8 were homogenous in all cell lines whereas differences in expression of other caspases were seen. In cytosolic fractions, all investigated caspases were processed in response to cytochrome c but the extent of processing varied between the cell lines. No stringent correlation between the amount of processing of caspase-9 and effector caspases was seen. Cytochrome cinduced effector caspase activity was quantitated by enzyme assay. Especially at low concentrations of added cytochrome c, this response varied greatly between the cell lines. These data demonstrate that the apoptotic system downstream of the mitochondria is qualitatively intact in pancreatic carcinoma. They further show that the response to cytochrome c can be quantitated in a cell-free system and that determinants other than mere expression of apoptotic molecules can regulate cytochrome c-induced apoptosis.

Zoledronic acid induces antiproliferative and apoptotic effects in human pancreatic cancer cells in vitro

“…In our study however, we have been unable to find a specific role for caspase-3, which is not cleaved/activated, and apoptosis was only slightly antagonised by selective caspase-3 inhibition. Our observations are in accordance with others who found no correlation between the amount of processing of caspase-9 and effector caspases in human pancreatic carcinoma (Gerhard et al, 2002). We can therefore hypothesise that in ZOL-exposed PC cells, a caspase-9-and caspase-6-dependent and caspase-3-independent pathway is operative, while in other tumour cell systems, as well as in normal osteoclast cells, execution of apoptosis induced by ZOL may occur by a caspase-3-dependent mechanism.…”

Analysis of apoptosome dysregulation in pancreatic cancer and of its role in chemoresistance

“…26 It should be mentioned that recent works postulated that experimental conditions may invalidate the evaluation of APAF1 expression levels by western blotting. 24,25 In spite of this finding, although APAF1 expression level has been considered constant in PDAC cell lines, 32 we decided to investigate this aspect by several approaches. Human samples of PDAC have been analyzed for APAF1 mRNA or protein dosage by quantitative RT-PCR and immunohistochemistry.…”

Disturbed balance of expression between XIAP and Smac/DIABLO during tumour progression in renal cell carcinomas

“…X-linked inhibitor of apoptosis is thought to act as a key determinant of apoptosis resistance by effectively inhibiting the activation of caspase-3, -7 and -9 (Deveraux et al, 1999;Holcik et al, 2001;Srinivasula et al, 2001;Suzuki et al, 2001). Thus, high expression of XIAP has been reported in many malignant tumour types, such as carcinomas of the breast, ovaries, lung, pancreas, cervix and prostate (Ferreira et al, 2001a, b;Liu et al, 2001;Gerhard et al, 2002;Hofmann et al, 2002;Mc Eleny et al, 2002;Parton et al, 2002;Sui et al, 2002) as well as leukaemias (Tamm et al, 2000). Moreover, upregulation of XIAP expression has been found to result in apoptosis resistance after exposure to anticancer drugs and ionising radiation (Li et al, 2000;Asselin et al, 2001;Holcik et al, 2001;Matsumiya et al, 2001), whereas downregulation of XIAP by antisense vectors increased the apoptosis sensitivity of carcinoma cell lines Holcik et al, 2001).…”