Protein kinase PKC delta and c-Abl are required for mitochondrial apoptosis induction by genotoxic stress in the absence of p53, p73 and Fas receptor.

Doxorubicin, cis-diamminedichloroplatinum (II) and 5-fluorouracil used in chemotherapy induce apoptosis in Hep3B cells in the absence of p53, p73, and functional Fas. Since mediators remain unknown, the requirement of PKC delta (PKCdelta) and c-Abl was investigated. Suppression of c-Abl or PKCdelta expression using SiRNAs impaired PARP cleavage, Gleevec and/or rottlerin inhibited the induction of the subG1 phase and the increase of reactive oxygen species level. Co-precipitations and phosphorylations to mitochondria of c-Abl, PKCdelta and Bcl-X(L/s) were induced. A depolarization of the mitochondrial membrane and activations of caspase-2 and -9 were observed. We propose that, in the absence of p53, p73 and Fas, genotoxic drugs could require both PKCdelta and c-Abl to induce apoptosis through the mitochondrial pathway.

Pleiotropic effects of PI-3' kinase/Akt signaling in human hepatoma cell proliferation and drug-induced apoptosis.

IGF-II and type I-IGF receptor (IGF-IR) gene expression is increased in primary liver tumors, and transgenic mice overexpressing IGF-II in the liver develop hepatocellular carcinoma (HCC) spontaneously, suggesting that alterations of IGF-IR signaling in vivo may play a role in the auto/paracrine control of hepatocarcinogenesis. We have addressed the contribution of PI-3'K/Akt signaling on the proliferation of HepG2 human hepatoma cells and on their protection against doxorubicin-induced apoptosis. Both basal HepG2 cell DNA replication and that stimulated by IGF-IR signaling were inhibited by the specific PI-3'K inhibitor Ly294002 (Ly). In the former case, PI-3'K signaling overcame cell cycle arrest in G1 via increased cyclin D1 protein and decreased p27kip1 gene expression. Doxorubicin treatment induced apoptosis in HepG2 cells and was concomitant with the proteolytic cleavage of Akt-1 and -2. Drug-induced apoptosis was reversed by IGF-I and this effect was (i) dependent on Akt-1 and -2 phosphorylation and (ii) accompanied by the inhibition of initiator caspase-9 activity, suggesting that IGF-IR signaling interferes with mitochondria-dependent apoptosis. Accordingly, Ly enhanced doxorubicin-induced apoptosis and suppressed its reversal by IGF-I. Altogether, the data emphasize the crucial role of PI-3'K/Akt signaling (i) in basal as well as IGF-IR-stimulated HepG2 cell proliferation and (ii) in controlling both doxorubicin-induced apoptosis (e.g., drug-induced cleavage of Akt) and its reversal by IGF-I (protection against apoptosis parallels the extent of Akt phosphorylation). They suggest that targeting Akt activity or downstream Akt effectors (e.g., GSK3-beta, FOXO transcription factors) may help define novel therapeutic strategies of increased efficacy in the treatment of HCC-bearing patients.

An evaluation of the role of insulin-like growth factors (IGF) and of type-I IGF receptor signalling in hepatocarcinogenesis and in the resistance of hepatocarcinoma cells against drug-induced apoptosis.

Strong evidence emphasizes the role of the insulin-like growth factor (IGF) system and of type-I IGF receptor (IGF-IR) signalling in tumourigenesis. In this connection: (i) changes in the expression pattern of components of the IGF system (autocrine/paracrine expression of IGF-I and -II, overexpression of IGF-IR, decreased expression of IGF-binding proteins (IGFBPs) and of type-II IGF receptor/cation-independent mannose-6-phosphate receptor (IGF-II/M6PR) and (ii) increased serum concentrations of proteases that cleave the IGFBPs (e.g., cathepsin D) were observed in patients with hepatocellular carcinomas (HCC), in human hepatoma cell lines and in their conditioned culture medium, as well as in rodent models of hepatocarcinogenesis. Accordingly, studies carried out with animal models do suggest that the IGF system and IGF-IR signalling may play a role in hepatocarcinogenesis and in deregulated proliferation and apoptosis of HCC cells. Finally the instrumental role of Raf/MEK/ERK, one of the signalling cascades stimulated by IGF-IR, in anthracycline-induced apoptosis of HepG2 and Huh-7 human hepatoma cell lines emphasizes that care must be taken when designing combinations of antitumoural molecules for antineoplastic treatment. This review addresses the putative roles of the IGF system in primary HCC, with a special focus on the underlying molecular mechanisms. In a second part it emphasizes the putative interference of IGF-IR signalling with chemotherapeutic drug-induced apoptosis in human hepatoma cells.

Role of constitutively activated and insulin-like growth factor-stimulated ERK1/2 signaling in human hepatoma cell proliferation and apoptosis: evidence for heterogeneity of tumor cell lines.

Enhanced insulin-like growth factor II (IGF-II) and type I IGF receptor (IGF-IR) gene expression in liver tumors and the development of liver tumors in transgenic mice overexpressing IGF-II in the liver suggest that the IGFs and underlying signaling cascades may play auto/paracrine roles in the control of hepatocarcinoma (HCC) cell proliferation and in their protection against apoptosis. We have focused on the role of mitogen-activated protein kinase (ERK1/2) signaling on human HepG2 and Huh-7 hepatoma cell proliferation and on the protection of these cells against drug-induced apoptosis. Physiological concentrations of IGF-I stimulated DNA replication in HepG2 cells (1.5-fold) but not in Huh-7 cells, and this effect was abolished by PD98059 (MEK-1 inhibitor). Doxorubicin or cisplatin treatment induced apoptosis (caspase-dependent poly[ADP-ribose]polymerase cleavage) in both cell lines, but dose-dependent reversion of drug-induced apoptosis (57-84%) by IGF-I was only observed in HepG2 cells. The very low level of IGF-IR at the plasma membrane of Huh-7 cells may account for their unresponsiveness to IGF-I. We have shown that drug treatment enhanced (17-fold) or did not modify constitutive ERK1/2 activity in cultured HepG2 or Huh-7 cells, respectively. In both cell lines, inhibition of constitutive and drug-induced ERK1/2 activity by PD98059 yielded a complete inhibition of drug-induced apoptosis. Altogether, our data demonstrate the heterogeneous response of human hepatoma cells to an IGF stimulus and suggest (1) that auto/paracrine effects of IGF-I/-II might contribute to the proliferation of HCC cells and to their protection against apoptosis in vivo and (2) that drug-induced activation of ERK1/2 plays a role in drug-induced apoptosis in human hepatoma cells.

Potent induction of apoptosis in human hepatoma cell lines by targeted cytotoxic somatostatin analogue AN-238.

BACKGROUND/AIMS: The efficacy of a targeted cytotoxic hybrid somatostatin analogue AN-238 and of its superactive radical 2-pyrrolinodoxorubicin (AN-201) to induce apoptosis of HepG2 and Hep3B human hepatoma cell lines were studied. AN-238 was designed to selectively target tumor cells expressing somatostatin receptor subtypes (sst(s)). Its effects on HepG2 or Hep3B cells displaying or lacking tumor suppressor p53, respectively, were compared. Normal rat isolated hepatocytes were also tested. METHODS: sst(s) were characterized by binding assays and RT-PCR. Cytotoxicity was quantified by flow cytometry. DNA fragmentation was studied by gel electrophoresis, PARP cleavage by Western blot and ROS formation using fluorescent probes. RESULTS: Specific binding of iodinated RC-160 to HepG2 and Hep3B cells, and its displacement by AN-238 was characterized. mRNA for hsst(2A) was found in both cell lines. Flow cytometry showed a stronger effect of AN-238 than AN-201 to induce sub-G1 phase. DNA fragmentation, nuclear bodies, and PARP cleavage were observed. In addition, AN-238 increased formation of ROS more potently than AN-201. However, no inductions of DNA fragmentation by AN-201 or AN-238 were observed on rat hepatocytes. CONCLUSIONS: Our results indicate that, in liver cancer, the cytotoxic somatostatin analogue AN-238 is a powerful agent that can induce apoptosis, through sst(s) and independently of p53.

Doxorubicin and octreotide induce a 40 kDa breakdown product of p53 in human hepatoma and tumoral colon cell lines.

The chemotherapeutic drug doxorubicin and the anti-proliferative long-acting somatostatin analogue octreotide, both used in cancer treatment, have been shown to increase the expression of the p53 tumour suppressor protein. In the present study, we demonstrate by Western-blot analysis that, in addition to the p53 protein, these molecules were able to induce the expression of a shorter protein with an apparent molecular mass of 40 kDa (p40), recognized by antibodies raised against the N-terminus of p53. This induction was present in tumoral and non-tumoral cells and did not depend on the status of the endogenous p53 protein. The p40 protein was significantly induced after 3 h of cell treatment with doxorubicin or octreotide, remained stable until 24 h and was located in the nuclear extract. Using reverse primers corresponding to each exon of the p53 gene, only one transcript was amplified by reverse transcriptase-PCR. This suggested that p40 was issued from a post-translational modification and not from an alternative splicing. This protein was not recognized by the PAb421 antibody, suggesting that it was issued from a cleavage of the p53 C-terminal region (p40deltaC). Furthermore, this cleavage was not dependent on caspase activity. In conclusion, these results support the hypothesis that this post-translational modification plays a significant role in the regulation of multiple p53 signalling pathways. These results also suggest that octreotide, a molecule with different signalling pathways, was able as doxorubicin to generate a p53 breakdown product.