The heavy metal cadmium, an environmental pollutant, has been widely demonstrated to be toxic, in particular for liver. In murines, cadmium induces apoptosis of hepatocytes and hepatomas. In human cells, apoptosis induced by cadmium has been exclusively demonstrated in tumoral cell lines. Nothing was known in normal liver, in vitro or in vivo. In the present study, we examined the effects of cadmium in nonmalignant human hepatocytes. For that purpose, we investigated whether cadmium was able to induce apoptosis of normal human hepatocytes (NHH) in primary culture and of a SV40-immortalized human hepatocyte (IHH) cell line. Treatment of IHH and NHH with cadmium induced the presence of a sub-G1 population at 10 and 100 μmol/L, respectively. DAPI staining of both cell types treated with cadmium 100 μmol/L revealed the induction of nuclear apoptotic bodies, supporting the hypothesis of apoptosis. In IHH and NHH, cadmium 100 μmol/L induced PARP cleavage into a 85 kDa fragment. In order to investigate the involvement of mitochondria in cadmium-induced apoptosis, we measured the mitochondrial membrane potential (ΔΨm). We observed that in IHH and NHH, cadmium 100 μmol/L induced a decrease of ΔΨm. As expected, cadmium under the same conditions enhanced caspase-9 and caspase-3 activities. In addition, cadmium from 1 to 100 μmol/L induced the expression of p53 and phosphorylation of its Ser15 in IHH and NHH. In conclusion, we showed in this study that human hepatocytes were sensitive to cadmium and apoptosis induced at concentrations suggested in the literature to inhibit p53 DNA-binding and DNA repair.

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 (PKCδ) and c‐Abl was investigated. Suppression of c‐Abl or PKCδ 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, PKCδ and Bcl‐XL/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 PKCδ and c‐Abl to induce apoptosis through the mitochondrial pathway.

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.

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 ssts and independently of p53. (C) 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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 (HCQ 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.

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.