Salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis through DR5 and survivin-dependent mechanisms.

Ras activation is a frequent event in human hepatocarcinoma that may contribute to resistance towards apoptosis. Salirasib is a ras and mTOR inhibitor that induces a pro-apoptotic phenotype in human hepatocarcinoma cell lines. In this work, we evaluate whether salirasib sensitizes those cells to TRAIL-induced apoptosis. Cell viability, cell death and apoptosis were evaluated in vitro in HepG2, Hep3B and Huh7 cells treated with DMSO, salirasib and YM155 (a survivin inhibitor), alone or in combination with recombinant TRAIL. Our results show that pretreatment with salirasib sensitized human hepatocarcinoma cell lines, but not normal human hepatocytes, to TRAIL-induced apoptosis. Indeed, FACS analysis showed that 25 (Huh7) to 50 (HepG2 and Hep3B) percent of the cells treated with both drugs were apoptotic. This occurred through activation of the extrinsic and the intrinsic pathways, as evidenced by a marked increase in caspase 3/7 (five to ninefold), caspase 8 (four to sevenfold) and caspase 9 (eight to 12-fold) activities in cells treated with salirasib and TRAIL compared with control. Survivin inhibition had an important role in this process and was sufficient to sensitize hepatocarcinoma cells to apoptosis. Furthermore, TRAIL-induced apoptosis in HCC cells pretreated with salirasib was dependent on activation of death receptor (DR) 5. In conclusion, salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis by a mechanism involving the DR5 receptor and survivin inhibition. These results in human hepatocarcinoma cell lines and primary hepatocytes provide a rationale for testing the combination of salirasib and TRAIL agonists in human hepatocarcinoma.

The Ras inhibitor farnesylthiosalicyclic acid (FTS) prevents nodule formation and development of preneoplastic foci of altered hepatocytes in rats.

BACKGROUND: Aberrant activation of oncogenes, such as Ras, likely contributes to the development of hepatocarcinoma (HCC). AIMS/METHODS: We evaluated in vivo the effect of intraperitoneal injections of the Ras inhibitor S-trans, trans-farnesylthiosalicyclic acid (FTS) on Ras activation and the development of preneoplastic liver lesions in rats receiving weekly diethylnitrosamine (DEN) injections for 16weeks. Western blotting, quantitative PCR, immunohistochemistry, Tunel and caspase activity assays were used. RESULTS: FTS prevents liver nodule formation and reduces foci expressing the tumour marker GSTp. FTS abrogates DEN-induced Ras membrane activity, increases Tunel positive cells in transformed, GSTp-expressing hepatocytes, up-regulates caspase 3 and 8 activity, induces Fas, Fas ligand and JNK phosphorylation that occurs independently of TNFalpha and Trail. Cytochrome C release, Bax, Bcl2, Bcl-xl, Ki67 and nuclear cyclin D expression is not affected by FTS. CONCLUSIONS: FTS inhibits Ras activation and prevents preneoplastic liver nodule development by inducing apoptosis in transformed hepatocytes through activation of the Fas/Fas ligand system. FTS might be new molecule for HCC treatment.

Alcohol dependence is associated with reduced plasma and fundic ghrelin levels.

BACKGROUND: Conflicting data concerning the involvement of ghrelin in the pathophysiology of alcohol dependence have been reported. The aim of this study is to investigate how chronic alcohol ingestion influences plasma ghrelin levels and whether potential changes observed in plasma relate to modifications in ghrelin production in the stomach where this peptide is primarily synthesized. MATERIALS AND METHODS: Fifty-one consecutive alcoholics admitted for alcohol withdrawal were prospectively enrolled and compared to a control group of 32 healthy volunteers matched for age, sex, height and weight. All subjects underwent fasting plasma ghrelin determination. Twenty-seven randomly selected alcoholics and 17 controls underwent gastroscopy for fundic and duodenal biopsies. Tissues were fixed for histology or frozen in liquid nitrogen for ghrelin protein and mRNA determinations by a radioimmunoassay and quantitative polymerase chain reaction, respectively. Alcohol consumption was normalized to body weight (BW) or body mass index (BMI) given the influence of BW and volume distribution on alcohol levels. RESULTS: Plasma and fundic ghrelin protein levels were significantly decreased in alcoholics. Fundic but not plasma ghrelin protein levels inversely correlated with alcohol consumption normalized to BW or BMI. Ghrelin mRNA levels in fundic biopsies were similar in alcoholics and controls. No significant differences in duodenal ghrelin protein and mRNA levels were found between both groups. CONCLUSIONS: Alcoholism was associated with decreased plasma ghrelin levels partly due to reduced ghrelin production in the stomach. Alcohol affected ghrelin production on the post-transcriptional level in the fundus, whereas duodenal ghrelin secretion did not respond in a similar manner to alcohol intake.

Expression of presumed specific early and late factors associated with liver regeneration in different rat surgical models.

Experiments performed on the portal branch ligation (PBL) model indicate that early changes observed after surgery are not related to the regenerative process because they also occur in atrophying lobes. To further confirm the lack of specificity of the early events and to exclude the influence of circulatory factors released by proliferating lobes on their occurrence, we investigated this response after sham operation (SO) and portacaval shunt (PCS), a model characterized by liver atrophy. We also attempted to determine expression of later events associated specifically with regeneration, ie, expression of p53 or c-Ha-ras, or inhibition of proliferation, ie, interleukin-1beta (IL-1beta) and transforming growth factor-beta1 (TGF-beta1) after partial (PH) and temporary partial (TPH) hepatectomy, SO and PCS. Nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription 3 (STAT3) DNA binding were assessed by electrophoretic mobility shift assay (EMSA), interleukin-6 (IL-6) mRNA by reverse transcription-polymerase chain reaction (RT-PCR), c-myc and c-jun mRNAs by Northern blot analysis at 0.5 and 2 hours, p53 and c-Ha-ras mRNAs by Northern blot analysis at 8 and 24 hours, and IL-1beta and TGF-beta1 by RT-PCR at 24 hours. The early response including an increase of NF-kappaB, STAT3, IL-6, and immediate-early genes expression was present after PH, PCS, and SO. In SO, slight differences were observed in comparison with PH: no NF-kappaB p65/p50 DNA binding was observed, only three of six SO rats were positive for IL-6, and immediate-early genes induction showed differences in the intensity of the response. At later times, p53 mRNA increased at 8 hours after PH and TPH, c-Ha-ras mRNA at 24 hours after PH, and IL-1beta mRNA at 24 hours after PCS. Early events are not specifically associated with the reduction of liver mass or with the regenerative process, are not predictive of future cell fate, and are most likely related to surgical stress. p53 and c-Ha-ras induction is closely associated with cell cycle progression whereas IL-1beta, but not TGF-beta1, appears to be one of the negative growth regulators that might play an important role in atrophy.

After portal branch ligation in the rat, cellular proliferation is associated with selective induction of c-Ha-ras, p53, cyclin E, and Cdk2.

BACKGROUND: In liver regeneration after portal branch ligation we previously showed that early cellular changes are observed in both the proliferating and atrophying liver lobes. They are therefore not indicative of future proliferative response. In this study we attempted to define precisely, in the same model, the time at which the cellular processes diverge between the lobes by measuring various parameters associated with cellular proliferation. We also investigated the possible role of inhibitors of cell proliferation in the absence of progression towards the S phase in the atrophying lobes. AIMS: Expression of p53, c-Ha-ras, cyclin E, cyclin dependent kinase (Cdk2), transforming growth factor (TGF)-beta, and interleukin (IL)-1alpha and IL-1beta were assessed in relation to their potential role in proliferating and atrophying cellular phenomenons. METHODS: Immunohistochemistry, northern blotting, western blotting, and reverse transcription-polymerase chain reaction were performed, mainly at time points corresponding to mid-G1/S phase progression (8-24 hours after surgery). RESULTS: The common and thus most likely non-specific response was still evident 5-8 hours after surgery and included an increase in IL-1 mRNA as well as p53 and cyclin E proteins. From 12 hours onwards, p53, c-Ha-ras, cyclin E, and Cdk2 were selectively induced in proliferating lobes whereas IL-1beta was predominantly activated in atrophying lobes. No changes in TGF-beta or IL-1alpha expression were observed at the same time points in any of the liver lobes. CONCLUSIONS: The initial response to portal branch ligation and thus probably to partial hepatectomy seems to be non-specific for at least eight hours. Thereafter, p53, c-Ha-ras, cyclin E, and Cdk2 seem to drive cellular proliferation while IL-1beta is associated with cellular atrophy. In contrast, TGF-beta and IL-1alpha do not seem to play a role in determining the commitment of cells towards atrophy or proliferation.

After portal branch ligation in rat, nuclear factor kappaB, interleukin-6, signal transducers and activators of transcription 3, c-fos, c-myc, and c-jun are similarly induced in the ligated and nonligated lobes.

Several studies have emphasized the involvement of transcription factors, cytokines, and proto-oncogenes in initiating the regenerative process after partial hepatectomy. To assess whether these events do specifically occur in a cellular system undergoing regeneration, we studied the induction of nuclear factor kappaB (NFkappaB), interleukin-6 (IL-6), signal transducers and activators of transcription 3 (Stat3), c-fos, c-myc, c-jun, after portal branch ligation (PBL), which produces atrophy of the deprived lobes (70% of the liver parenchyma), whereas the perfused lobes undergo compensatory regeneration. Nuclear extracts and total RNA were prepared from control livers as well as from atrophying and regenerating lobes at 0.5, 1, 2, 5, and 8 after PBL. NFkappaB and Stat3 induction were studied by electrophoretic mobility shift assays and Western blotting. IL-6 and proto-oncogenes expressions were assessed by reverse transcription polymerase chain reaction and Northern blotting, respectively. Assays were also performed after a sham operation. NFkappaB and Stat3 protein expression and DNA binding were rapidly and similarly induced in nuclear extracts from the atrophying and regenerating lobes. IL-6 was elevated in both lobes from 1 to 8 hours after PBL as well as c-fos, c-myc, and c-jun during the first 2 hours. IL-6 and Stat3 but not NFkappaB were also elevated after a sham operation. These findings suggest that the cellular and molecular changes occurring early in a regenerating liver are nonspecific, possibly stress-induced, cellular responses. They do not indicate the future evolution towards atrophy or regeneration.

CYP 3A proteins are expressed in human neutrophils and lymphocytes but are not induced by rifampicin.

Cytochrome P-450 3A (CYP 3A) enzymes, the prominent subfamily in the cytochrome system, are expressed in various extrahepatic tissues. Until now, their expression has been demonstrated in human polymorphic neutrophils (PMNs) but not in lymphocytes using immunohistochemistry and immunoblot analysis. Moreover, their potential modulation has not been determined yet. To study such an expression in different peripheral blood cell populations, rifampicin (600 mg/day during 6 days) was given to 8 healthy subjects. PMNs and lymphocytes were isolated by centrifugation of whole white blood cell fractions using Ficoll gradients before drug administration, immediately after, and 3 days after drug withdrawal. PMN and lymphocyte smears and homogenates were subjected to immunostaining and immunoblotting, respectively, with a mouse monoclonal antibody recognizing all CYP 3A proteins. These proteins were quantified by densitometric analysis. Before and after rifampicin administration, a positive cytoplasmic staining was observed in all PMNs and in about 50% of lymphocytes. CYP 3A expression in lymphocytes was further confirmed by positive immunoblots for lymphocyte homogenates. Neither in PMNs nor in lymphocytes, induction of CYP 3A protein expression was observed after rifampicin treatment despite overall induction of CYP 3A activity assessed by the urinary excretion of 6beta-hydroxycortisol. These results demonstrate that CYP 3A proteins are constitutively expressed not only in PMNs but also in lymphocytes. However, in both cell lineages CYP 3A protein expression was not induced by rifampicin.