Melatonin modulation of intracellular signaling pathways in hepatocarcinoma HepG2 cell line: role of the MT1 receptor.

Melatonin reduces proliferation in many different cancer cell lines. However, studies on the oncostatic effects of melatonin in hepatocarcinoma are limited. We have previously demonstrated that melatonin administration induces cycle arrest, apoptosis, and changes in the expression of its specific receptors in HepG2 human hepatocarcinoma cells. In this study, we used the receptor antagonist luzindole to assess the contribution of MT1 melatonin membrane receptor to melatonin effects on cell viability, mitogen-activated protein kinase (MAPKs) activation, and cAMP levels. Additionally, effects of MT1 inhibition on mRNA levels of cytosolic quinone reductase type-2 (NQO2) receptor and nuclear retinoic acid-related orphan receptor alpha (RORα) were tested. Melatonin, at 1000 and 2500 µm, significantly reduced cell viability. Pre-incubation with luzindole partially inhibited the effects of melatonin on cell viability. Melatonin at 2500 µm significantly reduced cAMP levels, and this effect was partially blocked by luzindole. Both melatonin concentrations increased the expression of phosphorylated p38, ERK, and JNK. ERK activation was completely abolished in the presence of luzindole. NQO2 but not RORα mRNA level significantly increased in luzindole-treated cells. Results obtained provide evidence that the melatonin effects on cell viability and proliferation in HepG2 cells are partially mediated through the MT1 membrane receptor, which seems to be related also with melatonin modulation of cAMP and ERK activation. This study also highlights a possible interplay between MT1 and NQO2 melatonin receptors in liver cancer cells.

Changes in the expression of melatonin receptors induced by melatonin treatment in hepatocarcinoma HepG2 cells.

Hepatocellular carcinoma (HCC) is one of the most common cancers and its incidence is increasing worldwide. Melatonin, an indoleamine hormone, exerts anti-oxidant, immunomodulatory, anti-aging, and antitumor effects. Previous studies have shown that melatonin can act through specific receptors, including MT(1), MT(2), MT(3) receptors as well as a nuclear receptor belonging to the orphan nuclear receptor family. Recently, we have described their role in the oncostatic and pro-apoptotic effects of melatonin on HepG2 human HCC cells. However, the potential role of the different melatonin cellular receptors on its antiproliferative effects remains unknown. In the present study, we examined the effect of melatonin treatment on HepG2 human HCC cells, analyzing cell cycle arrest and melatonin receptor expression. Melatonin was administered for 2, 4, and 6 days at 1000 or 2500 microm. Melatonin induced a dose- and time-dependent inhibition on cell proliferation. This treatment caused an alteration in the cell cycle, with an increase in the number of cells in G(2)/M phase at both 1000 and 2500 microm melatonin concentrations, and a significant increase on S phase cell percentage by the highest dose. Furthermore, increases in protein expression of MT(1), MT(3), and retinoic acid-related orphan receptor-alpha were found after melatonin treatments. These increases were coincident with a significant induction in the expression of p21 protein, which negatively regulates cell cycle progression. Our results confirm the antitumor effect of melatonin in HCC cells, suggesting that its oncostatic properties are related, at least in part, to changes on the expression of their different subtypes of receptors.

Melatonin induces cell cycle arrest and apoptosis in hepatocarcinoma HepG2 cell line.

Melatonin reduces proliferation in many different cancer cell lines. However, studies on the oncostatic effects of melatonin in the treatment of hepatocarcinoma are limited. In this study, we examined the effect of melatonin administration on HepG2 human hepatocarcinoma cells, analyzing cell cycle arrest, apoptosis and mitogen-activated protein kinase (MAPK) signalling pathways. Melatonin was dissolved in the cell culture media in 0.2% dimethyl sulfoxide and administered at different concentrations for 2, 4, 6, 8 and 10 days. Melatonin at concentrations 1000-10,000 microM caused a dose- and time-dependent reduction in cell number. Furthermore, melatonin treatment induced apoptosis with increased caspase-3 activity and poly(ADP-ribose) polymerase proteolysis. Proapoptotic effects of melatonin were related to cytosolic cytochrome c release, upregulation of Bax and induction of caspase-9 activity. Melatonin treatment also resulted in increased caspase-8 activity, although no significant change was observed in Fas-L expression. In addition, JNK 1,-2 and -3 and p38, members of the MAPK family, were upregulated by melatonin treatment. Growth inhibition by melatonin altered the percentage or cells in G0-G1 and G2/M phases indicating cell cycle arrest in the G2/M phase. The reduced cell proliferation and alterations of cell cycle were coincident with a significant increase in the expression of p53 and p21 proteins. These novel findings show that melatonin, by inducing cell death and cell cycle arrest, might be useful as adjuvant in hepatocarcinoma therapy.

Atorvastatin Decreases HBx-Induced Phospho-Akt in Hepatocytes via P2X Receptors.

Hepatocellular carcinoma (HCC) is rated as the fifth most common malignancy and third in cancer-related deaths worldwide. Statins, HMG-CoA reductase inhibitors, are potent cholesterol-lowering drugs, and recent epidemiologic evidence suggests that statins prevent aggressive HCC development. Previous experiments revealed that statins downregulate phosphorylated Akt (pAkt). Here, it is demonstrated that atorvastatin decreases nuclear pAkt levels in pancreatic and lung cancer cell lines within minutes, and this rapid effect is mediated by the purinergic P2X receptors. Akt is upregulated by hepatitis viruses and has oncogenic activity in HCC; therefore, we tested the possibility that the P2X-Akt pathway is important for the anticipated anticancer effects of statins in hepatocytes. Atorvastatin decreased hepatitis B virus X protein- and insulin-induced pAkt and pGsk3ß (Ser9) levels. Furthermore, Akt-induced lipogenesis was counteracted by atorvastatin, and these statin-induced effects were dependent on P2X receptors. Statin also decreased proliferation and invasiveness of hepatocytes. These data provide mechanistic evidence for a P2X receptor-dependent signaling pathway by which statins decrease pAkt, its downstream phosphorylation target pGsk3ß, and lipogenesis in hepatocytes.Implications: The Akt pathway is deregulated and may act as a driver in HCC development; the P2X-Akt signaling pathway may have a role in anticancer effects of statins. Mol Cancer Res; 15(6); 714-22. ©2017 AACR.

Methionine aminopeptidases as potential targets for treatment of gastrointestinal cancers and other tumours.

Methionine aminopeptidases (MetAP) are intracellular metalloproteins responsible for the removal of the initiator NH(2)-terminal methionine from newly synthesized proteins, thereby facilitating their intracellular translocation from the ribosome. Two types of MetAP enzymes, MetAP-1 (type-I) and MetAP-2 (type-II), which have a similar three-dimensional structure despite a low homology in their sequences, have been described. Since the discovery that fumagillin, an irreversible MetAP-2 inhibitor, prevents angiogenesis, different studies have been carried out to analyze the role of MetAP proteins as potential targets in cancer treatment. Data obtained indicate that anticancer effect of MetAP-2 inhibitors may be a result of the combined effect of MetAP-2 inhibition in endothelial cells (anti-angiogenesis) and in tumour cells directly. Moreover, it has been recently described that MetAP-1 has a potential role on cell division, and MetAP-1-specific inhibition is able to induce apoptosis in both HeLa and HT-1080 cell lines. A new subtype of MetAP-1, called MetAP-1D, has been found overexpressed in samples from colon cancer patients, its inhibition resulting in a decreased cell growth. Although molecular mechanisms of action of these proteins are largely unknown, a significant progress has been made to understand their structure-function relationships and their physiological roles. Their potential as promising targets in cancer treatment and in the development of new antitumour agents is analyzed focusing on MetAP irreversible inhibitors. The present review summarizes recent research data on different molecules able to induce MetAP inhibition in gastrointestinal cancers and other tumours.

Changes in the antioxidant system by TNP-470 in an in vivo model of hepatocarcinoma.

The objective of this study was to determine in a rat model of hepatocarcinoma (HCC) the effects of the antiangiogenic agent TNP-470 on antioxidant enzymes, including catalase (CAT), superoxide dismutases (Mn-SOD and Cu,Zn-SOD), and glutathione peroxidase (GPx). Tumor was induced in male Wistar rats by diethylnitrosamine and promoted by two-thirds hepatectomy plus acetaminofluorene administration. Experiments were carried out 28 weeks after initiating the treatment. TNP-470 was administered at 30 mg/kg, 2 times per week from weeks 20 to 28. Carcinomatous tissue was growing outside dysplastic nodules in rats with HCC. HCC caused oxidative stress demonstrated by increased lipid peroxidation and oxidized/reduced glutathione ratio that was accompanied by a reduced activity of antioxidant enzymes Cu,Zn-SOD, GPx, and CAT. In contrast, Mn-SOD activity and expression were higher in hepatocarcinoma than in control groups. These effects were absent in animals receiving TNP-470. No significant differences between untreated and TNP-470-treated rats were observed in the expression of the Cu,Zn-SOD, glutathione peroxidise, and CAT. We conclude that TNP-470 inhibits expression and activity of Mn-SOD induced by experimental hepatocarcinogenesis. Oxidative stress reduction by TNP-470 accounts for yet another anti-cancer effect of this molecule.