Arecoline induces HA22T/VGH hepatoma cells to undergo anoikis - involvement of STAT3 and RhoA activation.

BACKGROUND: Our previous study showed that, in basal cell carcinoma cells, arecoline reduces levels of the tumor cell survival factor interleukin-6 (IL-6), increases levels of tumor suppressor factor p53, and elicits cell cycle arrest, followed by apoptosis. In preliminarily studies, we observed that arecoline induces detachment of the human-derived hepatoma cell line HA22T/VGH from the extracellular matrix. In the present study, we explored the fate of the detached HA22T/VGH cells and investigated the underlying mechanism. METHODS: HA22T/VGH cells or primary cultured rat hepatocytes were treated with arecoline, then changes in morphology, viability, apoptosis, and the expression of surface beta1-integrin, apoptosis-related proteins, and IL-6 were examined. Furthermore, activation of the signal transducer and activator of transcription 3 (STAT3) pathway and the RhoA/Rock signaling pathway, including p190RhoGAP and Src homology-2 domain-containing phosphatase SHP2, was examined. RESULTS: A low concentration of arecoline (

Zinc at pharmacologic concentrations affects cytokine expression and induces apoptosis of human peripheral blood mononuclear cells.

OBJECTIVE: The present study examined the effect of zinc at concentrations of the apoptotic signaling pathway and immune function of peripheral blood mononuclear cells (PBMCs). METHODS: PBMCs from healthy subjects were treated in vitro with various zinc concentrations to imitate different serum statuses of physiologic (2 to 15 microM) and pharmacologic (15 to 100 microM) concentrations to higher than 100 microM and analyzed their expressions of cytokines and apoptotically related factors. RESULTS: Although a normal physiologic concentration of zinc had no effect on immunologic function or apoptosis of PBMCs, a pharmacologic concentration (100 microM) or higher affected both functions. Zinc decreased cell proliferation at concentrations higher than 100 microM and stimulated cytokine expression at concentrations of at least 100 microM. Further, at concentrations of at least 100 microM, apoptosis was induced, and expressions of caspase-3 and proapoptotic genes, including Fas (FasL) and c-fos, which trigger apoptosis through receptor-mediated extrinsic and mitochondrion-mediated apoptotic pathways, respectively, were increased. At concentrations at least 300 microM, expressions of antiapoptotic factors nuclear factor-kappaB, Bcl-2, and Bcl-X(L) were markedly decreased. CONCLUSIONS: Zinc stimulates cytokine expression and induces apoptosis of PBMCs from healthy subjects only at concentrations equal to or greater than the serum pharmacologic range. Receptor-mediated extrinsic and mitochondrial-mediated intrinsic pathways are involved in this zinc-induced apoptosis.

Combined arginine and ascorbic acid treatment induces apoptosis in the hepatoma cell line HA22T/VGH and changes in redox status involving the pentose phosphate pathway and reactive oxygen and nitrogen species.

Arginine is a physiological substrate for nitric oxide synthase to generate nitric oxide (NO), which can influence tumor cell survival, while ascorbic acid is selectively toxic for cancer cells. This study explored the effect of an arginine/ascorbic acid combination on human cancer cell lines. The hepatoma cell line HA22T/VGH was the most sensitive of the tested cells to combination treatment. A combination of 5.74 mM of arginine and 0.57 mM of ascorbic acid induced HA22T/VGH cell death through apoptosis and an increase in levels of reactive oxygen species and NO, as well as its stable products NO(2)(-) and NO(3)(-). The combination also reduced the activity of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and transaldolase in the pentose phosphate pathway, a major mechanism for producing NADPH, resulting in a marked decrease in intracellular NADPH levels. A dramatic decrease in intracellular glutathione (GSH) levels, a decrease in the mitochondrial membrane potential, ATP depletion and release of cytochrome c were also seen. Caspase-9 and caspase-3 were activated, apoptotic protein Bax expression increased and the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL decreased. These results suggest that this combination induced HA22T/VGH cell death by interfering with redox state regulation by a reduction in pentose phosphate pathway activity and increasing oxidative and nitrosative stress.

Hemeoxygenase-1 expression in response to arecoline-induced oxidative stress in human umbilical vein endothelial cells.

BACKGROUND: Arecoline, the most abundant areca alkaloid, has been reported to stimulate reactive oxygen species (ROS) production in several cell types. Overproduction of ROS has been implicated in atherogenesis. Hemeoxygenase-1 (HO-1) has cytoprotective activities in vascular tissues. This study investigated the effect of arecoline on adhesion molecule expression and explored the role of HO-1 in this process. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with arecoline, then ROS levels and the expression of adhesion molecules and HO-1 were analyzed and potential signaling pathways investigated. RESULTS: After 2h of arecoline treatment, ROS production was stimulated and reached a maximum at 12h. Expression of the adhesion molecules ICAM and VCAM was also induced. Glutathione pretreatment completely blocked arecoline-stimulated ROS production and VCAM expression, but not ICAM expression. Arecoline also induced HO-1 expression and this effect was partly due by ROS stimulation. Inhibition of c-jun N-terminal kinase (JNK) by SP600125, p38 by SB 203580, or tyrosine kinase by genistein reduced arecoline-induced HO-1 expression. In contrast, inhibition of ERK (extracellular signal-related MAP kinase) by PD98059 had no effect. Transfection of HUVECs with the GFP/HO-1 gene, which resulted in a 5-fold increase in HO-1 activity, markedly, but not completely, inhibited the decrease in cell viability caused by arecoline. CONCLUSIONS: This study demonstrates that, in HUVECs, arecoline stimulates ROS production and ICAM and VCAM expression. HO-1 expression is also upregulated through the ROS, tyrosine kinase, and MAPK (JNK and p38) signaling pathways.

Effects of soy isoflavones on apoptosis induction and G2-M arrest in human hepatoma cells involvement of caspase-3 activation, Bcl-2 and Bcl-XL downregulation, and Cdc2 kinase activity.

Genistein, biochanin-A, and daidzein, the predominant soy isoflavones, have been reported to lower the risk of cancer, but it is not known whether they protect against human hepatoma cancer. This study was designed to investigate their effects on cell growth, the cell cycle, and apoptosis induction in the human hepatoma cell lines, HepG2, Hep3B, Huh7, PLC, and HA22T. Genistein, biochanin-A, and daidzein inhibited growth of all five lines in a dose-dependent manner. DNA fragmentation studies and the TUNEL assay demonstrated that isoflavones caused tumor cell death by induction of apoptosis. Activation of caspase-3 and cleavage of the caspase-3 substrate, poly(ADP-ribose)polymerase, was seen in hepatoma cells after 24 hours' exposure to isoflavones. In addition, isoflavone cytotoxicity correlated with downregulation of Bcl-2 and Bcl-XL expression. Synergistic effects of the three isoflavones were observed on cell growth inhibition, apoptosis induction, and anti-apoptotic protein expression. Flow cytometry showed that genistein, but not biochanin-A or daidzein, induced progressive and sustained accumulation of hepatoma cancer cells in the G2/M phase as a result of inhibition of Cdc2 kinase activity. Coapplication of caffeine prevented this cell cycle arrest, but not apoptosis, showing that cell cycle arrest was not necessary for apoptosis. Furthermore, the isoflavones combination also had a significant tumor-suppressive effect in nude mice. These results suggest that isoflavones might be promising agents for the treatment of human hepatoma.