H+ /K+ ATPase expression in human parietal cells and gastric acid secretion in elderly individuals.

OBJECTIVE: To investigate whether the ultrastructure and hydrogen potassium adenosine triphosphate (H+ /K+ ATPase) expression of human parietal cells were associated with aging. METHODS: In all, 50 participants who underwent gastroscopy due to dyspepsia were divided into two age groups, with 19 in the younger group (YG, aged 20-59 years) and 31 in the elder group (EG, aged ≥60 years). The ultrastructure of their parietal cell was determined by electron microscopy (EM), and the expressions of H+ /K+ ATPase α-subunit mRNA and ß-unit protein were detected. Furthermore, 24-h esophageal pH monitoring was performed in the two groups. RESULTS: EM images showed no distinct difference in the morphology and distribution of parietal cells or the acid secretion-related organelle between the two groups. There were no differences between YG and EG in the proportion of mitochondria and the tubulovesicular system area. The expressions of H+ /K+ ATPase α-subunit mRNA and ß-subunit protein showed no age-related alteration between YG and EG. The expression of H+ /K+ ATPase α-subunit mRNA in EG was higher than that in YG, whereas the expression of ß-subunit protein was significantly higher in those aged ≥80 years than in the YG. No significant difference was found in the 24-h esophageal pH monitoring between YG and EG. CONCLUSION: Acid secretion-related organelles in parietal cells do not degenerate with aging, the expression of H+ /K+ ATPase even shows a trend to increase, indicating the existence of intact molecular biological basis for acid secretion in healthy elderly individuals.

Killing of Kras-mutant colon cancer cells via Rac-independent actin remodeling by the ßGBP cytokine, a physiological PI3K inhibitor therapeutically effective in vivo.

Activating mutations in Kras are the most frequent mutations in human cancer. They define a subset of patients who do not respond to current therapies and for whom prognosis is poor. Oncogenic Kras has been shown to deregulate numerous signaling pathways of which the most intensively studied are the Ras/extracellular signal-regulated kinase cascade and the phosphoinositide 3-kinase (PI3K)/Akt cascade. However, to date, there are no effective targeted therapies in the clinic against Kras-mutant cancers. Here, we report that the ß-galactoside-binding protein (ßGBP) cytokine, a physiologic inhibitor of class I PI3Ks, is a potent activator of apoptosis in Kras-mutant colorectal cancer cells, even when coharboring mutant-activated PIK3CA. Our study unveils an elective route to intrinsic and extrinsic apoptosis, which involves the cytoskeleton. Early events are inhibition of PI3K activity and Rac-independent actin rearrangement assignable to phosphoinositide changes at the plasma membrane. Cyclin E deregulation, arrest of DNA synthesis, and checkpoint kinase 2 activation underscore events critical to the activation of an intrinsic apoptotic program. Clustering of CD95/Fas death receptors underscore events critical to the activation of extrinsic apoptosis. In nude mice, we present the first evidence that xenograft tumor development is strongly inhibited by Hu-r-ßGBP. Taken together, our results open a new therapeutic opportunity to a subset of patients refractive to current treatments. This first demonstration of therapeutic efficacy against Kras-mutant colon cancer suggests that Hu-r-ßGBP may also be therapeutically effective against other cancers harboring activating Ras mutations as well as PIK3CA mutations.

Tanshinone IIA induces growth inhibition and apoptosis in gastric cancer in vitro and in vivo.

As a phytochemical derived from the roots of Salvia miltiorrhiza Bunge, Tanshinone IIA has been reported to possess anti-inflammatory and antioxidant activity. Studies in breast, colon, prostate and lung cancer indicate that Tanshinone IIA may exhibit a promising antitumor activity. However, systemic studies of the cytotoxic effects of Tanshinone IIA on gastric cancer have not been described. The present study offers a comprehensive evaluation of the antitumor effects of Tanshinone IIA in gastric cancer cells in vitro and in a mouse xenograft model. Cell viability and apoptosis in vitro were evaluated through the MTT assay and flow cytometry analysis. The results indicate that Tanshinone IIA can induce gastric cancer cell growth inhibition and apoptosis in a time- and concentration-dependent manner. Furthermore, we investigated the mechanism of the apoptotic effects induced by Tanshinone IIA. We found that Tanshinone IIA can not only cause cell cycle arrest in the G2/M phase, but also trigger the intrinsic apoptotic signaling pathway. The results suggest that Tanshinone IIA may serve as an effective adjunctive reagent in the treatment of gastric cancer.

The role of cellular oxidative stress in regulating glycolysis energy metabolism in hepatoma cells.

BACKGROUND: The Warburg effect has been found in a wide spectrum of human cancers, however the underlying mechanisms are still unclear. This study aims to explore the role of cellular oxidative stress in relation to glycolysis and the Warburg effect in hepatoma cells. METHODS: Various cell lines combining environmental hypoxia was used as an in vitro model to mimic tumor microenvironment in vivo. Superoxide dismutases (SOD) and xanthine oxidase (XO) gene transfection were used to produce various cellular redox levels. 2',7'-dichlorofluorescin (DCF) fluorescence and ESR spectrum were used to detect cellular reactive oxygen species (ROS). RESULTS: We found that endogenous or exogenous interference with the cellular oxidative stress can sensitively regulate glycolysis and the Warburg effect in hepatoma cells. Hepatoma cells displayed a high level of free radicals compared to immortalized normal hepatocyte cells. Increasing the level of ROS stress in hepatoma cells can directly upregulate HIF-1 and activate glycolysis without requirement of a hypoxic condition. This explains the mechanism whereby aerobic glycolysis, i.e. the Warburg effect arises. Either endogenously upregulating SOD or exogenously administration with antioxidant can, through downregulating ROS level, effectively regulate energy pathways in hepatoma cells and can inhibit the growth of tumor cells and xenograft tumors. CONCLUSION: This study suggests that the Warburg effect was related to an inherently high level of cellular ROS and HIF-1. Hepatoma cells adaptation to hypoxia for survival and rapid growth exploits oxidative stress ectopically activated glycolysis to compensate the energy supply. This specific mechanism in which tumor cells through cellular oxidative stress activate glycolysis to meet their energy metabolism requirement could be exploited to selectively kill tumor cells.

[Preparation of the spin trapping probe, N-tert-butyl-alpha-phenylnitrone, nanoparticle and its affinity to hepatoma cells].

This article describes the preparation of the N-tert-butyl-alpha-phenylnitrone (PBN) liposomes and their related characteristics. The PBN liposomes were prepared by film dispersion-supersonic method and the formula of liposomes was optimized by orthogonal uniform design. RP-HPLC was used to qualify the amount of PBN that entered into the hepatoma cells. Necrosis rate was also investigated by fluorescence activated cell sorter (FACS) after PBN liposomes transfection. Result showed that the mean particle size, entrapment efficiency, and polydispersity of the resulting PBN-liposome were 137.5 nm, 71.52% and 0.286, respectively. PBN liposomes can enter into the tumor cell stably and they have higher affinity to hepatoma cell compared with free PBN resulting in a higher necrosis rate after transfection. These results provide a potential method for early diagnosis and treatment of cancer using specific spin trapping probe targeting tumor cells.

Alpha-lipoic acid induces apoptosis in hepatoma cells via the PTEN/Akt pathway.

We report here that alpha-lipoic acid (alpha-LA), a naturally-occurring antioxidant, scavenges reactive oxygen species (ROS) followed by an increase in apoptosis of human hepatoma cells. Apoptosis induced by alpha-LA was dependent upon the activation of the caspase cascade and the mitochondrial death pathway. alpha-LA induced increases in caspase-9 and caspase-3 but had no significant effect on caspase-8 activity. Apoptosis induced by alpha-LA was found to be mediated through the tensin homologue deleted on chromosome 10 (PTEN)/Akt pathway. Prior to cell apoptosis, PTEN was activated and its downstream target Akt was inhibited. Our findings indicate that increasing ROS scavenging could be a therapeutic strategy to treat cancer.

Reduced cardiac output is associated with decreased mitochondrial efficiency in the non-ischemic ventricular wall of the acute myocardial-infarcted dog.

Cardiogenic shock is the leading cause of death among patients hospitalized with acute myocardial infarction (MI). Understanding the mechanisms for acute pump failure is therefore important. The aim of this study is to examine in an acute MI dog model whether mitochondrial bio-energetic function within non-ischemic wall regions are associated with pump failure. Anterior MI was produced in dogs via ligation of left anterior descending (LAD) coronary artery, that resulted in an infract size of about 30% of the left ventricular wall. Measurements of hemodynamic status, mitochondrial function, free radical production and mitochondrial uncoupling protein 3 (UCP3) expression were determined over 24 h period. Hemodynamic measurements revealed a > 50% reduction in cardiac output at 24 h post infarction when compared to baseline. Biopsy samples were obtained from the posterior non-ischemic wall during acute infarction. ADP/O ratios for isolated mitochondria from non-ischemic myocardium at 6 h and 24 h were decreased when compared to the ADP/O ratios within the same samples with and without palmitic acid (PA). GTP inhibition of (PA)-stimulated state 4 respiration in isolated mitochondria from the non-ischemic wall increased by 7% and 33% at 6 h and 24 h post-infarction respectively when compared to sham and pre-infarction samples. This would suggest that the mitochondria are uncoupled and this is supported by an associated increase in UCP3 expression observed on western blots from these same biopsy samples. Blood samples from the coronary sinus measured by electron paramagnetic resonance (EPR) methods showed an increase in reactive oxygen species (ROS) over baseline at 6 h and 24 h post-infarction. In conclusion, mitochondrial bio-energetic ADP/O ratios as a result of acute infarction are abnormal within the non-ischemic wall. Mitochondria appear to be energetically uncoupled and this is associated with declining pump function. Free radical production may be associated with the induction of uncoupling proteins in the mitochondria.

Reactive oxygen species stimulated human hepatoma cell proliferation via cross-talk between PI3-K/PKB and JNK signaling pathways.

Reactive oxygen species (ROS) are important for intracellular signaling mechanisms regulating many cellular processes. Manganese superoxide dismutase (MnSOD) may regulate cell growth by changing the level of intracellular ROS. In our study, we investigated the effect of ROS on 7721 human hepatoma cell proliferation. Treatment with H2O2 (1-10 microM) or transfection with antisense MnSOD cDNA constructs significantly increased the cell proliferation. Recently, the mitogen-activated protein kinases (MAPK) and the protein kinase B (PKB) were proposed to be involved in cell growth. Accordingly, we assessed the ability of ROS to activate MAPK and PKB. PKB and extracellular signal-regulated kinase (ERK) were both rapidly and transiently activated by 10 microM H2O2, but the activities of p38 MAPK and JNK were not changed. ROS-induced PKB activation was abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002, suggesting that PI3-K is an upstream mediator of PKB activation in 7721 cells. Transfection with sense PKB cDNA promoted c-fos and c-jun expression in 7721 cells, suggesting that ROS may regulate c-fos and c-jun expression via the PKB pathway. Furthermore we found that exogenous H2O2 could stimulate the proliferation of PKB-AS7721 cells transfected with antisense PKB cDNA, which was partly dependent on JNK activation, suggesting that H2O2 stimulated hepatoma cell proliferation via cross-talk between the PI3-K/PKB and the JNK signaling pathways. However, insulin could stimulate 7721 cell proliferation, which is independent of cross-talk between PI3-K/PKB and JNK pathways. In addition, H2O2 did not induce the cross-talk between the PI3-K/PKB and the JNK pathways in normal liver cells. Taken together, we found that ROS regulate hepatoma cell growth via specific signaling pathways (cross-talk between PI3-K/PKB and JNK pathway) which may provide a novel clue to elucidate the mechanism of hepatoma carcinogenesis.

[Influence of PKB on ROS regulation of proliferation in human 7721 hepatoma cells].

In the present study, the relationship between PKB signaling and reactive oxygen species (ROS) during the course of exogenous and endogenous ROS or antioxidants regulating human 7721 hepatoma cell proliferation was studied. To change endogenous ROS levels, 7721 cells were transfected with human manganese superoxide dismutase (MnSOD) construct containing sense or antisense MnSOD cDNA. Low level of exogenous ROS H2O2(1-10 mumol/L) significantly stimulated PKB activity and c-fos/c-jun expression and cell growth, which could be abolished by antioxidant danshensu (40 mg/L). It was observed that overexpression of MnSOD inhibited 7721 cell growth by inhibiting PKB activity and c-fos/c-jun expression; the PKB activity and c-fos/c-jun expression, however, were stimulated by down-regulated MnSOD expression. In addition, PKB-7721 cells (transfected with sense PKB cDNA) promoted c-fos/c-jun expression by stimulating PKB activity. These results suggest that the redox state stimulated hepatoma cell growth through PKB pathway, which modulates AP-1 expression.

Inhibition of Proliferation and Expression of N-ras in Hepatoma Cells by Antioxidation Treatment.

Kunming mice inoculated with hepatoma cell (H22) suspension subcutaneously at their right axilla were administered orally with antioxidants such as vitamine E, beta-carotene, glutamine, kappa-selenocarrageenan and polysaccharide-peptide of coriolus (PSP) solution. It was found that the inoculated hepatoma growth was suppressed to various extents. The two kinds of polysaccharide antioxidants improved non-specific immunity, enhanced the nitrogen monoxide (NO) content in plasma and strengthened the inhibition of hepatoma. Above antioxidants added in the culture of 7721 human hepatoma cells inhibited the cell proliferation and inducedits apoptosis. Meanwhile, the activity of glutathione peroxidase (GSH-Px) in the plasma of mice increased and the content of malondialdehyde (MDA) decreased. H(2)O(2) in low concentration improved the cancer cell proliferation and inhanced the expression of Mn-SOD c-fos and c-jun, but led to cells apoptosis or necrosis in high concentration. The mechanism of antioxidants inhibiting tumor growth and improving cancer cells apoptosis might be that, on the one hand, the antioxidants blocked the free radicals signal transduction on cancer cells proliferation, and on the other hand, they improved the release of NO through enhancing the non-specific immunity, so inhibiting the cancer cells proliferation directly.