SIRT3 participates in glucose metabolism interruption and apoptosis induced by BH3 mimetic S1 in ovarian cancer cells.

The Bcl-2 antiapoptotic proteins are important cancer therapy targets; however, their role in cancer cell metabolism remains unclear. We found that the BH3-only protein mimetic S1, a novel pan Bcl-2 inhibitor, simultaneously interrupted glucose metabolism and induced apoptosis in human SKOV3 ovarian cancer cells, which was related to the activation of SIRT3, a stress-responsive deacetylase. S1 interrupted the cellular glucose metabolism mainly through causing damage to mitochondrial respiration and inhibiting glycolysis. Moreover, S1 upregulated the gene and protein expression of SIRT3, and induced the translocation of SIRT3 from the nucleus to mitochondria. SIRT3 silencing reversed the effects of S1 on glucose metabolism and apoptosis through increasing the level of HK-II localized to the mitochondria, while a combination of the glycolysis inhibitor 2-DG and S1 intensified the cytotoxicity through further upregulation of SIRT3 expression. This study underscores an essential role of SIRT3 in the antitumor effect of Bcl-2 inhibitors in human ovarian cancer through regulating both metabolism and apoptosis. The manipulation of Bcl-2 inhibitors combined with the use of classic glycolysis inhibitors may be rational strategies to improve ovarian cancer therapy.

Suppression of chloride channel 3 expression facilitates sensitivity of human glioma U251 cells to cisplatin through concomitant inhibition of Akt and autophagy.

Cisplatin resistance is a difficult problem in clinical chemotherapy, and the mechanisms involved in cisplatin resistance require further study. In this study, we investigated the role of chloride channel-3 (ClC-3) in cisplatin resistance. Autophagy was demonstrated by accumulation of LC3-II, beclin 1 and Atg12-Atg5. The ultrastructure changes were observed under electron microscope. Chemical staining with acridine orange or MDC was used to detect acidic vesicular organelles. Quantification of apoptosis was detected by PI and Annexin V staining. The mechanisms involved in the Akt pathway and autophagy were studied by western blot analysis. Our results showed that Akt phosphorylation and autophagy were induced by cisplatin in human glioma U251 cells. Specific inhibition of ClC-3 by ClC-3 siRNA sensitized the apoptosis-resistant U251 cells to cisplatin-mediated cell death and downregulated phosphorylated Akt. Interestingly, ClC-3 suppression also inhibited induction of autophagy by cisplatin although the Akt/mTOR pathway was deregulated. Counteracting the autophagic process by 3-methylademine enhanced cytotoxicity of cisplatin, revealing that autophagy plays a key role in chemoresistance. Suppressing the Akt/mTOR pathway by the NADPH oxidase inhibitor diphenyl iodonium (DPI) indicated that cisplatin-induced activation of Akt/mTOR pathway requires generation of reactive oxygen species (ROS) through NADPH oxidase. Collectively, our results suggest that ClC-3 suppression causes the inhibition of Akt and autophagy, which can enhance the therapeutic benefit of cisplatin in U251 cells.

5-Nitro-2-(3-phenylpropylamino) benzoic acid induced drug resistance to cisplatin in human erythroleukemia cell lines.

Mechanisms of cisplatin resistance in cancer cells are not fully understood. Here, we showed a critical role for the chloride channel-3 (ClC-3) in cisplatin resistance in human erythroleukemia K562 and RK562 cells. We found that a chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) could protect cells from cisplatin-induced apoptosis. NPPB treatment decreased the mRNA and the protein expression of Bax/Bcl-2, decreased the protein expressions of cytochrome C and caspase-3, and increased the mRNA expressions of cyclin D1 and ClC-3 in cells treated with cisplatin. The caspase-3 activity was decreased significantly and the rate of cell apoptosis was decreased. NPPB treatment increased CIC-3 expression, which could increase acidification of intracellular compartments, and increased sequestration of cisplatin, inducing decreased effective drug concentrations, and subsequently cell death. Collectively, our data indicate that NPPB can induce drug resistance to cisplatin by upregulating the expression of CIC-3. NPPB-induced CIC-3 expression facilitates acidification of sequestrated cisplatin, and plays an important role in preventing cisplatin-induced apoptosis in human erythroleukemia K562 and RK562 cells.

[Significance and expression of FKHR and AKT after subarachnoid hemorrhage in rat brain cortex].

OBJECTIVE: To study the significance and expression of FKHR and AKT after subarachnoid hemorrhage (SAH) in rat brain cortex. METHODS: Twenty-four rats were randomly divided into three groups: sham, SAH and SAH plus nimodipine (n=8 each). A reliable SAH model was established by double injections of blood into cistern magna in Wistar rats. The neurological scores were measured by Loeffler and the expressions of FKHR, P-FKHR, AKT and P-FKHR detected by Western blot. RESULTS: Compared with sham group, the neurological score of SAH group obviously decreased (P < 0.05), the expression of FKHR became elevated in rat cortex (P < 0.01), the expression of AKT had no change and the expressions of P-AKT and P-FKHR obviously decreased (all P < 0.01). But the neurological score markedly increased (P < 0.01) and the expressions of P-AKT and P-FKHR became elevated (all P < 0.01) after administration of nimodipine. CONCLUSION: Both P-AKT and P-FKHR are involved in the process of brain cortex damage induced by SAH. The protective effects of nimodipine on brain injury induced by SAH may be related to the elevated expressions of P-AKT and P-FKHR in brain cortex.

The effects of dexamethasone on rat brain cortical nuclear factor kappa B (NF-kappaB) in endotoxic shock.

To explore the molecular mechanism of brain tissue injury induced by lipopolysaccharide (LPS), we studied the effects of endotoxic shock on rat brain cortex NF-kappaB and the effects of dexamethasone on these changes. Rats were randomly divided into LPS, LPS + dexamethasone, and control groups. The DNA-binding activity of NF-kappaB was observed using electrophoretic mobility shift assay (EMSA). Protein expression in nuclear extracts was studied using Western blots, and nuclear translocation was observed using immunohistochemistry. These indices were assayed at 1 h and 4 h after intravenous injection of LPS (4 mg x kg(-1)). EMSA showed significantly increased NF-kappaB DNA-binding activity in nuclear extracts from the LPS group at both 1 h and 4 h after LPS injection, compared with the control group (P < 0.01). For the LPS group, the NF-kappaB DNA-binding activity was greater at 1 h than at 4 h (P < 0.05). The expression of p65 and p50 protein in the nuclear extracts was also increased, as compared with the control group. However, the expression of p65 and p50 protein from cytosolic extracts did not show any significant change. Dexamethasone down-regulated not only NF-kappaB DNA-binding activity but also the expression of p65 protein in the nuclear extracts. From these data, we have concluded that NF-kappaB activation and nuclear translocation of NF-kappaB play a key role in the molecular mechanism of brain tissue injury in endotoxic shock. Dexamethasone may alleviate brain injury by inhibiting NF-kappaB activation.

Effects of ghrelin on the proliferation and secretion of splenic T lymphocytes in mice.

Ghrelin, a novel gut--brain peptide predominantly produced by the stomach, displays strong growth hormone (GH)-releasing activity mediated by the hypothalamus-pituitary GH secretagogue receptor (GHS-R). Recently, the ghrelin receptor has also been detected in peripheral systems including immune tissues, suggesting that ghrelin may play an important role in the regulation of immune function. In this paper, we assessed the presence and function of the ghrelin receptor in murine splenic T cells. The enriched T cells express the mRNA of ghrelin and ghrelin receptor mRNA, and there is a significantly positive correlation between them. Moreover, we showed that ghrelin dose-dependently inhibits proliferation of splenic T cells when they are costimulated by anti-CD3. In addition, ghrelin suppressed Th(1) (IL-2 and IFN-gamma) and Th(2) (IL-4 and IL-10) cytokines mRNA expression. These results demonstrate the presence of the ghrelin receptor in murine spleen T lymphocytes and a functional role of ghrelin as a modulator of lymphocyte function. This function of ghrelin may have some relevance to the pathophysiology of immunologic alterations related to metabolism.