Molecular targets of a human HNF1 alpha mutation responsible for pancreatic beta-cell dysfunction

The reverse tetracycline-dependent transactivator system was employed in insulinoma INS-1 cells to achieve controlled inducible expression of hepatocyte nuclear factor-1 alpha (HNF1 alpha)-P291fsinsC, the most common mutation associated with subtype 3 of maturity-onset diabetes of the young (MODY3). Nuclear localized HNF1 alpha-P291fsinsC protein exerts its dominant-negative effects by competing with endogenous HNF1 alpha for the cognate DNA-binding site. HNF1 alpha controls multiple genes implicated in pancreatic beta-cell function and notably in metabolism- secretion coupling. In addition to reduced expression of the genes encoding insulin, glucose transporter-2, L-pyruvate kinase, aldolase B and 3-hydroxy-3-methylglutaryl coenzyme A reductase, induction of HNF1 alpha-P291fsinsC also significantly inhibits expression of mitochondrial 2-oxoglutarate dehydrogenase (OGDH) E1 subunit mRNA and protein. OGDH enzyme activity and [(14)C]pyruvate oxidation were also reduced. In contrast, the mRNA and protein levels of mitochondrial uncoupling protein-2 were dramatically increased by HNF1 alpha-P291fsinsC induction. As predicted from this altered gene expression profile, HNF1 alpha-P291fsinsC also inhibits insulin secretory responses to glucose and leucine, correlated with impaired nutrient-evoked mitochondrial ATP production and mitochondrial membrane hyperpolarization. These unprecedented results suggest the molecular mechanism of HNF1 alpha-P291fsinsC causing beta-cell dysfunction.     

Thymosin alpha1 suppresses proliferation and induces apoptosis in human leukemia cell lines

Thymosin alpha1 (Talpha1), a 28-amino acid peptide, is a well-known immune system enhancer for the treatment of various diseases. In the present investigation, the effects of Talpha1 on the proliferation and apoptosis of human leukemia cell lines (HL-60, K562 and K562/ADM) were studied. The proliferation was significantly depressed after 96 h of treatment with Talpha1, and obvious signs of apoptosis, i.e., cell morphology, nuclei condensation and Annexin V binding, were observed thereafter. Moreover, the up-regulation of Fas/Apol (CD95) and decrease in bcl-2 anti-apoptotic gene expression were observed in apoptotic cells. The expression and the function of P-glycoprotein (P-gp) can be slightly inhibited by Talpha1. It is noteworthy that K562 and K562/ADM were more sensitive than HL-60 cells when subjected to Talpha1. Furthermore, HepG-2, the human hepatoma cell line, displayed significant less sensitivity to Talpha1 than all the human leukemia cell lines. D-Tubocurarine (TUB), a nicotinic acetylcholine receptors (nAChRs) antagonist, significantly antagonized the inhibition effects induced by Talpha1, whereas atropine, a muscarinic acetylcholine receptor antagonist, did not exhibit such effects. All the results indicate that Talpha1 was able to significantly suppress proliferation and induce apoptosis in human leukemia cell lines.     

MicroRNA-192 targeting retinoblastoma 1 inhibits cell proliferation and induces cell apoptosis in lung cancer cells

microRNAs play an important roles in cell growth, differentiation, proliferation and apoptosis. They can function either as tumor suppressors or oncogenes. We found that the overexpression of miR-192 inhibited cell proliferation in A549, H460 and 95D cells, and inhibited tumorigenesis in a nude mouse model. Both caspase-7 and the PARP protein were activated by the overexpression of miR-192, thus suggesting that miR-192 induces cell apoptosis through the caspase pathway. Further studies showed that retinoblastoma 1 (RB1) is a direct target of miR-192. Over-expression of miR-192 decreased RB1 mRNA and protein levels and repressed RB1-3'-UTR reporter activity. Knockdown of RB1 using siRNA resulted in a similar cell morphology as that observed for overexpression of miR-192. Additionally, RB1-siRNA treatment inhibited cell proliferation and induced cell apoptosis in lung cancer cells. Analysis of miRNA expression in clinical samples showed that miR-192 is significantly downregulated in lung cancer tissues compared to adjacent non-cancerous lung tissues. In conclusion, our results demonstrate that miR-192 is a tumor suppressor that can target the RB1 gene to inhibit cell proliferation and induce cell apoptosis in lung cancer cells. Furthermore, miR-192 was expressed at low levels in lung cancer samples, indicating that it might be a promising therapeutic target for lung cancer treatment.     

PanK4 inhibits pancreatic beta-cell apoptosis by decreasing the transcriptional level of pro-caspase-9

Dear Editor： Coenzyme A （CoA） is a primary and predominant acyl group carrier involved in a wide variety of important biochemical processes. The CoA biosynthetic pathway is composed of five enzymatic steps, of which Pantothenate kinase （PanK） is a key regulatory enzyme. The multiple isoforms of PanK are encoded by four different genes [1,2]. In our previous studies of SNP markers by genotyping the case-controlled DNAs, we found that one SNP within the hPANK4 gene on chromosome 1 was associated with type 2 diabetes [3-5]. We subsequently showed that rat PanK4 （rPanK4） was up-regulated when rats were challenged by high concentration of glucose [6]. M2-type pyruvate kinase （Pkm2） was found, both in vitro and in vivo, to be associated with rPanK4 [7]. These data suggest that PanK4 may have a role in diabetes pathogenesis. The development of type 2 diabetes is due partly to the loss of the pancreatic β-cell mass, therefore the secreted amount of insulin is insufficient to maintain the glucose homoestasis [8]. In the present study, we evaluated the effect ofrPanK4 on β-cell apoptosis. We aimed to determine the potential ofrPanK4 gene in β-cell apoptosis induced by the cytotoxic agent streptozotocin （STZ）.[第一段]     

Fatty acids potentiate interleukin-1beta toxicity in the beta-cell line INS-1E

Evidence for "lipotoxicity," i.e., negative effects of fatty acids (FA) on pancreatic beta-cells is incomplete. Here, we tested whether non-toxic concentrations of FA potentiate toxic effects of interleukin-1beta (IL-1beta). Culture of INS-1E clonal beta-cells for 2-6 days with 70 microM docosahexaenoic acid (DHA), eicosapentaenoic acid, arachidonic acid, 0.1mM linoleic acid, or 0.1-0.2mM oleic acid exerted no or minor negative effects on cell viability (MTT assay). Viability was reduced by 0.5 ng/ml IL-1beta. All tested FA significantly aggravated this effect after 6 days of culture. IL-1beta also exerted negative effects on cellular insulin content and DHA and oleic acid aggravated these effects. L-NAME, an inhibitor of constitutive nitric oxide (NO) synthase, reduced the negative effects of IL-1beta per se but did not abolish the potentiating effects of FA. CONCLUSION: FA potentiate toxic effects of IL-1beta on beta-cells by mechanisms that include NO-independent ones.     

Nitric oxide induces apoptosis via Ca2+-dependent processes in the pancreatic beta-cell line MIN6

An excessive production of nitric oxide (NO) in response to cytokines has been shown to be the major cause of the destruction of islet beta-cells associated with type 1 (insulin-dependent) diabetes mellitus. The NO-induced beta-cell death is the typical apoptosis. In the present study, we show evidence that supports a tight link between NO, Ca2+, protease and apoptosis in beta-cells. Three different NO donors, SNAP, NOR3 and NOC7, induced apoptosis in a beta-cell line, MIN6 cells, in a concentration-dependent manner. SNAP at 200 microM increased cytosolic Ca2+ concentration ([Ca2+]i) and induced apoptosis. The SNAP-induced apoptosis was blocked by a Ca2+ chelator, BAPTA-AM, and by an inhibitor of a Ca2+-dependent protease, calpain. In conclusion, an excessive NO production induces apoptosis, wherein an increase in [Ca2+]i and resultant activation of calpain play a key role.     

Real-time PCR: housekeeping genes in the INS-1E beta-cell line.

Investigation of gene expression is a developing area with several methods available. One method is quantitative PCR. A major pitfall in quantitative PCR is the normalisation procedure of the gene expression. Many experiments include a housekeeping gene, some use RNA concentration, and others use a geometric mean of several internal, stably expressed genes. This study demonstrates that real-time-PCR results differ with varying housekeeping genes and analysis protocols when applied to insulin-secreting INS-1E cells derived from the pancreas and stimulated by DEDTC (diethyldithiocarbamate, a zinc chelator) and GLP-1.     

Inhibitor of growth 4 inhibits cell proliferation,migration, and induces apoptosis of renal cell carcinoma cells

The inhibitor of growth 4 (ING4) is known as a tumor suppressor. The expressions of ING4 were markedly reduced in human renal clear cell carcinoma (ccRCC) tissues. However, the role of ING4 in renal cell carcinoma (RCC) remains unknown. The aim of the current study was to detect the ING4 expression level and its potential role in human RCC cell lines. Our results showed that ING4 was lowly expressed in human RCC cell lines compared with that in proximal tubular cell line. Ectopic overexpression of ING4 inhibited the proliferation, migration, and invasion properties, and as well as prevented epithelial-mesenchymal transition (EMT) phenotype of RCC cells. In addition, ING4 overexpression induced cell apoptosis and autophagy in RCC cells. Furthermore, ING4 overexpression suppressed the activation of PI3K/Akt pathway in RCC cells. The activator of PI3K/Akt, insulin-like growth factor 1, abolished the effects of ING4 on RCC cells. These findings indicated that ING4 presented anticancer activity in RCC cells. The effects of ING4 on RCC cells were mediated by regulating the PI3K/Akt pathway. These findings suggested that ING4 could be used for gene therapy of RCC.