Jinghuan Lv

Anti-inflammatory effect of resveratrol on TNF-α-induced MCP-1 expression in adipocytes

Chronic low-grade inflammation characterized by adipose tissue macrophage accumulation and abnormal cytokine production is a key feature of obesity and type 2 diabetes. Adipose-tissue-derived monocyte chemoattractant protein (MCP)-1, induced by cytokines, has been shown to play an essential role in the early events during macrophage infiltration into adipose tissue. In this study we investigated the effects of resveratrol upon both tumor necrosis factor (TNF)-alpha-induced MCP-1 gene expression and its underlying signaling pathways in 3T3-L1 adipocytes. Resveratrol was found to inhibit TNF-alpha-induced MCP-1 secretion and gene transcription, as well as promoter activity, which based on down-regulation of TNF-alpha-induced MCP-1 transcription. Nuclear factor (NF)-kappaB was determined to play a major role in the TNF-alpha-induced MCP-1 expression. Further analysis showed that resveratrol inhibited DNA binding activity of the NF-kappaB complex and subsequently suppressed NF-kappaB transcriptional activity in TNF-alpha-stimulated cells. Finally, the inhibition of MCP-1 may represent a novel mechanism of resveratrol in preventing obesity-related pathologies.

Activation of liver X receptors inhibits pancreatic islet beta cell proliferation through cell cycle arrest

Aims/hypothesisLiver X receptors (LXRs) are important transcriptional regulators of lipid homeostasis and proliferation in several cell types. However, the roles of LXRs in pancreatic beta cells have not been fully established. The aim of this study was to investigate the effects of LXRs on pancreatic beta cell proliferation.MethodsGene expression was analysed using real-time RT-PCR. Transient transfection and reporter gene assays were used to determine the transcriptional activity of LXRs in pancreatic beta cells. Cell viability and proliferation were analysed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DNA fluorometric, BrdU labelling and [3H]thymidine incorporation assays. Cell cycle distribution was investigated by flow cytometry analysis. Adenovirus-based RNA interference was used to knockdown LXRα, LXRβ and p27 in MIN6 cells and mouse islets.ResultsWe found that both Lxrα (also known as Nr1h3) and Lxrβ (also known as Nr1h2) were expressed and transactivated the LXR response element in HIT-T15 and MIN6 cells. Activation of LXRs dose-dependently inhibited pancreatic beta cell viability and proliferation. This was accompanied by beta cell cycle arrest at the G1 phase. Furthermore, LXR activation increased levels of the p27 protein by inhibiting its degradation. Knockdown of p27 reversed these effects of LXR activation on growth inhibition and cell cycle arrest.Conclusions/interpretationOur observations indicate that LXR activation inhibits pancreatic beta cell proliferation through cell cycle arrest. A well-known regulator of pancreatic beta cell cycle progression, p27, is upregulated and mediates the effects of LXRs on growth inhibition in beta cells. These observations suggest the involvement of aberrant activation of LXR in beta cell mass inadequacy, which is an important step in the development of type 2 diabetes.

Prostaglandin E2 regulates Foxo activity via the Akt pathway: implications for pancreatic islet beta cell dysfunction

Aims/hypothesisProstaglandin E2 (PGE2) is a well-recognised inhibitor of glucose-stimulated insulin secretion (GSIS). The aim of this study was to investigate the signalling pathway of PGE2 in beta cell function regulation in HIT-T15 cells and isolated rat islets.Materials and methodsmRNA levels of the prostaglandin E receptor 3 (Ptger3) were measured by real-time PCR. Western blot analysis was used to detect changes in the levels of PTGER3, phosphorylated and total Akt, phosphorylated and total forkhead box ‘Other’ (Foxo). Transient transfection and reporter assays were used to measure Foxo transcriptional activity. The biological significance of PGE2 in beta cell function was analysed using MTT, flow cytometry and GSIS assays.ResultsWe found that treating HIT-T15 cells with exogenous PGE2 stimulated Ptger3 gene expression specifically, and diminished cAMP generation. These were accompanied by the downregulation of Akt and Foxo phosphorylation in HIT-T15 cells and isolated rat islets. Moreover, PGE2 upregulated basal and partially reversed constitutively active Akt-inactivated Foxo transcriptional activity. Furthermore, GSIS was impaired in PGE2-treated HIT-T15 cells and isolated islets. However, the dosage used in the above experiments did not affect beta cell viability and apoptosis. In addition, insulin-like growth factor 1 (IGF-1) pretreatment reversed the effects of PGE2, and wortmannin treatment abolished the preventive effects of IGF-1.Conclusions/interpretationOur observations strongly suggest that PGE2 can induce pancreatic beta cell dysfunction through the induction of Ptger3 gene expression and inhibition of Akt/Foxo phosphorylation without impacting beta cell viability. These results shed light on the mechanisms of PGE2 actions in pancreatic beta cell dysfunction.

PP2A inhibitors induce apoptosis in pancreatic cancer cell line PANC-1 through persistent phosphorylation of IKKα and sustained activation of the NF-κB pathway.

Serine/threonine protein phosphatase 2A (PP2A), is thought to be a cancer suppresser, as inhibition of PP2A can induce phosphorylation and activation of substrate kinases, most of which can accelerate growth. Interestingly, cantharidin potently inhibits PP2A but efficiently represses various cancer cells. In the present study, we found that PP2A inhibitors, cantharidin or Okadaic acid, inhibited cell viability and triggered apoptosis in PANC-1 pancreatic cancer cell line dependent on PP2A/IKKα/IκBα/p65 NF-κB pathway. The activation of NF-κB pathway up-regulated downstream pro-apoptotic genes, TNF-α, TRAILR1 and TRAILR2, and triggered apoptosis through the extrinsic pathway, indicating that PP2A is a potential target for pancreatic cancer treatment.

Inhibition of Forkhead Box O1 Protects Pancreatic β-Cells against Dexamethasone-Induced Dysfunction

Forkhead Box O1 (FoxO1) is a key transcription regulator of insulin/IGF-I signaling pathway, and its activity can be increased by dexamethasone (DEX) in several cell types. However, the role of FoxO1 in DEX-induced pancreatic beta-cell dysfunction has not been fully understood. Therefore, in this study, we investigated whether FoxO1 could mediate DEX-induced beta-cell dysfunction and the possible underlying mechanisms in pancreatic beta-cell line RINm5F cells and primary rat islet. We found that DEX markedly increased FoxO1 mRNA and protein expression and decreased FoxO1 phosphorylation through the Akt pathway, which resulted in an increase in active FoxO1 in RINm5F cells and isolated rat islets. Activated FoxO1 subsequently inhibited pancreatic duodenal homeobox-1 expression and induced nuclear exclusion of pancreatic duodenal homeobox-1. Knockdown of FoxO1 by RNA interference restored the expression of pancreatic duodenal homeobox-1 and prevented DEX-induced dysfunction of glucose-stimulated insulin secretion in rat islets. Together, the results of present study demonstrate that FoxO1 is integrally involved in DEX-induced inhibition of pancreatic duodenal homeobox-1 and glucose-stimulated insulin secretion dysfunction in pancreatic islet beta-cells. Inhibition of FoxO1 can effectively protect beta-cells against DEX-induced dysfunction.

The BCL2 major breakpoint region (mbr) regulates gene expression

BCL2 expression is finely tuned by a variety of environmental and endogenous stimuli and regulated at both transcriptional and post-transcriptional levels. Our previous investigations demonstrated that the BCL2 major breakpoint region (mbr) in the 3′-UTR upregulates reporter gene expression, which implies that this region possessed intrinsic regulatory function. However, the effect of the mbr on BCL2 expression, and the underlying regulatory mechanisms, remain to be elucidated. To assess the direct effect of the mbr on the transcriptional activity of the BCL2 gene, we employed targeted homologous recombination to establish a mbr+/mbr− heterozygous Nalm-6 cell line and then compared the transcriptional activity and apoptotic effect on transcription between the wild type and targeted alleles. We found that deletion of the mbr significantly decreased the transcriptional activity of the corresponding allele in the mbr+/mbr− cell. The BCL2 allele deleted of the mbr had a slower response to apoptotic stimuli than did the wild type allele. The regulatory function of the mbr was mediated through SATB1. Overexpression of SATB1 increased BCL2 expression, while knockdown of SATB1 with RNAi decreased BCL2 expression. Our results clearly indicated that the mbr could positively regulate BCL2 gene expression and this regulatory function was closely related to SATB1.

Tissue inhibitor of metalloproteinase-1 decreased chemosensitivity of MDA-435 breast cancer cells to chemotherapeutic drugs through the PI3K/AKT/NF-кB pathway.

TIMP-1 is well known to be capable of inhibiting apoptosis. Elevated levels of TIMP-1 in tumor tissue have been shown to be strongly associated with a poor response to chemotherapy. In this study, using conventional cytotoxic drugs commonly used in the treatment of breast cancer, we investigated how TIMP-1 influenced the efficacy using breast cell lines. Our data demonstrated that overexpression of TIMP-1 could significantly decrease the sensitivity of MDA-435 breast cancer cells to epirubicin and paclitaxel. TIMP-1 can potently activate phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappaB (NF-кB) signaling. Furthermore, the TIMP-1-induced attenuation of the effect of epirubicin and paclitaxel was reversed by the PI3K/Akt chemical inhibitor LY294002 and the NF-кB inhibitor pyrrolidine dithiocarbamate (PDTC), showing that the PI3K/Akt and NF-кB signaling pathway was involved in the TIMP-1-induced effect on chemoresistance. Taken together, our results indicate that TIMP-1 decreased chemosensitivity through the PI3K/Akt/NF-кB signal transduction pathway in MDA-435 breast cancer cells.

Celecoxib Enhanced the Sensitivity of Cancer Cells to Anticancer Drugs by Inhibition of the Expression of P-Glycoprotein Through a COX-2-Independent Manner

The P‐glycoprotein (p170, P‐gp) encoded by human MDR1 gene functions as a pump to extrude anticancer drugs from cancer cells. Over‐expression of p170 is closely related to primary and induced drug resistance phenotype of tumor cells. Recent studies have demonstrated that expression of cyclooxygenase‐2 (COX‐2) is positively correlated with the p170 level, suggesting a potential of COX‐2 specific inhibitors in regulation of cytotoxicity of anticancer agents. Celecoxib is one of the specific inhibitors of COX‐2 and has been widely used in clinic. However, its function in the response of cancer cells to anticancer drugs and the related mechanism are still waiting to be investigated. To explore the correlation of celecoxib and the p170‐mediated drug resistance, the role of celecoxib in drug response of cancer cells was analyzed with flow cytometry, high performance liquid chromatography (HPLC), and colony formation experiments. Celecoxib (50 µM) was found to significantly enhance the sensitivity of MCF‐7 and JAR/VP16 cells to tamoxifen and etoposide, respectively, by inhibition of p170 expression and increase in intracellular accumulation of the drugs. However, celecoxib did not affect pump function of p170. Enzyme activity and methylation analyses demonstrated that the inhibitory effect of celecoxib on p170 was independent on COX‐2 but closely related to hypermethylation of MDR1 gene promoter. Our study suggested that celecoxib was a potential agent for enhancement of the sensitivity of cancer cells to anticancer drugs. It also provided a links between epigenetic change of MDR1 and drug response of cancer cells. J. Cell. Biochem. 108: 181–194, 2009.

Tissue inhibitor of metalloproteinase-1 protects MCF-7 breast cancer cells from paclitaxel-induced apoptosis by decreasing the stability of cyclin B1.

Paclitaxel (PTX) is a very effective drug in treating tumors. It disturbs microtubule dynamics and impairs the transition of cells from metaphase to anaphase in mitosis, leading to cell death by apoptosis. However, the effectiveness of PTX in cancer chemotherapy is hampered by drug resistance in some patients. Tissue inhibitor of metalloproteinase‐1 (TIMP‐1) is well known to be capable of inhibiting apoptosis. Elevated tumor tissue TIMP‐1 levels have been significantly associated with a poor response to chemotherapy. We hypothesized that TIMP‐1 could reduce the sensitivity of breast cancer cells to PTX by inhibiting apoptosis. To test this hypothesis, we first examined the effects of TIMP‐1 on the apoptosis induced by PTX and investigated the effects of TIMP‐1 on the expression and stability of cyclin B1 that critically regulates the metaphase to anaphase transition during mitosis in MCF‐7 breast cancer cells. Our data demonstrate that TIMP‐1 could significantly decrease the sensitivity of MCF‐7 cells to PTX‐induced apoptosis, attenuate mitotic blockage in G2/M, and enhance the degradation of cyclin B1. To further investigate whether the inhibitory effect of TIMP‐1 on PTX‐induced apoptosis is mediated by lowering levels of cyclin B1, a cyclin B1‐expression plasmid was transfected into clone overexpressing TIMP‐1. The levels of PTX‐induced apoptosis were then analyzed. The data showed that the TIMP‐1‐based decrease in PTX‐induced apoptosis was reversed by cyclin B1. Our data indicate that TIMP‐1 can protect breast cancer cells from PTX‐induced apoptosis by decreasing the stability of cyclin B1.

Forkhead Box O1/Pancreatic and Duodenal Homeobox 1 Intracellular Translocation Is Regulated by c-Jun N-Terminal Kinase and Involved in Prostaglandin E2-Induced Pancreatic β-Cell Dysfunction

Prostaglandin E(2) (PGE(2)) is a well-known mediator of beta-cell dysfunction in both type 1 and type 2 diabetes. We recently reported that down-regulation of the Akt pathway activity is implicated in PGE(2)-induced pancreatic beta-cell dysfunction. The aim of this study was to further dissect the signaling pathway of this process in pancreatic beta-cell line HIT-T15 cells and primary mouse islets. We found that PGE(2) time-dependently increased the c-Jun N-terminal kinase (JNK) pathway activity. JNK inhibition by the JNK-specific inhibitor SP600125 reversed PGE(2)-inhibited glucose-stimulated insulin secretion (GSIS). PGE(2) induced dephosphorylation of Akt and FOXO1, leading to nuclear localization and transactivation of FOXO1. Activation of FOXO1 induced nuclear exclusion but had no obvious effect on the whole-cell protein level of pancreatic and duodenal homeobox 1 (PDX1). However, these effects were all attenuated by JNK inhibition. Furthermore, adenovirus-mediated overexpression of dominant-negative (DN)-FOXO1 abolished whereas constitutively active (CA)-FOXO1 mimicked the effects of PGE(2) on GSIS in isolated mouse islets. In addition, we demonstrated that DN-JNK1 but not DN-JNK2 or CA-Akt abolished the PGE(2)-induced AP-1 luciferase reporter activity, whereas DN-JNK1 and CA-Akt but not DN-JNK2 reversed the effect of PGE(2) on FOXO1 transcriptional activity, and overexpression of DN-JNK1 rescued PGE(2)-impaired GSIS in mouse islets. Our results revealed that activation of the JNK is involved in PGE(2)-induced beta-cell dysfunction. PGE(2)-mediated JNK1 activation, through dephosphorylation of Akt and FOXO1, leads to nuclear accumulation of FOXO1 and nucleocytoplasmic shuttling of PDX1, finally resulting in defective GSIS in pancreatic beta-cells.