The PDZ domain of the E protein in SARS-CoV induces carcinogenesis and poor prognosis in LUAD.

BACKGROUND: In both lung adenocarcinoma (LUAD) and severe acute respiratory syndrome (SARS), uncontrolled inflammation can be detected in lung tissue. The PDZ-binding motif (PBM) in the SARS-CoV-1 E protein has been demonstrated to be a virulence factor that induces a cytokine storm. METHODS: To identify gene expression fluctuations induced by PBM, microarray sequencing data of lung tissue infected with wild-type (SARS-CoV-1-E-wt) or recombinant virus (SARS-CoV-1-E-mutPBM) were analyzed, followed by functional enrichment analysis. To understand the role of the screened genes in LUAD, overall survival and immune correlation were calculated. RESULTS: A total of 12 genes might participate in the initial and developmental stages of LUAD through expression variation and mutation. Moreover, dysregulation of a total of 12 genes could lead to a poorer prognosis. In addition, the downregulation of MAMDC2 and ITGA8 by PBM could also affect patient prognosis. Although the conserved PBM (-D-L-L-V-) can be found at the end of the carboxyl terminus in multiple E proteins of coronaviruses, the specific function of each protein depends on the entire amino acid sequence. CONCLUSIONS: In summary, PBM containing the SARS-CoV-1 E protein promoted the carcinogenesis of LUAD by dysregulating important gene expression profiles and subsequently influencing the immune response and overall prognosis.

Intrahepatic osteopontin signaling by CREBZF defines a checkpoint for steatosis-to-NASH progression.

BACKGROUND AND AIMS: NASH has emerged as a leading cause of chronic liver disease. However, the mechanisms that govern NASH fibrosis remain largely unknown. CREBZF is a CREB/ATF bZIP transcription factor that causes hepatic steatosis and metabolic defects in obesity. APPROACH AND RESULTS: Here, we show that CREBZF is a key mechanism of liver fibrosis checkpoint that promotes hepatocyte injury and exacerbates diet-induced NASH in mice. CREBZF deficiency attenuated liver injury, fibrosis, and inflammation in diet-induced mouse models of NASH. CREBZF increases HSC activation and fibrosis in a hepatocyte-autonomous manner by stimulating an extracellular matrix protein osteopontin, a key regulator of fibrosis. The inhibition of miR-6964-3p mediates CREBZF-induced production and secretion of osteopontin in hepatocytes. Adeno-associated virus -mediated rescue of osteopontin restored HSC activation, liver fibrosis, and NASH progression in CREBZF-deficient mice. Importantly, expression levels of CREBZF are increased in livers of diet-induced NASH mouse models and humans with NASH. CONCLUSIONS: Osteopontin signaling by CREBZF represents a previously unrecognized intrahepatic mechanism that triggers liver fibrosis and contributes to the severity of NASH.

CXCR7 Agonist TC14012 Improves Angiogenic Function of Endothelial Progenitor Cells via Activating Akt/eNOS Pathway and Promotes Ischemic Angiogenesis in Diabetic Limb Ischemia.

PURPOSE: Endothelial progenitor cells (EPCs) play a critical role in repairing damaged vessels and triggering ischemic angiogenesis, but their number is reduced and function is impaired under diabetic conditions. Improving EPC function has been considered a promising strategy to ameliorate diabetic vascular complications. In the present study, we aim to investigate whether and how CXCR7 agonist TC14012 promotes the angiogenic function of diabetic EPCs. METHODS: High glucose (HG) treatment was used to mimic the hyperglycemia in diabetes. Tube formation, cell scratch recovery and transwell assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and cleaved-caspase3 expression were used to evaluate the angiogenic capability, cell migration, and apoptosis of EPCs, respectively. Hind limb ischemia (HLI) model was used to appraise the ability of TC14012 in promoting diabetic ischemic angiogenesis in vivo. RESULTS: HG treatment impaired EPC tube formation and migration, and induced EPC apoptosis and oxidative damage, while TC14012 rescued tube formation and migration, and prevented HG-induced apoptosis and oxidative damage of EPCs. Furthermore, these beneficial effects of TC14012 on EPCs were attenuated by specific siRNAs against CXCR7, validating that CXCR7 is a functional target of TC14012 in EPCs. Mechanistic studies demonstrated that HG treatment reduced CXCR7 expression in EPCs, and impaired Akt and endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production; similarly, these signal impairments in HG-exposed EPCs could be rescued by TC14012. However, the protective effects of TC14012 on tube formation and migration, Akt and eNOS phosphorylation, and NO production in HG-treated EPCs were almost completely abolished by siRNAs against CXCR7 or Akt specific inhibitor wortmannin. More importantly, in vivo study showed that TC14012 administration enhanced blood perfusion recovery and angiogenesis in the ischemic hind limb and increased the EPC number in peripheral circulation of db/db mice, demonstrating the capability of TC14012 in promoting EPC mobilization and ischemia angiogenic function. CONCLUSION: TC14012 can prevent EPCs from HG-induced dysfunction and apoptosis, improve eNOS activity and NO production via CXCR7/Akt signal pathway, and promote EPC mobilization and diabetic ischemia angiogenesis.

Liraglutide Improves the Angiogenic Capability of EPC and Promotes Ischemic Angiogenesis in Mice under Diabetic Conditions through an Nrf2-Dependent Mechanism.

The impairment in endothelial progenitor cell (EPC) functions results in dysregulation of vascular homeostasis and dysfunction of the endothelium under diabetic conditions. Improving EPC function has been considered as a promising strategy for ameliorating diabetic vascular complications. Liraglutide has been widely used as a therapeutic agent for diabetes. However, the effects and mechanisms of liraglutide on EPC dysfunction remain unclear. The capability of liraglutide in promoting blood perfusion and angiogenesis under diabetic conditions was evaluated in the hind limb ischemia model of diabetic mice. The effect of liraglutide on the angiogenic function of EPC was evaluated by cell scratch recovery assay, tube formation assay, and nitric oxide production. RNA sequencing was performed to assess the underlying mechanisms. Liraglutide enhanced blood perfusion and angiogenesis in the ischemic hindlimb of db/db mice and streptozotocin-induced type 1 diabetic mice. Additionally, liraglutide improved tube formation, cell migration, and nitric oxide production of high glucose (HG)-treated EPC. Assessment of liraglutide target pathways revealed a network of genes involved in antioxidant activity. Further mechanism study showed that liraglutide decreased the production of reactive oxygen species and increased the activity of nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 deficiency attenuated the beneficial effects of liraglutide on improving EPC function and promoting ischemic angiogenesis under diabetic conditions. Moreover, liraglutide activates Nrf2 through an AKT/GSK3ß/Fyn pathway, and inhibiting this pathway abolished liraglutide-induced Nrf2 activation and EPC function improvement. Overall, these results suggest that Liraglutide represents therapeutic potential in promoting EPC function and ameliorating ischemic angiogenesis under diabetic conditions, and these beneficial effects relied on Nrf2 activation.

Nrf2 transcriptional upregulation of IDH2 to tune mitochondrial dynamics and rescue angiogenic function of diabetic EPCs.

Endothelial progenitor cells (EPCs) are reduced in number and impaired in function in diabetic patients. Whether and how Nrf2 regulates the function of diabetic EPCs remains unclear. In this study, we found that the expression of Nrf2 and its downstream genes were decreased in EPCs from both diabetic patients and db/db mice. Survival ability and angiogenic function of EPCs from diabetic patients and db/db mice also were impaired. Gain- and loss-of-function studies, respectively, showed that knockdown of Nrf2 increased apoptosis and impaired tube formation in EPCs from healthy donors and wild-type mice, while Nrf2 overexpression decreased apoptosis and rescued tube formation in EPCs from diabetic patients and db/db mice. Additionally, proangiogenic function of Nrf2-manipulated mouse EPCs was validated in db/db mice with hind limb ischemia. Mechanistic studies demonstrated that diabetes induced mitochondrial fragmentation and dysfunction of EPCs by dysregulating the abundance of proteins controlling mitochondrial dynamics; upregulating Nrf2 expression attenuated diabetes-induced mitochondrial fragmentation and dysfunction and rectified the abundance of proteins controlling mitochondrial dynamics. Further RNA-sequencing analysis demonstrated that Nrf2 specifically upregulated the transcription of isocitrate dehydrogenase 2 (IDH2), a key enzyme regulating tricarboxylic acid cycle and mitochondrial function. Overexpression of IDH2 rectified Nrf2 knockdown- or diabetes-induced mitochondrial fragmentation and EPC dysfunction. In a therapeutic approach, supplementation of an Nrf2 activator sulforaphane enhanced angiogenesis and blood perfusion recovery in db/db mice with hind limb ischemia. Collectively, these findings indicate that Nrf2 is a potential therapeutic target for improving diabetic EPC function. Thus, elevating Nrf2 expression enhances EPC resistance to diabetes-induced oxidative damage and improves therapeutic efficacy of EPCs in treating diabetic limb ischemia likely via transcriptional upregulating IDH2 expression and improving mitochondrial function of diabetic EPCs.

Correction: Dishevelled1-3 contribute to multidrug resistance in colorectal cancer via activating Wnt/ß-catenin signaling.

[This corrects the article DOI: 10.18632/oncotarget.23253.].

Procyanidin B2 improves endothelial progenitor cell function and promotes wound healing in diabetic mice via activating Nrf2.

One of the major reasons for the delayed wound healing in diabetes is the dysfunction of endothelial progenitor cells (EPCs) induced by hyperglycaemia. Improvement of EPC function may be a potential strategy for accelerating wound healing in diabetes. Procyanidin B2 (PCB2) is one of the major components of procyanidins, which exhibits a variety of potent pharmacological activities. However, the effects of PCB2 on EPC function and diabetic wound repair remain elusive. We evaluated the protective effects of PCB2 in EPCs with high glucose (HG) treatment and in a diabetic wound healing model. EPCs derived from human umbilical cord blood were treated with HG. The results showed that PCB2 significantly preserved the angiogenic function, survival and migration abilities of EPCs with HG treatment, and attenuated HG-induced oxidative stress of EPCs by scavenging excessive reactive oxygen species (ROS). A mechanistic study found the protective role of PCB2 is dependent on activating nuclear factor erythroid 2-related factor 2 (Nrf2). PCB2 increased the expression of Nrf2 and its downstream antioxidant genes to attenuate the oxidative stress induced by HG in EPCs, which were abolished by knockdown of Nrf2 expression. An in vivo study showed that intraperitoneal administration of PCB2 promoted wound healing and angiogenesis in diabetic mice, which was accompanied by a significant reduction in ROS level and an increase in circulating EPC number. Taken together, our results indicate that PCB2 treatment accelerates wound healing and increases angiogenesis in diabetic mice, which may be mediated by improving the mobilization and function of EPCs.

Endothelial Overexpression of Metallothionein Prevents Diabetes-Induced Impairment in Ischemia Angiogenesis Through Preservation of HIF-1α/SDF-1/VEGF Signaling in Endothelial Progenitor Cells.

Diabetes-induced oxidative stress is one of the major contributors to dysfunction of endothelial progenitor cells (EPCs) and impaired endothelial regeneration. Thus, we tested whether increasing antioxidant protein metallothionein (MT) in EPCs promotes angiogenesis in a hind limb ischemia (HLI) model in endothelial MT transgenic (JTMT) mice with high-fat diet- and streptozocin-induced diabetes. Compared with littermate wild-type (WT) diabetic mice, JTMT diabetic mice had improved blood flow recovery and angiogenesis after HLI. Similarly, transplantation of JTMT bone marrow-derived mononuclear cells (BM-MNCs) stimulated greater blood flow recovery in db/db mice with HLI than did WT BM-MNCs. The improved recovery was associated with augmented EPC mobilization and angiogenic function. Further, cultured EPCs from patients with diabetes exhibited decreased MT expression, increased cell apoptosis, and impaired tube formation, while cultured JTMT EPCs had enhanced cell survival, migration, and tube formation in hypoxic/hyperglycemic conditions compared with WT EPCs. Mechanistically, MT overexpression enhanced hypoxia-inducible factor 1α (HIF-1α), stromal cell-derived factor (SDF-1), and vascular endothelial growth factor (VEGF) expression and reduced oxidative stress in ischemic tissues. MT's pro-EPC effects were abrogated by siRNA knockdown of HIF-1α without affecting its antioxidant action. These results indicate that endothelial MT overexpression is sufficient to protect against diabetes-induced impairment of angiogenesis by promoting EPC function, most likely through upregulation of HIF-1α/SDF-1/VEGF signaling and reducing oxidative stress.

Interleukin-1ß augments the angiogenesis of endothelial progenitor cells in an NF-κB/CXCR7-dependent manner.

Endothelial progenitor cells (EPCs) are able to trigger angiogenesis, and pro-inflammatory cytokines have beneficial effects on angiogenesis under physiological and pathological conditions. C-X-C chemokine receptor type 7 (CXCR-7), receptor for stromal cell-derived factor-1, plays a critical role in enhancing EPC angiogenic function. Here, we examined whether CXCR7 mediates the pro-angiogenic effects of the inflammatory cytokine interleukin-1ß (IL-1ß) in EPCs. EPCs were isolated by density gradient centrifugation and angiogenic capability was evaluated in vitro by Matrigel capillary formation assay and fibrin gel bead assay. IL-1ß elevated CXCR7 expression at both the transcriptional and translational levels in a dose- and time-dependent manner, and blockade of the nuclear translocation of NF-κB dramatically attenuated the IL-1ß-mediated up-regulation of CXCR7 expression. IL-1ß stimulation significantly promoted EPCs tube formation and this effect was largely impaired by CXCR7-siRNA transfection. IL-1ß treatment stimulated extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation, and inhibition of Erk1/2 phosphorylation partially impaired IL-1ß-induced tube formation of EPCs but without significant effects on CXCR7 expression. Moreover, blocking NF-κB had no significant effects on IL-1ß-stimulated Erk1/2 phosphorylation. These findings indicate that CXCR7 plays an important role in the IL-1ß-enhanced angiogenic capability of EPCs and antagonizing CXCR7 is a potential strategy for inhibiting angiogenesis under inflammatory conditions.

Targeting Smox Is Neuroprotective and Ameliorates Brain Inflammation in Cerebral Ischemia/Reperfusion Rats.

Spermine oxidase (Smox) is a member of the polyamine oxidases and has been demonstrated to be involved in ischemic brain damage. In this study, we found that Smox expression was increased in a rat middle cerebral artery occlusion (MCAO) model and in cultured primary neurons after oxygen-glucose deprivation and reoxygenation (OGD/R). Smox downregulation by the adeno-associated virus RNA interference system significantly reduced the MCAO-induced brain infarct volume and neurological deficits and decreased neuronal apoptosis and inflammatory reactions. In addition, significant microglial activation and increased IL-6 and TNF-α expression were observed in microglia treated with supernatant from neurons after OGD/R. However, a significant reduction in microglial activation as well as IL-6 and TNF-α expression was observed in microglia treated with supernatant from Smox downregulated neurons after OGD/R. Therefore, the results indicated that Smox is an important mediator of cerebral ischemia injury and may be a therapeutic target for cerebral ischemia patients.

Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts.

Cardiac insulin resistance is a key pathogenic factor for diabetic cardiomyopathy (DCM), but the mechanism remains largely unclear. We found that diabetic hearts exhibited decreased phosphorylation of total Akt and isoform Akt2 but not Akt1 in wild-type (WT) male FVB mice, which was accompanied by attenuation of Akt downstream glucose metabolic signal. All of these signal changes were not observed in metallothionein cardiac-specific transgenic (MT-TG) hearts. Furthermore, insulin-induced glucose metabolic signals were attenuated only in WT diabetic hearts. In addition, diabetic hearts exhibited increased Akt-negative regulator tribbles pseudokinase 3 (TRB3) expression only in WT mice, suggesting that MT may preserve Akt2 function via inhibiting TRB3. Moreover, MT prevented tert-butyl hydroperoxide (tBHP)-reduced insulin-stimulated Akt2 phosphorylation in MT-TG cardiomyocytes, which was abolished by specific silencing of Akt2. Specific silencing of TRB3 blocked tBHP inhibition of insulin-stimulated Akt2 phosphorylation in WT cardiomyocytes, whereas overexpression of TRB3 in MT-TG cardiomyocytes and hearts abolished MT preservation of insulin-stimulated Akt2 signals and MT prevention of DCM. Most importantly, supplementation of Zn to induce MT preserved cardiac Akt2 signals and prevented DCM. These results suggest that diabetes-inhibited cardiac Akt2 function via TRB3 upregulation leads to aberrant cardiac glucose metabolism. MT preservation of cardiac Akt2 function by inhibition of TRB3 prevents DCM.

Sitagliptin-mediated preservation of endothelial progenitor cell function via augmenting autophagy enhances ischaemic angiogenesis in diabetes.

Recently, the dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin, a major anti-hyperglycaemic agent, has received substantial attention as a therapeutic target for cardiovascular diseases via enhancing the number of circulating endothelial progenitor cells (EPCs). However, the direct effects of sitagliptin on EPC function remain elusive. In this study, we evaluated the proangiogenic effects of sitagliptin on a diabetic hind limb ischaemia (HLI) model in vivo and on EPC culture in vitro. Treatment of db/db mice with sitagliptin (Januvia) after HLI surgery efficiently enhanced ischaemic angiogenesis and blood perfusion, which was accompanied by significant increases in circulating EPC numbers. EPCs derived from the bone marrow of normal mice were treated with high glucose to mimic diabetic hyperglycaemia. We found that high glucose treatment induced EPC apoptosis and tube formation impairment, which were significantly prevented by sitagliptin pretreatment. A mechanistic study found that high glucose treatment of EPCs induced dramatic increases in oxidative stress and apoptosis; pretreatment of EPCs with sitagliptin significantly attenuated high glucose-induced apoptosis, tube formation impairment and oxidative stress. Furthermore, we found that sitagliptin restored the basal autophagy of EPCs that was impaired by high glucose via activating the AMP-activated protein kinase/unc-51-like autophagy activating kinase 1 signalling pathway, although an autophagy inhibitor abolished the protective effects of sitagliptin on EPCs. Altogether, the results indicate that sitagliptin-induced preservation of EPC angiogenic function results in an improvement of diabetic ischaemia angiogenesis and blood perfusion, which are most likely mediated by sitagliptin-induced prevention of EPC apoptosis via augmenting autophagy.

A Novel CXCR4 antagonist enhances angiogenesis via modifying the ischaemic tissue environment.

Endothelial progenitor cells (EPCs) play a capital role in angiogenesis via directly participating in neo-vessel formation and secreting pro-angiogenic factors. Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 play a critical role in the retention and quiescence of EPCs within its niche in the bone marrow. Disturbing the interaction between SDF-1 and CXCR4 is an effective strategy for EPC mobilization. We developed a novel CXCR4 antagonist P2G, a mutant protein of SDF-1ß with high antagonistic activity against CXCR4 and high potency in enhancing ischaemic angiogenesis and blood perfusion. However, its direct effects on ischaemic tissue remain largely unknown. In this study, P2G was found to possess a robust capability to promote EPC infiltration and incorporation in neo-vessels, enhance the expression and function of pro-angiogenic factors, such as SDF-1, vascular endothelial growth factor and matrix metalloprotein-9, and activate cell signals involved in angiogenesis, such as proliferating cell nuclear antigen, protein kinase B (Akt), extracellular regulated protein kinases and mammalian target of rapamycin, in ischaemic tissue. Moreover, P2G can attenuate fibrotic remodelling to facilitate the recovery of ischaemic tissue. The capability of P2G in direct augmenting ischaemic environment for angiogenesis suggests that it is a potential candidate for the therapy of ischaemia diseases.

Elevating CXCR7 Improves Angiogenic Function of EPCs via Akt/GSK-3ß/Fyn-Mediated Nrf2 Activation in Diabetic Limb Ischemia.

RATIONALE: Endothelial progenitor cells (EPCs) respond to stromal cell-derived factor 1 (SDF-1) through chemokine receptors CXCR7 and CXCR4. Whether SDF-1 receptors involves in diabetes mellitus-induced EPCs dysfunction remains unknown. OBJECTIVE: To determine the role of SDF-1 receptors in diabetic EPCs dysfunction. METHODS AND RESULTS: CXCR7 expression, but not CXCR4 was reduced in EPCs from db/db mice, which coincided with impaired tube formation. Knockdown of CXCR7 impaired tube formation of EPCs from normal mice, whereas upregulation of CXCR7 rescued angiogenic function of EPCs from db/db mice. In normal EPCs treated with oxidized low-density lipoprotein or high glucose also reduced CXCR7 expression, impaired tube formation, and increased oxidative stress and apoptosis. The damaging effects of oxidized low-density lipoprotein or high glucose were markedly reduced by SDF-1 pretreatment in EPCs transduced with CXCR7 lentivirus but not in EPCs transduced with control lentivirus. Most importantly, EPCs transduced with CXCR7 lentivirus were superior to EPCs transduced with control lentivirus for therapy of ischemic limbs in db/db mice. Mechanistic studies demonstrated that oxidized low-density lipoprotein or high glucose inhibited protein kinase B and glycogen synthase kinase-3ß phosphorylation, nuclear export of Fyn and nuclear localization of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), blunting Nrf2 downstream target genes heme oxygenase-1, NAD(P)H dehydrogenase (quinone 1) and catalase, and inducing an increase in EPC oxidative stress. This destructive cascade was blocked by SDF-1 treatment in EPCs transduced with CXCR7 lentivirus. Furthermore, inhibition of phosphatidylinositol 3-kinase/protein kinase B prevented SDF-1/CXCR7-mediated Nrf2 activation and blocked angiogenic repair. Moreover, Nrf2 knockdown almost completely abolished the protective effects of SDF-1/CXCR7 on EPC function in vitro and in vivo. CONCLUSIONS: Elevated expression of CXCR7 enhances EPC resistance to diabetes mellitus-induced oxidative damage and improves therapeutic efficacy of EPCs in treating diabetic limb ischemia. The benefits of CXCR7 are mediated predominantly by a protein kinase B/glycogen synthase kinase-3ß/Fyn pathway via increased activity of Nrf2.

Dishevelled1-3 contribute to multidrug resistance in colorectal cancer via activating Wnt/ß-catenin signaling.

Multidrug resistance is a great obstacle in successful chemotherapy of colorectal cancer. However, the molecular mechanism underlying multidrug resistance is not fully understood. Dishevelled, a pivot in Wnt signaling, has been linked to cancer progression, while its role in chemoresistance remains unclear. Here, we found that Dishevelled1-3 was over-expressed in multidrug-resistant colorectal cancer cells (HCT-8/VCR) compared to their parental cells. Silencing Dishevelled1-3 resensitized HCT-8/VCR cells to multiple drugs including vincristine, 5-fluorouracil and oxaliplatin. Moreover, Dishevelled1-3 increased the protein levels of multidrug resistance protein 1 (P-gp/MDR1), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP), Survivin and Bcl-2 which are correlated with multidrug resistance. shß-catenin abolished Dishevelled-mediated these protein expressions. Unexpectedly, none of Dishevelled1-3 controlled ß-catenin accumulation and nuclear translocation. Furthermore, the nuclear translocations of Dishevelled1-3 were promoted in HCT-8/VCR cells compared to HCT-8. Dishevelled1-3 bound to ß-catenin in nucleus, and promoted nuclear complex formation and transcription activity of ß-catenin/TCF. Taken together, Dishevelled1-3 contributed to multidrug resistance in colorectal cancer via activating Wnt/ß-catenin signaling and inducing the expressions of P-gp, MRP2, BCRP, Survivin and Bcl-2, independently of ß-catenin accumulation and nuclear translocation. Silencing Dishevelled1-3 resensitized multidrug-resistant colorectal cancer cells, providing a novel therapeutic target for successful chemotherapy of colorectal cancer.

Plumbagin inhibits cell proliferation and promotes apoptosis in multiple myeloma cells through inhibition of the PI3K/Akt-mTOR pathway.

Plumbagin is the primary component of the traditional Chinese medicine Baihua Dan, and possesses anti-infection and anticancer effects with the ability to enhance the sensitivity of tumor cells to radiation therapy. The present study aimed to investigate the potential anticancer effect and mechanism of plumbagin on multiple myeloma (MM) cells. Human MM OPM1 cells were treated with plumbagin, and its impact on cell viability, cytotoxicity, apoptosis and caspase-3 activity was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, lactate dehydrogenase leakage, flow cytometry and colorimetric assays. In addition, the protein expression levels of phosphoinositide 3-kinase, phosphorylated (p)-Akt and p-mammalian target of rapamycin (mTOR) in OPM1 cells were analyzed by western blotting. The results demonstrated that plumbagin treatment inhibited cell viability, increased cell cytotoxicity, activated cell apoptosis and promoted caspase-3 activity in the OPM1 cells. Furthermore, pretreatment of plumbagin significantly suppressed PI3K, p-Akt and p-mTOR protein expression levels in the OPM1 cells. In conclusion, the present study indicates that plumbagin inhibits cell proliferation and promotes apoptosis in MM cells through inhibition of PI3K/Akt-mTOR expression.

From the Cover: Zinc Deficiency Worsens and Supplementation Prevents High-Fat Diet Induced Vascular Inflammation, Oxidative Stress, and Pathological Remodeling.

Obesity has become a common public health problem in the world and raises the risk of various cardiovascular diseases. Zinc is essential for multiple organs in terms of normal structure and function. The present study investigated the effects of high fat diet (HFD) induced obesity on the aorta in mice, and evaluated whether it can be affected by zinc deficiency or supplementation. Four-week-old male C57BL/6J mice were fed HFD with varied amounts of zinc (deficiency, adequate and supplementation) for 3 and 6 months. Results showed that HFD feeding induced a time-dependent aortic remodeling, demonstrated by increased vessel wall thickness, tunica cell proliferation and fibrotic responses, and inflammatory response, reflected by increased expression of inflammatory cytokines (tumor necrosis factor-α and vascular cell adhesion molecule 1). HFD feeding also caused aortic oxidative damage, reflected by 3-nitrotyrosine and 4-hydroxy-2-nonenal accumulation, and down-regulated nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression and function, shown by down-regulation of its downstream antioxidants, catalase, NAD(P)H dehydrogenase (quinone 1), and metallothionein expression. The vascular effects of obesity-induced by HFD was exacerbated by zinc deficiency but significantly improved by zinc supplementation. In addition, down-regulation of Nrf2 function and associated antioxidants expression were also worsened by zinc deficiency but improved by zinc supplementation. These results suggest that HFD induces aortic remodeling, which can be exacerbated by zinc deficiency and improved by zinc supplementation.

Effects of Proanthocyanidins on Intestinal Motility Disturbance Following Intestinal Ischemia/Reperfusion.

PURPOSE: To investigate the potential protective effects of Proanthocyanidins(PAs) on intestinal motility disturbance following intestinal ischemia/reperfusion (I/R). MATERIALS AND METHODS: Male rats were divided into four groups: Sham, I/R, I/R+PA100 and I/R+PA200. Sham group underwent laparotomy without ligation, the others were subjected to intestinal ischemia for 1 h and reperfusion 4 h. Rats in the I/R+PA100 group received PAs (100 mg/kg/d) for 5 days prior to I/R, while rats in the I/R+PA200 group received PAs (200 mg/kg/d). After reperfusion, using an electrophysiology instrument measured ileal slow wave. Ileal specimens were obtained to determine contractility, tissue levels of Bax, Bcl-2, and Caspase-3 and evaluate histopathological changes. In addition, blood sample was obtained to determine serum superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels. RESULTS: Intestinal I/R caused severe histopathological injury including mucosal erosions, inflammatory cell infiltration, necrosis, and hemorrhage. Both PAs treatment decreased mucosal pathological impairment in comparison with the I/R group (p < .05) in light microscopic evaluations. In both PAs-treated groups, Bax and Caspase-3 expression were decreased compared to I/R group, while the Bcl-2 expression increased (p < .05), which was similarly the case for serum SOD activity demonstrated significant enhance (p < .05) and decline in MDA levels in comparison with I/R group (both p < .05). Moreover, PAs treatment was more efficient in attenuating serum MDA levels of intestinal I/R (both p < .05). And the contractile amplitude and frequency of slow wave in I/R+PA100 and I/R+PA200 groups were higher than I/R group (both p < .05). CONCLUSIONS: PAs improve intestinal motility disturbance following intestinal I/R by alleviating oxidative stress and apoptosis.

MicroRNA-133b inhibits hepatocellular carcinoma cell progression by targeting Sirt1.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that function as critical gene regulators by targeting mRNAs for translational repression or degradation. In this study, we showed that the expression level of miR-133b was decreased, while Sirt1 mRNA expression levels were increased in hepatocellular carcinoma (HCC) and cell lines, and we identified Sirt1 as a novel direct target of miR-133b. The over-expression of miR-133b suppressed Sirt1 expression. In addition, miR-133b over-expression resulted in attenuating HCC cell proliferation and invasion together with apoptosis increase in vitro. HepG2 cell transplantation revealed that up-regulation of miR-133b could inhibit HCC tumor genesis in vivo. Forced expression of Sirt1 partly rescued the effect of miR-133b in vitro. Furthermore, our study showed that miR-133b over-expression or Sirt1 down-regulation elevated E-cadherin expression, and repressed glypican-3 (GPC3) and the anti-apoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1) expression. The inhibition of GPC3 expression repressed Bcl-2, Bcl-xL, and Mcl-1 expression, and elevated E-cadherin expression. Moreover, the Sirt1 up-regulation resulted in increases in HCC cell proliferation and invasion together with decreases apoptosis, and increases in the cytosolic accumulation and nuclear translocation of the transcription factor ß-catenin in vitro. But the effect of Sirt1 up-regulation was partly reversed by GPC3 down-regulation in vitro. Taken together, these findings provide insight into the role and mechanism of miR-133b in regulating HCC cell proliferation, invasion and apoptosis via the miR-133b/Sirt1/GPC3/Wnt ß-catenin axis, and miR-133b may serve as a potential therapeutic target in HCC in the future.

Silencing dishevelled-1 sensitizes paclitaxel-resistant human ovarian cancer cells via AKT/GSK-3ß/ß-catenin signalling.

OBJECTIVES: Expression of dishevelled-1 (DVL1) has recently been linked to cancer progression, however, its role in resistance to cancer therapy is unclear. In this study, we aimed to explore the function of DVL1 in paclitaxel-resistant human ovarian cancer cells. MATERIALS AND METHODS: The MTT assay was used to assess effects of DVL1 silencing on sensitivity of cells that were otherwise resistant to paclitaxel (Taxol). Western blotting and immunofluorescence staining were used to examine effects of DVL1 on AKT/GSK-3ß/ß-catenin signalling. RESULTS: Dishevelled-1 was found to be over-expressed in a paclitaxel-resistant cell line derived from human ovarian cancer cell line A2780 (A2780/Taxol line) as well as parental A2780 cells. Down-regulation of DVL1 (using the inhibitor 3289-8625 or siRNA (siDVL1) against DVL1) sensitized A2780/Taxol cells to paclitaxel. Over-expression of DVL1 in A2780 cells increased protein levels of P-gp, BCRP and Bcl-2, which are known targets of ß-catenin. Silencing DVL1 in A2780/Taxol cells also reduced levels of these proteins, and led to accumulation of ß-catenin. In addition, DVL1 aberrantly activated AKT/GSK-3ß/ß-catenin signalling. Inactivation of AKT signalling attenuated DVL1-mediated inhibition of GSK-3ß and accumulation of ß-catenin, in both A2780 and A2780/Taxol cells. CONCLUSIONS: Taken together, these results suggest that silencing DVL1 sensitized A2780/Taxol cells to paclitaxel, by down-regulating AKT/GSK-3ß/ß-catenin signalling, providing a novel strategy for chemosensitization of ovarian cancer to paclitaxel-induced cytotoxicity.