Unconjugated
Cholesteatoma is a benign keratinizing and hyper proliferative squamous epithelial lesion of the temporal bone. Epidermal growth factor (EGF) is one of the most important cytokines which has been shown to play a critical role in cholesteatoma. In this investigation, we studied the effects of EGF on the proliferation of keratinocytes and EGF-mediated signaling pathways underlying the pathogenesis of cholesteatoma. We examined the expressions of phosphorylated EGF receptor (p-EGFR), phosphorylated Akt (p-Akt), cyclinD1, and proliferating cell nuclear antigen (PCNA) in 40 cholesteatoma samples and 20 samples of normal external auditory canal (EAC) epithelium by immunohistochemical method. Furthermore, in vitro studies were performed to investigate EGF-induced downstream signaling pathways in primary external auditory canal keratinocytes (EACKs). The expressions of p-EGFR, p-Akt, cyclinD1, and PCNA in cholesteatoma epithelium were significantly increased when compared with those of control subjects. We also demonstrated that EGF led to the activation of the EGFR/PI3K/Akt/cyclinD1 signaling pathway, which played a critical role in EGF-induced cell proliferation and cell cycle progression of EACKs. Both EGFR inhibitor AG1478 and PI3K inhibitor wortmannin inhibited the EGF-induced EGFR/PI3K/Akt/cyclinD1 signaling pathway concomitantly with inhibition of cell proliferation and cell cycle progression of EACKs. Taken together, our data suggest that the EGFR/PI3K/Akt/cyclinD1 signaling pathway is active in cholesteatoma and may play a crucial role in cholesteatoma epithelial hyper-proliferation. This study will facilitate the development of potential therapeutic targets for intratympanic drug therapy for cholesteatoma.
The expression and function of P-glycoprotein (P-gp) is associated with the phenotype of multi-drug resistance (MDR), leading chemotherapy failure of patients suffered with cancer. Grape seed procyanidin(GSP) is a natural polyphenol supplement with anti-inflammatory effect. Present study assessed a new use of GSP on the MDR reversal activity and its possible molecular mechanisms in MDR1-overpressing paclitaxel resistant ovarian cancer cells. Our results showed GSP significantly enhanced the cytotoxicity of paclitaxel and adriamycin in paclitaxel resistant A2780/T cells but its parental A2780 cells. Furthermore, GSP strongly inhibited P-gp expression by blocking MDR1 gene transcription, as well as, increased the intracellular accumulation of the P-gp substrate rhodamine-123 in A2780/T cells. Nuclear factor-κB(NF-κB) activity, IκB degradation level and NF-κB/p65 nuclear translocation induced by lipopolysaccharide (LPS) and receptor activator for nuclear factor-κB ligand (RANKL) were markedly inhibited by pre-treatment with GSP. Meanwhile, GSP inhibited MAPK/ERK pathway by decreasing the phosphorylation of ERK1/2, resulting in reduced the Y-box binding protein 1 (YB-1) activation with blocking its nuclear translocation. Moreover, the up-regulation of P-gp expression, the activation of AKT/NF-κB and MAPK/ERK pathway induced by LPS was attenuated by GSP administration. Compared with PDTC and U1026, inhibitor of NF-κB and MAPK/ERK respectively, GSP showed the same tendency of down-regulating NF-κB and MAPK/ERK mediated YB-1 activities. Thus, GSP reverses P-gp associated MDR by inhibiting the function and expression of P-gp through down-regulation of NF-κB activity and MAPK/ERK pathway mediated YB-1 nuclear translocation, offering insight into the mechanism of reversing MDR by natural polyphenol supplement compounds. GSP could be a new potential MDR reversal agent used for combination therapy with chemotherapeutics in clinic.
Tectorigenin is a plant isoflavonoid originally isolated from the dried flower of Pueraria thomsonii Benth. Although its anti-inflammatory and anti-hyperglycosemia effects have been well documented, the effect of tectorigenin on endothelial dysfunction insulin resistance involved has not yet been reported. Herein, this study aims to investigate the action of tectorigenin on amelioration of insulin resistance in the endothelium. Palmitic acid (PA) was chosen as a stimulant to induce ROS production in endothelial cells and successfully established insulin resistance evidenced by the specific impairment of insulin PI3K signaling. Tectorigenin effectively inhibited the ability of PA to induce the production of reactive oxygen species and collapse of mitochondrial membrane potential. Moreover, tectorigenin presented strong inhibition effect on ROS-associated inflammation, as TNF-α and IL-6 production in endothelial cells was greatly reduced with suppression of IKKβ/NF-κB phosphorylation and JNK activation. Tectorigenin also can inhibit inflammation-stimulated IRS-1 serine phosphorylation and restore the impaired insulin PI3K signaling, leading to a decreased NO production. These results demonstrated its positive regulation of insulin action in the endothelium. Meanwhile, tectorigenin down-regulated endothelin-1 and vascular cell adhesion molecule-1 overexpression, and restored the loss of insulin-mediated vasodilation in rat aorta. These findings suggested that tectorigenin could inhibit ROS-associated inflammation and ameliorated endothelial dysfunction implicated in insulin resistance through regulating IRS-1 function. Tectorigenin might have potential to be applied for the management of cardiovascular diseases involved in diabetes and insulin resistance.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for the treatment of cancer, because it preferentially induces apoptosis in numerous cancer cells with little or no effect on normal cells. 5,7-Dihydroxyflavone is a dietary flavonoid commonly found in many plants. Here we show that the combined treatment with 5,7-dihydroxyflavone and TRAIL at subtoxic concentrations induced strong apoptotic response in human hepatocarcinoma HepG2 cells, acute leukemia Jurkat T cells, and cervical carcinoma HeLa cells. We further investigated the mechanisms by which 5,7-dihydroxyflavone augments TRAIL-induced apoptosis in HepG2 cells. 5,7-Dihydroxyflavone up-regulated the expression of pro-apoptotic protein Bax, attenuated the expression of anti-apoptotic proteins Bcl-2, Mcl-1, and IAPs, and reduced the phosphorylation levels of Akt and STAT3, weakening the anti-apoptotic signals thus facilitating the process of apoptosis. Moreover, 5,7-dihydroxyflavone and TRAIL were well tolerated in mice, and the combination of 5,7-dihydroxyflavone and TRAIL reduced tumor burden in vivo in a HepG2 tumor xenograft model. Interestingly, 5,7-dihydroxyflavone-mediated sensitization to TRAIL-induced cell death was not observed in normal human hepatocytes L-O2. These results suggest that the 5,7-dihydroxyflavone in combination with TRAIL might be used for cancer prevention and/or therapy.
Integrin is important in migration and metastasis of tumor cells. Changes of integrin expression and distribution will cause an alteration of cellular adhesion and migration behaviors. In this study, we investigated sulfatide regulation of the integrin αV subunit expression in hepatoma cells and observed that either exogenous or endogenous sulfatide elicited a robust upregulation of integrin αV subunit mRNA and protein expression in hepatoma cells. This regulatory effect occurred with a corresponding phosphorylation (T739) of the transcription factor Sp1. Based on the electrophoretic mobility shift assay, sulfatide enhanced the integrin αV promoter activity and strengthened the Sp1 complex super-shift. The results of chromatin immunoprecipitation analysis also indicated that sulfatide enhanced Sp1 binding to the integrin αV promoter in vivo. Silence of Sp1 diminished the stimulation of integrin αV expression by sulfatide. In the early stage of sulfatide stimulation, phosphorylation of Erk as well as c-Src was noted, and inhibition of Erk activation with either U0126 or PD98059 significantly suppressed Sp1 phosphorylation and integrin αV expression. We demonstrated that sulfatide regulated integrin αV expression and cell adhesion, which was associated with Erk activation.
Treatment trends of retinoblastoma (RB) have gradually evolved from eye enucleation and external radiation to local treatment. Combined treatment with an oncolytic virus and chemotherapy is currently a new method in RB treatment. To investigate the therapeutic effect of oncolytic adenovirus SG600 in combination with vincristine (VCR) on retinoblastoma in vitro, the cell viability, cell cycle effects and apoptotic activity of HXO-RB(44) cells treated with SG600, VCR or SG600 plus VCR were measured using a cell counting kit-8-based procedure and flow cytometry. Western blot analysis for Akt, p-Akt, p-p53 and p-Rb protein was performed to investigate the underlying mechanisms of combined therapy. The combination therapy exerted a synergistic antitumor effect via a type of G(2)/M and S phase arrest rather than the induction of apoptosis. The combination of VCR and SG600 further reduced Akt phosphorylation compared with cells treated with VCR alone, suggesting that SG600 could overcome chemoresistance, perhaps by down-regulating Akt in RB cells. An increase in the expression of p-p53 and decrease in p-Rb expression in HXO-RB(44) after co-treatment might be associated with cell cycle block. Western blot examination revealed that VCR might enhance SG600 replication. These results suggest that viro-chemo combination therapy is a feasible and potentially promising approach for the treatment of retinoblastoma.
To study the roles of microRNA-223 (miR-223) in regulation of cell growth, we established a miR-223 over-expression model in HeLa cells infected with miR-223 by Lentivirus pLL3.7 system. We observed in this model that miR-223 significantly suppressed the proliferation, growth rate, colony formation of HeLa cells in vitro, and in vivo tumorigenicity or tumor formation in nude mice. To investigate the mechanisms involved, we scanned and examined the potential and putative target molecules of miR-223 by informatics, quantitative PCR and Western blot, and found that insulin-like growth factor-1 receptor (IGF-1R) was the functional target of miR-223 inhibition of cell proliferation. Targeting IGF-1R by miR-223 was not only seen in HeLa cells, but also in leukemia and hepatoma cells. The downstream pathway, Akt/mTOR/p70S6K, to which the signal was mediated by IGF-1R, was inhibited as well. The relative luciferase activity of the reporter containing wild-type 3'UTR(3'untranslated region) of IGF-1R was significantly suppressed, but the mutant not. Silence of IGF-1R expression by vector-based short hairpin RNA resulted in the similar inhibition with miR-223. Contrarily, rescued IGF-1R expression in the cells that over-expressed miR-223, reversed the inhibition caused by miR-223 via introducing IGF-1R cDNA that didn't contain the 3'UTR. Meanwhile, we also noted that miR-223 targeted Rasa1, but the downstream molecules mediated by Rasa1 was neither targeted nor regulated. Therefore we believed that IGF-1R was the functional target for miR-223 suppression of cell proliferation and its downstream PI3K/Akt/mTOR/p70S6K pathway suppressed by miR-223 was by targeting IGF-1R.
PURPOSE:
Celecoxib, an inhibitor of cyclooxygenase-2 (COX2), was investigated for enhancement of chemotherapeutic efficacy in cancer clinical trials. This study aimed to determine whether celecoxib combined with 5-fluorouracil or sorafenib or gefitinib is beneficial in HepG2 multicellular spheroids (MCSs), as well as elucidate the underlying mechanisms.
METHODS:
The human hepatocellular carcinoma cell line HepG2 MCSs were used as in vitro models to investigate the effects of celecoxib combined with 5-fluorouracil or sorafenib or gefitinib treatment on cell growth, apoptosis, and signaling pathway.
RESULTS:
MCSs showed resistance to drugs compared with monolayer cells. Celecoxib combined with 5-fluorouracil or sorafenib exhibited a synergistic action. Exposure to celecoxib (21.8 μmol/L) plus 5-fluorouracil (8.1 × 10(-3) g/L) or sorafenib (4.4 μmol/L) increased apoptosis but exerted no effect on COX2, phosphorylated epidermal growth-factor receptor (p-EGFR) and phosphorylated (p)-AKT expression. Gefitinib (5 μmol/L), which exhibits no growth-inhibition activity as a single agent, increased the inhibitory effect of celecoxib. Gefitinib (5 μmol/L) plus celecoxib (21.8 μmol/L) increased apoptosis. COX2, p-EGFR, and p-AKT were inhibited.
CONCLUSION:
Celecoxib combined with 5-fluorouracil or sorafenib or gefitinib may be superior to single-agent therapy in HepG2 MCSs. Our results provided molecular evidence to support celecoxib combination-treatment strategies for patients with human hepatocellular carcinoma. MCSs provided a good model to evaluate the interaction of anticancer drugs.
BACKGROUND:
The dipeptidyl peptidase-4 inhibitor sitagliptin, a new anti-diabetic medicine, is effective in treating type 2 diabetes mellitus by increasing the activation and duration of action of glucagon-like peptide-1. Since atherosclerosis is the main pathological feature of diabetic cardiovascular complications, it is important to investigate the anti-atherosclerotic effect of sitagliptin and explore the relevant mechanisms.
METHODS:
Male apolipoprotein-E-knockout mice were randomly divided into two groups and fed either high-fat diet (HFD) or HFD plus sitagliptin at a concentration of 0.3% for 16 weeks. Body weight, food intake, blood glucose, serum lipids and adhesion molecules were measured. The atherosclerotic plaque area and its histological composition were analyzed using Sudan staining and immunohistochemistry. The expression of inflammatory cytokines (monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-6) and the activation of AMP-activated protein kinase (AMPK) and mitogen-activated protein kinase (MAPK) in the aortas were determined using quantitative polymerase chain reaction and western blot, respectively.
RESULTS:
Mice treated with sitagliptin developed fewer atherosclerotic plaques than the control group (7.64 ± 1.98% vs 12.91 ± 1.15%, p < 0.001), particularly in the aortic arch and abdominal aorta, where plaques were decreased 1.92- and 2.74-fold, respectively (p < 0.05 and p < 0.01). Sitagliptin significantly reduced the content of collagen fiber in plaques 1.2-fold (p < 0.05). Moreover, sitagliptin significantly reduced the expression of monocyte chemoattractant protein-1 and interleukin-6 in the aorta (p < 0.01 and p < 0.05), as well as the serum levels of soluble vascular cell adhesion molecule-1 and P-selectin (both p < 0.05). In addition, Sitagliptin induced phosphorylation of AMPK and Akt (p < 0.05 and p < 0.01), while suppressed phosphorylation of p38 and extracellular signal-regulated kinase (Erk) 1/2 (p < 0.05 and p < 0.01) in aortas.
CONCLUSIONS:
Our present study indicates that sitagliptin can reduce the area of the atherosclerotic lesion, possibly by regulating the AMPK and MAPK pathways and then reducing leukocyte -endothelial cell interaction and inflammation reactions. These actions are independent of weight loss and glucose-reducing effects.
BACKGROUND:
MT1G inactivation mediated by promoter methylation has been reported in thyroid cancer. However, the role of MT1G in thyroid carcinogenesis remains unclear. The aim of this study is to examine the biological functions and related molecular mechanisms of MT1G in thyroid cancer.
METHODS:
Methylation-specific PCR (MSP) was performed to analyze promoter methylation of MT1G and its relationship with clinicopathological characteristics of papillary thyroid cancer (PTC) patients. Conventional and real-time quantitative RT-PCR assays were used to evaluate mRNA expression. The functions of ectopic MT1G expression were determined by cell proliferation and colony formation, cell cycle and apoptosis, as well as cell migration and invasion assays.
RESULTS:
MT1G expression was frequently silenced or down-regulated in thyroid cancer cell lines, and was also significantly decreased in primary thyroid cancer tissues compared with non-malignant thyroid tissues. Promoter methylation, along with histone modification, contributes to MT1G inactivation in thyroid tumorigenesis. Moreover, our data showed that MT1G hypermethylation was significantly positively associated with lymph node metastasis in PTC patients. Importantly, restoring MT1G expression in thyroid cancer cells dramatically suppressed cell growth and invasiveness, and induced cell cycle arrest and apoptosis through inhibiting phosphorylation of Akt and Rb.
CONCLUSIONS:
We have for the first time revealed that MT1G appears to be functional tumor suppressor involved in thyroid carcinogenesis mainly through modulating the phosphatidylinositol-3-kinase (PI3K)/Akt pathway and partially through regulating the activity of Rb/E2F pathway in this study.
BACKGROUND:
Hepatocellular carcinoma (HCC) usually has a dismal prognosis because of its limited response to current pharmacotherapy and high metastatic rate. Sulfated oligosaccharide has been confirmed as having potent antitumor activities against solid tumors. Here, we explored the preclinical effects and molecular mechanisms of isomalto oligosaccharide sulfate (IMOS), another novel sulfated oligosaccharide, in HCC cell lines and a xenograft model.
METHODS:
The effects of IMOS on HCC proliferation, apoptosis, adhesion, migration, and invasiveness in vitro were assessed by cell counting, flow cytometry, adhesion, wound healing, and transwell assays, respectively. The roles of IMOS on HCC growth and metastasis in xenograft models were evaluated by tumor volumes and fluorescent signals. Total and phosphorylated protein levels of AKT, ERK, and JNK as well as total levels of c-MET were detected by Western blotting. IMOS-regulated genes were screened by quantitative reverse-transcription PCR (qRT-PCR) array in HCCLM3-red fluorescent protein (RFP) xenograft tissues and then confirmed by qRT-PCR in HepG2 and Hep3B cells.
RESULTS:
IMOS markedly inhibited cell proliferation and induced cell apoptosis of HCCLM3, HepG2, and Bel-7402 cells and also significantly suppressed cell adhesion, migration, and invasion of HCCLM3 in vitro. At doses of 60 and 90 mg/kg/d, IMOS displayed robust inhibitory effects on HCC growth and metastasis without obvious side effects in vivo. The levels of pERK, tERK, and pJNK as well as c-MET were significantly down-regulated after treatment with 16 mg/mL IMOS. No obvious changes were found in the levels of pAkt, tAkt, and tJNK. Ten differentially expressed genes were screened from HCCLM3-RFP xenograft tissues after treatment with IMOS at a dose of 90 mg/kg/d. Similar gene expression profiles were confirmed in HepG2 and Hep3B cells after treatment with 16 mg/mL IMOS.
CONCLUSIONS:
IMOS is a potential anti-HCC candidate through inhibition of ERK and JNK signaling independent of p53 and worth studying further in patients with HCC, especially at advanced stages.
Second heart field (SHF) progenitors exhibit continued proliferation and delayed differentiation, which are modulated by FGF4/8/10, BMP and canonical Wnt/β-catenin signaling. PTEN-Akt signaling regulates the stem cell/progenitor cell homeostasis in several systems, such as hematopoietic stem cells, intestinal stem cells and neural progenitor cells. To address whether PTEN-Akt signaling is involved in regulating cardiac progenitors, we deleted Pten in SHF progenitors. Deletion of Pten caused SHF expansion and increased the size of the SHF derivatives, the right ventricle and the outflow tract. Cell proliferation of cardiac progenitors was enhanced, whereas cardiac differentiation was unaffected by Pten deletion. Removal of Akt1 rescued the phenotype and early lethality of Pten deletion mice, suggesting that Akt1 was the key downstream target that was negatively regulated by PTEN in cardiac progenitors. Furthermore, we found that inhibition of FOXO by Akt1 suppressed the expression of the gene encoding the BMP ligand (BMP7), leading to dampened BMP signaling in the hearts of Pten deletion mice. Cardiac activation of Akt also increased the Ser552 phosphorylation of β-catenin, thus enhancing its activity. Reducing β-catenin levels could partially rescue heart defects of Pten deletion mice. We conclude that Akt signaling regulates the cell proliferation of SHF progenitors through coordination of BMP signaling and β-catenin activity.
To explore the effect and mechanism of action of different ω-6/ω-3 polyunsaturated fatty acids (PUFAs) ratio on the expression of AKT and mTOR in mice bearing endometrial carcinoma. Once the human endometrial carcinoma xenograft models were successfully established, 40 BALB/C mice were randomized into five groups: group A (ω-6 PUFAs), group B (10:1 ω-6/ω-3 PUFAs), group C (control group), group D (1:1 ω-6/ω-3 PUFAs), and group E (ω-3 PUFAs). Six weeks post-treatment, mice were sacrificed and the xenograft tissues were harvested for immunohistochemical SP analysis of AKT and mTOR expression. AKT and mTOR mRNA expression was determined by reverse transcription polymerase chain reaction. Group A and group B had the highest positive expression of AKT and mTOR, with increased mRNA expression. Group D and group E had the lowest positive expression of AKT and mTOR, with decreased mRNA expression. There was a positive correlation between the expression of AKT and that of mTOR (r = 0.92). Thus, ω-6/ω-3 PUFAs in different proportions are associated with the mRNA expression of AKT and mTOR in the tissues of mouse xenograft model of human endometrial cancer.
A gemcitabine (GEM)-resistant human pancreatic cancer cell line (PANC-1RG7) was established in vitro by gradually increasing GEM concentrations and cloning cell cultures to develop a cellular model of acquired drug resistance studies. We found that PANC-1RG7 cells exhibited significantly different morphological characteristics from parental cells. PANC-1RG7 cells grew slowly (p<0.05), yet the cell cycle remained unchanged (p>0.05). PANC-1RG7, with a resistance index to GEM of 39.9, showed cross-resistance characteristics to methotrexate, gefitinib, cisplatin and 5-fluorouracil. The proliferation inhibition of GEM was significantly reduced in vivo (p<0.05). The known resistance-associated genes and proteins we detected remained unchanged, with the exception of cytidine deaminase, multidrug resistance-related protein and breast cancer resistance protein genes, which decreased; by contrast, 5'-nucleotidase, ribonucleotide reductase (RRM) 1 and RRM2 proteins increased (p<0.05). Therefore, a cell line with acquired GEM resistance was established successfully. Resistance was acquired by overexpressing RRM1 and RRM2 proteins.
Hemangioendotheliomas could be repressed by various anti-angiogenic agents in animal models. It was unclear whether the agents target hemangioendothelioma cells directly. This study elucidated the mechanism by which endostatin inhibited hemangioendothelioma progression. Expression of the endostatin receptors nucleolin and integrin α5β1 in hemangioendothelioma was assessed by immunohistochemistry. The effects of endostatin on the hemangioendothelioma-derived cells (EOMA) were evaluated by proliferation and apoptosis assays and by angiogenesis array screening. This revealed the contribution of the Chemokine (C-X-C motif) ligand 1 (CXCL1) to hemangioendothelioma progression, which was explored in vitro and in vivo. The clinical relevance of CXCL1 expression in hemangioendothelioma was also evaluated using tissue array. EOMA cells expressed nucleolin and integrin α5β1 and bound to endostatin. Endostatin did not alter proliferation or hypoxia-induced apoptosis in EOMA cells but it did impair the pro-angiogenic capacity of the cells. Endothelial cell migration was induced by CXCL1 produced by EOMA cells and endostatin downregulated CXCL1 production by inactivating its transcriptional factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). In vivo, the knockdown of CXCL1 significantly impaired EOMA cell growth in nude mice; endostatin had no effect when CXCL1 was overexpressed. A strong correlation was observed between CXCL1 levels and hemangioendothelioma occurrence in patients. CXCL1, which was responsible for hemangioendothelioma progression by stimulating angiogenesis, was impaired by endostatin via inactivation of NF-κB in an animal model. In vascular lesions in patients, CXCL1 expression was a negative prognostic factor. CXCL1-inhibting agents such as endostatin may constitute a useful approach to treat the malignant or intermediate vascular lesions.
Tenuifoliside A (TFSA) is a bioactive oligosaccharide ester component of Polygala tenuifolia Wild, a traditional Chinese medicine which was used to manage mental disorders effectively. The neuroprotective and anti-apoptotic effects of TFSA have been demonstrated in our previous studies. The present work was designed to study the molecular mechanism of TFSA on promoting the viability of rat glioma cells C6. We exposed C6 cells to TFSA (or combined with ERK, PI3K and TrkB inhibitors) to examine the effects of TFSA on the cell viability and the expression and phosphorylation of key proteins in the ERK and PI3K signaling pathway. TFSA increased levels of phospho-ERK and phospho-Akt, enhanced release of BDNF, which were blocked by ERK and PI3K inhibitors, respectively (U0126 and LY294002). Moreover, the TFSA caused the enhanced phosphorylation of cyclic AMP response element binding protein (CREB) at Ser133 site, the effect was revoked by U0126, LY294002 and K252a. Furthermore, when C6 cells were pretreated with K252a, a TrkB antagonist, known to significantly inhibit the activity of brain-derived neurotrophic factor (BDNF), blocked the levels of phospho-ERK, phospho-Akt and phosphor-CREB. Taking these results together, we suggested the neuroprotection of TFSA might be mediated through BDNF/TrkB-ERK/PI3K-CREB signaling pathway in C6 glioma cells.
This study was carried out to investigate the impact of tripterygium glycosides (TGs) on ovarian function of female rats in vitro and in vivo. In vitro studies showed that TG induced cells decrease at G1 phase and inhibited cell proliferation in rat granulosa cells. In vivo, female rats were intragastrically administered with TG at the dose of 60 mg/kg/day for consecutive 50 days. TG caused a prolonged estrous cycle, and a significant reduction in ovarian index, serum E2 level, and numbers of secondary and antral follicles (p < 0.05) in these rats. A significant reduction of viable embryos was demonstrated in TG-treated female rats after mating (p < 0.01). Further, we observed observed the reduced expression level of TGF-β1 after TG treatment in vitro and in vivo. Moreover, the expression of Smad2 and AKT was also decreased after TG treatment. These results suggest that TG can impair ovarian function through Smads-mediated TGF-β1 signal pathway.
Human ribonuclease inhibitor (RI), a cytoplasmic protein, is constructed almost entirely of leucine rich repeats. RI could suppress activities of ribonuclease and angiogenin (ANG) through closely combining with them. ANG is a potent inducer of blood vessel growth and has been implicated in the establishment, growth, and metastasis of tumors. ILK/PI3K/AKT signaling pathway also plays important roles in cell growth, cell-cycle progression, tumor angiogenesis, and cell apoptosis. Our previous experiments demonstrated that RI might effectively inhibit some tumor growth and metastasis. Our recent study showed that ILK siRNA inhibited the growth and induced apoptosis in bladder cancer cells as well as increased RI expression, which suggest a correlation between RI and ILK. However, the exact molecular mechanism of RI in anti-tumor and in the cross-talk of ANG and ILK signaling pathway remains largely unknown. Here we investigated the effects of up-regulating RI on the growth and apoptosis in murine melanoma cells through angiogenin and ILK/PI3K/AKT signaling pathway. We demonstrated that up-regulating RI obviously decreased ANG expression and activity. We also discovered that RI overexpression could remarkably inhibit cell proliferation, regulate cell cycle and induce apoptosis. Furthermore, up-regulation of RI inhibited phosphorylation of ILK downstream signaling targets protein kinase B/Akt, glycogen synthase kinase 3-beta (GSK-3β), and reduced β-catenin expression in vivo and vitro. More importantly, RI significant inhibited the tumor growth and angiogenesis of tumor bearing C57BL/6 mice. In conclusion, our findings, for the first time, suggest that angiogenin and ILK signaling pathway plays a pivotal role in mediating the inhibitory effects of RI on melanoma cells growth. This study identifies that RI may be a useful molecular target for melanoma therapy.
The RNA-binding protein Musashi2 (Msi2) has been identified as a master regulator within a variety of stem cell populations via the regulation of translational gene expression. A recent study has suggested that Msi2 is strongly expressed in leukemic cells of acute myeloid leukemia patients, and elevated Msi2 is associated with poor prognosis. However, the potential role of Msi2 in leukemogenesis is still not well understood. Here, we investigated the effect of Msi2 knockdown on the biological properties of leukemic cells. High expression of Msi2 was found in K562 and KG-1a leukemic cell lines, and low expression was observed in the U937 cell line. We transduced K562 cells with two independent adenoviral shRNA vectors targeting Msi2 and confirmed knockdown of Msi2 at the mRNA and protein levels. Msi2 silencing inhibited cell growth and caused cell cycle arrest by increasing the expression of p21 and decreasing the expression of cyclin D1 and cdk2. In addition, knockdown of Msi2 promoted cellular apoptosis via the upregulation of Bax and downregulation of Bcl-2 expression. Furthermore, Msi2 knockdown resulted in the inactivation of the ERK/MAPK and p38/MAPK pathways, but no remarkable change in p-AKT was observed. These data provide evidence that Msi2 plays an important role in leukemogenesis involving the MAPK signaling pathway, which indicates that Msi2 may be a novel target for leukemia treatment.
Excessive βAR stimulation is an independent factor in inducing pathological cardiac hypertrophy. Here, we report miR-145 regulates both expression and localization of GATA6, thereby protecting the heart against cardiomyocyte hypertrophy induced by isoproterenol (ISO). The protective activity of miR-145 was associated with down-regulation of ANF, BNP and β-MHC expression, a decreased rate of protein synthesis, inhibited cardiomyocyte growth and the modulation of several signaling pathways including ERK1/2, JNK and Akt-GSK3β. The anti-hypertrophic effect was abrogated by exogenous over-expression of transcription factor GATA6 which was further identified as a direct target of miR-145. In addition, GSK3β antagonists, LiCl and TDZD8, restored the nuclear accumulation of GATA6, which was attenuated by miR-145 Finally, we observed a dynamic pattern of miR-145 expression in ISO-treated NRCMs and in the hearts of TAC mice. Together, our results identify miR-145 as an important regulator in cardiac hypertrophy.
Angiogenesis has become an attractive target for the treatment of certain diseases such as cancer and rheumatoid arthritis. Our previous studies demonstrated that the saponin fraction from Gleditsia sinensis fruits had anti-angiogenic potential, and Gleditsiosides B (GB) was probably the main active constituent. In the present study, we assessed the effect of GB on endothelial cell migration, a crucial event in angiogenesis, and explored the underlying mechanisms. The migration of endothelial cells was assessed by transwell. The expressions of MMP-2/-9 and TIMP-1/-2 were analyzed by Western blotting, and the activities of MMP-2/-9 were detected by gelatin zymography assay. Moreover, migration-related proteins and signaling pathways, including FAK, MAPKs and PI3K/AKT, were analyzed by Western blotting. It was shown that GB, at a concentration of 10 μM without significant cytotoxicity, could effectively abrogate the migration of human umbilical vein endothelial cells (HUVECs) induced by bFGF. GB also inhibited the expression and activity of MMP-2, elevated the expression of TIMP-1, and restrained the phosphorylations of FAK, ERK, PI3K and AKT in a concentration-dependent manner. The findings suggest that GB was able to abrogate the migration of endothelial cells through down-regulating the activation of MMP-2 and FAK via preventing ERK and PI3K/AKT signaling pathways.
Genistein is an isoflavone phytoestrogen with biological activities in management of metabolic disorders. This study aims to evaluate the regulation of insulin action by genistein in the endothelium. Genistein inhibited insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and attenuated downstream Akt and endothelial nitric oxide synthase (eNOS) phosphorylation, leading to a decreased nitric oxide (NO) production in endothelial cells. These results demonstrated its negative regulation of insulin action in the endothelium. Palmitate (PA) stimulation evoked inflammation and induced insulin resistance in endothelial cells. Genistein inhibited IKKβ and nuclear factor-кB (NF-кB) activation with down-regulation of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production and expression. Genistein inhibited inflammation-stimulated IRS-1 serine phosphorylation and restored insulin-mediated tyrosine phosphorylation. Genistein restored insulin-mediated Akt and eNOS phosphorylation, and then led to an increased NO production from endothelial cells, well demonstrating its positive regulation of insulin action under insulin-resistant conditions. Meanwhile, genistein effectively inhibited inflammation-enhanced mitogenic actions of insulin by down-regulation of endothelin-1 and vascular cell adhesion protein-1 overexpression. PA stimulation impaired insulin-mediated vessel dilation in rat aorta, while genistein effectively restored the lost vasodilation in a concentration-dependent manner (0.1, 1 and 10 μM). These results suggested that genistein inhibited inflammation and ameliorated endothelial dysfunction implicated in insulin resistance. Better understanding of genistein action in regulation of insulin sensitivity in the endothelium could be beneficial for its possible applications in controlling endothelial dysfunction associated with diabetes and insulin resistance.
Cardiac hypertrophy is a response of the myocardium to increased workload and is characterised by an increase of myocardial mass and an accumulation of extracellular matrix (ECM). As an ECM protein, an integrin ligand, and an angiogenesis inhibitor, all of which are key players in cardiac hypertrophy, mindin is an attractive target for therapeutic intervention to treat or prevent cardiac hypertrophy and heart failure. In this study, we investigated the role of mindin in cardiac hypertrophy using littermate Mindin knockout (Mindin ( -/- )) and wild-type (WT) mice. Cardiac hypertrophy was induced by aortic banding (AB) or angiotensin II (Ang II) infusion in Mindin ( -/- ) and WT mice. The extent of cardiac hypertrophy was quantitated by echocardiography and by pathological and molecular analyses of heart samples. Mindin ( -/- ) mice were more susceptible to cardiac hypertrophy and fibrosis in response to AB or Ang II stimulation than wild type. Cardiac function was also markedly exacerbated during both systole and diastole in Mindin ( -/- ) mice in response to hypertrophic stimuli. Western blot assays further showed that the activation of AKT/glycogen synthase kinase 3β (GSK3β) signalling in response to hypertrophic stimuli was significantly increased in Mindin ( -/- ) mice. Moreover, blocking AKT/GSK3β signalling with a pharmacological AKT inhibitor reversed cardiac abnormalities in Mindin ( -/- ) mice. Our data show that mindin, as an intrinsic cardioprotective factor, prevents maladaptive remodelling and the transition to heart failure by blocking AKT/GSK3β signalling.
Platelets play a key role in hemostasis and in the initiation and propagation of thrombus formation. New peptide pGlu-Asn-Trp (pENW), initially extracted from snake venom, shows a concentration-dependent antithrombotic activity, significantly attenuated thrombus formation in the arterial and venous vessel systems. This study was designed to further reveal the mechanisms underlying its antithrombotic effect by focusing on its in vitro antiplatelet effect after precluding its influence on coagulation factors. It showed that pENW concentration-dependently inhibited ADP-, collagen- and platelet activating factor (PAF)-induced platelet aggregation, inversely depending upon the intensity of stimulation induced by agonists. Furthermore, data obtained by ELISA and flow cytometry presented that pENW also suppressed ADP-mediated serotonin secretion and P-selectin expression in a concentration-dependent manner. As shown by Western blot assay, ADP-induced platelet Akt phosphorylation was attenuated by the priming incubation with pENW, demonstrating the influence on platelet intracellular signaling. It provided the explaining information for its activity of inhibiting platelet activation in vitro. These results suggested pENW attenuated thrombus formation in part by inhibiting platelet activation instead of coagulation factors, presented evidence of pENW interfering intracellular signaling system in the process of platelet activation and indicated the possibility that pENW could potentially be developed as a novel therapeutic agent in the prevention and treatment of thrombotic disorders.
Polybrominated diphenyl ethers (PBDEs) had been used extensively in electrical and electronic products as brominated flame retardants. PBDEs are widely distributed in environment media and wildlife since they are lipophilic and persistent, resulting in bioaccumulation and bioamplification through food chains. Accumulation of PBDEs in the environment and human tissues will consequently cause potential negative effects on the ecological environment and human health. To date, some in vitro and in vivo studies have reported that PBDEs possess neurotoxicity, hepatotoxicity, immunotoxicity, reproduction toxicity, endocrine disrupting activity and carcinogenicity. BDE-47 is one of the most predominant PBDE congeners detected in human tissues. The objective of this study is to investigate whether low concentration of BDE-47 could cause hormesis effect in the human hepatoma HepG(2) cells, and to explore the possible molecular mechanism. The results showed that low concentration of BDE-47 (10(-10), 10(-9) and 10(-8) M) could promote cell proliferation and cause no obvious change in DNA damage or cell apoptosis, while the high concentration significantly inhibit cell proliferation. Meanwhile, the reactive oxygen species (ROS) in low concentration BDE-47 (10(-10), 10(-9) and 10(-8) M) treated groups significantly elevated compared with the control group. After low concentration BDE-47 treatment, the expression of proliferating cell nuclear antigen (PCNA), Cyclin D1, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and phosphorylated protein kinase B (p-Akt) in the HepG(2) cells was markedly up-regulated. However, in DNA-PKcs inhibited cells, the promotion effect on cell proliferation was significantly suppressed. Cell cycle analysis showed a significant decrease in G1 phase after exposure to low concentration of BDE-47. Moreover, pre-exposure to low concentration BDE-47 seemed alleviate the negative effects of high concentration (50 μM) exposure to cause DNA damage and apoptosis. These results suggested that BDE-47 has a hormesis effect in HepG(2) cells and DNA-PKcs/Akt pathway may be involved in regulation of cell proliferation and apoptosis.
We examined the effects of anti-six-transmembrane epithelial antigen of the prostate-4 (STEAP4) antibodies on glucose transport in mature adipocytes and determined the mechanism of insulin resistance in obesity. Western blotting was performed to determine STEAP4 expression, to assess translocation of insulin-sensitive glucose transporter 4 (GLUT4), and to measure phosphorylation and total protein content of insulin-signaling proteins. Confocal laser microscopy and flow cytometry were used to detect intracellular reactive oxygen species (ROS) and fluctuations in mitochondrial membrane potential (ΔΨ). ATP production was measured by using a luciferase-based luminescence assay kit. After the application of anti-STEAP4 antibodies at 0.002 mg/mL, adipocytes exhibited reduced insulin-stimulated glucose transport by attenuating the phosphorylation of IRS-1, PI3K (p85), and Akt. The antibodies also potentially increase the level of ROS and decrease cellular ATP production and ΔΨ. In conclusion, (i) STEAP4 regulates the function of IRS-1, PI3K, and Akt and decreases insulin-induced GLUT4 translocation and glucose uptake; (ii) ROS-related mitochondrial dysfunction may be related to a reduced IRS-1 correlation with the PI3K signaling pathway, leading to insulin resistance. These observations highlight the potential role of STEAP4 in glucose homeostasis and possibly in the pathophysiology of type 2 diabetes related to obesity and may provide new insights into the mechanisms of insulin resistance in obesity.
Integrin-linked kinase (ILK), an intracellular serine/threonine kinase, is implicated in cell growth and survival, cell-cycle progression, tumor angiogenesis, and cell apoptosis. Recent studies showed that the expression and activity of ILK increased significantly in many types of solid tumors. However, the exact molecular mechanism of ILK underlie tumor has not been fully ascertained. The purpose of our study was to determine whether knockdown of ILK would inhibit cell growth and induce apoptosis in bladder cancer cells using a plasmid vector based small interfering RNA (siRNA). The experiments showed that knockdown of ILK could remarkably inhibit cell proliferation and growth, regulate cell cycle and induce apoptosis of bladder cancer BIU-87 and EJ cells. We demonstrated that knockdown of ILK inhibited phosphorylation of downstream signaling targets protein kinase B/Akt, glycogen synthase kinase 3-beta (GSK-3β), and reduced expression of β-catenin in BIU-87 as well as EJ cells by Western blot and Immunofluorescence analysis. In addition, down-regulation of ILK also could increase expression of Ribonuclease inhibitor (RI), an important acidic cytoplasmic protein with many functions. BALB/C nude mice injected with the BIU-87 cells transfected ILK siRNA showed a significant inhibition of the tumor growth with lighter tumor weight, lower microvessels density and higher apoptosis rate than those in the other two control groups. In conclusion, these results suggest that ILK might be involved in the development of bladder cancer, and could be served as a novel potential therapy target for human bladder cancer. Our study may be of biological and clinical importance.
CaN induces the apoptosis in neurons, but the influence of CIPC and the intervention of pretreament with Ca(2+)-regulated factors, such as nimodipine, MK801 and cyclosporine A, on CaN expression is not clear. We also do not know whether cbl-b takes part in the induction of ischemia or induces an expression change of cbl-b in CIPC. So we will discuss the effect of CIPC, pretreatment with nimodipine, MK801 and cyclosporine A on the expression of the CaN, cbl-b and p-AKT in the hippocampus neurons. In our study, we established rat models including sham, ischemia, CIPC, nimodipine, MK801 and cyclosporine A. The neurological deficit scores were processed. The right hippocampus was removed and stained with TTC, and the volume of cerebral infarction was calculated. The apoptotic neurons were detected by TUNEL staining. The expressions of CaN, cbl-b and p-AKT at the protein level were examined by Western blotting, and the transcription of cbl-b by RT-PCR, respectively. The results showed that the neurological deficit scores, the volume of the cerebral infarction, the numbers of the apoptotic neurons, the protein expression of CaN, cbl-b and the transcription of cbl-b were the highest in the ischemia and MK801 groups, there were no difference between the two groups(P>0.05); these factors in CIPC group were all lower than those in the ischemia group(P<0.05); and much lower in the nimodipine and cyclosporine A group than those in the CIPC group (among them, the volume of the cerebral infarction in the nimodipine and cyclosporine A groups P<0.01, the expression of CaN in nimodipine group P<0.01, others were P<0.05), but no significant difference existed between the nimodipine and cyclosporine A groups(P>0.05). The expression of p-AKT was the lowest in the ischemia and MK801 groups, and there was no difference between the two groups (P>0.05), This factor was higher in CIPC group than that in the ischemia group (P<0.05); it was the highest in the nimodipine and cyclosporine A groups among these groups (the nimodipine group P<0.01, the cyclosporine A group P<0.05), no significant difference existed between the nimodipine and cyclosporine A groups (P>0.05. Continuous ischemia increases the expression of CaN, and the transcription and the protein expression of cbl-b. Cbl-b reduces the phosphorylated expression of AKT, ultimately activating apoptosis. CIPC inhibits above process and reduces the expression of CaN and cbl-b, and increases the expression of p-AKT, thereby inhibiting apoptosis in neurons. Nimodipine and cyclosporine A can reduce the expression of CaN and cbl-b, and increase the expression of p-AKT, via a moderate increase in the concentration of intracellular calcium and inhibition of the activity of CaN; MK801 counteracts the effect of CIPC.
Oleanolic acid (OA), a widely used plant-derived triterpenoid, has been shown to possess potent antiatherosclerotic effects, which may be associated with the induction of heme oxygenase-1 (HO-1). However, the underlying mechanisms involved in the effect of OA on HO-1 expression are unclear. In the current study, primary rat vascular smooth muscle cells (VSMCs) were exposed to OA and we found that it enhanced HO-1 expression in a concentration- and time-dependent manner, accompanied by increased HO-1 activity. VSMCs treated with OA exhibited activation of Akt, p38 and extracellular-signal-regulated kinase (ERK). Wortmannin (a PI3K inhibitor) and PD98059 (an ERK inhibitor) attenuated OA-induced HO-1 expression, whereas SB203580 (a p38 inhibitor) had no effect. The transcription factor NF-E2-related factor 2 (Nrf2) is a key regulator of HO-1 expression. OA treatment increased Nrf2 nuclear translocation, which was also inhibited by wortmannin and PD98059. Furthermore, transfection of VSMCs with the Nrf2 siRNA-expressing lentiviral vector decreased HO-1 expression induced by OA. Finally, pretreatment of VSMCs with OA remarkably reduced hydrogen peroxide-induced cell apoptotic death, and this effect was greatly attenuated in the presence of ZnPP (a HO-1 inhibitor), wortmannin or PD98059. Taken together, these results suggest that activation of Akt and ERK is required for OA-induced activation of Nrf2 followed by upregulation of HO-1 expression in VSMCs, which may confer an adaptive survival response in atherosclerosis.
Oxidative stress is a major cause in neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and cerebral ischemia. Ginsenoside Rg1, a natural product extracted from Panax ginseng C.A. Meyer, has been reported to exert notable neuroprotective activities, which partly ascribed to its antioxidative activity. However, its molecular mechanism against oxidative stress induced by exogenous hydrogen peroxide (H(2)O(2)) remained unclear. In this study, we investigated its effect on H(2)O(2)-induced cell death and explored possible signaling pathway in PC12 cells. We proved that pretreatment with Rg1 at concentrations of 0.1-10 μM remarkably reduced the cytotoxicity induced by 400 μM of H(2)O(2) in PC12 cells by MTT and Hoechst and PI double staining assay. Of note, we demonstrated the activation of NF-κB signaling pathway induced by H(2)O(2) thoroughly in PC12 cells, and Rg1 suppressed phosphorylation and nuclear translocation of NF-κB/p65, phosphorylation and degradation of inhibitor protein of κB (IκB) as well as the phosphorylation of IκB-kinase complex (IKK) by western blotting or indirect immunofluorescence assay. Besides, Rg1 also inhibited the activation of Akt and the extracellular signal-regulated kinase 1/2 (ERK1/2). Furthermore, the protection of Rg1 on H(2)O(2)-injured PC12 cells was attenuated by pretreatment with two NF-κB pathway inhibitors (JSH-23 or BOT-64). In conclusion, our results suggest that Rg1 could rescue the cell injury by H(2)O(2) via down-regulation NF-κB signaling pathway as well as Akt and ERK1/2 activation, which put new evidence on the neuroprotective mechanism of Rg1 against the oxidative stress and the regulatory role of H(2)O(2) in NF-κB pathway in PC12 cells.
SCOPE:
Quercetin is a dietary flavonoid whose role in the regulation of the activity of insulin remains controversial. Our study aimed to investigate how quercetin and its major metabolite quercetin-3-glucuronide (Q-3-G) regulate insulin-mediated glucose disposal in skeletal muscle under normal and inflammatory conditions.
METHODS AND RESULTS:
Under normal conditions, quercetin impaired glucose and insulin tolerance and attenuated insulin-mediated phosphorylation of Akt substrate of 160 kDa (AS160) and TBC1D1 without affecting Akt activity in male Institute of Cancer Research (ICR) mice. However, under inflammatory conditions, quercetin exhibited an opposite effect in these animals. In C2C12 cells, quercetin also decreased insulin-stimulated AS160 and TBC1D1 phosphorylation and glucose uptake in the absence of an inflammatory insult, whereas it improved the action of insulin under inflammatory conditions. Knockdown of adenosine 5'-monophosphate-activated protein kinase α (AMPKα) blocked the differential effects of quercetin under both conditions. Unlike quercetin, Q-3-G had no influence on insulin-induced phosphorylation of AS160 and TBC1D1 and glucose uptake in C2C12 myotubes under normal conditions. Q-3-G displayed a similar regulation with quercetin in glucose disposal under inflammatory conditions.
CONCLUSION:
Quercetin suppressed insulin-mediated glucose disposal in skeletal muscle tissue/cells under normal conditions while it ameliorated impaired glucose uptake under inflammatory conditions with activation of AMPK. In contrast, Q-3-G ameliorated insulin resistance in skeletal cells under inflammatory conditions without affecting glucose disposal under normal conditions.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
BACKGROUND:
Considering the key role of TF in coagulation of sepsis or acute lung injury (ALI), we investigated whether berberine (BBR) could inhibit TF expression and procoagulant activity and explored its possible mechanism.
METHODS:
The effects of berberine on the expression, procoagulant activity of TF and related signal pathways induced by lipopolysaccharide (LPS) were observed in THP-1 cells.
RESULTS:
Our results showed that berberine could inhibit LPS-induced TF activity and expression, and down-regulate NF-κB, Akt and MAPK/JNK/p38/ERK pathways.
CONCLUSION:
Berberine inhibits TF expression and related pathway, which provides some new insights on its mechanism for sepsis treatment.
Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
ETHNOPHARMACOLOGICAL RELEVANCE:
Cyclocarya paliurus Batal., a Chinese native plant, is the sole species in its genus and its leaves have been widely used as a remedy for diabetes in traditional folk medicine. The study was undertaken to evaluate the effects of Cyclocarya paliurus leaves extracts (CPE) on adipokine expression and insulin sensitivity in mice.
MATERIALS AND METHODS:
Mice were stimulated with conditioned medium (prepared from activated macrophages, Mac-CM) to induce adipose dysfunction and insulin resistance. Then mice were treated with CPE (100, 200 and 500 mg/kg, ig.) or metformin (200 mg/kg, ig.), followed by glucose and insulin intolerance, adipokine expression, phosphorylation of insulin receptor substrate (IRS-1) and glucose consumption measurement.
RESULTS:
CPE, as well as metformin effectively promoted glucose disposal in oral glucose tolerance test in normal mice. Mac-CM challenge induced glucose and insulin intolerance, but CPE reversed these alternations with increased glycogen content in muscle and liver, well demonstrating its beneficial effects on glucose homeostasis. RT-qPCR analysis showed that CPE inhibited TNF-a, IL-6, MCP-1 and resistin overexpression and effectively enhanced adiponectin expression in adipose tissue when mice were exposed to Mac-CM stimulation. Inflammation impaired insulin signaling in muscle, whereas CPE inhibited inflammation-induced serine phosphorylation of IRS-1 and effectively restored the phosphorylation of both IRS-1 at tyrosine residues and downstream Akt phosphorylation in response to insulin. Moreover, independently of insulin, CPE promoted glucose consumption in adipocytes under normal and inflammatory conditions.
CONCLUSION:
Above-mentioned results demonstrated that CPE beneficially regulated adipokines expression and ameliorated insulin resistance through inhibition of inflammation in mice.
Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
SCOPE:
Quercetin is the most abundant dietary flavonol with beneficial regulation of glucose homeostasis, but its regulation of insulin action remains uncertain. This study aims to investigate the effects of quercetin on insulin-mediated glucose transporter 4 (GLUT4) translocation under basal and inflammatory conditions as well as the molecular mechanisms in adipocytes.
METHODS AND RESULTS:
The effects of quercetin on insulin-mediated GLUT4 translocation in 3T3-L1 cells under basal and insulin resistant conditions were investigated. Meanwhile, we investigated the effect of quercetin on AMP-activated protein kinase (AMPK) activation implicated in regulation of insulin action. Quercetin inhibited insulin-mediated GLUT4 translocation by inhibiting AS160 phosphorylation. Differently, when inflammatory challenge impaired insulin action in 3T3-L1 cells, quercetin inhibited IκB kinase β (IKKβ) phosphorylation and facilitated insulin signaling, leading to the restoration of insulin-mediated AS160 phosphorylation and downstream GLUT4 translocation. AMPK inhibitor Compound C or knockdown of AMPKα by small interfering RNA (siRNA) abolished both actions of quercetin. Results from mice adipose tissue (AT) further confirmed its positive regulation of AMPK phosphorylation and opposite effects on AS160 phosphorylation in vivo.
CONCLUSION:
Quercetin demonstrated divergent effects on insulin-mediated GLUT4 translocation in adipocytes under basal and insulin resistant conditions, which were related to its regulation of AMPK activity.
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
BACKGROUND:
Pancreatic cancer treatment is limited and effective drugs are needed. We investigated cucurmosin (CUS)-induced apoptosis in cystic fibrosis pancreatic adenocarcinoma cells (CFPAC-1) and a possible mechanism of action to evaluate the clinical application potential of this new Type I ribosome-inactivating protein.
METHODS:
We analyzed the growth inhibition and apoptosis of CFPAC-1 cells via methylthiazol tetrazolium assay and fluorescence-activated cell sorting. Western blot was used to analyze the protein levels of caspase 3, bcl-2, caspase 9, platelet-derived growth factor receptor (PDGFR)-β, PI3K, Akt, p-Akt, the mammalian target of rapamycin (mTOR), p-mTOR, P70S6K-α, p-P70S6K-α, 4E-BP1, p-4E-BP1 and p-Bad after CUS intervention. The mRNA expression of PDGFR-β was analyzed using reverse transcription polymerase chain reaction.
RESULTS:
CUS inhibited the proliferation of pancreatic cancer cells. The induction of apoptosis depended on the CUS dose and incubation time. The drug inhibited all of the examined proteins in the PI3K/Akt/mTOR signalling pathway and induced the active fragments of caspase 3 and caspase 9. CUS downregulated PDGFR-β expression but no significant change was observed at the mRNA level.
CONCLUSION:
CUS strongly inhibits the growth of CFPAC-1 by inducing cell apoptosis. CUS downregulated the expression of PDGFR-β at the protein level and induced the apoptosis of CFPAC-1 through the PI3K/Akt/mTOR signalling pathway.
BACKGROUND:
Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) presents great challenges in the treatment of non-small cell lung cancer (NSCLC) patients, while the mechanisms are still not well understood. The β-catenin signaling pathway has been found to be associated with chemoresistance and can activate the EGFR and its downstream pathways. This study aimed to investigate the role of β-catenin in acquired resistance to EGFR-TKIs in NSCLC cell lines.
METHODS:
The expression and transcriptional activity of β-catenin were measured in both the NSCLC cell line PC9 and its sub-line PC9/AB(2) which has acquired resistance to gefitinib. Knockdown and overexpression of β-catenin in the PC9/AB(2) and PC9 cells were performed. The cell survival rate and the activation of the EGFR and its downstream pathways were detected in the two cell lines after transfection.
RESULTS:
Nuclear translocation of β-catenin was increased in the PC9/AB(2) cells and the baseline expression of members of the β-catenin signaling pathway was also higher in the PC9/AB(2) cells. Knocking down the expression of β-catenin increased the sensitivity of the PC9/AB(2) cells to gefitinib by blocking the activation of the EGFR downstream pathways, while β-catenin overexpression improved PC9 cells resistance to gefitinib by enhancing the activation of the EGFR and its downstream signaling.
CONCLUSION:
β-catenin plays an important role in acquired resistance to EGFR-TKIs in NSCLC cell lines and may be a potential therapeutic target for NSCLC patients who have failed to respond to targeted therapy.
Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.
SCOPE:
Quercetin represents antioxidative/antiinflammatory flavonoids widely distributed in the human diet. Quercetin is efficiently metabolized during absorption to quercetin-3-O-glucuronide. This study aims to parallelly investigate whether quercetin and quercetin-3-O-glucuronide exert protection against palmitate (PA)-induced inflammation and insulin resistance in the endothelium.
METHODS AND RESULTS:
Human umbilical vein endothelial cells were pretreated with quercetin and quercetin-3-O-glucuronide for 30 min, and then incubated with 100 μM PA for 30 min or 12 h with or without insulin. PA stimulation led to reactive oxygen species (ROS) production with collapse of mitochondrial membrane potential (Δψm). Quercetin and quercetin-3-O-glucuronide inhibited ROS overproduction and effectively restored Δψm, demonstrating their chemorpotection of mitochondrial function through antioxidative actions. Also, quercetin and quercetin-3-O-glucuronide inhibited ROS-associated inflammation by inhibition of interleukin-6 and tumor necrosis factor-α production with suppression of IKKβ/NF-κB phosphorylation. Inflammation impaired insulin PI3K signaling and reduced insulin-mediated nitric oxide (NO) production. Quercetin and quercetin-3-O-glucuronide facilitated PI3K signaling by positive regulation of serine/tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and restoration of downstream Akt/eNOS activation, leading to an increased insulin-mediated NO level.
CONCLUSION:
The above-mentioned evidence indicates that quercetin and quercetin-3-O-glucuronide are equally effective in inhibiting ROS-associated inflammation and ameliorating insulin resistant endothelial dysfunction by beneficial regulation of IRS-1 function.
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
SCOPE:
Flavonoids have well-known antioxidant, anti-inflammatory, and anti-cancer activities. Isoflavone genistein is considered a potent antioxidant agent against oxidative stress. Although several mechanisms have been proposed, a clear antioxidant mechanism of genistein is still remained to be answered.
METHODS AND RESULTS:
In this study, we focused on the concerted effects on expression of Nrf2 and phase II enzyme pathway components. Transient transfection assays, RT-PCR and immunoblot analysis were performed to study its molecular mechanisms of action. In Caco-2 cells, treatment with genistein markedly attenuated H(2)O(2) -induced peroxide formation; this amelioration was reversed by buthionine sulfoximine(GCLC inhibitor) and zinc protoporphyrin(HO-1 inhibitor). Genistein increased HO-1 and GCLC mRNA and protein expression. Genistein treatment activated the ERK1/2 and PKC signaling pathway; therefore increased Nrf2 mRNA and protein expression. The roles of the ERK1/2 and PKC signaling pathway were determined using PD98059 (ERK1/2 inhibitor) and GF109203X (PKC inhibitor) and RNA interference directed against Nrf2. Both inhibitors and siNrf2 abolished genistein-induced HO-1 and GCLC protein expression. These results suggest the involvement of ERK1/2, PKC, and Nrf2 in inducing HO-1 and GCLC by genistein.
CONCLUSION:
Our studies show that genistein up-regulated HO-1 and GCLC expression through the EKR1/2 and PKC /Nrf2 pathways during oxidative stress.
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
OBJECTIVE:
To investigate the effect of cucurmosin (CUS) on proliferation inhibition in the human pancreatic cancer cell line SW-1990 in vitro and in vivo.
METHODS:
1. MTT assay was used to analyse the proliferation inhibition of CUS in SW-1990 cells compared with gemcitabine (GEM) in vitro. 2. We established an NOD-SCID mice orthotopic transplantation model and estimated the proliferation inhibition effect of CUS in SW-1990 cells in vivo. 3. Western blot was used to determine the protein expressions of Caspase 3, Bcl-2, Caspase 9, PI3K, Akt, mTOR, P70S6k, and 4E-BP1 after CUS intervention.
RESULTS:
1. CUS inhibited the proliferation of pancreatic cancer cells and induced apoptosis in CUS dose- and time-dependent manners. 2. NOD-SCID mice models were established successfully, and the tumour proliferation inhibition rates of these models increased compared with the control group. 3. CUS inhibited all of the examined proteins in the PI3K/Akt/mTOR signalling pathway and induced active fragments of Caspase 3 and Caspase 9.
CONCLUSION:
1. CUS can inhibit the growth of SW-1990 cells in vitro and in vivo. 2. CUS can induce apoptosis in SW-1990 cells to inhibit cell growth.
AIM:
Our previous investigation demonstrated that plasminogen activator inhibitor-1 (PAI-1) siRNA ameliorated bleomycin (BLM)-induced rat lung fibrosis. The present study was undertaken to explore the effect and the mechanism of PAI-1 siRNA and plasmid pcDNA on the proliferation and apoptosis of cultured fibroblasts from BLM-induced fibrotic lung tissues.
MATERIALS AND METHODS:
The fibroblasts from BLM-induced fibrotic lung tissue were isolated and transfected using PAI-1 siRNA and plasmid pcDNA-PAI-1. The techniques of real time RT-PCR and/or western blot were used to determine the expression of PAI-1, α-smooth muscle actin (α-SMA) (real time RT-PCR only), collagen type-1 and type-3 (real time RT-PCR only), and the levels of caspase-3, ERK and AKT signal molecules. The proliferation of fibroblasts was measured by cell cycle with flow cytometry. The intracellular concentration of Ca(2+) was examined by confocal laser microscopy.
RESULTS:
PAI-1 siRNA downregulated the PAI-1 mRNA expression by 70%±7% at 24h and protein expression by 73.5%±10% and 42%±3% at 48h and 72h compared to Non-specific siRNA group. Flow cytometry showed that the fibroblasts at the G(2)M+S phase were significantly reduced by 20.56±1.03% after transfecting PAI-1 siRNA and were significantly increased by 43.8±1.21% after transfecting plasmid pcDNA-PAI-1. The mRNA expressions of α-SMA, collagen type-1and type-3 were downregulated after transfecting the PAI-1 siRNA, while upregulated after the transfection of pcDNA-PAI-1. PAI-1 siRNA increased the level of caspase-3, inhibited the expressions of p-ERK and p-AKT protein molecules, while the pcDNA-PAI-1 transfection showed a reversal effect on these expressions. Intracellular Ca(2+) concentration was decreased after transfecting PAI-1 siRNA, whereas increased after transfecting pcDNA-PAI-1.
CONCLUSION:
PAI-1 promotes the proliferation, transforming into myofibroblasts, collagen synthesis, and inhibits apoptosis of pulmonary fibroblasts by activating Ca(2+), ERK and AKT signaling pathway. Decreasing PAI-1 expression is an available strategy in inhibiting the progression of pulmonary fibrosis.
Copyright © 2012 Elsevier Ltd. All rights reserved.
OBJECTIVE:
We investigated whether diosgenin, a widely used steroidal sapogenin, exerted protection against palmitate (PA)-induced inflammation and insulin resistance in the endothelium.
METHODS:
Human umbilical vein endothelial cells (HUVECs) were pretreated with diosgenin for 30 min, and then incubated with 100 μmol/L PA for 30 min or 24 h with or without insulin. IKKβ, p65 phosphorylation, serine phosphorylation of insulin receptor substrate-1 (IRS-1) at S307, tyrosine phosphorylation of IRS-1, Akt and eNOS activation were determined by Western blot analysis. Levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), endothelin-1 (ET-1) and plasminogen activator inhibitor-1 (PAI-1) were measured with ELISA Kits. Intracellular nitric oxide (NO) was viewed with fluorescence microscopy. Effects of diosgenin on insulin-mediated vasodilation was investigated in the isolated rat aortic rings.
RESULTS:
Diosgenin significantly reduced PA-enhanced IKKβ and NF-κB phosphorylation with inhibition of TNF-α and IL-6 production in endothelial cells at the concentrations of 0.1, 1 and 10 μmol/L, well demonstrating its anti-inflammatory activity in an IKKβ/NF-κB-dependent fashion. Meanwhile, diosgenin attenuated PA-induced serine phosphorylation (S307) of IRS-1 and restored IRS-1 tyrosine phosphorylation in response to insulin. The beneficial modulation of serine/tyrosine phosphorylation of IRS-1 by diosgenin contributed to the improvement of insulin signaling along PI3K/Akt/eNOS pathways and thereby increased insulin-mediated NO production. Salicylate (5 mmol/L), an inhibitor of IKKβ, showed similar activities as diosgenin. Diosgenin also remarkably inhibited ET-1 and PAI-1 production in the endothelial cells, and markedly restored the loss of insulin-mediated vasodilation in the presence of PA.
CONCLUSION:
The above-mentioned evidence suggests that diosgenin ameliorated endothelial dysfunction involved in insulin resistance through an IKKβ/IRS-1-dependent manner, shows potential application in the treatment for the cardiovascular diseases including atherosclerosis.
Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.
PURPOSE:
This study was to investigate the clinicopathologic significance and potential role of HOXB7 in the development and progression of colorectal cancer (CRC).
EXPERIMENTAL DESIGN:
The relationship between HOXB7 expression and clinical characteristics of CRC was analyzed in 224 paraffin-embedded archived CRC specimens by immunohistochemistry (IHC). The effects of HOXB7 on cell growth and proliferation, as well as on tumorigenesis, were examined both in vitro and in vivo, using MTT assay, colony formation assay, cell cycle analysis, soft agar assay, and tumorigenesis in nude mice. Western blotting and real-time reverse transcriptase-PCR were performed to examine the impact of HOXB7 on the PI3K/Akt and MAPK signaling pathways.
RESULTS:
HOXB7 protein level was significantly correlated with advanced Dukes stage (P < 0.001), T stage (P = 0.012), distant metastasis (P = 0.042), higher proliferation index (P = 0.007) and poor survival of patients (P = 0.005). Enforced expression of HOXB7 in CRC cell lines significantly enhanced cell growth, proliferation and tumorigenesis. Conversely, knockdown of HOXB7 caused an inhibition of cell growth, proliferation, and tumorigenesis. We also showed that HOXB7 accelerated G(0)-G(1) to S-phase transition concomitantly with upregulation of cyclin D1 and downregulation of p27Kip1. On the contrary, knockdown of HOXB7 caused G(1)-S-phase arrest, downregulation of cyclin D1 and upregulation of p27Kip1. Enforced expression of HOXB7 could enhance PI3K/AKT and MAPK pathway activity.
CONCLUSION:
Our findings suggest that HOXB7 protein, as a valuable marker of CRC prognosis, plays an important role in the development and progression of human CRC.
©2011 AACR.
BACKGROUND:
Previous studies showed that connective tissue growth factor (CTGF)-induced proliferation of lung fibroblasts and production of chemokines in mesangial cells could be inhibited by lipoxin A(4) (LXA(4)). It is speculated that LXA(4) could modulate the CTGF-induced epithelial to mesenchymal transition.
METHODS:
The expressions of alpha-smooth muscle actin (alpha-SMA), E-cadherin, integrin-linked kinase (ILK), extracellular signal-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3-K), Akt and Smad signaling were assessed by Western blot and/or real-time RT-PCR, and activation of Ras or ILK by activity assay, expressions of alpha-SMA and zonula occludens-1 by immunofluorescence assay in proximal tubular epithelial cells (HK-2).
RESULTS:
Pretreatment of HK-2 cells with LXA(4) inhibited the morphological fibroblast-like changes and alpha-SMA expression induced by CTGF but not by transforming growth factor-beta(1) (TGF-beta(1)). The expressions of E-cadherin and zonula occludens-1 reduced by CTGF but not by TGF-beta(1) were increased by LXA(4). LXA(4) inhibited the expression and activity of ILK and activation of Ras, ERK1/2, PI3-K and Akt in HK-2 cells stimulated by CTGF. LXA(4) did not affect TGF-beta(1)-induced expression of ILK, Smad-2/3 phosphorylation and Smad-2's binding to Smad-4 and subsequent nuclear translocation.
CONCLUSION:
LXA(4) inhibits the tubular epithelial to mesenchymal transition, initiated by CTGF but not by TGF-beta(1), via downregulation of ILK, Ras/MEK/ERK1/2 and PI3-K/Akt-dependent signal pathway stimulated by CTGF.
2010 S. Karger AG, Basel.
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