Upregulation Of p-AKT Research Articles

Mechanisms of Antiulcer Effect of an Active Ingredient Group of Modified Xiao Chaihu Decoction.

The present study aimed to investigate the antiulcer activities and mechanisms of action of an active ingredient group (AIG) of Modified Xiao Chaihu Decoction (MXCD). The gastroprotective action of the AIG was studied in ethanol-induced, pylorus ligature-induced, and acetic acid-induced in vivo gastric ulcer models. The enzyme-linked immunoadsorbent assay (tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and epidermal growth factor (EGF)), nitrate reductase assay (nitric oxide (NO)), western blot analysis (Bax, Bcl-2, cleaved-caspase-3, and cleaved-PARP (poly (ADP-Ribose) polymerase)), histological analysis (HE), and immunohistochemical analysis (HSP-70, p-AKT, and PCNA) were used to evaluate the anti-inflammatory, antiapoptotic, and healing properties of AIG. Numerous mechanisms are involved in the antiulcer activity of AIG, including the increase of PGE2, NO, and EGF content and a reduction in TNF-α levels. The upregulation of HSP-70, p-AKT, and PCNA seems to be directly linked to the healing effect of AIG. Bax, Bcl-2, cleaved-caspase-3, and cleaved-PARP also play a key role in this process. The AIG exerted gastroprotective effects by reducing antisecretory, anti-inflammatory, and antiapoptotic mechanisms. In addition, it promotes cell proliferation. Therefore, activation of the PI3K/AKT signaling pathway may play an important role in cell proliferation.

Linking CREB function with altered metabolism in murine fibroblast-based model cell lines.

The cAMP-responsive element binding protein CREB is frequently overexpressed and activated in tumors of distinct histology, leading to enhanced proliferation, migration, invasion and angiogenesis as well as reduced apoptosis. The de-regulated expression of CREB might be linked with transcriptional as well as post-transcriptional regulation mechanisms. We show here that altered CREB expression levels and function are associated with changes in the cellular metabolism. Using comparative proteome-based analysis an altered expression pattern of proteins involved in the cellular metabolism in particular in glycolysis was found upon CREB down-regulation in HER-2/neu-transfected cell lines. This was associated with diminished expression levels of the glucose transporter 1, reduced glucose uptake and reduced glycolytic activity in HER-2/neu-transfected cells with down-regulated CREB when compared to HER-2/neu+ cells. Furthermore, hypoxia-induced CREB activity resulted in changes of the metabolism in HER-2/neu transfected cells. Low pH values in the supernatant of HER-2/neu transformants were restored by CREB down-regulation, but further decreased by hypoxia. The altered intracellular pH values were associated with a distinct expression of lactate dehydrogenase, and its substrate lactate. Moreover, enhanced phosphorylation of CREB on residue Ser133 was accompanied by a down-regulation of pERK and an up-regulation of pAKT. CREB promotes the detoxification of ROS by catalase, therefore protecting the mitochondrial activity under oxidative stress. These data suggest that there might exists a link between CREB function and the altered metabolism in HER-2/neu-transformed cells. Thus, targeting these altered metabolic pathways might represent an attractive therapeutic approach at least for the treatment of patients with HER-2/neu overexpressing tumors.

GSK3α/β: A Novel Therapeutic Target for Neuroendocrine Tumors

Introduction: Glycogen synthase kinase 3α/β (GSK3α/β) is a serine/threonine kinase that plays a critical role in cancer. Aims: In this study, we evaluated the effects of the specific GSK3α/β inhibitor AR-A014418 in vitro to gain novel insights into GSK3α/β signaling in neuroendocrine tumors (NETs). Materials and Methods: Human NET cell lines (BON1, QGP1, H727, and GOT1) were treated with different concentrations of AR-A014418 alone and in combination with lovastatin, everolimus, 5-fluorouracil (5-FU), and γ-irradiation. Results: AR-A014418 significantly dose- and time-dependently decreased cell viability in all 4 NET cell lines through inhibition of epithelial growth factor receptor and mTORC1/p70S6K signaling, as well as cyclin D3 downregulation and induction of pChk1. In all cell lines tested, FACS analysis showed an AR-A014418-induced increase in the sub-G1 phase, reflecting cell death. Apoptosis induction was observed in H727, GOT1 and QGP1 cells, but not in BON1 cells. Furthermore, significant antimigratory effects upon GSK3α/β inhibition were found and were associated with β-catenin downregulation in all cell lines tested. Compensatory upregulation of pAkt and pERK in response to GSK3α/β inhibition was prevented by combining AR-A014418 with the ERK and Akt inhibitor lovastatin. Accordingly, the lovastatin/AR-A014418 combination was synergistic in BON1 and QGP1 cells. Moreover, AR-A014418 displayed promising chemosensitizing effects on 5-FU in QGP1 and slight radiosensitizing properties in BON1 and QGP1 cells. Conclusion: Our data provide new insights into the role of GSK3α/β in NETs and suggest that GSK3α/β inhibition could be a novel therapeutic option in NETs, especially in combination with lovastatin or 5-FU, depending on tumor entity.

Upregulation of pAKT(Ser473) expression in progression of HPV-positive oropharyngeal squamous cell carcinoma.

PIK3CA alterations have been shown to be a frequent event in oropharyngeal squamous cell cancer (SCC), especially in human papillomavirus (HPV)-related tumors. Tissue microarrays (TMAs) were used to evaluate pAKT(Ser473)/(Thr308), total protein kinase B (AKT)(pan) and phosphatase and tensin homolog (PTEN) expression in primary tumors and corresponding nodal disease in oropharyngeal SCC. The HPV status was determined in regard of HPV16 DNA and RNA. Survival analysis was performed by using Kaplan-Meier curves, log-rank testing, and multivariate Cox regression analysis. HPV16 is a prognostic predictive marker for advanced oropharyngeal SCC. pAKT(Ser473) and PTEN are highly expressed in HPV-related oropharyngeal SCCs in contrast to pAKT(Thr308). The pAKT(Ser473) expression increased from primary tumors to progressive nodal disease (21.1%; P < .011). Activation of phosphoinositide 3-kinase (PI3K)/pAKT(Ser473) frequently occurs in advanced HPV-positive oropharyngeal SCC and elevated pAKT(Ser473) levels represent a feature during progression of oropharyngeal SCC, indicating a critical role of the mammalian target of rapamycin (mTOR) complex. Further studies are required to evaluate specific drugs targeting PI3K/AKT/mTOR in consideration of PIK3CA alterations.

Antagonist of thromboxane A2 receptor by SQ29548 lowers DOCA-induced hypertension in diabetic rats

Diabetes is one of high risk factors for cardiovascular diseases, including atherosclerosis and hypertension. This study was conducted to elucidate whether and how thromboxane receptor (TPr) activation contributes to hypertension in diabetes. Human umbilical vein endothelial cells (HUVECs) were cultured. The phosphorylated levels of endothelial nitric oxide synthase (eNOS) and Akt were monitored by western blot. Endothelial function was determined by organ bath. High glucose (HG) or thromboxane A2 mimetic U46619 significantly reduced the levels of p-eNOS and p-Akt in cultured HUVECs, which were reversed by inhibition of TPr. HG/U46619-induced reductions of p-eNOS and p-Akt were accompanied with increases of total and phosphorylated tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN). PTEN siRNA restored Akt-eNOS signaling in cells treated with HG. In rats, streptozotocin-induced hyperglycemia was associated with aortic PTEN upregulation and reductions of p-Akt and p-eNOS. TPr antagonist SQ29548 ablated these alterations and reduced blood pressure in rats with DOCA-induced hypertensive. In conclusion, hyperglycemia activates thromboxane A2 receptor to augment DOCA-induced high blood pressure in rats via the PTEN-Akt-eNOS signaling.

Induction of Plac8 promotes pro-survival function of autophagy in cadmium-induced prostate carcinogenesis

Chronic exposure to cadmium is known to be a risk factor for human prostate cancer. Despite over-whelming evidence of cadmium causing carcinogenicity in humans, the specific underlying molecular mechanisms that govern metal-induced cellular transformation remain unclear. Acute exposure (up to 72 h) to cadmium induces apoptosis in normal prostate epithelial cells (RWPE-1), while chronic exposure (>1 year) transforms these cells to a malignant phenotype (cadmium-transformed prostate epithelial cells; CTPE). Increased expression of autophagy-regulated genes; Plac8, LC3B and Lamp-1; in CTPE cells was associated with cadmium-induced transformation. Increased expression of Plac8, a regulator of autophagosome/autolysosome fusion, facilitates the pro-survival function of autophagy and upregulation of pAKT(ser473) and NF-κβ, to allow CTPE to proliferate. Likewise, inhibition of Plac8 suppresses CTPE cell growth. Additionally, overexpression of Plac8 in RWPE-1 cells induces resistance to cadmium toxicity. Pharmacological inhibitors and an inducer of autophagy failed to affect Plac8 expression and CTPE cell viability, suggesting a unique role for Plac8 in cadmium-induced prostate epithelial cell transformation. These results support a role for Plac8 as an essential component in the cadmium-induced transformation of normal prostate epithelial cells to a cancerous state.

Downregulation of HADH promotes gastric cancer progression via Akt signaling pathway.

HADH is a key enzyme in fatty acid oxidation. The aim of this study was to identify the role of HADH in gastric cancer. We analyzed the expression of HADH in 102 pairs of gastric cancer samples. Western blot analysis revealed that HADH was decreased in stage I/II gastric cancer samples compared to matched adjacent normal gastric tissue, and its expression was further decreased in stage III/IV samples. Importantly, the reduced expression of HADH was associated with increased expression of p-Akt and reduced expression of PTEN in the gastric carcinoma tumor samples. To determine the significance of HADH downregulation in gastric cancer progression, we tested the impact of HADH knockdown or overexpression on the migration and invasion of the gastric cancer cells using a transwell assay. Knockdown of HADH significantly promoted gastric cancer cell migration and invasion, which was associated with increased expression of p-Akt. The PI3K inhibitor LY294002 inhibited HADH shRNA induced migration/invasion, and abolished the upregulation of p-Akt. By contrast, HADH overexpression inhibited the migration and invasion of MKN45 cells. Herein, for the first time, we demonstrate that downregulation of HADH promotes gastric cancer progression via activation of Akt signaling pathway.

Abstract 4157: Co-blockade of mTORC1, ERBB and estrogen receptor signalling pathways in endocrine resistance breast cancer: Combating tumor plasticity

Abstract Introduction The majority of breast cancers (BC) are estrogen (E) receptor positive (ER+). Endocrine therapies target E stimulation of tumour growth but resistance remains problematic, often a result of enhanced crosstalk between ER and growth factor pathways. Previously we reported the antiproliferative efficacy of combining everolimus (RAD001, mTORC1 inhibitor) with endocrine therapy in resistance models, but potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB and mTORC1) may target tumour rewiring and provide enhanced long-term clinical utility in endocrine resistant BCs. Methods Several ER+ BC lines adapted to long term E deprivation (LTED), modelling relapse to an aromatase inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan ERBB inhibitor) in the presence or absence of estradiol (E2), tamoxifen or fulvestrant. Effects on proliferation, cell signalling, cell cycle and transcription were assessed. Additionally, an in vivo model of AI resistance was treated with monotherapies or combinations to evaluate efficacy in delaying tumour progression. Results All cell lines showed dose dependent decreases in proliferation in response to RAD001 (IC50 0.6-50nM without E2; 1-10nM with E2). Neratinib showed a wide range of IC50 values in the presence of E2 (300-1000nM). In the absence of E2, wild type (wt) cell lines showed IC50 values in excess of 1800nM with hormetic response curves, whilst in the LTED IC50 values ranged between 400-900nM. Combination of either agent with endocrine therapy caused a concentration dependent decrease in proliferation in both wt and LTED lines but the maximum effect was observed with a triple combination of RAD001, neratinib and endocrine therapy. Expression of pS6 was suppressed by RAD001 in all cells tested, whilst neratinib caused a cell specific reduction in expression of ERBB family proteins. Upregulation of pAKT was observed in all cell lines upon treatment with RAD001. Combination of RAD001 with neratinib suppressed the upregulation of pAKT and reduced cell cycle progression. In the absence of E2, RAD001 reduced ER mediated transcription and recruitment of ER and CREB binding protein to the TFF1 promoter, contrasting with neratinib, which caused a marked increase. In vivo study using LTED tumour xenografts showed the triple combination of RAD001, neratinib and fulvestrant was the most effective at reducing tumour volume (89%, p&amp;lt;0.001). Antitumor effects persisted after therapy removal with exception of RAD001 alone or in combination with neratinib where a slow increase in tumour growth was observed. Conclusion The combination of RAD001, neratinib and endocrine agents may be effective in patients who have relapsed on endocrine therapy but retain a functional ER by combating ERBB pathway upregulation. Citation Format: Ricardo Ribas, Sunil Pancholi, Stephanie K. Guest, Aradhana Rani, Joanna Nikitorowicz-Buniak, Nikiana Simigdala, Allan Thornhill, Richard E. Cutler Jr, Alshad S. Lalani, Francesca Avogadri-Connors, Mitch Dowsett, Stephen R. Johnston, Lesley-Ann Martin. Co-blockade of mTORC1, ERBB and estrogen receptor signalling pathways in endocrine resistance breast cancer: Combating tumor plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4157. doi:10.1158/1538-7445.AM2017-4157

MicroRNA-590-3p promotes cell proliferation and invasion by targeting inositol polyphosphate 4-phosphatase type II in human prostate cancer cells.

Inositol polyphosphate 4-phosphatase type II emerges as a tumor suppressor in prostate cancer, and its loss of expression is associated with poor prognosis for prostate cancer. However, the mechanism of downregulation of inositol polyphosphate 4-phosphatase type II in prostate cancer development has not yet been fully clarified. In this study, microRNA-590-3p was found to be upregulated in both prostate cancer tissues and cell lines. Overexpression of microRNA-590-3p by microRNA-590-3p mimics promoted prostate cancer cell proliferation and invasion and accelerated the growth of xenografted tumors, while microRNA-590-3p inhibitors contributed to inhibition of cellular proliferation and invasion as well as tumor growth. A dual-luciferase reporter assay and expression analysis further confirmed that inositol polyphosphate 4-phosphatase type II was a direct target of microRNA-590-3p. Enforced expression of microRNA-590-3p led to repression of inositol polyphosphate 4-phosphatase type II messenger RNA and protein expression, as well as upregulation of p-Akt, p-FoxO3a, and cyclin D1 and downregulation of p21 expression in prostate cancer cell lines. Overexpression of inositol polyphosphate 4-phosphatase type II could reduce microRNA-590-3p-induced cell proliferation and invasion as well as tumor growth, and decrease microRNA-590-3p-mediated upregulation of cyclin D1 and downregulation of p21 expression in prostate cancer cells. Taken together, our findings reveal that microRNA-590-3p is a potential onco-microRNA that participates in carcinogenesis of human prostate cancer by suppressing inositol polyphosphate 4-phosphatase type II expression and involving the Akt/FoxO3a pathway. MicroRNA-590-3p may represent a potential therapeutic target for prostate cancer patients.

Erythropoietin Modification Enhances the Protection of Mesenchymal Stem Cells on Diabetic Rat-Derived Schwann Cells: Implications for Diabetic Neuropathy.

Diabetes-triggered apoptosis of Schwann cells (SC) contributes to the degradation of diabetic peripheral neuropathy (DNP). In recent years, mesenchymal stem cells (MSC) were applied to DPN repair and it was demonstrated that paracrine secretion played a key role in neuroprotection exerted by MSC. Erythropoietin (EPO) is a potent cytokine capable of reducing apoptosis of SC. However, the expression of EPO in MSC is limited. In this study, we hypothesized that overexpression of EPO in MSC (EPO-MSC) may significantly improve their neuroprotective potentials. The EPO overexpression in MSC was achieved by lentivirus transduction. SC derived from the periphery nerve of diabetic rats were cocultured with MSC or EPO-MSC in normal or high glucose culture condition, respectively. In normal glucose culture condition, the overexpression of EPO in MSC promoted the MSC-induced restoration of SC from diabetic rats, including increases in GSH level and cell viability, decrease in TUNEL apoptosis, upregulation of antiapoptotic proteins, p-Akt, and Bcl-2, and downregulation of proapoptotic proteins, cleaved caspase-3, and Bax. The subsequent results in high glucose culture condition showed similar promotions achieved by EPO-MSC. Thus, it could be concluded that EPO-MSC possessed a potent potential in hampering apoptosis of SC, and the suppression was probably attributed to attenuating oxidative stress and regulating apoptosis related protein factors.

Insulin and Insulin-like growth factor-1 can activate the phosphoinositide-3-kinase /Akt/FoxO1 pathway in T cells in vitro.

ABSTRACTHyper-glycemic food increases insulin-like growth factor 1 (IGF-1) and insulin signaling and regulates endocrine responses and thereby may modulate the course of acne. Inflammation and adaptive immune responses have a pivotal role in all stages of acne. Recent hypothesis suggests that hyperglycemic food reduces nuclear forkhead box-O1 (FoxO1) transcription factor and may eventually induces acne. The aim of our study was to investigate the role of IGF-1 and insulin on the phosphoinositide-3-kinase (PI3K)/Akt/FoxO1 pathway in human primary T cells and on the molecular functions of T cells in vitro. T cells were stimulated with 0.001 μM IGF-1 or 1 μM insulin +/− 20 μM PI3K inhibitor LY294002. T cells were also exposed to SZ95 sebocyte supernatants which were pre-stimulated with IGF-1 or insulin. We found that 0.001 µM IGF-1 and 1 µM insulin activate the PI3K pathway in T cells leading to up-regulation of p-Akt and p-FoxO1 at 15 and 30 minutes. Nuclear FoxO1 was decreased and FoxO transcriptional activity was reduced. 0.001 µM IGF-1 and 1 µM insulin increased T cell proliferation but have no significant effect on Toll-like receptor2/4 (TLR) expression. Interestingly, supernatants from IGF-1- or insulin-stimulated sebocytes activated the PI3K pathway in T cells but reduced T cell proliferation. Taken together, this study helps to support that high glycemic load diet may contribute to induce activation of the PI3K pathway and increase of proliferation in human primary T cells. Factors secreted by IGF-1- and insulin-stimulated sebocytes induce the PI3K pathway in T cells and reduce T cell proliferation, which probably can reflect a protective mechanism of the sebaceous gland basal cells.

Lycium barbarum polysaccharides inhibit proliferation and migration of bladder cancer cell lines BIU87 by suppressing Pi3K/AKT pathway.

The aim of this study was to verify whether Lycium barbarum polysaccharides inhibits proliferation and migration of BIU87 cells through Pi3K/AKT pathway. Different concentrations of Lycium barbarum polysaccharides were used to incubate with BIU87cells. LY-294002 and IGF-1 were used to inhibit and activate Pi3K/AKT pathway respectively. MTT were used to investigate the proliferation of BIU87cells. Transwell chambers and wound healing were used to test the migratory ability of BIU87cells. Western blotting were used to investigate the expressions of P21,P27,MMP-2, MMP-9, AKT and p-AKT in BIU87cells. Compared with the control group, the proliferation and migration of BIU87cells and the expression of p-AKT were significantly decreased in the study group; the inhibitory effect of the downregulation of p-AKT by LY-294002on the induction of BIU87cells proliferation and migration was identical to that of Lycium barbarum polysaccharides; upregulation of p-AKT by IGF-1 reversed the Lycium barbarum polysaccharides-induced inhibition of BIU87cells dedifferentiation. In conclusion, LBP inhibits the proliferation and migration of BIU87 cells by suppressing Pi3K/AKT signaling pathway.

Aspirin enhances the cytotoxic activity of bortezomib against myeloma cells via suppression of Bcl-2, survivin and phosphorylation of AKT.

In our previous study, it was found that aspirin (ASA) exerted antimyeloma actions in vivo and in vitro. The resistance to bortezomib (BTZ) in multiple myeloma (MM) is partly due to AKT activation and the upregulation of survivin induced by BTZ, which are the targets of ASA in gastric and ovarian cancer, respectively. Thus, the present study investigated the interaction between ASA and BTZ in MM and further clarified the underlying mechanisms. MM1.S and RPMI-8226 cell lines harboring the N- and K-Ras mutations, respectively, were treated with 2.5 mM ASA, 10 nM BTZ and ASA+BTZ for different durations. The proliferation and apoptosis of the cells were determined, and the underlying mechanisms governing the interaction of ASA and BTZ were examined in the MM cells. Treatment with ASA+BTZ caused higher rates of proliferative inhibition and apoptosis in the MM1.S and RPMI-8226 cells in time-dependent manner, compared with either agent alone. A drug interaction assay revealed the additive effect of ASA and BTZ on the myeloma cells. ASA alone inhibited the levels of phosphorylated AKT (p-AKT) and survivin, whereas BTZ alone augmented the levels of p-AKT and survivin. Of note, ASA markedly decreased the upregulation of p-AKT and survivin induced by BTZ. Treatment with ASA+BTZ significantly suppressed the level of Bcl-2, compared with either agent alone. ASA may potentiate the antimyeloma activity of BTZ against myeloma cells via suppression of AKT phosphorylation, survivin and Bcl-2, indicating the potential of ASA+BTZ in treating MM, particularly for cases of BTZ-refractory/relapsed MM.

Effects of fibroblast growth factor-2 on cell proliferation of cementoblasts

Background/purposeFibroblast growth factor (FGF)-2 is known as a signaling molecule that induces tissue regeneration. Little is known about the effect of FGF-2 on cementoblasts for periodontal and periapical regeneration. The aim of this study was to investigate the effects of FGF-2 on murine immortalized cementoblast cell line (OCCM.30). Materials and methodsCell growth and proliferation was judged by using alamar blue reduction assay. Flow cytometry analysis was used to evaluate Stro-1 positive cells expression with or without FGF-2. Western blot was used to evaluate the expression of phosphorylated serine–threonine kinase Akt (p-Akt) and extracellular signal-regulated protein kinase (p-ERK) in cementoblasts. ResultsFGF-2 was found to increase cell growth in a dose-dependent manner (P<0.05). The concentration of 10 ng/mL FGF-2 enhanced cell proliferation in a time-dependent manner (P<0.05). In addition, 10 ng/mL FGF-2 significantly increased the number of Stro-1 positive cells in the first 24 hours (P<0.05). Moreover, 10 ng/mL FGF-2 was found to upregulate p-Akt and p-ERK in a time-dependent manner (P<0.05). ConclusionTaken together, FGF-2 could increase cementoblast growth, proliferation, and Stro-1 positive cells. These enhancements are associated with the upregulation of p-Akt and p-ERK expression. The application of FGF-2 may provide benefit for periodontal and periapical regeneration during the early phase of wound healing.

PIK3CD promoted proliferation in diffuse large B cell lymphoma through upregulation of c-myc.

Despite PIK3CD has been extensively reported in cancers, however, little evidence has been available regarding its role in the setting of diffuse large B cell lymphoma (DLBCL). In the present study, to investigate the role of PIK3CD in DLBCL, relevant experiments were carried out on both in vivo clinical tissue level and in vitro cell line level. Prognostic and clinicopathological significance were analyzed after immunohistochemical assay of PIK3CD expression on DLBCL tissue microarray. MTT assay and flow cytometry were employed to evaluate the proliferative variation, cell cycle, and apoptosis. Athymic nude mice xenografted with DLBCL cell line were employed to confirm the role of PIK3CD. It was found that there was a significant difference between expression of PIK3CD and international prognosis index (IPI), performance state (PS), and inferior overall prognosis. Furthermore, PIK3CD can promote proliferation and prevent apoptosis in DLBCL cells in vitro through upregulation of c-myc and p-AKT and in contrast downregulation of p21 and p27. In nude mice model, knock-down of PIK3CD was shown to be able to suppress the proliferation of DLBCL but not significantly compared with control group. Taken together, our study showed that PIK3CD can promote proliferation of DLBCL cells both in vitro and in vivo, suggesting that PIK3CD could be druggable in the therapy of DLBCL.

Effects of Low-dose Triamcinolone Acetonide on Rat Retinal Progenitor Cells under Hypoxia Condition.

Background:Retinal degenerative diseases are the leading causes of blindness in developed world. Retinal progenitor cells (RPCs) play a key role in retina restoration. Triamcinolone acetonide (TA) is widely used for the treatment of retinal degenerative diseases. In this study, we investigated the role of TA on RPCs in hypoxia condition.Methods:RPCs were primary cultured and identified by immunofluorescence staining. Cells were cultured under normoxia, hypoxia 6 h, and hypoxia 6 h with TA treatment conditions. For the TA treatment groups, after being cultured under hypoxia condition for 6 h, RPCs were treated with different concentrations of TA for 48–72 h. Cell viability was measured by cell counting kit-8 (CCK-8) assay. Cell cycle was detected by flow cytometry. Western blotting was employed to examine the expression of cyclin D1, Akt, p-Akt, nuclear factor (NF)-κB p65, and caspase-3.Results:CCK-8 assays indicated that the viability of RPCs treated with 0.01 mg/ml TA in hypoxia group was improved after 48 h, comparing with control group (P < 0.05). After 72 h, the cell viability was enhanced in both 0.01 mg/ml and 0.02 mg/ml TA groups compared with control group (all P < 0.05). Flow cytometry revealed that there were more cells in S-phase in hypoxia 6 h group than in normoxia control group (P < 0.05). RPCs in S and G2/M phases decreased in groups given TA, comparing with other groups (all P < 0.05). There was no significant difference in the total Akt protein expression among different groups, whereas upregulation of p-Akt and NF-κB p65 protein expression and downregulation of caspase-3 and cyclin D1 protein expression were observed in 0.01 mg/ml TA group, comparing with hypoxia 6 h group and control group (all P < 0.05).Conclusion:Low-dose TA has anti-apoptosis effect on RPCs while it has no stimulatory effect on cell proliferation.

Methylation-mediated repression of microRNA-129-2 suppresses cell aggressiveness by inhibiting high mobility group box 1 in human hepatocellular carcinoma.

Aberrant expression of microRNAs (miRNAs) and its dysfunction have been revealed as crucial modulators of cancer initiation and progression. MiR-129-2 has been reported to play a tumor suppressive role in different human malignancies. Here, we demonstrated that miR-129-2 was significantly decreased in hepatocellular carcinoma (HCC) tissues and cell lines. Furthermore, miR-129-2 was expressed at significant lower levels in aggressive and recurrent tumor tissues. Clinical analysis indicated that miR-129-2 expression was inversely correlated with venous infiltration, high Edmondson-Steiner grading and advanced tumor-node-metastasis (TNM) stage in HCC. Notably, miR-129-2 was an independent prognostic factor for indicating overall survival (OS) and disease-free survival (DFS) of HCC patients. Ectopic expression of miR-129-2 inhibited cell migration and invasion in vitro and in vivo. Furthermore, we confirmed that high mobility group box 1 (HMGB1) was a direct target of miR-129-2, and it abrogated the function of miR-129-2 in HCC. Mechanistic investigations showed that miR-129-2 overexpression inhibited AKT phosphorylation at Ser473 and decreased the expression of matrix metalloproteinase2/9 (MMP2/9). Upregulation of p-AKT abolished the decreased cell migration and invasion induced by miR-129-2 in HCC. Whereas inhibition of Akt phosphorylation significantly decreased HMGB1-enhanced HCC cell migration and invasion. Moreover, we found that miR-129-2 was downregulated by DNA methylation, and demethylation of miR-129-2 increased miR-129-2 expression in HCC cells and resulted in significant inhibitory effects on cell migration and invasion. In conclusion, miR-129-2 may serve as a prognostic indicator for HCC patients and exerts tumor suppressive role, at least in part, by inhibiting HMGB1.

Mir-765 promotes cell proliferation by downregulating INPP4B expression in human hepatocellular carcinoma.

microRNAs (miRNAs) dysregulation is widely involved in cancer progression and contributed to sustained cell proliferation by directly targeting multiple targets. Therefore, better understanding the underlying mechanism of miRNA in carcinogenesis may improve diagnostic and therapeutic strategies for malignancy. In our study, we found that mir-765 is upregulated in both hepatocellular carcinoma (HCC) cell lines and tissues, compared to human normal liver cell line and adjacent non-cancerous tissues, respectively. Overexpression of mir-765 increased HCC cells proliferation and tumorigenicity, whereas inhibition of mir-765 reverses this effect. Furthermore, we demonstrated that INPP4B as a direct target of mir-765 and ectopic expression of mir-765 repressed INPP4B expression, resulting in upregulation of p-AKT, Cyclin D1, and downregulation of p-FOXO3a, p21 expression in HCC. Strikingly, we found that silencing the expression of INPP4B is the essential biological function of miR-765 during HCC cell proliferation. Collectively, our findings reveal that miR-765 is a potential onco-miR that participates in carcinogenesis of human HCC by suppressing INPP4B expression, and might represent a potential therapeutic target for HCC patients.

Upregulated ATF6 contributes to chronic intermittent hypoxia-afforded protection against myocardial ischemia/reperfusion injury.

In the present study, we investigated the role of activating transcription factor 6 (ATF6) in the mechanism by which chronic intermittent hypoxia (CIH) increases tolerance to myocardial ischemia/reperfusion (I/R). Experiments were conducted using a rat model of I/R injury in vivo and isolated Langendorff-perfused rat hearts ex vivo. The role of Akt in this process was also investigated in vitro using rat myoblast H9c2 cells. Cell viability was measured using a cell counting kit-8 assay. Lactate dehydrogenase (LDH) and creatine kinase cardiac isoenzyme activity were also measured as markers of cellular damage. ATF6, Akt and phosphorylated (p)-Akt expression was analyzed by western blot analysis. RNA interference (RNAi) was used to suppress ATF6 expression. We noted that ATF6 expression in the ventricular myocardium was significantly increased in rats exposed to CIH. Furthermore, we noted that CIH preserved cardiac function after I/R in vivo and improved post-ischemic recovery of myocardial performance in isolated rat hearts. ATF6 and p-Akt expression was upregulated in cultured H9c2 cells exposed to chronic mild hypoxia compared with those cultured under normoxic conditions. Chronic mild hypoxia attenuated subsequent simulated I/R injury in H9c2 cells (48 h), as evidenced by increased cell viability and decreased LDH activity. By contrast, decreased cell viability and increased LDH activity were observed in siRNA-ATF6-transfected H9c2 cells, with a concomitant reduction in p-Akt levels. These results indicated that ATF6 upregulation is involved in the mechanism by which CIH attenuates myocardial I/R injury, possibly through upregulation of p-Akt, which is a key regulator of cardiomyocyte survival.

Abstract P3-05-05: Targeting tumor re-wiring by triple blockade of mTORC1, ERBB and ER signaling pathways in endocrine resistant breast cancer

Abstract AIM To target tumor re-wiring by combined mTORC1 inhibition plus hormonal treatment with or without co-blockade of ERBB signaling in endocrine resistant models of human breast cancer (BC). BACKGROUND Around 80% of BCs are estrogen receptor positive (ER+). Endocrine therapies target estrogenic stimulation of tumor growth but resistance remains problematic. Several strategies have shown that resistance often depends on the acquisition of enhanced cross-talk between ER and growth-factor pathways, allowing the disease to circumvent the need for steroid hormones. We have previously reported the antiproliferative effects of the combination of everolimus (RAD001-mTORC1 inhibitor) with endocrine therapy in resistance models, but potential routes of escape from treatment via ERBB2/3 signaling were observed. We hypothesised that combined targeting of three signaling pathways, namely ER, ERBB and mTORC1 may provide enhanced anti-tumor activity. METHODS ER+ BC cell lines (MCF7, SUM44 and HCC1428) adapted to long term estrogen-deprivation (LTED) which model relapse on an aromatase inhibitor, along with their wild-type (wt) cell lines were treated with neratinib, a pan-ERBB tyrosine kinase inhibitor, in combination with RAD001 ± estradiol (E2), tamoxifen or fulvestrant. End points included proliferation, cell signaling, cell cycle and effect on ER-mediated transactivation and recruitment by ChIP. RESULTS All cell lines showed a concentration-dependent decrease in proliferation in response to RAD001 (IC50 0.6-50nM in absence of E2 and 1-10nM in presence of E2). A wide range of IC50 values (300-1000nM) was observed with neratinib treatment in the presence of E2. However, in the absence of E2, wt cell lines showed IC50 values in excess of 1800nM with hormetic dose response curves, in which lower concentrations induced cell proliferation. In contrast, LTED IC50 values ranged between 400-900nM. Combination of either agent with endocrine therapy caused a concentration dependent decrease in proliferation in all wt cell lines and their LTED derivatives, but the maximum effect was observed when a triple combination of RAD001, neratinib and ER-blockade was used. Expression of pS6 was dramatically suppressed by RAD001 ± neratinib in all cell lines tested, whilst neratinib caused a cell line specific reduction in expression of ERBB family proteins. Upregulation of pAKT was observed in all cell lines following treatment with RAD001, indicating both inhibitors were effectively suppressing their respective targets. Combination of RAD001 with neratinib suppressed the upregulation of pAKT and significantly reduced cell cycle progression. In the absence of E2, RAD001 caused a reduction in ER-mediated transcription and decrease in recruitment of ER and the CREB-binding protein (CBP) to the TFF1 promoter. In contrast, neratinib induced a marked increase in ER-recruitment and concomitant rise in ER-mediated transactivation, which was reduced by the addition of RAD001. CONCLUSION Targeting tumor re-wiring by triple blockade of ERBB, ER and mTORC1 signaling pathways significantly reduces cell proliferation supporting the potential combination in patients who have relapsed on endocrine therapy and retain a functional ER. Citation Format: Nikitorowicz-Buniak J, Ribas R, Rani A, Pancholi S, Guest SK, Cutler Jr RE, Lalani A, Dowsett M, Johnston SR, Martin L-A. Targeting tumor re-wiring by triple blockade of mTORC1, ERBB and ER signaling pathways in endocrine resistant breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-05.