Expression Of p-Akt Proteins Research Articles

CAPE-pNO2 Inhibited the Growth and Metastasis of Triple-Negative Breast Cancer via the EGFR/STAT3/Akt/E-Cadherin Signaling Pathway.

Overexpressed epidermal growth factor receptor (EGFR) and overactivated epithelial-mesenchymal transition (EMT) in triple-negative breast cancer (TNBC) can enhance tumorigenesis and tumor recurrence and metastasis. Caffeic acid p-nitro-phenethyl ester (CAPE-pNO2) has various pharmacological activities in our previous research, but its effect on metastasis and growth of TNBC has not been studied. In this study, Caffeic acid phenethyl ester (CAPE) was as a positive control. in vitro, MTT, Transwell, wound healing, colony formation and cell adhesion assays were performed to examine the effect on viability, invasion, migration, colony formation and adhesion of MDA-MB-231 cells by CAPE-pNO2, the results indicated that CAPE-pNO2 significantly dose-dependently inhibited metastasis of MDA-MB-231 cells (p < 0.05). in vivo, TNBC xenograft mice were established by subcutaneously injected with MDA-MB-231 cells, and they were used to estimate the effect on metastasis and growth of CAPE-pNO2 after 38 days of treatment. HE staining and TUNEL staining were carried out in tumor tissues, results showed that CAPE-pNO2 obviously suppressed the tumor growth, induced cells apoptosis (p < 0.01) and decreased pulmonary and splenic metastatic tumor cells. The results of IHC demonstrated that the VEGFA and Ki-67 proteins expression were downregulated (p < 0.01) in tumor tissues. Furthermore, western blot analysis was used to quantify key metastasis- and growth-associated proteins expression in vitro and in vivo, the results suggested that CAPE-pNO2 downregulated the proteins expression of p-EGFR, p-STAT3, p-Akt, MMP-2, MMP-9, Survivin, and key EMT-related proteins (Vimentin and N-cadherin) (p < 0.01), and increased the expression of E-cadherin (p < 0.01) in vivo and in vitro. Besides, CAPE-pNO2 had a similar effect as erlotinib in regulating the EGFR downstream proteins in EGF-induced MDA-MB-231cells. Collectively, these results indicated that CAPE-pNO2 possessed inhibitory effect on the growth and metastasis of TNBC may via the EGFR/STAT3/Akt/E-cadherin signaling pathway, and CAPE-pNO2 is better than CAPE in inhibiting growth and metastasis.

Bmi-1-siRNA Regulates the Proliferation of K562 Leukemia Cells in vitro and in vivo by PTEN/pAKT Pathway

To investigate the effect of Bmi-1 gene silence on the proliferation ability of K562 cells in vitro and in vivo, and to explore the relation of molecular mechanism between proliferation ability of K562 cells in vitro and in vivo with PTEN/pAKT signaling pathway. The Bmi-1 small interference RNA (siRNA) sequences were transfected into K562 cells for decreasing Bmi-1 expression. The effect of Bmi-1 siRNA on the proliferation of K562 cells in vitro and in vivo was detected by MTT method and colony-forming test, the effect of Bmi-1 siRNA on the tumorogenicity of K562 cells was observed by subcutaneous inoculation of K562 cells, LY294002 and Bpv treated K562 cells in nude mice, the expression of Bmi-1, PTEN and pAKT proteins were detected by Western blot. The Bmi-1 siRNA could inhibit the proliferation activity, colony-forming and tumor-forming abilities of K562 cells. After the silence of Bmi-1 gene, the PTEN expression in Bmi-1 gene-silenced group was significantly enhanced. While the pAKT expression in Bmi-1 gene-silenced group was significantly reduced; after the K562 cells were treated with LY294002 (an inhibitor of pAKT), the pAKT expression colony-forming and tumor forming abilities were reduced in comparison with untreated K562 cells; after the K562-S1 cells were treated with Bpv (an inhibitor of PTEN), the PTEN expression decreased, while the pAKT expression, colony forming and tumor-forming abilities were restored. The Bmi-1 gene possibly involves in regulation of K562 proliferation in vivo and in vitro, the effect of PTEN/pAKT signaling pathway maybe one of molecular mechanisms mediating this regulation.

Role of tea polyphenols in delaying hyperglycemia-induced senescence in human glomerular mesangial cells via miR-126/Akt-p53-p21 pathways.

The aim of this study was to investigate the effects and possible mechanism of tea polyphenols (TPs) on the senescence of human glomerular mesangial cells (HGMCs) under high glucose conditions. HGMCs were divided into the normal group (NG, 5.5mmol/L glucose), mannitol group (MNT, 5.5mmol/L glucose and 24.5mmol/L mannitol), TP group (TP, 30mmol/L glucose and 5 μg/mL TP) and high-dose D-glucose group (HG, 30mmol/L glucose). The effects of TP on the cell morphology of HGMCs; the percentage of cells positive for senescence-associated β-galactosidase (SA-β-gal); the ratio of G1 phase of cell cycle; telomere length; and the expression of p-Akt, p53, p21 and Rb proteins of the Akt-p53-p21 signaling pathway and the expression miR-126 were examined. High glucose led to premature senescence of HGMCs, as evident from the increase in the percentage of SA-β-gal-positive cells, decrease in telomere length, cell cycle arrest at G1 phase,decrease in the expression of miR-126 and p-Akt and increase in the expression of p53, p21 and Rb proteins in the HG group. In contrast, in the TP group, these effects of high glucose treatment were abrogated and this indicates that TP had a protective effect on HGMCs. High glucose induces the senescence of HGMCs in vitro via the miR-126 and Akt-p53-p21 signaling pathways. TP can delay the high glucose-induced senescence of HGMCs by regulating the activity of these signaling pathways. Thus, the polyphenols present in tea may have potential for the treatment of diabetic nephropathies associated with premature senescence.

Apelin-13 attenuates early brain injury following subarachnoid hemorrhage via suppressing neuronal apoptosis through the GLP-1R/PI3K/Akt signaling

Apelin, an endogenous ligand for the orphan G-protein-coupled receptor APJ, possesses anti-apoptotic and neuroprotective properties. It has been shown to be a protective factor for different types of central nervous system insults, such as ischemia and traumatic brain injury. Here, we investigated the effects of apelin-13 on early brain injury (EBI) following subarachnoid hemorrhage (SAH), and the underlying molecular mechanisms involved. Apelin-13 was delivered to rats via intracerebroventricular administration. Neurological scores, brain water content and neuronal apoptosis were measured 24 h after SAH. The PI3K/Akt inhibitor LY294002 or GLP-1R siRNA were injected into the lateral cerebral ventricle before induction of SAH. Changes in the expression of p-Akt, GLP-1R and apoptosis-associated proteins (Bax, Bcl-2, cleaved caspase-3) were then investigated. Results showed that the levels of GLP-1R in neurons increased significantly, reaching a peak at 24 h after the induction of SAH. Treatment with apelin-13 improved neurological deficits, as well as alleviated brain edema and apoptotic cell death. Apelin-13 was also able to increase the levels of p-Akt, GLP-1R and Bcl-2, while inhibiting the expression levels of Bax and cleaved caspase-3. The anti-apoptotic and neuroprotective effects of apelin-13 were partially reversed by addition of LY294002 or GLP-1R siRNA. These results provide evidence that apelin-13 attenuates EBI following SAH via suppressing neuronal apoptosis, and that this effect may act partially via the activation of the GLP-1R/PI3K/Akt signaling pathway.

Melatonin receptor stimulation by agomelatine prevents Aβ-induced tau phosphorylation and oxidative damage in PC12 cells.

PurposeAs a novel antidepressant drug, agomelatine has good therapeutic effect on the mood disorder and insomnia in Alzheimer’s disease (AD). Recent studies have shown the neuroprotective function of agomelatine, including anti-oxidative and anti-apoptosis effect. However, it remains unclear whether agomelatine exerts neuroprotection in AD. Thus, the neuroprotective effect of agomelatine against amyloid beta 25–35 (Aβ25–35)-induced toxicity in PC12 cells was evaluated in this study.MethodsThe concentration of malondialdehyde (MDA), LDH, and ROS was investigated to evaluate oxidative damage. The expression of P-tau, tau, PTEN, P-Akt, Akt, P-GSK3β, and GSK3β proteins was assessed by Western blotting. Our results demonstrated that Aβ25–35 significantly increased the content of MDA, LDH, and ROS. Meanwhile, Aβ25–35 upregulated the expression of P-tau and PTEN as well as downregulated P-Akt and P-GSK3β expression. These effects could be blocked by agomelatine pretreatment. Furthermore, luzindole, the melatonin receptor (MT) antagonist, could reverse the neuroprotective effect of agomelatine.ConclusionThe results demonstrated that antidepressant agomelatine might prevent the tau protein phosphorylation and oxidative damage induced by Aβ25–35 in PC12 cells by activating MT-PTEN/Akt/GSK3β signaling. This study provided a novel therapeutic target for AD in the future.

Hyperoside protects against cerebral ischemia-reperfusion injury by alleviating oxidative stress, inflammation and apoptosis in rats

This study investigated the effects of hyperoside pre-treatment on the oxidative stress, inflammatory response and apoptosis in rats with cerebral ischemia-reperfusion (I/R) injury. SD rats were randomly divided into sham, I/R, hyperoside (I/R + Hyp) and nimodipine (I/R + Nim) groups. The rats in the hyperoside group and the nimodipine group were intra-gastrically given hyperoside (50 mg/kg/day) and nimodipine (15 mg/kg/day), respectively, 15 days before operation. A rat cerebral ischemia model was established by suture-occlusion 1 h after the last administration. The thread embolus was removed 2 h after the ischemia for the 24 h reperfusion. The results showed that compared with that in the I/R model group, the neurologic function of the hyperoside group rats was significantly improved and the cerebral infarction volume ratio decreased significantly. The total antioxidant capacity, the activity of superoxide dismutase and glutathione peroxidase in the brain tissue of the hyperoside group rats were significantly higher than those in the I/R model group. The content of malondialdehyde, interleukin-1β and tumour necrosis factor-α in the brain tissue of the hyperoside group rats was significantly lower than that in the I/R model group. Compared with that in the I/R model group, the expression of Bcl-2 mRNA, and p-PI3K and p-AKT proteins in the hyperoside group increased significantly, whereas that of Bax and caspase-3 mRNA decreased significantly. These results suggest that hyperoside had a significant protective effect against cerebral I/R injury in rats, which is related to its inhibiting the oxidative stress, alleviating the inflammatory response and having anti-apoptotic effects.

MiR-3188 Inhibits Non-small Cell Lung Cancer Cell Proliferation Through FOXO1-Mediated mTOR-p-PI3K/AKT-c-JUN Signaling Pathway.

This study investigated the role of miR-3188 on the proliferation of non-small cell lung cancer cells and its relationship to FOXO1-modulated feedback loop. Two non-small cell lung cancer (NSCLC) cell lines A549 and H1299 were used. RNA silencing was achieved by lentiviral transfection. Cell proliferation was assessed by immunohistochemical staining of Ki67 and PCNA, Edu incorporation, and colony formation assay. Western blotting was used to examine expression of FOXO1, mTOR, p-mTOR, CCND1, p21, c-JUN, AKT, pAKT, PI3K, p-PI3K, and p27 proteins. It was found that miR-3188 reduced cell proliferation in NSCLC cells. Molecular analyses indicated that the effect of mammalian target of rapamycin (mTOR) was directly mediated by miR-3188, leading to p-PI3K/p-AKT/c-JUN inactivation. The inhibition of this signaling pathway further caused cell-cycle suppression. Moreover, FOXO1 was found to be involved in regulating the interaction of miR-3188 and mTOR through p-PI3K/p-AKT/c-JUN signaling pathway. Taken together, our study demonstrated that miR-3188 interacts with mTOR and FOXO1 to inhibit NSCLC cell proliferation through a mTOR-p-PI3K/AKT-c-JUN signaling pathway. Therefore, miR-3188 might be a potential target for the treatment of NSCLC.

Chinese herbal decoction of Wenshen Yangxue formula improved fertility and pregnancy rate in mice through PI3K/Akt signaling.

Traditional Chinese medicine (TCM) is an effective management to infertility. The association between TCM-mediated fertility and inhibition of phosphatidylinositol-3-kinase (PI3K) would be investigated. Institute of Cancer Research mice were treated with three herbal decoctions, named Wenshen Yangxue formula, Wenshen formula, and Yangxue formula, plus with human gonadotropins. PI3K inhibitor wortmannin was administrated to half of mice. Some index such as body weight, fertility ability would be investigated. The expression of P13K/Akt signaling was detected by using Western blot analysis. No difference was observed in body weight among groups. Mice receiving the administration of human gonadotropins and herbal decoctions showed increased follicle numbers, percentage of fertilization, and promoted embryonic development. The treatment of Wenshen Yangxue formula decoction showed the highest efficiency, significant higher than Wenshen and Yangxue formulas. And increased the expression of p-PI3K and p-Akt proteins. These results suggested the herbal decoctions promoted the fertilization of mice, which was related to the charge of PI3K/Akt activation.

Effect of Acupuncture on Reproductive Endocrine and p-Akt Protein Expression in Polycystic Ovary Syndrome Rats with Insulin Resistance

To observe the effect of manual acupuncture intervention on the reproductive endocrine and p-Akt protein expression in polycystic ovary syndrome with insulin resistance (PCOS-IR) rats, so as to reveal its mechanism in relieving PCOS-IR. Female Wistar rats were randomly divided into control, model, medication (Metformin) and acupuncture groups (n＝8 in each). The PCOS-IR model was established by subcutaneous injection of Dehydroepiandrosterone (DHEA, 6 mg/100 g), once daily for 20 days. Rats of the medication group were treated by gavage of Metformin solution (100 mg/kg), once daily for 28 days. For rats of the acupuncture group, manual acupuncture stimulation (lifting-thrusting and rotating of needles) was applied to the bilateral "Shenshu"(BL 23), "Yishu"(EX-B 3), "Zigong" (EX-CA 1), "Sanyinjiao"(SP 6), "Fenglong"(ST 40) and "Guanyuan"(CV 4) for 5 min, once a day for 28 days. H．E. staining was used to observe the histopathologic changes of the ovary tissue. Serum levels of follicle-stimulating hormone (FSH), estradiol (E2), luteinizing hormone (LH), testosterone (T) and fasting insulin (FINS) were measured by ELISA. Fasting plasma glucose (FPG) was measured by blood glucose meter, and homeostasis model assessment-insulin resistance index (HOMA-IR) was calculated. Western blot was used to detect the expression of Akt and p-Akt proteins in ovarian tissues. Following modeling, the ovaries showed polycystic changes and an increase of atresia follicles with a larger diameter and no corpus luteum in the model group. After the intervention, there were various types of follicles and a small amount of corpus luteum at different stages of development in both acupuncture and medication groups. In comparison with the control group, serum FSH and E2 levels as well as ovarian Akt and p-Akt protein expression were considerably decreased (P<0.05, P<0.01), and serum LH, T and FINS contents and HOMA-IR were significantly increased in the model group (P<0.01). Following the intervention, the decreased serum FSH and E2 levels as well as ovarian Akt and p-Akt protein expression, and the increased serum LH, T and FINS contents and HOMA-IR were remarkably reversed in both acupuncture and medication groups (P<0.05，P<0.01), without significant differences between the two groups in the levels of those serum and ovary indexes (P>0.05)．. Acupuncture intervention can regulate the levels of reproductive endocrine hormones and reduce insulin resistance in PCOS-IR rats, which may be related to its effect in up- regulating the expression of p-Akt protein in the ovaries.

Ethanol-induced cognitive dysfunction is associated with alterations in the mammalian target of rapamycin signalling pathway in the hippocampus of male mice.

The aim of the present study is to investigate the effect of acute excessive administration of ethanol on the expression of proteins related to the PI3K/Akt/mTOR signalling pathway in the mouse hippocampus and to reveal the possible molecular mechanism of learning and memory deficits induced by ethanol. A total of 120 8-week-old Kunming mice (half male and half female) were randomly assigned into low-dose, moderate-dose, and high-dose male and female groups with intragastric administration of 12.5, 25 and 50% ethanol, respectively, at the dosage of 0.1 ml/10 g·day for 14 days. The male and female control groups received an equal volume of distilled water. Then, the spatial learning and memory of the mice were evaluated by the Morris water maze task. The expression of p-mTOR, p-Akt, mTOR and Akt proteins was tested by western blotting and immunohistochemical staining methods in the hippocampal formation in each group, and haematoxylin-eosin stain was used to identify morphological changes in the hippocampal region. Our results indicated that 25 and 50% ethanol administration led to cognitive dysfunction and hippocampal pyramidal cell impairment in the female and male mice, with the male mice showing more severe impairment. In the 50% ethanol group, the male mice exhibited low expression levels of p-Akt and p-mTOR, but the female mice had no significant differences compared with the respective control group. Interestingly, the male expression levels of p-Akt and p-mTOR were significantly lower than those of females. Overall, these findings suggested that the cognitive deficits induced by ethanol are more serious in male mice than in female mice, and the PI3K/Akt/mTOR signalling pathway in the hippocampus might be involved in the impairment process.

Galangin Induces Apoptosis in MCF-7 Human Breast Cancer Cells Through Mitochondrial Pathway and Phosphatidylinositol 3-Kinase/Akt Inhibition

Aims: The study aimed to investigate the molecular mechanism of inhibition of proliferation and apoptosis induction by galangin against MCF-7 human breast cancer cells. Methods: Cell Counting Kit-8 assay was used to assess cell viability and flow cytometry was used to detect cell apoptosis. The expression level of apoptosis-related proteins (cleaved-caspase-9, cleaved-caspase-8, cleaved-caspase-3, Bad, cleaved-Bid, Bcl-2, Bax, p-phosphatidylinositol 3-kinase [PI3K], and p-Akt) and cell cycle-related proteins (cyclin D3, cyclin B1, cyclin-dependent kinases CDK1, CDK2, CDK4, p21, p27, p53) were evaluated by Western blotting. Results: Galangin increased the expression of Bax and decreased the expression of Bcl-2 in a concentration-dependent manner, inhibited cell viability, and induced apoptosis. Meanwhile, the expression of cleavage of caspase-9, caspase-8, caspase-3, Bid, and Bad increased significantly while the expression of p-PI3K and p-Akt proteins decreased. In addition, the protein levels of cyclin D3, cyclin B1, CDK1, CDK2, and CDK4 were downregulated while the expression levels of p21, p27, and p53 were upregulated significantly. Conclusion: Galangin could suppress the viability of MCF-7 cells and induce cell apoptosis via the mitochondrial pathway and PI3K/Akt inhibition as well as cell cycle arrest.

Expressions of octamer-binding transcription factor 4 and phosphorylated-protein kinase B in colorectal cancer and their clinical significances

Objective To investigate the expressions of octamer-binding transcription factor 4 (Oct4) and phosphorylated-protein kinase B (p-Akt) proteins in colorectal cancer and their clinical significances, in order to explore the roles of Oct4 and p-Akt in the staging and grading of colorectal cancer. Methods Immunohistochemical technique was used to examine the expression of Oct4 and p-Akt proteins in 78 cases of colorectal cancer in Wuxi Second Hospital of Traditional Chinese Medicine and Wuxi First People's Hospital from January 2011 to December 2016. Relationship between expressions of Oct4 and p-Akt proteins and clinicopathological parameters were analyzed. Results The positive rate of Oct4 in tumors was 74.36 % (58/78), which was obviously higher than that in normal tissues [35.90 % (20/78), χ2 = 23.32, P < 0.01]; and the positive rate of p-Akt in tumors was 67.95 % (53/78), which was obviously higher than that in normal tissues[28.21 % (25/78), χ2 = 24.68, P < 0.01]. The double positive and negative expression rate of these two proteins accounted for 80.8 % (63/78), with a linear positive correlation (r = 0.455, P < 0.000 1). In 78 cases of colorectal cancer, the expression of Oct4 protein was correlated with histological grade, lymph node metastasis, and Duke staging (all P < 0.05), and the expression of p-Akt protein was correlated with histological grade and lymph node metastasis (both P < 0.05). The multivariate logistic regression analysis showed that the expression of Oct4 protein was related to histological grade and Duke staging (both P < 0.05), and the expression of p-Akt protein was only related to lymph node metastasis (P < 0.05). Conclusion The combined detection of Oct4 protein and p-Akt protein has reliable and important clinical significance for judging the histological grade, lymph node metastasis and Duke staging of colorectal cancer. Key words: Colorectal neoplasms; Octamer-binding transcription factor 4; Phosphorylated-protein kinase B

Inhibition of PTEN protects PC12 cells against oxygen-glucose deprivation induced cell death through mitoprotection

Mitochondria involve in the determination of ischemic neuronal cell fate through regulation of apoptotic and necrotic cell death. Phosphatase and tensin homolog (PTEN) protein negatively regulates Akt/PKB signaling which is the major cell survival pathway. The current study aimed to examine the impact of SF1670, a potent PTEN inhibitor, on mitochondria-mediated cell survival pathways in an in vitro stroke-like model. PC12 cells were exposed to one hour oxygen and glucose deprivation (OGD) followed by different time points of reperfusion (0, 30, 60, 120 and 180 min) and SF1670 treatments. Our findings showed that OGD/R exposure increased reactive oxygen species (ROS) levels, reduced phosphorylated Akt (p-Akt), ratios of Bcl-2/BAX, intracellular ATP, mitochondrial vital activity and mitochondrial membrane potential (Δψm). OGD/R exposure also increased cleaved caspase 3/pro-caspase 3 and cleaved caspase 9/pro-caspase 9 ratios associated with low cell viability, high lactate dehydrogenase (LDH) release, and greater apoptotic cell death in the TUNEL assay. Conversely, inhibition of PTEN by SF1670 were associated with increased expression of p-Akt and anti-apoptotic proteins (Bcl-2), attenuated pro-apoptotic proteins (BAX) and oxidative stress index (ROS), improved mitochondrial function (restored MMP and ATP), and decreased apoptotic cell death. These results strongly suggest that neuroprotective effect of SF1670 against OGD/R-induced cell death at least is partially mediated through mitoprotective properties of SF1670.

Elevated expression of IRS-1 associates with phosphorylated Akt expression and predicts poor prognosis of breast invasive ductal carcinoma

Overexpression of insulin receptor substrate 1 (IRS-1) has been reported to promote cell growth, atypical hyperplasia, and carcinogenesis, and phosphorylated Akt (p-Akt) is certified to be involved in many types of cancers such as breast invasive ductal carcinoma (BIDC). However, the relationship between IRS-1 and Akt, as well as the role of expression of IRS-1 in BIDC, has never been reported. The purpose of this research is to investigate the association between expression of IRS-1 and p-Akt proteins and clinicopathological features of BIDC by immunohistochemistry, as well as the survival status. The results showed that the percentage of either elevated expression of IRS-1 or positive p-Akt expression in BIDC was significantly higher than that in control breast tissue from noncancer patients (P < .001 and P = .001, respectively). Overexpression of IRS-1 was evidently associated with positive expression of p-Akt (r = 0.337, P < .001). Also, positive percentage of p-Akt expression was statistically different among different molecular subtypes of BIDC (highest in luminal B BIDC, P = .009). Furthermore, significantly worse overall survival was found in BIDC patients with high expression of IRS-1 and p-Akt than in patients with low expression (P = .006 and P = .004, respectively). The multivariate Cox proportional hazard regression analysis showed that high expression of IRS-1 and positive expression of p-Akt protein were independent poor prognostic factors for patients with BIDC (P = .022 and P = .046, respectively). In conclusion, we report for the first time that overexpression of IRS-1 protein is associated with expression of p-Akt, and overexpression of IRS-1 and positive expression of p-Akt might be independent biomarkers for poor prognosis in BIDC.

Expression and clinical significance of proline-rich tyrosine kinase 2 and phospho-protein kinase B in tongue squamous cell carcinoma

This study aimed to investigate the expression and clinical significance of proline-rich tyrosine kinase 2 (Pyk2) and phospho-protein kinase B (p-AKT) in tongue squamous cell carcinoma (TSCC) and adjacent nontumor tissues. The Pyk2 and p-AKT protein levels were detected via immunohistochemistry in 45 cases of TSCC tissues and 30 cases of adjacent nontumor tissues. The relationships of the two protein levels and clinicopathological characteristics were also analyzed. Pyk2 and p-AKT levels were significantly higher in the TSCC tissues than in the adjacent nontumor tissues (P<0.05). Nontumor tissues showed poor or no expression. The expression levels of the two proteins were positively correlated (γs=0.412). The expression of Pyk2 was associated with histopathological differentiation type, regional lymph node metastasis, and TNM staging (P<0.05), but not with age and gender. The expression of p-AKT was only related to histopathological differentiation types (P<0.05). The abnormal expression of Pyk2 and p-AKT proteins might be closely related to the development and progression of TSCC. Joint detection can be used as an indicator to estimate the degree of TSCC.

Upregulation of Neural Precursor Cell Expressed Developmentally Downregulated 4-1 is Associated with Poor Prognosis and Chemoresistance in Lung Adenocarcinoma.

Background:The E3 ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-1 (NEDD4-1) negatively regulates phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein levels through polyubiquitination and proteolysis, but its significance in lung cancer is still unclear. This study investigated the expression and the role of NEDD4-1 in tumor development and chemosensitivity of lung adenocarcinoma (ADC).Methods:We retrospectively investigated the expression and significance of NEDD4-1, PTEN, and p-Akt proteins in 135 paired ADC and adjacent noncancerous tissue specimens using immunohistochemistry. Furthermore, we evaluated the relationship between NEDD4-1 expression and clinicopathologic characteristics and prognosis. The effects of small interfering RNA against NEDD4-1 on proliferation and chemosensitivity were examined in A549 cells in vitro using 3-(4,5-dimethylthiazol-2-yl) -5-(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)- 2H-tetrazolium method. The ability of migration and invasion of A549 cells was tested by transwell assay. Moreover, reverse-transcription quantitative polymerase chain reaction and Western blotting analyses were used to determine the expression of NEDD4-1, PTEN, phosphoinositide 3-kinase (PI3K)/Akt activity, and its downstream target proteins.Results:NEDD4-1 protein was significantly upregulated in lung ADC tissues, whereas it was weak or negative in normal lung epithelial cells. The expression of NEDD4-1 in ADC (78.5%, 106/135) was significantly much higher than that in adjacent normal lung tissue (13.3%, 29/135, P < 0.01), and it was associated with lymph node metastasis, tumor-node-metastasis (TNM) stage, and chemotherapy resistance. PTEN expression was downregulated in lung ADC (60.7% vs. 100.0% in noncancerous specimens, P = 0.007), and was negatively correlated with lymph node metastasis, histological variants, clinical stage, chemoresistance. In addition, expression of p-Akt in ADC tissues (71.1% 96/135) was much higher than that in adjacent lung epithelial cells (6.7%, 9/135, P < 0.01). Kaplan-Meier and multivariate analysis demonstrated that expressions of NEDD4-1 and PTEN were both independent risk factors for survival in patients with lung ADC. NEDD4-1 knockdown in vivo decreased proliferation, migration, and invasion and improved chemosensitivity to cisplatin and paclitaxel in A549 cells. NEDD4-1 knockdown also significantly enhanced PTEN expression and inhibited p-Akt activity and downstream target proteins.Conclusions:NEDD4-1 upregulation may contribute to the progression of lung ADC. NEDD4-1 may regulate the proliferation, invasion, migration, and chemoresistance of lung ADC cells through the PI3K/Akt pathway, suggesting that it may be regarded as a therapeutic target for the treatment of lung ADC.

Mitofusin2 Induces Cell Autophagy of Pancreatic Cancer through Inhibiting the PI3K/Akt/mTOR Signaling Pathway.

Aim Pancreatic cancer is one of the most quickly fatal cancers around the world. Burgeoning researches have begun to prove that mitochondria play a crucial role in cancer treatment. Mitofusin2 (Mfn2) plays an indispensable role in mitochondrial fusion and adjusting function. However, the role and underlying mechanisms of Mfn2 on cell autophagy of pancreatic cancer is still unclear. Our aim was to explore the effect of Mfn2 on multiple biological functions involving cell autophagy in pancreatic cancer. Methods Pancreatic cancer cell line, Aspc-1, was treated with Ad-Mfn2 overexpression. Western blotting, caspase-3 activity measurement, and CCK-8 and reactive oxygen species (ROS) assay were used to examine the effects of Mfn2 on pancreatic cancer autophagy, apoptosis, cell proliferation, oxidative stress, and PI3K/Akt/mTOR signaling. The expression of tissue Mfn2 was detected by immunohistochemical staining. Survival analysis of Mfn2 was evaluated by OncoLnc. Results Mfn2 improved the expression of LC3-II and Bax and downregulated the expression of P62 and Bcl-2 in pancreatic cancer cells. Meanwhile, Mfn2 also significantly inhibited the expression of p-PI3K, p-Akt, and p-mTOR proteins in pancreatic cancer cells. In addition, Mfn2 inhibited pancreatic cancer cell proliferation and ROS production. Assessment of Kaplan-Meier curves showed that Mfn2− pancreatic cancer has a worse prognosis than Mfn2+ pancreatic cancer has. Conclusions Our finding suggests that Mfn2 induces cell autophagy of pancreatic cancer through inhibiting the PI3K/Akt/mTOR signaling pathway. Meanwhile, Mfn2 also influences multiple biological functions of pancreatic cancer cells. Mfn2 may act as a therapeutic target in pancreatic cancer treatment.

Galangin Alleviates Liver Ischemia-Reperfusion Injury in a Rat Model by Mediating the PI3K/AKT Pathway

Background/Aims: Liver ischemia-reperfusion (I/R) injury is a pathological process that often occurs during liver and trauma surgery. There are numerous causes of liver I/R injury, but the mechanism is unknown. Galangin (GA) is a flavonoid, a polyphenolic compound widely distributed in medicinal herbs that has anti-inflammatory, antioxidant, and antitumor activity. This study evaluated the protective effect of GA on hepatic I/R injury. Methods: An I/R model was created in male Wistar rats by clamping the hepatoportal vein, hepatic artery and hepatic duct for 30 min followed by reperfusion for 2 h. A hypoxia/restoration (H/R) model was established in buffalo rat liver (BRL) cells by hypoxia for 4 h followed by normoxic conditions for 10 h. The extent of liver injury was assayed by serum ALT/AST, hepatic histology, and MPO activity. Oxidative stress was assayed by serum superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and malondialdehyde (MDA). Expression of apoptosis-related proteins in BRL cells was assayed in western blots. Expression of AKT and p-AKT proteins in vivo and vitro were assayed in western blots. Results: GA significantly decreased ALT/AST expression, reversed changes in oxidative stress markers induced by I/R, and mediated caspase-3 activity expression of apoptosis-related proteins in vivo and in vitro. Methylthiazol tetrazolium (MTT) assay, flow cytometry, and Hoechst 33258 staining confirmed that GA inhibited apoptosis of BRL cells. GA also increased the expression of phosphorylated AKT after H/R. Conclusion: GA reduced liver I/R injury both in vivo and vitro and inhibited BRL cell apoptosis. PI3K/AKT signaling have been involved. GA may protect against liver I/R and be a potential therapeutic candidate.

Timosaponin AIII inhibits the growth of human leukaemia cells HL-60 by down-regulation of PI3K/AKT and Wnt/β-catenin pathways

ABSTRACTThe effect of timosaponin AIII (TSA-III) on human leukaemia HL-60 cells and the underlying mechanism of action were explored in this study. HL-60 cells were treated with different concentrations of TSA-III, and then the survival rate and apoptosis rate of the cells, expression of apoptosis-related genes, activation of caspase-3, cell distribution in the cell cycle phases, and expression level of the related signal pathway proteins were detected. The results showed that TSA-III could dose- and time-dependently inhibit the proliferation of HL-60 cells. TSA-III could also increase the apoptosis rate of HL-60 cells, the level of Bax and Bak mRNA and the activation rate of caspase-3, and decrease the level of Bcl-2 mRNA. At the same time, the proportion of HL-60 cells treated with TSA-III in G0/G1 phase increased, whereas the proportion of the cells in S phase decreased. In addition, TSA-III could elevate the expression of PTEN protein, and reduce the expression of p-AKT, β-catenin, Cyclin D1 and C-myc proteins. These results indicate that TSA-III should have a cytotoxic effect on HL-60 cells, mainly showing its inhibition on the proliferation, inducing the apoptosis and cell cycle arrest, and the mechanism may be related to its inhibition of PI3K/AKT and Wnt/β-catenin pathways.

Neuroprotection of dexmedetomidine against propofol-induced neuroapoptosis partly mediated by PI3K/Akt pathway in hippocampal neurons of fetal rat

The aim was to investigate how the PI3K/Akt pathway is involved in the protection of dexmedetomidine against propofol. The hippocampal neurons from fetal rats were separated and cultured in a neurobasal medium. Cell viability was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Then neurons were pretreated with different concentrations of dexmedetomidine before 100 μmol/L propofol was added. Akt, phospho-Akt (p-Akt), Bad, phospho-Bad (p-Bad), and Bcl-xL were detected by Western blot. Also, neurons were pretreated with dexmedetomidine alone or given the inhibitor LY294002 before dexmedetomidine pretreatment, and then propofol was added for 3 h. The results demonstrated that propofol decreased the cell viability and the expression of p-Akt and p-Bad proteins, increased the level of Bad, and reduced the ratio of Bcl-xL/Bad. Dexmedetomidine pretreatment could reverse these effects. The enhancement of p-Akt and p-Bad induced by dexmedetomidine was prevented by LY294002. These results showed that dexmedetomidine potently protected the developing neuron and this protection may be partly mediated by the PI3K/Akt pathway.