Objective: To explore the protective effects of dexmedetomidine (Dex) against high glucose-induced epithelial-mesenchymal transition in HK-2 cells and relevant mechanisms. Methods: HK-2 cells were exposed to either glucose or glucose+Dex for 6 h. The production of ROS, morphology of HK-2 cells, and cell cycle were detected. Moreover, the expression of AKT, p-AKT, ERK, p-ERK, PI3K, E-Cadherin, Claudin-1, and α-SMA were determined and compared between HK-2 cells exposed to glucose and those exposed to both glucose and Dex with or without PI3K/AKT pathway inhibitor LY294002 and ERK pathway inhibitor U0126. Results: Compared with HK-2 cells exposed to high level of glucose, the HK-2 cells exposed to both high level of glucose and Dex showed: (1) lower level of ROS production; (2) cell morphology was complete; (3) more cells in G1 phase; (4) lower expression of p-AKT, p-ERK and α-SMA, higher expression of E-Cadherin and Claudin-1. PI3K/AKT inhibitor LY294002 and ERK inhibitor U0126 decreased the expression of p-AKT, p-ERK and α-SMA, and increased the expression of E-Cadherin and Claudin-1. Conclusion Dex can attenuate high glucose-induced HK-2 epithelial-mesenchymal transition by inhibiting AKT and ERK.
Adrenergic alpha-2 Receptor Agonists ; pharmacology ; Cell Line ; Dexmedetomidine ; pharmacology ; Epithelial-Mesenchymal Transition ; drug effects ; Glucose ; metabolism ; Humans ; MAP Kinase Signaling System ; drug effects ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; Signal Transduction ; drug effects

BACKGROUND: Long noncoding RNAs (lncRNAs) play pivotal roles in various malignant tumors. Epidermal growth factor receptor (EGFR) signaling is associated with the pathogenesis of cutaneous squamous cell carcinoma (cSCC). This study aimed to explore the role of LINC00520 in the development of cSCC via EGFR and phosphoinositide 3-kinase-protein kinase B (PI3K/Akt) signaling pathways. METHODS: A microarray analysis was applied to screen differentially expressed lncRNAs in cSCC samples. The A431 cSCC cell line was transfected and assigned different groups. The expression patterns of LINC00520, EGFR, and intermediates in the PI3K/Akt pathway were characterized using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting analysis. Cell proliferation, migration, and invasion were detected using the MTT assay, scratch test, and Transwell assay, respectively. Cell-based experiments and a tumorigenicity assay were conducted to assess the effect of LINC00520 on cSCC progression. This study was ended in September 2017. Comparisons between two groups were analyzed with t-test and comparisons among multiple groups were analyzed using one-way analysis of variance. The nonparametric Wilcoxon rank sum test was used to analyze skewed data. The enumerated data were analyzed using the chi-square test or Fisher exact test. RESULTS: Data from chip GSE66359 revealed depletion of LINC00520 in cSCC. Cells transfected with LINC00520 vector and LINC00520 vector + si-EGFR showed elevated LINC00520 level but decreased levels of the EGFR, PI3K, AKT, VEGF, MMP-2 and MMP-9 mRNAs and proteins, and inhibition of the growth, migration and adhesion of cSCC cells, while the si-LINC00520 group showed opposite trends (all P < 0.05). Compared with the LINC00520 vector group, the LINC00520 vector + si-EGFR group showed decreased levels of the EGFR, PI3K, AKT, VEGF, MMP-2 and MMP-9 mRNAs and proteins, and inhibition of the growth, migration and adhesion of cSCC cells, while the LINC00520 vector + EGFR vector group showed opposite results (all P < 0.05). CONCLUSION Based on our results, LINC00520-targeted EGFR inhibition might result in the inactivation of the PI3K/Akt pathway, thus inhibiting cSCC development.
Animals ; Carcinoma, Squamous Cell ; pathology ; prevention & control ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Disease Progression ; ErbB Receptors ; antagonists & inhibitors ; Female ; Humans ; Lymphatic Metastasis ; Mice ; Neoplasm Invasiveness ; Phosphatidylinositol 3-Kinases ; physiology ; Proto-Oncogene Proteins c-akt ; physiology ; RNA, Long Noncoding ; physiology ; Signal Transduction ; physiology ; Skin Neoplasms ; pathology ; prevention & control

OBJECTIVE: To study the effects of the overexpression of autophagy-related gene 3 (ATG3) on autophagy and salinomycin-induced apoptosis in breast cancer cells and explore the underlying mechanisms. METHODS: We used the lentivirus approach to establish a breast cancer cell line with stable overexpression of ATG3. Western blotting, immunofluorescence staining and transmission electron microscopy were used to analyze the effect of ATG3 overexpression on autophagy in breast cancer MCF-7 cells. Using the AKT/mTOR agonists SC79 and MHY1485, we analyzed the effect of AKT/mTOR signal pathway activation on ATG3 overexpression-induced autophagy. Western blotting and flow cytometry were used to analyze the effect of autophagy on apoptosis of the ATG3-overexpressing cells treated with salinomycin and 3-MA (an autophagy inhibitor). RESULTS: In ATG3-overexpressing MCF-7 cells, ATG3 overexpression obviously promoted autophagy, inhibited the AKT/mTOR signaling pathway, significantly weakened salinomycin-induced apoptosis ( < 0.01), caused significant reduction of the levels of the pro-apoptotic proteins cleaved-caspase 3 ( < 0.01) and Bax ( < 0.05), and enhanced the expression of the anti-apoptotic protein Bcl-2 ( < 0.05). The inhibition of autophagy obviously weakened the inhibitory effect of ATG3 overexpression on salinomycin-induced apoptosis. CONCLUSIONS ATG3 overexpression promotes autophagy possibly by inhibiting the AKT/mTOR signaling pathway to decrease salinomycin-induced apoptosis in MCF-7 cells, suggesting that autophagy induction might be one of the mechanisms of drug resistance in breast cancer cells.
Acetates ; pharmacology ; Apoptosis ; drug effects ; genetics ; Autophagy ; drug effects ; Autophagy-Related Proteins ; metabolism ; Benzopyrans ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Drug Resistance, Neoplasm ; Female ; Gene Expression Regulation ; Humans ; MCF-7 Cells ; Morpholines ; pharmacology ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; metabolism ; Pyrans ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism ; Triazines ; pharmacology ; Ubiquitin-Conjugating Enzymes ; metabolism

Accumulating data have revealed that abnormal activity of the mTOR (mammalian target of rapamycin) pathway plays an important role in epileptogenesis triggered by various factors. We previously reported that pretreatment with perifosine, an inhibitor of Akt (also called protein kinase B), abolishes the rapamycin-induced paradoxical increase of S6 phosphorylation in a rat model induced by kainic acid (KA). Since Akt is an upstream target in the mTOR signaling pathway, we set out to determine whether perifosine has a preventive effect on epileptogenesis. Here, we explored the effect of perifosine on the model of temporal epilepsy induced by KA in rats and found that pretreatment with perifosine had no effect on the severity or duration of the KA-induced status epilepticus. However, perifosine almost completely inhibited the activation of p-Akt and p-S6 both acutely and chronically following the KA-induced status epilepticus. Perifosine pretreatment suppressed the KA-induced neuronal death and mossy fiber sprouting. The frequency of spontaneous seizures was markedly decreased in rats pretreated with perifosine. Accordingly, rats pretreated with perifosine showed mild impairment in cognitive functions. Collectively, this study provides novel evidence in a KA seizure model that perifosine may be a potential drug for use in anti-epileptogenic therapy.
Animals ; Anticonvulsants ; pharmacology ; Brain ; drug effects ; pathology ; Convulsants ; toxicity ; Disease Models, Animal ; Epilepsy, Temporal Lobe ; chemically induced ; pathology ; Kainic Acid ; toxicity ; Male ; Neurons ; drug effects ; pathology ; Phosphorylcholine ; analogs & derivatives ; pharmacology ; Protein Kinase Inhibitors ; pharmacology ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; Rats ; Rats, Sprague-Dawley ; Status Epilepticus ; chemically induced ; pathology

OBJECTIVETo observe the effects of estrogen on epidermis growth of mice and proliferation of keratinocytes (human epidermal cell line HaCaT), and to explore its mechanism.

METHODS(1) Five adult C57BL/6 mice in estrus cycle were identified by vaginal exfoliative cytology diagnosis and set as estrus group, while another 5 adult C57BL/6 mice with ovary resected before sexual development were set as ovariectomized group. The full-thickness skin from the tail root of mice in two groups were collected. The thickness of epidermis was observed and measured after HE staining. The distribution of proliferating cell nuclear antigen (PCNA)-positive cells in epidermis was observed by immunohistochemical staining, the number of which was counted. (2) HaCaT cells in logarithmic growth phase were cultured with RPMI 1640 nutrient solution containing 10% fetal bovine serum, and they were divided into negative control group (NC), pure estradiol group (PE), protein kinase B (Akt) inhibitor group (AI), and extracellular signal-regulated kinase (ERK) inhibitor group (EI) according to the random number table, with 20 wells in each group. To nutrient solution of each group, 1 μL dimethyl sulfoxide, 1 μL 17β-estradiol (100 nmol/L), 1 μL LY294002 (10 μmol/L), and 1 μL PD98059 (30 μmol/L) were added in group NC, group PE, group AI, and group EI respectively, and the last two groups were added with 1 μL 17β-estradiol (100 nmol/L) in addition. At post culture hour (PCH) 0 (immediately after culture), 24, 48, 72, 5 wells of cells from each group were collected to detect the proliferation activity of cells by cell counting kit 8 and microplate reader. (3) HaCaT cells in logarithmic growth phase were collected, grouped, and treated with the above-mentioned methods, with 3 wells in each group. At PCH 72, cell cycle distribution was detected by flow cytometer to calculate proliferation index (PI) of cells. (4) HaCaT cells in logarithmic growth phase were collected, grouped, and treated with the above-mentioned methods, with 3 dishes in each group. At PCH 72, the protein levels of phosphorylated Akt (p-Akt), phosphorylated ERK (p-ERK), and PCNA were determined with Western blotting. The cell experiments were repeated for 3 times. Data were processed with t test, one-way analysis of variance, analysis of variance of factorial design, and LSD test.

RESULTS(1) The epidermis thickness of mice in ovariectomized group was (33.5±3.0) μm, which was obviously thinner than that in estrus group [(51.4±3.1) μm, t=20.7, P<0.01]. The PCNA-positive cells mainly aggregated in the basal layer of epidermis of mice in two groups. The number of PCNA-positive cells in epidermis of mice in ovariectomized group was 37±12 per 200 fold visual field, obviously fewer than that in estrus group (96±15 per 200 fold visual field, t=15.3, P<0.01). (2) During PCH 0 to 48, there were no significant differences in the proliferation activity of cells between group PE and group NC (with P values above 0.05). At PCH 72, compared with that in group NC, the proliferation activity of cells in group PE was obviously increased (P<0.01). The proliferation activity of cells in groups AI and EI was obviously lower than that in the previous two groups (with P values below 0.01). (3) Compared with that in group NC [(51.6±1.1)%], the PI of cells in group PE was obviously increased [(58.5±0.8)%, P<0.05]. The PI values of cells in groups AI and EI were (34.9±0.8)% and (48.2±0.4)% respectively, both obviously lower than those in the previous two groups (with P values below 0.01). (4) Compared with that of group NC (0.566±0.034), the protein level of p-Akt in cells of group PE was significantly increased (1.048±0.077, P<0.01). Compared with that of group PE, the protein level of p-Akt was obviously decreased in cells of groups AI and EI (respectively 0.682±0.095 and 0.672±0.019, with P values below 0.01). Compared with that of group NC (0.469±0.013), the protein level of p-ERK obviously increased in cells of groups PE, AI, and EI (respectively 1.064±0.089, 1.010±0.038, 0.778±0.065, with P values below 0.01). The protein level of p-ERK in cells of group EI was obviously lower than that in group PE (P<0.01). Compared with that of group NC (0.386±0.053), the protein level of PCNA was obviously increased in cells of group PE (0.743±0.043, P<0.01). The protein levels of PCNA in cells of groups AI and EI were 0.264±0.019 and 0.223±0.065 respectively, both obviously lower than those in the previous two groups (with P values below 0.01).

CONCLUSIONSLack of estrogen damages the growth ability of epidermis of mice. Estrogen (17β-estradiol) can promote the proliferation of HaCaT cells by increasing the expression of PCNA via activating ERK/Akt signaling pathway.

Animals ; Cell Cycle ; Cell Line ; Cell Proliferation ; drug effects ; Epidermis ; cytology ; drug effects ; growth & development ; Estradiol ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Female ; Humans ; Keratinocytes ; cytology ; drug effects ; Mice ; Mice, Inbred C57BL ; Phosphorylation ; Proliferating Cell Nuclear Antigen ; metabolism ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; Signal Transduction

β3-adrenoceptor (β3-AR) has been shown to promote myocardial apoptosis. However, the exact physiological role and importance of this receptor in the human myocardium, and its underlying mode of action, have not been fully elucidated. The present study aimed to determine the effects of β3-AR on the promotion of myocardial apoptosis and on norepinephrine (NE) injury. We analyzed NE-induced cardiomyocyte (CM) apoptosis by using a TUNEL and an annexin V/propidium iodide apoptosis assay. Furthermore, we investigated the NE-induced expression of the apoptosis marker genes Akt and p38MAPK, their phosphorylated counterparts p-Akt and p-p38MAPK, caspase-3, Bcl-2, and Bax. In addition, we determined the effect of a 48-h treatment with a β3-AR agonist and antagonist on expression of these marker genes. β3-AR overexpression was found to increase CM apoptosis, accompanied by an increased expression of caspase-3, bax/bcl-2, and p-p38MAPK. In contrast, the β3-blocker reduced apoptosis of CMs and the associated elevated Akt expression. We identified a novel and potent anti-apoptosis mechanism via the PI3K/Akt pathway and a pro-apoptosis pathway mediated by p38MAPK.
Adrenergic Agonists ; pharmacology ; Adrenergic Antagonists ; pharmacology ; Animals ; Apoptosis ; Cells, Cultured ; Myocytes, Cardiac ; drug effects ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Adrenergic, beta-3 ; genetics ; metabolism ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases ; metabolism

SIRT6 is a NAD(+)-dependent histone deacetylase and has been implicated in the regulation of genomic stability, DNA repair, metabolic homeostasis and several diseases. The effect of SIRT6 in cerebral ischemia and oxygen/glucose deprivation (OGD) has been reported, however the role of SIRT6 in oxidative stress damage remains unclear. Here we used SH-SY5Y neuronal cells and found that overexpression of SIRT6 led to decreased cell viability and increased necrotic cell death and reactive oxygen species (ROS) production under oxidative stress. Mechanistic study revealed that SIRT6 induced autophagy via attenuation of AKT signaling and treatment with autophagy inhibitor 3-MA or knockdown of autophagy-related protein Atg5 rescued H2O2-induced neuronal injury. Conversely, SIRT6 inhibition suppressed autophagy and reduced oxidative stress-induced neuronal damage. These results suggest that SIRT6 might be a potential therapeutic target for neuroprotection.
Adenine ; analogs & derivatives ; toxicity ; Autophagy ; drug effects ; Autophagy-Related Protein 5 ; antagonists & inhibitors ; genetics ; metabolism ; Blotting, Western ; Cell Line, Tumor ; Humans ; Hydrogen Peroxide ; toxicity ; Microtubule-Associated Proteins ; metabolism ; Oxidative Stress ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; metabolism ; Reactive Oxygen Species ; metabolism ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; drug effects ; Sirtuins ; antagonists & inhibitors ; genetics ; metabolism ; Transfection

The phosphatidylinositol 3 kinase (PI3K) pathway is frequently altered in cancer, including ovarian cancer (OC). Unfortunately, despite a sound biological rationale and encouraging activity in preclinical models, trials of first-generation inhibitors of mammalian target of rapamycin (mTOR) in OC have demonstrated negative results. The lack of patient selection as well as resistance to selective mTOR complex-1 (mTORC1) inhibitors could explain the disappointing results thus far. Nonetheless, a number of novel agents are being investigated, including dual mTORC1/mTORC2, Akt, and PI3K inhibitors. Although it is likely that inhibition of the PI3K/Akt/mTOR pathway may have little effect in unselected OC patients, certain histological types, such as clear cell or endometrioid OC with frequent phosphatidylinositol-4,5-biphosphate 3-kinase, catalytic subunit alpha (PIK3CA) and/or phosphatase and tensin homolog (PTEN) alterations, may be particularly suited to this approach. Given the complexity and redundancy of the PI3K signaling network, PI3K pathway inhibition may be most useful in combination with either chemotherapy or other targeted therapies, such as MEK inhibitors, anti-angiogenic therapy, and hormonal therapy, in appropriately selected OC patients. Here, we discuss the relevance of the PI3K pathway in OC and provide an up-to-date review of clinical trials of novel PI3K inhibitors alone or in combination with cytotoxics and novel therapies in OC. In addition, the challenges of drug resistance and predictive biomarkers are addressed.
Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Drug Resistance, Neoplasm ; Female ; Humans ; Ovarian Neoplasms ; drug therapy ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; physiology ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; physiology ; Signal Transduction ; drug effects ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; physiology

PURPOSE: To investigate the effects of resveratrol on the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in human adult retinal pigment epithelial (ARPE-19) cells, and on experimental choroidal neovascularization (CNV) in mice. MATERIALS AND METHODS: ARPE-19 cells were treated with different concentrations of resveratrol and then incubated under hypoxic conditions with subsequent evaluation of cell viability, expression of HIF-1alpha, and expression of VEGF. The effects of resveratrol on the synthesis and degradation of hypoxia-induced HIF-1alpha were evaluated using inhibitors of the PI3K/Akt/mTOR and the ubiquitin proteasome pathways. In animal studies, CNV lesions were induced in C57BL/6 mice by laser photocoagulation. After 7 days of oral administration of resveratrol or vehicle, which began one day after CNV induction, image analysis was used to measure CNV areas on choroidal flat mounts stained with isolectin IB4. RESULTS: In ARPE-19 cells, resveratrol significantly inhibited HIF-1alpha and VEGF in a dose-dependent manner, by blocking the PI3K/Akt/mTOR signaling pathway and by promoting proteasomal HIF-1alpha degradation. In mice experiments, orally administered resveratrol significantly inhibited CNV growth in a dose-dependent manner. CONCLUSION: Resveratrol may have therapeutic value in the management of diseases involving pathological neovascularization.
Adult ; Animals ; Anoxia/metabolism/physiopathology ; Cell Survival/drug effects ; Choroidal Neovascularization/*metabolism/pathology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/*drug effects/metabolism ; Mice ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases/antagonists & inhibitors/*physiology ; Proteasome Endopeptidase Complex ; Proto-Oncogene Proteins c-akt/antagonists & inhibitors/*physiology ; Retinal Pigment Epithelium/*drug effects/metabolism ; Signal Transduction ; Stilbenes/administration & dosage/*pharmacology ; TOR Serine-Threonine Kinases/antagonists & inhibitors/*physiology ; Ubiquitin ; Vascular Endothelial Growth Factor A/*drug effects/metabolism

Humans ; Leukemia ; drug therapy ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; TOR Serine-Threonine Kinases ; antagonists & inhibitors