Protein Expression Levels Of p-Akt Research Articles

Sevoflurane postconditioning attenuates cardiomyocytes hypoxia/reoxygenation injury via PI3K/AKT pathway mediated HIF-1α to regulate the mitochondrial dynamic balance

BackgroundMyocardial ischemia-reperfusion injury (I/RI) is a major cause of perioperative cardiac-related adverse events and death. Studies have shown that sevoflurane postconditioning (SpostC), which attenuates I/R injury and exerts cardioprotective effects, regulates mitochondrial dynamic balance via HIF-1α, but the exact mechanism is unknown. This study investigates whether the PI3K/AKT pathway in SpostC regulates mitochondrial dynamic balance by mediating HIF-1α, thereby exerting myocardial protective effects.MethodsThe H9C2 cardiomyocytes were cultured to establish the hypoxia-reoxygenation (H/R) model and randomly divided into 4 groups: Control group, H/R group, sevoflurane postconditioning (H/R + SpostC) group and PI3K/AKT blocker (H/R + SpostC + LY) group. Cell survival rate was determined by CCK-8; Apoptosis rate was determined by flow cytometry; mitochondrial membrane potential was evaluated by Mito Tracker™ Red; mRNA expression levels of AKT, HIF-1α, Opa1and Drp1 were detected by quantitative real-time polymerase chain reaction (qRT-PCR); Western Blot assay was used to detect the protein expression levels of AKT, phosphorylated AKT (p-AKT), HIF-1α, Opa1 and Drp1.ResultsCompared with the H/R group, the survival rate of cardiomyocytes in the H/R + SpostC group increased, the apoptosis rate decreased and the mitochondrial membrane potential increased. qRT-PCR showed that the mRNA expression of HIF-1α and Opa1 were higher in the H/R + SpostC group compared with the H/R group, whereas the transcription level of Drp1 was lower in the H/R + SpostC group. In the H/R + SpostC + LY group, the mRNA expression of HIF-1α was lower than the H/R + SpostC group. There was no difference in the expression of Opa1 mRNA between the H/R group and the H/R + SpostC + LY group. WB assay results showed that compared with the H/R group, the protein expression levels of HIF-1α, Opa1, P-AKT were increased and Drp1 protein expression levels were decreased in the H/R + SpostC group. HIF-1α, P-AKT protein expression levels were decreased in the H/R + SpostC + LY group compared to the H/R + SpostC group.ConclusionSpostC mediates HIF-1α-regulated mitochondrial fission and fusion-related protein expression to maintain mitochondrial dynamic balance by activating the PI3K/AKT pathway and increasing AKT phosphorylation, thereby attenuating myocardial I/R injury.

Semaphorin‑3A alleviates cardiac hypertrophy by regulating autophagy.

Cardiac hypertrophy, characterized by cardiomyocyte enlargement, is an adaptive response of the heart to certain hypertrophic stimuli; however, prolonged hypertrophy results in cardiac dysfunction and can ultimately cause heart failure. The present study evaluated the role of semaphorin-3A (Sema3A), a neurochemical inhibitor, in cardiac hypertrophy, utilizing an isoproterenol (ISO) induced H9c2 cell model. Cells were stained with rhodamine-phalloidin to assess the cell surface area and reverse transcription-quantitative PCR was performed to quantify mRNA expression levels of Sema3A, brain natriuretic factor (BNF) and β-myosin heavy chain (β-MHC). The protein expression levels of the autophagy-related proteins light chain 3 (LC3), p62 and Beclin-1, and the Akt/mTOR signaling pathway associated proteins Akt, phosphorylated (p)-Akt, mTOR, p-mTOR, 4E-binding protein 1 (4EBP1) and p-4EBP1 were semi-quantified using western blotting. Rapamycin, a canonical autophagy inducer, was administered to H9c2 cells to elucidate the regulatory mechanism of Sema3A. The results indicated significantly increased cell surface area and elevated BNF and β-MHC mRNA expression levels, increased LC3II/I ratio and Beclin-1 protein expression levels and significantly decreased p62 protein expression levels after treatment of H9c2 cardiomyocytes with ISO for 24 h. Sema3A overexpression improved ISO-induced hypertrophy in H9c2 cells, indicated by decreased cell surface area and reduced BNF and β-MHC mRNA expression levels. Moreover, Sema3A overexpression inhibited ISO-induced autophagy in H9c2 cells, indicated by decreased LC3II/I ratio and Beclin-1 protein expression levels and increased p62 protein expression levels. The autophagy activator rapamycin partially inhibited the protective effect of Sema3A on ISO-induced hypertrophy. Sema3A overexpression suppressed the decrease of the protein expression levels of p-Akt, mTOR and their downstream target 4EBP1, which is induced by ISO. Collectively, these results suggested Sema3A prevented ISO-induced cardiac hypertrophy by inhibiting autophagy via the Akt/mTOR signaling pathway.

HGG-08. COMBINATORIAL ACTIVITY OF CDK4/6-INHIBITORS AND TRAMETINIB IN PEDIATRIC HIGH-GRADE GLIOMA WITH BRAFV600E MUTATION AND HOMOZYGOUS CDKN2A DELETION

Abstract BRAFV600E mutations occur in 10-15% and homozygous CDKN2A deletions in approximately 40% of adolescent and pediatric high-grad glioma (HGG) and is associated with significantly decreased overall survival. Moreover, a subset of pHGG is defined by combined BRAFV600E mutation and concomitant CDKN2A homozygous deletion. Standard therapy has remained unchanged and consists of maximal safe resection and focal radiotherapy still resulting in poor overall survival. Therefore, new therapeutic approaches to increase patient survival are of utmost importance. Targeted therapy of BRAFV600E mutant HGG has emerged as effective treatment but remains challenging due to therapy resistance. CDK4/6 inhibitors represent a promising target in CDKN2A altered HGG. However, palbociclib demonstrated only low efficacy in HGG patients. Therefore, combination approaches may be more effective, and we investigated CDK4/6-inhibitors alone and in combination with trametinib in HGG cell models with BRAFV600E mutation and homozygous CDKN2A deletion. Efficacy of CDK4/6-inhibitor monotherapy and combinatorial approach with trametinib was assessed by short- and long-term viability assays in four patient-derived HGG cell models with homozygous CDKN2A deletion and BRAFV600E mutation. Furthermore, effects of CDK4/6 inhibitor therapy alone or in combination with trametinib on downstream signaling pathways (MAPK, PI3K) were analyzed with Western blots. Abemaciclib showed the highest activity in all cell models with IC50-values ranging from 0,5 – 2µM. Combined treatment approaches with trametinib showed synergistic effects across all cell models. Long-term viability assays revealed distinct sensitivity in the nanomolar range for CDK4/6-inhibitors and combined treatment with trametinib in all cell models. Exposure to abemaciclib and trametinib significantly decreased pRB, pERK, pS6, and pAKT protein expression levels when compared to monotherapy alone. Summarizing, combined treatment with CDK4/6-inhibitors and trametinib showed promising therapeutic effects on HGG models with homozygous CDKN2A deletion and BRAFV600E mutation. Currently, effects on cell cycle distribution and downstream molecular mechanisms are investigated to identify potential predictive biomarkers.

Elemene inhibits the growth and promotes apoptosis of bladder cancer cells through PTEN-Akt signaling pathway

Purpose: To investigate the influence of elemene on growth and apoptotic changes in bladder cancer cells, and the involvement of the phosphatase and tensin homologous protein (PTEN)-serine/threonine kinase (Akt) signal pathway in the process.
 Methods: Human bladder cell line SV-HUC-1 and human bladder cancer cell line T24 were randomly assigned to blank control group and elemene groups. Protein expression levels of apoptosis-related factors (Bcl-2, Bax, and caspase-3] and PTEN, Akt, and p-Akt in both cells were determined using western blot assay.
 Results: In T24 cells, the protein level of Bcl-2 was significantly higher in elemene group than in blank control, while the protein expression levels of Bax and caspase-3 in elemene group were significantly lower than the corresponding levels in blank control group (p < 0.05). The protein expression of PTEN level in T24 cells was significantly higher in elemene group than in blank control group, while protein expression level of p-Akt was significantly lower in elemene group than in blank control (p < 0.05). The Akt protein expression was comparable in elemene and blank control groups.
 Conclusion: Elemene inhibits the growth and enhances apoptosis of bladder cancer cells through a mechanism involving up-regulation of the expression of PTEN and suppression of the expression of pAkt.

Effect of aerobic exercise on AKT/GSK3β mediated hepatocyte apoptosis in nonalcoholic fatty liver disease

To investigate the effect of aerobic exercise on AKT/GSK3β pathway-mediated hepatocyte apoptosis in non-alcoholic fatty liver diseases(NAFLD). A total of 30 6-week-old male C57BL/6J mice, and mice were fed adaptively for one week. The control group was fed with ordinary diet, and the model group and model exercise group were fed with high-fat diet until 18 weeks. At the 10th week of the experiment, the model exercise group received aerobic exercise intervention for 8 consecutive weeks until the end of the experiment at the 18th week. Automatic biochemical analyzer to detect serum total cholesterol(TC), triglycerides(TG), alanine aminotransferase(ALT), aspartate aminotransferase(AST), low-density lipoprotein(LDL-C) and high-density lipoprotein(HDL-C) levels. Liver pathological morphology was observed by staining with oil red O and HE. The expression changes of AKT, P-AKT~( Ser473), GSK3β, P-GSK3β~(Ser9) and Caspase-3 proteins were detected by western blot, and the apoptosis of hepatocytes was detected by in situ terminal transferase labeling(TUNEL). (1) After intervention, compared with control group, body weight, liver index, serum TC, TG, ALT, AST and LDL-C levels in model group were significantly increased(P<0.01 or P<0.05), while HDL-C level was significantly decreased(P<0.01). Compared with model group, body weight, liver index, serum TC, TG, ALT, AST and LDL-C levels in model exercise group were significantly decreased(P<0.01 or P<0.05), while HDL-C level was significantly increased(P<0.01). (2) Compared with the control group, hepatocyte steatosis and the number of lipid droplets in model group were significantly increased. Compared with the model group, the degree of hepatic adipose degeneration was significantly improved and the number of hepatic lipid droplets was significantly decreased in the model exercise group. (3) Compared with control group, the protein expression levels of P-AKT~(Ser473) and P-GSK3β~(Ser9) in model group were significantly decreased(P<0.01 or P<0.05), the protein expression levels of Caspase-3 were significantly increased(P<0.05), and the number of hepatocyte apoptosis was significantly increased(P<0.05). Compared with model group, the expression of P-AKT~(Ser473) and P-GSK3β~(Ser9) protein in model exercise group was significantly increased(P<0.01 or P<0.05), the expression of Caspase-3 protein was significantly decreased(P<0.05), and the number of hepatocyte apoptosis was significantly decreased(P<0.01). Aerobic exercise can effectively improve NAFLD, by activating AKT/GSK3β pathway and increasing the expression of AKT/GSK3β pathway related molecules, thereby reducing caspase-3 expression and hepatocyte apoptosis.

Insulin ameliorates dim blue light at night-induced apoptosis in hippocampal neurons via the IR/IRS1/AKT/GSK3β/β-catenin signaling pathway

In recent years, the damaging effects of night light pollution, one of the environmental pollutions, on memory has been attracting attention. However, the underlying molecular mechanisms by which light at night, especially blue light at night, impairs memory remains unclear. Here, a total of 42 C57BL6/J mice that exposed to no light at night, dim white light at night (dLAN-WL), or dim blue light at night (dLAN-BL) for 28 days. Behavioral data indicated that exposure to dLAN-BL resulted in severe recognition memory impairment, as evidenced by the reduced recognition index and discrimination index in the novel object recognition test. At the same time, we observed a decrease in plasma insulin levels. Consistent with these changes, we also observed that dLAN-BL reduced the number of neurons in the CA1, CA3 and DG regions of the hippocampus, up-regulated the mRNA expression levels of Bax, down-regulated the mRNA expression levels of Bcl-2, Bcl-xl and the protein expression level of pIRS1, pAKT, pGSK3β, β-catenin in the hippocampus. In vitro experiments, we found that insulin (10 nM) inhibited apoptosis and up-regulated the protein expression levels of pAKT, pGSK3β, β-catenin of HT22 cells induced by H2O2 (200 μM). However, these changes disappeared when the insulin receptors (IR) in HT22 cells were silenced. Taken together, our findings suggested that the impairment of memory in mice induced by dLAN-BL was mediated by insulin via the IR/IRS1/AKT/GSK3β/β-catenin pathway. Data AvailabilityAll data generated or analyzed during this study are included in this published article.

Secukinumab alleviates cognitive impairment by attenuating oxidative stress and neuronal apoptosis via the IL-17RA/AKT/ERK1/2 pathway in a rat model of sepsis

BackgroundSeptic-associated encephalopathy (SAE) is a critical manifestation of sepsis that leads to long-term cognitive impairment. Interleukin (IL)-17A has been shown to mediate neuronal apoptosis in central nervous system diseases, while oxidative stress has been found to have a detrimental effect in SAE. However, the relationship between IL-17A and oxidative stress in SAE remains unclear. This study aimed to investigate the effects of secukinumab on alleviating cognitive impairment in a rat model of sepsis, as well as examine its underlying molecular mechanism of action. MethodsA total of 282 male 8-week-old Sprague-Dawley rats were randomly subjected to cecal ligation and puncture (CLP) or sham treatment followed by volume resuscitation immediately after surgery. Secukinumab was administered intranasally 1 h post-CLP. Rats were given the p-ERK activator ceramide C6 intracerebroventricularly (i.c.v) 24 h before CLP surgery. Recombinant rIL-17A was administered i.c.v. at 0 h in naive rats, followed by intraperitoneal injection of the AKT inhibitor GDC0068 1 h post-rIL-17A injection. Clinical scores, body weight, and survival rate were assessed. In addition, immunofluorescence staining, neurobehavioral tests, Nissl staining, and western blotting were performed. Cognitive function was assessed 15–20 days post-CLP using the Morris water maze test. ResultsIL-17A and IL-17RA protein expression levels in the rat hippocampus increased and peaked 24 h post-CLP. Furthermore, IL-17RA was found to be expressed in neurons. The survival rate after CLP was 50%. Following CLP, an increased clinical score and significant decrease in body weight were observed. However, treatment with secukinumab led to a decrease in the clinical score of rats 24 h post-CLP. CLP resulted in spatial and memory impairment and anxiety-like behaviors in rats, while secukinumab treatment significantly alleviated cognitive impairment compared to the CLP group (p < 0.05). In addition, oxidative stress and neuronal apoptosis were found to be increased in the CLP group, while secukinumab significantly reduced oxidative stress and neuronal apoptosis in the hippocampus following CLP. Furthermore, secukinumab treatment led to a significant decrease in the protein expression levels of p-AKT, p-ERK1/2, Romo1, and Bax, together with increased Bcl-2 protein expression. Finally, treatment with ceramide C6 and GDC0068 abolished the neuroprotective effects of secukinumab post-CLP. ConclusionOur results demonstrated that secukinumab attenuated oxidative stress and neuronal apoptosis and partially ameliorated cognitive impairment via the IL-17RA/AKT/ERK1/2 pathway in a rat model of sepsis. Thus, secukinumab may be a potential therapeutic strategy for septic patients.

P2X7R promotes migration and invasion of Lewis lung cancer cells by activating the AKT signaling pathway

To explore the role of P2X7 receptor (P2X7R) in migration and invasion of mouse Lewis lung cancer (LLC) cells and examine the tumorigenic ability of LLC cells in P2X7R-knockout mice. RT-PCR was used to examine P2X7R mRNA expression in LLC cells. LLC cells were treated with ATP (as a P2X7R agonist) or 2'- 3'- O- (4-benzoyl- benzoyl)-ATP (BzATP) (a P2X7R agonist) with or without pretreatment with P2X7R antagonist oxATP or A438079. The changes in migration and invasive abilities of the cells were evaluated using wound healing assay and Transwell assay; Western blotting was performed to determine the activation level of the key proteins in the AKT signaling pathway. The effects of BzATP, A438079, and LY294002 (a inhibitor of the PI3K/AKT pathway) on migration and invasion of LLC cells were also examined. In wild-type (WT) and P2X7R knockout (P2X7-/-) C57BL/6 mice, the growth of subcutaneous LLC cell xenografts were observed by measuring tumor volume and weight. P2X7R expression was detected in LLC cells. Treatment with P2X7R agonist significantly enhanced migration and invasion abilities of LLC cells, and this effect was inhibited by application of P2X7R antagonists (P < 0.001). Western blotting showed that BzATP treatment of LLC cells significantly increased the expression level of p-AKT protein, which was obviously lowered by treatment with P2X7R antagonist (P < 0.01). P2X7R antagonist strongly inhibited BzATP-induced enhancement of LLC cell migration and invasion (P < 0.001). In the tumor- bearing mice, the tumor volume and weight were significantly lower in P2X7-/- mice than in WT mice (P < 0.05). P2X7R promotes migration and invasion of LLC cells by activating the AKT signaling pathway, and LLC cells show lowered tumorigenic capacity in P2X7-/- mice.

SPARC Overexpression Enhances the Sensitivity of SKM-1 Cells to Ara-C by Regulating CPBP/MLKL

To investigate the effect of SPARC gene overexpression on the chemotherapeutic sensitivity of AML-MDS cell line SKM-1 to Ara-C and to further explore its mechanism. Subjects were divided into 6 groups: SKM-1 cells (Control), Negative control (LV-NC), SPARC overexpression (LV-SPARC), SKM-1 cells+30 ng/ml Ara-C (30 ng/ml Ara-C), LV-NC+30 ng/ml Ara-C and LV-SPARC+30 ng/ml Ara-C. Cell activity was detected by CCK-8 assay, cell cycle distribution and apoptosis were detected by flow cytometry, mRNA expression levels of SPARC, CPBP and MLKL were detected by RT-qPCR, and the expression levels of related protein were detected by Western blot. After co-treatment with SPARC overexpression and Ara-C, the cell viability decreased and apoptosis increased significantly, with obvious up-regulation of Bax and down-regulation of BCL-2 (P<0.05). Compared with the control group, the cell cycle of LV-SPARC+30 ng/ml Ara-C group was significantly arrested in S phase with obvious down-regulation of CDK2 and up-regulation of p27KIP1 (P<0.05). Compared with LV-SPARC group and 30 ng/ml Ara-C group, the mRNA and protein expression levels of CPBP and MLKL (p-MLKL) were significantly elevated in LV-SPARC+30 ng/ml Ara-C group (P<0.05). In addition, after co-treatment with SPARC overexpression and Ara-C, the protein expression level of p-AKT decreased and the protein expression level of p53 increased (P<0.05). SPARC overexpression enhanced the sensitivity of SKM-1 cells to Ara-C and promoted cell cycle arrest and apoptosis, the mechanism of which may be related to the regulation of CPBP/MLKL pathway.

A Novel miRNA Y-56 Targeting IGF-1R Mediates the Proliferation of Porcine Skeletal Muscle Satellite Cells Through AKT and ERK Pathways.

As a key regulator of gene transcription and post-transcriptional modification, miRNAs play a wide range of roles in skeletal muscle development. Skeletal muscle satellite cells contribute to postnatal growing muscle fibers. Thus, the goal of this study was to explore the effects of novel miRNA Y-56 on porcine skeletal muscle satellite cells (PSCs). We found that Y-56 was highly expressed in porcine muscle tissues, and its expression was higher in Bama Xiang pigs than in Landrace pigs. The EdU assay, cell counting kit-8, and flow cytometry results showed that Y-56 overexpression suppressed cell proliferation and cell cycle, whereas Y-56 inhibition resulted in the opposite consequences. The results of qRT-PCR and Western blot showed that Y-56 remarkably inhibited the expression levels of cyclin-dependent kinase 4 (CDK4), proliferating cell nuclear antigen (PCNA), and cyclin D1. We identified that IGF-1R was a direct target of Y-56 by dual-luciferase reporter assay. Moreover, IGF-1R overexpression promoted the proliferation and cell cycle process of PSCs and upregulated the expression of CDK4, PCNA, and cyclin D1. Conversely, IGF-1R knockdown had the opposite effect. Furthermore, IGF-1R overexpression partially reversed the inhibition of the cell proliferation and cell cycle process of PSCs and the downregulation of the expression of CDK4, PCNA, and Cyclin D1 caused by Y-56 overexpression. Finally, Y-56 inhibited the protein expression levels of p-AKT and p-ERK. Collectively, our findings suggested that Y-56 represses the proliferation and cell cycle process of PSCs by targeting IGF-1R-mediated AKT and ERK pathways.

Effect of ginsenoside Rg3 on proliferation and apoptosis of 786-0 cells and AktmTORSTAT3 signaling in renal carcinoma

Surgery is currently the major approach treating kidney cancer, which is insensitive to chemo-/radio-therapy. However, due to the limitation of current diagnostic technique, certain number of patients has lost the opportunity of surgery. Therefore the searching for novel effective treatment approach is of critical importance. Ginsenoside Rg3 can inhibit proliferation and induce apoptosis of various tumor cells. It effect in kidney cancer, however, is still lacked. This study thus treated kidney cancer cell line 786-O with ginsenoside Rg3 to illustrate its effect on cell proliferation and apoptosis, and investigated possible mechanism.Gradient concentrations of ginsenoside Rg3 was used to treat 786-O cells, whose proliferation was measured by CCK8 assay. The apoptotic rate was quantified using flow cytometry. Western blotting was used to measure protein expression levels of p-Akt, p-mTOR and p-STAT3. Ginsenoside Rg3 significantly inhibited proliferation of kidney cancer cell 786-O in a dose- and time-dependent manner: higher concentration and longer treatment time caused more significant inhibition on cell proliferation. Similar patterns also occurred for the induction for cell apoptosis by ginsenoside Rg3. The expression levels of p-Akt, p-mTOR and p-STAT3, however, were decreased with higher dosages of ginsenoside Rg3, and were negatively correlated with apoptotic ratio. Ginsenoside Rg3 can exert anti-tumor role via inducing apoptosis and inhibiting proliferation of 786-O cells, possibly via the blocking of Akt/mTOR/STAT3 pathway.

Neuroprotective effects of Senkyunolide I against glutamate-induced cells death by attenuating JNK/caspase-3 activation and apoptosis

Glutamate-induced neurotoxicity is one of the most important pathogenic mechanisms in neurological diseases and is widely used as an in vitro model for ischemic stroke. Senkyunolide I (SEI), an active constituent derived from traditional Chinese medicine Ligusticum chuanxiong Hort. and Angelica sinensis (Oliv.) Diels, has been shown to have beneficial effects against focal cerebral ischemia-reperfusion in rats. However, the mechanisms underlying SEI-mediated neuroprotection remain not well understood. Thus, we explored the influence of SEI in glutamate-mediated injury to mouse neuroblastoma (Neuro2a) cells and determined the mechanisms involved. Neuro2a cells were treated with SEI under exposure to glutamate for 24 h. Cell viability was assessed by using WST-1 reagents, and apoptosis was evaluated using Annexin V-FITC and a PI double staining kit. The protein expression levels of p-AKT, AKT, p-GSK3β, GSK3β, p-p38, p38, p-ERK, ERK, p-JNK, JNK, Bcl-2, Bax, Bcl-xl, p-Bad, Bad, p53, and cleaved caspase-3 were determined by Western blot analysis. Glutamate significantly decreased cell viability and elevated the level of apoptosis. Treatment with SEI reversed those effects. Furthermore, the expression of p-JNK/JNK and cleaved caspase-3 were also reduced after treatment with SEI. Our findings demonstrate that SEI protected Neuro2a cells against glutamate toxicity by regulating JNK/caspase-3 pathway and apoptosis. Thus, SEI maybe a promising candidate for neuroprotection.

Antiprostate Cancer Activity of Ineupatolide Isolated from Carpesium cernuum L.

Objective The aim of the study was to investigate the antiprostate cancer effects and mechanism of ineupatolide (T-21), a natural product isolated from the Compositae plant Carpesium cernuum L., on PC-3 human prostate cancer cells. Methods The effect of T-21 on the proliferation of PC-3 cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, cell migration, and invasion experiments; the morphology of cell apoptosis was observed by Hoechst-propidium iodide staining; the effects of T-21 on PC-3 cell apoptosis and the cell cycle were evaluated by flow cytometry; and the effect of T-21 on the expression levels of phosphorylated protein kinase B (p-AKT), AKT, X-linked inhibitor of apoptosis protein (xlAP), procaspase-3, and poly (ADP-ribose) polymerase (PARP) in PC-3 cells was measured by western blotting. Results T-21 significantly inhibited the proliferation of cells, and its half-maximal inhibitory concentrations at 12, 24, and 48 h were 38.46 ± 1.01, 24.63 ± 0.70, and 7.36 ± 0.58 μM, respectively. T-21 may promote cell apoptosis in a concentration-dependent manner and block the cell cycle in the G2 and S phases. In addition, T-21 significantly reduced the protein expression levels of p-AKT, AKT, xlAP, procaspase-3, and PARP. Conclusion T-21 exhibits antiproliferation effects on PC-3 cells by promoting apoptosis and arresting the cell cycle in the G2 and S phases. The possible mechanism underlying its potential therapeutic effects against prostate cancer is related to the AKT/xlAP pathway.

MiR-335-5p inhibits proliferation of Huh-7 liver cancer cells via targeting the Oct4/Akt pathway.

The aim of this study was to detect the expression of micro ribonucleic acid (miR)-335-5p in the liver tissues of patients with liver cancer, and to explore its effect on liver cancer and mechanism using Huh7 human liver cancer cells. Liver tissues were collected from patients with liver cancer. The expression of miR-335-5p in tissues was detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Subsequently, Huh7 cells were transfected with miR-335-5p in vitro. After overexpressing miR-335-5p, changes in the expression of octamer-binding transcription factor 4 (Oct4) gene were observed via qRT-PCR. Furthermore, the proliferation of Huh7 cells and the protein expressions of protein kinase B (Akt) and phosphorylated Akt (p-Akt) were detected using cell counting kit (CCK)-8 assay and Western blotting (WB), respectively. Compared with Control group, the expression of miR-335-5p increased significantly in the liver tissues of liver cancer patients (p<0.01). In comparison with those in negative group, the messenger RNA (mRNA) expression of Oct4 and the proliferation rate of Huh7 cells were both significantly inhibited in miR-335-5p group (p<0.01, p<0.05). After overexpression of miR-335-5p, the protein expression level of p-Akt decreased remarkably (p<0.01). MiR-335-5p directly binds to the 3' untranslated region (3'UTR) of Oct4 mRNA to restrain the phosphorylation of Akt, thereby inhibiting Huh7 cell proliferation.

Differential Protein Metabolism and Regeneration in Gastrocnemius Muscles in High-fat Diet Fed Mice and Pre-hibernation Daurian Ground Squirrels: A Comparison between Pathological and Healthy Obesity.

We focused on pathological obesity induced by excessive fat intake (nutritional obesity) in non-hibernator and healthy obesity due to pre-hibernation (PRE) fat storage in hibernator to study the effects of different types of obesity on skeletal muscle protein metabolism and cell regeneration. Kunming mice were fed with high-fat diet for 3 months to construct a pathological obesity model. Daurian ground squirrels fattened naturally before hibernation were used as a healthy obesity model. Body weight, adipose tissue wet weight, gastrocnemius muscle wet weight, muscle fiber cross-sectional area (CSA) and fiber type distribution were measured. The protein expression levels related to protein degradation (MuRF-1, atrogin-1, calpain1, calpain2, calpastatin, desmin, troponin T, Beclin-1, LC3-II), protein synthesis (P-Akt, P-mTORC1, P-S6K1, P-4E-BP1) and cell regeneration (MyoD, myogenin, myostatin) were detected by Western blot. As a result, the body weight and adipose tissue wet weight were both significantly increased in high fat obese (OB) mice and pre-hibernation fat (PRE) ground squirrels. The muscle wet weight, ratio of muscle wet weight to body weight, and muscle fiber CSA were significantly decreased, while the percentage of MHC I fiber isoform was significantly increased in gastrocnemius muscle of OB mice compared with the control (CON) group. The protein expression levels of P-Akt, P-mTORC1, P-4E-BP1 and myogenin were significantly decreased, while those of calpain1, calpain2, MuRF-1 and myostatin were significantly increased in the OB mice. In the ground squirrels, the muscle wet weight, muscle fiber CSA and percentage of MHC I fiber isoform all showed no change in the gastrocnemius muscle in the PRE group compared with the summer active (SA) group. The protein expression levels of P-Akt, P-mTORC1, P-S6K1 and MyoD were significantly increased, while those of Beclin-1 and LC3-II were significantly decreased in the PRE ground squirrels. This study demonstrated that the decrease in protein expression levels in the Akt/mTOR pathway (P-Akt, P-mTORC1 and P-4E-BP1) and cell regeneration (myogenin) and the increase in protein expression levels of the calpain pathway (calpain1 and calpain2) and ubiquitin-proteasome pathway (MuRF-1) were involved in the mechanism of muscle atrophy in gastrocnemius muscle of the pathologically obese Kunming mice induced by high-fat diet. In contrast, the increased protein expression levels of the Akt/mTOR pathway (P-Akt, P-mTORC1 and P-S6K1) and cell regeneration (MyoD), and the decreased protein expression levels of the autophagy lysosomal pathway (Beclin-1 and LC3-II) were involved in the mechanism of anti-atrophy in gastrocnemius muscle of the healthy obese ground squirrels fattened before hibernation.

Implications of cell division cycle associated 4 on the Wilm’s tumor cells viability via AKT/mTOR signaling pathway

Objective The aim of present report was to elucidate the effect of cell division cycle associated 4 (CDCA4) on the proliferation and apoptosis of Wilm’s tumor cells, and to further evaluate its underlying mechanism. Methods The expression profiles of CDCA4 and clinical information of Wilm’s tumor patients were obtained from public Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database portal. Real-time qPCR and western blot analyses were utilized to determine the expression levels of CDCA4. Gain- and loss-of-function of CDCA4 assays were conducted with transfection technology to investigate the biological role of CDCA4 in Wilm’s tumor cells. Cell counting kit 8 and flow cytometer assays were employed to examine the effect of CDCA4 on the cells proliferation and apoptosis. Protein expression levels of indicated markers in each group of Wilm’s tumor cells were measured by western blot. Results The transcriptional expression of CDCA4 was drastically upregulated in Wilm’s tumor tissues according to the public TARGET database and in Wilm’s tumor cells. The cells viability was remarkably reduced whereas the cells apoptosis was increased in CDCA4-knockdown group compared with negative control group. However, CDCA4-overexpression group promoted the cells proliferation and suppressed the cells apoptosis. Furthermore, the protein expression levels of p-AKT, p-mTOR, and Cyclin D1 were significantly reduced after depletion of CDCA4, whereas overexpression of CDCA4 dramatically elevated these markers’ expression levels. Conclusions CDCA4 is highly expressed in Wilm’s tumor and promoted the proliferation whereas inhibited the apoptosis of Wilm’s tumor cells through activating the AKT/mTOR signaling pathway.

Pyrotinib Sensitizes 5-Fluorouracil-Resistant HER2+ Breast Cancer Cells to 5-Fluorouracil.

5-Fluorouracil (5-FU) is a widely used chemotherapeutic agent for breast cancer. However, acquired chemoresistance leads to a loss of its efficacy; methods to reverse are urgently needed. Some studies have shown that pyrotinib, an ErbB receptor tyrosine kinase inhibitor, is effective against HER2+ breast cancer. However, whether pyrotinib sensitizes 5-FU-resistant breast cancer cells to 5-FU is unknown. We hypothesized that the combination of pyrotinib and 5-FU would show synergistic antitumor activity, and pyrotinib could reverse 5-FU resistance in HER2+ breast cancer cells in vitro and in vivo. Our data showed that pyrotinib inhibited the growth of 5-FU-resistant SKBR-3/FU and MDA-MB-453/FU cell lines and the parental cell lines. 5-FU remarkably suppressed the growth of SKBR-3 and MAD-MB-453 cells. However, SKBR-3/FU and MAD-MB-453/FU cells showed resistance to 5-FU. A combination of pyrotinib and 5-FU resulted in the synergistic inhibition of the growth of the 5-FU-resistant SKBR-3/FU and MDA-MB-453/FU cell lines and the parental cell lines. Pyrotinib decreased significantly the IC50 values of 5-FU and the thymidylate synthase (TS) mRNA expression levels in the 5-FU-resistant SKBR-3/FU and MDA-MB-453/FU cell lines and the parental cell lines and increased significantly the intracellular concentration of 5-FU in SKBR-3/FU and MDA-MB-453/FU cells. In addition, pyrotinib reduced the ABCG2 mRNA and protein expression levels in SKBR-3/FU and MDA-MB-453/FU cells and downregulated the protein expression levels of pAKT, pHER2, and pHER4 in all four cell lines. After TS or ABCG2 in 5-FU-resistant breast cancer cells was knocked down, the sensitivity of SKBR-3/FU and MDA-MB-453/FU cells to 5-FU was restored. Moreover, in vivo experiments demonstrated that pyrotinib in combination with 5-FU more effectively inhibited SKBR-3/FU tumor growth than either pyrotinib or 5-FU alone. In conclusion, our findings suggest that pyrotinib could restore sensitivity of 5-FU-resistant HER2+ breast cancer cells to 5-FU through downregulating the expression levels of TS and ABCG2.

Up-Regulation of PI3K/Akt Signaling Pathway in H929 Cell Line by Doxycycline Not Beneficial to Its Anti-Myeloma Effect

To investigate the effect and possible mechanism of up-regulation of p-Akt by doxycycline (DOX) on myeloma cell line H929. Multiple myeloma cell line H929 was treated with DOX at different concentrations for different times, and cell proliferation rate was measured by CCK-8 assay. The protein expression level of p-Akt, PTEN, p-PDK1, p-mTOR, p-GSK-3β, and p-BAD was analyzed by Western blot. The mRNA levels of mTOR, BCL-2, and NF-κB was analyzed by RT-PCR. PI3K inhibitor Wortmannin was used to antagonize the up-regulation of p-Akt, and the cell proliferation and p-Akt protein expression level were analyzed by CCK-8 assay and Western blot respectively. DOX could inhibit the proliferation of H929 cells and up-regulate the expression of p-Akt at the same time. The protein levels of both p-PDK1 and PTEN in H929 cells did not alter significantly during DOX treatment. The expressions of p-BAD and p-GSK-3β were up-regulated in H929 cells after treated with DOX, but the expression of p-mTOR was not altered. The mRNA levels of mTOR, BCL-2, and NF-κB in H929 were all down-regulated in H929 cells during DOX treatment. The effect up-regulating p-Akt level by DOX was suppressed when DOX combined with PI3K inhibitor Wortmannin and Wortmannin could enhance the inhibitory effect of DOX in H929 cells. DOX can activate PI3K/Akt signaling pathway in H929 cells, and antagonizing this effect of DOX may enhance its cytotoxicity to myeloma cells.

Effects of Twist1 on drug resistance of chronic myeloid leukemia cells through the PI3K/AKT signaling pathway

This study aimed to investigate the effects of Twist1 on the drug resistance of chronic myeloid leukemia (CML) cells through the PI3K/AKT signaling pathway. K562 and KCL-22 cells were modeled for imatinib resistance, so as to analyze the effects of inhibiting Twist1 and the pathway on the therapeutic effect of imatinib on imatinib-resistant CML cells, and to find the mechanism of action of Twist1 on affecting the resistance. After the CML cells were successfully resistant to imatinib, Twist1 expression increased again in the cells and the PI3K/AKT signaling pathway was further activated. After the silence of the Twist1 expression, the imatinib-resistant CML cells were more sensitive to imatinib, and the PI3K/AKT signaling pathway was inhibited, and the expression level of p-AKT protein significantly reduced. According to further experiments, imatinib enhanced its inhibitory effect on the growth of the imatinib-resistant CML cells after the activation of the pathway was inhibited by an LY3023414 inhibitor. In conclusion, Twist1 and the PI3K/AKT signaling pathway are over-activated during the formation of the CML cells resistant to imatinib. The silence of Twist1 can reverse the resistance through the pathway.

BW373U86 upregulates autophagy by inhibiting the PI3K/Akt pathway and regulating the mTOR pathway to protect cardiomyocytes from hypoxia-reoxygenation injury.

The purpose of this study was to explore the protective effect of BW373U86 (a δ-opioid receptor (DOR) agonist) on ischemia-reperfusion (I/R) injury in rat cardiomyocytes and its underlying mechanism. Primary rat cardiomyocytes were cultured and pretreated with BW373U86 for intervention. The cardiomyocytes were cultured under the condition of 94% N2 and 5% CO2 for 24 h to perform hypoxia culture and conventionally cultured for 12 h to perform reoxygenation culture. The cell viability of cardiomyocytes was detected by an MTT assay (Sigma-Aldrich). The autophagy lysosome levels in cardiomyocytes were evaluated by acidic vesicular organelles with dansylcadaverine (MDC) staining (autophagy test kit, Kaiji Biology, kgatg001). The protein expression levels of LC3, p62, and factors in the PI3K/Akt/mTOR signaling pathway were detected by Western blot. Pretreatment with BW373U86 could improve the cell viability of cardiomyocytes with hypoxia-reoxygenation (H/R) injury (p < 0.05). Interestingly, after coculture of BW373U86 and PI3K inhibitor (3-methyladenine), the protein expression levels of p-Akt in cardiomyocytes were markedly increased in comparison with those in the BW373U86 group (p < 0.05). However, there were no significant differences in the protein expression levels of mTOR between the coculture group and the BW373U86 group (p > 0.05). BW373U86 upregulated autophagy to protect cardiomyocytes from H/R injury, which may be related to the PI3K/Akt/m TOR pathway.