Osteopontin promotes gastric cancer progression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.

Cytotoxicity of nonylphenol on spermatogonial stem cells via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway

BACKGROUND With continuous advancement of industrial society,environmental pollution has become more and more serious.There has been an increase in infertility caused by environmental factors.Nonylphenol(NP)is a stable degradation product widely used in daily life and production and has been proven to affect male fertility.However,the underlying mechanisms therein are unclear.Thus,it is necessary to study the effect and mechanism of NP on spermatogonial stem cells(SSCs).AIM To investigate the cytotoxic effect of NP on SSCs via the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway.METHODS SSCs were treated with NP at 0,10,20 or 30μmol.MTT assay was performed to evaluate the effect of NP on the proliferation of SSCs.Flow cytometry was conducted to measure SSC apoptosis.The expression of Bad,Bcl-2,cytochrome-c,pro-Caspase 9,SOX-2,OCT-4,Nanog,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,PLZF and PI3K/AKT/mTOR-related proteins was observed by western blot,and the mRNA expression of SOX-2,OCT-4 and Nanog was detected by quantitative reverse transcription polymerase chain reaction.RESULTS Compared with untreated cells(0μmol NP),SSCs treated with NP at all concentrations showed a decrease in cell proliferation and expression of Bcl-2,Nanog,OCT-4,SOX-2,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,and PLZF(P<0.05),whereas the expression of Bad,cytochrome-c,and pro-Caspase 9 increased significantly(P<0.05).We further examined the PI3K/AKT/mTOR pathway and found that the phosphorylation of PI3K,AKT,mTORC1,and S6K was significantly decreased by NP at all concentrations compared to that in untreated SSCs(P<0.05).NP exerted the greatest effect at 30μmol among all NP concentrations.CONCLUSION NP attenuated the proliferation,differentiation and stemness maintenance of SSCs while promoting apoptosis and oxidative stress.The associated mechanism may be related to the PI3K/AKT/mTOR pathway.

Influence of Phosphatidylinositol-3-Kinase/Protein Kinase B-Mammalian Target of Rapamycin Signaling Pathway on the Neuropathic Pain Complicated by Nucleoside Reverse Transcriptase Inhibitors for the Treatment of HIV Infection

Background： Nucleoside reverse transcriptase inhibitors （NRTIs） are the earliest and most commonly used anti-human immunodeficiency virus drugs and play an important role in high active antiretroviral therapy. However, NRTI drug therapy can cause peripheral neuropathic pain. In this study, we aimed to investigate the mechanisms ofrapamycin on the pain sensitization of model mice by in vivo experiments to explore the effect of mammalian target of rapamycin （mTOR） in the pathogenesis ofneuropathic pain caused by NRTIs. Methods： Male Kun Ming （KM） mice weighing 20-2 g were divided into control, 2 mg/kg rapamycin, 12 mg/kg stavudine, and CMC-Na groups. Drugs were orally administered to mice for 42 consecutive days. The von Frey filament detection and thermal pain tests were conducted on day 7, 14, 21, 28, 35, and 42 after drug administration. After the last behavioral tests, immunohistochemistry and western blotting assay were used for the measurement of mTOR and other biomarkers. Multivariate analysis of variance was used. Results： The beneficial effects ofrapamycin on neuropathic pain were attributed to a reduction in mammalian target of rapamycin sensitive complex 1 （mTORC1）-positive cells （70.80± 2.41 vs. 112.30 ± 5.66, F = 34.36, P 〈 0.01 ） and mTORC1 activity in the mouse spinal cord. Mechanistic studies revealed that Protein Kinase B （Akt）/mTOR signaling pathway blockade with rapamycin prevented the phosphorylation of mTORC1 in stavudine-intoxicated mice （0.72 ± 0.04 vs. 0.86 ± 0.03, F=4.24, P = 0.045）, as well as decreased the expression of phospho-pTOS6K （0.47 ± 0.01 vs. 0.68 ± 0.03, F=6.01, P = 0.022） and phospho-4EBP1 （0.90 ± 0.04 vs. 0.94 ± 0.06, F= 0.28, P = 0.646）. Conclusions： Taken together, these results suggest that stavudine elevates the expression and activity of mTORC1 in the spinal cord through activating the Akt/mTOR signaling pathway. The data also provide evidence that rapamycin might be useful for the treatment of peripheral neuropathic pain.

TopoisomeraseⅡalpha promotes gallbladder cancer proliferation and metastasis through activating phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

Background:TopoisomeraseⅡalpha(TOP2A)has been reported to play a crucial role in the tumorigenesis of various cancer types.However,the biological role of TOP2A in gallbladder cancer(GBC)remains unknown.The current study aimed to explore the function and potential mechanism of TOP2A in GBC.Methods:Based on Gene Expression Profiling Interactive Analysis data,we found TOP2A was significantly up-regulated in GBC tissues and resulting in shorter overall survival.Quantitative real-time polymerase chain reaction and immunohistochemistry were conducted to detect the expression of TOP2A in 45 pairs of GBC tissues and adjacent non-tumor tissues.In vitro,cell proliferation,migration,and invasion ability were examined by cell counting kit-8 and transwell assay,respectively.Epithelial-mesenchymal transition(EMT)related and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway-related markers were measured by Western blotting.Xenograft model assay was performed to evaluate the effect of TOP2A in vivo.Results:TOP2A was found up-regulated in GBC(tumor vs.normal,12.62 vs.0.34)and correlated with the late tumor node metastasis stage(P=0.0032),present of lymph node metastasis(P=0.0273),and poor prognosis in GBC patients(log-rank P=0.028).In vitro and in vivo assays showed that knockdown of TOP2A notably inhibited cell proliferation,migration,invasion,EMT process,and tumor growth in GBC.In addition,TOP2A down-regulation significantly decreased the protein levels of phosphor(p)-PI3K,p-Akt,and p-mTOR.Conclusion:Our study demonstrates that TOP2A was overexpressed in GBC and associated with poor prognosis in GBC patients.TOP2A promotes GBC cell proliferation,migration,invasion,EMT process,and tumor growth through activating PI3K/Akt/mTOR signaling pathway,and may serve as a novel prognostic biomarker and therapeutic target for GBC.

Regulatory Effects of Zuogui Pill on Apoptosis of Follicles in Rats Injured by 60Co-γRays Based on PI3K/Akt/m TOR Signaling Pathway

[Objectives]To explore the protective effects of Zuogui Pill on ^(60)Co-γ-ray-induced premature aging of rats based on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway.[Methods]Sixty sexually mature female SD rats were irradiated with ^(60)Co-γ-ray(6.0 Gy,LD 40)for 24 h at one time.These rats were randomly divided into model group,Progynova group[0.18(g·kg)/d],Progynova[0.09(g·kg)/d]+Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill medium dose[9.45(g·kg)/d)]group and Zuogui Pill low dose[4.725(g·kg)/d]group.The administration(once a day)lasted 21 d.The rat serum[follicle-stimulating hormone(FSH),luteinizing hormone(LH)and estradiol(E_(2))]were detected by Enzyme-linked immunosorbent assay(ELISA).The morphological changes of ovary were observed by hematoxylin-eosin(HE)staining.The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL).The protein expression of phosphorylated(p)-PI3K,p-Akt,p-mTOR,B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax)in ovarian tissues were detected by Western blot.[Results]Compared with the normal group,the model group showed significant increase in the serum FSH(P<0.01),significant decrease in serum E_(2)(P<0.05),and decrease in the number of early follicles and luteum in the ovary(P<0.01).Besides,the apoptosis rate of granulosa cells increased significantly(P<0.01);the expression of p-PI3K,p-Akt,p-mTOR and Bcl-2 in ovarian tissue decreased significantly,while the expression of Bax increased significantly(P<0.01).Compared with the model group,the number of early follicles in the ovary increased and the apoptosis rate of granulosa cells decreased after intervention in each administration group.In addition,the protein expressions of p-PI3K,p-Akt,p-mTOR and Bcl-2 increased,while the expression of Bax decreased,especially in Progynova+Zuogui Pill high dose group,the differences were statistically significant(P<0.05,P<0.01).[Conclusions]Zuogui Pill may protect the radiation-injured ovary through activating the expression of PI3K/Akt/mTOR protein in ovarian tissue,increasing the amount of Bcl-2 protein and inhibiting the expression of Bax protein.

Tongxinluo Activates PI3K/AKT Signaling Pathway to Inhibit Endothelial Mesenchymal Transition and Attenuate Myocardial Fibrosis after Ischemia-Reperfusion in Mice

Objective To investigate the potential role of Tongxinluo(TXL)in attenuating myocardial fibrosis after myocardial ischemia-reperfusion injury(MIRI)in mice.Methods A MIRI mouse model was established by left anterior descending coronary artery ligation for 45 min.According to a random number table,66 mice were randomly divided into 6 groups(n=11 per group):the sham group,the model group,the LY-294002 group,the TXL group,the TXL+LY-294002 group and the benazepril(BNPL)group.The day after modeling,TXL and BNPL were administered by gavage.Intraperitoneal injection of LY-294002 was performed twice a week for 4 consecutive weeks.Echocardiography was used to measure cardiac function in mice.Masson staining was used to evaluate the degree of myocardial fibrosis in mice.Qualitative and quantitative analysis of endothelial mesenchymal transition(EndMT)after MIRI was performed by immunohistochemistry,immunofluorescence staining and flow cytometry,respectively.The protein expressions of platelet endothelial cell adhesion molecule-1(CD31),α-smoth muscle actin(α-SMA),phosphatidylinositol-3-kinase(PI3K)and phospho protein kinase B(p-AKT)were assessed using Western blot.Results TXL improved cardiac function in MIRI mice,reduced the degree of myocardial fibrosis,increased the expression of CD31 and inhibited the expression ofα-SMA,thus inhibited the occurrence of EndMT(P<0.05 or P<0.01).TXL significantly increased the protein expressions of PI3K and p-AKT(P<0.05 or P<0.01).There was no significant difference between TXL and BNPL group(P>0.05).In addition,the use of the PI3K/AKT pathway-specific inhibitor LY-294002 to block this pathway and combination with TXL intervention,eliminated the protective effect of TXL,further supporting the protective effect of TXL.Conclusion TXL activated the PI3K/AKT signaling pathway to inhibit EndMT and attenuated myocardial fibrosis after MIRI in mice.

Scorpiones,Scolopendra and Gekko Inhibit Lung Cancer Growth and Metastasis by Ameliorating Hypoxic Tumor Microenvironment via PI3K/AKT/mTOR/HIF-1αSignaling Pathway

Objective:To investigate whether Buthus martensii karsch(Scorpiones),Scolopendra subspinipes mutilans L.Koch(Scolopendra)and Gekko gecko Linnaeus(Gekko)could ameliorate the hypoxic tumor microenvironment and inhibit lung cancer growth and metastasis by regulating phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin/hypoxia-inducible factor-1α(PI3K/AKT/mTOR/HIF-1α)signaling pathway.Methods:Male C57BL/6J mice were inoculated with luciferase labeled LL/2-luc-M38 cell suspension to develop lung cancer models,with rapamycin and cyclophosphamide as positive controls.Carboxy methyl cellulose solutions of Scorpiones,Scolopendra and Gekko were administered intragastrically as 0.33,0.33,and 0.83 g/kg,respectively once daily for 21 days.Fluorescent expression were detected every 7 days after inoculation,and tumor growth curves were plotted.Immunohistochemistry was performed to determine CD31 and HIF-1αexpressions in tumor tissue and microvessel density(MVD)was analyzed.Western blot was performed to detect the expression of PI3K/AKT/mTOR/HIF-1αsignaling pathway-related proteins.Enzyme-linked immunosorbent assay was performed to detect serum basic fibroblast growth factor(bFGF),transforming growth factor-β1(TGF-β1)and vascular endothelial growth factor(VEGF)in mice.Results:Scorpiones,Scolopendra and Gekko prolonged the survival time and inhibited lung cancer metastasis and expression of HIF-1α(all P<0.01).Moreover,Scorpiones,Scolopendra and Gekko inhibited the phosphorylation of AKT and ribosomal protein S6 kinase(p70S6K)(P<0.05 or P<0.01).In addition,they also decreased the expression of CD31,MVD,bFGF,TGF-β1 and VEGF compared with the model group(P<0.05 or P<0.01).Conclusion:Scorpiones,Scolopendra and Gekko all showed beneficial effects on lung cancer by ameliorating the hypoxic tumor microenvironment via PI3K/AKT/mTOR/HIF-1αsignaling pathway.

Treating non-small cell lung cancer by targeting the PI3K signaling pathway

The phosphosphatidylinositol-3-kinase(PI3K)signaling pathway is one of the most important intracellular signal transduction pathways affecting cell functions,such as apoptosis,translation,metabolism,and angiogenesis.Lung cancer is a malignant tumor with the highest morbidity and mortality rates in the world.It can be divided into two groups,non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).NSCLC accounts for>85%of all lung cancers.There are currently many clinical treatment options for NSCLC;however,traditional methods such as surgery,chemotherapy,and radiotherapy have not been able to provide patients with good survival benefits.The emergence of molecular target therapy has improved the survival and prognosis of patients with NSCLC.In recent years,there have been an increasing number of studies on NSCLC and PI3K signaling pathways.Inhibitors of various parts of the PI3K pathway have appeared in various phases of clinical trials with NSCLC as an indication.This article focuses on the role of the PI3K signaling pathway in the occurrence and development of NSCLC and summarizes the current clinical research progress and possible development strategies.

基于磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物西罗莫司靶蛋白通路途径髓样细胞触发受体2对胶质细胞炎症的影响

目的探究基于磷脂酰肌醇-3-激酶(phosphoinositide-3-kinase,PI3K)/蛋白激酶B(protein kinase B,AKT)/哺乳动物西罗莫司靶蛋白(mammalian target of sirolimus,mTOR)通路途径髓样细胞触发受体2(triggering receptors expressed on myeloid cells-2,TREM2)对胶质细胞炎症的影响。方法对胶质细胞采用脂多糖(lipopolysaccharide,LPS)进行诱导造模后完成分组检测,分组如下:对照组、LPS处理组和TREM2组,通过RT-PCR方法检测白细胞介素(interleukin)-6、IL-1β和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)中mRNA表达水平,采用免疫印迹法检测各组细胞PI3K、AKT、mTOR蛋白表达水平。结果与对照组比较,LPS处理组、TREM2组IL-6、IL-1β和TNF-α中mRNA表达水平均升高,TREM2组IL-6、IL-1β、TNF-α中mRNA表达水平高于LPS处理组,差异有统计学意义(P<0.05)。LPS处理组和TREM2组细胞PI3K、AKT和mTOR蛋白表达水平均高于对照组,TREM2组细胞PI3K、AKT和mTOR蛋白表达水平高于LPS处理组,差异有统计学意义(P<0.05)。结论小胶质细胞过度活化后诱导神经炎症,P13K/AKT/mTOR信号通路参与多种神经退行性疾病的发生发展过程,其在细胞炎症反应中都发挥着重要作用。

当归黄芪胶囊对心力衰竭大鼠心肌自噬的影响

目的探讨当归黄芪胶囊是否通过磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/西罗莫司靶蛋白(mTOR)信号通路调控心力衰竭大鼠心肌的自噬。方法用2.5 mg·kg^(-1)阿霉素腹腔注射构建心力衰竭大鼠模型,另取8只为空白组。将造模成功的大鼠随机分为模型组、对照组和低、中、高剂量实验组。对照组给予腹腔注射30 mg·kg^(-1)的3-甲基腺嘌呤;低、中、高剂量实验组分别灌胃给予150、300、450 mg·kg^(-1)的当归黄芪胶囊;空白组及模型组均灌胃给予等量无菌蒸馏水。6组大鼠每天给药1次,持续6周。用超声多普勒检测心功能,用蛋白印迹法检测心肌组织中PI3K、Akt、mTOR、选择性自噬接头蛋白(P62)、微管相关轻链蛋白3Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ)的表达水平。结果空白组、模型组、对照组和高剂量实验组的左心室射血分数分别为(85.00±3.63)%、(56.75±4.83)%、(75.63±3.70)%和(72.75±4.23)%,PI3K相对表达水平分别为1.00±0、0.28±0.05、0.64±0.08和0.74±0.16,磷酸化Akt/Akt分别为1.00±0、0.49±0.06、0.90±0.16和0.95±0.10,磷酸化mTOR/mTOR分别为1.00±0、0.42±0.09、0.73±0.13和0.83±0.08,P62相对表达水平分别为1.00±0、0.24±0.12、0.57±0.09和0.96±0.10,LC3Ⅱ/Ⅰ相对表达水平分别为1.00±0、4.31±0.75、2.20±0.76和1.59±0.24。与模型组比较,高剂量实验组和对照组的上述指标在统计学上差异均有统计学意义(均P<0.05)。结论当归黄芪胶囊能通过调控PI3K/Akt/mTOR通路,抑制心力衰竭大鼠心肌的自噬,从而改善大鼠的心功能。

Role and mechanism of matrine alone and combined with acitretin for HaCaT cells and psoriasis-like murine models

Background: Acitretin and matrine have been used in the treatment of psoriasis in China. This study was designed to investigate the role and related mechanisms of matrine alone and in combination with acitretin in the treatment of psoriasis in vitro and in vivo. Methods: HaCaT cells were treated with matrine at different concentrations of 0 (blank control), 0.2, 0.4, 0.8, and 1.6 mg/mL for 24, 48, 72 h, respectively. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay was used to assess the growth and proliferation of HaCaT cells. Cell cycle and apoptosis were detected by flow cytometry. Expression of protein was detected by Western blotting. Autophagy was observed by transmission electron microscopy. Then HaCaT cells were assigned to normal saline (NS) control group, matrine (0.4 mg/mL) group, acitretin (10 μmol/L) group, and matrine plus acitretin group, and the above methods were repeated. In animal experiments, the cumulative score (erythema, scaling, thickening) as a measure of the severity of inflammation was used to measure the skin performance of mice after treated with matrine 50 mg/kg, acitretin 4.5 mg/kg or combination of the two drugs on the psoriasis-like mouse models, respectively. Pathological findings of the lesions were observed, and the protein expressions in the lesions were detected by immunohistochemistry. Results: Cell proliferation inhibition was seen in HaCaT cells with treatment of matrine in a dose- and time-dependent manner (P < 0.01, respectively). Cell cycle G0/G1 phase arrest was observed in a dose-dependent way (P < 0.01). The expression of p21 (P < 0.05), LC3II/I (P < 0.01), and Beclin 1 (P < 0.01) increased and the expression of cyclin D1 (P < 0.05) decreased with increasing doses of matrine. Compared with the blank control, more autophagosomes were seen in HaCaT cells treated with matrine at 0.4 mg/mL by transmission electron microscopy (2.667 ± 1.202 vs. 21.33 ± 1.453, t = 9.899, P < 0.01). Cell proliferation inhibition and degree of the G0/G1 phase arrest was significantly higher in matrine plus acitretin group than those in matrine, acitretin, or the NS control group (P < 0.01, respectively). Compared with matrine or acitretin group, the expression of p21 (P < 0.05, P < 0.05) and LC3II/I (P < 0.01, P < 0.05) in matrine plus acitretin group increased significantly and the expression of cyclin D1 (P < 0.01, P < 0.05) and p62 (P < 0.05, P < 0.05) was reduced significantly. Compared with matrine or acitretin, matrine plus acitretin significantly down-regulated the phosphorylation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway (P < 0.05) and its downstream p-p70S6K (P < 0.05). In addition, the cumulative score of mice in the matrine plus acitretin group was significantly better than that in the matrine or acitretin group (1.480 ± 0.230 vs. 2.370 ± 0.241, P < 0.01;1.480 ± 0.230 vs. 2.888 ± 0.341, P < 0.01). The expression of LC3 protein in the matrine plus acitretin group was also higher than that in the matrine, acitretin, or the NS control group (P < 0.05, respectively). Conclusions: Matrine has therapeutic potentials for psoriasis. Matrine and acitretin show synergistic effect via cell cycle arrest and autophagy induction by PI3K/Akt/mTOR pathway.