Tumor-related factor complement Clq/TNF-related protein 6 affects the development of digestive system tumors through the phosphatidylinositol 3-kinase pathway

In this editorial,we review the work of Razali et al published in World J Gas-troenterology,with a particular focus on the effect of rs10889677 variation in the phosphatidylinositol 3-kinase(PI3K)pathway and buparlisib on colitis-associated cancer.The role of PI3K in promoting cancer progression has been widely recognized,as it is involved in regulating the survival,differentiation,and prolif-eration of cancer cells.The complement Clq/TNF-related protein 6(CTRP6)is a newer tumor-associated factor.Recent studies have revealed the pro-tumor effect of CTRP6 in gastric cancer,hepatocellular carcinoma,colorectal cancer,and other gastrointestinal tumors through the PI3K pathway.This article attempts to reveal the mechanism through which the CTRP6 affects the development of digestive system tumors through the PI3K pathway by summarizing recent research.

Phosphatidylinositol 3-kinase/Akt pathway regulates hepatic stellate cell apoptosis

AIM: To investigate the role of phosphatidylinositol 3-kinase (PI 3-K)/Akt signaling pathway in the balance of HSC activation and apoptosis in rat hepatic stellate cells (HSC). METHODS: An activated HSC cell line was used in this study. LY 294002, the PI 3-K/Akt signal pathway block-er was used to investigate the molecular events on apoptosis in HSC and to interpret the role of this path-way in HSC apoptosis. Immunocytochemistry, Western blot and reverse transcription polymerase chain reac-tion (RT-PCR) analysis were applied to detect the ex-pression of PI 3-K, and simultaneously phosphorylated-Akt (p-Akt) and total-Akt were determined by Western blot. The HSC apoptosis was examined by annexin-V/ propidium iodide double-labelled flow cytometry and transmission electron microscopy. RESULTS: The apoptosis rates in LY 294002 (30.82% ± 2.90%) and LY 294002 + PDGF-BB (28.16% ± 2.58%) groups were signif icantly increased compared with those of control (9.02% ± 1.81%) and PDGF-BB (4.35% ± 1.18%). PDGF-BB augmented PI 3-K and p-Akt expres-sion. LY 294002 signif icantly reduced the contents of PI 3-K and p-Akt. mRNA transcription evaluated by RT-PCR showed similar tendencies as protein expression. CONCLUSION: Inhibition of PI 3-K/Akt signaling path-way induces apoptosis in HSC.

Alterations in the human epidermal growth factor receptor 2-phosphatidylinositol 3-kinase-v-Akt pathway in gastric cancer

AIM:To investigate human epidermal growth factor receptor 2(HER2)-phosphatidylinositol 3-kinase(PI3K)-vAkt murine thymoma viral oncogene homolog signaling pathway.METHODS:We analyzed 231 formalin-fixed,paraffinembedded gastric cancer tissue specimens from Japanese patients who had undergone surgical treatment.The patients' age,sex,tumor location,depth of invasion,pathological type,lymph node metastasis,and pathological stage were determined by a review of the medical records.Expression of HER2 was analyzed by immunohistochemistry(IHC) using the HercepTest TM kit.Standard criteria for HER2 positivity(0,1+,2+,and 3+) were used.Tumors that scored 3+ were considered HER2-positive.Expression of phospho Akt(pAkt) was also analyzed by IHC.Tumors were considered pAkt-positive when the percentage of positive tumor cells was 10% or more.PI3K,catalytic,alpha polypeptide(PIK3CA) mutations in exons 1,9 and 20 were analyzed by pyrosequencing.Epstein-Barr virus(EBV) infection was analyzed by in situ hybridization targeting EBV-encoded small RNA(EBER) with an EBER-RNA probe.Microsatellite instability(MSI) was analyzed by polymerase chain reaction using the mononucleotide markers BAT25 and BAT26.RESULTS:HER2 expression levels of 0,1+,2+ and 3+ were found in 167(72%),32(14%),12(5%) and 20(8.7%) samples,respectively.HER2 overexpression(IHC 3+) significantly correlated with intestinal histological type(15/20 vs 98 /205,P = 0.05).PIK3CA mutations were present in 20 cases(8.7%) and significantly correlated with MSI(10/20 vs 9/211,P < 0.01).The mutation frequency was high(21%) in T4 cancers and very low(6%) in T2 cancers.Mutations in exons 1,9 and 20 were detected in 5(2%),9(4%) and 7(3%) cases,respectively.Two new types of PIK3CA mutation,R88Q and R108H,were found in exon1.All PIK3CA mutations were heterozygous missense singlebase substitutions,the most common being H1047R(6/20,30%) in exon20.Eighteen cancers(8%) were EBV-positive and this positivity significantly correlated with a diffuse histological type(13/18 vs 93/198,P = 0.04).There were 7 cases of lymphoepithelioma-like carcinomas(LELC) and 6 of those cases were EBV-positive(percent/EBV:6/18,33%;percent/all LELC:6/7,86%).pAkt expression was positive in 119(53%) cases but showed no correlation with clinicopathological characteristics.pAkt expression was significantly correlated with HER2 overexpression(16/20 vs 103/211,P < 0.01) but not with PIK3CA mutations(12/20 vs 107/211,P = 0.37) or EBV infection(8/18 vs 103/211,P = 0.69).The frequency of pAkt expression was higher in cancers with exon20 mutations(100%) than in those with exon1(40%) or exon9(56%) mutations.One case showed both HER2 overexpression and EBV infection and 3 cases showed both PIK3CA mutations and EBV infection.However,no cases showed both PIK3CA mutations and HER2 overexpression.One EBVpositive cancer with PIK3CA mutation(H1047R) was MSI-positive.Three of these 4 cases were positive for pAkt expression.In survival analysis,pAkt expression significantly correlated with a poor prognosis(hazard ratio 1.75;95%CI:1.12-2.80,P = 0.02).CONCLUSION:HER2 expression,PIK3CA mutations and EBV infection in gastric cancer were characterized.pAkt expression significantly correlates with HER2 expression and with a poor prognosis.

Herbal cake-partitioned moxibustion inhibits colonic autophagy in Crohn’s disease via signaling involving distinct classes of phosphatidylinositol 3-kinases

BACKGROUND Autophagy is an evolutionarily conserved biological process in eukaryotic cells that involves lysosomal-mediated degradation and recycling of related cellular components.Recent studies have shown that autophagy plays an important role in the pathogenesis of Crohn’s disease(CD).Herbal cake-partitioned moxibustion(HM)has been historically practiced to treat CD.However,the mechanism by which HM regulates colonic autophagy in CD remains unclear.AIM To observe whether HM can alleviate CD by regulating colonic autophagy and to elucidate the underlying mechanism.METHODS Rats were randomly divided into a normal control(NC)group,a CD group,an HM group,an insulin+CD(I+CD)group,an insulin+HM(I+HM)group,a rapamycin+CD(RA+CD)group,and a rapamycin+HM(RA+HM)group.2,4,6-trinitrobenzenesulfonic acid was administered to establish a CD model.The morphology of the colonic mucosa was observed by hematoxylin-eosin staining,and the formation of autophagosomes was observed by electron microscopy.The expression of autophagy marker microtubule-associated protein 1 light chain 3 beta(LC3B)was observed by immunofluorescence staining.Insulin and rapamycin were used to inhibit and activate colonic autophagy,respectively.The mRNA expression levels of phosphatidylinositol 3-kinase class I(PI3KC1),Akt1,LC3B,sequestosome 1(p62),and mammalian target of rapamycin(mTOR)were evaluated by RT-qPCR.The protein expression levels of interleukin 18(IL-18),tumor necrosis factor-α(TNF-α),nuclear factorκB/p65(NF-κB p65),LC3B,p62,coiled-coil myosin-like BCL2-interacting protein(Beclin-1),p-mTOR,PI3KC1,class III phosphatidylinositol 3-kinase(PI3KC3/Vps34),and p-Akt were evaluated by Western blot analysis.RESULTS Compared with the NC group,the CD group showed severe damage to colon tissues and higher expression levels of IL-18 and NF-κB p65 in colon tissues(P<0.01 for both).Compared with the CD group,the HM group showed significantly lower levels of these proteins(PIL-18<0.01 and Pp65<0.05).There were no significant differences in the expression of TNF-αprotein in colon tissue among the rat groups.Typical autophagic vesicles were found in both the CD and HM groups.The expression of the autophagy proteins LC3B and Beclin-1 was upregulated(P<0.01 for both)in the colon tissues of rats in the CD group compared with the NC group,while the protein expression of p62 and p-mTOR was downregulated(P<0.01 for both).However,these expression trends were significantly reversed in the HM group compared with the CD group(PLC3B<0.01,PBeclin-1<0.05,Pp62<0.05,and Pm-TOR<0.05).Compared with those in the RA+CD group,the mRNA expression levels of PI3KC1,Akt1,mTOR,and p62 in the RA+HM group were significantly higher(PPI3KC1<0.01 and PAkt1,mTOR,and p62<0.05),while those of LC3B were significantly lower(P<0.05).Compared with the RA+CD group,the RA+HM group exhibited significantly higher PI3KC1,p-Akt1,and pmTOR protein levels(PPI3KC1<0.01,Pp-Akt1<0.05,and Pp-mTOR<0.01),a higher p62 protein level(P=0.057),and significantly lower LC3B and Vps34 protein levels(P<0.01 for both)in colon tissue.CONCLUSION HM can activate PI3KC1/Akt1/mTOR signaling while inhibiting the PI3KC3(Vps34)-Beclin-1 protein complex in the colon tissues of CD rats,thereby inhibiting overactivated autophagy and thus exerting a therapeutic effect.

Alisol B acetate induces apoptosis of SGC7901 cells via mitochondrial and phosphatidylinositol 3-kinases/Akt signaling pathways

AIM： To examine the effect of alisol B acetate on the growth of human gastric cancer cell line SGC7901 and its possible mechanism of action.METHODS： The cytotoxic effect of alisol B acetate on SGC7901 cells was measured by 3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide （MI-I-） assay. Phase-contrast and electron microscopy were used to observe the morphological changes. Cell cycle and mitochondrial transmembrane potential （A^Pm） were determined by flow cytometry. Western blotting was used to detect the expression of apoptosis-regulated gene Bcl-2, Bax, Apaf-1, caspase-3, caspase-9, Akt, P-Akt and phosphatidylinositol 3-kinases （PI3K）.RESULTS： Alisol B acetate inhibited the proliferation of SGC7901 cell line in a time- and dose-dependent manner. PI staining showed that alisol B acetate can change the cell cycle distribution of SGC7901, increase the proportion of cells in G0-G1 phase and decrease the proportion of S phase cells and G2-M phase cells. Alisol B acetate at a concentration of 30 pmol/L induced apoptosis after 24, 48 and 72 h incubation, with occurrence rates of apoptotic cells of 4.36%, 14.42% and 21.16%, respectively. Phase-contrast and electron microscopy revealed that the nuclear fragmentation and chromosomal condensed, cells shrank and attachment loss appeared in the SGC7901 treated with alisol B acetate. Apoptosis of SGC7901 cells was associated with cell cycle arrest, caspase-3 and caspase-9 activation, loss of mitochondrial membrane potential and up-regulation of the ratio of Bax/Bcl-2 and inhibition of the PI3K/Akt.CONCLUSION： Alisol B acetate exhibits an antiproliferative effect in SGC7901 cells by inducing apoptosis. Apoptosis of SGC7901 cells involves mitochondria-caspase and PI3K/Akt dependent pathways.

Phosphatidylinositol 3-kinase CB association with preoperative radiotherapy response in rectal adenocarcinoma

AIM: To examine the correlation of phosphatidylinositol 3-kinase (PIK3) CB expression with preoperative radiotherapy response in patients with stage II/III rectal adenocarcinoma.

Analysis of Phosphatidylinositol 3-kinase Activation in the Adipose Tissue of Gestational Diabetes Mellitus Patients and Insulin Resistance

The P85 regulatory subunit protein and gene expression and P110 catalylic subunit activity of phosphatidylinositol 3-kinase (PI-3K) were investigated in adipose tissue of patients with gestational diabetes mellitus (GDM) in order to explore the molecular mechanisms of insulin resistance (IR) of GDM. Samples from patients with GDM (n=50), and controls (n=50) were collected. Fasting insulin (FIN) was determined by radioimmunoassay. Fasting plasma glucose (FPG) was measured by oxidase assay. Western blot technique was used to detect the levels of PI-3K P85 subunit in adipose tissues of patients with GDM. The mRNA expression of PI-3K P85 subunit was detected by reverse transcription polymerase chain reaction (RT-PCR) method in the adipose tissue. PI-3K activity was examined by immunoprecipitation, thin-layer chromatography and gamma scintillation counting. The results were analyzed statistically. It was found that the levels of FPG, FIN and HOMA-IR in GDM group were significantly higher than those in control group (all P0.05). PI-3K activity was significantly decreased to 82.89% in GDM group as compared with control group (P<0.01) and negatively correlated with HOMA-IR (r=-0.75, P<0.01). It was concluded that PI-3K in GDM patients may be involved in the insulin signaling pathway, resulting in IR of GDM.

Glycine-extended gastrin activates two independent tyrosine-kinases in upstream of p85/p110 phosphatidylinositol 3-kinase in human colonic tumour cells

AIM： To investigate whether Src, JAK2 and phosphatidylinositol 3-kinase （PI3K） pathways are involved in the proliferation of human colonic tumour cells induced by glycine-extended gastrin （G-gly）, the precursor of the mature amidated gastrin and to elucidate the molecular interaction between these three kinases in response to this peptide. METHODS： Using the human colonic tumour cell line HCT116 as a model, we first measured the activation of PI3K, p60-Src and JAK2 in response to G-gly by in vitro kinase assays. Then we investigated the involvement of these kinases in G-gly-induced cell proliferation by MTT test. RESULTS： G-gly stimulation induced p60-Src, JAK2 and PI3K activation in HCT116. The different pathways were involved in proliferation of human colon cancer cells induced by G-gly. Furthermore, we found that both Src and JAK2 were necessary to PI3K regulation by this peptide. However, we did not find any cross-talk between the two tyrosine kinases. CONCLUSION： Our results suggest that the p60-Src/ PI3K and JAK2/PI3K pathways act independently to mediate G-gly proliferative effect on human colonic tumour cells.

Micro RNA-21 promotes phosphatase gene and protein kinase B/phosphatidylinositol 3-kinase expression in colorectal cancer

AIM: To explore the regulatory mechanism of the target gene of micro RNA-21(mi R-21), phosphatase gene(p TEN), and its downstream proteins, protein kinase B(AKT) and phosphatidylinositol 3-kinase(p I3K), in colorectal cancer(CRC) cells. METHODS: Quantitative real-time p CR(q RT-p CR) and Western blot were used to detect the expression levels of mi R-21 and p TEN in HCT116, HT29, Colo32 and SW480 CRC cell lines. Also, the expression levels of p TEN m RNA and its downstream proteins AKT and p I3 K in HCT116 cells after downregulating mi R-21 were investigated. RESULTS: Comparing the mi R-21 expression in CRC cells, the expression levels of mi R-21 were highest in HCT116 cells, and the expression levels of mi R-21 were lowest in SW480 cells. In comparing mi R-21 and p TEN expression in CRC cells, we found that the protein expression levels of mi R-21 and p TEN were inversely correlated(p < 0.05); when mi R-21 expression was reduced, m RNA expression levels of p TEN did not significantly change(p > 0.05), but the expression levels of its protein significantly increased(p < 0.05). In comparing the levels of p TEN protein and downstream AKT and p I3 K in HCT116 cells after downregulation of mi R-21 expression, the levels of AKT and p I3 K protein expression significantly decreased(p < 0.05). CONCLUSION: p TEN is one of the direct target genesof mi R-21. Thus, phosphatase gene and its downstream AKT and p I3 K expression levels can be regulated by regulating the expression levels of mi R-21, which in turn regulates the development of CRC.

Anti-silencing function 1B knockdown suppresses the malignant phenotype of colorectal cancer by inactivating the phosphatidylinositol 3-kinase/AKT pathway

BACKGROUND Mounting studies have highlighted the pivotal influence of anti-silencing function 1B(ASF1B)on the malignancy of cancers.AIM To explore the influence and mechanism of ASF1B in colorectal cancer(CRC).METHODS Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect mRNA expression of ASF1B.Immunohistochemical staining was performed to detect protein expression of ASF1B and Ki67 in tumor tissues.Western blot analysis was used to determine levels of ASF1B and proliferation/epithelial mesenchymal transition(EMT)/stemness-related proteins.In addition,the proliferation of CRC cells was assessed using Cell Counting Kit-8 and 5-Ethynyl-2’-Deoxyuridine assays.The migration and invasion of CRC cells were evaluated using transwell assays.Stemness of CRC cells was tested using the sphere formation assay.To construct a xenograft tumor model,HCT116 cells were introduced into mouse flanks via subcutaneous injection.RESULTS ASF1B expression was markedly increased in CRC tissues and cells,and it was inversely correlated with overall survival of CRC patients and was positively associated with the tumor node metastasis(TNM)stage of CRC patients.Silencing of ASF1B suppressed proliferation,migration,invasion,stemness and EMT of CRC cells as well as tumorigenesis of xenograft mice.Furthermore,protein levels of Pphosphatidylinositol 3-kinase(p-PI3K)and p-AKT were decreased after silencing of ASF1B in CRC cells.The inhibitory effects of ASF1B knockdown on cell proliferation,stemness and EMT were partly abolished by PI3K activator in CRC cells.CONCLUSION Silencing of ASF1B inactivated the PI3K/AKT pathway to suppress CRC malignancy in vitro.

Response of Subcutaneous Xenografts of Endometrial Cancer in Nude Mice to Inhibitors of Phosphatidylinositol 3-Kinase/Akt and Mitogen-Activated Protein Kinase (MAPK) Pathways: An Effective Therapeutic Strategy for Endometrial Cancer

Objective: This study was designed to explore whether inhibition of the extracellular-regulated kinase (ERK) and phosphatidylinositol-3-kinase (PI3K) signaling pathways can inhibit the growth of xenografts of endometrial cancer cell lines with different estrogen receptors (ER) profiles in vivo and to provide preliminary laboratory basis for the probability of endometrial adenocarcinoma treatment with blockage of the two pathways, especially to endometrial cancer with low ER status. Methods: Human endometrial cancer Ishikawa bearing ER and HEC-1Awith low ER status cells were subcutaneously injected into BALB/c nude mice to establish endometrial cancer xenograft tumor models. The effects of PI3K/Akt inhibitor LY294002, MAPK/ERK1/2 inhibitor PD-98059 and their combinations on the growth of the xenograft tumors and apoptotic state of Ishikawa and HEC-1Acells were tested in vivo using the inhibitory rate, the terminal deoxynucleotidyl transferase-mediated nick-end labeling assay, H/E-stain. Western blot analysis was used to detect the alterations of activated ERK (P-ERK) and AKT (P-AKT) during this process. Results: LY294002, a PI3K/Akt pathway inhibitor, induced significant suppression in the growth of both Ishikawa and HEC-1Acell xenograft tumors, concomitant with increased apoptosis in xenografts as evidenced by TUNEL. A similar effect was also observed when the MAPK/ERK1/2 signaling pathway was inhibited by PD98059. Concurrent inhibition of the PI3K/Akt and MAPK/ERK1/2 pathways showed enhanced anti-tumor effects in vivo as indicated by increased apoptosis. At the same time, the levels of P-ERK and P-AKT in both xenograft tumors decreased, and their levels in combination group was the lowest. Conclusions: PD98059, LY294002 and their combinations showed remarkable inhibitory effects on xenograft tumors of endometrial carcinoma cell lines with different expression status of ER in vivo through blockage of PI3K/Akt and MAPK/ERK1/2 signaling pathways. This suggests that targeting these pathways may be an effective therapeutic strategy against endometrial carcinomas, especially for ER-negative cancers which show poor response to endocrinal therapy.

Roles of phosphatidylinositol-3-kinases signaling pathway in inflammation-related cancer:Impact of rs10889677 variant and buparlisib in colitis-associated cancer

BACKGROUND Phosphatidylinositol-3-kinases(PI3K)is a well-known route in inflammationrelated cancer.Recent discovery on PI3K-related genes revealed a potential variant that links ulcerative colitis(UC)and colorectal cancer(CRC)with colitisassociated cancer(CAC).PI3K/AKT pathway has been recommended as a potential additional therapeutic option for CRC due to its substantial role in modifying cellular processes.Buparlisib is a pan-class I PI3K inhibitor previously shown to reduce tumor growth.AIM To investigate the regulation of rs10889677 and the role of buparlisib in the PI3K signaling pathway in CAC pathogenesis.METHODS Genomic DNA from 32 colonic samples,including CAC(n=7),UC(n=10)and CRC(n=15),was sequenced for the rs10889677 mutation.The mutant and wildtype fragments were amplified and cloned in the pmirGLO vector.The luciferase activity of cloned vectors was assessed after transfection into the HT29 cell line.CAC mice were induced by a mixture of a single azoxymethane injection and three cycles of dextran sulphate sodium,then buparlisib was administered after 14 d.The excised colon was subjected to immunohistochemistry for Ki67 and Cleaved-caspase-3 markers and quantitative real-time polymerase chain reaction analysis for Pdk1 and Sgk2.RESULTS Luciferase activity decreased by 2.07-fold in the rs10889677 mutant,confirming the hypothesis that the variant disrupted miRNA binding sites,which led to an increase in IL23R expression and the activation of the PI3K signaling pathway.Furthermore,CAC-induced mice had a significantly higher disease activity index(P<0.05).Buparlisib treatment significantly decreased mean weight loss in CAC-induced mice(P<0.05),reduced the percentage of proliferating cells by 5%,and increased the number of apoptotic cells.The treatment also caused a downward trend of Pdk1 expression and significantly decreased Sgk2 expression.CONCLUSION Our findings suggested that the rs10889677 variant as a critical initiator of the PI3K signaling pathway,and buparlisib had the ability to prevent PI3K-non-AKT activation in the pathophysiology of CAC.

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.

Class I phosphatidylinositol 3-kinase inhibitors for cancer therapy

The phosphatidylinositol 3-kinase(PI3K) pathway is frequently activated in human cancers.Class I PI3 Ks are lipid kinases that phosphorylate phosphatidylinositol 4,5-bisphosphate(PIP2) at the 3-OH of the inositol ring to generate phosphatidylinositol 3,4,5-trisphosphate(PIP3), which in turn activates Akt and the downstream effectors like mammalian target of rapamycin(m TOR) to play key roles in carcinogenesis. Therefore, PI3 K has become an important anticancer drug target, and currently there is very high interest in the pharmaceutical development of PI3 K inhibitors. Idelalisib has been approved in USA and Europe as the first-in-class PI3 K inhibitor for cancer therapy. Dozens of other PI3 K inhibitors including BKM120 and ZSTK474 are being evaluated in clinical trials. Multifaceted studies on these PI3 K inhibitors are being performed, such as single and combinational efficacy, resistance, biomarkers,etc. This review provides an introduction to PI3 K and summarizes key advances in the development of PI3 K inhibitors.

Jiaotai Pill(交泰丸) Enhances Insulin Signaling through Phosphatidylinositol 3-Kinase Pathway in Skeletal Muscle of Diabetic Rats

objective： To investigate the effect of Jiaotai Pill （交泰丸, JTP） at different constitutional proportions on insulin signaling through phosphatidylinositol 3-kinase （PI3K） pathway in the skeletal muscle of diabetic rats. Methods： The rat model of type 2 diabetes mellitus （T2DM） was established by intravenous injection of a small dose of streptozotoein plus high fat diet feeding. JTP at the same dosage of cinnamon and the increasing dosage of Coptis chinensis was administered to diabetic rats for nine weeks respectively. Plasma glucose and insulin levels were assayed. The expressions of proteins were determined by Western blot method. Results： All the three formulations of JTP decreased plasma glucose and fasting insulin levels as well as increased the protein expressions of insulin receptor β （InsR β） subunit, insulin receptor substrate-1 （IRS-1）, PI3K p85 subunit and glucose transporter 4 （GLUT4） in skeletal muscle. Meanwhile, JTP increased the tyrosine phosphorylation of InsRβ subunit and IRS-1, and reduced the serine phosphorylation of IRS-1 in skeletal muscle. Interestingly, the effect of JTP on improving insulin sensitivity was not dose-dependent. In contrast, JTP containing the least amount of Coptis chinensis exhibited the best effect. Conclusion： JTP at different constitutional proportions attenuates the development of diabetes in a rat model of T2DM. The mechanism might be associated with enhancing insulin signaling through PI3K pathway in the skeletal muscle.

Electroacupuncture Pretreatment at Neiguan (PC 6) attenuates autophagy in rats with myocardial ischemia reperfusion through the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway

OBJECTIVE: To investigate the protective efficacy of electroacupuncture(EA) pretreatment at Neiguan(PC6) on myocardial ischemia-reperfusion(I/R) in rats.METHODS: Fifty rats were randomly divided into five groups(n = 10): sham operation group, model group(underwent in vivo myocardial I/R), EA pretreatment group [EA at Neiguan(PC 6) 1 week before I/R], wortmannin group(1 week before I/R, the PI3K inhibitor, wortmannin, was injected), EA pretreatment + wortmannin group(both pretreatments were performed simultaneously). After establishing the I/R model, 2,3,5-triphenyltetrazolium chloride(TTC) staining was used to analyze the weight and area of the myocardial infarction tissue.The biosignal and pressure test system was used to determine the left ventricular systolic mean pressure(LVSP), left ventricular end-diastolic pressure(LVEDP), fractional shortening(FS), and ejection fraction(EF). Ultraviolet spectrophotometry was used to determine the expression of creatine kinase(CK)-MB, inducible nitric oxide synthase(i NOS), and total antioxidant capacity(T-AOC) in the serum. The expression of autophagy-related protein 13(ATG13), mammalian target of rapamycin(m TOR), and phosphatidylinositol 3-kinase(PI3K) in cardiac muscle cells was determined by immunofluorescence. Hematoxylin and eosin staining was used to observe autophagy-related pathological changes in rat cardiomyocytes, and ultrastructural changes of cardiomyocytes were examined by transmission electron microscopy.RESULTS: In this study, the infarction size and tissue weight of the EA pretreatment group were decreased compared with the model group(P <0.0001). Furthermore, compared with the model group, the LVEDP values of the EA pretreatment group were significantly reduced(P = 0.0091), and the LVSP, FS, and EF values were slightly increased (P = 0.0007, 0.0020, 0.0031). EA pretreatment also significantly decreased the expression of CK-MB and i NOS, while it increased the expression of T-AOC in the serum of rats with I/R injury(P <0.0001). Furthermore, EA pretreatment slightly widened the myocardial fiber space, reduced necrosis and myocardial cell swelling and maintained the nucleus and mitochondria structure intact.CONCLUSION: EA pretreatment promoted autophagy flux and alleviated myocardial I/R injury through the PI3K-Akt-m TOR pathway.

Effects of Panax Quinquefolium Saponin on Phosphatidylinositol 3-Kinase/Serine Threonine Kinase Pathway of Neonatal Rat Myocardial Cells Subjected to Hypoxia

Objective： To explore the effects of Panax Quinquefolium Saponin （PQS） on phosphatidylinositol 3-kinase/serine threonine kinase （P13/Akt） pathway of neonatal rat myocardial cells subjected to hypoxia. Methods： Neonatal rat myocardial cells were cultured in vitro. After the myocardial cell injury was induced by hypoxia, the cells were randomized into 5 groups： the normal group, the model group, the positive control group （Ciclosporin A, 2 p, mol/L）, the low-dose PQS group （PQSL, 25mg/L）, and the high-dose PQS group （PQSH, 50 mg/L）. Morphology and behavior of myocardial cells were observed under an inverted microscope. Apoptosis rate and lactate dehydrogenase （LDH） leakage rate of myocardial cells were determined by colorimetry. Mitochondrial transmembrane potential was assessed using a fluorexon laser. Phospho-glycogen synthase kinase （GSK）-3β and phospho-Akt as well as cytochrome C were determined by Western blot. Results： LDH leakage in the Ciclosporin A group, PQSH group and PQSL group reduced progressively compared with the model group （P〈0.05）. Akt and GSK-3β was strongly phosphorylated after treatment with Ciclosporin A and PQS compared with the model group （P〈0.05, P〈0.01）. Compared with the model group （16.41 ± 1.74; 35.28 ± 6.30）, both the integrated optical density of mitochondrial permeability transition pore （MPTP） and the mitochondrial transmembrane potential significantly increased in the PQSH group （42.74± 2.12; 71.36 ± 6.54） and the PQSL group （39.58± 1.49; 66.99± 5.45; P〈0.05, P〈0.01）. However, the protein of cytochrome C outside the mitochonddon decreased in the PQSH group （273.66 ± 14.61） and the PQSL group （259.62 ± 17.31） compared with the model group （502.41 ± 17.76; P〈0.05）. Conclusion： Through activation of the P13K/Akt pathway and inhibition of the MPTP, PQS might protect the heart against ischemia injury and apoptosis of myocardial cells.

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.

Efficacy of self-made Gengnian decoction on phosphatidylinositol 3-kinases/protein kinase B/mammalian target of rapamycin signaling pathway in perimenopausal rats

OBJECTIVE:To investigate the efficacy of self-made Gengnian decoction on expressions of phosphatidylinositol 3-kinase(PI3 K),protein kinase B(Akt)and mammalian target of rapamycin(m TOR)in ovarian tissues of perimenopausal rats.They were identified with symptom pattern of kidney-Yang deficiency in terms of Traditional Chinese Medicine.METHODS:Female Sprague-Dawley rats aged10-12 months were selected.Estrous cycle was observed by vaginal smears of keratinocytes to screen the perimenopausal model rats.The chosen rats were randomly divided into five groups,including perimenopausal model of kidney-Yang deficiency group(24 rats),self-made Gengnian decoction of high-dose group(24 rats),self-made Gengnian decoction of middle-dose group(24 rats),self-made Gengnian decoction of low dose group(24 rats)and tibolone control group(24 rats).In addition,rats aged 4-6 months were selected as young control group.The perimenopausal model rats of kidney-Yang deficiency were prepared by alternative intramuscular injection of hydrocortisone 5 mg·kg^-1·d^-1The successfully prepared models in self-made Gengnian decoction of high-dose,middle-dose and low-dose groups and tibolone control group were given self-made Gengnian decoction 26.4,13.2 and 6.6 mg·kg^-1·d^-1,and tibolone tablets solvent 0.22 mg·kg^-1·d^-1,respectively,through intragastric administration.Models group and young control group were given the same dose of normal saline,1 time a day for 15 consecutive days.24 h after the last administration,blood and ovarian tissues were collected after anesthesia with 20%ethyl carbamate.The follicles of different levels in ovarian tissue were observed and counted by histopathological hematoxylin-eosin staining.Enzyme linked immunosorbent assay was applied to test insulin-like growth factor-1(IGF-1)level in the serum of experimental rats.The expression levels of PI3 K,phosphorylated-Akt(p-Akt)and phosphorylated-m TOR(p-m TOR)m RNA in ovarian tissue were detected by quantitative real-time polymerase chain reaction.RESULTS:The total follicle counts of perimenopausal model rats with kidney-Yang deficiency were significantly reduced,and the number of follicles(mainly increased in preantral follicles and antral follicles)in perimenopausal model rats with kidney-Yang deficiency was significantly increased after intervention of high and middle doses of Gengnian decoction and tibolone(P<0.05).Compared with normal rats in young control group,the levels of IGF-1 in serum of perimenopausal rats with kidney-Yang deficiency were significantly decreased(P<0.01),and those intervened by high dose of Gengnian decoction and tibolone were significantly up-regulated.The relative expression levels of PI3 K,p-Akt,p-m TOR m RNA in ovarian tissues of perimenopausal rats with kidney-Yang deficiency were significantly lower than those of young rats(P<0.01),and those intervened by high dose of Gengnian decoction and tibolone were significantly up-regulated(P<0.05).CONCLUSION:Self-made Gengnian decoction can increase the levels of IGF-1,PI3 K,Akt and m TOR m RNA expression in serum.

Phosphatidylinositol 3-kinase inhibitor, LY294002, induced senescence-like changes in human diploid fibroblasts

Objective To reveal the role of Phosphatidylinositol 3-kinases (PI3Ks) in regulating human diploid fibroblast (2BS cell) senescence as well as the possible mechanisms involved.Methods Using a PI3Ks specific inhibitor, LY294002, cell cycle, apoptosis, proliferation, senescence association β-galactosidase staining as well as senescence association CKIs, p16INK4 and p21Cip1 protein expressions were all measured in the low passages of 2BS cells.Results Both 25 μmol/L and 50 μmol/L concentrations of LY294002 could cause a significant decrease in cells entering into S phase, and this cell cycle of G, phase arrest was dose-dependent. Meanwhile, LY294002 contributed to apoptosis, caused 2BS cell growth arrest, and activated senescence association p-galactosidase (P<0. 05). In addition, LY294002 could induce time-course expressions of p16INK4 and p21Cip1 in 2BS cell lines.Conclusions PI3Ks inhibitor LY294002 could induce senescence-like changes in 2BS cell lines. Two senescence associated CKIs, p16INK4 and p21Cip1, might be involved in this senescence phenotype proceeding in 2BS cell lines.