BACKGROUND: Hyperglycemia, a characteristic feature of diabetes, induces glucotoxicity in pancreatic beta-cells, resulting in further impairment of insulin secretion and worsening glycemic control. Thus, preservation of insulin secretory capacity is essential for the management of type 2 diabetes. In this study, we evaluated the ability of an Orthosiphon stamineus (OS) extract to prevent glucotoxicity in insulin-producing cells. METHODS: We measured insulin mRNA expression and glucose-stimulated insulin secretion (GSIS) in OS-treated INS-1 cells after exposure to a high glucose (HG; 30 mM) concentration. RESULTS: The hexane extract of OS elevated mRNA expression of insulin as well as pancreatic and duodenal homeobox-1 of INS-1 cells in a dose-dependent manner. The hexane OS extract also increased the levels of phosphorylated phosphatidylinositol 3-kinase (PI3K) in a concentration-dependent manner. Additionally, Akt phosphorylation was elevated by treatment with 100 and 200 micromol of the hexane OS extract. Three days of HG exposure suppressed insulin mRNA expression and GSIS; these expressions were restored by treatment with the hexane OS extract. HG elevated peroxide levels in the INS-1 cells. These levels were unaffected by OS treatment under both normal and hyperglycemic conditions. CONCLUSION: Our results suggested that the hexane extract of OS elevates insulin mRNA expression and prevents glucotoxicity induced by a 3-day treatment with HG. This was associated with the activation of PI-3K and Akt.
Glucose ; Hyperglycemia ; Insulin* ; Orthosiphon* ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases ; Phosphorylation ; RNA, Messenger

Alzheimer's disease (AD) is the most common form of dementia. Although uncountable clinical trials have been done to develop the treatment of AD, there are a couple of drugs that can be used only for symptomatic treatment. Therefore, many studies based on the amyloid cascade hypothesis and the tauopathy hypothesis are still ongoing. After the failure of numerous huge Phase III clinical trials, arguments on those hypotheses have arisen and efforts to establish other possible therapeutic strategies based on diverse plausible mechanisms associated with AD have been done as well. One of the new therapeutic targets for AD is the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. In this review, questions on the two hypotheses, the definition of the PI3K/AKT pathway, the relationship between the pathway and AD, and the possibility of the modulation of the pathway as a new therapeutic strategy for AD will be discussed briefly.
Alzheimer Disease* ; Amyloid ; Dementia ; Phosphatidylinositol 3-Kinase ; Tauopathies

Activation of the mammalian target of rapamycin (mTOR) signaling pathway is an important mechanism of resistance to endocrine therapy in breast cancer. Everolimus, an mTOR inhibitor, has been shown to increase the efficacy of endocrine therapy and overcome resistance to endocrine therapies. Clinical studies have suggested that everolimus combined with endocrine therapy prolongs progression-free survival in hormone receptor-positive breast cancer patients. However, because breast cancer includes a group of highly heterogeneous tumors, patients may have different responses to everolimus. Therefore, finding biomarkers that can predict a patient's positive response or resistance to everolimus is critical. Numerous preclinical studies have shown that PIK3CA/PTEN mutations are predictive of sensitivity to everolimus; however, clinical trials have not confirmed the correlation between mutation status and clinical response. KRAS or BRAF mutations can bypass the phosphatidylinositol 3-kinase pathway; therefore, mutations in KRAS or BRAF may lead to resistance to mTOR inhibitors, and preclinical studies have shown that PIK3CA mutant cells which also contain KRAS mutations are resistant to everolimus. However, there are no clinical data in breast cancer patients to support this conclusion. Therefore, large-scale clinical studies are needed to identify biomarkers of efficacy and resistance to everolimus.
Biomarkers* ; Breast Neoplasms* ; Breast* ; Disease-Free Survival ; Everolimus* ; Humans ; Phosphatidylinositol 3-Kinase ; Sirolimus

Bone marrow mesenchymal stem cells (BM MSCs) can differentiate into multi-lineage tissues. However, obtaining BM MSCs by aspiration is difficult and can be painful; therefore peripheral blood (PB) MSCs might provide an easier alternative for clinical applications. Here, we show that circulating PB MSCs proliferate as efficiently as BM MSCs in the presence of extracellular matrix (ECM) and that differentiation potential into osteoblast in vitro and in vivo. Both BM MSCs and PB MSCs developed into new bone when subcutaneously transplanted into immune-compromised mice using hydroxyapatite/tricalcium phosphate as a carrier. Furthermore, LY294002 and Wortmannin blocked mesenchymal stem cell attachment in a dose-dependent manner, suggesting a role of phosphatidylinositol 3-kinase in MSC attachment. Our data showed that the growth of PB MSCs could be regulated by interaction with the ECM and that these cells could differentiate into osteoblasts, suggesting their potential for clinical applications.
Animals ; Bone Marrow ; Extracellular Matrix ; In Vitro Techniques ; Mesenchymal Stromal Cells ; Mice ; Osteoblasts ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositols

OBJECTIVE: To investigate the effect of glycolytic enzyme pyruvate kinase type 2 (PKM2) on the proliferation and apoptosis of human leukemia HL-60 cells. METHODS: si-PKM2 plasmid was transfected into HL-60 cells (set as si-PKM2 group), and blank vector transfected cells were set as control group (si-Ctl group). The expression levels of PKM2 mRNA and protein in si-Ctl group and si-PKM2 group were detected by RT-qPCR and Western blot. CCK-8 cell detection kit was used to detect the proliferation ability of the cells in the two groups. Flow cytometry was used to detect the changes of cell cycle and apoptosis. Western blot and RT-qPCR were used to detect the changes of p-Akt and p-mTOR protein levels in PI3K/Akt/mTOR signaling pathway and the changes of glycolysis-related mRNA levels of the cells in the two groups. The changes in glucose consumption and lactic acid production of the cells were assayed. Over expressed PKM2, HL-60 cells were treated with PI3K inhibitor LY294002 or galactose, the changes in cell proliferation ability, cell cycle and apoptosis, as well as changes in glucose consumption and lactic acid production were detected. RESULTS: Interfered by si-PKM2, mRNA and protein levels of PKM2 in si-PKM2 group significantly decreased, and proliferation ability of the cells was also reduced (P<0.05). After PKM2 knockdown, the cells were significantly blocked at G CONCLUSION PKM2 knockdown can inhibit the proliferation and induce apoptosis of HL-60 cells, and its molecular mechanism may be related to the PKM2-mediated PI3K/Akt/mTOR-glycolysis, which suggesting that PKM2 may serve as a molecular target for the prevention and treatment of leukemia.
Apoptosis ; Cell Proliferation ; Glycolysis ; Humans ; Phosphatidylinositol 3-Kinases/metabolism* ; Pyruvate Kinase

OBJECTIVE: To determine of the regulation of cyclooxygenase-2 (COX-2) expression by Interleukin-1beta in WISH cells. METHODS: Amnion WISH cells were incubated in media containing increasing concentrations of IL-1beta or with various inhibitors. Increased COX-2 expression was determined by Western blot analysis with anti-COX-2 antibody. Concomitant measurements of culture media PGE2 were made by an enzyme immunoassay. RESULTS: The COX-2 and prostaglandin E2 production induced by IL-1beta increased in a dose- and time-dependent manner. One of the regulating factors that induced COX-2 by IL-1beta was protein kinase C (PKC). PKC inhibitor, Ro 31-8220 was pretreated and continued treating by IL-1beta. Then, PKC inhibitor completely blocked COX-2 protein induction by IL-1beta. In contrast, COX-2 induction by IL-1beta after pretreating PKC stimulator, phobol 12-myristate 13-acetate was potentiated with synergism. Another factor in controlling COX-2 protein induction was identified as phosphatidylinositol 3-kinase (PI 3K). COX-2 protein induction by IL-1beta after pretreating PI 3K inhibitors, wortmannin and LY294002 strongly increased. This kind of result reflected that PI 3K act as negative regulator. COX-2 induction by IL-1beta was known to be regulated in not only transcription step, but also translation step after performing experiment of actinomycin and cycloheximide treatment. CONCLUSION: COX-2 protein and prostaglandin E2 production induced by IL-1beta were controlled by many factors in amnion cell. Among those factors, PKC and PI 3K have an important role, but their control mechanism act as positive and negative, respectively.
Amnion ; Blotting, Western ; Culture Media ; Cycloheximide ; Cyclooxygenase 2* ; Dactinomycin ; Dinoprostone ; Immunoenzyme Techniques ; Interleukin-1beta* ; Phosphatidylinositol 3-Kinase ; Protein Kinase C

Cell-matrix interactions have major effects upon phenotypic features such as gene regulation, cytoskeletal structure, differentiation, and aspects of cell growth control. Upon detachment from the matrix epithelial cells enter into programmed cell death and this cell detachment-induced apoptosis has been referred to as "anoikis". This study was undertaken to determine whether apoptosis is induced by inhibition of contact with extracellular matrix in mouse inner medullary collecting duct cells (mIMCD-3), what are signaling mechanisms of the process and whether EGF protects detachment-induced apoptosis. Upon detachment from the extracellular matrix, MDCK and mIMCD-3, which were derived from inner medulla of SV40 transgenic mouse, entered into programmed cell death as a time-dependent manner. Apoptotic cell death induced by cell detachment increased in serum-free medium, which was partly protected by the addition of epidermal growth factor (EGF). Ly294002 and wortmannin, inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), negated the EGF effect, whereas PD98059, a MEK inhibitor, did not. The addition of SB203580, an inhibitor of p38 kinase, did not protect apoptosis in suspended mIMCD-3 cells. These results indicate that apoptosis is induced by inhibition of contact with extracellular matrix in mouse inner medullary collecting duct cells and that PI 3-kinase, not MAPK, is a key mediator of the EGF-induced survival of renal epithelial cells in the absence of attachment.
Animals ; Apoptosis ; Cell Death* ; Epidermal Growth Factor ; Epithelial Cells* ; Extracellular Matrix* ; Mice* ; Mice, Transgenic ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases ; Phosphotransferases

OBJECTIVE: To investigate the expressions and time-dependent changes of phosphatidylinositol-3-kinase (PI3K), phospho-PI3K (p-PI3K), protein kinase B (PKB/Akt) and phospho-Akt (p-Akt) during wound healing process of mice skin. METHODS: The changes of PI3K, p-PI3K, Akt and p-Akt expression in skin wound were detected by immunohistochemistry, Western blotting and real-time PCR. RESULTS: Immunohistochemistry showed the expression of PI3K and p-Akt were observed in mononuclear and fibroblast after skin wound, and reached peak in reconstruction. The positive bands of PI3K, p-PI3K, Akt and p-Akt were observed in all time points of the wound healing process by Western blotting. The expression peak of p-PI3K and p-Akt showed in inflammation and proliferation; the expression peak of PI3K and Akt in reconstruction. Real-time PCR showed the expression peak of PI3K mRNA in inflammation and reconstruction and the peak of Akt mRNA in reconstruction. CONCLUSION During the wound healing process, the expressions of PI3K, Akt, p-PI3K and p-Akt show different changes with significant correlation to wound time. The expression of PI3K/Akt may be a valuable marker for wound time estimation.
Animals ; Blotting, Western ; Class I Phosphatidylinositol 3-Kinases ; Fibroblasts/metabolism* ; Mice ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases/metabolism* ; Phosphorylation ; Proto-Oncogene Proteins c-akt/metabolism* ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Skin/injuries* ; Wound Healing

BACKGROUND: Sjögren's syndrome (SS) is an autoimmune disorder characterized by sicca syndrome and/or systemic manifestations. The treatment is still challenging. This study aimed to explore the therapeutic role and mechanism of exosomes obtained from the supernatant of stem cells derived from human exfoliated deciduous teeth (SHED-exos) in sialadenitis caused by SS. METHODS: SHED-exos were administered to the submandibular glands (SMGs) of 14-week-old non-obese diabetic (NOD) mice, an animal model of the clinical phase of SS, by local injection or intraductal infusion. The saliva flow rate was measured after pilocarpine intraperitoneal injection in 21-week-old NOD mice. Protein expression was examined by western blot analysis. Exosomal microRNA (miRNAs) were identified by microarray analysis. Paracellular permeability was evaluated by transepithelial electrical resistance measurement. RESULTS: SHED-exos were injected into the SMG of NOD mice and increased saliva secretion. The injected SHED-exos were taken up by glandular epithelial cells, and further increased paracellular permeability mediated by zonula occluden-1 (ZO-1). A total of 180 exosomal miRNAs were identified from SHED-exos, and Kyoto Encyclopedia of Genes and Genomes analysis suggested that the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway might play an important role. SHED-exos treatment down-regulated phospho-Akt (p-Akt)/Akt, phospho-glycogen synthase kinase 3β (p-GSK-3β)/GSK-3β, and Slug expressions and up-regulated ZO-1 expression in SMGs and SMG-C6 cells. Both the increased ZO-1 expression and paracellular permeability induced by SHED-exos were abolished by insulin-like growth factor 1, a PI3K agonist. Slug bound to the ZO-1 promoter and suppressed its expression. For safer and more effective clinical application, SHED-exos were intraductally infused into the SMGs of NOD mice, and saliva secretion was increased and accompanied by decreased levels of p-Akt/Akt, p-GSK-3β/GSK-3β, and Slug and increased ZO-1 expression. CONCLUSION Local application of SHED-exos in SMGs can ameliorate Sjögren syndrome-induced hyposalivation by increasing the paracellular permeability of glandular epithelial cells through Akt/GSK-3β/Slug pathway-mediated ZO-1 expression.
Mice ; Animals ; Humans ; Sjogren's Syndrome/therapy* ; Proto-Oncogene Proteins c-akt/metabolism* ; Tight Junctions/metabolism* ; Glycogen Synthase Kinase 3 beta ; Mice, Inbred NOD ; Phosphatidylinositol 3-Kinases/metabolism* ; Exosomes/metabolism* ; Xerostomia ; Phosphatidylinositol 3-Kinase ; MicroRNAs/genetics*

Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway (PAM pathway) plays an important role in the development of breast cancer and are closely associated with the resistance to endocrine therapy in advanced breast cancer. Therefore, anti-cancer treatment targeting key molecules in this signaling pathway has become research hot-spot in recent years. Randomized clinical trials have demonstrated that PI3K/AKT/mTOR inhibitors bring significant clinical benefit to patients with advanced breast cancer, especially to those with hormone receptor (HR)-positive, human epidermal growth factor receptor (HER) 2-negative advanced breast cancer. Alpelisib, a PI3K inhibitor, and everolimus, an mTOR inhibitor, have been approved by Food and Drug Administration. Based on their high efficacy and relatively good safety profile, expanded indication of everolimus in breast cancer have been approved by National Medical Products Administration. Alpelisib is expected to be approved in China in the near future. The members of the consensus expert panel reached this consensus to comprehensively define the role of PI3K/AKT/mTOR signaling pathway in breast cancer, efficacy and clinical applications of PI3K/AKT/mTOR inhibitors, management of adverse reactions, and PIK3CA mutation detection, in order to promote the understanding of PI3K/AKT/mTOR inhibitors for Chinese oncologists, improve clinical decision-making, and prolong the survival of target patient population.
Breast Neoplasms/metabolism* ; Consensus ; Everolimus/therapeutic use* ; Female ; Humans ; MTOR Inhibitors ; Phosphatidylinositol 3-Kinase/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Phosphoinositide-3 Kinase Inhibitors ; Proto-Oncogene Proteins c-akt/metabolism* ; Sirolimus/therapeutic use* ; TOR Serine-Threonine Kinases/metabolism*