HSP70 inhibition suppressed glioma cell viability during hypoxia/reoxygenation by inhibiting the ERK1/2 and PI3K/AKT signaling pathways

Journal of Bioenergetics and Biomembranes - Heat shock protein 70 (HSP70) can regulate astrocyte viability under hypoxic and ischemic conditions. However, the protective mechanism involved is not...     

Orexin-A protects against oxygen-glucose deprivation/reoxygenation-induced cell damage by inhibiting endoplasmic reticulum stress-mediated apoptosis via the Gi and PI3K signaling pathways

The neuropeptide orexin-A (OXA) has a neuroprotective effect, acting as an anti-apoptotic factor in response to multiple stimuli. Apoptosis induced by endoplasmic reticulum stress (ERS) underlies oxygen-glucose deprivation and reoxygenation (OGD/R)-induced cell damage, an in vitro model of ischemia/reperfusion injury. However, that OXA inhibits ERS-induced apoptosis in the OGD/R model has not been reported. In the present study, we investigated the neuroprotective effect of OXA (0.1 μM) on OGD/R-induced damage in the human neuroblastoma cell line SH-SY5Y. After OXA treatment following 4 h oxygen-glucose deprivation (OGD) and then 4 h reoxygenation (R), cell morphology, viability, and apoptosis were analyzed by histology, Cell Counting Kit-8 assay, and flow cytometry, respectively. Western blotting was used to measure expression levels of ERS- and apoptosis-related proteins. To determine signaling pathways involved in OXA-mediated neuroprotection, the Gi pathway inhibitor pertussis toxin (PTX; 100 ng/mL) and PI3K inhibitor LY294002 (LY; 10 μM) were added. In addition, in order to prove the specificity of these characteristics, the OXA antagonist Suvorexant (DORA; Ki of 0.55 nM and 0.35 nM for OX1R and OX2R) was used for intervention. Our results showed that OGD/R induced cell damage, manifested as morphological changes and a significant decrease in viability. Furthermore, Western blotting detected an increase in ERS-related proteins GRP78, p-IRE1α, p-JNK, and Cleaved caspase-12, as well as apoptosis-related proteins Cleaved caspase-3 and Bax, and a decrease in the anti-apoptosis factor Bcl-2. OXA intervention alleviated the degree of cellular damage, and protein expression was also reversed. In addition, the protective effect of OXA was reduced by adding PTX and LY. Meanwhile, after the use of DORA, changes in the expression of related proteins were detected, and it was found that the protective effect of OXA was weakened. Collectively, our results indicate that OXA has a neuroprotective effect on OGD/R-induced cell damage by inhibiting ERS-induced apoptosis through the combined action of Gi and PI3K signaling pathways. These findings help to clarify the mechanism underlying the neuroprotective action of OXA, which should aid the development of further candidate drugs, and provide a new therapeutic direction for the treatment of ischemic stroke.     

GRP94 promotes muscle differentiation by inhibiting the PI3K/AKT/mTOR signaling pathway

The glucose-regulated endoplasmic reticulum chaperone protein 94 (GRP94) is required for many biological processes, such as secretion of immune factors and mesoderm induction. Here, we demonstrated that GRP94 promotes muscle differentiation in vitro and in vivo. Moreover, GRP94 inhibited the PI3K/AKT/mTOR signaling pathway. Using both in vitro and in vivo approaches, in myoblasts, we found that this inhibition resulted in reduced proliferation and increased differentiation. To further investigate the mechanism of GRP94-induced muscle differentiation, we used co-immunoprecipitation and proximity ligation assays and found that GRP94 interacted with PI3K-interacting protein 1 (Pik3ip1). The latter protein promoted muscle differentiation by inhibiting the PI3K/AKT/mTOR pathway. Furthermore, GRP94 was found to regulate Pik3ip1 expression. Finally, when Pik3ip1 expression was inhibited, GRP94-induced promotion of muscle differentiation was diminished. Taken together, our data demonstrated that GRP94 promoted muscle differentiation, mediated by Pik3ip1-dependent inhibition of the PI3K/AKT/mTOR signaling pathway.     

Participation of PI3K and ERK1/2 pathways are required for human brain vascular smooth muscle cell migration

Human brain vascular smooth muscle cell (HBVSMC) migration contributes to angiogenesis and several pathological processes in the brain. However, the molecular mechanism of angiogenesis, in which smooth muscle cell contributes, remains unclear. Our study investigates the role of vascular endothelial growth factor (VEGF) in the HBVSMC migration and elucidates the chemotactic signaling pathway mediating this action. We used the in vitro 'scratch' wound method to detect the HBVSMC migration. VEGF(165) (1-40ng/ml) induced the HBVSMC migration in a dose-dependent manner (P<0.05). VEGF(165) does not induce HBVSMC proliferation. Wortmannin, a specific phosphatidylinositol 3-kinase (PI3K) inhibitor, significantly inhibited serine/threonine kinase Akt/protein kinase B (PKB) phosphorylation and reduced HBVSMC migration into the wound edge following VEGF(165) stimulation (P<0.05). PD98059, an extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor, also significantly inhibited ERK1/2 phosphorylation and reduced the numbers of SMC migration. Parallel distance measurement showed that VEGF(165) induced HBVSMC migration significantly reduced due to inhibition of PI3K or ERK1/2 phosphorylation (P<0.05). Our results demonstrate that VEGF(165) could induce HBVSMC migration but not proliferation in vitro. Inhibiting Akt/PKB or ERK1/2 phosphorylation could reduce VEGF(165) induced HBVSMC migration. We provide the first evidence that activation of PI3K or ERK1/2 pathways are a crucial event in VEGF(165) mediated signal transduction leading to HBVSMC migration.     

Apelin suppresses apoptosis of human vascular smooth muscle cells via APJ/PI3-K/Akt signaling pathways

Apoptosis of vascular smooth muscle cells (VSMCs) plays an important role in regulating vascular remodeling during cardiovascular diseases. Apelin is the endogenous ligand for the G-protein-coupled receptor APJ and plays an important role in the cardiovascular system. However, the mechanisms of apelin on apoptosis of VSMCs have not been elucidated. Using a culture of human VSMCs as a model for the study of apoptosis, the relationship between apelin and apoptosis of human VSMCs and the signal pathway involved were investigated. Using western blotting, we confirmed that VSMCs could express APJ. To evaluate the possible role of apelin in VSMC apoptosis, we assessed its effect on apoptosis of human VSMCs. The results showed that apelin inhibited human VSMCs apoptosis induced by serum deprivation. Suppression of APJ with small-interfering RNA (siRNA) abolished the anti-apoptotic activity of apelin. Apelin increased Bcl-2 protein expression, but decreased Bax protein expression. An increase in activation of extracellular signal-regulated protein kinase (ERK) and Akt (a downstream effector of phosphatidylinositol 3-kinase) was shown after apelin stimulation. Suppression of APJ with siRNA abolished the apelin-induced activation of ERK and Akt. LY294002 (a PI3-K inhibitor) blocked apelin-induced activation of Akt and abolished the apelin-induced antiapoptotic activity. Our study suggests that apelin suppresses serum deprivation-induced apoptosis of human VSMCs, and that the anti-apoptotic action is mediated through the APJ/PI3-K/Akt signaling pathways.     

Retracted: MicroRNA-99b suppresses human cervical cancer cell activity by inhibiting the PI3K/AKT/mTOR signaling pathway

Cervical cancer is common cancer among women with high morbidity. MicroRNAs (miRs) are involved in the progression and development of cervical cancer. This study aimed to explore the effect of miR-99b-5p (miR-99b) on invasion and migration in cervical cancer through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway. The microarray-based analysis was used to screen out differentially expressed miRNAs. Expression of miR-99b, PI3K, AKT, mTOR, and ribosomal protein S6 kinase (p70S6K) was determined in both cervical cancer tissues and paracancerous tissues. Next, alteration of miR-99b expression in cervical cancer was conducted to evaluate levels of PI3K, AKT, mTOR, p70S6K matrix metallopeptidase 2, epithelial cell adhesion molecule, and intercellular adhesion molecule 1, as well as the effect of miR-99b on cell proliferation, invasion, migration, cell cycle distribution, and apoptosis. The results demonstrated that miR-99b expression was decreased and levels of PI3K, AKT, mTOR, and p70S6K were elevated in cervical cancer tissues. More important, overexpressed miR-99b repressed the PI3K/AKT/mTOR signaling pathway, inhibited cell proliferation, invasion, and migration, blocked cell cycle entry, and promoted apoptosis in cervical cancer. These results indicate that miR-99b attenuates the migration and invasion of human cervical cancer cells through downregulation of the PI3K/AKT/mTOR signaling pathway, which provides a therapeutic approach for cervical cancer treatment.     

A novel matrine derivative WM622 inhibits hepatocellular carcinoma by inhibiting PI3K/AKT signaling pathways

Nasopharyngeal carcinoma (NPC) is a unique subtype of head and neck cancer, with tendency to spread to regional lymph nodes and distant organs at early stage. Vimentin, a major cytoskeletal protein constituent of the intermediate filament, plays a critical role in the epithelial–mesenchymal transition. Overexpression of vimentin is considered to be a critical prerequisite for metastasis in numerous human cancers. Therefore, targeting vimentin for cancer therapy has gained a lot of interest. In the present study, we detected vimentin expression in NPC tissues and found that overexpression of vimentin is associated with poor prognosis of NPC patients. Silencing of vimentin in NPC CNE2 cells by RNAi suppresses cells migration and invasion in vitro. However, blocking vimentin did not affect cell proliferation of CNE2 cells. In addition, the in vivo metastatic potential of CNE2 cells transfected with Vimentin shRNA was suppressed in a nude mouse model of pulmonary metastasis. Silencing of Vimentin in CNE2 cells leads to a decrease of microvessel density and VEGF, CD31, MMP2, and MMP9 expressions in pulmonary metastatic tumors. Importantly, we found that it is easier for the tumor cells from the high vimentin-expressing pulmonary metastatic tumors to reinvade the microvessel and to form stable tumor plaques attached to the endothelial cells, which resemble the resource of circulating tumor cells and are very hard to eliminate. However, depletion of vimentin inhibits the formation of vascular tumor plaques. Our findings suggest that RNAi-based vimentin silencing may be a potential and promising anti-metastatic therapeutic strategy for NPC.     

Mollugin induces tumor cell apoptosis and autophagy via the PI3K/AKT/mTOR/p70S6K and ERK signaling pathways

Mollugin, a bioactive phytochemical isolated from Rubia cordifolia L., has shown preclinical anticancer efficacy in various cancer models. However the effects of mollugin in regulating cancer cell survival and death remains undefined. In the present study we found that mollugin exhibited cytotoxicity on various cancer models. The suppression of cell viability was due to the induction of mitochondria apoptosis. In addition, the presence of autophagic hallmarks was observed in mollugin-treated cells. Notably, blockade of autophagy by a chemical inhibitor or RNA interference enhanced the cytotoxicity of mollugin. Further experiments demonstrated that phosphatidylinositide 3-kinases/protein kinase B/mammalian target of rapamycin/p70S6 kinase (PI3K/AKT/mTOR/p70S6K) and extracellular regulated protein kinases (ERK) signaling pathways participated in mollugin-induced autophagy and apoptosis. Together, these findings support further studies of mollugin as candidate for treatment of human cancer cells.     

PI3-kinase/Akt modulates vascular smooth muscle tone via cAMP signaling pathways.

Phosphatidylinositol 3-kinase (PI3-kinase) activates protein kinase B (also known as Akt), which phosphorylates and activates a cyclic nucleotide phosphodiesterase 3B. Increases in cyclic nucleotide concentrations inhibit agonist-induced contraction of vascular smooth muscle. Thus we hypothesized that the PI3-kinase/Akt pathway may regulate vascular smooth muscle tone. In unstimulated, intact bovine carotid artery smooth muscle, the basal phosphorylation of Akt was higher than that in cultured smooth muscle cells. The phosphorylation of Akt decreases in a time-dependent manner when incubated with the PI3-kinase inhibitor, LY-294002. Agonist (serotonin)-, phorbol ester (phorbol 12,13-dibutyrate; PDBu)-, and depolarization (KCl)-induced contractions of vascular smooth muscles were all inhibited in a dose-dependent fashion by LY-294002. However, LY-294002 did not inhibit serotonin- or PDBu-induced increases in myosin light chain phosphorylation or total O(2) consumption, suggesting that inhibition of contraction was not mediated by reversal or inhibition of the pathways that lead to smooth muscle activation and contraction. Treatment of vascular smooth muscle with LY-294002 increased the activity of cAMP-dependent protein kinase and increased the phosphorylation of the cAMP-dependent protein kinase substrate heat shock protein 20 (HSP20). These data suggest that activation of the PI3-kinase/Akt pathway in unstimulated smooth muscle may modulate vascular smooth muscle tone (allow agonist-induced contraction) through inhibition of the cyclic nucleotide/HSP20 pathway and suggest that cyclic nucleotide-dependent inhibition of contraction is dissociated from the myosin light chain contractile regulatory pathways.     

Ether-linked diglycerides inhibit vascular smooth muscle cell growth via decreased MAPK and PI3K/Akt signaling

Diglycerides (DGs) are phospholipid-derived second messengers that regulate PKC-dependent signaling pathways. Distinct species of DGs are generated from inflammatory cytokines and growth factors. Growth factors increase diacyl- but not ether-linked DG species, whereas inflammatory cytokines predominately generate alkyl, acyl- and alkenyl, acyl-linked DG species in rat mesenchymal cells. These DG species have been shown to differentially regulate protein kinase C (PKC) isotypes. Ester-linked diacylglycerols activate PKC-epsilon and cellular proliferation in contrast to ether-linked DGs, which lead to growth arrest through the inactivation of PKC-epsilon. It is now hypothesized that ether-linked DGs inhibit mitogenesis through the inactivation of ERK and/or Akt signaling cascades. We demonstrate that cell-permeable ether-linked DGs reduce vascular smooth muscle cell growth by inhibiting platelet-derived growth factor-stimulated ERK in a PKC-epsilon-dependent manner. This inhibition is specific to the ERK pathway, since ether-linked DGs do not affect growth factor-induced activation of other family members of the MAPKs, including p38 MAPK and c-Jun NH(2)-terminal kinases. We also demonstrate that ether-linked DGs reduce prosurvival phosphatidylinositol 3-kinase (PI3K)/Akt signaling, independent of PKC-epsilon, by diminishing an interaction between the subunits of PI3K and not by affecting protein phosphatase 2A or lipid (phosphatase and tensin homologue deleted in chromosome 10) phosphatases. Taken together, our studies identify ether-linked DGs as potential adjuvant therapies to limit vascular smooth muscle migration and mitogenesis in atherosclerotic and restenotic models.     

Resveratrol suppresses the epithelial-to-mesenchymal transition in PC-3 cells by down-regulating the PI3K/AKT signaling pathway

Resveratrol possesses a wide spectrum of pharmacological properties and has been an ideal alternative drug for the treatment of different cancers, including prostate cancer. However, the mechanisms by which resveratrol inhibits the growth of prostate cancer are still not fully elucidated. To understand the effect of resveratrol on the apoptosis and the epithelial-to-mesenchymal transition (EMT) of prostate cancer as well as its related mechanism, we investigated the potential use of resveratrol in PC-3 prostate cancer cells in vitro using real-time PCR, fluorescence-activated cell sorting, Western blotting, etc. Resveratrol suppresses the PC-3 prostate cancer cell growth and induces apoptosis. Resveratrol also influences the expression of EMT-related proteins (increased E-cadherin and decreased Vimentin expression). Finally, resveratrol also suppressed Akt phosphorylation in PC-3 cells. This study indicates that resveratrol may be a potential anti-cancer treatment for prostate cancer; moreover, it provides new evidence that resveratrol suppresses prostate cancer growth and metastasis.     

bFGF attenuates endoplasmic reticulum stress and mitochondrial injury on myocardial ischaemia/reperfusion via activation of PI3K/Akt/ERK1/2 pathway

Extensive research focused on finding effective strategies to prevent or improve recovery from myocardial ischaemia/reperfusion (I/R) injury. Basic fibroblast growth factor (bFGF) has been shown to have therapeutic potential in some heart disorders, including ischaemic injury. In this study, we demonstrate that bFGF administration can inhibit the endoplasmic reticulum (ER) stress and mitochondrial dysfunction induced in the heart in a mouse model of I/R injury. In vitro, bFGF exerts a protective effect by inhibiting the ER stress response and mitochondrial dysfunction proteins that are induced by tert‐Butyl hydroperoxide (TBHP) treatment. Both of these in vivo and in vitro effects are related to the activation of two downstream signalling pathways, PI3K/Akt and ERK1/2. Inhibition of these PI3K/Akt and ERK1/2 pathways by specific inhibitors, LY294002 and PD98059, partially reduces the protective effect of bFGF. Taken together, our results indicate that the cardioprotective role of bFGF involves the suppression of ER stress and mitochondrial dysfunction in ischaemic oxidative damage models and oxidative stress‐induced H9C2 cell injury; furthermore, these effects underlie the activation of the PI3K/Akt and ERK1/2 signalling pathways.     

PKC-delta-dependent pathways contribute to PDGF-stimulated ERK1/2 activation in vascular smooth muscle

Platelet-derived growth factor (PDGF) is an important regulator of vascular smooth muscle (VSM) cell growth and migration and has been identified as a key mediator of neointima formation resulting from vascular injury. PDGF exerts its effects, in part, through activation of ERK1/2. Previously, we reported that PKC-delta, specifically compared with PKC-alpha, mediated phorbol ester- and ATP-dependent activation of ERK1/2 in VSM cells. The purpose of this study was to determine whether PKC-delta was involved in PDGF-dependent activation of ERK1/2 in VSM cells. The addition of PDGF resulted in the activation, and Src family kinase-dependent tyrosine phosphorylation, of PKC-delta. Treatment with rottlerin (0.1-10 microM), a selective PKC-delta inhibitor, or adenoviral overexpression of kinase-negative PKC-delta significantly attenuated PDGF-induced activation of ERK1/2. The effects of the PKC-delta inhibitors decreased with increasing concentrations of activator PDGF. Interestingly, treatment with Go6976 (0.1-3 microM), a selective inhibitor of cPKCs, or adenoviral overexpression of kinase-negative PKC-alpha also inhibited PDGF-stimulated ERK1/2. Furthermore, inhibition of cPKC activity with Go6976 or overexpression of kinase-negative PKC-alpha attenuated PKC-delta activation and tyrosine phosphorylation in response to PDGF. These studies indicate involvement of both PKC-delta and PKC-alpha isozymes in PDGF-stimulated signaling in VSM and suggest an unexpected role for PKC-alpha in the regulation of PKC-delta activity.     

Isovalerylspiramycin I suppresses non-small cell lung carcinoma growth through ROS-mediated inhibition of PI3K/AKT signaling pathway

Novel drugs are required for non-small cell lung cancer (NSCLC) treatment urgently. Repurposing old drugs as new treatments is a practicable approach with time and cost savings. Some studies have shown that carrimycin, a Chinese Food and Drug Administration (CFDA)-approved macrolide antibiotic, possesses potent anti-tumor effects against oral squamous cell carcinoma. However, its detailed component and underlying mechanisms in anti-NSCLC remain unknown. In our study, isovalerylspiramycin I (ISP-I) was isolated from carrimycin and demonstrated a remarkable anti-NSCLC efficacy in vitro and in vivo with a favorable safety profile. It has been proven that in NSCLC cell lines H460 and A549, ISP-I could induce G2/M arrest and apoptosis, which was mainly attributed to ROS accumulation and subsequently PI3K/AKT signaling pathway inhibition. Numerous downstream genes including mTOR and FOXOs were also changed correspondingly. An observation of NAC-induced reverse effect on ISP-I-leading cell death and PI3K/AKT pathway inhibition, emphasized the necessity of ROS signaling in this event. Moreover, we identified ROS accumulation and PI3K/AKT pathway inhibition in tumor xenograft models in vivo as well. Taken together, our study firstly reveals that ISP-I is a novel ROS inducer and may act as a promising candidate with multi-target and low biological toxicity for anti-NSCLC treatment.     

PSMC6 promotes osteoblast apoptosis through inhibiting PI3K/AKT signaling pathway activation in ovariectomy-induced osteoporosis mouse model

There is now increasing evidence which suggests a key role for osteoblast apoptosis in the pathogenesis of postmenopausal osteoporosis. Here, we evaluated the role and mechanism of proteasome 26S subunit, ATPase (PSMC) 6, a protein that is highly expressed in bone. Gene expression pattern had been extracted based on database of Gene Expression Omnibus (GEO). GEO2R was employed for analyses, while the DAVID database was adopted to further analyze the gene ontology (GO) as well as Kyoto Encyclopedia of Genomes pathway (KEGG) enrichment. Then, the Search Tool Retrieval of Interacting Genes (STRING) was utilized to carry out interaction regulatory network for the top 200 differentially expressed genes (DEGs). A key gene, called PSMC6, was identified by Cytoscape 3.6.0. The OVX osteoporosis model was established in female C57BL/6 mice by full bilateral ovariectomy. According to our findings, PSMC6 gene knockout would elevate bone mineral density (BMD) and the phosphorylation level of PI3K protein and increased the protein level of cleaved caspase-3/-9 in OVX osteoporosis mice. Further, MTT, bromodeoxyuridine, and flow cytometry assays revealed that PSMC6 inhibition promoted the progression of cell cycle and cell proliferation, whereas, PSMC6 overexpression promoted the apoptosis and inhibited cell cycle progression and cell proliferation in vitro. Besides, we found that PI3K activation significantly decreased PSMC6-induced osteoblast apoptosis and promoted cell proliferation through regulating the protein levels of p53, cyclinD1, and cleaved caspase-3/9. In conclusion, PSMC6 aggravated the degree of OVX-induced osteoporosis by inhibiting the PI3K/AKT signal transduction pathway, thereby promoting the apoptosis of osteoblasts.     

DMBT1 suppresses progression of gallbladder carcinoma through PI3K/AKT signaling pathway by targeting PTEN

ABSTRACT

Gallbladder carcinoma (GBC) is a highly lethal malignancy of the gastrointestinal tract. Despite extensive research, the underlying molecular mechanism of GBC remains largely unclear. Deleted in malignant brain tumors 1 (DMBT1) is low-expression during cancer progression and as a potential tumor-suppressor gene in various types of cancer. However, its role in Gallbladder cancer remains poorly understood. Here, we found that DMBT1 was significantly low-expression and deletion of copy number in GBC tissues by qRT-PCR and Western blot. Overexpression of DMBT1 impaired survival, promoted apoptosis in GBC cells in vitro, and inhibited tumor progression in vivo. Further study of underlying mechanisms demonstrated that DMBT1 combined with PTEN which could stabilize PTEN protein, resulting in inhibiting the activation of PI3K/AKT signaling pathway. Our study revealed a new sight of DMBT1 as a tumor-suppressor gene on the PI3K/AKT pathway in GBC, which may be a potential therapeutic target for improving treatment.