Qiliqiangxin attenuates hypoxia‐induced injury in primary rat cardiac microvascular endothelial cells via promoting HIF‐1α‐dependent glycolysis

Protection of cardiac microvascular endothelial cells (CMECs) against hypoxia injury is an important therapeutic strategy for treating ischaemic cardiovascular disease. In this study, we investigated the effects of qiliqiangxin (QL) on primary rat CMECs exposed to hypoxia and the underlying mechanisms. Rat CMECs were successfully isolated and passaged to the second generation. CMECs that were pre‐treated with QL (0.5 mg/mL) and/or HIF‐1α siRNA were cultured in a three‐gas hypoxic incubator chamber (5% CO₂, 1% O₂, 94% N₂) for 12 hours. Firstly, we demonstrated that compared with hypoxia group, QL effectively promoted the proliferation while attenuated the apoptosis, improved mitochondrial function and reduced ROS generation in hypoxic CMECs in a HIF‐1α‐dependent manner. Meanwhile, QL also promoted angiogenesis of CMECs via HIF‐1α/VEGF signalling pathway. Moreover, QL improved glucose utilization and metabolism and increased ATP production by up‐regulating HIF‐1α and a series of glycolysis‐relevant enzymes, including glucose transport 1 (GLUT1), hexokinase 2 (HK2), 6‐phosphofructokinase 1 (PFK1), pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). Our findings indicate that QL can protect CMECs against hypoxia injury via promoting glycolysis in a HIF‐1α‐dependent manner. Lastly, the results suggested that QL‐dependent enhancement of HIF‐1α protein expression in hypoxic CMECs was associated with the regulation of AMPK/mTOR/HIF‐1α pathway, and we speculated that QL also improved HIF‐1α stabilization through down‐regulating prolyl hydroxylases 3 (PHD3) expression.     

Maternal diabetes up‐regulates NOX2 and enhances myocardial ischaemia/reperfusion injury in adult offspring

Offspring of diabetic mothers are at risk of cardiovascular diseases in adulthood. However, the underlying molecular mechanisms are not clear. We hypothesize that prenatal exposure to maternal diabetes up‐regulates myocardial NOX2 expression and enhances ischaemia/reperfusion (I/R) injury in the adult offspring. Maternal diabetes was induced in C57BL/6 mice by streptozotocin. Glucose‐tolerant adult offspring of diabetic mothers and normal controls were subjected to myocardial I/R injury. Vascular endothelial growth factor (VEGF) expression, ROS generation, myocardial apoptosis and infarct size were assessed. The VEGF‐Akt (protein kinase B)‐mammalian target of rapamycin (mTOR)‐NOX2 signalling pathway was also studied in cultured cardiomyocytes in response to high glucose level. In the hearts of adult offspring from diabetic mothers, increases were observed in VEGF expression, NOX2 protein levels and both Akt and mTOR phosphorylation levels as compared to the offspring of control mothers. After I/R, ROS generation, myocardial apoptosis and infarct size were all significantly higher in the offspring of diabetic mothers relative to offspring of control mothers, and these differences were diminished by in vivo treatment with the NADPH oxidase inhibitor apocynin. In cultured cardiomyocytes, high glucose increased mTOR phosphorylation, which was inhibited by the PI3 kinase inhibitor LY294002. Notably, high glucose‐induced NOX2 protein expression and ROS production were inhibited by rapamycin. In conclusion, maternal diabetes promotes VEGF‐Akt‐mTOR‐NOX2 signalling and enhances myocardial I/R injury in the adult offspring. Increased ROS production from NOX2 is a possible molecular mechanism responsible for developmental origins of cardiovascular disease in offspring of diabetic mothers.     

Altering PI3K—Akt signalling in zebrafish embryos affects PTEN phosphorylation and gastrulation

Background information. PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a negative regulator of the PI3K (phosphoinositide 3‐kinase)–Akt (also called protein kinase B) signalling pathway and is essential for embryogenesis, but its function in early vertebrate embryos is unclear. Results. To address how PTEN functions in early embryos, we overexpressed one of the four zebrafish PTEN isoforms at the 1–2‐cell stage. Overexpression of Ptena454 alters phospho‐Akt levels and impairs cell movements associated with gastrulation. Heat shocking embryos increases phospho‐Akt levels and lowers phospho‐Ptena454 levels. Inhibiting CK2 (protein kinase CK2) activity reduces phospho‐Pten levels and augments the effects due to Ptena454 overexpression. Low phospho‐Akt and corresponding low phospho‐GSK‐3 (glycogen synthase kinase‐3) and high phospho‐Pten levels accompany wortmannin or LY294002 treatment, which inhibit PI3K activity. Conclusions. These results suggest that Ptena454 regulation is correlated to changes in phospho‐Akt levels. We propose a model in which homoeostasis in rapidly dividing and migrating embryonic cells depends on a counterbalance between pro‐survival signalling employing CK2 and GSK‐3 and the pro‐apoptotic activity of Ptena454.     

MFTZ‐1 reduces constitutive and inducible HIF‐1α accumulation and VEGF secretion independent of its topoisomerase II inhibition

The macrolide compound MFTZ‐1 has been identified as a novel topoisomerase II (Top2) inhibitor with potent in vitro and in vivo anti‐tumour activities. In this study, we further examined the effects of MFTZ‐1 on hypoxia‐inducible factor‐1α (HIF‐1α) accumulation, vascular endothelial growth factor (VEGF) secretion and angiogenesis. MFTZ‐1 reduced HIF‐1α accumulation driven by hypoxia or growth factors in human cancer cells. Mechanistic studies revealed that MFTZ‐1 did not affect the degradation of HIF‐1α protein or the level of HIF‐1α mRNA. By contrast, MFTZ‐1 apparently inhibited constitutive and inducible activation of both phosphatidylinositol‐3‐kinase (PI3K)‐Akt and p42/p44 mitogen‐activated protein kinase (MAPK) pathways. Further studies revealed that MFTZ‐1 abrogated the HIF‐1α‐driven increase in VEGF mRNA and protein secretion. MFTZ‐1 also lowered the basal level of VEGF secretion. The results reveal an important feature that MFTZ‐1 can reduce constitutive, HIF‐1α‐independent VEGF secretion and concurrently antagonize inducible, HIF‐1α‐dependent VEGF secretion. Moreover, MFTZ‐1 disrupted tube formation of human umbilical vein endothelial cells (HUVECs) stimulated by hypoxia with low‐concentration serum or by serum at normoxia, and inhibited HUVECs migration at normoxia. MFTZ‐1 also prevented microvessel outgrowth from rat aortic ring. These data reflect the potent anti‐angiogenesis of MFTZ‐1 under different conditions. Furthermore, using specific small interfering RNA targeting Top2α or Top2‐defective HL60/MX2 cells, we showed that MFTZ‐1 affected HIF‐1α accumulation and HUVECs tube formation irrelevant to its Top2 inhibition. Taken together, our data collectively reveal that MFTZ‐1 reduces constitutive and inducible HIF‐1α accumulation and VEGF secretion possibly via PI3K‐Akt and MAPK pathways, eliciting anti‐angiogenesis independently of its Top2 inhibition.     

Follistatin could promote the proliferation of duck primary myoblasts by activating PI3K/Akt/mTOR signalling

FST (follistatin) is essential for skeletal muscle development, but the intracellular signalling networks that regulate FST-induced effects are not well defined. We sought to investigate whether FST promotes the proliferation of myoblasts through the PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signalling. In the present study, we transfected the pEGFP-duFST plasmid and added PI3K and mTOR inhibitors to the medium of duck primary myoblasts. Then, we analysed the cellular phenotypic changes that occurred and analysed the expression of target genes. The results showed that FST promoted myoblast proliferation, induced the mRNA expression of PI3K, Akt, mTOR, 70-kDa ribosomal protein S6K (S6 kinase) and the protein expression of phospho-Akt (Thr³⁰⁸), mTOR, phospho-mTOR (serine 2448), phospho-S6K (Ser⁴¹⁷), inhibited the mRNA expression of FoxO1, MuRF1 (muscle RING finger-1) and the protein expression of phospho-FoxO1 (Ser²⁵⁶). Moreover, we found that the overexpression of FST could alleviate the inhibitory effect of myoblast proliferation caused by the addition of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor. Additionally, the overexpression of duck FST also relieved the inhibition of myoblast proliferation caused by the addition of rapamycin (an mTOR inhibitor) through PI3K/Akt/mTOR signalling. In light of the present results, we hypothesize that duck FST could promote myoblast proliferation, which is dependent on PI3K/Akt/mTOR signalling.     

Hypoxia induces BMP‐2 expression via ILK,Akt, mTOR,and HIF‐1 pathways in osteoblasts

It has been shown that hypoxia stimulation regulates bone formation, maintenance, and repair. Bone morphogenetic protein (BMP) plays important roles in osteoblastic differentiation and bone formation. However, the effects of hypoxia exposure on BMP‐2 expression in cultured osteoblasts are largely unknown. Here we found that hypoxia stimulation increased mRNA and protein levels of BMP‐2 by qPCR, Western blot and ELISA assay in osteoblastic cells MG‐63, hFOB and bone marrow stromal cells M2‐10B4. Integrin‐linked kinase (ILK) inhibitor (KP‐392), Akt inhibitor (1L‐6‐hydroxymethyl‐chiro‐inositol‐2‐[(R)‐2‐O‐methyl‐3‐O‐octadecylcarbonate]) or mammalian target of rapamycin (mTOR) inhibitor (rapamycin) inhibited the potentiating action of hypoxia. Exposure to hypoxia increased the kinase activity of ILK and phosphorylation of Akt and mTOR. Furthermore, hypoxia also increased the stability and activity of HIF‐1 protein. The binding of HIF‐1α to the HRE elements after exposure to hypoxia was measured by EMSA assay. Moreover, the use of pharmacological inhibitors or genetic inhibition revealed that both ILK/Akt and mTOR signaling pathway were potentially required for hypoxia‐induced HIF‐1α activation and subsequent BMP‐2 up‐regulation. Taken together, our results provide evidence that hypoxia enhances BMP‐2 expression in osteoblasts by an HIF‐1α‐dependent mechanism involving the activation of ILK/Akt and mTOR pathways. J. Cell. Physiol. 223:810–818, 2010. © 2010 Wiley‐Liss, Inc.     

miR‐148a inhibits early relapsed colorectal cancers and the secretion of VEGF by indirectly targeting HIF‐1α under non‐hypoxia/hypoxia conditions

Vascular endothelial growth factor (VEGF) is correlated with angiogenesis and early relapse of colorectal cancer (CRC). This study investigated the role of miR‐148a in the regulation of VEGF/angiogenesis and early relapse of CRC. We established a stable clone with miR‐148a expression in HCT116 and HT29 cell lines and created a hypoxic condition by using CoCl₂ to determine the underlying mechanism of miR‐148a. The effects of miR‐148a on the phosphoryl‐ERK (pERK)/hypoxia‐inducible factor‐1α (HIF‐1α)/VEGF pathway were evaluated through Western blotting and the inhibitory effect of miR‐148a on angiogenesis was demonstrated through a tube formation assay. Sixty‐three CRC tissues (28 early relapse and 35 non‐early relapse) were analysed to assess the relationship between miR‐148a and HIF‐1α/VEGF. The protein expression of pERK/HIF‐1α/VEGF in HCT116 and HT29 cells was significantly decreased by miR‐148a (all P < 0.05). The protein expression of VEGF/HIF‐1α was strongly inversely associated with the expression of miR‐148a in the 63 CRC tissue samples (all P < 0.05). Tube formation assay demonstrated that miR‐148a significantly obliterated angiogenesis. miR‐148a suppresses VEGF through down‐regulation of the pERK/HIF‐1α/VEGF pathway and might lead to the inhibition of angiogenesis; miR‐148a down‐regulation increased the early relapse rate of CRC. This demonstrates that miR‐148a is a potential diagnostic and therapeutic target.     

Penehyclidine hydrochloride protects against anoxia/reoxygenation injury in cardiomyocytes through ATP‐sensitive potassium channels,and the Akt/GSK‐3β and Akt/mTOR signaling pathways

Penehyclidine hydrochloride (PHC) can protect against myocardial ischemia/reperfusion (I/R) injury. However, the possible mechanisms of PHC in anoxia/reoxygenation (A/R)‐induced injury in H9c2 cells remain unclear. In the present study, H9c2 cells were pretreated with PI3K/Akt inhibitor LY294002, ATP‐sensitive K⁺ (KATP) channel blocker 5‐hydroxydecanoate (5‐HD), PHC, or KATP channel opener diazoxide (DZ) before subjecting to A/R injury. Cell viability and cell apoptosis were determined by cell counting kit‐8 assay and annexin V/PI assay, respectively. Myocardial injury was evaluated by measuring creatine kinase (CK) and lactate dehydrogenase (LDH) activities. Intracellular Ca²⁺ levels, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨ_m), and mitochondrial permeability transition pore (mPTP) were measured. The levels of cytoplasmic/mitochondrial cytochrome c (Cyt‐C), Bax, Bcl‐2, cleaved caspase‐3, KATP channel subunits (Kir6.2 and SUR2A), and the members of the Akt/GSK‐3β and Akt/mTOR signaling pathways were determined by western blotting. We found that PHC preconditioning alleviated A/R‐induced cell injury by increasing cell viability, reducing CK and LDH activities, and inhibiting cell apoptosis. In addition, PHC preconditioning ameliorated intracellular Ca²⁺ overload and ROS production, accompanied by inhibition of both mPTP opening and Cyt‐C release into cytoplasm, and maintenance of ΔΨ_m. Moreover, PHC preconditioning activated mitochondrial KATP channels, and modulated the Akt/GSK‐3β and Akt/mTOR signaling pathways. Similar effects were observed upon treatment with DZ. Pretreatment with LY294002 or 5‐HD blocked the beneficial effects of PHC. These results suggest that the protective effects of PHC preconditioning on A/R injury may be related to mitochondrial KATP channels, as well as the Akt/GSK‐3β and Akt/mTOR signaling pathways.     

HIF‐1α mediates the induction of IL‐8 and VEGF expression on infection with Afa/Dr diffusely adhering E. coli and promotes EMT‐like behaviour

Microbes regulate a large panel of intracellular signalling events that can promote inflammation and/or enhance tumour progression. Indeed, it has been shown that infection of human intestinal cells with the Afa/Dr diffusely adhering E. coli C1845 strain induces expression of pro‐angiogenic and pro‐inflammatory genes. Here, we demonstrate that exposure of cryptic‐like intestinal epithelial cells to C1845 bacteria induces HIF‐1α protein levels. This effect depends on the binding of F1845 adhesin to the membrane‐associated DAF receptor that initiates signalling cascades promoting translational mechanisms. Indeed, inhibition of MAPK and PI‐3K decreases HIF‐1α protein levels and blocks C1845‐induced phosphorylation of the ribosomal S6 protein. Using RNA interference we show that bacteria‐induced HIF‐1α regulates the expression of IL‐8, VEGF and Twist1, thereby pointing to a role for HIF‐1 in angiogenesis and inflammation. In addition, infection correlates with a loss of E‐cadherin and cytokeratin 18 and a rise in fibronectin, suggesting that bacteria may induce an epithelial to mesenchymal transition‐like phenotype. Since HIF‐1α silencing results in reversion of bacteria‐induced EMT markers, we speculate that HIF‐1α plays a key role linking bacterial infection to angiogenesis, inflammation and some aspects of cancer initiation.     

Overexpression of microRNA‐138 alleviates human coronary artery endothelial cell injury and inflammatory response by inhibiting the PI3K/Akt/eNOS pathway

This study aimed to investigate the role of miR‐138 in human coronary artery endothelial cell (HCAEC) injury and inflammatory response and the involvement of the PI3K/Akt/eNOS signalling pathway. Oxidized low‐density lipoprotein (OX‐LDL)‐induced HCAEC injury models were established and assigned to blank, miR‐138 mimic, miR‐138 inhibitor, LY294002 (an inhibitor of the PI3K/Akt/eNOS pathway), miR‐138 inhibitor + LY294002 and negative control (NC) groups. qRT‐PCR and Western blotting were performed to detect the miR‐138, PI3K, Akt and eNOS levels and the protein expressions of PI3K, Akt, eNOS, p‐Akt, p‐eNOS, Bcl‐2, Bax and caspase‐3. ELISAs were employed to measure the expressions of TNF‐α, IL‐4, IL‐6, IL‐8, IL‐10 and nitric oxide (NO) and the activities of lactate dehydrogenase (LDH) and eNOS. MTT and flow cytometry were performed to assess the proliferation and apoptosis of HCAECs. Compared to the blank group, PI3K, Akt and eNOS were down‐regulated in the miR‐138 mimic and LY294002 groups but were up‐regulated in the miR‐138 inhibitor group. The miR‐138 mimic and LY294002 groups showed decreased concentrations of TNF‐α, IL‐6, IL‐8 and NO and reduced activities of LDH and eNOS, while opposite trends were observed in the miR‐138 inhibitor group. The concentrations of IL‐4 and IL‐10 increased in the miR‐138 mimic and LY294002 groups but decreased in the miR‐138 inhibitor group. The miR‐138 mimic and LY294002 groups had significantly decreased cell proliferation and increased cell apoptosis compared to the blank group. These findings indicate that up‐regulation of miR‐138 alleviates HCAEC injury and inflammatory response by inhibiting the PI3K/Akt/eNOS signalling pathway.     

Deferoxamine promotes mesenchymal stem cell homing in noise‐induced injured cochlea through PI3K/AKT pathway

Objective

Over 5% of the world's population suffers from disabling hearing loss. Stem cell homing in target tissue is an important aspect of cell‐based therapy, which its augmentation increases cell therapy efficiency. Deferoxamine (DFO) can induce the Akt activation, and phosphorylation status of AKT (p‐AKT) upregulates CXC chemokine receptor‐4 (CXCR4) expression. We examined whether DFO can enhance mesenchymal stem cells (MSCs) homing in noise‐induced damaged cochlea by PI3K/AKT dependent mechanism.

Materials and Methods

Mesenchymal stem cells were treated with DFO. AKT, p‐AKT protein and hypoxia inducible factor 1‐ α (HIF‐1α) and CXCR4 gene and protein expression was evaluated by RT‐ PCR and Western blot analysis. For in vivo assay, rats were assigned to control, sham, noise exposure groups without any treatment or receiving normal, DFO‐treated and DFO +LY294002 (The PI3K inhibitor)‐treated MSCs. Following chronic exposure to 115 dB white noise, MSCs were injected into the rat cochlea through the round window. Number of Hoechst‐ labelled cells was determined in the endolymph after 24 hours.

Results

Deferoxamine increased P‐AKT, HIF‐1α and CXCR4 expression in MSCs compared to non‐treated cells. DFO pre‐conditioning significantly increased the homing ability of MSCs into injured ear compared to normal MSCs. These effects of DFO were blocked by LY294002.

Conclusions

Pre‐conditioning of MSCs by DFO before transplantation can improve stem cell homing in the damaged cochlea through PI3K/AKT pathway activation.

     

Salidroside suppressing LPS‐induced myocardial injury by inhibiting ROS‐mediated PI3K/Akt/mTOR pathway in vitro and in vivo

The purpose of the present study was to investigate the effect of salidroside (Sal) on myocardial injury in lipopolysaccharide (LPS)‐induced endotoxemic in vitro and in vivo. SD rats were randomly divided into five groups: control group, LPS group (15 mg/kg), LPS plus dexamethasone (2 mg/kg), LPS plus Sal groups with different Sal doses (20, 40 mg/kg). Hemodynamic measurement and haematoxylin and eosin staining were performed. Serum levels of creatine kinase (CK), lactate dehydrogenase, the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH‐px), glutathione, tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), and interleukin‐1β (IL‐1β) were measured after the rats were killed. iNOS, COX‐2, NF‐κB and PI3K/Akt/mTOR pathway proteins were detected by Western blot. In vitro, we evaluated the protective effect of Sal on rat embryonic heart‐derived myogenic cell line H9c2 induced by LPS. Reactive oxygen species (ROS) in H9c2 cells was measured by flow cytometry, and the activities of the antioxidant enzymes CAT, SOD, GSH‐px, glutathione‐S‐transferase, TNF‐α, IL‐6 and IL‐1β in cellular supernatant were measured. PI3K/Akt/mTOR signalling was examined by Western blot. As a result, Sal significantly attenuated the above indices. In addition, Sal exerts pronounced cardioprotective effect in rats subjected to LPS possibly through inhibiting the iNOS, COX‐2, NF‐κB and PI3K/Akt/mTOR pathway in vivo. Furthermore, the pharmacological effect of Sal associated with the ROS‐mediated PI3K/Akt/mTOR pathway was proved by the use of ROS scavenger, N‐acetyl‐l ‐cysteine, in LPS‐stimulated H9C2 cells. Our results indicated that Sal could be a potential therapeutic agent for the treatment of cardiovascular disease.     

Effects and mechanisms of ghrelin on cardiac microvascular endothelial cells in rats

Ghrelin is thought to directly exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function. Our study demonstrates the ability of ghrelin to promote rat CMEC (cardiac microvascular endothelial cell) proliferation, migration and NO (nitric oxide) secretion. CMECs were isolated from left ventricle of adult male Sprague—Dawley rat by enzyme digestion and maintained in endothelial cell medium. Dil‐ac‐LDL (1,1′‐dioctadecyl‐3,3,3′,3′‐ tetramethylindocarbocyanine‐labelled acetylated low‐density lipoprotein) intake assays were used to identify CMECs. Cells were split into five groups and treated with varying concentrations of ghrelin as follows: one control non‐treated group; three ghrelin dosage groups (1×10^?9, 1×10^?8, 1×10^?7 mol/l) and one ghrelin+PI3K inhibitor group (1×10^?7 mol/l ghrelin+20 μmol/l LY294002). After 24 h treatment, cell proliferation capability was measured by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assay and Western blot for PCNA (proliferating cell nuclear antigen) protein expression. Migration of CMECs was detected by transwell assays, and NO secretion of CMECs was measured via nitrate reduction. Protein expression of AKT and phosphorylated AKT in CMECs was measured by Western blot after exposure to various concentrations of ghrelin and the PI3K inhibitor LY294002. Our results indicate that ghrelin significantly enhanced cell growth at concentrations of 10^?8 mol/l (0.271±0.041 compared with 0.199±0.021, P=0.03) and 10^?7 mol/l (0.296±0.039 compared with 0.199±0.021, P<0.01). However, addition of the PI3K/AKT inhibitor LY294002 inhibited the ghrelin‐mediated enhancement in cell proliferation (0.227±0.042 compared with 0.199±0.021, P=0.15). At a concentration between 10^?8 and 10^?7 mol/l, ghrelin caused a significant increase in the number of migrated cells compared with the control group (126±9 compared with 98±7, P=0.02; 142±6 compared with 98±7, P<0.01), whereas no such change could be observed in the presence of 20 μmol/l of the PI3K/Akt inhibitor LY294002 (103±7 compared with 98±7, P=0.32). Ghrelin treatment significantly enhanced NO production in a dose‐dependent fashion compared with the untreated control group [(39.93±2.12) μmol/l compared with (30.27±2.71) μmol/l, P=0.02; (56.80±1.98) μmol/l compared with (30.27±2.71) μmol/l, P<0.01]. However, pretreatment with 20 μmol/l LY294002 inhibited the ghrelin‐stimulated increase in NO secretion [(28.97±1.64) μmol/l compared with (30.27±2.71) μmol/l, P=0.37]. In summary, we have found that ghrelin treatment promotes the proliferation, migration and NO secretion of CMECs through activation of PI3K/AKT signalling pathway.     

Cross‐communication between histone H3 and H4 acetylation and Akt‐mTOR signalling in prostate cancer cells

Molecular tumour targeting has significantly improved anti‐cancer protocols. Still, the addition of molecular targeting to the treatment regime has not led to a curative breakthrough. Combined mammalian target of Rapamycin (mTOR) and histone deacetylase (HDAC) inhibition has been shown not only to enhance anti‐tumour potential, but also to prevent resistance development seen under mono‐drug therapy. This investigation was designed to evaluate whether cross‐communication exists between mTOR signalling and epigenetic events regulated by HDAC. DU‐145 prostate cancer cells were treated with insulin‐like growth factor (IGF) to activate the Akt‐mTOR cascade or with the HDAC‐inhibitor valproic acid (VPA) to induce histone H3 and H4 acetylation (aH3, aH4). Subsequently, mTOR, Rictor, Raptor, p70s6k, Akt (all: total and phosphorylated), H3 and H4 (total and acetylated) were analysed by western blotting. Both techniques revealed a link between mTOR and the epigenetic machinery. IGF activated mTOR, Rictor, Raptor, p70s6k and Akt, but also enhanced aH3 and aH4. Inversely, IGFr blockade and knock‐down blocked the Akt‐mTOR axis, but simultaneously diminished aH3 and aH4. VPA treatment up‐regulated histone acetylation, but also activated mTOR‐Akt signalling. HDAC1 and 2 knock‐down revealed that the interaction with the mTOR system is initiated by histone H3 acetylation. HDAC‐mTOR communication, therefore, is apparent whereby tumour‐promoting (Akt/mTOR^high, aH3/aH4^low) and tumour‐suppressing signals (Akt/mTOR^low, aH3/aH4^high) are activated in parallel. Combined use of an HDAC‐ and mTOR inhibitor might then diminish pro‐tumour effects triggered by the HDAC‐ (Akt/mTOR^high) or mTOR inhibitor (aH3/aH4^low) alone.     

RhoGDI2与PI3K/Akt/mTOR信号通路在肺癌细胞中的相关性研究

目的探讨肿瘤转移相关因子RhoGDI2与PI3K/Akt/mTOR信号通路在肺癌侵袭转移过程中的作用及相关机制。方法利用PI3K/Akt/mTOR信号通路上特异性的抑制剂,采用MTT法,伤口愈合实验及侵袭实验观察不同浓度药物对肺癌95D细胞生长侵袭转移能力的影响,通过Western Blot方法观察RhoGDI2蛋白水平的变化。结果PI3K抑制剂LY294002及mTOR抑制剂Rapamycin都能抑制肺癌细胞95D的侵袭转移能力,联合应用抑制作用更强。PI3K抑制剂LY294002处理组RhoGDI2蛋白的表达量增加,且随浓度增加RhoGDI2蛋白表达也增加。mTOR抑制剂Rapamycin组,在低浓度时增加RhoGDI2蛋白的表达,但增大Rapamycin的浓度,RhoGDI2蛋白的表达反而降低。低浓度LY294002组和Rapa-mycin组联合应用可以明显增加RhoGDI2蛋白的表达。结论PI3K/Akt/mTOR信号通路中Akt的活化与RhoGDI2密切相关,RhoGDI2可能直接或间接通过与Akt的相互作用参与调节肺癌的侵袭转移的过程。     

HMGB1‐induced angiogenesis in perforated disc cells of human temporomandibular joint

High mobility group 1 protein (HMGB1), a highly conserved nuclear DNA‐binding protein and inflammatory mediator, has been recently found to be involved in angiogenesis. Our previous study has demonstrated the elevation of HMGB1 in the tissue of perforated disc of temporomandibular joint (TMJ). Here, we investigated a novel mediator of HMGB1 in regulating hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) to mediate angiogenesis in perforated disc cells of TMJ. HMGB1 increased the expression of HIF‐1α and VEGF in a dose‐ and time‐dependent manner in these cells. Moreover, immunofluorescence assay exhibits that the HIF‐1α were activated by HMGB1. In addition, HMGB1 activated extracellular signal‐related kinase 1/2 (Erk1/2), Jun N‐terminal kinase (JNK), but not P38 in these cells. Furthermore, both U0126 (ErK inhibitor) and SP600125 (JNK inhibitor) significantly suppressed the enhanced production of HIF‐1α and VEGF induced by HMGB1. Tube formation of human umbilical vein endothelial cells (HUVECs) was significantly increased by exposure to conditioned medium derived from HMGB1‐stimulated perforated disc cells, while attenuated with pre‐treatment of inhibitors for VEGF, HIF‐1α, Erk and JNK, individually. Therefore, abundance of HMGB1 mediates activation of HIF‐1α in disc cells via Erk and JNK pathway and then, initiates VEGF secretion, thereby leading to disc angiogenesis and accelerating degenerative change of the perforated disc.