Akt Signal Transduction Pathway Research Articles

Lung ischemia reperfusion injury: the therapeutic role of dipeptidyl peptidase 4 inhibition.

Dipeptidyl peptidase 4 (DPP4) is a cell surface protease that has been reported to play a role in glucose homeostasis, cancer, HIV, autoimmunity, immunology and inflammation. A role for DPP4 in ischemia-reperfusion injury (IRI) in the heart has been established. Dipeptidyl peptidase 4 inhibition (DPP4i) appeared to decrease infarct size, improves cardiac function and promotes myocardial regeneration. Lung ischemia reperfusion injury is caused by a complex mechanism in which macrophages and neutrophils play an important role. Generation of reactive oxygen species (ROS), uncoupling of nitric oxide synthase (NOS), activation of nuclear factor-κB (NF-κB), activation of nicotinamide adenine dinucleotide phosphate metabolism, and generation of pro-inflammatory cytokines lead to acute lung injury (ALI). In this review we present the current knowledge on DPP4 as a target to treat IRI in the lung. We also provide evidence of the roles of the DPP4 substrates glucagon-like peptide 1 (GLP-1), vasoactive intestinal peptide (VIP) and stromal cell-derived factor-1α (SDF-1α) in protection against oxidative stress through activation of the mitogen-activated protein kinase (MAPK) 1/2 and phosphatidylinositol 3'-kinase (PI3K)/Akt signal transduction pathways.

Akt Signal Transduction Pathways and Nuclear Factor-kappa B (NF-κB) Transcription as a Molecular Target of Oral Tongue Squamous Cell Carcinoma (SP-C1) Using Papua's Anthill Plant ( Myrmecodia pendans ).

Squamous cell carcinoma is a malignant tumor derived from epithelial tissue with cell structure group, capable to infiltrate through the bloodstream and lymphatic tissue, spreading throughout the body. This study aim to complete theoretical foundation of flavonoid compound from anthill plant (Myrmecodia pendans) which contribute in growing cell line oral tongue squamous cell carcinoma through proliferation inhibition, inhibition mechanism transduction Akt signal and NF-κB in tongue cancer cell Supri's-clone (SP-C1). Application benefit to explore potential fractionation anthill plant use herbal ingredients for chemo protective therapy. This whole study conducted with experiment laboratorium method utilized tongue cancer human cell SP-C1. This study consist 2 steps, first to determinate, extraction and fractionation anthill plant and carry out tonicity test to get flavonoid fraction from anthill plant which has anticancer potential against tongue cancer cell SP-C1. The second stage held with invasion inhibition test, proliferation and inhibition test against protein Akt expression and NF-κB in tongue cancer cell SP-C1. The barriers to proliferation through the test of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, barriers to invasion through Boyden chamber assay and the membrane polycarbonate, ELISA analysis and Western blotting analysis on the obstacle Akt signal transduction pathways and transcription factor of nuclear factor-kappa B (NF-κB). Data analysis conducted with 2 way ANOVA followed with LSD post hoc test with significance is set on 95%. Pearson correlation conduct to find strong relationship intervariable. This study showed that the average cell growth inhibition SP-C1 based on the time and concentration using the MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The resistance of cancer cell in vitro SP-C1. On ELISA testing and Western blotting analysis, inhibiton of protein expression of Akt signal transduction and transcription factor of nuclear factor-kappa B (NF-κB) showed increased protein expression was significantly obstacles and prove that the ethyl acetate fraction flavonoid inhibits translocation and activation of transcription pathway NF-κB and growth factors that induces the phosphorylation of Akt signal transduction pathway. Ethyl acetate fraction flavonoid anthill has antitumor activity in multiple molecular targets transduction pathway including Akt and nuclear factor-kappa B (NF-κB) squamous cell carcinoma of the tongue.

IL-8 induces the epithelial-mesenchymal transition of renal cell carcinoma cells through the activation of AKT signaling.

The epithelial-mesenchymal transition (EMT) process has increasingly been examined due to its role in the progression of human tumors. Renal cell carcinoma (RCC) is one of the most common urological tumors that results in patient mortality. Previous studies have demonstrated that the EMT process is closely associated with the metastasis of RCC; however, the underlying molecular mechanism has not been determined yet. The present study revealed that interleukin (IL)-8 was highly expressed in metastatic RCC. IL-8 could induce the EMT of an RCC cell line by enhancing N-cadherin expression and decreasing E-cadherin expression. Furthermore, IL-8 could induce AKT phosphorylation, and the phosphatidylinositol-4,5-bisphosphate 3-kinase inhibitor LY294002 could inhibit the EMT of RCC cells that was induced by IL-8. Therefore, these results suggest that IL-8 is able to promote the EMT of RCC through the activation of the AKT signal transduction pathway, and this may provide a possible molecular mechanism for RCC metastasis.

Pentoxifylline modulates the AKT/mTOR/AMPK pathway: potential role as an adjunct in cancer chemotherapy

Targeting specific pathways involved in the growth and death of cells, such as AKT (signal transduction pathway promoting growth), mTOR (intracellular signaling pathway regulating cell cycle), and AMPK (coordination of growth, autophagy, and metabolism), is an established, effective strategy to inhibit cancer cell growth. Previously, we have shown that pentoxifylline (PTX), a clinically‐utilized non‐specific phosphodiesterase inhibitor, can inhibit growth and induce G1 arrest in prostate cancer cells. In the present studies, we examined the mechanism of PTX induced growth inhibition via modulation of growth/metabolic pathways, and the potential use of PTX as an adjunct in cancer chemotherapy. In prostate adenocarcinoma cells (LNCaP), we show PTX modulates the AKT pathway, as evidenced by the decreased phosphorylation of AKT. Downstream modulation of the mTOR pathway is indicated by decreased phosphorylation of mTOR, ribosomal S6K, and 4EBP1, and total cyclin D1. P‐mTOR levels return to baseline by 24h, while p‐S6K, p‐4EBP1, and cyclin D1 levels are suppressed for at least 48h, indicating a sustained decrease in the activity of cell growth pathway proteins. PTX also modulates the AMPK pathway, as revealed by modulation of downstream p‐RAPTOR, p‐ACC and p‐ULK. Both the AKT and AMPK pathways regulate the activation of cellular autophagy, an intracellular degradation system and determinant of cell survival. Treatment of LNCaP cells with PTX alone, and in combination with bafilomycin, a lysosomal degradation inhibitor, increased LC3‐II expression, indicating an induction of autophagic flux. Overall, PTX modulates both AKT and AMPK pathways, decreasing cell growth and increasing autophagy. Moreover, LNCaP cells treated with PTX were more susceptible to docetaxel (DOC; first‐line chemotherapy) induced cytotoxicity than DOC alone. Interestingly, the PTX/DOC combination decreased the expression of LC3 relative to PTX alone, suggesting activation of an alternate cell death pathway. Indeed, assessment of caspase cleavage, pBcl‐2, and annexin staining revealed a synergistic effect of PTX/DOC treatment on the induction of apoptosis. In summary, the findings provide a mechanistic basis for the potential use of PTX as an adjunct in the chemotherapy of prostate cancer. The ability of PTX to potentiate the effects of DOC on tumor growth inhibition in a mouse xenograft model is currently being assessed.Support or Funding InformationASPET SURF, P30ES006694, T32ES006694

Fyn kinase genetic ablation causes structural abnormalities in mature retina and defective Müller cell function.

Fyn kinase is widely expressed in neuronal and glial cells of the brain, where it exerts multiple functional roles that affect fundamental physiological processes. The aim of our study was to investigate the, so far unknown, functional role of Fyn in the retina. We report that Fyn is expressed, in vivo, in a subpopulation of Müller glia. We used a mouse model of Fyn genetic ablation and Müller-enriched primary cultures to demonstrate that Fyn deficiency induces morphological alterations in the mature retina, a reduction in the thickness of the outer and inner nuclear layers and alterations in postnatal Müller cell physiology. These include shortening of Müller cell processes, a decrease in cell proliferation, inactivation of the Akt signal transduction pathway, a reduced number of focal adhesions points and decreased adhesion of these cells to the ECM. As abnormalities in Müller cell physiology have been previously associated to a compromised retinal function we evaluated behavioral responses to visual stimulation. Our results associate Fyn deficiency with impaired visual optokinetic responses under scotopic and photopic light conditions. Our study reveals novel roles for Fyn kinase in retinal morphology and Müller cell physiology and suggests that Fyn is required for optimal visual processing.

Repeated Estradiol Treatment Attenuates Chronic Cerebral Hypoperfusion-Induced Neurodegeneration in Rat Hippocampus.

Although a substantial number of pre-clinical and experimental studies have investigated effects of 17β-estradiol, its precise molecular mechanism of action in the early state of chronic cerebral hypoperfusion remains controversial. The present study attempted to verify whether post-ischemic estradiol treatment (33.3μg/kg for seven consecutive days) affects previously reported number of hippocampal apoptotic cells and amount of DNA fragmentation characteristic for apoptosis as well as the expression of key elements within synaptosomal Akt and Erk signal transduction pathways (NF-κB, Bax, Bcl-2, cytochromeC, caspase3, and PARP). Additionally, alterations of aforementioned molecules linked to protection in various neurodegenerative disorders were monitored in the cytosolic, mitochondrial, and nuclear fractions associating investigated kinases and NF-κB with gene expression of their downstream effectors-Bcl-2, Bax, and caspase3. The results revealed that an initial increase in the number of apoptotic cells and amount of DNA fragmentation induced by chronic cerebral hypoperfusion was significantly reduced by 17β-estradiol. In synaptic regions, an altered profile with respect to the protein expression of Bcl-2 and phosphorylated Akt was detected, although the level of other examined proteins was not modified. In other investigated sub-cellular fractions, 17β-estradiol elicited phosphorylation and translocation of Akt and Erk along with modulation of the expression of their subsequent effectors. Our findings support the concept that repeated post-ischemic 17β-estradiol treatment attenuates neurodegeneration induced by chronic cerebral hypoperfusion in hippocampus through the activation of investigated kinases and regulation of their downstream molecules in sub-cellular manner indicating a time window and regime of its administration as a valid therapeutic intervention.

Multiwalled carbon nanotube buckypaper induces cell cycle arrest and apoptosis in human leukemia cell lines through modulation of AKT and MAPK signaling pathways

MWCNT buckypaper (BP) shows physico-chemical and mechanical properties that make it potentially useful as a substrate in nano-bio interface research including in tissue engineering. When used as a scaffold material, BP comes into contact with host cells and surrounding tissues; therefore it is critical to determine its biocompatibility and interaction with living systems. The aim of this study was to investigate BP effects on cell growth, apoptosis and reactive oxygen species (ROS) production in three human leukemia cell lines HL-60, U-937 and K-562. BP was able to induce both the reduction of cell proliferation, associated with an arrest in G0/G1 phase of cell cycle and the increase of apoptosis in leukemic cell lines, thus exerting both cytostatic and cytotoxic effects. The growth inhibitory effect was likely mediated by the decrease of cyclins D, E, A, B1 levels and CDK4 expression; meanwhile, the apoptotic effect, not mediated by ROS production, was presumably due to the combined action of the survival and pro-apoptotic AKT and MAPK signal transduction pathways. These results raised the issue of biocompatibility of MWCNT BP for the creation of carbon nanotubes based scaffolds to utilize as prostheses in tissue engineering.

Cloning, expression, and purification of cold inducible RNA-binding protein and its neuroprotective mechanism of action

Background: Cold-inducible RNA-binding protein (CIRP) is induced in response to hypothermia, where it exerts neuroprotective effects. Our preliminary studies revealed that it inhibits H2O2-induced apoptosis in rat neurons. In the current study, we report effective expression and purification approaches for the synthesis of CIRP, and assess its potential protective effects against oxidative stress. Methods: CIRP-encoding was expressed using the prokaryotic expression system pGEX-4T-1, and SP-Sepharose and Sephacryl S-200 columns were used to purify rCIRP. To mimic ischemia/reperfusion injury-associated oxidative stress, neuro2a cells (N2a) were pre-treated with rCIRP for 2h, followed by hydrogen peroxide (H2O2 60μmol/ml) for 24h. Cell viability was then quantified using an MTT assay. In addition, western blotting was performed to measure the cell cycle related signal transduction pathways. Results: N2a cells exhibited decreased viability following H2O2 treatment, whereas rCIRP significantly improved viability following H2O2 treatment. CIRP also accelerated cell cycle progression from S to G2/M phase in cultured mouse neuroblastoma cells. In addition, CIRP increased levels of p-ERK and p-Akt, and also re-activated the cell cycle-related protein cyclin D1 and c-Myc. These results suggest that CIRP activated the Akt and ERK signal transduction pathways in N2a cells. Conclusions: Our findings suggest that CIRP could exert protective effects against oxidative stress, and that it might be a novel neuroprotective agent.

Effects of VBMDMP on the reversal of cisplatin resistance in human lung cancer A549/DDP cells.

Tumor drug resistance is a major obstacle to cancer chemotherapy. We previously constructed a fusion protein based on two tumstatin-derived sequences named recombinant VBMDM (rVBMDMP). We preliminarily confirmed its inhibition of HUVEC and colon cancer cell growth. The present study further systematically observed the inhibitory effect of rVBMDMP on lung cancer cell growth and analyzed a possible mechanism to provide a theoretical basis for the development of new antitumor protein drugs. The effect of rVBMDMP on human lung adenocarcinoma (A549) and cisplatin-resistant human lung adenocarcinoma (A549/DDP) cell proliferation was evaluated by MTS assay. Hoechst 33342 staining performed together with fluorescence microscopy and immunoblot analysis were used to examine the effects of rVBMDMP on the apoptosis of A549/DDP cells. A protein phosphorylation chip was used to identify changes in rVBMDMP-induced signaling protein phosphorylation. Changes in the phosphatidylinositol 3 kinase (PI3K)/Akt signal transduction pathway and expression of multidrug resistance protein (MRP-2)-related molecules following rVBMDMP treatment in A549/DDP cells were evaluated by western blot analysis. A lung cancer xenograft model was used to evaluate the reversal effect of rVBMDMP on drug-resistance of A549/DDP cell tumors to cisplatin in vivo. The results demonstrated that rVBMDMP increased the phosphorylation of 79 signaling proteins, including focal adhesion kinase (FAK), caspase-6, Fas, FasL and FAF1 and downregulated 30 signaling proteins, including integrin αV, integrin β3, PI3K/Akt, NF-κB and MRP-2 compared with the controls. rVBMDMP also increased the sensitivity of A549 and A549/DDP cells to cisplatin and directly induced apoptosis, which may be related to MRP-2 and Bcl-2 downregulation. The effects of growth inhibition and apoptosis induction of rVBMDMP on A549/DDP cells may be related to the inhibition of integrin αVβ3 and PI3K/Akt protein phosphorylation. Finally, we observed an increase in cancer cell sensitivity to cisplatin by rVBMDMP using the A549/DDP cell xenograft model in nude mice. Our study suggests that rVBMDMP may be an effective potential chemotherapy sensitizer and may be a viable drug candidate in anticancer therapies.

Expression and clinical significance of the phosphatidylinositol 3-kinase/protein kinase B signal transduction pathway in non-small cell lung carcinoma.

The overactivation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signal transduction pathway has been examined in various carcinomas and is reported to be significantly correlated with prognosis. However, little is known with regard to the PI3K/Akt signal transduction pathway in advanced non-small cell lung carcinoma (NSCLC). The present study investigated the expression of PI3K and phosphorylated (p)-Akt protein and its clinical significance in NSCLC. The clinical records of 157 patients with NSCLC (70 stage I–IIIA and 87 stage IIIB–IV cases), consisting of 75 cases of squamous cell carcinoma and 82 cases of adenocarcinoma, together with 30 resected lung cancer tumor-adjacent tissue samples, were retrospectively evaluated. PI3K and p-Akt expression in the NSCLC and tumor-adjacent tissues were measured using an immunohistochemical method, and its correlation with the clinicopathological data and prognosis in advanced NSCLC was evaluated. PI3K and p-Akt expression was significantly higher in the cancer tissues (χ2=14.8455; P=0.001) than in the tumor-adjacent tissues (χ2=14.2615; P=0.001). The overexpression of p-Akt in stage I–IIIA NSCLC was associated with lymph node metastasis (χ2=6.1189; P=0.013) and tumor-node-metastasis (TNM) stage (χ2=8.9752; P=0.011), however, no correlation was observed with gender, age, pathological type and histological grade. The overexpression of p-Akt in stage IIIB–IV NSCLC was only associated with TNM stage (χ2=5.7501; P=0.016), and no correlation was observed with gender, age, pathological type, histological grade and Eastern Cooperative Oncology Group (ECOG) performance status (PS). The overexpression of PI3K was not found to correlate with the aforementioned clinicopathological variables in all patients. Survival was significantly improved in advanced NSCLC with PI3K- and p-Akt-negative expression compared with PI3K- and p-Akt-positive expression [P13K: 17.70 months (95% confidence interval (CI), 15.11–20.28 months) vs. 13.43 months (95% CI, 11.83–15.02 months); P=0.004; and p-Akt: 17.13 months (95% CI, 14.93–19.34 months) vs. 13.07 months (95% CI, 11.32–14.82 months); P=0.007]. Multivariate analysis showed that PI3K [hazard ratio (HR)=2.143; 95% CI, 1.211–3.790; P=0.009], p-Akt (HR=1.991; 95% CI, 1.009–3.927; P=0.047), TNM stage (HR=4.788; 95% CI, 2.591–8.848; P=0.001) and ECOG-PS (HR=3.272; 95% CI, 1.701–6.296; P=0.001) were independent predictors for survival in stage IIIB–IV NSCLC. These results indicated that p-Akt overexpression closely correlates with factors of an unfavorable prognosis in NSCLC. PI3K and p-Akt overexpression are independent markers of a poor prognosis in advanced NSCLC.

Erk/MAP Kinase Signaling Pathway and Neuroendocrine Differentiation of Non–Small-Cell Lung Cancer

Almost all small-cell lung cancers (SCLC) and carcinoid tumors express neuroendocrine differentiation (NED), and 10% to 20% of non-small-cell lung cancers (NSCLC) are associated with NED. Although distinct clinical features and histology of SCLC and carcinoid tumors are well recognized, the clinical significance and the molecular basis of NED in NSCLC remain unclear. To explore the potential molecular pathway involved in NED of NSCLC and its clinical relevance, we conducted investigations using an NSCLC cell line (NCI-H157) as a NED induction model, and explored the potential intracellular signal transduction pathways involved in NED of NSCLC. We confirmed our findings using activators versus inhibitors to these signal transduction pathways in vitro. We also performed immunohistochemical stains of phospho-Erk1/2 of lung cancer specimens known to have NED and explored its clinical relevance. We discovered that NED of NSCLC was associated with the activation of Erk1/2-mitogen-activated protein kinases (MAPK) signal transduction pathway, and the inhibition of the Akt signal transduction pathway. Using specific activator (Pb) and inhibitors (siRNA-Erk1/2 and U0126) to the Erk1/2-MAP-kinase pathway, as well as the inhibitor (LY294002) to the Akt pathway, we found that Erk1/2-MAP-kinase activation was essential for NED of NCI-H157 cells. Staining of Erk1/2-MAP-kinase pathway revealed a high rate of positivity in NSCLC tumors with NED when compared with other neuroendocrine lung tumors. To our knowledge, our findings are the first to describe the potential involvement of Erk/MAPK signal transduction pathway of NSCLC in the association with NED. Further investigation of the Erk/MAPK signal transduction pathway of NSCLC may yield discoveries in identifying specific molecular targets for the treatment of NSCLC with NED.

Recent progress on nutraceutical research in prostate cancer

Recently, nutraceuticals have received increasing attention as the agents for cancer prevention and supplement with conventional therapy. Prostate cancer (PCa) is the most frequently diagnosed cancer and second leading cause of cancer-related death in men in the US. Growing evidences from epidemiological studies, in vitro experimental studies, animal studies, and clinical trials have shown that nutraceuticals could be very useful for the prevention and treatment of PCa. Several nutraceuticals including isoflavone, indole-3-carbinol, 3,3'-diindolylmethane, lycopene, (-)-epigallocatechin-3-gallate, and curcumin are known to downregulate the signal transductions in AR, Akt, NF-κB, and other signal transduction pathways which are vital for the development of PCa and the progression of PCa from androgen-sensitive to castrate-resistant PCa. Therefore, nutraceutical treatment in combination with conventional therapeutics could achieve better treatment outcome in prostate cancer therapy. Interestingly, some nutraceuticals could regulate the function of cancer stem cell (CSC)-related miRNAs and associated molecules, leading to the inhibition of prostatic CSCs which are responsible for drug resistance, tumor progression, and recurrence of PCa. Hence, nutraceuticals may serve as powerful agents for the prevention of PCa progression and they could also be useful in combination with chemotherapeutics or radiotherapy. Such strategy could become a promising newer approach for the treatment of metastatic PCa with better treatment outcome by improving overall survival.

Mutations in the PTEN tumor gene and risk of endometriosis: a case–control study

Are mutations in the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene associated with endometriosis? Loss of heterozygosity (LOH) at the 10q23.3 locus, PTEN somatic mutations and changes in the levels and distribution of proteins in the PTEN-PI3K/Akt signal transduction pathway are associated with endometriosis. Endometriosis has a strong genetic basis. Recent genome-wide association and linkage studies have reported a significant association of endometriosis with 7p15.2, 9p21 and 10q23-26 loci. PTEN, which maps to 10q23.3, acts as a tumor suppressor gene through the action of its phosphatase protein product, phosphatase and tensin homolog (PTEN). This phosphatase is involved in the regulation of the cell cycle, and mutations of PTEN are a step in the development of many cancers. A total of 1252 subjects of Indian origin (endometriosis patients = 752; controls = 500) were recruited to participate in this case-control study. Recruitment took place from 2001 to 2009 at Institute of Reproductive Medicine (IRM), Kolkata, India; Infertility Institute and Research Centre (IIRC), Secundrabad, India and Vasavi Medical and Research Centre, Hyderabad, India. LOH on 10q, 9p and 7p was analyzed in analogous ectopic-eutopic endometria along with blood samples from 32 advanced stage endometriosis patients by PCR-GeneScan analysis. Genotyping of PTEN was carried out on genomic DNA of analogous ectopic-eutopic endometria (n = 32) as well as blood samples from 720 patients and 500 controls by PCR-sequencing analysis to explore somatic and germ-line mutations, respectively. The levels and distribution of PTEN, p-Akt, p-Bad and p27 were analyzed in the eutopic endometria of patients (n = 5) and controls (n = 5) using western-blot and immunohistochemistry. PCR-GeneScan analysis revealed a higher LOH frequency at 10q23.3 (84.4%) compared with other loci analyzed, hence we focused our attention on PTEN. PCR-sequencing analysis revealed seven novel somatic mutations and 23 germ-line polymorphisms in patients. Among somatic mutations, a frame-shift insertion at 10:89692992-89692993 (in the functionally important N-terminal phosphatase domain of PTEN) occurred in 11 of the 32 ectopic endometria. Western-blot and immunohistochemical analysis revealed decreased PTEN and increased p-Akt and p-Bad levels in eutopic endometria of patients compared with controls (all comparisons, P < 0.0001). Furthermore, PTEN loss was more frequent in the nucleus than in the cytoplasm. Expression of p27 did not differ between patients and controls. Protein analysis was performed in eutopic endometrial samples from only a small number of patients and controls. In future investigations, a larger sample size should be used and the role of the other genes involved in the PTEN-PI3K/Akt signal transduction pathway should be analyzed. Our findings revealed a possible involvement of the PTEN-PI3K/Akt-Bad axis in the pathogenesis of endometriosis, which may facilitate the discovery of suitable pathway inhibitors for disease treatment. This study was supported by grants from the Science & Engineering Research Board (SERB), India (Lr No: SR/FT/LS-188/2009) to BM. The authors have no competing interests to declare.

Hexabromocyclododecane facilitates FSH activation of ERK1/2 and AKT through epidermal growth factor receptor in rat granulosa cells

The toxicity of hexabromocyclododecane (HBCDD) has been extensively studied; however, the mechanism and the effects of HBCDD on female reproductive system have been less frequently reported. In this study, we exposed rat granulosa cells to HBCDD during in vitro follicle-stimulating hormone (FSH)-driven cell proliferation and differentiation. Here, we show that HBCDD affects the FSH-driven signal transduction and ovulatory competence of granulosa cells. We found that HBCDD over-activates the FSH-stimulated extracellular-regulated kinase 1/2 (ERK1/2) and protein kinase B (PKB, also known as AKT). Inactivation of the epidermal growth factor receptor (EGFR) kinase activity with AG1478 and the mitogen-regulated kinase activity with U0126 completely prevented ERK1/2 activation in the FSH-stimulated and HBCDD-exposed granulosa cells. Moreover, AG1478 restored the HBCDD-induced AKT activation to the level observed in the FSH-stimulated cells. Western blot shows that HBCDD potentiates FSH-stimulated EGFR phosphorylation in granulosa cells. Real-time PCR demonstrates that HBCDD decreases the FSH-induced luteinizing hormone receptor (Lhr) expression. Inadequate level of LHR in the HBCDD-exposed granulosa cells prevented human chorionic gonadotropin in stimulating expression of the ovulatory genes such as amphiregulin (Areg), epiregulin (Ereg), and progesterone receptor (Pgr). Addition of U0126 and AG1478 restored Lhr level in the FSH-stimulated and HBCDD-exposed granulosa cells. These results indicate a direct effect of HBCDD on EGFR activation, resulting in over-activation of ERK1/2 and AKT signal transduction pathways in the FSH-treated cells. Increased activity of the EGFR-ERK1/2 pathway above physiological level prevents sufficient acquisition of LHR in proliferating granulosa cells, thus compromising ovulation.

Therapeutic Effects of IGF-1 on Stress Urinary Incontinence in Rats with Simulated Childbirth Trauma

We examined the effect of IGF-1 in a rat model of stress urinary incontinence induced by simulated childbirth trauma. Simulated birth trauma was induced by vaginal distension in female Sprague Dawley® rats. Four, 7, 14 and 28 days after distension we performed functional assessment by measuring leak point pressure, urethral baseline pressure and the urethral response during a passive increment in intravesical pressure. The expression of IGF-1 and IGF1R mRNA and protein in damaged tissues was examined by real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry. Thereafter hrIGF-1 (50 and 150 μg/kg per day) was continuously delivered from 1 day before distension using subcutaneous osmotic pumps. Four and 7 days after distension the effect of hrIGF-1 treatment was examined by functional analysis of leak point pressure, urethral baseline pressure and the urethral response as well as Western blot and histological analysis. After 4 and 7 days rats with vaginal distension had significantly decreased leak point pressure, urethral baseline pressure and urethral responses. IGF-1 and IGF1R mRNA and protein levels were significantly increased in urethral and pudendal nerves 4 and 7 days after distension. IGF-1 treated groups showed significant improvement in leak point pressure, urethral baseline pressure and urethral responses 4 and 7 days after distension. Moreover, IGF-1 treatment increased Akt phosphorylation and induced cellular proliferation and antiapoptotic effects in the urethra. IGF-1 treatment accelerated recovery from stress urinary incontinence induced by simulated childbirth trauma in association with activation of the Akt signal transduction pathway in rats. This suggests that IGF-1 has therapeutic potential for stress urinary incontinence in women.

Study on molecular target promoting human neural stem cells of ginsenoside Rg1 by gene chip

To screen out main molecular target promoting human neural stem cells (NSCs) of ginsenoside Rg1 by using the gene chip technology. First, MTT assay was adopted to screen out the optimal concentration of Rg1-promoted NSC proliferation (120 mg x L(-1)). Then, on the 7th day after the Rg1-promoted NSC proliferation, the expression of target genes was observed by the gene chip technology. The most important target gene and signal transduction pathways were screened out through the data calculations. On the 7th day after the Rg1-promoted NSC proliferation, obtained 440 differential genes, 266 significantly upregulated genes and 174 significantly down-regulated genes. HES1 gene, CAMP (cyclic adenosine monophosphate)-PKA (protein kinase A) and PI3K (phosphatidylinositol 3 kinase)-AKT signal transduction pathways were closely related to the NSC proliferation. The differentially expressed genes screened out by gene chip may provide new clues for studies on molecular mechanism of ginsenoside Rg1-promoted NSCs proliferation.

TACC3 Is Essential for EGF-Mediated EMT in Cervical Cancer

The third member of transforming acidic coiled-coil protein (TACC) family, TACC3, has been shown to be an important player in the regulation of centrosome/microtubule dynamics during mitosis and found to be deregulated in a variety of human malignancies. Our previous studies have suggested that TACC3 may be involved in cervical cancer progression and chemoresistance, and its overexpression can induce epithelial-mesenchymal transition (EMT) by activating the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated protein kinases (ERKs) signal transduction pathways. However, the upstream mechanisms of TACC3-mediated EMT and its functional/clinical importance in human cervical cancer remain elusive. Epidermal growth factor (EGF) has been shown to be a potent inducer of EMT in cervical cancer and associated with tumor invasion and metastasis. In this study, we found that TACC3 is overexpressed in cervical cancer and can be induced upon EGF stimulation. The induction of TACC3 by EGF is dependent on the tyrosine kinase activity of the EGF receptor (EGFR). Intriguingly, depletion of TACC3 abolishes EGF-mediated EMT, suggesting that TACC3 is required for EGF/EGFR-driven EMT process. Moreover, Snail, a key player in EGF-mediated EMT, is found to be correlated with the expression of TACC3 in cervical cancer. Collectively, our study highlights a novel function for TACC3 in EGF-mediated EMT process and suggests that targeting of TACC3 may be an attractive strategy to treat cervical cancers driven by EGF/EGFR signaling pathways.

Targeting phosphatidylinositol 3-kinase-Akt through hepatocyte growth factor for cardioprotection

Several growth factors have been shown to protect the cardiomyocyte from the detrimental effects of acute ischemia-reperfusion injury, through the activation of a variety of cell-surface receptors and the subsequent recruitment of a number of intracellular signal transduction pathways. Among these growth factors, hepatocyte growth factor (HGF), also named as scatter factor, acts by recruiting the phosphatidylinositol 3-kinase (PI3K)-Akt signal transduction pathway, linked to cardioprotection, at the time of myocardial infarction and myocardial reperfusion. HGF has been reported to increase in the early phase of myocardial infarction, and has been shown to have mitogenic, angiogenic, antiapoptotic and antifibrotic activities in cardiac myocytes and endothelial cells. Also, endogenous HGF may play an important role in the regeneration of endothelial cells and cardiomyocytes by promoting angiogenesis and inhibiting apoptosis during remodeling of the ischemic myocardium. Thus, HGF has the potential to emerge as a cardioprotective agent for the treatment of several pathological cardiac conditions. Here we review the role of HGF with respect to its ability to confer direct myocardial protection in the setting of ischemia-reperfusion injury, focusing on the main underlying signaling pathway involved.

Oleocanthal Inhibits Proliferation and MIP-1&amp;#945; Expression in Human Multiple Myeloma Cells

Multiple myeloma (MM) is a plasma cell malignancy that causes devastating bone destruction by activating osteoclasts in the bone marrow milieu. MM is the second of all hematological malignancies. Thus, the search for new pharmacological weapons is under intensive investigation being MM a critically important public health goal. Recently, it has been demonstrated that macrophage inflammatory protein 1- alpha (MIP-1 α) is crucially involved in the development of osteolytic bone lesions in MM. Phenolic components of extra virgin olive oil are reported to have anti tumor activity. However, the underlying mechanisms and specific targets of extra virgin olive oil remain to be elucidated. In the present study, we investigated the effects of a recently isolated novel extra virgin olive oil polyphenol, oleocanthal, on the human multiple myeloma cell line ARH-77. Here we report that this natural compound has a remarkable in vitro activity by inhibiting MIP-1 α expression and secretion in MM cells. In addition, we also demonstrated that oleocanthal inhibits MM cells proliferation by inducing the activation of apoptosis mechanisms and by down-regulating ERK1/2 and AKT signal transduction pathways. This in vitro study suggests a therapeutic potential of oleocanthal in treating multiple myeloma.

Reactive oxygen species via redox signaling to PI3K/AKT pathway contribute to the malignant growth of 4-hydroxy estradiol-transformed mammary epithelial cells.

The purpose of this study was to investigate the effects of 17-β-estradiol (E2)-induced reactive oxygen species (ROS) on the induction of mammary tumorigenesis. We found that ROS-induced by repeated exposures to 4-hydroxy-estradiol (4-OH-E2), a predominant catechol metabolite of E2, caused transformation of normal human mammary epithelial MCF-10A cells with malignant growth in nude mice. This was evident from inhibition of estrogen-induced breast tumor formation in the xenograft model by both overexpression of catalase as well as by co-treatment with Ebselen. To understand how 4-OH-E2 induces this malignant phenotype through ROS, we investigated the effects of 4-OH-E2 on redox-sensitive signal transduction pathways. During the malignant transformation process we observed that 4-OH-E2 treatment increased AKT phosphorylation through PI3K activation. The PI3K-mediated phosphorylation of AKT in 4-OH-E2-treated cells was inhibited by ROS modifiers as well as by silencing of AKT expression. RNA interference of AKT markedly inhibited 4-OH-E2-induced in vitro tumor formation. The expression of cell cycle genes, cdc2, PRC1 and PCNA and one of transcription factors that control the expression of these genes – nuclear respiratory factor-1 (NRF-1) was significantly up-regulated during the 4-OH-E2-mediated malignant transformation process. The increased expression of these genes was inhibited by ROS modifiers as well as by silencing of AKT expression. These results indicate that 4-OH-E2-induced cell transformation may be mediated, in part, through redox-sensitive AKT signal transduction pathways by up-regulating the expression of cell cycle genes cdc2, PRC1 and PCNA, and the transcription factor – NRF-1. In summary, our study has demonstrated that: (i) 4-OH-E2 is one of the main estrogen metabolites that induce mammary tumorigenesis and (ii) ROS-mediated signaling leading to the activation of PI3K/AKT pathway plays an important role in the generation of 4-OH-E2-induced malignant phenotype of breast epithelial cells. In conclusion, ROS are important signaling molecules in the development of estrogen-induced malignant breast lesions.