Phosphatase And Tensin Homolog Deleted On Chromosome Ten Research Articles

Effect of Jianpi Jiedu Recipe on angiogenesis and the PTEN/PI3K/AKT signaling pathway in the course of Helicobacter pylori-induced gastric cancer in C57BL/6 mice

Highlights Jianpi Jiedu Recipe can reduce the infection rate of Helicobacter pylori in mice gastric mucosa by inhibiting the expression of MVD and VEGF, and reducing the inactivation of PTEN. Editor’s Summary In addition to use triple antibiotics to treat the infection of Helicobacter pylori , Jianpi Jiedu Recipe can reduce the infection rate of Helicobacter pylori and further prevent the occurrence of gastric cancer. Objective: To reveal the effect of Jianpi Jiedu recipe (JPJDR) on angiogenesis and the PTEN (Phosphatase and tensin homolog deleted on chromosome ten)/PI3K/AKT signaling pathway in the course of H. pylori infection-induced carcinogenesis of gastric mucosa in C57BL/6 mice. Methods: Two-hundred C57BL/6 mice were randomly divided into five groups (control group, model group, JPJDR low-dose group, JPJDR medium-dose group, and JPJDR high-dose group), 40 in each group. A mouse model of gastric cancer, induced by H. pylori standard strain infection, was established. The mice of JPJDR low-dose, middle-dose, and high-dose groups were intragastrically administered 250, 500, and 1000 mg/kg JPJDR per day, respectively. After 72 weeks, the H. pylori infection in gastric mucosa of the mice was analyzed by rapid urease test; the pathological changes in the gastric mucosa of mice were assessed by histopathological examination, and micro-vessel density (MVD), vascular endothelial growth factor (VEGF), and PTEN/PI3K/AKT levels were determined. Results: The incidence of gastric cancer in each group (control group, model group, JPJDR low-dose, medium-dose, high-dose group) was 0%, 26.3%, 13.2%, 10%, and 7.5% respectively. The incidence of gastric cancer in the Chinese medicine group was significantly lower than that of the model group ( P = 0.020, P = 0.023, P = 0.007). The expression of MVD and VEGF in the model group was significantly higher than that in the control group ( P = 0.002, P < 0.001), while the expression of MVD and VEGF decreased in the Chinese medicine group. The expression of p-PTEN and p-AKT in the model group was significantly higher than that in the control group (All P < 0.001), while Chinese medicine could reduce the expression of p-PTEN and p-AKT to varying extents. Conclusion: Long-term infection of C57BL/6 mice with H. pylori induces gastric carcinogenesis, by increasing gastric mucosal MVD, promoting the expression of VEGF, inhibiting the activity of PTEN, and activating the PI3K/AKT signaling pathway. JPJDR can reduce the infection rate of H. pylori in mouse gastric mucosa, inhibit the expression of MVD and VEGF, and reduce the inactivation of PTEN.

Effect of matrine on the growth of human breast cancer MCF-7/ADR cell xenograft model in nude mice by regulating apoptosis-related gene expression though phosphatidylinositol 3 kinase/protein kinase B signal pathway

Objective To investigate the mechanism and effect of matrine on the growth of human breast cancer MCF-7/ADR cell xenograft model in nude mice. Methods A total of 20 nude mice were subcutaneously inoculated with MCF-7/ADR cells. After the successful establishment of xenograft model, the 20 nude mice were randomly divided into 4 groups, 5 rats in each group. Low- and high-dose matrine groups were intraperitoneally injected with matrine at dose of 50 and 100 mg/kg respectively. Positive control group was treated orally with MK2206 at dose of 120 mg/kg, and negative control group received normal saline. At 2st day after treatment, the tumor inhibition rate was measured, the mRNA and protein expression of multidrug resistance (MDR)1, phosphatase and tensin homologue deleted on chromosometen (PTEN), protein kinase B (Akt), B cell lymphoma/leukemia-2 associated X protein (bax), B cell lymphoma/leukemia-2 (bcl-2) and cysteinyl aspartate-specific protease-3 (Caspase-3) was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting respectively. Results The tumor inhibition rate in positive control group, low-dose and high-dose matrine groups was 37.9%, 42.1% and 60.7% respectively. RT-PCR showed the MDR1 gene relative expression in low- and high-dose matrine groups was 0.548±0.054, and 0.869±0.058, and the Caspase-3 gene relative expression was 1.642±0.062 and 1.327±0.068 respectively (P 0.05). Western blotting showed the protein expression levels of PTEN, bax and Caspase-3 in positive control group, low-dose and high-dose matrine groups were significantly increased, and those of permeability glycoprotein (P-gp), p-Akt and bcl-2 were significantly decreased as compared with the negative control group (P<0.05). Conclusion Matrine could inhibit the growth of MCF-7/ADR cell xenograft model in nude mice by affecting Akt key gene and regulating the expression of apoptosis-related genes though PI3K/Akt signal pathway. Key words: Matrine; Breast cancer; Apoptosis; Phosphatidylinositol 3 kinase/protein kinase B signal pathway; Nude mice

Exosome-transmitted microRNA-19a promotes drug resistance in breastcancer by targeting phosphatase and tensin homolog/protein kinase B/p53 axis

Objective To investigate the specific effect and underlying molecular mechansim of microRNA (miRNA, miR)-19a through MCF-7/A secrete dexosomes in drug resistance transfer. Methods Exosomes were extracted from the supernatant of cultured MCF-7/A cells by fractionation ultracentrifugation and were identified by transmission electron microscopy and Nanosight. GFP-MCF-7/S, a breast cancer sensitive cell line stably expressing green fluorescent protein (GFP), was constructed by recombinant lentiviral vector with GFP. The co-cultured experiments of cell-cell and cell-exosomewere designed to observe thephenomenon of drug resistance transfer. Real-time quantitative polymerase chain reaction(Real-time PCR), Western blotting and cell transfection techniquewere used to determine the specific effects of miR-19a and phosphatase and tensin homologue deleted on chromosometen (PTEN)/protein kinase B (Akt)/p53 axis in the progress of drug resistance. Results Compared to MCF-7/S, the expression of miR-19a was significantly increased in MCF-7/A cells (1.03±0.16 vs. 3.56±0.58, P=0.002), down-regulaion of miR-19a apparently increased sensitivity to Adriamycin (ADM) in MCF-7/A cells [(41.17±10.65)% vs. (23.45±8.89)%, P=0.011]. Transmission electron microscopy showed that the exosomes cells had adiameter of 40-100 nm and exhibited round or elliptic shape secreted by both two type cancer cells. The cell inhibition ratio of ADM detected by the intensity of GFP and cell counting kit-8 (CCK-8) assay showed that cell inhibition rate of MCF-7/S+ MCF-7/A and MCF-7/S+ MCF-7/A exos group were significantly lower than the MCF-7/S+ MCF-7/S [(31.50±6.68)% vs. (51.20±12.44)%, P=0.006] and MCF-7/S+ MCF-7/S exos group [(27.67±6.02)% vs. (49.65±10.52)%, P=0.000], which companied with the increasing expression miR-19a (1.01±0.13 vs. 2.67±0.62, P=0.000). Western blotting assay showed that miR-19 in exosomes secreted by drug resistant cancer cells significantly inhibited the expression of PTEN (1.15±0.18 vs. 0.67±0.13, P=0.000) and p53 protein (0.99±0.18 vs. 0.49±0.11, P=0.005), whereas increases phosphorylation of Akt (1.15±0.18 vs. 0.67±0.13, P=0.000), which promoted the occurrence of drug resistance in receptor cells. Conclusion MiR-19a transfers drug resistance between breast cancer cells by exosome secreted from drug-resistance cancer cells through the regulation of PTEN/Akt/p53 axis in receptor cancer cells. Key words: Exosome; MicroRNA-19a; Breast cancer; Drug assistant; Phosphatase and tensin homologue deleted on chromosometen/protein kinase B/p53 axis

PI3K/Akt/mTOR signaling &amp; its regulator tumour suppressor genes PTEN &amp; LKB1 in human uterine leiomyomas

Background & objectives:Despite their high occurrence and associated significant level of morbidity manifesting as spectrum of clinical symptoms, the pathogenesis of uterine leiomyomas (ULs) remains unclear. We investigated expression profile of tumour suppressor genes PTEN (phosphatase and tensin homolog deleted on chromosome ten) and LKB1 (liver kinase B1), and key signaling components of P13K (phosphatidylinositol 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) pathway in leiomyomas and adjacent normal myometrium in women of reproductive age, to explore the possibility of targeting this pathway for future therapeutic implications.Methods:Real time PCR (qPCR) was used to quantify relative gene expression levels of PTEN, Akt1, Akt2, mTOR, LKB1 and VEGFA (vascular endothelial growth factor A) in leiomyoma as compared to adjacent normal myometrium. Immunohistochemistry was subsequently performed to analyze expression of PTEN, phospho-Akt, phospho-mTOR, phospho-S6, LKB1 and VEGFA in leiomyoma and adjacent normal myometrium.Results:Significant upregulation of PTEN (2.52 fold; P=0.03) and LKB1 (3.93 fold; P=0.01), and downregulation of VEGFA (2.95 fold; P=0.01) genes were observed in leiomyoma as compared to normal myometrium. Transcript levels of Akt1, Akt2 and mTOR did not vary significantly between leiomyoma and myometrium. An increased immunoexpression of PTEN (P=0.015) and LKB1 (P<0.001) and decreased expression of VEGFA (P=0.01) was observed in leiomyoma as compared to myometrium. Immunostaining for activated (phosphorylated) Akt, mTOR and S6 was absent or low in majority of leiomyoma and myometrium.Interpretation & conclusions:Upregulation of PTEN and LKB1 in concert with negative or low levels of activated Akt, mTOR and S6 indicates that PI3K/Akt/mTOR pathway may not play a significant role in pathogenesis of leiomyoma.

Gambogenic acid alters chemosensitivity of breast cancer cells to Adriamycin.

BackgroundBreast cancer remains a major health problem worldwide, and is becoming increasingly resistant to traditional drug treatments. For instance, Adriamycin (ADR) is beneficial for the treatment of breast cancer. However, its wide application often leads to drug resistance in clinic practice, which results in treatment failure. Gambogenic acid (GNA), a polyprenylated xanthone isolated from the traditional medicine gamboge, has been reported to effectively inhibit the survival and proliferation of cancer cells. Its effects on ADR resistance have not yet been reported in breast cancer. In this study, we examined the ability of GNA to modulate ADR resiatance and the molecular mechanisms underlying this process using a cell based in vitro system.MethodsAn MTT assay was used to evaluate the inhibitory effect of the drugs on the growth of MCF-7 and MCF-7/ADR cell lines. The effects of drugs on apoptosis were detected using Annexin-V APC/7-AAD double staining. The expression of apoptosis-related proteins and the proteins in the PTEN/PI3K/AKT pathway were evaluated by Western blot analysis.ResultsIn the MCF-7/ADR cell lines, the IC50 (half maximal inhibitory concentration) of the group that received combined treatment with GNA and ADR was significantly lower than that in the ADR group, and this value decreased with an increasing concentration of GNA. In parallel, GNA treatment increased the chemosensitivity of breast cancer cells to ADR. The cell apoptosis and cell cycle anaysis indicated that the anti-proliferative effect of GNA is in virtue of increased G0/G1 arrest and potentiated apoptosis. When combined with GNA in MCF-7/ADR cell lines, the expression levels of the tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome ten) and the apoptosis-related proteins caspase-3 and capsese-9 were significantly increased, while the expression of phosphorylated AKT was decreased.ConclusionsOur study has indicated a potential role for GNA to increase the chemosensitivity of breast cancer cells to ADR. This modulatory role was mediated by suppression of the PTEN/PI3K/AKT pathway that led to apoptosis in MCF-7/ADR cells. This work suggests that GNA may be used as a regulatory agent for treating ADR resistance in breast cancer patients.

Expression and clinical evidence of miR-494 and PTEN in non-small cell lung cancer.

The aim of this study was to explore the expression and clinical significance of miR-494 and PTEN (phosphatase and tensin homologue deleted on chromosome ten) in non-small cell lung cancer (NSCLC). Immunohistochemistry for PTEN and in situ hybridization (ISH) for miR-494 were performed in 92 NSCLC tissues and 10 normal lung tissues to detect their expression, and correlation between their expression with clinical characteristics and prognosis was analyzed. The expression of miR-494 was significantly higher in NSCLC than in normal lung tissues (P = 0.004). The positive expression of PTEN protein in the lung carcinoma tissues was significantly lower than that in the normal lung tissues (P = 0.013), while the level of miR-494 expression was negatively correlated with PTEN expression (r = -0.577, P < 0.01). The high positive rate of miR-494 was positively correlated with pathological TNM (p-TNM) staging and lymph node metastasis. The expression of miR-494 was negatively correlated with grade of differentiation. However, the expression of PTEN was positively correlated with grade of differentiation. Patients with over-expression of miR-494 had a shorter overall survival (OS), while the negative group of PTEN was correlated with poor OS. MiR-494 over-expression and low PTEN expression are closely related to tumor p-TNM staging and lymph node metastasis, differentiation, and OS. Combined detection of PTEN and miR-494 can aid in determining malignancy degree and the prognosis of patients with NSCLC. MiR-494 may be served as a novel prognostic factor and may lead to new treatment strategies for NSCLC.

PI3K/AKT/mTOR pathway is activated after imatinib secondary resistance in gastrointestinal stromal tumors (GISTs).

Phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of the rapamycin (mTOR) pathway activation may be related to imatinib resistance; however, no study has focused on whether signal conduction of this pathway will change after imatinib resistance. A total of 111 GIST samples from 91 patients were used in this study, including 20 pairs of samples before and after imatinib treatment. Immunohistochemistry was performed on tissue for p-KIT (phospho-KIT), PTEN (phosphatase and tensin homolog deleted on chromosome ten), PI3K, phospho-AKT (p-AKT), phospho-4EBP1 (p-4EBP1) and phospho-S6 (p-S6RP). The activation of AKT/mTOR was significantly higher in imatinib secondary resistant GIST (53.1%) than in imatinib-sensitive (27.1%) and primary resistant GIST (33.3%) (P=0.049). In the analysis of 20 pairs of samples, comparing pre-imatinib GIST with on-treatment ones, the PI3K status was changed from inactivated to activated in four cases each in eight patients with effective imatinib and 12 patients whose secondary resistance happened, respectively. AKT/mTOR status was inactivated in pre-imatinib and on-treatment samples in eight patients with effective imatinib; however, the status of six patients was changed from inactivated to activated in 12 patients at the time of tumor progression. The negative expression of p-KIT was accompanied with PI3K pathway and/or AKT/mTOR pathway activity in some GISTs with secondary resistance. PI3K/AKT/mTOR pathway can be partly activated after imatinib secondary resistance in GIST. In this pathway, activation of AKT/mTOR is a more crucial factor, and PI3K activation may be the early part of secondary resistance.

The expression of phosphatase and tensin homologue deleted on chromosometen/mammalian target of rapamycin signaling pathway in the spinal cord of rats during the development

Objective To observe the expression pattern of phosphatase and tensin homologue deleted on chromosometen(PTEN)/mammalian target of rapamycin(mTOR)pathway in the spinal cord of SD rats. Methods We dissected spinal cords of rats at developmental stages E15, P0, P7 and P56,and applied Western blotting to detect the expression levels of critical target proteins on the pathway. Furthermore, immunofluorescence was used to detect the cellular localization of these targets in the spinal cord. Results The ratio of gray value for PTEN to actin was 0.281±0.086,0.462±0.002,0.591±0.022 and 0.392±0.001 from E15 to P56. There was statistically significant difference between each two groups except for P0 and P7. The ratio of gray value for S6 to actin was 0.646±0.116,0.450±0.129,0.824±0.082 and 0.174±0.038 from E15 to P56. There was statistically significant difference between P56 to E15 and P7.Meanwhile neurons in the spinal cord of rats were found to be abundant of PTEN and S6 expression with immunofluorescence method. Conclusion Our data showed a development-related expression pattern of PTEN/mTOR signaling pathway in the spinal cord of rats. These results may provide a factual basis for the etiology of the decrease of intrinsic regeneration ability of central nervous system(CNS)along with development, and supply some thread for therapy after spinal cord injury. Key words: Phosphatase and tensin homologue deleted on chromosometen; S6; Spinal cord injury; Axonal regeneration

ASSA14-03-43 Novel therapeutic uses of triptolide in reversing isoprenaline-induced cardiac remodelling in Wistar rats

Background Nowadays, chronic heart failure (CHF) is a global health problem in the elderly. Over 650 thousand new cases are diagnosed every year in the United States. Previous studies indicate that immune-inflammatory activation plays an important role in the pathogenesis of cardiac remodelling in CHF patients. PG-490, triptolide (TPL), is a diterpene triepoxide with potent immunosuppressive and anti-inflammatory properties and has been used in China for centuries to treat immune-related disorders. However, the effects of TPL on CHF and cardiac remodelling remain unclear. Objective To determine whether TPL can protect myocardial fibrosis and improve cardiac function in isoprenaline-induced CHF rats. Methods Age matched male Wistar rats were used in this study. CHF and cardiac remodelling model was established by hypodermic injection of isoprenaline for ten days. The rats were randomly divided to receive placebo, TPL (20 or 100 μg/kg/d, i.g.) or captopril for six weeks. Cardiac remodelling markers including ventricular mass, collagen volume fraction, perivascular collagen area, collagenI and hydroxyproline concentration were studied. Echocardiography, Masson staining, immunohistochemistry, western blot and real-time PCR were performed. The protein and mRNA expression of PTEN (phosphatase and tensin homolog deleted on chromosome ten) were analysed. Results Compared to the untreated CHF rats, the cardiac remodelling markers, LVEDD (8.40 ± 0.52 mm vs 6.11 ± 0.58 mm) and LVESD (6.44 ± 0.93 mm vs 4.49 ± 0.38 mm) were significantly lower in TPL-treated (100 μg/kg/d) rats (p Figure 1 ) and mRNA (0.22 ± 0.06 vs 0.52 ± 0.01) were higher in TPL-treated rats (p in vitro . Conclusions Our findings indicate that TPL could alleviate myocardial remodelling and improve cardiac function in rats by upregulating PTEN signalling pathway. TPL may be a potential therapeutic drug for CHF.

ASSA14-03-02 Protective Effects of Paeoniflorin against Myocardial Fibrosis in Isoprenaline-induced Chronic Heart Failure Rats

<h3>Background</h3> The life time risk of developing heart failure is 20% for Americans over 40 years old. Chronic heart failure (CHF) is becoming a global health problem in the elderly. Previous studies indicate that immune-inflammatory activation plays an important role in the pathogenesis of myocardial fibrosis in CHF. Paeoniflorin (PF) is one of the main effective components of peony, which has been widely used in traditional Chinese medicine for its potent immunomodulatory and anti-inflammatory properties. However, the effects of PF on myocardial fibrosis remain unclear. <h3>Objective</h3> To exam whether PF protects myocardial fibrosis and improves cardiac function in isoprenaline-induced CHF rats. <h3>Methods</h3> Age matched male Wistar rats were used in this study. CHF and cardiac remodelling model was established by hypodermic injection of isoprenaline for ten days. After the CHF model establishment, the rats were randomly divided to receive placebo, PF (50 or 100 mg/kg/d, i.g.) or captopril for six weeks. Cardiac remodelling markers including ventricular mass, collagen volume fraction, perivascular collagen area and hydroxyproline concentration were studied. Echocardiography, Masson staining, immunohistochemistry, western blot and real-time PCR were performed. The protein and mRNA expression of PTEN (phosphatase and tensin homolog deleted on chromosome ten) were analysed. <h3>Results</h3> Compared to the untreated CHF rats, the perivascular collagen area, collagen volume fraction (30.97 ± 4.22% vs 13.75 ± 3.77%), hydroxyproline concentration (4.92 ± 0.78 μg/mg wet weight vs 2.08 ± 0.43 μg/mg wet weight), LVIDd (8.40 ± 0.52 mm vs 6.11 ± 0.58 mm) and LVIDs (6.44 ± 0.93 mm vs 4.49 ± 0.38 mm) were significantly lower in PF-treated (100 mg/kg/d) rats (p &lt; 0.05, n = 8). PF treatment also improved LVEF levels (46.44 ± 3.50% vs 60.89 ± 3.10%). Besides, the expressions of PTEN protein (0.11 ± 0.05 vs 0.63 ± 0.19, p &lt; 0.05) and mRNA (0.22 ± 0.06 vs 0.39 ± 0.02, p &lt; 0.01) were higher in PF-treated rats (n = 3). No damage of renal or liver function was detected in these rats (p &gt; 0.05). Furthermore, we also found that PF can accelerate myocardial fibroblast cell apoptosis and upregulate PTEN signalling <i><i>in vitro</i></i>. <h3>Conclusions</h3> Our findings indicate that PF could alleviate myocardial fibrosis and improve cardiac function in isoprenaline-induced CHF rats by upregulating PTEN signalling pathway. PF may be a potential therapeutic drug for CHF.

Sequestosome 1/p62 facilitates HER2-induced mammary tumorigenesis through multiple signaling pathways.

Previous studies have shown that increased levels of the adaptor protein Sequestosome 1/p62 are observed in human breast cancers and significantly correlate with HER2 overexpression. However, the role of p62 in the pathophysiology of HER2-induced mammary tumorigenesis has not yet been investigated. In this study, we report that p62 facilitates HER2-mediated cell survival in both two-dimensional and three-dimensional cell culture and that HER2-induced cellular transformation requires p62, as well as NRF2, which is known to become stabilized by its release from Kelch-like ECH-associated protein 1 (KEAP1) via p62-KEAP1 interaction. In agreement with these results, genetic ablation of p62 delays HER2-induced mammary tumorigenesis in tumor cell allografts in nude mice, and in MMTV-Neu transgenic mice. We also report that ablation of p62 impairs AKT and β-catenin activation in association with PTEN (phosphatase and tensin homolog deleted on chromosome ten) accumulation, both in vitro and in vivo. Further in vivo studies suggest that loss of p62 also impairs NF-κB and NRF2 activation. Collectively, our results provide compelling evidence that p62 contributes to HER2-induced mammary tumorigenesis through multiple signaling pathways, including the PTEN/phosphoinositide-3-kinase/AKT axis, WNT/β-catenin signaling, the NF-κB pathway and the NRF2-KEAP1 axis, and offer novel insights into the potential role of p62 in the regulation of the tumor suppressor PTEN.

MiR-205 inhibits cell apoptosis by targeting phosphatase and tensin homolog deleted on chromosome ten in endometrial cancer Ishikawa cells.

BackgroundMicroRNAs (miRNAs) are frequently dysregulated in human cancers and can act as either potent oncogenes or tumor suppressor genes. In the present study, we intend to prove that the gene PTEN (phosphatase and tensin homolog deleted on chromosome ten) is a target gene of miR-205 and to investigate the suppressive effects on PTEN transcriptional activity by enhancing miR-205 expression in endometrial cancer Ishikawa cells.MethodsUsing Ishikawa cells as model systems, we up-regulated miR-205 expression by transient transfection with miR-205 mimics. A luciferase reporter assay, qRT-PCR and western blotting assays were used to verify whether PTEN is a direct target of miR-205. Meanwhile, the modulatory role of miR-205 in the AKT (protein kinase B) pathway was evaluated by determining the AKT phosphorylation. As a biological counterpart, we investigated cell apoptosis using flow cytometry.ResultsOur data indicate that miR-205 down-regulates the expression of PTEN through direct interaction with the putative binding site in the 3′-untranslated region (3′-UTR) of PTEN. Moreover, we documented the functional interactions of miR-205 and PTEN, which have a downstream effect on the regulation of the AKT pathway, explaining, at least in part, the inhibitory effects on Ishikawa cell apoptosis of enhancing miR-205 expression.ConclusionsFor the first time, we demonstrate that the expression of PTEN is directly regulated by miR-205 in endometrial cancer cells and leads the inhibition of cellular apoptosis. This relationship could be targeted for new therapeutic strategies for endometrial cancer.

MicroRNA-21 Regulates hTERT via PTEN in Hypertrophic Scar Fibroblasts

BackgroundAs an important oncogenic miRNA, microRNA-21 (miR-21) is associated with various malignant diseases. However, the precise biological function of miR-21 and its molecular mechanism in hypertrophic scar fibroblast cells has not been fully elucidated.Methodology/Principal FindingsQuantitative Real-Time PCR (qRT-PCR) analysis revealed significant upregulation of miR-21 in hypertrophic scar fibroblast cells compared with that in normal skin fibroblast cells. The effects of miR-21 were then assessed in MTT and apoptosis assays through in vitro transfection with a miR-21 mimic or inhibitor. Next, PTEN (phosphatase and tensin homologue deleted on chromosome ten) was identified as a target gene of miR-21 in hypertrophic scar fibroblast cells. Furthermore, Western-blot and qRT-PCR analyses revealed that miR-21 increased the expression of human telomerase reverse transcriptase (hTERT) via the PTEN/PI3K/AKT pathway. Introduction of PTEN cDNA led to a remarkable depletion of hTERT and PI3K/AKT at the protein level as well as inhibition of miR-21-induced proliferation. In addition, Western-blot and qRT-PCR analyses confirmed that hTERT was the downstream target of PTEN. Finally, miR-21 and PTEN RNA expression levels in hypertrophic scar tissue samples were examined. Immunohistochemistry assays revealed an inverse correlation between PTEN and hTERT levels in high miR-21 RNA expressing-hypertrophic scar tissues.Conclusions/SignificanceThese data indicate that miR-21 regulates hTERT expression via the PTEN/PI3K/AKT signaling pathway by directly targeting PTEN, therefore controlling hypertrophic scar fibroblast cell growth. MiR-21 may be a potential novel molecular target for the treatment of hypertrophic scarring.

Turning Off AKT: PHLPP as a Drug Target

Precise control of the balance between protein phosphorylation, catalyzed by protein kinases, and protein dephosphorylation, catalyzed by protein phosphatases, is essential for cellular homeostasis. Dysregulation of this balance leads to pathophysiological states, driving diseases such as cancer, heart disease, and diabetes. Aberrant phosphorylation of components of the pathways that control cell growth and cell survival are particularly prevalent in cancer. One of the most studied tumor suppressors in these pathways is the lipid phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome ten), which dephosphorylates the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3), thus preventing activation of the oncogenic kinase AKT (v-akt murine thymoma viral oncogene homolog). In 2005, the discovery of a family of protein phosphatases whose members directly dephosphorylate and inactivate AKT introduced a new negative regulator of the phosphoinositide 3-kinase (PI3K) oncogenic pathway. Pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) isozymes comprise a novel tumor suppressor family whose two members, PHLPP1 and PHLPP2, are deleted as frequently as PTEN in cancers such as those of the prostate. PHLPP is thus a novel therapeutic target to suppress oncogenic pathways and is a potential candidate biomarker to stratify patients for the appropriate targeted therapeutics. This review discusses the role of PHLPP in terminating AKT signaling and how pharmacological intervention would impact this pathway.

Hypertrophied Myocardium Is Refractory to Sevoflurane-Induced Protection With Alteration of Reperfusion Injury Salvage Kinase/Glycogen Synthase Kinase 3β Signals

Recent studies have demonstrated that volatile anesthetic postconditioning confers myocardial protection against ischemia-reperfusion injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been shown to be impaired by ventricular hypertrophy, we investigate whether anesthetic-induced cardiac protection was maintained in rat hearts with ventricular hypertrophy. Transverse aortic constriction operation was performed on male Sprague-Dawley rats to induce left ventricular (LV) hypertrophy, then sham-operated or hypertrophied rat hearts were subjected to 40 min of global ischemia and 2 h of reperfusion. The isolated hearts received 3% sevoflurane for 10 min or six cycles of 10-s ischemia/10-s reperfusion after reperfusion. The hemodynamics, infarct size, PTEN (phosphatase and tensin homolog deleted on chromosome ten), phosphorylated Akt, phosphorylated extracellular regulated protein kinase (ERK) 1/2, and phosphorylated glycogen synthase kinase 3β (GSK3β) were determined. We found the myocardial expression of PTEN, phosphorylated Akt, ERK1/2, and phosphorylated GSK3β did not significantly differ between sham-operated and transverse aortic constriction-control groups. Both sevoflurane and ischemic postconditioning significantly improved LV hemodynamics, reduced infarct size, and increased the phosphorylation of Akt, ERK1/2, and their downstream target of GSK3β in the sham-operated rat hearts. In contrast, neither sevoflurane nor ischemic postconditioning improved LV hemodynamic, reduced infarct size, and increased the phosphorylated Akt, ERK1/2, and GSK3β in hypertrophied myocardium. All the results above indicate that ventricular hypertrophy abrogated sevoflurane-induced cardioprotection against ischemia-reperfusion injury by alteration of RISK/GSK3β signals.

Critical Role of Increased PTEN Nuclear Translocation in Excitotoxic and Ischemic Neuronal Injuries

Stroke is the leading cause of disability in developed countries. However, no treatment is available beyond 3 h post-ictus. Here, we report that nuclear translocation of PTEN (phosphatase and tensin homolog deleted on chromosome TEN) is a delayed step causatively leading to excitotoxic (in vitro) and ischemic (in vivo) neuronal injuries. We found that excitotoxic stimulation of N-methyl-d-aspartate (NMDA) resulted in PTEN nuclear translocation in cultured neurons, a process requiring mono-ubiquitination at the lysine 13 residue (K13), as the translocation was prevented by mutation of K13 or a short interfering peptide (Tat-K13) that flanks the K13 residue. More importantly, using a rat model of focal ischemia, we demonstrated that systemic application of Tat-K13, even 6 h after stroke, not only reduced ischemia-induced PTEN nuclear translocation, but also strongly protected against ischemic brain damage. Our study suggests that inhibition of PTEN nuclear translocation may represent a novel after stroke therapy.

Abstract 4014: The therapeutic potential of ICG-001 and β-catenin specific RNAi in liver cancer development.

Abstract Liver cancer is one of the most common malignant tumors. It is reported to be the third most lethal malignancy worldwide. Recent studies including our own identified CD133+ cell population as the tumor initiating cells for liver cancer. Tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten) is aberrantly expressed in liver cancers. Liver specific Pten (Pm) null mice develop liver cancer following an extensive phase of chronic lipid accumulation and demonstrate escalating levels of hepatic injury markers from 6-12M, prior to hepatic progenitor cell proliferation. Concomitantly, expression of mRNA levels for Wnt ligands and receptors also increased progressively. Wnt/β-Catenin signaling pathway has various roles in regulating embryonic development and tumorigenesis. In Pten null liver progenitor cell line and tissues; we observed high levels of βcatenin, a downstream target component of the Wnt signaling pathway compared to control cells. In this study, we investigate the role of the Wnt/β-Catenin pathway in the activation of hepatic progenitor cells in the Pten deletion liver cancer model using two methods: a novel Wnt/β-Catenin inhibitor ICG-001 and β-Catenin specific RNAi. ICG-001 is a small molecule, which specifically inhibits β-Catenin/CBP interaction. Coactivator CBP/β-Catenin/T cell factor (TCF) mediated transcription has been reported as critical for stem cell/progenitor cell proliferation. ICG-001 significantly attenuates the proliferation of hepatic cancer stem cells in vitro and in vivo. Our preliminary results demonstrate inhibition of Wnt/β-Catenin pathway using sh-β-Catenin in vitro can effectively inhibit the proliferation of CD133+ hepatic cancer stem cells. These observations indicate that Wnt/β- Catenin pathway likely mediates proliferation of hepatic cancer stem cell. Targeting this pathway using ICG-001 and/or β-Catenin directed shRNA holds promise for the treatment and eradication of liver cancer. Citation Format: Anketse D. Kassa, Vivian G. Medina, Ni Zeng, Lina He, Cu Nguyen, Goar Smbatyan, Megan Rieger, Michael Kahn, Bangyan L. Stiles. The therapeutic potential of ICG-001 and β-catenin specific RNAi in liver cancer development. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4014. doi:10.1158/1538-7445.AM2013-4014

The roles of Notch and phosphatidylinositol 3 kinase/protein kinase B signaling pathways in the pathogenesis of bladder cancer

Objective To investigate the roles of Notch and phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathways in the pathogenesis of bladder cancer.Methods The expression levels of key members of Notch and PI3K/Akt signaling pathways in 36 cases of bladder cancer tissues and paired paritumour tissues were deteccted by using real-time quantitative polymerase chain reaction (PCR)and Western blotting,respectively.After pharmacal inhibition of Notch or PI3K/Akt signaling pathway,proliferation abilities of bladder cancer cells 5637 and T24 were measured by using MTS.The expression of phosphorylated Akt was tested when Notch signaling pathway was inhibited by N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butylester (DAPT).Experiments were repeated three times.Results Both Notch and PI3K/Akt signaling pathways were activated in bladder cancer.As Compared with paired paritumour tissues,Notch1 was up-regulated (2.60 ±0.37) folds in bladder cancer tissue,Hes1 was upregulated (1.80 ± 0.24) folds,and phosphorylated Akt was up-regulated (all P ＜ 0.05).After pharmacal inhibition of Notch or PI3K/Akt signaling pathway,proliferation abilities of 5637 and T24 cells were reduced.Furthermore,when Notch signaling pathway was inhibited by DAPT,Hes1 was down-regulated (3.60 ± 0.53) folds,phosphatase and tensin homologue deleted on chromosometen (PTEN) was downregulated (2.30 ± 0.24) folds,and.phosphorylated Akt was also down-regulated significantly (all P ＜0.05).Conclusion Notch pathway signaling promotes proliferation of bladder cancer cells by activating PI3K/Akt signaling pathway. Key words: Bladder neoplasms; Cell proliferation; Notch signaling pathway; Phosphatidylinositol 3 kinase

The Safe Use of a PTEN Inhibitor for the Activation of Dormant Mouse Primordial Follicles and Generation of Fertilizable Eggs

BackgroundPrimordial ovarian follicles, which are often present in the ovaries of premature ovarian failure (POF) patients or are cryopreserved from the ovaries of young cancer patients who are undergoing gonadotoxic anticancer therapies, cannot be used to generate mature oocytes for in vitro fertilization (IVF). There has been very little success in triggering growth of primordial follicles to obtain fertilizable oocytes due to the poor understanding of the biology of primordial follicle activation.Methodology/Principal FindingsWe have recently reported that PTEN (phosphatase and tensin homolog deleted on chromosome ten) prevents primordial follicle activation in mice, and deletion of Pten from the oocytes of primordial follicles leads to follicular activation. Consequently, the PTEN inhibitor has been successfully used in vitro to activate primordial follicles in both mouse and human ovaries. These results suggest that PTEN inhibitors could be used in ovarian culture medium to trigger the activation of primordial follicle. To study the safety and efficacy of the use of such inhibitors, we activated primordial follicles from neonatal mouse ovaries by transient treatment with a PTEN inhibitor bpV(HOpic). These ovaries were then transplanted under the kidney capsules of recipient mice to generate mature oocytes. The mature oocytes were fertilized in vitro and progeny mice were obtained after embryo transfer.Results and ConclusionsLong-term monitoring up to the second generation of progeny mice showed that the mice were reproductively active and were free from any overt signs or symptoms of chronic illnesses. Our results indicate that the use of PTEN inhibitors could be a safe and effective way of generating mature human oocytes for use in novel IVF techniques.

Disruption of E3 ligase NEDD4 in peripheral neurons interrupts axon outgrowth: Linkage to PTEN

Exploiting molecules and pathways important in developmental axon behaviour may offer new insights into regenerative behaviour of adult peripheral neurons after injury. In previous work, we have provided evidence that inhibition or knockdown of PTEN (phosphatase and tensin homolog deleted on chromosome ten) dramatically increases adult peripheral axon outgrowth, especially in preconditioned neurons (Christie et al., 2010). PTEN appears to operate as an endogenous brake to regeneration. Recent reports from Drinjakovic et al. (2010) have highlighted a role for the ubiquitin proteasome system (UPS) during neurite outgrowth in developing Xenopus retinal ganglion cells. Specifically, disruption of the UPS E3 ligase Nedd4 (neural precursor cell-expressed developmentally down-regulated protein 4) inhibited neurite branching through up-regulation of PTEN. We explored the potential role of Nedd4 in the peripheral neurons of adult rat dorsal root ganglia (DRG), particularly its impact on regenerative behaviour. Global inhibition of the UPS in vitro was associated with a severe decrease in neurite branching, both in preconditioned (injured) and control DRG sensory neurons. These involved neurons however maintained or qualitatively increased their PTEN expression, suggesting ongoing PTEN activity during UPS inhibition. Considering component's of UPS more specifically, Nedd4 co-localized with PTEN within sensory neurons in vivo and in vitro. Nedd4 also co-localized with PTEN and NF200 labelled regenerating axons at the injury site in the periphery following a 3 day sciatic nerve cut. A significant role for this unique co-expression was observed with fluorescently tagged siRNA inhibition of Nedd4, which decreased neurite outgrowth, an impact associated with greater expression of PTEN and that was completely reversed with application of a PTEN inhibitor. Overall, our results suggest an important role for Nedd4 regulation of PTEN in the response of peripheral neurons to injury. By degrading PTEN among other potential actions, Nedd4 supports axonal outgrowth whereas its inhibition facilitates PTEN inhibition of regeneration.