Prophylactic treatment with BX795 blocks activation of AKT and its downstream targets to protect vaginal keratinocytes and vaginal epithelium from HSV-2 infection.

Genital herpes infections in humans are usually caused by herpes simplex virus type-2 (HSV-2), which result in recurrent lesions in the anogenital region. Past studies have shown that a viral protein translation inhibitor, BX795 is capable of mitigating HSV-2 infection both in vitro and in vivo when dosed therapeutically. However, any preventative benefits of this compound against HSV-2 infection remain poorly understood. In this study, we show that BX795 when added prophylactically to human vaginal keratinocytes generates strong preventative effects against a future HSV-2 infection. As a possible mechanism for this action, we found that BX795 efficiently reduces phosphorylation of AKT and its downstream targets p70S6K and 4EBP1. Our in-silico protein docking studies support our immunoblotting results and provide further credence to the proposed mechanism. Using a murine model of vaginal infection, we show that prior treatment with BX795 is also protective in vivo and leads to lower viral replication in the vaginal tissue.

Olive polyphenols attenuate TNF-α-stimulated M-CSF and IL-6 synthesis in osteoblasts: Suppression of Akt and p44/p42 MAP kinase signaling pathways.

BACKGROUND: Olive oil polyphenols, which possess cytoprotective activities like anti-oxidant and anti-inflammatory effects, could modulate osteoblast functions. The aim of this study is to elucidate the effects and the underlying mechanisms of hydroxytyrosol and oleuropein on the tumor necrosis factor-α (TNF-α)-induced macrophage colony-stimulating factor (M-CSF) and interleukin-6 (IL-6) synthesis in osteoblasts. METHODS: Osteoblast-like MC3T3-E1 cells were pretreated with hydroxytyrosol, oleuropein, deguelin, PD98059 or wedelolactone, and then stimulated by TNF-α. The levels of M-CSF and IL-6 in the conditioned medium were determined with ELISA. The mRNA expression levels of M-CSF or IL-6 were determined with real-time RT-PCR. The phosphorylation levels of Akt, p44/p42 mitogen-activated protein (MAP) kinase or NF-κB in the cell lysates were determined with Western blot analysis. RESULTS: Hydroxytyrosol and oleuropein attenuated the TNF-α-stimulated M-CSF release. Deguelin, an inhibitor of Akt, significantly suppressed the TNF-α-stimulated M-CSF release, which failed to be affected by the MEK1/2 inhibitor PD98059 or the IκB inhibitor wedelolactone. Hydroxytyrosol and oleuropein suppressed the TNF-α-induced phosphorylation of Akt and p44/p42 MAP kinase. Hydroxytyrosol and oleuropein attenuated the TNF-α-stimulated IL-6 release. Hydroxytyrosol suppressed the TNF-α-induced mRNA expressions of M-CSF and IL-6. Hydroxytyrosol or oleuropein failed to affect the cell viability. CONCLUSION: Our present findings strongly suggest that olive oil polyphenols hydroxytyrosol and oleuropein down-regulates TNF-α signaling at the points upstream of Akt and p44/p42 MAP kinase in osteoblasts, leading to the attenuation of M-CSF and IL-6 synthesis.

Phytochemicals in cancer and their effect on the PI3K/AKT-mediated cellular signalling.

Protein kinases belong to the largest family of enzymes controlling every aspect of cellular activity including gene expression, cell division, differentiation and metabolism. They are part of major intracellular signalling pathways. Hence, it is not surprising that they are involved in the development of major diseases such as cardiovascular disorders, diabetes, dementia and, most importantly, cancer when they undergo mutations, modifications and unbalanced expression. This review will explore the possibility to draw a connection between the application of natural phytochemicals and the treatment of cancer. We have chosen to focus on the PI3K/AKT cellular signalling pathway which has been shown to be a major target by natural compounds in cell cultures and animal models.

Central and peripheral emetic loci contribute to vomiting evoked by the Akt inhibitor MK-2206 in the least shrew model of emesis.

Akt (protein kinase B) signaling is frequently activated in diverse cancers. Akt inhibitors such as perifosine and MK-2206 have been evaluated as potential cancer chemotherapeutics. Although both drugs are generally well tolerated, among their most common side-effects vomiting is a major concern. Here we investigated whether these Akt inhibitors evoke emesis in the least shrew model of vomiting. Indeed, both perifosine and MK-2206 induced vomiting with maximal efficacies of 90% at 50 mg/kg (i.p.) and 100% at 10 mg/kg (i.p.), respectively. MK-2206 (10 mg/kg, i.p.) increased c-Fos immunoreactivity both centrally in the shrew brainstem dorsal vagal complex (DVC) emetic nuclei, and peripherally in the jejunum. MK-2206 also evoked phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in both the DVC emetic nuclei and the enteric nervous system in the jejunum. The ERK1/2 inhibitor U0126 suppressed MK-2206-induced emesis dose-dependently. We then evaluated the suppressive efficacy of diverse antiemetics against MK-2206-evoked vomiting including antagonists/inhibitors of the: L-type Ca2+ channel (nifedipine at 2.5 mg/kg, subcutaneously (s.c.)); glycogen synthase kinase 3 (GSK-3) (AR-A014418 at 10 mg/kg and SB216763 at 0.25 mg/kg, i.p.); 5-hydroxytryptamine 5-HT3 receptor (palonosetron at 0.5 mg/kg, s.c.); substance P neurokinin NK1 receptor (netupitant at 10 mg/kg, i.p.) and dopamine D2/3 receptor (sulpride at 8 mg/kg, s.c.). All tested antagonists/blockers attenuated emetic parameters to varying degrees. In sum, this is the first study to demonstrate how pharmacological inhibition of Akt evokes vomiting via both central and peripheral mechanisms, a process which involves multiple emetic receptors.

KNSTRN promotes tumorigenesis and gemcitabine resistance by activating AKT in bladder cancer.

KNSTRN is a component of the mitotic spindle, which was rarely investigated in tumorigenesis. AKT plays an essential role in tumorigenesis by modulating the phosphorylation of various substrates. The activation of AKT is regulated by PTEN and PIP3. Here, we prove KNSTRN is positively correlated with malignancy of bladder cancer and KNSTRN activates AKT phosphorylation at Thr308 and Ser473. More importantly, our study reveals that both KNSTRN and PTEN interact with PH domain of AKT at cell membrane. The amount of KNSTRN interacted with AKT is negatively related to PTEN. Furthermore, PIP3 pull-down assay proves that KNSTRN promoted AKT movement to PIP3. These data suggest KNSTRN may activate AKT phosphorylation by promoting AKT movement to PIP3 and alleviating PTEN suppression. Based on the activation of AKT phosphorylation, our study demonstrates that KNSTRN promotes bladder cancer metastasis and gemcitabine resistance in vitro and in vivo. Meanwhile, the effect of KNSTRN on tumorigenesis and gemcitabine resistance could be restored by AKT specific inhibitor MK2206 or AKT overexpression. In conclusion, we identify an oncogene KNSTRN that promotes tumorigenesis and gemcitabine resistance by activating AKT phosphorylation and may serve as a therapeutic target in bladder cancer.

Synthesis, In Silico and In Vitro Assessment of New Quinazolinones as Anticancer Agents via Potential AKT Inhibition.

A series of novel quinazolinone derivatives (2-13) was synthesized and examined for their cytotoxicity to HepG2, MCF-7, and Caco-2 in an MTT assay. Among these derivatives, compounds 4 and 9 exhibited significant cytotoxic activity against Caco-2, HepG2, and MCF-7 cancer cells. Compound 4 had more significant inhibitory effects than compound 9 on Caco-2, HepG2, and MCF-7 cell lines, with IC50 values of 23.31 ± 0.09, 53.29 ± 0.25, and 72.22 ± 0.14µM, respectively. The AKT pathway is one of human cancer's most often deregulated signals. AKT is also overexpressed in human cancers such as glioma, lung, breast, ovarian, gastric, and pancreas. A molecular docking study was performed to analyze the inhibitory action of newly synthetic quinazolinone derivatives against Homo sapiens AKT1 protein. Molecular docking simulations were found to be in accordance with in vitro studies, and hence supported the biological activity. The results suggested that compounds 4 and 9 could be used as drug candidates for cancer therapy via its potential inhibition of AKT1 as described by docking study.

Geldanamycin-Derived HSP90 Inhibitors Are Synthetic Lethal with NRF2.

Activating mutations in KEAP1-NRF2 are frequently found in tumors of the lung, esophagus, and liver, where they are associated with aggressive growth, resistance to cancer therapies, and low overall survival. Despite the fact that NRF2 is a validated driver of tumorigenesis and chemotherapeutic resistance, there are currently no approved drugs which can inhibit its activity. Therefore, there is an urgent clinical need to identify NRF2-selective cancer therapies. To this end, we developed a novel synthetic lethal assay, based on fluorescently labeled isogenic wild-type and Keap1 knockout cell lines, in order to screen for compounds which selectively kill cells in an NRF2-dependent manner. Through this approach, we identified three compounds based on the geldanamycin scaffold which display synthetic lethality with NRF2. Mechanistically, we show that products of NRF2 target genes metabolize the quinone-containing geldanamycin compounds into more potent HSP90 inhibitors, which enhances their cytotoxicity while simultaneously restricting the synthetic lethal effect to cells with aberrant NRF2 activity. As all three of the geldanamycin-derived compounds have been used in clinical trials, they represent ideal candidates for drug repositioning to target the currently untreatable NRF2 activity in cancer.

Long noncoding RNA PTCSC1 drives esophageal squamous cell carcinoma progression through activating Akt signaling.

Long noncoding RNAs (lncRNAs) have critical roles in various malignancies. However, the specific expression and roles of lncRNA PTCSC1 in esophageal squamous cell carcinoma (ESCC) are still unknown. Here, we identified that lncRNA PTCSC1 was elevated in ESCC tissues and cell lines compared with adjacent noncancerous tissues and normal esophageal epithelial cell line, respectively. Enhanced expression of PTCSC1 facilitated ESCC cells proliferation and migration in vitro and ESCC xenograft growth in vivo. Conversely, deficiency of PTCSC1 suppressed ESCC cells proliferation and migration in vitro and ESCC tumor growth in vivo. Furthermore, PTCSC1 was found to activate Akt signaling in ESCC cells. Blocking Akt signaling with MK-2206 abolished the pro-proliferative and pro-migratory roles of PTCSC1. In summary, our findings demonstrated PTCSC1 as an oncogenic lncRNA in ESCC via activating Akt signaling and suggested that targeting PTCSC1 represents a promising therapeutic strategy against ESCC.

Differential effects of the Akt pathway on the internalization of Klebsiella by lung epithelium and macrophages.

Host response to lung infection includes coordinated efforts of multiple cell types, including the lung epithelium and macrophages. Importantly, both the lung epithelium and macrophages can internalize and clear invading pathogens. However, the mechanisms and their ability to internalize or phagocytose differ. Akt is a key cellular pathway that controls cell proliferation and survival, in addition to its role in host defense. The role of the Akt pathway was assessed using pharmacological Akt modulators in lung epithelial (A549) and macrophage (RAW 264.7) cell lines during Klebsiella bacterial infection. Our data show that the inhibition of the Akt pathway using specific Akt inhibitor MK2206 increased the phagocytic ability of lung epithelial cells but not of macrophages. In contrast, the activation of Akt using specific activator SC-79 decreased the phagocytic ability of epithelial cells, while it increased the phagocytic ability of macrophages. The altered phagocytic ability in both cell types using Akt modulators was not due to changes in bacterial adhesion to the host cell. The clinical usefulness of these Akt modulators may vary based on the type of infection and on the relative contribution of epithelial cells and macrophages in clearing the particular bacterial infection. The Akt pathway has differential roles in the internalization of Klebsiella bacteria by respiratory epithelial cells and immune cells.

Radiation-induced Adaptive Response: New Potential for Cancer Treatment.

Radiotherapy is highly effective due to its ability to physically focus the treatment to target the tumor while sparing normal tissue and its ability to be combined with systemic therapy. This systemic therapy can be utilized before radiotherapy as an adjuvant or induction treatment, during radiotherapy as a radiation "sensitizer," or following radiotherapy as a part of combined modality therapy. As part of a unique concept of using radiation as "focused biology," we investigated how tumors and normal tissues adapt to clinically relevant multifraction (MF) and single-dose (SD) radiation to observe whether the adaptations can induce susceptibility to cell killing by available drugs or by immune enhancement. We identified an adaptation occurring after MF (3 × 2 Gy) that induced cell killing when AKT-mTOR inhibitors were delivered following cessation of radiotherapy. In addition, we identified inducible changes in integrin expression 2 months following cessation of radiotherapy that differ between MF (1 Gy × 10) and SD (10 Gy) that remain targetable compared with preradiotherapy. Adaptation is reflected across different "omics" studies, and thus the range of possible molecular targets is not only broad but also time, dose, and schedule dependent. While much remains to be studied about the radiation adaptive response, radiation should be characterized by its molecular perturbations in addition to physical dose. Consideration of the adaptive effects should result in the design of a tailored radiotherapy treatment plan that accounts for specific molecular changes to be targeted as part of precision multimodality cancer treatment.

Lactic acidosis induces resistance to the pan-Akt inhibitor uprosertib in colon cancer cells.

BACKGROUND: Akt signalling regulates glycolysis and drives the Warburg effect in cancer, thus decreased glucose utilisation is a pharmacodynamic marker of Akt inhibition. However, cancer cells can utilise alternative nutrients to glucose for energy such as lactate, which is often elevated in tumours together with increased acidity. We therefore hypothesised that lactic acidosis may confer resistance to Akt inhibition. METHODS: The effect of the pan-Akt inhibitor uprosertib (GSK2141795), on HCT116 and LS174T colon cancer cells was evaluated in the presence and absence of lactic acid in vitro. Expression of downstream Akt signalling proteins was determined using a phosphokinase array and immunoblotting. Metabolism was assessed using 1H nuclear magnetic resonance spectroscopy, stable isotope labelling and gas chromatography-mass spectrometry. RESULTS: Lactic acid-induced resistance to uprosertib was characterised by increased cell survival and reduced apoptosis. Uprosertib treatment reduced Akt signalling and glucose uptake irrespective of lactic acid supplementation. However, incorporation of lactate carbon and enhanced respiration was maintained in the presence of uprosertib and lactic acid. Inhibiting lactate transport or oxidative phosphorylation was sufficient to potentiate apoptosis in the presence of uprosertib. CONCLUSIONS: Lactic acidosis confers resistance to uprosertib, which can be reversed by inhibiting lactate transport or oxidative metabolism.

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

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

A new anti-HER2 antibody that enhances the anti-tumor efficacy of trastuzumab and pertuzumab with a distinct mechanism of action.

The majority of patients with metastatic breast cancer who are treated with the anti-HER2 monoclonal antibody, trastuzumab, generally develop resistance to the drug within a year after initiation of the treatment. Here we describe a new anti-HER2 humanized monoclonal antibody, 19H6-Hu, which binds to HER2 extracellular domain (ECD) with high affinity and inhibits proliferation of multiple HER2-overexpressing cancer cell lines as a single agent or in combination with trastuzumab. 19H6-Hu binds to the domain III in proximity to the domain IV of HER2 ECD, which differs from trastuzumab and pertuzumab. 19H6-Hu in combination with trastuzumab was more effective at blocking phosphorylation of ERK1/2, AKT(S473)and HER2 (Y1248) in HER2-positive cancer cells compared to trastuzumab alone or in combination with pertuzumab. Combination of three antibodies, 19H6-Hu, inetetamab (a trastuzumab analog) and pertuzumab exhibited much stronger inhibition of large NCI-N87 tumor xenografts (>400mm3) than the current standard of care, inetetamab (trastuzumab) plus Docetaxel (DTX), as well as the combination of 19H6-Hu, inetetamab and DTX. Our results highlight the functional variability of HER2 domains and provide a new insight into the design of HER2-targeting agents.

Recombinant OX40 attenuates neuronal apoptosis through OX40-OX40L/PI3K/AKT signaling pathway following subarachnoid hemorrhage in rats.

Subarachnoid hemorrhage (SAH) is the most devastating form of stroke. Reducing neuronal apoptosis is an important countermeasure against early brain injury (EBI) after SAH. Recent evidence indicates that OX40-OX40L coupling is critical for cell survival and proliferation. Current study was performed to detect the role of recombinant OX40 (ReOX40) against neuronal apoptosis after SAH. The endovascular perforation model of SAH was performed on Sprague-Dawley (SD) rats. ReOX40 was injected intracerebroventricularly (i.c.v) 1 h after SAH induction and the following methods were employed: neurological function evaluation, immunofluorescence staining, fluoro-Jade C staining, and western blot. To study the underlying precise molecular mechanism, small interfering ribonucleic acid (siRNA) for OX40L and a specific inhibitor of PI3K, LY294002, were injected i.c.v. into SAH + ReOX40 rats before induction of SAH. When compared with sham rats, the expression of OX40 and OX40L was seen to decrease in the brain at 24 h after SAH induction. Administration of ReOX40 (5 µg/kg) increased expression of the OX40L, reduced the neuronal apoptosis, and improved short and long-term neurological function deficits. Furthermore, ReOx40 heightened activation of OX40L/PI3K/AKT axis, increased the downstream anti-apoptotic protein (Bcl2, Bcl-XL), and depressed the apoptotic protein (cleaved caspase 3, Bax). However, the protective effects of ReOX40 were abolished by the administration of OX40L siRNA and LY294002, respectively. These results demonstrate that ReOX40 attenuates neuronal apoptosis through OX40-OX40L/PI3K/AKT pathway in EBI after SAH.

Blockade of Transient Receptor Potential Vanilloid 4 Enhances Antioxidation after Myocardial Ischemia/Reperfusion.

Antioxidative stress provides a cardioprotective effect during myocardial ischemia/reperfusion (I/R). Previous research has demonstrated that the blockade of transient receptor potential vanilloid 4 (TRPV4) attenuates myocardial I/R injury. However, the underlying mechanism remains unclear. The current study is aimed at investigating the antioxidative activity of TRPV4 inhibition and elucidating the underlying mechanisms in vitro and ex vivo. We found that the inhibiting TRPV4 by the selective TRPV4 blocker HC-067047 or specific TRPV4-siRNA significantly reduces reactive oxygen species (ROS) and methane dicarboxylic aldehyde (MDA) levels in H9C2 cells exposed to hypoxia/reoxygenation (H/R). Meanwhile, the activity of antioxidative enzymes, particularly superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), is enhanced. Furthermore, after H/R, HC-067047 treatment increases the expression of P-Akt and the translocation of nuclear factor E2-related factor 2 (Nrf2) and related antioxidant response element (ARE) mainly including SOD, GSH-Px, and catalase (CAT). LY294002, an Akt inhibitor, suppresses HC-067047 and specific TRPV4-siRNA-induced Nrf2 expression and its nuclear accumulation. Nrf2 siRNA attenuates HC-067047 and specific TRPV4-siRNA-induced ARE expression. In addition, treatment with LY294002 or Nrf2 siRNA significantly attenuates the antioxidant and anti-injury effects of HC-067047 in vitro. Finally, in experiments on isolated rat hearts, we confirmed the antioxidative stress roles of TRPV4 inhibition during myocardial I/R and the application of exogenous H2O2. In conclusion, the inhibition of TRPV4 exerts cardioprotective effects through enhancing antioxidative enzyme activity and expressions via the Akt/Nrf2/ARE pathway.

microRNA-613 exerts anti-angiogenic effect on nasopharyngeal carcinoma cells through inactivating the AKT signaling pathway by down-regulating FN1.

Background: Nasopharyngeal carcinoma (NPC) is a disease highly sensitive to radiotherapy with the unclear etiology. However, the specific effects of microRNA-613 (miR-613) on NPC still remain elusive. Therefore, the present study probes into the underlying mechanism of miR-613 in NPC via AKT signaling pathway by regulating Fibronectin 1 (FN1).Methods: First, microarray analysis was used to screen differentially expressed genes (DEGs) and regulatory miRs associated with NPC. Next, miR-613 and FN1 expression in NPC cells was determined, followed by verification of target relationship between miR-613 and FN1. With NPC cells exposed to miR-613 mimic, si-FN1 and LY294002 (inhibitor of AKT signaling pathway), the regulatory effects of miR-613 on proliferation, apoptosis, invasion, migration and angiogenesis of NPC cells were detected with ratio of B-cell lymphoma 2/Bcl-2-associated X protein (Bcl-2/Bax), Cleaved-caspase3, matrix metallopeptidase 2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), and cell adhesion molecule-1 (CD31) expression measured. Then, tumorigenesis and MVD were determined after Xenograft in nude mice.Results: FN1 modulated by miR-613 was critical for NPC via the AKT signaling pathway. NPC cells exhibited down-regulated miR-613 and up-regulated FN1. Besides, miR-613 was verified to target FN1. Moreover, overexpressed miR-613, silenced FN1 or LY294002 treatment suppressed proliferation, invasion, migration, and angiogenesis in NPC cells, which was indicated by reduced expression of AKT, mTOR, MMP-2, MMP-9, VEGF, and CD31 as well as decreased ratio of Bcl-2/Bax and increased expression of Cleaved-caspase3. Furthermore, cell apoptosis was promoted and tumorigenesis and MVD in nude mice were inhibited with overexpression of miR-613, silenced FN1 or LY294002 treatment.Conclusion: Taken together, miR-613 inhibits angiogenesis in NPC cells through inactivating FN1-dependent AKT signaling pathway.

Design, synthesis, in silico pharmacokinetics prediction and biological evaluation of 1,4-dihydroindeno[1,2-c]pyrazole chalcone as EGFR /Akt pathway inhibitors.

In an attempt to develop potent and selective anticancer agents, a series of 15 conjugates of 1,4-dihydroindeno[1,2-c]pyrazole chalcone (12a-o) were designed, synthesized and evaluated for their antiproliferative activity against MCF7, A549, MDA-MB-231, HCT116 and SKBR3 human cancer cell lines. Among them, 12h, 12l and 12m showed IC50 values: 3.82, 5.33 and 4.21 µM, respectively, on A549 cell with respect to the positive control, Erlotinib (IC50 value: 10.26 µM). Detailed biological assays showed accumulation of mitotic cells in G2/M phase. In addition, Western blot analysis and immunofluorescence study revealed inhibition of EGFR and Akt pathways. In silico computational studies were also carried out to predict the binding modes and pharmacokinetic parameters of these conjugates.

The effects of kinase modulation on in vitro maturation according to different cumulus-oocyte complex morphologies.

Successful production of transgenic pigs requires oocytes with a high developmental competence. However, cumulus-oocyte complexes (COCs) obtained from antral follicles have a heterogeneous morphology. COCs can be classified into one of two classes: class I, with five or more layers of cumulus cells; and class II, with one or two layers of cumulus cells. Activator [e.g., epidermal growth factor (EGF)] or inhibitors (e.g., wortmannin and U0126) are added to modulate kinases in oocytes during meiosis. In the present study, we investigated the effects of kinase modulation on nuclear and cytoplasmic maturation in COCs. Class I COCs showed a significantly higher developmental competence than class II COCs. Moreover, the expression of two kinases, AKT and ERK, differed between class I and class II COCs during in vitro maturation (IVM). Initially, inhibition of the PI3K/AKT signaling pathway in class I COCs during early IVM (0-22 h) decreased developmental parameters, such as blastocyst formation rate, blastomere number, and cell survival. Conversely, EGF-mediated AKT activation in class II COCs enhanced developmental capacity. Regarding the MAPK signaling pathway, inhibition of ERK by U0126 in class II COCs during early IVM impaired developmental competence. However, transient treatment with U0126 in class II COCs increased oocyte maturation and AKT activity, improving embryonic development. Additionally, western blotting showed that inhibition of ERK activity negatively regulated the AKT signaling pathway, indicative of a relationship between AKT and MAPK signaling in the process underlying meiotic progression in pigs. These findings may help increase the developmental competence and utilization rate of pig COCs with regard to the production of transgenic pigs and improve our understanding of kinase-associated meiosis events.

Anticancer Activity of Metformin, an Antidiabetic Drug, Against Ovarian Cancer Cells Involves Inhibition of Cysteine-Rich 61 (Cyr61)/Akt/Mammalian Target of Rapamycin (mTOR) Signaling Pathway.

BACKGROUND Ovarian cancer is considered one of the lethal cancers responsible for high mortality and morbidity across the world. The prognosis and the survival rate of ovarian cancer is far from decent. Cysteine-rich 61 (Cyr61) also known as CCN1, is a member of CCN-family of growth factors, reported to be significantly overexpressed in several malignancies which include, but are not limited to, ovarian cancer. Recent studies have revealed that women with type 2 diabetes mellitus have an elevated risk of ovarian cancer. Hence, administration of an antidiabetic drug with anticancer effects such as metformin may act as an effective therapeutic regime against ovarian cancer. MATERIAL AND METHODS Cell viability and apoptosis were examined by MTT and Annexin V/PI double staining respectively. Cell migration was determined by Boyden Chamber assay. Transient knockdown of Cyr61 in ovarian cancer cells was achieved by transecting the cells with siRNA for Cyr61using Lipofectamine 2000. RESULTS Our results indicated that treatment of ovarian cancer cells with metformin caused significant downregulation of Cyr61 protein expression levels ultimately favoring apoptosis. Transient knockdown of Cyr61 resulted in the inhibition of cell proliferation and migration. This was also associated with the concomitant downregulation of pAkt and pmTOR confirming the role of Cyr61 as an upstream modulator of Akt signaling. Conversely the extracellular supplementation of recombinant Cyr61 attenuates the cytotoxic properties of metformin in ovarian cancer cells. CONCLUSIONS Taken together, we concluded that metformin exhibits anticancer effects and Cyr61 acts as a direct target for metformin in ovarian cancer cells.

PIKfyve inhibitor cytotoxicity requires AKT suppression and excessive cytoplasmic vacuolation.

PIKfyve phosphoinositide kinase produces PtdIns(3,5)P2 and PtdIns5P and governs a myriad of cellular processes including cytoskeleton rearrangements and cell proliferation. The latter entails rigorous investigation since the cytotoxicity of PIKfyve inhibition is a potential therapeutic modality for cancer. Here we report the effects of two PIKfyve-specific inhibitors on the attachment/spreading and viability of mouse embryonic fibroblasts (MEFs) and C2C12 myoblasts. Importantly, 18-h treatment of adherent cells with YM201636 (800 nM) and apilimod (20 nM) in serum-containing culture media did not affect cell viability despite the presence of multiple cytoplasmic vacuoles, a hallmark of PIKfyve inhibition. Strikingly, at the same dose and duration the inhibitors caused excessive cytoplasmic vacuolation, initial suppression of cell attachment/spreading and subsequent marked detachment/death in serum-deprived cells. The remaining adherent cells under serum-deprived conditions had smaller surface area, lacked vinculin/actin-positive focal adhesions and displayed vacuoles occupying the entire cytoplasm. Serum or growth factors protected against PIKfyve inhibitor cytotoxicity. This protection required Akt activation evidenced by the abrogated beneficial effect of serum upon treatment with the clinically-relevant Akt inhibitor MK-2206. Moreover, Akt inhibition triggered cell detachment/death even in serum-fed adherent MEFs treated with apilimod. Intriguingly, BafilomycinA1 (H+-vacuolar ATPase inhibitor), which prevents the cytoplasmic vacuolation under PIKfyve perturbations, rescued all defects in attaching/spreading as well as in adherent cells under serum-starved or serum-fed conditions, respectively. Together, the results indicate that the cytotoxicity of PIKfyve inhibitors in MEFs and C2C12 myoblasts requires Akt suppression and excessive cytoplasmic vacuolation.