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Background/Aims: Cellular senescence, or permanent growth arrest, is known as an effective tumor suppressor mechanism that can be induced by different stressors, such as oncogenes, chemotherapeutics or cytokine cocktails. Previous studies demonstrated that the growth-repressing state of oncogene-induced senescent cells depends on argonaute protein 2 (Ago2)-mediated transcriptional gene silencing and Ago2/Rb corepression of E2F-dependent cell cycle genes. Cytokine-induced senescence (CIS) likewise depends on activation of the p16Ink4a/Rb pathway, and consecutive inactivation of the E2F family of transcription factors. In the present study, we therefore analyzed the role of Ago2 in CIS. Methods: Human cancer cell lines were treated with interferon-gamma (IFN-γ) and tumor necrosis factor (TNF) to induce senescence. Senescence was determined by growth assays and measurement of senescence-associated β-galactosidase (SA-β-gal) activity, Ago2 translocation by Ago2/ Ki67 immunofluorescence staining and western blot analysis, and gene transcription by quantitative polymerase chain reaction (qPCR). Results: IFN-γ and TNF permanently stopped cell proliferation and time-dependently increased SA-β-gal activity. After 24 – 48 h of cytokine treatment, Ago2 translocated from the cytoplasm into the nucleus of Ki67-negative cells, an effect which was shown to be reversible. Importantly, the proinflammatory cytokine cocktail suppressed Ago2-regulated cell cycle control genes, and siRNA-mediated depletion of Ago2 interfered with cytokine-induced growth inhibition. Conclusion: IFN-γ and TNF induce a stable cell cycle arrest of cancer cells that is accompanied by a fast nuclear Ago2 translocation and repression of Ago2-regulated cell cycle control genes. As Ago2 downregulation impairs cytokine-induced growth regulation, Ago2 may contribute to tissue homeostasis in human cancers. [ABSTRACT FROM AUTHOR]

Background/Aims: Platinum-based chemotherapy is one of the most important strategies for treatment of colorectal cancer. To improve the therapeutic efficiency, adjuvant drugs were sought to sensitize colorectal cancer cells to platinum-based agents such as cisplatin. As previous research has shown that miRNAs are associated with chemosensitivity, we aimed to alter miRNA regulation in colorectal cancer cells to increase their chemosensitivity. Methods: MTT assays were performed to determine the viability of HT29, SW480, and LoVo cells. Quantitative real time polymerase chain reaction (qRT-PCR) was performed to examine the expression of miR-20a in these cell lines. Regulation of the miR-20a/ASK1 axis was confirmed by western blotting and luciferase reporter assays. After treatment with miR-20a inhibitor (anti-miR-20a) and cisplatin, production of reactive oxygen species (ROS), mitochondrial membrane potential, and apoptosis were measured by flow cytometry. Activation of ASK1, Bcl-xl, JNK, and caspase-9, -7, and -3 was detected by western blotting. Results: miR-20a was overexpressed in colorectal cancer cell lines. Furthermore, knockdown of miR-20a increased the sensitivity of colorectal cancer cells to cisplatin treatment in vitro and in vivo. We demonstrated that the ASK1 gene was the target of miR-20a, and knockdown of miR-20a increased the expression of ASK1 in colorectal cancer cells. As cisplatin treatment induced production of ROS, knockdown of miR-20a enhanced ROS signaling through promoting the phosphorylation of ASK1. Phosphorylation of JNK and the subsequent mitochondrial apoptosis were triggered by the combination of cisplatin and anti-miR-20a. Conclusions: Knockdown of miR-20a enhanced sensitivity of colorectal cancer cells to cisplatin through the ROS/ASK1/JNK pathway. [ABSTRACT FROM AUTHOR]

Background/Aims: To explore the effect of sulforaphane (SFN) treatment in rats through the induction of Stress Urinary Incontinence (SUI) via the Nrf2-ARE pathway. Methods: A total of 18 female rats (Sprague-Dawley) were assigned to three groups: a control group, an SUI group, and an SUI+SFN group (six rats per group). Rats in the treatment groups were induced via postpartum vaginal balloon dilation and bilateral ovariectomy. Rats in the SUI+SFN group were treated via intraperitoneal injection once per day for a total of one month. Urethral sphincter muscle histological was observed by HE and Masson staining. Peak voiding pressure and interval of micturition were measured by cystometry. Oxidative stress markers and protein expression in the Nrf2-ARE pathway were examined by immunohistochemical staining and western blotting. Results: Prolonged micturition interval and higher peak voiding pressure were observed in the SUI+SFN group. Disturbance of muscle morphology was ameliorated, muscle content was elevated, and collagen content was restrained in response to SFN treatment. The SOD, GSH-Px, and CAT activities were elevated in the SUI+SFN group compared to those in the control group. The level of cell apoptosis was decreased in SUI rats after SFN treatment; however, apoptosis was mainly located in the urethral mucosa instead of the muscle layer. SFN reduced the Bax/Bcl-2 expression ratio. Nrf2 and Nrf2 target antioxidant proteins were elevated in the SFN group. Conclusions: SFN was effective for SUI treatment via decreasing oxidative stress and activating the Nrf2-ARE pathway. [ABSTRACT FROM AUTHOR]

Objective: This study explored the protective effects of the microRNA-126 (miR-126)- mediated PI3K/Akt/eNOS signaling pathway on human cardiac microvascular endothelial cells (HCMECs) against hypoxia/reoxygenation (H/R)-induced injury and the inflammatory response. Methods: Untreated HCMECs were selected for the control group. After H/R treatment and cell transfection, the HCMECs were assigned to the H/R, miR-126 mimic, mimicnegative control (NC), miR-126 inhibitor, inhibitor-NC, wortmannin (an inhibitor of PI3K) and miR-126 mimic + wortmannin groups. Super oxide dismutase (SOD), nitric oxide (NO), vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS) were measured utilizing commercial kits. Quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were performed to detect miR-126 expression and the mRNA and protein expression of inflammatory factors. Western blotting was used to determine the expression of key members in the PI3K/Akt/eNOS signaling pathway. ACCK- 8 assay and flow cytometry were employed to examine cell proliferation and apoptosis, respectively. The angiogenic ability in each group was detected by the lumen formation test. Results: Compared to the control group, p/t-PI3K, p/t-Akt and p/t-eNOS expression, NO, VEGF and SOD levels, cell proliferation and in vitro lumen formation ability were decreased, while the ROS content, interleukin (IL)-6, IL-10 and tumor necrosis factor (TNF)-α expression and cell apoptosis were significantly increased in the H/R, mimic-NC, miR-126 inhibitor, inhibitor- NC, wortmannin and miR-126 mimic + wortmannin groups. Additionally, in comparison with the H/R group, the miR-126 mimic group had elevated p/t-PI3K, p/t-Akt and p/t-eNOS expression, increased NO, VEGF and SOD contents, and strengthened cell proliferation and lumen formation abilities but also exhibited decreased ROS content, reduced IL-6, IL-10 and TNF-α expressions, and weakened cell apoptosis, while the miR-126 inhibitor and wortmannin group exhibited the opposite results. Furthermore, decreased p/t-PI3K, p/t-Akt and p/t-eNOS expressions, decreased NO, VEGF and SOD contents, cell proliferation and lumen formation abilities, as well as increased ROS content, increased IL-6, IL-10 and TNF-α expression, and increased cell apoptosis were observed in the miR-126 mimic + wortmannin group compared to themiR-126 mimic group. Conclusions: These findings indicated that miR-126 protects HCMECs from H/R-induced injury and inflammatory response by activating the PI3K/Akt/ eNOS signaling pathway. [ABSTRACT FROM AUTHOR]

Background/Aims: Colorectal cancer (CRC) is the third most common type of cancer worldwide. Sprouty proteins are modulators of mitogeninduced signal transduction processes and therefore can influence the process of cancerogenesis. The encoded protein of Sprouty homolog 4 (SPRY4) is associated with various human cancers. However, its biological role and clinical significance in CRC development and progression are unknown. Methods: The aim of this study was to evaluate the expression and biological role of SPRY4 in colorectal cancer. qRT-PCR was performed to investigate the expression of SPRY4 in tumor tissues and corresponding non tumor colorectal tissues from 70 patients. The effect of SPRY4 on proliferation was evaluated by MTT and colony formation assays. CRC cells transfected with SPRY4 were injected into nude mice to study the effect of SPRY4 on tumorigenesis in vivo. Results: The lower expression of SPRY4 was remarkably correlated with deep tumor invasion and advanced TNM stage. Multivariate analyses revealed that SPRY4 expression served as an independent predictor for overall survival. Using 5-aza treatment, we also observed that SPRY4 expression can be affected by DNA methylation. Further experiments revealed that overexpressed SPRY4 significantly inhibited CRC cell proliferation both in vitro and in vivo. Conclusion: Our study demonstrated that SPRY4 is involved in the development and progression of colorectal cancer by regulating cell proliferation and shows that SPRY4 may be a potential diagnostic and prognostic target in patients with colorectal cancer. [ABSTRACT FROM AUTHOR]

Background/Aims: Cryptococcus neoformans infections are becoming increasingly prevalent and remain a life-threatening clinical issue in immune-compromised hosts. The microorganism evades a variety of endogenous anti-fungal mechanims of host immune cells. The signaling pathways in human immune cells that become activated in response to Cryptococcus neoformans infection have yet to be fully characterized. Methods: Human monocytes were incubated with Cryptococcus neoformans, and the whole transcriptome of monocytes was sequenced before and after exposure to Cryptococcus neoformans using mass parallel sequencing techniques. Based on the genes that demonstrated altered expression patterns, we performed GO and KEGG enrichment analysis to further characterize the pathways involved in monocyte activation by Cryptococcus neoformans. Results: We found that immune and inflammatory responses, as well as chemotaxis, were the most heavily activated cellular events. Specifically, the toll-like receptor, tumor necrosis factor, NF-kB and Jak-STAT pathways were the most active pathways in response to Cryptococcus neoformans infection. The sequencing data of selected genes from the transcriptome analysis were further validated by real-time polymerase chain reactions. Conclusion: Taken together, our study is the first characterization of the transcriptome alterations in human immune cells upon C. neoformans infection, providing additional information that may be helpful in discovering novel anti-fungal targets. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Aims: To investigate global proteomic changes induced in CD44+CD24- stem cells isolated from the prostate cancer cell lines, LNCaP and DU145, post prolonged TGF-β treatment in order to understand underlying mechanisms that promote stemness in prostate cancer cells. Methods: CD44+CD133+α2β1Integrin+CD24- population was isolated from mock or TGF-β treated (7 days) prostate cancer cell line, LNCaP, through fluorescent activated cell sorting. Cell lysates were obtained from the ±TGF-β cell population and proteomics profiling (MS/MS) was performed by mass spectrometry. Relative enrichment or depletion in the CD44+CD24-population post-TGF-β treatment was determined relative to mock-treated CD44+CD24- cells post normalization to GAPDH expression levels. Results obtained from MS/MS were validated using immunoblotting. Functional validation of one putative regulator was performed using gain-of-function strategy to investigate its role in rendering stemness in LNCaP and DU145 cells in vitro and in promoting tumorigenicity in vivo. Results: TGF-β treatment caused significant enrichment of CD44+CD24- population in LNCaP cells (22.35 ± 0.94% in mock treated vs 95.23 ± 2.34% in TGF-β treated cells; P < 0.01), which were also positive for CD133 and α2β1Integrin. Mass spectrometry analysis of the enriched cell population revealed that sixty-three proteins were either up- or down-regulated greater than five folds, out of which the poly r(C) binding protein (PCBP)-1 was the most down-regulated (9.31 ± 0.05 folds). Ectopic overexpression of PCBP1 in LNCaP and DU145 cells not only attenuated enrichment of CD44+CD133+CD24- population in these cells following TGF-β treatment, but also significantly decreased tumorigenicity of the stem cell subset, as assessed by in vitro soft agar colony formation and in vivo xenograft assays. Conclusion: Our proteomic profiling and subsequent validation indicate that PCBP1 is central to CSCs enrichment and functionality in prostate cancer. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: Growth arrest-specific protein 6 (Gas6) is a cytokine that can be synthesized by a variety of cell types and secreted into the extracellular matrix. Previous studies have confirmed that Gas6 is involved in certain pathophysiological processes of the cardiovascular system through binding to its receptor, Axl. In the present study, we investigated the role of Gas6 in cellular senescence and explored the mechanisms underlying its activity. Methods: We used vascular smooth muscle cells (VSMCs) to create two cellular senescence models, one for replicative senescence (RS) and one for induced senescence (IS), to test the hypothesis that Gas6 delays senescence. Results: Gas6-treated cells appear relatively younger compared with non-Gas6-treated cells. In particular, Gas6-treated cells displayed decreased staining for SA-β-Gal, fewer G1 phase cells, and decreased levels of p16INK4a and p21Cip1 expression; conversely, Gas6-treated cells displayed more S phase cells and significantly increased proliferation indexes. Furthermore, in both the IS and RS models with Gas6 treatment, the levels of PI3K, p-Akt, and p-FoxO3a decreased following Axl inhibition by R428; similarly, the levels of p-Akt and p-FoxO3a also decreased following PI3K inhibition by LY294002. Conclusion: Gas6/Axl signaling is essential for delaying the cellular senescence process regulated by the PI3K/Akt/FoxO signaling pathway. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We investigated the anti-tumor efficiency of sonodynamic therapy (SDT) on human tongue squamous carcinoma SAS cell line using low intensity ultrasound (LIU) of 0.6 and 0.8 W/cm2, plus 5-aminolevulinic acid (ALA). Xenograft in vivo experiments using Balb/ca nude mice and MTT assays in vitro showed that ALA-LIU therapy significantly suppressed the proliferation of SAS cells. ALA-LIU therapy markedly enhanced SAS cell apoptosis rate compared to LIU alone. Based on TEM and fluorescence microscopy observations, there are notably morphology changes and seriously swollen mitochondria in xenograft tissues, and ALA-induced PpIX bond strongly to mitochondria of SAS cells. Immunohistochemical staining and western blotting demonstrated upregulation of Bax, cytochrome c and caspase-3, and downregulation of Bcl-2 for both in vivo and in vitro cases after ALA-LIU treatment. Increase of reactive oxygen species (ROS) in the ALA-LIU treatment groups were found using 2, 7-dichlorofluorescin diacetate (DCFH-DA) staining. Administration of the ROS scavenger, N-acetylcysteine (NAC), suppressed ALA-LIU-induced apoptosis and the expression of mitochondria apoptosis-related proteins, which confirmed that the ALA-LIU induced SAS cell apoptosis is through the generation of ROS. The process initially damaged mitochondria, activated pro-apoptotic factors Bax and cytochrome c and supressed the anti-apoptotic factor Bcl-2, activated caspase-3 to executed apoptosis through mitochondrial signaling pathway. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]