TNFα activates MAPK and Jak-Stat pathways to promote mouse Müller cell proliferation.

Mouse Müller cells, considered as dormant retinal progenitors, often respond to retinal injury by undergoing reactive gliosis rather than displaying neural regenerative responses. Tumor necrosis factor alpha (TNFα) is a key cytokines induced after injury and implicated in mediating inflammatory and neural regenerative responses in zebrafish. To investigate the involvement of TNFα in mouse retinal injury, adult C57BL/6J mice were subjected to light damage for 14 consecutive days. TNFα was elevated in the retina of mice exposed to light damage, which induced Müller cell proliferation in vitro. Affymetrix microarray showed that, in Müller cells, TNFα induces up-regulation of inflammatory and proliferation-related genes, including NFKB2, leukemia inhibitory factor, interleukin-6, janus kinase (Jak) 1, Jak2, signal transducer and activator of transcription (Stat) 1, Stat2, mitogen-activated protein kinase (MAPK) 7, and MAP4K4 but down-regulation of neuroprogenitor genes, including Sox9, Ascl1, Wnt2 and Hes1. Blocking the Jak/Stat and MAPK pathways attenuated TNFα-induced Müller cell proliferation. These results suggest that TNFα may drive the proliferation and inflammatory response, rather than the neural regenerative potential, of mouse Müller cells.

A novel family of expression vectors with multiple affinity tags for wheat germ cell-free protein expression.

BACKGROUND: Cell-free protein expression has become a widely used alternative of in vivo, cell-based systems in functional and structural studies of proteins. The wheat germ-based method outstands from the commercially available eukaryotic in vitro translation systems by its flexibility, high translation efficiency and success rate of properly folded eukaryotic protein synthesis. The original T7 promoter containing pEU3-NII vector was improved previously by addition of a ligation-independent cloning site, His6- and GST-tags, and a TEV protease cleavage site to facilitate the creation of recombinant plasmids, permit affinity purification, and enable production of purified, tag-free target proteins, respectively. RESULTS: Here, we describe a further development of pEU3-NII vector by inserting the rare-cutting, NotI restriction enzyme cleavage site to simplify vector linearization step prior to in vitro transcription. Additionally, His12, FLAG, and Halo affinity tag coding vectors have been created to increase detection sensitivity, specificity of interaction studies, and provide covalently linkable ligands for pull-down assays, respectively. Finally, the presented GST-His6, and GST-biotin double-tagging vectors could broaden the range of possibilities of protein-protein interaction studies. CONCLUSIONS: The new generation of pEU3-NII vector family allows a more rapid production of translationally active mRNA and wheat germ cell-free expression of target proteins with a wide variety of affinity tags thus enables designing flexible and diverse experimental arrangement for in vitro studies of proteins.

Anti-Apoptotic Effects of Docosahexaenoic Acid in IL-1ß-Induced Human Chondrosarcoma Cell Death through Involvement of the MAPK Signaling Pathway.

Osteoarthritis (OA) is a degenerative disease characterized by progressive articular cartilage destruction and joint marginal osteophyte formation with different degrees of synovitis. Docosahexaenoic acid (DHA) is an unsaturated fatty acid with anti-inflammatory, antioxidant, and antiapoptotic functions. In this study, the human chondrosarcoma cell line SW1353 was cultured in vitro, and an OA cell model was constructed with inflammatory factor IL-1ß stimulation. After cells were treated with DHA, cell apoptosis was measured. Western blot assay was used to detect protein expression of apoptosis-related factors (Bax, Bcl-2, and cleaved caspase-3) and mitogen-activated protein kinase (MAPK) signaling pathway family members, including extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), and p38 MAPK. Our results show that IL-1ß promotes the apoptosis of SW1353 cells, increases the expression of Bax and cleaved caspase-3, and activates the MAPK signaling pathway. In contrast, DHA inhibits the expression of IL-1ß, inhibits IL-1ß-induced cell apoptosis, and has a certain inhibitory effect on the activation of the MAPK signaling pathway. When the MAPK signaling pathway is inhibited by its inhibitors, the effects of DHA on SW1353 cells are weakened. Thus, DHA enhances the apoptosis of SW1353 cells through the MAPK signaling pathway.

Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na+, K+-ATPase control of cell adhesion, proliferation, and survival.

The ion pump Na+, K+-ATPase (NKA) is a receptor for the cardiotonic steroid ouabain. Subsaturating concentration of ouabain triggers intracellular calcium oscillations, stimulates cell proliferation and adhesion, and protects from apoptosis. However, it is controversial whether ouabain-bound NKA is considered a signal transducer. To address this question, we performed a global analysis of protein phosphorylation in COS-7 cells, identifying 2580 regulated phosphorylation events on 1242 proteins upon 10- and 20-min treatment with ouabain. Regulated phosphorylated proteins include the inositol triphosphate receptor and stromal interaction molecule, which are essential for initiating calcium oscillations. Hierarchical clustering revealed that ouabain triggers a structured phosphorylation response that occurs in a well-defined, time-dependent manner and affects specific cellular processes, including cell proliferation and cell-cell junctions. We additionally identify regulation of the phosphorylation of several calcium and calmodulin-dependent protein kinases (CAMKs), including 2 sites of CAMK type II-γ (CAMK2G), a protein known to regulate apoptosis. To verify the significance of this result, CAMK2G was knocked down in primary kidney cells. CAMK2G knockdown impaired ouabain-dependent protection from apoptosis upon treatment with high glucose or serum deprivation. In conclusion, we establish NKA as the coordinator of a broad, tightly regulated phosphorylation response in cells and define CAMK2G as a downstream effector of NKA.-Panizza, E., Zhang, L., Fontana, J. M., Hamada, K., Svensson, D., Akkuratov, E. E., Scott, L., Mikoshiba, K., Brismar, H., Lehtiö, J., Aperia, A. Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na+, K+-ATPase control of cell adhesion, proliferation, and survival.

Differential molecular pathways expression in HER2 positive early breast cancer according to hormone receptor status.

PURPOSE: Hormone receptors (HR) status in HER2 + breast cancer (BC) is a recognized stratification factor with relevant clinical implication. According to HR expression, HER2 + BC show different clinical characteristics, treatment sensitivity and prognosis. The interaction between HR and HER2 pathways remains incompletely understood. METHODS: Thirty-four HER2 + BC were included: 18 tumors with HER2+/HR + and 16 with HER2+/HR-. The expression of 770 genes and 13 molecular pathways were evaluated using Nanostring PanCancer Pathway panel performed on FFPE BC biopsies. RESULTS: Gene expression analysis identified 127 genes with significantly different expression between the two cohorts. 83% of these genes were overexpressed in HER2+/HR- cohort. Globally, 23% of them belonged to PI3K pathway (41 genes), 15% to Trascriptional regulation (26 genes) and 12% to MAPK (22 genes). Regarding pathway expression, PI3K, MAPK and NOTCH were significantly differently expressed between the two groups (p = 0.003, p = 0.0018 and p = 0.02, respectively), all of them were overexpressed in HER2+/HR- tumors. CONCLUSIONS: According to HR status, HER2 + tumors express different pathways profiles: the overexpression of PI3K, MAPK and NOTCH pathways in HER2+/HR- group could justify different survival outcomes and treatment sensitivity. The identification of tumor driver pathways may be a useful instrument for individualized pathway-directed therapies. Further clinical implications are warranted.

Tannic acid protects against experimental acute lung injury through downregulation of TLR4 and MAPK.

Acute lung injury (ALI) and its severe form acute respiratory distress syndrome (ARDS) remain a major cause of morbidity and mortality in critically ill patients, and no specific therapies are still available to control the mortality rate. Thus, we explored the preventive and therapeutic effects of tannic acid (TA), a natural polyphenol in the context of ALI. We used in vivo and in vitro models, respectively, using lipopolysaccharide (LPS) to induce ALI in mice and exposing J774 and BEAS-2B cells to LPS. In both preventive and therapeutic approaches, TA attenuated LPS-induced histopathological alterations, lipid peroxidation, lung permeability, infiltration of inflammatory cells, and the expression of proinflammatory mediators. In addition, in-vitro study showed that TA treatment could reduce the expression of proinflammatory mediators. Further studies revealed that TA-dampened inflammatory responses by downregulating the LPS-induced toll-like receptor 4 (TLR4) expression and inhibiting extracellular-signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) activation. Furthermore, cells treated with the inhibitors of ERK1/2 (PD98059) and p38 (SB203580) mitigated the expression of cytokines induced by LPS, thus suggesting that ERK1/2 and p38 activity are required for the inflammatory response. In conclusion, TA could attenuate LPS-induced inflammation and may be a potential therapeutic agent for ALI-associated inflammation in clinical settings.

Involvement of vascular endothelial growth factor (VEGF) and mitogen-activated protein kinases (MAPK) in the mechanism of neuroleptic drugs.

BACKGROUND: Recent evidence suggests that the mitogen activated protein kinase (MAPK)-associated signaling pathway in the frontal cortical areas demonstrates abnormal activity in cases of schizophrenia. Moreover, schizophrenia patients often display alterations in the regional cellular energy metabolism and blood flow of the brain; these are shown to parallel changes in angiogenesis primarily mediated by vascular endothelial growth factor (VEGF). METHODS: The present study examines the differential effects of time-dependent treatment with haloperidol, olanzapine and amisulpride (20µM) on VEGF and MAPK mRNA expression and VEGF level, using the T98 cell line as an example of nerve cells. For the purposes of comparison, the effect of neuroprotective pituitary adenylate cyclase-activating polypeptide (PACAP) on the expression of VEGF mRNA and secretion were also evaluated in this cell model. RESULTS: RT-PCR analysis revealed that all the tested neuroleptics increased VEGF mRNA expression after 72-h incubation; however, only haloperidol and olanzapine also increased the level of VEGF detected by ELISA, and they demonstrated significantly stronger effects than PACAP. Haloperidol and olanzapine, but not amisulpride, decreased MAPK14 mRNA expression in T98G cells after 72-h incubation. CONCLUSION: The obtained results suggest that haloperidol and olanzapine can trigger the MAPK and VEGF signaling pathway, which may contribute to their neuroprotective mechanism of action.

Influence of ochratoxin A on adaptation of Penicillium nordicum on a NaCl-rich dry-cured ham-based medium.

Iberian dry-cured ham is an important meat product with high consumption worldwide. The special ecological conditions occurring throughout its ripening favour surface colonisation of filamentous fungi. Normally, moulds contribute to the development of the sensory qualities of the ham; however, some toxigenic species, such as Penicillium nordicum, are able to successfully adapt to the NaCl-rich environment found in dry-cured ham and produce ochratoxin A (OTA) in this product. Moreover, it was suggested that the biosynthesis of OTA by P. nordicum itself may support the adaptation to this food environment. However, this mechanism has not been completely elucidated yet. The objective of this work was to evaluate the influence of different concentrations of commercial OTA (cOTA, at 0, 0.2, 1 and 5 ppb) on growth rate, biosynthetic- and stress-related gene expression and OTA production by two P. nordicum strains (Pn15 and Pn69) on dry-cured ham based-media. Two NaCl conditions (0% and 10%) were evaluated for each cOTA level. In general, no intra-strain and inter-strain differences in growth rates were found among the conditions tested. The stress-related Hog1 gene expression of the strain Pn15 was affected by cOTA and NaCl concentration whilst the strain Pn69 was not affected by these variables. The expression of OTA-related otapks and otanps genes of the strain Pn15 was affected by several NaCl and cOTA combinations. However, the strain Pn69 showed no differences in relative gene expression. Regarding to OTA production, different behaviours were displayed by the two strains. The strain Pn15, which produced high OTA amounts by itself, produced OTA without the necessity of the presence of NaCl or cOTA as stressors. However, the presence of cOTA triggers OTA production by the weak OTA producing Pn69 in the absence of NaCl. In addition, although a moderate correlation was found between the expression of the OTA-related genes and mycotoxin produced by P. nordicum in the absence of NaCl, none was obtained between Hog1 gene expression and mycotoxin production. This study is a step forward for a better understanding of the ability of P. nordicum producers of OTA to colonise NaCl-rich habitats such as Iberian ham for proposing actions to minimise OTA contamination in this meat product.

Mitogen activated protein kinase 6 and MAP kinase phosphatase 1 are involved in the response of Arabidopsis roots to L-glutamate.

KEY MESSAGE: The function and components of L-glutamate signaling pathways in plants have just begun to be elucidated. Here, using a combination of genetic and biochemical strategies, we demonstrated that a MAPK module is involved in the control of root developmental responses to this amino acid. Root system architecture plays an essential role in plant adaptation to biotic and abiotic factors via adjusting signal transduction and gene expression. L-Glutamate (L-Glu), an amino acid with neurotransmitter functions in animals, inhibits root growth, but the underlying genetic mechanisms are poorly understood. Through a combination of genetic analysis, in-gel kinase assays, detailed cell elongation and division measurements and confocal analysis of expression of auxin, quiescent center and stem cell niche related genes, the critical roles of L-Glu in primary root growth acting through the mitogen-activated protein kinase 6 (MPK6) and the dual specificity serine-threonine-tyrosine phosphatase MKP1 could be revealed. In-gel phosphorylation assays revealed a rapid and dose-dependent induction of MPK6 and MPK3 activities in wild-type Arabidopsis seedlings in response to L-Glu. Mutations in MPK6 or MKP1 reduced or increased root cell division and elongation in response to L-Glu, possibly modulating auxin transport and/or response, but in a PLETHORA1 and 2 independent manner. Our data highlight MPK6 and MKP1 as components of an L-Glu pathway linking the auxin response, and cell division for primary root growth.

Association of S100 calcium-binding protein A12, receptor for advanced glycation endproducts, and nuclear factor-κB expression with inflammation in pulp tissues from tooth caries.

AIM: S100 calcium-binding protein A1 (S100A12) is a pro-inflammatory molecule which is secreted during inflammation and induces chemotaxis and the production of pro-inflammatory cytokines via interaction with receptor for advanced glycation endproducts (RAGE) and subsequent, activation of nuclear factor-κB (NF-κB). The present study was designed to determine the expression levels of S100A12, RAGE, and NF-κB in the inflamed pulp of carried teeth. METHODS: In the present study, mRNA from 50 inflamed pulp and 50 healthy pulp were used for expression studies using real-time polymerase chain reaction. The expression levels of S100A12, RAGE, and NF-κB were compared between inflamed and healthy tissues. RESULTS: The results revealed that the expression of S100A12, but not of RAGE or NF-κB, was significantly decreased in inflamed pulp when compared to healthy pulp. mRNA levels of RAGE were also increased in the inflamed pulp taken from men when compared with women. CONCLUSION: The results suggest that S100A12 does not participate in the induction of inflammation in dental pulp. However, RAGE can participate in the inflammation in the pulp of males.

Hydrogen sulfide alleviates the cold stress through MPK4 in Arabidopsis thaliana.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that mediates physiological processes in animals and plants. In this study, we investigated the relationship of H2S and mitogen activated protein kinase (MAPK) under cold stress in Arabidopsis. H2S up-regulated MAPK expression levels and was involved in the cold stress-related upregulation of MAPK genes expression. We then chose MPK4 whose expression level was influenced the most by H2S as a target and found that H2S's ability to alleviate cold stress required MPK4. Both H2S and MPK4 regulated the expression levels of the cold response genes inducer of CBF expression 1 (ICE1), C-repeat-binding factors (CBF3), cold responsive 15A (COR15A) and cold responsive 15B (COR15B). H2S inhibited the opening of stomata under cold stress, which required the participation of MPK4. In conclusion, MPK4 is a downstream component of H2S-related cold-stress resistance, and H2S and MPK4 both regulated the cold response genes and stomatal movement to response the cold stress.

Expression of the receptor for advanced glycation end products in acquired reactive perforating collagenosis.

BACKGROUND: Acquired reactive perforating collagenosis (ARPC) is a rare skin disorder characterized by transepidermal elimination of dermal collagen. There is little data regarding the pathogenesis of ARPC. The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor that plays an important role in inflammatory responses and may be involved in the pathogenesis of ARPC. AIM: To explore the expression of RAGE in ARPC. METHODS: Paraffin-embedded punch biopsy specimens of 41 patients with ARPC and of 11 healthy controls with normal skin were obtained from the Department Of Pathology. Clinical data of all patients were reviewed from the medical files. All specimens were stained immunohistochemically with antibody to RAGE (Anti-RAGE). The intensity of RAGE expression was assessed semi-quantitatively on epidermal cells, microvascular endothelium, dermal fibroblasts and inflammatory cells and graded as 0 (no staining), 1 (weak), 2 (moderate) and 3 (strong). The patients were divided into diabetic and nondiabetic groups for analysis. RESULTS: RAGE expression in microvascular endothelium, inflammatory cells and fibroblasts of patients with ARPC was more intense than normal tissues of healthy participants (P values are 0.005, 0.017 and P > 0.05). LIMITATIONS: Our method of assessment of RAGE expression was semi-quantitative. CONCLUSION: We observed an overexpression of RAGE in lesional samples of patients with ARPC which was independent of the presence of diabetes.

High ASMA+ Fibroblasts and Low Cytoplasmic HMGB1+ Breast Cancer Cells Predict Poor Prognosis.

INTRODUCTION: The influence of cancer-associated fibroblasts (CAFs) and high mobility group box 1 (HMGB1) has been recognized in several cancers, although their roles in breast cancer are unclear. The present study aimed to determine the levels and prognostic significance of α-smooth muscle actin-positive (ASMA+) CAFs, plus HMGB1 and receptor for advanced glycation end products (RAGE) in cancer cells. MATERIALS AND METHODS: A total of 127 breast samples, including 96 malignant and 31 benign, were examined for ASMA, HMGB1, and RAGE by immunohistochemistry. The χ2 test and Fisher's exact test were used to test the association of each protein with clinicopathologic parameters. The Kaplan-Meier method or log-rank test and Cox regression were used for survival analysis. RESULTS: ASMA+ fibroblast infiltration was significantly increased in the tumor stroma compared with that in benign breast tissue. The levels of cytoplasmic HMGB1 and RAGE were significantly greater in the breast cancer tissue than in the benign breast tissues. High ASMA expression correlated significantly with large tumor size, clinical stage III-IV, and angiolymphatic and perinodal invasion. In contrast, increased cytoplasmic HMGB1 correlated significantly with small tumor size, pT stage, early clinical stage, luminal subtype (but not triple-negative subtype), and estrogen receptor and progesterone receptor expression. The levels of ASMA (hazard ratio, 14.162; P = .010) and tumor cytoplasmic HMGB1 (hazard ratio, 0.221; P = .005) could serve as independent prognostic markers for metastatic relapse in breast cancer patients. The ASMA-high/HMGB1-low profile provided the most reliable prediction of metastatic relapse. CONCLUSION: We present for the first time, to the best of our knowledge, the potential clinical implications of the combined assessment of ASMA+ fibroblasts and cytoplasmic HMGB1 in breast cancer.

The long non-coding RNA TP73-AS1 modulates HCC cell proliferation through miR-200a-dependent HMGB1/RAGE regulation.

BACKGROUND: P73 antisense RNA 1 T (non-protein coding), also known as TP73-AS1, is a long non-coding RNA (lncRNA) which is involved in cell proliferation and the development of tumors. However, the exact effects and molecular mechanisms of TP73-AS1 in hepatocellular carcinoma (HCC) progression are still unknown. The present study is aimed to investigate the detailed functions and the mechanism of TP73-AS1 in regulation of HCC cell proliferation. METHODS: TP73-AS1 expression in HCC tissues and cell lines was determined using real-time PCR assays; the correlation of TP73-AS1 expression with clinicopathological features of HCC was analyzed. The functions of TP73-AS1 in regulation of HCC cell proliferation was evaluated using MTT and BrdU assays. The candidate upstream miRNAs of HMGB1 were screened using miRcode, miRWalk, miRanda and Target scan, verified using real-time PCR assays. The interaction between TP73-AS1 and miR-200a was confirmed using Luciferase report gene assays. The proten levels of HMGB1 signaling-related factors in response to co-processing TP73-AS1 knockdown and miR-200a inhibition were determined using Western blot assays and ELISA. Further, miR-200a, HMGB1 mRNA and RAGE mRNA and their correlations in HCC tissues were determined. RESULTS: TP73-AS1 was upregulated in HCC tissues and cell lines. High TP73-AS1 expression was correlated with worse clinicopathological features, poorer prognosis and shorter survival. Knockdown of TP73-AS1 inhibited the HCC proliferation and the expression levels of HMGB1, RAGE and NF-κB in HCC cells. By using online tools, we screened out several candidate upstream miRNAs of HMGB1, among which miR-200a overexpression inhibited HMGB1 mRNA expression the most significantly. By using luciferase assays, we confirmed that miR-200a could directly bind to TP73-AS1 and the 3'UTR of HMGB1; TP73-AS1 competed with HMGB1 for miR-200a binding. MiR-200a inhibition could up-regulate HMGB1, RAGE, NF-κB expression as well as NF-κB regulated cytokines levels, which could be partially restored by si-TP73-AS1. In HCC tissues, miR-200a was down-regulated while HMGB1 and RAGE were up-regulated; TP73-AS1 was inversely correlated with miR-200a, while positively correlated with HMGB1 and RAGE, respectively. CONCLUSION: Our data indicated that TP73-AS1 might be an oncogenic lncRNA that promoted proliferation of HCC and could be regarded as a therapeutic target in human HCC.

ERK1/2, JNK and STAT3 activation and correlation with tumor differentiation in oral SCC.

Signal transducer and activator of transcription 3 (STAT3) and mitogen activated protein kinases (MAPKs), including ERK and JNK, have been implicated in oral squamous cell carcinoma (OSCC) development and progression. Our purpose was to evaluate the levels of activated STAT3, ERK1/2 and JNK by immunohistochemistry in OSCC and to investigate possible correlations of these molecules with each other as well as with the degree of tumor differentiation. Immunohistochemical assessment of the phosphorylated levels of STAT3(tyrosine/ serine), ERK1/2 and JNK was performed in 60 OSCC, including well, moderately and poorly differentiated tumors. Semiquantitative scoring system was used, by calculating intensity of immunostaining, percentage of positive cells and combined scores. Statistics included Fisher's test, Student's T-Test and Kruskal-Wallis analysis, Spearman's correlation coefficient and multivariate logistic regression analyses. Immunohistochemical levels of both pSTAT3(tyr) and pERK1/2 showed statistically significant differences between well and poorly differentiated tumors with the latter receiving higher mean percentage, intensity and total scores. On the other hand, pJNK showed statistically significantly higher intensity levels in moderately compared to poorly differentiated tumors. pSTAT3(ser) immunoexpression did not appear to correlate with tumor differentiation. Between different molecules, more pronounced, pERK1/2 levels exhibited statistically significant positive correlation with pSTAT3(ser), pSTAT3(tyr) and pJNK expression. ERK1/2 and STAT3 activation (as assessed by tyrosine but not serine phosphorylation) could contribute to a less differentiated phenotype in OSCC, while JNK activation may have an opposite, although possibly less pronounced, effect. Positive correlations between MAPK and STAT3 levels may indicate a direct crosstalk and/or regulation by common upstream pathways.

Receptor for advanced glycation end product blockade enhances the chemotherapeutic effect of cisplatin in tongue squamous cell carcinoma by reducing autophagy and modulating the Wnt pathway.

Tongue squamous cell carcinoma (TSCC) is one of the most severe types of cancer with poor outcomes. Cisplatin is used widely to treat cancer cells, but many patients develop acquired drug resistance. The receptor for advanced glycation end products (RAGE) is expressed widely in TSCC and associated with drug-induced chemotherapy resistance. However, the effect of RAGE and cisplatin on Tca-8113 cells remains unknown. We assayed the combined use of RAGE blockade and cisplatin effect on Tca-8113 cells' viability by MTT and apoptosis rate of Tca-8113 cells on RAGE blockade+cisplatin treatment; cisplatin alone; or RAGE blockade alone by flow cytometry. We observed the expressions of autophagy-related proteins beclin1, LC3II, p62; Wnt signaling-related proteins ß-catenin, GSK3ß, WNT5A, ROR-2; and apoptosis-related protein cleaved caspase-3, bcl-2-associated X proteins using western blot. We determined WNT5A and beclin1 expression on Tca-8113 cells by immunofluorescence. We further observed autophagy vacuoles by monodansylcadaverine staining. We found that RAGE blockade and cisplatin significantly decreased cell viability and increased the cell apoptosis rate compared with cisplatin alone. Furthermore, RAGE blockade suppressed the canonical Wnt pathway proteins ß-catenin and GSK-3ß, but upregulated noncanonical WNT5A and receptor ROR-2. We show that RAGE blockade suppressed the levels of autophagy-related protein LC3II/I, beclin1, accelerated degradation of autophagy for the increasing p62 expression, and increased cell apoptosis for the increasing expressions of cleaved caspase-3 and bcl-2-associated X proteins. We observed the location of WNT5A and beclin1 expressions on cells by immunofluorescence and their trends were consistent with western blotting. Taken together, our findings suggested that RAGE blockade+cisplatin improved chemotherapeutic effects by reducing autophagy and regulating Wnt/ß-catenin to suppress the progression of TSCC.

Overexpression of receptor for advanced glycation end products (RAGE) in ovarian cancer.

BACKGROUND: Ovarian cancer is one of the important challenges in the field of gynecologic oncology because of some problems in understanding its etiology and pathogenesis. Receptor for advanced glycation end products (RAGE) is a multiligand trans-membranous receptor which is upregulated in some human cancers. Mechanisms of RAGE involvement in carcinogenesis of ovarian cancer are unknown. OBJECTIVE: This study aimed to investigate the expression of RAGE in ovarian cancers and its association with clinicopathological characteristics. METHODS: The RAGE expression level in ovarian cancer and corresponding noncancerous tissues were analyzed by real time quantitative RT-PCR and immunohistochemistry techniques. RESULTS: Results indicated that RAGE gene was overexpressed in ovarian cancer tissue compared with adjacent noncancerous tissue (p < 0.001). A significant association between RAGE expression and tumor size (p = 0.04), depth of stromal invasion (p = 0.031), lymphovascular invasion (p = 0.041) and stage of cancer (p = 0.041) was observed. The receiver operating characteristic (ROC) analyses yielded the area under the curve (AUC) values of 0.86 for RAGE in discriminating ovarian cancer samples from non-cancer controls. CONCLUSIONS: In conclusion overexpression of RAGE in ovarian cancer may be a useful biomarker to predict tumor progression.

Pim1 Kinase Overexpression Enhances ckit+ Cardiac Stem Cell Cardiac Repair Following Myocardial Infarction in Swine.

BACKGROUND: Pim1 kinase plays an important role in cell division, survival, and commitment of precursor cells towards a myocardial lineage, and overexpression of Pim1 in ckit+ cardiac stem cells (CSCs) enhances their cardioreparative properties. OBJECTIVES: The authors sought to validate the effect of Pim1-modified CSCs in a translationally relevant large animal preclinical model of myocardial infarction (MI). METHODS: Human cardiac stem cells (hCSCs, n = 10), hckit+ CSCs overexpressing Pim1 (Pim1+; n = 9), or placebo (n = 10) were delivered by intramyocardial injection to immunosuppressed Yorkshire swine (n = 29) 2 weeks after MI. Cardiac magnetic resonance and pressure volume loops were obtained before and after cell administration. RESULTS: Whereas both hCSCs reduced MI size compared to placebo, Pim1+ cells produced a ∼3-fold greater decrease in scar mass at 8 weeks post-injection compared to hCSCs (-29.2 ± 2.7% vs. -8.4 ± 0.7%; p < 0.003). Pim1+ hCSCs also produced a 2-fold increase of viable mass compared to hCSCs at 8 weeks (113.7 ± 7.2% vs. 65.6 ± 6.8%; p <0.003), and a greater increase in regional contractility in both infarct and border zones (both p < 0.05). Both CSC types significantly increased ejection fraction at 4 weeks but this was only sustained in the Pim1+ group at 8 weeks compared to placebo. Both hCSC and Pim1+ hCSC treatment reduced afterload (p = 0.02 and p = 0.004, respectively). Mechanoenergetic recoupling was significantly greater in the Pim1+ hCSC group (p = 0.005). CONCLUSIONS: Pim1 overexpression enhanced the effect of intramyocardial delivery of CSCs to infarcted porcine hearts. These findings provide a rationale for genetic modification of stem cells and consequent translation to clinical trials.

All-trans retinoic acid suppresses the adhering ability of ARPE-19 cells via mitogen-activated protein kinase and focal adhesion kinase.

This study investigated the signaling mechanism underlying the anti-adhesive effect of all-trans retinoic acid (ATRA) on retinal pigment epithelial ARPE-19 cells. Adhesion kinetics with or without ATRA treatment were profiled by adhesion assay. Surface coating with type IV collagen, fibronectin, laminin, but not type I collagen, significantly enhanced adhesion and spreading of ARPE-19 cells, while ATRA at subtoxic doses (ranging from 10-7 to 10-6 M) profoundly suppressed the extracellular matrix-enhanced adhesion ability. Cell attachment on FN activated PI3K/Akt and MAPK cascades, whereas ATRA pretreatment blunted the early phosphorylation of Akt and MAPK signaling mediators including p38 MAPK, JNK1/2, and ERK1/2. Mechanistically, signaling blockade with selective kinase inhibitors demonstrated that all MAPK pathways were involved in the anti-adhesive effect of ATRA, whereas the PI3K inhibitor treatment significantly potentiated the ATRA-suppressed RPE cell adhesion. Moreover, ATRA treatment did not affect intracellular F-actin distribution, but remarkably reduced focal adhesion kinase (FAK) expression and its nuclear localization during ARPE-19 cell attachment. In conclusion, ATRA suppresses the adhering ability of ARPE-19 cells at least in part through MAPK and FAK pathways. Signaling blockade with PI3K inhibitor could be regarded as an alternative modality for treating proliferative vitreoretinopathy.