Markers Cyclin D1 Research Articles

Bladder cancer therapy without toxicity-A dose-escalation study of alpha1-oleate.

Potent chemotherapeutic agents are required to counteract the aggressive behavior of cancer cells and patients often experience severe side effects, due to tissue toxicity. Our study addresses if a better balance between efficacy and toxicity can be attained using the tumoricidal complex alpha1-oleate, formed by a synthetic, alpha-helical peptide comprising the N-terminal 39 amino acids of alpha-lactalbumin and the fatty acid oleic acid. Bladder cancer was established, by intravesical instillation of MB49 cells on day 0 and the treatment group received five instillations of alpha1-oleate (1.7-17 mM) on days 3 to 11. A dose-dependent reduction in tumor size, bladder size and bladder weight was recorded in the alpha1-oleate treated group, compared to sham-treated mice. Tumor markers Ki-67, Cyclin D1 and VEGF were inhibited in a dose-dependent manner, as was the expression of cancer-related genes. Remarkably, toxicity for healthy tissue was not detected in alpha1-oleate-treated, tumor-bearing mice or healthy mice or rabbits, challenged with increasing doses of the active complex. The results define a dose-dependent therapeutic effect of alpha1-oleate in a murine bladder cancer model.

Effects of Mitofusin2 on astrocytes proliferation in vitro induced by scratch injury

Reactive astrogliosis, a common phenomenon after central nervous system (CNS) injury, exerts negative effects on neuronal repair and recovery by forming a glial scar. Mitofusin2 (Mfn2), a hyperplasia suppression gene, is a potential target of therapeutics to better control astrogliosis. To simulate traumatic injury of the CNS in vivo, an in vitro scratch injury model was established to investigate the role of Mfn2 in the proliferation of astrocytes in this study. We demonstrated that scratch-injury stimulation upregulated the expression of the markers cyclin D1, PCNA and GFAP and turned quiescent astrocytes into mitotic cells, which may have been via activation of Ras-Raf1-ERK1/2 and PI3K-Akt signaling. Meanwhile, both the gene and protein of Mfn2 were markedly inhibited. Furthermore, overexpression of Mfn2 effectively attenuated astrocyte proliferation and halted the cell cycle, concomitant with marker downregulation and wound healing suppression. Our results demonstrate that overexpression of Mfn2 inhibits the reactive astrogliosis process by blocking the Raf1-ERK1/2 and PI3K-Akt signal pathways. Therapeutic approaches that target Mfn2 may have protective effects against reactive gliosis and glia formation.

Clinico-pathological significance of Drp1 dysregulation and its correlation to apoptosis in oral cancer patients

Dysregulation in mitochondrial dynamics has been associated with several diseases including cancer. Present study assessed the alteration in mitochondrial fission protein (Drp1) in oral epithelial cells collected from clinically confirmed pre-cancer and cancer patients and further correlates it with the cellular apoptosis signaling. Results indicate the ROS accumulation in OSCC patients is accompanied by several changes including increase in mitochondrial mass, expression of mitochondrial fission protein (Drp1) and alteration in apoptotic signaling. The positive co-relation has been observed between the expressions of anti-apoptotic Bcl-2proteinswith mitochondrial fission protein Drp1. Higher mitochondrial fission in oral cancer cells was also correlated with the increased expression of cell cycle marker CyclinD1 indicating highly proliferative stage of oral cancer cells. The clinical correlation can be extended to develop biomarker for diagram and program in oral cancer management.

Circ_0001971 regulates oral squamous cell carcinoma progression and chemosensitivity by targeting miR-194/miR-204 in vitro and in vivo.

Circular RNAs (circRNAs) play a wide role in human cancers, including oral squamous cell carcinoma (OSCC). The purpose of this study was to investigate the biological functions of circ_0001971 and associated mechanisms in OSCC. The expression of circ_0001971, miR-194, and miR-204 was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Cell proliferation and viability were assessed using cell counting kit-8 (CCK-8) assay. Cell migration and invasion were examined using the transwell assay. Cell apoptosis was monitored by flow cytometry assay. The protein levels of proliferation marker (CyclinD1), epithelial mesenchymal-transition (EMT) markers (E-cadherin (E-cad) and N-cadherin (N-cad)) and apoptosis markers (Cleaved-caspase-3 (Cleaved-cas-3) and Cleaved-caspase-9 (Cleaved-cas-9)) were measured by Western blot. The relationship between circ_0001971 and miR-194 or miR-204 was predicted by online tool starBase and verified by the Dual-Luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Tumor formation assay in nude mice was conducted to observe the role of circ_0001971 in vivo. The expression of circ_0001971 was significantly increased in tumor tissues and cell lines. Circ_0001971 knockdown inhibited cell proliferation, migration, and invasion but promoted cisplatin (DDP) sensitivity and cell apoptosis. It was confirmed that miR-194 and miR-204 were targets of circ_0001971, and miR-194 inhibition or miR-204 inhibition reversed the effects of circ_0001971 knockdown in OSCC cells. Moreover, circ_0001971 knockdown impeded tumorigenesis and development in vivo. Circ_0001971 regulates cell proliferation, migration, invasion, apoptosis, and chemosensitivity of OSCC by interacting with miR-194 and miR-204 in vitro and in vivo. We provided a theoretical basis for the action mechanism of circ_0001971 on OSCC progression and chemosensitivity.

Antitumor Activity of Ficus deltoidea Extract on Oral Cancer: An In Vivo Study.

Background The aim of this study is to evaluate the chemopreventive and chemotherapeutic activities of Ficus deltoidea (FD) in an animal model induced for oral cancer using 4-nitroquinoline-1-oxide (4NQO). Methods Male Sprague-Dawley (SD) rats were randomized into six groups (n = 7 per group): Group 1 (untreated group); Group 2 (control cancer group) received 4NQO only for 8 weeks in their drinking water; Groups 3 and 4 (chemopreventive) received 4NQO for 8 weeks and were simultaneously treated with FD extract at 250 and 500 mg/kg, respectively, by oral gavage; Groups 5 and 6 (chemotherapeutic) received 4NQO for 8 weeks followed by the administration of FD extract at 250 and 500 mg/kg, respectively, for another 10 weeks. The incidence of oral cancer was microscopically evaluated. Moreover, immunohistochemical expression was analysed in tongue specimens using an image analyser computer system, while the RT2 profiler PCR array method was employed for gene expression analysis. Results The results of the present study showed a beneficial regression effect of the FD extract on tumor progression. The FD extract significantly reduced the incidence of oral squamous cell carcinoma (OSCC) from 100% to 14.3% in the high-dose groups. The immunohistochemical analysis showed that the FD extract had significantly decreased the expression of the key tumor marker cyclin D1 and had significantly increased the expression of the β-catenin and e-cadherin antibodies that are associated with enhanced cellular adhesion. Based on the gene expression analysis, FD extract had reduced the expression of the TWIST1 and RAC1 genes associated with epithelial-mesenchymal transition (EMT) and had significantly downregulated the COX-2 and EGFR genes associated with cancer angiogenesis, metastasis, and chemoresistance. Our data suggest that the FD extract exerts chemopreventive and chemotherapeutic activities in an animal model induced for oral cancer using 4NQO, thus having the potential to be developed as chemopreventive and chemotherapeutic agents.β-catenin and e-cadherin antibodies that are associated with enhanced cellular adhesion. Based on the gene expression analysis, FD extract had reduced the expression of the TWIST1 and RAC1 genes associated with epithelial-mesenchymal transition (EMT) and had significantly downregulated the COX-2 and EGFR genes associated with cancer angiogenesis, metastasis, and chemoresistance. Our data suggest that the FD extract exerts chemopreventive and chemotherapeutic activities in an animal model induced for oral cancer using 4NQO, thus having the potential to be developed as chemopreventive and chemotherapeutic agents.TWIST1 and RAC1 genes associated with epithelial-mesenchymal transition (EMT) and had significantly downregulated the COX-2 and EGFR genes associated with cancer angiogenesis, metastasis, and chemoresistance. Our data suggest that the FD extract exerts chemopreventive and chemotherapeutic activities in an animal model induced for oral cancer using 4NQO, thus having the potential to be developed as chemopreventive and chemotherapeutic agents.RAC1 genes associated with epithelial-mesenchymal transition (EMT) and had significantly downregulated the COX-2 and EGFR genes associated with cancer angiogenesis, metastasis, and chemoresistance. Our data suggest that the FD extract exerts chemopreventive and chemotherapeutic activities in an animal model induced for oral cancer using 4NQO, thus having the potential to be developed as chemopreventive and chemotherapeutic agents.COX-2 and EGFR genes associated with cancer angiogenesis, metastasis, and chemoresistance. Our data suggest that the FD extract exerts chemopreventive and chemotherapeutic activities in an animal model induced for oral cancer using 4NQO, thus having the potential to be developed as chemopreventive and chemotherapeutic agents.EGFR genes associated with cancer angiogenesis, metastasis, and chemoresistance. Our data suggest that the FD extract exerts chemopreventive and chemotherapeutic activities in an animal model induced for oral cancer using 4NQO, thus having the potential to be developed as chemopreventive and chemotherapeutic agents.

A Polyphenol-Rich Extract From Muscadine Grapes Inhibits Triple-Negative Breast Tumor Growth.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that tends to affect young women and has a high propensity to metastasize. No targeted treatments are available for this type of breast cancer due to a lack of estrogen or progesterone receptors or overexpression of human epidermal growth factor receptor type 2 overexpression. Currently, patients have no therapeutic options once standard of care is complete, indicating a need for safe and effective therapies to slow or prevent the progression of TNBC to metastatic disease. Studies showed that isolated polyphenols or polyphenol-rich muscadine grape extracts polyphenols inhibit the proliferation of various cancer cells including breast cancer. A proprietary muscadine grape extract (MGE) was administered to nude mice with human MDA-MB-231 TNBC atumors for 4 weeks to determine the effect of the extract on tumor growth. MGE decreased tumor volume in association with a reduction in the proliferative markers Ki67 and cyclin D1. To determine the molecular mechanisms for the MGE-induced reduction in tumor growth, mouse 4T1, MDA-MB-231, or human BT-549 TNBC cells were treated with MGE, and various signaling pathways were investigated. MGE reduced c-Met, differentially abrogated ERK/MAPK and AKT signaling, and decreased a downstream targets of ERK/MAPK and AKT pathways, cyclin D1. Cyclin D1 reduction was associated with retinoblastoma activation and cell cycle arrest in MDA-MB-231 TNBC cells. MGE-regulated molecular signaling pathways were functionally associated with a dose-dependent reduction in cell proliferation. The pluripotency of MGE and high index of safety and tolerability suggest that the extract may serve as a therapeutic to reduce TNBC progression to metastatic disease.

Roles of Id1/HIF-1 and CDK5/HIF-1 in cell cycle reentry induced by amyloid-beta peptide in post-mitotic cortical neuron

One neurotoxic mechanism of amyloid-beta peptide (Aβ), the major component of senile plaques in the brains of Alzheimer's disease (AD) patients, is to trigger cell cycle reentry in fully differentiated neurons. However, the detailed underlying mechanisms remain unclear. Using Aβ25-35–treated primary rat cortical neurons as the experimental system, in the present study we tested whether Aβ-induced inhibitor of differentiation-1 (Id1)/hypoxia-inducible factor-1alpha (HIF-1α) and cyclin-dependent kinase-5 (CDK5) contribute to cell cycle reentry in fully differentiated post-mitotic neurons. We found that Id1-induced HIF-1α mediated Aβ25-35–dependent expression of the cell cycle markers cyclin D1 and proliferating cell nuclear antigen (PCNA), both colocalized with microtubule-associated protein-2 (MAP-2) + cells, indicative of cell cycle reentry in the mature neurons. Aβ25-35 also enhanced p35 cleavage to p25 without affecting CDK5 expression. The CDK5 inhibitor roscovitine and the siRNA targeting CDK5 both suppressed Aβ25-35–dependent HIF-1α expression and cell cycle reentry. Intriguingly, Aβ25-35–induced Id1 repressed p25 production while CDK5/p25 reciprocally inhibited Id1 expression, despite the observation that both Id1 and CDK5/p25 acted upstream of HIF-1α. These results demonstrated that both Id1/HIF-1 and CDK5/HIF-1 contribute to Aβ-induced cell cycle reentry in post-mitotic neurons; furthermore, Id1 and CDK5/p25 reciprocally suppress expression of each other.

Activation of β-Catenin Signaling and its Crosstalk With Estrogen and Histone Deacetylases in Human Uterine Fibroids.

Uterine fibroids (UF) are the most common benign tumor of the myometrium (MM) in women of reproductive age. However, the mechanism underlying the pathogenesis of UF is largely unknown. To explore the link between nuclear β-catenin and UF phenotype and β-catenin crosstalk with estrogen and histone deacetylases (HDACs). Protein/RNA levels of β-catenin (CTNNB1 gene), its responsive markers cyclin D1 and c-Myc, androgen receptor (AR), p27, and class-I HDACs were measured in matched UF/MM tissues or cell populations. The effects of chemical inhibition/activation and genetic knockdown of CTNNB1 on UF phenotype were measured. The anti-UF effect of 2 HDAC inhibitors was evaluated. β-catenin nuclear translocation in response to β-catenin inhibition/activation, estrogen, and HDAC inhibitors in UF cells. UF tissues/cells showed significantly higher expression of nuclear β-catenin, cyclin D1, c-Myc, and HDACs 1, 2, 3, and 8 than MM. Estradiol induced β-catenin nuclear translocation and consequently its responsive genes in both MM and UF cells, while an estrogen receptor antagonist reversed this induction effect. Treatment with β-catenin or HDAC inhibitors led to dose-dependent growth inhibition, while Wnt3a treatment increased proliferation compared with control. Chemical inhibition of β-catenin decreased cyclin D1 and c-Myc expression levels, while β-catenin activation increased expression of the same markers. Genetic knockdown of CTNNB1 resulted in a marked decrease in β-catenin, cyclin D1, c-Myc, and AR expression. Treatment of UF cells with HDAC inhibitors decreased nuclear β-catenin, cyclin D1, and c-Myc expression. Moreover, HDAC inhibitors induced apoptosis of UF cells and cell cycle arrest. β-catenin nuclear translocation contributes to UF phenotype, and β-catenin signaling is modulated by estradiol and HDAC activity.

Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6

BackgroundLong non-coding RNAs (lncRNAs) are tumor-associated biological molecules and have been found to be implicated in the progression of colorectal cancer (CRC). This study aims to examine the effects of lncRNA RP11-468E2.5 and its target genes (STAT5 and STAT6) on the biological activities of CRC cells via the Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway.MethodsWe initially screened the GEO database for differentially expressed lncRNAs related to CRC and then made a prediction of the implicated target genes. Then we collected CRC tissues and adjacent normal tissues from 169 CRC patients. Human CRC HCT116 and SW480 cells were treated with small interference RNA (siRNA) against RP11-468E2.5, AG490 (an inhibitor of the JAK/STAT signaling pathway), or both in combination. Next, we measured the effects of RP11-468E2.5 treatment on cellular activities such as cell viability, cycle distribution and cell apoptosis, and studied interactions among RP11-468E2.5, STAT5/STAT6, and the JAK/STAT signaling pathway. Finally, an in vivo tumor formation assay was performed to observe the effect of RP11-468E2.5 on tumor growth.ResultsThe CRC-related gene microarray data showed low expression of RP11-468E2.5 in CRC surgical specimens. However, RP11-468E2.5 was confirmed to target STAT5 and STAT6, which participate in the JAK/STAT signaling pathway. CRC tissues showed lower expression of RP11-468E2.5, higher expression of STAT5, STAT6 and of the cell cycle marker Cyclin D1 (CCND1), compared to the findings in adjacent normal tissues. The treatment of siRNA against RP11-468E2.5 increased expression of JAK2, STAT3, STAT5, STAT6, CCND1 and Bcl-2 along with the extent of STAT3, STAT5 and STAT6 phosphorylation, while lowering expression of P21 and P27. Treatment with AG490 exhibited approximately opposite effects, whereas siRNA against RP11-468E2.5 treatment stimulated CRC cell proliferation and reduced cell apoptosis, while promoting cell cycle entry; AG490 treatment reversed these results.ConclusionsAltogether, we conclude that up-regulation of RP11-468E2.5 inhibits the JAK/STAT signaling pathway by targeting STAT5 and STAT6, thereby suppressing cell proliferation and promoting cell apoptosis in CRC.

Role of molecular markers in glioblastoma patients of Pakistan

Abstract Background Glioma is the most common primary brain malignancy with a largely unknown pathogenesis, although complex genetic abnormalities are thought to contribute to disease development. Several independent studies have investigated the expression of Epidermal Growth Factor Receptor (EGFR), Cyclooxygenase 2 (COX-2), p53 and Cyclin D1 in gliomas. However, conflicting evidence exists for expression and prognostic significance of these markers in glioma. Furthermore, no work relating to these genes has been performed on the glioma population of Pakistan. Our objective was to investigate the expression and prognostic significance of molecular markers p53, EGFR, COX-2 and Cyclin D1 in glioma in the Pakistani population. Methods Immunohistochemistry of markers p53, EGFR, COX-2 and Cyclin D1 was performed on 100 formalin-fixed paraffin-embedded specimens of patients diagnosed and treated atAga Khan University Hospital, Karachi, Pakistan. Expression was correlated with patient clinicopathological characteristics and overall survival. Results The prevalence of expression of Cyclin D1, EGFR, COX-2, and p53 was 57%, 52%, 54% and 39%, respectively. p53 and EGFR expression were positively correlated with younger and older age respectively. Chi squared test revealed a significant association between EGFR and Cyclin D1, with EGFR expression decreasing with increasing strength of Cyclin D1 staining. Only patient age was found to be a significant predictor for overall survival. Conclusions There is a relatively low prevalence of COX-2 and EGFR in the Pakistani population as compared to published literature. We also observed a correlation between EGFR and Cyclin D1 expression. The use of these biomarkers as targets for personalized therapies is worth investigating in the Pakistani population. Legal entity responsible for the study Aga Khan University, Karachi, Pakistan. Funding University Research Council, Aga Khan University, Karachi, Pakistan. Disclosure All authors have declared no conflicts of interest.

In vitro evaluation of Naltrexone HCl 1% Topical Cream in XemaTop™ for psoriasis.

Psoriasis is a multifactorial skin disease involving abnormal cell proliferation and inflammation; an efficacious topical treatment is yet to be identified. A formulation containing 1% Naltrexone HCl in XemaTop™ base was compounded, characterized and evaluated in vitro as a possible treatment for psoriasis. A three-dimensional psoriasis tissue model was exposed to the formulation for 2 or 5days and analyzed for the level of markers of cellular proliferation, and inflammatory cytokine IL-6. Using immunohistochemical staining, the level of Ki67 protein significantly decreased in the drug-treated tissues. Western blot analysis showed 86% and 53% down-regulation of other proliferation markers PCNA and CYCLIN D1, respectively, after 5-day exposure. The pro-survival Wnt/β-catenin pathway was compromised as indicated by 57% decrease in the level of β-CATENIN and down-regulation of its down-stream targets including CYCLIN D1 (decreased by 53%), c-MYC (63%), c-JUN (92%) and MET (96%) proteins. Likewise, the PI3K/AKT/mTOR pathway was significantly inhibited by 1% Naltrexone HCl in XemaTop™, suggesting protein synthesis was affected. The production of IL-6 was inhibited by 70% in drug-treated tissues. These results suggest that the compounded drug is efficacious in down-regulating molecular markers associated with the pathogenesis of psoriasis. Low-dose Naltrexone in XemaTop™ was stable within 180days when stored under refrigerated or ambient conditions. These results provide a basis for a clinical evaluation of 1% Naltrexone HCl in XemaTop™ in psoriasis patients.

Beyond its antioxidant properties: Quercetin targets multiple signalling pathways in hepatocellular carcinoma in rats

AimsHepatocellular carcinoma (HCC) pathogenesis involves the interplay of multiple signalling pathways. Notch and Hedgehog (Hh) are two major developmental pathways that act in concert to regulate adult cell repair. CK2α -serine-threonine kinase-down-regulation enhanced apoptotic activity and was proven beneficial for HCC patients. Quercetin is a bioactive flavonoid and has been shown to protect against HCC through its antioxidant activity. This study was carried out to elucidate the antineoplastic effect of quercetin through regulating both Notch and Hh pathways, apoptosis, cell proliferation and CK2α activity. Main methodsHepatocellular carcinoma was induced in male Sprague Dawley rats by thioacetamide. Quercetin was administered in both protective and curative doses. Parameters of liver function and oxidative stress were assessed. CK2α, Notch and Hh pathways were evaluated using RT-PCR and ELISA. Apoptosis was investigated by detecting caspase-3, caspase-8 and p53. Proliferative and cell cycle markers as cyclin D1 and Ki-67 were detected immunohistochemically. Key findingsQuercetin inhibited CK2α and downregulated mRNA and protein expression of Notch1 and Gli2. Quercetin also suppressed caspase-3 expression but not caspase-8. Quercetin elevated p53 expression whereas proliferative and cell cycle markers cyclin D1 and Ki-67 were downregulated. Markers of hepatic cellular integrity such as AST, ALT, ALP, GGT, albumin and bilirubin were significantly ameliorated. This was confirmed by histological examination. Quercetin also alleviated oxidative stress as shown by SOD, GSH, MDA and NO levels. SignificanceWe can conclude that in addition to its antioxidant power, quercetin blocked Notch, Hedgehog, regulated the apoptotic and proliferative pathways and inhibited CK2α in HCC.

1,25 Dihydroxyvitamin D3 Enhances the Antifibroid Effects of Ulipristal Acetate in Human Uterine Fibroids.

Both European and American trials showed superior effect of ulipristal acetate (UPA) to placebo. However, the latter, which included black patients with known higher vitaminD3 deficiency risk, showed lower rate of amenorrhea responders and insignificant uterine fibroid (UF) size reduction. Our objective is to investigate whether adding vitamin D3 to UPA can enhance UPA potency on UF phenotype in vitro. We screened the antiproliferative effect of different (UPA/vitaminD3) combinations on UF cell proliferation using dimethylthiazolyl diphenyltetrazolium bromide assay. Cells then were treated with UPA 100 nM in the presence or absence of vitamin D3 100 nM, and expression level of several markers related to proliferation, apoptosis, fibrosis, inflammation, and angiogenesis was measured on RNA or at protein level using quantitative real-time polymerase chain reaction, Western blot, immunofluorescence, or multiplex enzyme-linked immunosorbent assay techniques. Significant dose- and time-dependent growth inhibitory effects of UPA/vitamin D3 combinations were observed compared to untreated cells at 2 and 4 days (P < .05). Importantly, vitamin D3/UPA combination significantly reduced cell proliferation as compared to UPA at 2, 4, 6, and 8 days (P < .05). Combination treatment significantly decreased protein expression of proliferation markers Ki-67, PCNA, and CyclinD1 by more than 50% compared to UPA (P < .05) along with a significant increase in apoptosis induction. Combination treatment resulted in a 2-fold decrease in protein levels of extracellular matrix markers collagen-1 and fibronectin besides pro-fibrogenic cytokine transforming growth factor β3 (P < .05). Moreover, it significantly decreased the production of pro-inflammatory cytokines interleukins 6, 8, 1α, and 1β compared to UPA (P < .05). Combination of vitamin D3 with UPA exhibits additional and orchestrated anti-UF effects, therefore might offer a more favorable clinical option.

Selected molecular markers as indicators of clinical profile, tumor characteristics and treatment outcome in squamous cell carcinoma of the larynx.

e17522 Background: Laryngeal Squamous cell carcinoma is widely prevalent in India and is the fourth leading cancer in males. The possibility that specific tumour characteristics (demonstrable by immunohistochemistry) may aid the clinician in choosing optimal treatments for a specific tumour, holds great promise. Objectives of the present study were to study the expression of select molecular markers p53, Bcl-2 (Apoptotic markers), EGFR, Ki67, Cyclin D1 (Proliferation markers) and Cox-2 (Inflammatory marker) in LSCC so as to form a hypothesis that a particular group of markers will be useful in determining the prognosis of LSCC. Methods: We studied 72 cases of laryngeal squamous cell carcinoma in an attempt to determine relationship between their clinicopathological characteristics and treatment outcome with the six molecular markers. Samples from patients who underwent laryngectomy from Jan 2006 to Dec 2010 were taken and analyzed prospectively and followed up till Dec 2015 or till death. . Immunoreactivity in tissue sections was evaluated as negative when no positive cells were observed within the tumor, weak (1+) , moderate (2+) , and strong or intense (3+) . Data were analyzed using SPSS software package. Chi squared test or exact test were used to test the associations. Logistic regression analysis was used to estimate the odds ratios and 95% confidence intervals. The disease free survival time of each patient was calculated by taking the difference between the date of surgery and the recurrence date/ death date/ the last follow up date whichever is applicable. Results: Proliferation markers EGFR, Cyclin D1 and Ki 67, individually and collectively were predictive of extracapsular spread and perineural spread of tumour. Node negative patients having intense expression of Cyclin D1 or Ki 67 has bad prognosis.. The significant expression of Cox-2 was highly predictive of Node positivity. In older patients and in T4 stage the presence of P53 / Bcl-2 resulted in worse overall survival. Markers of aggressiveness were identified as p53, Bcl-2 Cox-2. Markers of invasiveness were EGFR, Cyclin D1 and Ki 67. Markers predicting survival were p53, BCl-2, Cyclin D1 and Ki 67. Conclusions: If Proliferation markers are detected preoperatively in biopsy specimens, has important implications in planning aggressive management Cyclin D1 or Ki 67 in Node negative patients indicate need for a neck dissection or irradiation. Cox-2 assessment will be useful in predicting the occult nodal metastasis for prophylactic treatment

A Novel lncRNA IHS Promotes Tumor Proliferation and Metastasis in HCC by Regulating the ERK- and AKT/GSK-3β-Signaling Pathways

Long noncoding RNAs (lncRNAs) are involved in a variety of biological processes such as tumor proliferation and metastasis. A close relationship between hepatitis B virus X protein (HBx) and SMYD3 in promoting the proliferation and metastasis of hepatocellular carcinoma (HCC) was recently reported. However, the exact oncogenic mechanism of HBx-SMYD3 remains unknown. In this study, by performing lncRNA microarray analysis, we identified a novel lncRNA that was regulated by both HBx and SMYD3, and we named it lncIHS (lncRNA intersection between HBx microarray and SMYD3 microarray). lncIHS was overexpressed in HCC and decreased the survival rate of HCC patients. Knockdown of lncIHS inhibited HCC cell migration, invasion, and proliferation, and vice versa. Further study showed that lncIHS positively regulated the expression of epithelial mesenchymal transition (EMT)-related markers c-Myc and Cyclin D1, as well as the activation of the ERK- and AKT-signaling pathways. lncIHS exerted its oncogenic effect through ERK and AKT signaling. Moreover, results from transcriptome-sequencing analysis and mass spectrometry showed that lncIHS regulated multiple genes that were the upstream molecules of the ERK- and AKT-signaling pathways. Therefore, our findings suggest a regulatory network of ERK and AKT signaling through lncIHS, which is downstream of HBx-SMYD3, and they indicate that lncIHS may be a potential target for treating HCC.

Secukinumab attenuates reactive astrogliosis via IL-17RA/(C/EBPβ)/SIRT1 pathway in a rat model of germinal matrix hemorrhage.

AimsReactive astrogliosis plays a critical role in neurological deficits after germinal matrix hemorrhage (GMH). It has been reported that interleukin‐17A and IL‐17A receptor IL‐17RA/(C/EBPβ)/SIRT1 signaling pathway enhances reactive astrogliosis after brain injuries. We evaluated the effects of secukinumab on reactive astrogliosis in a rat pup model of GMH.MethodsA total of 146 Sprague Dawley P7 rat pups were used. GMH was induced by intraparenchymal injection of collagenase. Secukinumab was administered intranasally 1 hour post‐GMH. C/EBPβ CRISPR or SIRT1 antagonist EX527 was administrated intracerebroventricularly (icv) 48 hours and 1 hour before GMH induction, respectively. Neurobehavior, Western blot, histology, and immunohistochemistry were used to assess treatment regiments in the short term and long term.ResultsThe endogenous IL‐17A, IL‐17RA, C/EBPβ, and GFAP and proliferation marker CyclinD1 were increased, while SIRT1 expression was decreased after GMH. Secukinumab treatment improved neurological deficits, reduced ventriculomegaly, and increased cortical thickness. Additionally, treatment increased SIRT1 expression and lowered proliferation proteins PCNA and CyclinD1 as well as GFAP expression. C/EBPβ CRISPR activation plasmid and EX527 reversed the antireactive astrogliosis effects of secukinumab.ConclusionSecukinumab attenuated reactive astrogliosis and reduced neurological deficits after GMH, partly by regulating IL‐17RA/(C/EBPβ)/SIRT1 pathways. Secukinumab may provide a promising therapeutic strategy for GMH patients.

691 In vitro evaluation of naltrexone HCl 1% topical cream in xematop for psoriasis

Psoriasis is a multifactorial skin disease involving abnormal cell proliferation and inflammation; an efficacious topical treatment is yet to be identified. A topical formulation containing 1% naltrexone HCl in xematop base was compounded, characterized and evaluated in vitro as a possible treatment for psoriasis. A three-dimensional psoriasis tissue model was exposed to the formulation for 2 or 5 days and analyzed for the level of markers of cellular proliferation, and inflammatory cytokine IL-6. Using immunohistochemical staining, the level of Ki67 protein significantly decreased in the drug-treated tissues. Western blot analysis showed 86% and 53% down-regulation of other proliferation markers PCNA and CYCLIN D1, respectively, after 5-day exposure. The pro-survival Wnt/β-catenin pathway was compromised as indicated by 57% decrease in the level of β-CATENIN and down-regulation of its downstream targets including CYCLIN D1 (decreased by 53%), c-MYC (63%), c-JUN (92%) and MET (96%) proteins. Likewise, the PI3K/AKT/mTOR pathway was significantly inhibited by 1% naltrexone HCl in xematop, suggesting protein synthesis was affected. The production of IL-6 was inhibited by 70% in drug-treated tissues. These results suggest that the compounded drug is efficacious in down-regulating molecular markers associated with the pathogenesis of psoriasis. Low-dose naltrexone in xematop was stable within 180 days when stored under refrigerated or ambient conditions. These results provide a basis for a clinical evaluation of 1% naltrexone HCl in xematop in psoriasis patients.

The absence of the adaptor proteins CARDIF/STING negatively regulates the prolfieration of liver regeneration after partial hepatectomy in mice

Background: Nucleic acid sensors are known to play a major role during liver regeneration, however, there is only limited knowledge on the role of central adaptor proteins, such as CARDIF and STING. Our hypothesis states that these modulators of the innate immune system have an influence on liver regeneration. Methods: We use a newly established CARDIF/STING knockout mouse model and a C57BL/6 control cohort. A 2/3 partial hepatectomy (PH) was performed in both groups (n=60 mice in total). Liver regeneration was quantified by liver-to-body weight ratio, proliferation was determined by BrdU staining, RNA- and protein levels of proliferation markers were analyzed in resected tissues and pro-inflammatory cytokines were measured in murine serum. Results: CARDIF/STING knockout mice showed a significantly impaired liver regeneration after PH. Expression analysis of Interleukin-6 revealed a significant decrease in the wild type cohort after PH. In CARDIF/STING knockout mice, the proliferation marker Cyclin D1 was decreased until 24 hours after surgery, while p21Cip1 expression was increased 8h post-PH. Conclusion: Together, these data provide the first experimental evidence that CARDIF/STING knockout mice show a delayed liver regeneration. In the absence of these two proteins, we observed a modulated immune response which lead to a negative regulation of the proliferative capacity after partial hepatectomy. This finding could impact on the future development of molecular therapies making use of the innate immune system.

The absence of the adaptor proteins CARDIF/STING negatively regulates the prolfieration of liver regeneration after partial hepatectomy in mice

Background: Nucleic acid sensors are known to play a major role during liver regeneration, however, there is only limited knowledge on the role of central adaptor proteins, such as CARDIF and STING. Our hypothesis states that these modulators of the innate immune system have an influence on liver regeneration. Methods: We use a newly established CARDIF/STING knockout mouse model and a C57BL/6 control cohort. A 2/3 partial hepatectomy (PH) was performed in both groups (n=60 mice in total). Liver regeneration was quantified by liver-to-body weight ratio, proliferation was determined by BrdU staining, RNA- and protein levels of proliferation markers were analyzed in resected tissues and pro-inflammatory cytokines were measured in murine serum. Results: CARDIF/STING knockout mice showed a significantly impaired liver regeneration after PH. Expression analysis of Interleukin-6 revealed a significant decrease in the wild type cohort after PH. In CARDIF/STING knockout mice, the proliferation marker Cyclin D1 was decreased until 24 hours after surgery, while p21Cip1 expression was increased 8h post-PH. Conclusion: Together, these data provide the first experimental evidence that CARDIF/STING knockout mice show a delayed liver regeneration. In the absence of these two proteins, we observed a modulated immune response which lead to a negative regulation of the proliferative capacity after partial hepatectomy. This finding could impact on the future development of molecular therapies making use of the innate immune system.

CREB activation in hypertrophic chondrocytes is involved in the skeletal overgrowth in epiphyseal chondrodysplasia Miura type caused by activating mutations of natriuretic peptide receptor B.

Natriuretic peptide receptor B (NPRB) produces cyclic guanosine monophosphate (cGMP) when bound by C-type natriuretic peptide (CNP). Activating mutations in NPRB cause a skeletal overgrowth disorder, which has been named epiphyseal chondrodysplasia, Miura type (ECDM; OMIM #615923). Here we explored the cellular and molecular mechanisms for the skeletal overgrowth in ECDM using a mouse model in which an activating mutant NPRB is specifically expressed in chondrocytes. The mutant mice (NPRB[p.V883M]-Tg) exhibited postnatal skeletal overgrowth and increased cGMP in cartilage. Both endogenous and transgene-derived NPRB proteins were localized at the plasma membrane of hypertrophic chondrocytes. The hypertrophic zone of growth plate was thickened in NPRB[p.V883M]-Tg. An in vivo BrdU-labeling assay suggested that some of the hypertrophic chondrocytes in NPRB[p.V883M]-Tg mice continued to proliferate, although wild-type (WT) chondrocytes stopped proliferating after they became hypertrophic. In vitro cell studies revealed that NPRB activation increased the phosphorylation of cyclic AMP-responsive element binding protein (CREB) and expression of cyclin D1 in matured chondrocytes. Treatment with cell-permeable cGMP also enhanced the CREB phosphorylation. Inhibition of cyclic adenosine monophosphate (cAMP)/protein kinase A pathway had no effects on the CREB phosphorylation induced by NPRB activation. In immunostaining of the growth plates for the proliferation marker Ki67, phosphorylated CREB and cyclin D1, most signals were similarly observed in the proliferating zone in both genotypes, but some cells in the hypertrophic zone of NPRB[p.V883M]-Tg were also positively stained. These results suggest that NPRB activation evokes its signal in hypertrophic chondrocytes to induce CREB phosphorylation and make them continue to proliferate, leading to the skeletal overgrowth in ECDM.