High-glucose-induced hyperosmolar stress sensitizes HepG2 cell lines to sorafenib.

Sorafenib is an FDA approved chemotherapeutic against hepatocellular carcinoma (HCC) yet associated with various resistance mechanisms. The role of high glucose status on sorafenib action is still to be elucidated. This study clarifies such interaction, taking HepG2 cell lines as HCC models, MALAT1 and H19 as molecular players. HepG2 cell lines were purchased and classified into 8 groups. High glucose status was set by using d-glucose (33 mM) with insulin (1 µM). Mannitol (27.5 mM) was used as a negative osmotic control. Sorafenib was prepared at 15 µM and 20 µM. Cellular viability was assessed with MTT viability assay. Then, with trypan blue viability assay, the results were double checked and HepG2 morphology was examined by optical microscopy. MALAT1 and H19 RQs were assessed by real time PCR (RT-PCR). Results show that in comparison with sorafenib impact on HepG2, high glucose status drops cellular viability to 83.13 % (p < 0.01). With hyperosmolar mannitol, it decreases cellular viability to 72.89 % (p < 0.001). Regarding the molecular impact, hyperosmolar mannitol with sorafenib elevates both MALAT1 and H19 RQs. Yet, high glucose status elevates MALAT1and declines H19 (p < 0.05 and p < 0.001 for MALAT1 and H19 comparisons respectively). Therefore, the impact of high glucose status could be, in part, attributed to the hyperosmolar stress it induces on HepG2. Also, hyperosmolar mannitol, owing to its cytotoxic impact, is recommended for further confirmatory studies either as a separate therapeutic or as an adjuvant to sorafenib.

A novel protective role of sacubitril/valsartan in cyclophosphamide induced lung injury in rats: impact of miRNA-150-3p on NF-κB/MAPK signaling trajectories.

Cyclophosphamide (CP) is a chemotherapeutic agent that induces oxidative stress causing multiple organ damage. Sacubitril/valsartan, is a combined formulation of neprilysin inhibitor (sacubitril) and angiotensin II receptor blocker (valsartan), that induces the protective effect of brain natriuretic peptide. The aim of the current study is to investigate the prophylactic impacts of sacubitril/valsartan versus valsartan against CP-induced lung toxicity in rats. Rats were assigned randomly into 6 groups; control; received corn oil (2 ml/kg/day; p.o. for 6 days), sacubitril/valsartan (30 mg/kg; p.o. for 6 days), valsartan (15 mg/kg; p.o. for 6 days), CP (200 mg/kg; i.p. on day 5), sacubitril/valsartan + CP (30 mg/kg; p.o. for 6 days, 200 mg/kg; i.p. single dose on day 5, respectively), valsartan + CP (15 mg/kg; p.o. for 6 days, 200 mg/kg; i.p. single dose on day 5, respectively). Both sacubitril/valsartan and valsartan produced a significant decrease in the inflammation and fibrosis markers in the BALF, in comparison with the CP group. Both sacubitril/valsartan and valsartan produced an apparent decrease in the relative genes expression of miR-150-3p and NF-κB, as well as a significant decrease in the relative expression of P38 and ERK1/2 MAPKs and an increase in the relative gene expression of Nrf-2, compared to CP group. Intriguingly, sacubitril/valsartan , showed subtle superiority in almost all investigated parameters, compared to valsartan. In conclusion, sacubitril/valsartan effectively abrogated the CP induced lung inflammation and fibrosis, providing a potential promising protection that could be linked to their ability to inhibit miR-150-3p via inhibition of NF-κB and MAPK signaling pathways.

Long non-coding RNAs (CASC2 and TUG1) in hepatocellular carcinoma: Clinical significance.

BACKGROUND: The biology of hepatocellular carcinoma remains poorly understood. Long non-coding RNAs (lncRNAs) have been confirmed to be key regulators of most cell processes and cancer. The lncRNA cancer susceptibility candidate 2 (CASC2) was originally identified as a downregulated gene in endometrial cancer and acted as a tumor suppressor. The lncRNA taurine up-regulated gene 1 (TUG1) has been shown to play an oncogenic role in various cancers. However, the relative expression of CASC2 and TUG1 in hepatocellular carcinoma (HCC) on top of hepatitis C virus (HCV) and the relationship between both remains unclear. The present study aimed to evaluate both lncRNA CASC2 and TUG1 relative gene expression in whole blood of HCC/HCV patients in relation to HCV and healthy subjects and to relate them to each other and to different clinicopathological factors. METHODS: The relative expression of CASC2 and TUG1 was estimated by a quantitative reverse transcriptase-polymerase chain reaction in 30 HCC/HCV patients and compared with 20 cases of HCV patients and 20 controls. RESULTS: CASC2 was downregulated in HCC/HCV patients, whereas TUG1 was overexpressed in relation to HCV and the control group, indicating their antagonistic effect. This suggests their role in the pathogenesis of HCC on top of HCV. Their expression was correlated to Barcelona Clinic Liver Cancer stage and serum alpha-fetoprotein level. CONCLUSIONS: CASC2 and TUG1 could be new potential biomarkers with a valid non-invasive technique.

MicroRNA-21 as a predictor and prognostic factor for trastuzumab therapy in human epidermal growth factor receptor 2-positive metastatic breast cancer.

Breast cancer is the second most common cancer diagnosed worldwide. Human epidermal growth factor receptor 2 (HER2)-positive breast cancer represents about 20% to 30% of all breast cancers. Trastuzumab is used in the treatment of HER2-positive breast cancer. MicroRNA-21 (miR-21) is an oncomiR that acts by inhibiting many tumor-suppressor genes. We analyzed the relative expression levels of serum miR-21 in 20 HER2-positive metastatic breast cancer patients before and after 3 months of treatment with trastuzumab. miR-21 levels decreased with a high significant difference after trastuzumab therapy (P = 0.001). Although miR-21 expression levels were lower in responders than in nonresponders, the difference was not statistically significant ( P = 0.6). Our results demonstrated a significant negative correlation between its basal expression, expression levels after treatment, and time to progression ( P = 0.03 and 0.01, respectively). These results make miR-21 a potential prognostic factor for HER2-positive metastatic breast cancer patients. Additionally, it can be an interesting potential target in therapy using antisense oligonucleotides for miR-21.

Autophagy in osteoporosis: Relation to oxidative stress.

Impaired autophagy and oxidative stress are implicated in the development of many diseases. This study aimed to investigate the involvement of autophagy represented by autophagy-related gene 7 (Atg7) and oxidative stress represented by superoxide dismutase 2 (SOD2) gene expression and enzyme activity in the pathogenesis of osteoporosis. Atg7 and SOD2 gene relative expression were evaluated by SYBR green quantitative real-time-polymerase chain reaction in the osteoporotic group (n = 26) versus the osteoporosis free group (n = 14). SOD2 enzyme activity was evaluated by colorimetric method in both study groups. Both Atg7 and SOD2 relative expression showed highly significant decrease (P < 0.01) between both groups. However, SOD2 enzyme activity showed no significant difference between the two groups. There was a significant direct correlation between Atg7 and SOD2 gene expression in both study groups. Atg7 relative expression showed significant ( P < 0.01) direct correlation with vitamin D serum levels and body mass index in osteoporotic group. In conclusion, both genes are involved in the pathogenesis of osteoporosis and this could be amenable to future therapeutic intervention.

Vitamin D receptor gene methylation in hepatocellular carcinoma.

Worldwide, hepatocellular carcinoma (HCC) is the major subtype of primary liver cancers. HCC is typically diagnosed late in its course. With respect to cancer, the genomic actions of vitamin D are mediated through binding to the Vitamin D Receptor (VDR), which allows it to modulate the expression of genes in a cell-and tissue-specific manner. Epigenetics is a rapidly evolving field of genetic study applicable to HCC. Changes in DNA methylation patterns are thought to be early events in hepatocarcinogenesis. Curcumin has great potential as an epigenetic agent. Accordingly, the current study has been designed to study the methylation status of VDR gene promoter for the first time in HCC aiming to find its clinical significance and potential screening role in chronic Liver Disease (CLD). Additionally, we aimed to investigate, the effect of Curcumin on HCC cell line, aiming to discover new therapeutic targets through epigenetics. This study was conducted on 45 formalin-fixed, paraffin-embedded liver tissue blocks including 15 HCC samples (group A), 15 CLD samples (group B) and 15 apparently normal tissue taken from around benign lesions (group C). Methylation Specific Restriction Digestion and qPCR were done on all samples after DNA extraction. The percentage of VDR gene promoter methylation was significantly higher in the HCC group compared to both CLD and control groups (p < 0.01). VDR promoter methylation by (MS-qPCR) was decreased and the relative expression of VDR by (qRT-PCR) was markedly increased in a dose-dependent fashion in cells grown in Curcumin-adequate medium. In conclusion, this study may open a new gate for the use of VDR promoter methylation as a potential biomarker in HCC.

Metformin potentiates cognitive and antidepressant effects of fluoxetine in rats exposed to chronic restraint stress and high fat diet: potential involvement of hippocampal c-Jun repression.

Several hypotheses link high fat diet (HFD) with the pathophysiology of depression and its response to antidepressants. This study aimed to determine the effect of metformin (MET) on the cognitive and antidepressant activity of fluoxetine (FLU) through its effect on c-Jun expression. Behavioral, cognitive function, biochemical, and histopathological studies were performed in non-HFD- and HFD-fed rats exposed to chronic restraint stress (CRS). Stressed group showed cognitive impairment, depressive-like symptoms, disturbed glucose homeostasis and lipid profile, reduced adiponectin level, brain-derived neurotrophic factor (BDNF) expression, and increased corticosterone and c-Jun. All these were aggravated by HFD. MET, FLU and their combination produced significant improvement in lipid profile with significant increase in adiponectin and BDNF expression. Corticosterone, body weight and insulin resistance showed significant decrease in the treated groups. Moreover, there was a significant decrease in hippocampal c Jun expression. There was a significant preferable effect toward the combination. Conclusion, MET may decrease the refractoriness to FLU and improves the cognition in individuals who are fed on HFD.

Vitamin D and K signaling pathways in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a primary liver malignancy, and is now the six most common in between malignancies. Early diagnosis of HCC with prompt treatment increases the opportunity of patients to survive. With the advances in understanding the molecular biology of HCC, new therapeutic strategies to treat HCC have emerged. There is a growing consensus that vitamins are important for the control of various cancers. Biochemical evidence clearly indicates that HCC cells are responsive to the inhibitory effect of vitamin D, vitamin D analogues and vitamin K. In this review, we summarize the mechanisms used by vitamin D and K to influence the development of HCC and the latest development of vitamin analogues for potential HCC therapy.

MicroRNA-181a and its target Smad 7 as potential biomarkers for tracking child acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common pediatric hematologic tumor. MiR-181a was expected to have a role in the development of hematological malignancies; it might act as tumor suppressor or oncogene. Smad7 was selected as miR-181a target pair. It is a negative regulator for the TGF-ß1 signaling pathway. In this study, relative expression levels of miR-181a by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), both Smad 7 and TGF-ß1 proteins levels by enzyme linked immunosorbent assay (ELISA) were all measured in serum of 60 child, 30 with ALL and 30 age and sex matched healthy child as control group. MiR-181a expression showed highly significant decrease; plus a significant increase and decrease of Smad7 and TGF-ß1 protein levels respectively, in serum samples of ALL as compared to control group. MiR-181a expression achieved a highly significant positive and a significant negative correlation with TGF-ß1 and Smad7 respectively. Furthermore, the levels of Smad7 and TGF-ß1 were negatively correlated with each other (p<0.05). Although, positivity rate of both Smad7 and TGF-ß1 in ALL group increased with presence of hepatosplenomegaly, still there was no statistical significance. In conclusion, miR-181a could act as a tumor suppressor in pediatric ALL with over expression of its target pair, Smad7. Smad7 regulates TGF-ß1 signaling via a negative feedback loop and mediates the interaction between TGF-ß1 and other signaling pathways; suggesting that Smad7 over expression may have therapeutic potential in ALL.

Role of microRNA-7 and selenoprotein P in hepatocellular carcinoma.

There is an obvious need to diagnose hepatocellular carcinoma using novel non-invasive and sensitive biomarkers. In this regard, the aim of this study was to evaluate and correlate both relative quantification of microRNA-7 using quantitative real time polymerase chain reaction and quantitative analysis of selenoprotein P using enzyme-linked immunosorbent assay in sera of hepatocellular carcinoma patients, chronic liver disease patients, as well as normal healthy subjects in order to establish a new diagnostic biomarker with a valid non-invasive technique. In addition, this study aimed to investigate whether changes in selenium supply affect microRNA-7 expression and selenoprotein P levels in human hepatocarcinoma cell line (HepG2). The results showed a highly significant decrease in serum microRNA-7 relative quantification values and selenoprotein P levels in malignant group in comparison with benign and control groups. The best cutoff for serum microRNA-7 and selenoprotein P to discriminate hepatocellular carcinoma group from benign and control groups was 0.06 and 4.30 mg/L, respectively. Furthermore, this study showed that changes in selenium supply to HepG2 cell line can alter the microRNA-7 profile and are paralleled by changes in the concentration of its target protein (selenoprotein P). Hence, serum microRNA-7 and selenoprotein P appear to be potential non-invasive diagnostic markers for hepatocellular carcinoma. Moreover, the results suggest that selenium could be used as an anticancer therapy for hepatocellular carcinoma by affecting both microRNA-7 and selenoprotein P.

Evaluation of MicroRNA-210 and Protein tyrosine phosphatase, non-receptor type 2 in Pre-eclampsia.

BACKGROUND: The precise origin of Pre-eclampsia (PE) remains elusive. Multiple pieces of evidence support the existence of hypoxia in PE. MiRNA-210 (miR-210), which is induced by Hypoxia-Inducible Factor-1α (HIF-1α) during hypoxia, is one of the most hypoxia sensitive miRNAs. MiR-210 mediates these functions by regulating a lot of target mRNAs. Protein tyrosine phosphatase, non-receptor type 2 (PTPN2) was one of miR-210 targets and was found to be down regulated by hypoxia. OBJECTIVE: To assess the levels of relative expression of miR-210 and its target PTPN2 in Egyptian women with PE. This is in order to clarify their possible role in the progression of PE and their relation to each other and to different clinicopathological factors. STUDY DESIGN: Group1 included 35 normal primigravida and group 2 included 35 primigravida patients with PE. PE group was subdivided into-mild and severe (PE). Total RNA was extracted from placental tissue samples and Real-Time PCR was performed on the extracted RNA. RESULTS: There was a highly significant difference between the studied groups as regards fold change of placental miR-210 and PTPN2 (P<0.01). There was a highly significant negative correlation between miR-210 and PTPN2 RQ among the studied groups and among the preeclampsia group (P<0.01). CONCLUSION: The results of this study demonstrated that placental expression of miR-210 was up regulated in pregnancies complicated with PE in comparison to normal pregnancies. This increase in miR-210 resulted in down regulation of its target PTPN2 mRNA and this can have a direct role in the pathogenesis of the PE disease. Additionally, both miR-210 & PTPN2 relative expression could differentiate between mild & severe PE.

Expression of nucleostemin gene in primary osteoarthritis.

INTRODUCTION: The identification of genes associated with osteoarthritis can help reveal underlying biological mechanisms that may lead to development of new therapeutic targets or biomarkers for early detection and risk stratification. Nucleostemin (GNL3) is a nucleolar GTPase initially identified in the nucleolus of rat neural stem cells. The current study was conducted to determine the expression of nucleostemin gene in the synovium and synovial fluid of patients with primary osteoarthritis and to correlate its expression to the different clinicopathological factors of the disease. PATIENTS AND METHODS: It included 31 patients with primary knee osteoarthritis and 25 osteoarthritis free patients served as a control group. Synovial tissue and synovial fluid samples were obtained directly from each patient for real time PCR of GNL3. RESULTS: Relative expression of GNL3 in synovial tissue and fluid samples was significantly higher in the osteoarthritic group as compared to the non-osteoarthritic group. GNL3 relative expression in both samples showed a significant difference among different BMI categories and among different radiographic grades of osteoarthritis. A high significant correlation was found between GNL3 relative expression levels in synovial tissue samples and those of synovial fluid samples with concordance of 85.7%. CONCLUSION: Nucleostemin could serve as a powerful prognostic marker for clinical use in osteoarthritis and its usefulness needs to be standardized and validated in a large-scale prospective multicentric study.

Involvement of fibroblast-specific protein 1 (S100A4) and matrix metalloproteinase-13 (MMP-13) in CCl4-induced reversible liver fibrosis.

INTRODUCTION: The intense basic research on the molecular and cellular mechanisms of liver fibrosis regression intends to translate these findings into new therapies targeting such pathways in human liver disease. Fibrosis regression is rapidly initiated in mouse models of fibrosis within days after termination of the cause, so in this study, we investigated the expression of S100A4 and MMP-13 during liver fibrogenesis and remodeling. METHODS: Thirty rats were divided into three groups: control group, fibrotic group, and fibrotic resolution group (10 each). The rats were sacrificed 48h and 96h after cessation of CCL-4, respectively. Liver tissue levels of S100A4 mRNA and S100A4 protein, MMP-13 mRNA and serum levels of serum TGF-ß1, ALT and AST were determined. RESULTS: Expression of S100A4 was increased during fibrotic stage and declined during resolution which was in correlation with the pro-fibrotic marker TGF-ß1 with concordance about 90%, while MMP-13 expression increased in both stages reaching to 40 fold during resolution. CONCLUSION: These findings suggested that S100A4 level in the liver tissue was related positively with liver fibrosis making it a good marker for liver fibrogenesis and also a good target for novel antifibrotic strategies.

Validation of analytical breast cancer microarray analysis in medical laboratory.

A previously reported microarray data analysis by RISS algorithm on breast cancer showed over-expression of the growth factor receptor (Grb7) and it also highlighted Tweety (TTYH1) gene to be under expressed in breast cancer for the first time. Our aim was to validate the results obtained from the microarray analysis with respect to these genes. Also, the relationship between their expression and the different prognostic indicators was addressed. RNA was extracted from the breast tissue of 30 patients with primary malignant breast cancer. Control samples from the same patients were harvested at a distance of ≥5 cm from the tumour. Semi-quantitative RT-PCR analysis was done on all samples. There was a significant difference between the malignant and control tissues as regards Grb7 expression. It was significantly related to the presence of lymph node metastasis, stage and histological grade of the malignant tumours. There was a significant inverse relation between expression of Grb7 and expression of both oestrogen and progesterone receptors. Grb7 was found to be significantly related to the biological classification of breast cancer. TTYH1 was not expressed in either the malignant or the control samples. The RISS by our group algorithm developed was laboratory validated for Grb7, but not for TTYH1. The newly developed software tool needs to be improved.

Evaluation of matrix metalloproteinase-2 in lung cancer.

PURPOSE: There is an obvious need to diagnose lung cancer using novel noninvasive and sensitive biomarkers. In this regard, the aim of the present study was to evaluate and compare sputum matrix metalloproteinase 2 (MMP-2) in relation to serum MMP-2 of lung cancer patients and other nonmalignant lung diseases in order to establish a new diagnostic and prognostic biomarker with a valid noninvasive technique. EXPERIMENTAL DESIGN: Group 1 included 32 newly diagnosed lung cancer patients and group 2 included 20 patients with benign pulmonary diseases. In addition, 38 healthy subjects served as control group. MMP-2 activity levels were evaluated in serum and sputum samples of the studied groups using ELISA and zymography techniques. RESULTS: There was a highly significant increase in serum and sputum MMP-2 levels in malignant group in comparison with benign and control groups. In addition, there was a significant difference in the levels of serum and sputum MMP-2 as regards the different histopathological types of lung cancer and advanced stages of lung cancer. Gelatin zymography was used to confirm the enzymatic activity of MMP-2. A higher MMP-2 activity was detected in lung cancer group in comparison with benign and control groups. CONCLUSIONS AND CLINICAL RELEVANCE: Serum and to a larger extent sputum MMP-2 appear to be potential noninvasive markers for detecting lung cancer.

Evaluation of nestin in lung adenocarcinoma: relation to VEGF and Bcl-2.

CONTEXT: Nestin is a marker of multipotent precursor cells that is up regulated in cancer. OBJECTIVE: To explore its diagnostic role and its relationship to vascular endothelial growth factor (VEGF) and Bcl-2 in lung adenocarcinoma. MATERIALS AND METHODS: Evaluation of nestin expression in lung biopsies by real-time PCR and serum VEGF and Bcl-2 by ELISA in 27 adenocarcinoma patients and 15 control subjects. RESULTS: Nestin was significantly higher in lung adenocarcinoma patients especially with advanced grade and stage and it was significantly correlated to VEGF and Bcl-2. CONCLUSION: Nestin can be considered as a potential diagnostic marker in lung adenocarcinoma.

6-gingerol ameliorated doxorubicin-induced cardiotoxicity: role of nuclear factor kappa B and protein glycation.

PURPOSE: Doxorubicin is a widely used antitumour drug. Cardiotoxicity is considered a major limitation for its clinical use. The present study was designed to assess the possible antioxidant and antiapoptotic effects of 6-gingerol in attenuating doxorubicin-induced cardiac damage. METHOD: Male albino rats were treated with either intraperitoneal doxorubicin (18 mg/kg divided into six equal doses for 2 weeks) and/or oral 6-gingerol (10 mg/kg starting 5 days before and continued till the end of the experiment). RESULTS: 6-gingerol significantly ameliorated the doxorubicin-induced elevation in the cardiac enzymes. The stimulation of oxidative stress by doxorubicin was evidenced by the significant decrease in the serum soluble receptor for advanced glycation endproduct allowing unopposed serum advanced glycation endproduct availability. Moreover, doxorubicin activated nuclear factor kappa B (NF-κB) which was indicated by an increase in its immunohistochemical staining in the nucleus. In addition, doxorubicin-induced cardiotoxicity was accompanied by elevation of cardiac caspase-3. Notably, pretreatment with 6-gingerol significantly ameliorated the changes in sRAGE, NF-κB and cardiac caspase-3. Cardiac enzymes showed significant positive correlation with NF-κB and caspase-3 but negative with serum sRAGE, suggesting their role in doxorubicin-induced cardiac injury. These findings were confirmed by cardiac tissue histopathology. CONCLUSION: 6-gingerol, a known single compound from ginger with anticancer activity, was shown to have a promising role in cardioprotection against doxorubicin-induced cardiotoxicity. This study suggested a novel mechanism for 6-gingerol cardioprotection, which might be mediated through its antioxidative effect and modulation of NF-κB as well as apoptosis.

STUDY OF THE ROLE OF SERUM FOLIC ACID IN ATOPIC DERMATITIS: A CORRELATION WITH SERUM IgE AND DISEASE SEVERITY.

BACKGROUND: Most atopic dermatitis (AD) patients have elevated serum immunoglobulin E (IgE). Impaired folic acid (FA) metabolism was found to reduce the intracellular methyl donor pool, associated with a higher prevalence of atopy. AIM: To assess serum IgE and FA in AD patients and to correlate their levels with the disease severity, and with each other. MATERIALS AND METHODS: Twenty patients with AD were assessed for serum FA and IgE, compared with 20 age- and sex-matched controls. Patients were classified into three groups (mild, moderate, and severe AD) based on clinical severity according to Nottingham index. In both patients and controls, serum IgE was measured using Enzyme-linked immunosorbent assay technique and serum FA was measured using Microparticle Enzyme Immunoassay technique. RESULTS: Serum FA levels were lower in AD patients compared with controls, but the difference was not statistically significant. FA levels did not show statistically significant difference among disease severity groups and did not correlate with serum IgE levels. On the other hand, serum IgE levels were significantly elevated in AD patients compared with controls, and among AD patients, its levels were significantly elevated in severe AD compared with mild and moderate disease. CONCLUSION: Serum IgE is useful in assessment of AD severity and activity. FA contribution to AD needs further investigations.