George Hilal

TNF-α messenger ribonucleic acid (mRNA) in patients with nonalcoholic steatohepatitis

BACKGROUND AND AIM tumor necrosis factor (TNF)-α plays a significant role in the pathogenesis of nonalcoholic steatohepatitis (NASH). A few studies have confirmed high TNF-α plasma protein levels in patients with NASH compared to healthy volunteers. We herein aimed to revisit these findings using other molecular techniques. METHODS a cross-sectional evaluation of patients newly diagnosed with NASH. A quantitative assay for the measurement of TNF-α messenger ribonucleic acid (mRNA) was performed for NASH patients and controls using real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS in 39 patients with NASH (mean age 38.6 ± 9.4 years, range 28-60 years; 79% males), the mean TNF-α mRNA level was significantly higher than that found for controls (137.6 ± 102.3 ng/mL versus 83.5 ± 43.8 ng/mL, respectively; P = 0.012). A TNF-α mRNA cut-off of 100 ng/mL predicted NASH most optimally (AUC 0.685 ± 0.066, P = 0.01; with 66.7% sensitivity and 74.1% specificity). Serum TNF-α and soluble TNF-α receptor II (sTNFRII) levels were significantly higher in patients compared to controls using ELISA. CONCLUSION high TNF-α mRNA levels, determined by RT-PCR, characterize patients with NASH.

Platelet rich plasma (PRP) induces chondroprotection via increasing autophagy, anti-inflammatory markers, and decreasing apoptosis in human osteoarthritic cartilage

Objectives: Autophagy constitutes a defense mechanism to overcome aging and apoptosis in osteoarthritic cartilage. Several cytokines and transcription factors are linked to autophagy and play an important role in the degradative cascade in osteoarthritis (OA). Cell therapy such as platelet rich plasma (PRP) has recently emerged as a promising therapeutic tool for many diseases including OA. However, its mechanism of action on improving cartilage repair remains to be determined. The purpose of this study is to investigate the effect of PRP on osteoarthritic chondrocytes and to elucidate the mechanism by which PRP contributes to cartilage regeneration. Methods: Osteoarthritic chondrocytes were co‐cultured with an increasing concentration of PRP obtained from healthy donors. The effect of PRP on the proliferation of chondrocytes was performed using cell counting and WST8 proliferation assays. Autophagy, apoptosis and intracellular level of IL‐4, IL‐10, and IL‐13 were determined using flow cytometry analyses. Autophagy markers BECLIN and LC3II were also determined using quantitative polymerase chain reaction (qPCR). qPCR and ELISA were used to measure the expression of ADAMDTS‐5, MMP3, MMP13, TIMP‐1–2–3, aggregan, Collagen type 2, TGF‐&bgr;, Cox‐2, Il‐6, FOXO1, FOXO3, and HIF‐1 in tissues and co‐cultured media. Results: PRP increased significantly the proliferation of chondrocytes, decreased apoptosis and increased autophagy and its markers along with its regulators FOXO1, FOXO3 and HIF‐1 in osteoarthritic chondrocytes. Furthermore, PRP caused a dose‐dependent significant decrease in MMP3, MMP13, and ADAMTS‐5, IL‐6 and COX‐2 while increasing TGF‐&bgr;, aggregan, and collagen type 2, TIMPs and intracellular IL‐4, IL‐10, IL‐13. Conclusion: These results suggest that PRP could be a potential therapeutic tool for the treatment of OA. HIGHLIGHTSPlatelet Rich Plasma is suggested as a new treatment for osteoarthritis.The proposed therapeutic effect is chondroprotection.Chondroprotection is assumed via two major mechanisms:1‐by increasing the major players in cartilage protection as as autophagy 2‐by decreasing the markers of cartilage degradation such as MMPs.

The chemokine CCL20 induces proinflammatory and matrix degradative responses in cartilage

IntroductionLocal inflammation plays a role in the pathophysiology of osteoarthritis (OA) and chemokines exert catabolic effects on articular cartilage either through paracrine and/or autocrine mechanisms. We sought to compare the expression levels of the chemokine (C–C motif) ligand 20 (CCL20) and its chemokine receptor 6 (CCR6) in donor and osteoarthritic (OA) cartilage and to investigate the role of CCL20 in the pathogenesis of OA and chondrocyte phenotype.MethodsCartilage/chondrocytes from donor and OA knee joints was analyzed for CCL20 and CCR6 expression by RT-PCR and immunohistochemistry. Effects of CCL20 on cytokines and mediators of cartilage degradation were examined by RT-PCR for mRNA expression levels, enzyme-linked immunosorbent assays, and proteoglycan (GAG) assays.ResultsCCL20 and CCR6 proteins were abundantly expressed in OA cartilage sections compared to donor sections as judged by immunohistochemistry. RT-PCR of cartilage extracts confirmed the predominance of CCL20/CCR6 mRNA expression in OA cartilage. CCL20 mRNA expression was low in donor chondrocytes but increased after stimulation with proinflammatory cytokines. mRNA expression levels of IL-6, cyclooxygenase-2, and iNOS were elevated in donor chondrocyte cultures treated with rhCCL20. The release of MMP1/13, PGE2, proteoglycan GAG fragments, and IL-6 from cartilage explant cultures was markedly augmented in the presence of CCL-20. CCL-20 stimulated MMP-13, ADAMTS-5, and col type X mRNA but inhibited col type II mRNA expression in freshly explanted and cultured cartilage specimens.ConclusionsCCL20/CCR6 may play an important role in the pathogenesis of OA by inducing changes in phenotype and catabolic gene expression in chondrocytes.

Telomerase Inhibition Decreases Alpha-Fetoprotein Expression and Secretion by Hepatocellular Carcinoma Cell Lines: In Vitro and In Vivo Study

Alpha-fetoprotein (AFP) is a diagnostic marker for hepatocellular carcinoma (HCC). A direct relationship between poor prognosis and the concentration of serum AFP has been observed. Telomerase, an enzyme that stabilizes the telomere length, is expressed by 90% of HCC. The aim of this study was to investigate the effect of telomerase inhibition on AFP secretion and the involvement of the PI3K/Akt/mTOR signaling pathway. Proliferation and viability tests were performed using tetrazolium salt. Apoptosis was determined through the Annexin V assay using flow cytometry. The concentrations of AFP were measured using ELISA kits. The AFP mRNA expression was evaluated using RT-PCR, and cell migration was evaluated using a Boyden chamber assay. The in vivo effect of costunolide on AFP production was tested in NSG mice. Telomerase inhibition by costunolide and BIBR 1532 at 5 and 10 μM decreased AFP mRNA expression and protein secretion by HepG2/C3A cells. The same pattern was obtained with cells treated with hTERT siRNA. This treatment exhibited no apoptotic effect. The AFP mRNA expression and protein secretion by PLC/PRF/5 was decreased after treatment with BIBR1532 at 10 μM. In contrast, no effect was obtained for PLC/PRF/5 cells treated with costunolide at 5 or 10 μM. Inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP concentration. In contrast, the MAPK/ERK pathway appeared to not be involved in HepG2/C3A cells, whereas ERK inhibition decreased the AFP concentration in PLC/PRF/5 cells. Modulation of the AFP concentration was also obtained after the inhibition or activation of PKC. Costunolide (30 mg/kg) significantly decreased the AFP serum concentration of NSG mice bearing HepG2/C3A cells. Both the inhibition of telomerase and the inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP production of HepG2/C3A and PLC/PRF/5 cells, suggesting a relationship between telomerase and AFP expression through the PI3K/Akt/mTOR pathway

Gastrointestinal cancer cells treatment with bevacizumab activates a VEGF autoregulatory mechanism involving telomerase catalytic subunit hTERT via PI3K-AKT, HIF-1α and VEGF receptors

Background Targeting angiogenesis has been considered a promising treatment of choice for a large number of malignancies, including gastrointestinal cancers. Bevacizumab is an anti-vascular endothelial growth factor (anti-VEGF) being used for this purpose. However, treatment efficacy is largely questioned. Telomerase activity, responsible for cancer cell immortality, is detected in 85–95% of human cancers and is considered a potential regulator of VEGF. The aim of our study was to investigate the interrelationship between VEGF and hTERT in gastrointestinal cancers and to explore cell response to a combined inhibition of telomerase and VEGF. Methods AGS (gastric cancer), Caco-2 (colorectal cancer) and HepG2/C3A (hepatocellular carcinoma), were treated with telomerase inhibitors BIBR-1232 (10μM) and costunolide (10μM), with bevacizumab (Avastin® at 5 ng/ml or 100μg/ml) or with a combination of both types of inhibitors. VEGF and hTERT mRNA levels, and telomerase activity were detected by RT-PCR. VEGF levels were quantified by ELISA. Telomerase was knocked down using hTERT siRNA and hTERT was overexpressed in the telomerase negative cell line, Saos-2 (osteosarcoma), using constructs expressing either wild type hTERT (hTERT-WT) or dominant negative hTERT (hTERT-DN). Tube formation by HUVECs was assessed using ECMatrix™ (EMD Millipore). Results Our results showed that telomerase regulates VEGF expression and secretion through its catalytic subunit hTERT in AGS, Caco2, and HepG2/C3A, independent of its catalytic activity. Interestingly, VEGF inhibition with bevacizumab (100μg/ml) increased hTERT expression 42.3% in AGS, 94.1% in Caco2, and 52.5% in HepG2/C3A, and increased telomerase activity 30-fold in AGS, 10.3-fold in Caco2 and 8-fold in HepG2/C3A. A further investigation showed that VEGF upregulates hTERT expression in a mechanism that implicates the PI3K/AKT/mTOR pathway and HIF-1α. Moreover, bevacizumab treatment increased VEGFR1 and VEGFR2 expression in cancer cells and human umbilical vein endothelial cells (HUVECs) through hTERT. Thus, the combination of bevacizumab with telomerase inhibitors decreased VEGF expression and secretion by cancer cells, inhibited VEGFR1 and VEGFR2 upregulation, and reduced tube formation by HUVECs. Conclusions Taken together, our results suggest that bevacizumab treatment activates a VEGF autoregulatory mechanism involving hTERT and VEGF receptors and that an inhibition of this pathway could improve tumor cell response to anti-VEGF treatment.

Telomerase as a potential marker for inflammation and cancer detection in bronchial washing: a prospective study.

OBJECTIVES The diagnosis of lung cancer remains difficult especially in peripheral tumors, given the absence of relevant markers and of sensitive imaging techniques. Telomerase is a ribonucleotide enzyme responsible for the immortalization of cancerous cells and seems to increase in bronchial aspirates of lung cancer patients. The purpose of our study is to further investigate the value of telomerase measurement in bronchial aspirates as a diagnostic tool for lung cancer. DESIGN AND METHODS Random 82 bronchial aspirates were obtained from patients undergoing bronchoscopy to diagnose any lung illness including inflammation and cancer. Cytology examination, quantification of proteins by Bradford method, and telomerase activity measurement by quantitative Real-time PCR were performed. Out of 82 specimens, 11 were excluded because of hemolysis, absence of elements or lack of final diagnosis. ROC curve analysis was done. RESULTS A significant difference in telomerase activity average was noted between normal patients and those with inflammation and cancer. Discriminatory capacity of telomerase activity was: for cancer vs. non cancer, AUC =0.74 (95% CI: 0.62-0.84), sensitivity=78%, specificity=72%, Negative Predictive Value=87%, at cut-off >0.46 atmol/mg protein/20 min; for cancer vs. normal, AUC=0.87 (95% CI: 0.72-0.96), se=78%, sp=92%, NPV=71%, at cut-off >0.46; for cancer vs. inflammation, AUC=0.69 (95% CI: 0.55-0.80), se=74%, sp=70%, NPV=79%, at cut-off >1.03, and for inflammation vs. normal, AUC=0.76 (95% CI: 0.62-0.88), se=79%, sp=77%, NPV=59%, at cut-off >0. CONCLUSION Telomerase activity in bronchial aspirates is a promising diagnostic marker for lung cancer and inflammation detection.

Different TP53 mutants in p53 overexpressed epithelial ovarian carcinoma can be associated both with altered and unaltered glycolytic and apoptotic profiles

Backgroundp53 is a tumor suppressor and key regulator of glycolysis in cancer cells, however highly mutated in tumors. In ovarian cancer, studies concerning p53 mutations focus on the DNA binding domain since the majority of hotspot mutations affects this region. Yet, mutations in other regions such as the proline rich domain may also affect the protein’s expression and activity. The aim of this study is to investigate the effect of various positions of mutations in TP53 gene on glycolysis, apoptosis and transcription of p53 target genes.MethodsMutations frequency and their effect on p53 expression were assessed by PCR-SSCP, sequencing and immunohistochemistry on 30 ovarian cancer biopsies. Six tumors were cultured, as well as SK-OV-3, OVCAR-3 and Igrov-1. SK-OV-3 cells were transfected with 2 TP53 mutants. p53 transcriptional activity was assayed by qPCR, apoptosis by flow cytometry and glycolysis by glucose and lactate measurements, with quantification of glycolytic enzymes expression.ResultsOur results showed a high frequency of the P72R mutant, associated with p53 overexpression in the ovarian biopsies. However, P72R mutant cells showed similar apoptosis and glycolysis as WT cells. DNA binding domain mutations decreased the transcriptional activity of the protein and increased glucose consumption and lactate production.ConclusionDespite the overexpression of the P72R mutated protein in the biopsies, it showed a similar apoptotic activity and glucose regulation ability as WT p53. Knowing that p53 expression status is used for chemotherapeutic approaches and prognosis in ovarian cancer, the results obtained highlight the importance of locating TP53 mutations.

Novel plasma telomerase detection method to improve cancer diagnostic assessment

Background The activity levels of telomerase and its mRNA have been found to be more diagnostically sensitive than cytological results in many cancerous tissues and correlate well with the clinical disease stage. Currently, there are several methods of detecting telomerase in tissues and in blood. The most commonly used method is a conventional quantitative real-time polymerase chain reaction (PCR) which is time and labor exhausting. Methods We have developed a simple and innovative blood test method that allows us to diagnose cancer and relapsed cancer in a cost- and time -effective manner. We had evaluated our novel method in two populations: 1) in vivo in three mice with pancreatic ductal adenocarcinoma (PDAC) versus one control mouse and 2) clinically in 30 cancer patients versus 10 individuals without cancer. We compared our novel method with the old conventional method. At least one sample was obtained from each patient included in the study. Results The novel method substantially increased the sensitivity (from 37% to 77%, p<0.001) and negative predictive value (from 32% to 56%, p = 0.005) of the telomerase test for all cancer patients (those who were substantially treated and those who were not). There was no significant difference in telomerase activity between cancer patients and healthy volunteers using the conventional method (p = 0.13), whereas there was a significant difference using the novel method (p = 0.001). Conclusion Conventional method shows no significant difference in telomerase activity between cancer patients and healthy volunteers (p = 0.13), whereas there was a significant difference using the novel method (p = 0.001).

An Evaluation of the Stemness, Paracrine, and Tumorigenic Characteristics of Highly Expanded, Minimally Passaged Adipose-Derived Stem Cells.

The use of adipose-derived stem cells (ADSC) in regenerative medicine is rising due to their plasticity, capacity of differentiation and paracrine and trophic effects. Despite the large number of cells obtained from adipose tissue, it is usually not enough for therapeutic purposes for many diseases or cosmetic procedures. Thus, there is the need for culturing and expanding cells in-vitro for several weeks remain. Our aim is to investigate if long- term proliferation with minimal passaging will affect the stemness, paracrine secretions and carcinogenesis markers of ADSC. The immunophenotypic properties and aldehyde dehydrogenase (ALDH) activity of the initial stromal vascular fraction (SVF) and serially passaged ADSC were observed by flow cytometry. In parallel, the telomerase activity and the relative expression of oncogenes and tumor suppressor genes were assessed by q-PCR. We also assessed the cytokine secretion profile of passaged ADSC by an ELISA. The expanded ADSC retain their morphological and phenotypical characteristics. These cells maintained in culture for up to 12 weeks until P4, possessed stable telomerase and ALDH activity, without having a TP53 mutation. Furthermore, the relative expression levels of TP53, RB, and MDM2 were not affected while the relative expression of c-Myc decreased significantly. Finally, the levels of the secretions of PGE2, STC1, and TIMP2 were not affected but the levels of IL-6, VEGF, and TIMP 1 significantly decreased at P2. Our results suggest that the expansion of passaged ADSC does not affect the differentiation capacity of stem cells and does not confer a cancerous state or capacity in vitro to the cells.

PO-038 PKC isoforms distinctively modulate telomerase expression and AFP secretion in hepatocellular carcinoma

Introduction Despite its role as a diagnostic and prognosis marker for Hepatocellular carcinoma (HCC), alpha fetoprotein (AFP) plays a key role in advancing tumorigenesis and tumour expansion. Telomerase, an enzyme elongating telomere length, is upregulated in 80% of cancers including HCC. Our team had elucidated a modulation of AFP by telomerase and protein kinase C (PKC). PKC family is formed of several isoforms, each with a specific cellular function and expression. The aim of this study is to investigate the interrelation between PKC isoforms, telomerase and AFP in HCC. Material and methods PKC isoforms were quantified by RT-qPCR in two AFP secretory cell lines, HepG2/C3A and PLC/PRF/5 and two non-secretory AFP cell lines SNU-387 (hTERT+) and SKOV-3 (hTERT-). According to the results, the expression of four isoforms was suppressed by si-RNAs in HepG2/C3A and PLC cells. AFP and telomerase mRNA levels were quantified in transfected cells by q-PCR, and AFP secretion by ELISA. Toxicity and cell proliferation were assessed by WST-1. In order to examine the effect of the dual presence of AFP and hTERT on PKC isoforms, SNU-387 and SKOV-3 were transfected with AFP expression plasmid pCMV3-AFP, then PKC isoforms mRNA was assessed by qPCR. Results and discussions Four PKC isoforms, alpha, beta, delta and epsilon exhibited the highest expression levels in all cells compared to the remaining isoforms. An increase in telomerase expression, AFP expression and secretion and cell proliferation reaching a maximum of two folds was observed after individual gene silencing of the four isoforms in HepG2/C3A cells; however, an inverse pattern was noticed when using PKC pan inhibitor Go6983. Similar results were observed in PLC cell line. The expression of the four isoforms increased in SNU-387 and SKOV-3 cells after 24 hour transfection. The effect persisted after 48 hour in SNU-387, contrary to SKOV-3 where the levels decreased returning to the controls expression levels. Conclusion Taken together, decreased individual PKC isoforms rise telomerase expression and AFP secretion. However, PKC isoforms overexpression requires the presence of hTERT in AFP secretory cells. Thus, these results show for the first time the possible inter relation linking PKC isoforms to both AFP and hTERT in HCC.