All-trans-retinoic acid modulates glycolysis via H19 and telomerase: the role of mir-let-7a in estrogen receptor-positive breast cancer cells.

BACKGROUND: Breast cancer (BC) is the most commonly diagnosed cancer in women. Treatment approaches that differ between estrogen-positive (ER+) and triple-negative BC cells (TNBCs) and may subsequently affect cancer biomarkers, such as H19 and telomerase, are an emanating delight in BC research. For instance, all-trans-Retinoic acid (ATRA) could represent a potent regulator of these oncogenes, regulating microRNAs, mostly let-7a microRNA (miR-let-7a), which targets the glycolysis pathway, mainly pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA) enzymes. Here, we investigated the potential role of ATRA in H19, telomerase, miR-let-7a, and glycolytic enzymes modulation in ER + and TNBC cells. METHODS: MCF-7 and MDA-MB-231 cells were treated with 5 µM ATRA and/or 100 nM fulvestrant. Then, ATRA-treated or control MCF-7 cells were transfected with either H19 or hTERT siRNA. Afterward, ATRA-treated or untreated MDA-MB-231 cells were transfected with estrogen receptor alpha ER(α) or beta ER(ß) expression plasmids. RNA expression was evaluated by RT‒qPCR, and proteins were assessed by Western blot. PKM2 activity was measured using an NADH/LDH coupled enzymatic assay, and telomerase activity was evaluated with a quantitative telomeric repeat amplification protocol assay. Student's t-test or one-way ANOVA was used to analyze data from replicates. RESULTS: Our results showed that MCF-7 cells were more responsive to ATRA than MDA-MB-231 cells. In MCF-7 cells, ATRA and/or fulvestrant decreased ER(α), H19, telomerase, PKM2, and LDHA, whereas ER(ß) and miR-let-7a increased. H19 or hTERT knockdown with or without ATRA treatment showed similar results to those obtained after ATRA treatment, and a potential interconnection between H19 and hTERT was found. However, in MDA-MB-231 cells, RNA expression of the aforementioned genes was modulated after ATRA and/or fulvestrant, with no significant effect on protein and activity levels. Overexpression of ER(α) or ER(ß) in MDA-MB-231 cells induced telomerase activity, PKM2 and LDHA expression, in which ATRA treatment combined with plasmid transfection decreased glycolytic enzyme expression. CONCLUSIONS: To the best of our knowledge, our study is the first to elucidate a new potential interaction between the estrogen receptor and glycolytic enzymes in ER + BC cells through miR-let-7a.

Evaluating synergistic effects of metformin and simvastatin on ovarian cancer cells.

BACKGROUND: Ovarian Cancer (OC) stands as the most lethal gynecological malignancy, presenting an urgent clinical challenge in the quest to improve response rates. One approach to address this challenge is through drug repurposing, exemplified by the investigation of metabolic-modulating drugs such as Metformin (MTF) and Simvastatin (SIM). This study aims to explore the molecular mechanisms contributing to the potential synergistic anti-cancer effects between MTF and SIM on ovarian cancer cells. METHODS: We assessed the effects of the combination on the proliferation and viability of two cell lines OVCAR-3 and SKOV-3. IC50 concentrations of MTF and SIM were determined using a proliferation assay, followed by subtoxic concentrations to explore the potential synergistic effects on the viability of both cell lines. Transcriptomic analysis was conducted on OVCAR-3 treated cells, and the findings were validated by assessing the expression levels of differentially expressed genes (DEGs) through real-time PCR in both cell lines SK-OV-3 and OVCAR-3. RESULTS: Cytotoxicity analysis guided the selection of treatment concentrations as such MTF 10 mM and SIM 5 µM. The combined treatment of MTF and SIM demonstrated a synergistic inhibition of proliferation and viability in both cell lines. In OVCAR-3, exclusive identification of 507 DEGs was seen in the combination arm. Upregulation of FOXO3, RhoA, and TNFα, along with downregulation of PIK3R1, SKP2, and ATP6V1D levels, was observed in OVCAR-3 treated cells. Real-time PCR validation confirmed the consistency of expression levels for the mentioned DEGs. CONCLUSION: Our data strongly supports the presence of synergy between MTF and SIM in OC cells. The combination's effect is associated with the dysregulation of genes in the key regulators AMPK and mTOR alongside other interconnected pathways.

HSP27 modulates tumoural immune evasion by enhancing STAT3-mediated upregulation of PD-L1 and NLRC5 in ovarian cancer.

Multiple preclinical studies have demonstrated that the addition of hyperthermia (HT) to immunotherapy could enhance tumour immunogenicity and stimulate an antitumour immune response, primarily via heat shock proteins (HSPs). However, antitumour immune responses are often impeded by immune evasion mechanisms, such as the overexpression of programmed death-ligand1 (PD-L1) and the loss of major histocompatibility complex class 1 (MHC-1) expression. In this context, we sought to investigate the effect of HT on PD-L1 and NOD-like receptor family CARD domain containing 5 (NLRC5) identified as the key transcriptional activator of MHC-1 genes, and their interaction in ovarian cancer. A coculture of ovarian cancer cell lines (IGROV1 and SKOV3) with peripheral blood mononuclear cells was set up. Then, culture media conditioned with IGROV1 or SKOV3 subjected to HT was tested on untreated cell cultures. Knocking down heat shock protein B1 (HSPB1 or HSP27), heat shock protein A1 (HSPA1 or HSP70), and pharmacological inhibition of STAT3 phosphorylation were performed. Subsequently, we measured expression levels of PD-L1, NLRC5, and proinflammatory cytokines. The correlation between PD-L1 and NLRC5 expression in ovarian cancer was evaluated using the Cancer Genome Atlas database. We found that HT produces a concomitant decrease in PD-L1 and NLRC5 expression in coculture. Notably, however, the conditioned media by heat-shocked cells increases their expression. HSP27 knockdown can reverse this increase. Adding STAT3 phosphorylation inhibitor significantly enhanced the expression inhibition of PD-L1 and NLRC5 induced by HSP27 silencing. Correlation analysis showed a positive correlation in ovarian cancer between NLRC5 and PD-L1. These findings demonstrate that HSP27 modulates PD-L1 and NLRC5 expression through the activation of a common regulator 'STAT3'. Moreover, the positive correlation between PD-L1 and NLRC5 led us to conclude that the upregulation of PD-L1 and the downregulation of MHC class I are two mutually exclusive mechanisms of immune evasion in ovarian cancer.

Enhancing cancer treatment and understanding through clustering of gene responses to categorical stressors.

Cancer cells have a unique metabolic activity in the glycolysis pathway compared to normal cells, which allows them to maintain their growth and proliferation. Therefore, inhibition of glycolytic pathways may be a promising therapeutic approach for cancer treatment. In this novel study, we analyzed the genetic responses of cancer cells to stressors, particularly to drugs that target the glycolysis pathway. Gene expression data for experiments on different cancer cell types were extracted from the Gene Expression Omnibus and the expression fold change was then clustered after dimensionality reduction. We identified four groups of responses: the first and third were most affected by anti-glycolytic drugs, especially those acting on multiple pathways at once, and consisted mainly of squamous and mesenchymal tissues, showing higher mitotic inhibition and apoptosis. The second and fourth groups were relatively unaffected by treatment, comprising mainly gynecologic and hormone-sensitive groups, succumbing least to glycolysis inhibitors. Hexokinase-targeted drugs mainly showed this blunted effect on cancer cells. This study highlights the importance of analyzing the molecular states of cancer cells to identify potential targets for personalized cancer therapies and to improve our understanding of the disease.

Microarray analysis of breast cancer gene expression profiling in response to 2-deoxyglucose, metformin, and glucose starvation.

BACKGROUND: Breast cancer (BC) is the most frequently diagnosed cancer in women. Altering glucose metabolism and its effects on cancer progression and treatment resistance is an emerging interest in BC research. For instance, combining chemotherapy with glucose-lowering drugs (2-deoxyglucose (2-DG), metformin (MET)) or glucose starvation (GS) has shown better outcomes than with chemotherapy alone. However, the genes and molecular mechanisms that govern the action of these glucose deprivation conditions have not been fully elucidated. Here, we investigated the differentially expressed genes in MCF-7 and MDA-MB-231 BC cell lines upon treatment with glucose-lowering drugs (2-DG, MET) and GS using microarray analysis to study the difference in biological functions between the glucose challenges and their effect on the vulnerability of BC cells. METHODS: MDA-MB-231 and MCF-7 cells were treated with 20 mM MET or 4 mM 2-DG for 48 h. GS was performed by gradually decreasing the glucose concentration in the culture medium to 0 g/L, in which the cells remained with fetal bovine serum for one week. Expression profiling was carried out using Affymetrix Human Clariom S microarrays. Differentially expressed genes were obtained from the Transcriptome Analysis Console and enriched using DAVID and R packages. RESULTS: Our results showed that MDA-MB-231 cells were more responsive to glucose deprivation than MCF-7 cells. Endoplasmic reticulum stress response and cell cycle inhibition were detected after all three glucose deprivations in MDA-MB-231 cells and only under the metformin and GS conditions in MCF-7 cells. Induction of apoptosis and inhibition of DNA replication were observed with all three treatments in MDA-MB-231 cells and metformin-treated MCF-7 cells. Upregulation of cellular response to reactive oxygen species and inhibition of DNA repair mechanisms resulted after metformin and GS administration in MDA-MB-231 cell lines and metformin-treated MCF-7 cells. Autophagy was induced after 2-DG treatment in MDA-MB-231 cells and after metformin in MCF-7 cells. Finally, inhibition of DNA methylation were observed only with GS in MDA-MB-231 cells. CONCLUSION: The procedure used to process cancer cells and analyze their expression data distinguishes our study from others. GS had the greatest effect on breast cancer cells compared to 2-DG and MET. Combining MET and GS could restrain both cell lines, making them more vulnerable to conventional chemotherapy.

Poor Biointegration of Porcine Acellular Dermal Matrix Associated with Unfavorable Gingival Healing: A Report of Three Cases.

AIM: The aim of the present work was to explain the poor biointegration of acellular dermal xenogeneic matrix, leading to an unfavorable gingival healing following a grafting procedure for the treatment of soft tissue deficiencies. BACKGROUND: Numerous works have demonstrated the successful use of acellular dermal matrix (ADM) in soft tissue augmentation procedures. However, spare human investigations reported adverse healing outcomes at microscopic level. CASE DESCRIPTION: Three patients showing various soft tissue deficiencies (recession, gingival thickening) requiring a gingival augmentation were grafted using an ADM porcine acellular dermal matrices (pADM) as a soft tissue substitute. For this purpose, appropriate soft tissue augmentation surgeries were performed and the grafted pADM was left for proper healing. Biopsies were harvested from two out of the three patients, respectively, at 11 and 27 weeks in order to conduct a histological evaluation of the pADM's doubtful biointegration. Moreover, the ultrastructural analysis of pADM was performed using scanning electron microscopy, and additional histological procedures were used to assess its ability to support human gingival fibroblast cultures. Signs of gingival inflammation persisted several months postoperatively. Histologically, numerous inflammatory cells characterized the grafted site. Indeed, the high number of foreign body giant cell granulomas and the very densified newly formed collagen fibers highlighted a fibrotic process within gingival connective tissue. The ultrastructural and histological analysis showed that pADM was characterized by very thick and dense collagen bundles demonstrating a nonphysiological collagen network organization. Cell culture experiments showed fibroblasts proliferating on the matrix surface, sparing its deeper part, even though the collagen matrix degradation seemed to occur following a gradient from the pADM surface inward. CONCLUSION: The unfavorable clinical results may be caused by the poor colonization of matrix cells and poor angiogenesis leading to the inadequate biointegration of pADM. Hence, the pADM structure in terms of porosity and degradability should be further investigated. CLINICAL SIGNIFICANCE: The present cases highlighted a poor integration of pADM following soft tissue grafting procedures, which was caused by the inadequate ultrastructure of the used pADM. Therefore, despite the utility of such tissue substitutes, their manufacturing improvement could be required to obtain a better biointegration.

The long non coding RNA H19 as a biomarker for breast cancer diagnosis in Lebanese women.

Breast cancer is the most common cancer in women worldwide. Minimally invasive percutaneous image-guided biopsies are the current cornerstone in the diagnosis of breast lesions detected on mammography/ultrasonography/MRI or palpable clinically. However, apparently benign breast disease seen on benign biopsies is a limiting factor for diagnosis and a risk factor of breast cancer especially in the high-risk category patients. Hypothesizing that molecular changes often occur before morphological variations, the levels of the LncRNA H19 were measured in anonymous tissues obtained from 79 women's image guided breast biopsies, and correlated with cancer progression and aggressiveness. Using a double-blinded approach, H19 might be attributed an interesting role of a more sensitive biomarker in core breast biopsies, independently of the radiological/clinical classification and distant from the clinical management. We established different thresholds for H19 levels in normal versus proliferative, versus malignant tissues. Additionnally, H19 could act as an intra-group risk marker categorizing the biopsies in normal versus benign, versus precancerous breast tissue, and as a prognostic factor in cancerous lesions discriminating aggressive versus nonaggressive lesions. Our study suggests that the lncRNA H19 could be a potential marker for breast cancer diagnosis, prognosis and risk management.

Peritoneal Carcinomatosis of Colorectal Origin in Cyclosporine Immunosuppressed Rats.

OBJECTIVE: We evaluate the seeding step of peritoneal carcinomatosis cancer as a surrogate for the role of the omentum in colorectal tumors. METHODS: The study included 5 groups of adult male Sprague Dawley rats: immunocompetent rats (group 1), immunosuppressed rats without omentectomy (group 2), immunosuppressed rats with omentectomy (group 3), immunosuppressed rats with omentectomy receiving NSAID (group 4), and immunosuppressed rats without omentectomy receiving NSAID (group 5). Except for group 1, the rats were immunosuppressed using cyclosporine orally at a dose of 25 mg/kg/day that was started 48 hours before tumor cell infiltration in the peritoneum. All the rats received an intraperitoneal suspension of 10 million Caco-2 cancer cells. Rats in groups 1, 2, and 3 were followed up without further interventions and rats in groups 4 and 5 received naproxen 180mg/kg until rat sacrifice. Cyclosporine and naproxen were continued in the corresponding groups until the killing after 21 days of tumor cell infiltration. RESULTS: Fourteen rats survived the experiment during the observation period and remained in good clinical condition except for one rat (from group 4) that deceased at week 2. At day 21 before sacrifice, mean weight variations showed a +4% in group 0, -9% in group 1, -18% in group 2, -31% in group 3 and -36% in group 4. Light microscopy did not identify any tumor cells in the abdominal cavity or thorax solid organs but showed a granulomatous reaction that involved the majority of the organs. CONCLUSION: The conclusions of this study are limited by the small number of rats as it is a pilot study to design an animal model with peritoneal carcinomatosis. Further steps in this study will include more aggressive cancer cell lines such as HT29 and more aggressive immunosuppression in a larger number of rats.

Glucose restriction combined with chemotherapy decreases telomere length and cancer antigen-125 secretion in ovarian carcinoma.

Although chemotherapy is the standard treatment for ovarian cancer (OC), recent studies have focused on its coupling with hypoglycemic drugs to decrease glucose availability. Similarly to cancer antigen 125 (Ca-125), telomerase, the key protein for telomere lengthening, is overexpressed in 90% of OC cases. The aim of the present study was to investigate the effect of the combination of glucose restriction and chemotherapy on telomere length and Ca-125 secretion in OC cells. SKOV-3, OVCAR-3 and Igrov-1 cells were treated with 20 µM cisplatin and 100 nM paclitaxel for 48 h in three different glucose concentrations: i) 4.5 g/l, ii) 1 g/l and iii) 0.5 g/l. The same treatment was repeated once per week for 6 consecutive weeks. The surviving cells were considered platinum-taxane escape (PTES) cells. The expression levels of telomerase and Ca-125 in treated and PTES cells were quantified by qPCR, and Ca-125 secretion by ELISA. Telomere length was evaluated by qPCR according to the Cawthon method. The modulation of Ca-125 by telomerase was assessed using inhibitors, small interfering RNA and transfection with human telomerase reverse transcriptase (hTERT) vectors. The implication of phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B/mechanistic target of rapamycin (PI3K/Akt/mTOR) in Ca-125 modulation was investigated using specific inhibitors. An increase in hTERT and Ca-125 expression levels (range, 1.5-3 fold) was observed in short-term treated cells. However, an opposite effect was detected in PTES cells, where the rate of decrease in the expression levels of hTERT and Ca-125 reached 60% after treatment in 0.5 g/l glucose. Moreover, telomere length was decreased by 30% in cells treated with 0.5 g/l glucose. Inhibition of hTERT expression significantly decreased Ca-125 secretion, suggesting a potential modulation of Ca-125 by hTERT. The inhibition of the PI3K/Akt/mTOR pathway also decreased Ca-125 secretion; however, the effect of this treatment was not enhanced when coupled with telomerase inhibitors. In conclusion, the combination of chemotherapy and glucose restriction was observed to decrease Ca-125 secretion and telomerase expression leading to shortening in telomere length. Thus, decreasing glucose availability for OC cells during treatment may lead to a better clinical outcome and potentially improve the prognosis of patients with OC.

Glucose restriction reverses the Warburg effect and modulates PKM2 and mTOR expression in breast cancer cell lines.

Aerobic glycolysis, known as the "Warburg effect", is one of several hallmarks of cancer cells. The conversion of phosphoenolpyruvate (PEP) to pyruvate can be down regulated by the re-expression of the embryonic isoform 2 of pyruvate kinase (PKM2). This mechanism allows the accumulation of glycolytic intermediates for the biosynthesis of macromolecules, such as proteins, lipids and nucleic acids. PKM2 is favored by the well-known PI3K/Akt/mTOR proliferative pathway. This pathway is induced by high glucose levels, and the mTOR kinase is the central activator of the Warburg effect. In this study, we investigated the role of glucose restriction (GR) and mTOR inhibition  in reversing the Warburg effect in MDA-MB 231 and MCF-7 breast cancer cell lines. PKM2 expression was measured by western blot. Lactate production by cells was determined by a colorimetric assay. The concentration of glucose in the supernatant of cells was measured using the Trinder method. ATP level  was evaluated by using a Colorimetric/Fluorometric ATP Assay Kit. Our results showed that MDA-MB 231 cells increased glucose consumption when the glucose concentration was 0 g/L (P <0.01). In MCF-7 cells, glucose deprivation reduced lactate secretion by 80% (P =0.0001) but tripled glucose consumption (P = 0.0041). ATP concentration increased approximately when MCF-7 cells were deprived of glucose (P = 0.02). GSK1059615 does not significantly modulate lactate secretion and glucose uptake in both cell lines. Glucose restriction contribute to the reduction of the Warburg effect through mTOR inhibition and regulation of PKM2 kinases.

DKK1 promotes hepatocellular carcinoma inflammation, migration and invasion: Implication of TGF-ß1.

Dickkopf-1 (DKK1), an inhibitor of the most frequently impaired signaling pathway in hepatocellular carcinoma (HCC), the Wnt/beta-catenin pathway, seems to fulfill contradictory functions in the process of tumorigenesis, acting either as an oncogenic promoter of metastasis or as a tumor suppressor. Elevated serum levels of DKK1 have been reported in HCC; however, little is known about its functional significance. In the current study, we treated HepG2/C3A and PLC/PRF/5 with the recombinant protein DKK1. Cytotoxicity was first determined by the WST-8 assay. AFP expression was measured at both the mRNA and protein levels. Expression of the oncogenes MYC, CCND1, hTERT, and MDM2 and the tumor suppressor genes TP53, P21 and RB was assessed. Western blot analysis of non-phosphorylated ẞ-catenin and Sanger sequencing were performed to explain the functional differences between the two cell lines. Subsequently, inflammation, migration and invasion were evaluated by qPCR, ELISA, the Boyden chamber assay, zymography, and MMP-2 and MMP-9 western blot analysis. Knockdown of DKK1 and TGF-ß1 were also performed. Our results suggest that DKK1 exerts an oncogenic effect on HepG2/C3A cell line by upregulating the expression of oncogenes and downregulating that of tumor suppressor genes, whereas the opposite effect was demonstrated in PLC/PRF/5 cells. This differential impact of DKK1 can be explained by the mutations that affect the canonical Wnt pathway that were detected in exon 3 of the CTNNB1 gene in the HepG2 cell line. We further confirmed that DKK1 promotes inflammation, tumor invasion and migration in both cell types. The canonical pathway was not responsible for the DKK1 proinvasive effect, as indicated by the active ẞ-catenin levels in the two cell lines upon DKK1 treatment. Interestingly, knockdown of TGF-ß1 negatively affected the DKK1 proinvasive effect. Taken together, DKK1 appears to facilitate tumor invasion and migration through TGF- ß1 by remodeling the tumor microenvironment and inducing inflammation. This finding endorses the relevance of TGF-ß1 as a therapeutic target.

Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis.

AIM: To investigate the effect of adipose-derived mesenchymal stem cells (ADMSCs) and their conditioned media (CM) on hepatocellular carcinoma (HCC) cell tumorigenesis. METHODS: The proliferation rate of HepG2 and PLC-PRF-5 HCC cancer cells was measured using the trypan blue exclusion method and confirmed using the cell-counting kit 8 (commonly known as CCK-8) assay. Apoptosis was detected by flow cytometry using annexin V-FITC. Protein and mRNA expression was quantified by ELISA and real time PCR, respectively. Migration and invasion rates were performed by Transwell migration and invasion assays. Wound healing was examined to confirm the data obtained from the migration assays. RESULTS: Our data demonstrated that when co-culturing HCC cell lines with ADMSCs or treating them with ADMSC CM, the HCC cell proliferation rate was significantly inhibited and the apoptosis rate increased. The decreased proliferation rate was accompanied by an upregulation of P53 and Retinoblastoma mRNA and a downregulation of c-Myc and hTERT mRNA levels. More notably, ADMSCs and their CM suppressed the expression of the two important markers of HCC carcinogenicity, alpha-fetoprotein and Des-gamma-carboxyprothrombin. In addition, the migration and invasion levels of HepG2 and PLC-PRF-5 cells significantly decreased, potentially through increased expression of the tissue inhibitor metalloproteinases TIMP-1, TIMP-2 and TIMP-3. CONCLUSION: These findings shed new light on a protective and therapeutic role for ADMSCs and their CM in controlling HCC invasiveness and carcinogenesis.

Telomerase regulation by the long non-coding RNA H19 in human acute promyelocytic leukemia cells.

BACKGROUND: Since tumor growth requires reactivation of telomerase (hTERT), this enzyme is a challenging target for drug development. Therefore, it is of great interest to identify telomerase expression and activity regulators. Retinoids are well-known inducers of granulocytic maturation associated with hTERT repression in acute promyelocytic leukemia (APL) blasts. In a maturation-resistant APL cell line, we have previously identified a new pathway of retinoid-induced hTERT transcriptional repression independent of differentiation. Furthermore, we reported the isolation of a cell variant resistant to this repression. Those cell lines could serve as unique tools to identify new telomerase regulators. METHODS: Using a microarray approach we identified the long non-coding RNA, H19 as a potential candidate playing a role in telomerase regulation. Expression of H19, hTERT, and hTR were examined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Telomerase activity was quantified by quantitative telomeric repeats amplification protocol (qTRAP). In vitro and in vivo assays were performed to investigate H19 function on telomerase expression and activity. RESULTS: We showed both in retinoid-treated cell lines and in APL patient cells an inverse relationship between the expression of H19 and the expression and activity of hTERT. Exploring the mechanistic link between H19 and hTERT regulation, we showed that H19 is able to impede telomerase function by disruption of the hTERT-hTR interaction. CONCLUSIONS: This study identifies a new way of telomerase regulation through H19's involvement and thereby reveals a new function for this long non-coding RNA that can be targeted for therapeutic purpose.

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

BACKGROUND: p53 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. METHODS: Mutations 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. RESULTS: Our 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. CONCLUSION: Despite 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.

Effect of age and body mass index on the yield of stromal vascular fraction.

OBJECTIVE: The aim of this study was to determine the influence of age, body mass index, and site of liposuction on the cell yield of SVF. METHODS: A prospective study was performed on 58 patients. The average age was 39 years old, with BMI ≤ 25 or BMI ≥ 25. Fat tissue was harvested from the abdominal region, flanks, or thighs and SVF was isolated. RESULTS: The yield of viable SVF was evaluated by trypan blue, and the markers of stem cells were evaluated by flow cytometry. The cells were positive for stem cells markers, the age, sex of the patient had no impact on SVF cell yield with an average of 1.17 × 10^8. However, the BMI > 25 had resulted in higher cell numbers, and the harvest site had a significant impact on cell yield with abdomen being the site of interest. CONCLUSION: These data demonstrate that the age of the person does not affect the cell yield of SVF; nevertheless, the donor site and BMI might be important factors in affecting cell number.

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.

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).

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-ß, 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-ß, 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.

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.

Nonalcoholic Steatohepatitis: Involvement of the Telomerase and Proinflammatory Mediators.

Nonalcoholic steatohepatitis or NASH is an excessive accumulation of fat in hepatocytes accompanied by inflammation and hepatic injury. Proinflammatory molecules such as IL-17, CCL20, S100A8, S100A9, and S100A8/A9 have been shown to be implicated in many types of cancer. Telomerase activity has been found to be associated with chronic inflammation and cancer. NASH can progress to fibrosis then cirrhosis and finally to hepatocellular carcinoma (HCC). Our objective is to try to find a relation between inflammation and the progression of NASH into HCC. We found that there was a significant elevation in the telomerase activity, detected by real-time PCR, between NASH and fibrotic NASH in the liver biopsies of patients. The expression of S100A8, S100A9, S100A8/A9, CCL20, and IL-17, detected by ELISA, is significantly increased in NASH patients with fibrosis in comparison with controls. But, in NASH patients, S100A9, S100A8/A9, and IL-17 only are significantly elevated in comparison with controls. The same, on the mRNA level, expression of IL-17, detected by RT-PCR, is significantly elevated in NASH patients in comparison with controls. Therefore, there is a direct link between the expression of IL-17, CCL20, telomerase, S100A8, and S100A9 in the fibrotic condition and the progression towards cancer.