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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.

Protein kinase C (PKC) family has been an alluring objective for new cancer drug discovery. It has been reported to regulate telomerase in several cancer types. Our team had previously used telomerase to elucidate alpha-fetoprotein (AFP) modulation in hepatocellular carcinoma (HCC). The aim of this study was to investigate the interrelationships among PKC isoforms, telomerase and AFP in HCC. PKCα and PKCδ were the most expressed isoforms in HepG2/C3A, PLC/PRF/5, SNU-387 and SKOV-3 cells. Following the upregulation of AFP using pCMV3-AFP and the human telomerase reverse transcriptase (hTERT) using a construct expressing a wild-type hTERT, and after their inhibition with all-trans retinoic acid and hTERT siRNA each respectively, we found that the expression of PKCα, PKCβI, PKCβII and PKCδ was affected by the variation of AFP and hTERT mRNA levels. An increase in AFP expression and secretion was observed after gene silencing of PKCα, PKCβ, PKCδ, and PKCε in HepG2/C3A. A similar pattern was observed in transfected PLC/PRF/5 cells, however PKCδ isoform silencing decreased AFP expression. Furthermore, telomerase activity was quantified using quantitative telomeric repeat amplification protocol. The variations in hTERT expression and telomerase activity were similar to those of AFP. Further investigation showed that PKC isoforms regulate AFP and hTERT expression levels through PI3K/AKT/mTOR pathway in HepG2/C3A and PLC/PRF/5 cells. Thus, these results show for the first time a possible interrelationship that links PKC isoforms to both AFP and hTERT via PI3K/AKT/mTOR pathway in HCC.

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.

Introduction/Background Telomeres are DNA structures protecting the linear ends of eukaryotic chromosomes against degradation and fusion, thereby maintaining genome stability. Telomerase is an enzyme that stabilizes the length of linear chromosomes by de novo synthesizing telomeric repeats during incomplete DNA replication, thus ensuring immortalization. This enzyme is expressed in 80% of cancers, including ovarian carcinoma. The human telomerase reverse transcriptase has been investigated as a detection marker for cancers in early stages, and a prognosis marker in late stages disease. The aim of this study is to investigate telomere length as a marker for survival and recurrence in ovarian carcinoma. Methodology 37 ovarian cancer biopsies have been isolated from Lebanese patients. The biopsies were subjected to DNA extraction, and then telomere length was assayed in cancer cells following the method elaborated by R. Cawthon (2002). Clinical data of each patient have been retrieved, including survival, recurrence, lymph node ratio, age, weight. Results The results obtained revealed a direct relation between telomere length and survival. In fact, patients with shorter survival rates (less than 1 year) exhibited 30% longer telomeres compared with patients with longer survival rates (3 to 5 years). Moreover, recurrence rates increased with longer telomeres. Higher ratios (35%) were detected in patients with early recurrence (less than 1 year) compared to patients with late or absent recurrence. However, no direct effect between telomere length and the other parameters was spotted. Conclusion Telomere length could be used as a prognostic marker in patients with ovarian cancer by predicting better survival and lower recurrence in case of shorter telomeres. This will be of a great value when making therapeutic decisions in a heterogeneous and aggressive disease as ovarian cancer. Disclosure Nothing to disclose.

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

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.

Background: Even in the presence of oxygen, cancer cells tend to metabolize glucose into lactate instead of undergoing oxidative phosphorylation. Knowing that glucose is crucial for tumor's proliferation, new patient's treatment strategies tend to combine chemotherapy to hypoglycemic drugs. p53 is a tumor suppressor and key regulator of glycolysis in cancer cells, however highly mutated in tumors. In ovarian cancer, the majority of studies concerning p53 mutations focus on the DNA binding domain since the majority of hotspot mutations affects this region of the gene. However, mutations affecting other regions such as the proline rich domain linked directly to apoptosis 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. Material and Methods: this study was divided into two parts. The in vitro study was done using 3 ovarian cancer cell lines SKOV-3 (p53 null), Igrov-1 (WT p53) and Ovcar-3 (DNA domain mutant p53), along with the transfection of SKOV-3 cells by three different vectors: WTp53, P72R p53 and R249S p53. The ex vivo part was performed on 30 ovarian cancer biopsies. The biopsies were subjected to Tp53 immunohistochemistry and sequencing to study the mutations and their effect on p53 expression. The effect of Tp53 mutations on glucose metabolism was assayed by glucose consumption measurements, lactate production and mRNA expression of various glycolytic enzymes. To study the effect of mutations on the transcriptional activity of p53, p21 and MDM2 expression was quantified in these cells. Annexin V assay was performed on transfected SKOV-3 to study the apoptotic activity of WT and mutant p53. Results: DNA binding domain mutant cells (Ovcar-3 and R249S SKOV-3) presented a 30% increase in glucose consumption and lactate production when compared to IGROV-1 and WT SKOV-3. P72R SKOV-3 exhibited the same pattern as WTp53 cells. The increase in the mRNA expression of glucose transporters GLUT-1 and 3 and of the glycolytic enzymes PK, PFK, G6PDH and aldolase in cells with DNA binding domain mutations varied from 20 to 30%, whereas the levels of PDHa and TIGAR decreased significantly (40%). The transcriptional activity of DNA binding domain mutant cells showed a 50% decrease in comparison to WT and P72R mutant cells. The apoptotic activity of p53 was unaltered in P72R transfected SKOV-3 cells. The ex vivo results revealed a high frequency of the P72R variant in the ovarian biopsies (69%), associated with an overexpression of the p53 protein. The glycolytic and transcriptional patterns of cells extracted from these biopsies were similar to those obtained in transfected cells. Conclusion: Though both DNA-binding and proline-rich domains mutations lead to p53 overexpression, only the first type promotes Warburg effect and could have the worst patient's prognosis. Citation Format: Stephanie Antoun, David Atallah, Roula Tahtouh, Malak Moubarak, Nada Alaeddine, George Hilal. TP53 gene mutations differently regulate ovarian cancer metabolism: Ex vivo and in vitro studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2413.

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.

Background: Hepatocellular carcinoma (HCC) is the third largest contributor to cancer mortality in the world. Alphafetoprotein (AFP) is synthetized and secreted by the majority of HCCs. Despite its controversial value as classical HCC diagnostic and follow-up marker, the AFP was lately shown to correlate with the volume of liver cancer. It is also useful as a predictive marker for radiotherapy treatment response and patients prognosis. On the molecular level, AFP has a proliferative role in HCC since it acts on its own receptor on the surface of hepatocytes; however, its mechanism of action remains unclear. Our laboratory have previously demonstrated that telomerase and protein kinase C (PKC) modulate AFP secretion in HCC cell lines and this effect is additive. The aim of this study is to elucidate the PKC isoforms involved in AFP secretion modulation and telomerase expression. Methods: Two AFP secretory cell lines, HepG2/C3a and PLC/PRF/5, and one non-secretory AFP cell line, SNU-387, were cultured in DMEM (RPMI-1640 for SNU-387) media with 10% FBS and 1% Penicillin/Streptomycin and incubated in humid 5% CO2 incubator. The RNA was extracted; the RT-PCR was performed to characterize the PKC isoforms and the modulation of hTERT by these isoforms. The role of each isoform on AFP secretion was elucidated using different PKC isoform modulators; the AFP concentration was measured using ELISA technique. The viability and toxicity of cells were assessed using WST-1. Results: The three cell lines express the PKC alpha, delta and epsilon isoforms. Whereas the other isoforms Beta 1 and 2 are slightly expressed in C3a, the gamma and beta isoforms are slightly expressed in PLC and SNU respectively. Dose-dependent inhibition of PKC alpha and beta by GO6976 decreased AFP secretion by 40% at 10 uM by the HepG2/C3a cell line. The inhibition of PKC delta by Rottlerin dose-dependently decreased AFP secretion by 30% at 5 uM. The PKC epsilon modulator FR236924 dose-dependently inhibited AFP secretion by 50% at 1 uM. Pan PKC inhibitor, GO6983, decreased AFP secretion by 45% at 5 uM. The same profile was obtained for PLC/PRF/5 with a maximum 30% of AFP down-regulation except for Rottlerin which showed a 75% of AFP inhibition. The catalytic subunit expression of telomerase, hTERT, was only inhibited by Rottlerin at 5 uM. Our experiments did not show any cell death nor cell proliferation modulation when incubating the three cell lines with PKC inhibitors. Conclusion: Combining the above results with our previous ones, we suggest for the first time, that PKC delta modulates AFP secretion through telomerase however the mechanism by which PKC alpha and epsilon modulate AFP secretion is to be elucidated. However, further experiments are needed to support this statement. Citation Format: Raia Doumit, Roula Tahtouh, Rita Ammoury, Riad Sarkis, Nada Alaaddine, George Hilal. The role of protein kinase C isoforms in telomerase activity and alphafetoprotein secretion by the hepatocellular carcinoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 940.