Raquel Alves

Selective cytotoxicity and cell death induced by human amniotic membrane in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a worldwide high incidence and mortality. For this reason, it is essential to invest in new therapies for this type of cancer. Our team already proved that human amniotic membrane (hAM) is able to inhibit the metabolic activity of several human cancer cell lines, including HCC cell lines. Taking into account the previously performed work, this experimental study aimed to investigate the pathways by which hAM protein extracts (hAMPEs) act on HCC. Our results showed that hAMPE reduce the metabolic activity, protein content and DNA content in a dose- and time-dependent manner in all HCC cell lines. This therapy presents selective cytotoxicity, since it was not able to inhibit a non-tumorigenic human cell line. In addition, hAMPE induced cell morphology alterations in all HCC cell lines, but death type is cell line dependent, as proved by in vitro and in vivo studies. In conclusion, hAMPE have a promising role in HCC therapy, since it is capable of inducing HCC cytotoxicity and cell death.

Genetic variants involved in oxidative stress, base excision repair, DNA methylation, and folate metabolism pathways influence myeloid neoplasias susceptibility and prognosis.

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) share common features: elevated oxidative stress, DNA repair deficiency, and aberrant DNA methylation. We performed a hospital‐based case‐control study to evaluate the association in variants of genes involved in oxidative stress, folate metabolism, DNA repair, and DNA methylation with susceptibility and prognosis of these malignancies. To that end, 16 SNPs (one per gene: CAT, CYBA, DNMT1, DNMT3A, DNMT3B, GPX1, KEAP1, MPO, MTRR, NEIL1, NFE2F2, OGG1, SLC19A1, SOD1, SOD2, and XRCC1) were genotyped in 191 patients (101 MDS and 90 AML) and 261 controls. We also measured oxidative stress (reactive oxygen species/total antioxidant status ratio), DNA damage (8‐hydroxy‐2′‐deoxyguanosine), and DNA methylation (5‐methylcytosine) in 50 subjects (40 MDS and 10 controls). Results showed that five genes (GPX1, NEIL1, NFE2L2, OGG1, and SOD2) were associated with MDS, two (DNMT3B and SLC19A1) with AML, and two (CYBA and DNMT1) with both diseases. We observed a correlation of CYBA TT, GPX1 TT, and SOD2 CC genotypes with increased oxidative stress levels, as well as NEIL1 TT and OGG1 GG genotypes with higher DNA damage. The 5‐methylcytosine levels were negatively associated with DNMT1 CC, DNMT3A CC, and MTRR AA genotypes, and positively with DNMT3B CC genotype. Furthermore, DNMT3A, MTRR, NEIL1, and OGG1 variants modulated AML transformation in MDS patients. Additionally, DNMT3A, OGG1, GPX1, and KEAP1 variants influenced survival of MDS and AML patients. Altogether, data suggest that genetic variability influence predisposition and prognosis of MDS and AML patients, as well AML transformation rate in MDS patients.

L744,832 and Everolimus Induce Cytotoxic and Cytostatic Effects in Non-Hodgkin Lymphoma Cells

Non-Hodgkin Lymphoma (NHL) constitutes a very heterogeneous group of diseases with different aggressiveness. Diffuse large B-cell lymphoma (DLBCL) and Burkitt’s lymphoma (BL) are two clinically aggressive lymphomas from the germinal center, very heterogeneous and with different genetic signatures. Several intracellular pathways are involved in lymphomagenesis, being BCR/PI3K/AKT/mTOR and RAS/RAF pathways the most frequently ones. In this context the therapeutic potential of a mTOR inhibitor – everolimus – and a RAS/RAF pathway inhibitor – L744,832 – was evaluated in two NHL cell lines. Farage and Raji cells were cultured in the absence and presence of several concentrations of everolimus and L744,832 in monotherapy and in combination with each other, as well as in association with the conventional chemotherapy drug vincristine. Our results show that everolimus and L744,832 induce antiproliferative and cytotoxic effect in a time-, dose-, and cell line-dependent manner, inducing cell death mainly by apoptosis. A potentiation effect was observed when the drugs were used in combination. In conclusion, the results suggest that everolimus and L744,832, alone or in combination, could provide therapeutic benefits in these subtypes of NHL.

Intestinal Epithelial Stem Cells: Distinct Behavior After Surgical Injury and Teduglutide Administration

ABSTRACT Background: Previous studies suggest that intestinal epithelial stem cells (IESC), critical drivers of homeostasis and regeneration, include two subpopulations: crypt-based columnar and “position +4” stem cells, identified by Lgr5 and Bmi1 biomarkers, respectively. Teduglutide is an enterotrophic counterpart of glucagon-like peptide 2. This study aimed to investigate the response of putative IESC to surgical injury and teduglutide administration on an animal model of intestinal resection and anastomosis. Methods: Wistar rats (n = 59) were distributed into four groups: “Ileal Resection” versus “Laparotomy”, subsequently subdivided into “Postoperative Teduglutide Administration” versus “No Treatment”; and sacrificed at third or seventh days, with ileal sample harvesting. Flow cytometry was used to analyze epithelial stem cells with monoclonal antibodies against Lgr5, Bmi1 and also CD44, CD24, CD166, and Grp78 surface markers. Results: Surgical trauma induced an increase of epithelial stem cells population at third day (9.0 ± 0.3 versus 5.7 ± 0.3%, p = 0.0001), which was more intense and involved all subpopulations after ileal resection. At seventh day, teduglutide was significantly associated with higher proportion of Lgr5+/Bmi1− cells (5.8 ± 0.1 versus 2.9 ± 0.3%, p = 0.005) and, on the contrary, lower percentage of Lgr5−/Bmi1+ cells (0.03 ± 0.01 versus 1.9 ± 0.1%, p = 0.049) after ileal resection; and higher proportion of Lgr5+/Bmi1+ cells (1.7 ± 0.1 versus 1.1 ± 0.2%, p = 0.028) after isolated laparotomy. After surgery, Lgr5+/Bmi1− and Lgr5−/Bmi1+ subpopulations demonstrated an inverse correlation and both correlated negatively with Grp78 labeling index. Lgr5−/Bmi1+ and CD44+/CD24low/CD166+/Grp78+ cells proportions exhibited a high grade positive correlation. Conclusion: Those observations support the existence of two epithelial stem cells subpopulations with distinct behavior after surgical injury and teduglutide treatment.

975PGENE METHYLATION IN MYELODYSPLASTIC SYNDROME – A COMPARATIVE STUDY BETWEEN BONE MARROW AND PERIPHERAL BLOOD

ABSTRACT Aim: Blood-based specimens may be a potential source of non-invasive DNA methylation cancer biomarkers. Peripheral blood leukocytes from patients with solid tumors exhibit complex and distinct cancer-associated DNA methylation patterns, which might be seen as epigenetic biomarkers with significant clinical potential. However, peripheral blood cell methylation profiles are largely unknown in hematopoietic cancers. Our aim was to compare DNA methylation status in bone marrow (BM) aspirate and peripheral blood (PB) of Myelodysplastic Syndrome (MDS) patients at diagnosis. Methods: We compare DNA methylation status of the tumor suppressor genes, p15, p16, p53, DAPK and MGMT, and of TRAIL (TNF-Related Apoptotic Inducing Ligand) receptor genes, TRAIL-DcR1, -DcR2, -DR4 and -DR5), in 45 Myelodysplastic Syndrome (MDS) patients at diagnosis, in genomic DNA obtained from BM aspirate and PB samples, after informed consent. Genomic DNA was isolated by standard protocols and modified by sodium bissulphite. The MS-PCR for each gene was performed using two sets of primers, one for methylated DNA and other for unmethylated DNA. k2 Test was used to analyses association between groups and Kappa statistics to evaluate concordance, results were considered statistically significant when p Results: We observed a good concordant results between BM and PB samples in 77,8% of patients for p16 (p = 0,017), 75,6% for p15 (p = 0,028), 62,2% for DAPK (p = 0,031), 84,4% for TRAIL-DcR1, 68,9% for TRAIL-DcR2, 66,7% for TRAIL-DR4 and a discordant results for TRAIL-DR5 gene (p = 0,012), since only 37,8% of the tested samples were concordant. In some cases discrepancies were also bidirectional, with cases presenting demethylated PB and methylated BM aspirate and vice versa. No patient presented p53 and MGMT gene methylated. Conclusions: Our results show a correlation between gene methylation patterns in PB and BM aspirate in MDS patients. Although DNA methylation patterns measured in PB may have great potential as informative biomarkers of cancer risk and prognosis, large systematic and prospective studies will be needed. Disclosure: All authors have declared no conflicts of interest.