José M Nascimento-Costa

Delocalized lipophilic cations as a new therapeutic approach in cancer

Delocalized lipophilic cations (DLCs) penetrate plasma and mitochondrial membranes and accumulate in mitochondria. The higher mitochondrial membrane potentials of neoplastic vs normal cells, in general, account for greater uptake and may be a way to selectively target these cells since. Dequalinium (DQA) is a DLC and so our goal is to evaluate the therapeutic potential of DQA in cancer, namely in B-cell Chronic Lymphocytic Leukaemia (B-CLL), Acute Promyelocytic Leukaemia (APL) and Hepatocellular Carcinoma (HCC). For this we used 3 cell lines, EHEB (B-CLL), HL-60 (APL) and HUH-7 (HCC), to evaluate the effect of different concentrations of DQA either by single dose administration, by daily dose administration and by association with conventional anticarcinogenic agents. Cell viability and death was determined by the resazurin assay, optical microscopy and by flow cytometry. The latter was also used to evaluate the mitochondrial membrane potential, the levels of ROS (H2O2; O2•-) and the antioxidant defense, Reduced Glutathione (GSH), using fluorescent probes. We found that DQA induced a decrease in cell viability inducing cell death by late apoptosis/necrosis in a time, dose and cell type dependent manner, with and IC50 of 2.5, 4.7 and 7.5 μM at 48h of exposure, respectively to HL-60, HUH-7 and EHEB. These effects may be mediated by oxidative stress as we have observed and increase in ROS production and a decrease in GSH levels and in mitochondrial membrane potential. We also observed that if DQA is administered on a daily basis a much lower concentration is required to induce the same effect. On the other hand, the association of DQA with the conventional drug induces a synergistic effect, because lower concentration of both drugs is required to obtain the some effect.

1664PCELLULAR AND MOLECULAR MECHANISMS OF MECHANOTRANSDUCTION INVOLVED IN METASTASIS - AN IN VITRO STUDY IN HEPATOCARCINOMA AND BREAST CANCER CELL LINES

ABSTRACT Introduction: Invasiveness of tumors requires several distinct cellular functions including lost of cellular adhesion, cell detachment, altered motility and extracellular matrix interaction. This interaction between cells and its surrounding tissues involves mechanical stimuli that are transmitted into cells and translated into a biochemical response - mechanotransduction. The increased mechanical forces made by a stiff microenvironment would contribute to the aggressiveness of solid tumors. Objectives: To study the effects of increased pressure on hepatocarcinoma and breast cancer cells prolifera-tion, migration, adhesion, cytoskeleton organization and invasiveness. Methods: A breast cancer (MCF7) and hepatocellular carcinoma (HUH7) cell line were submitted to a constant increase of atmospheric pressure, 40 mmHg and 60 mmHg, in an airtight box during 4 hours. We analyzed: 1, Cell viability followed by propidium iodide cell cycle evaluation assay; 2, Cell migration by the scratch assay; 3, the expression of integrins by western blot using specific antibodies; 4, the actin cytoskeleton by fluorescent microscopy; and 5, Invasiveness using an invasion kit with a polycarbonate basement membrane. Results: Increasing pressure caused an increased in the viability at 4h after the mechanical stimulation, and that was sustained for 24h. Those results were complemented with cell cycle analysis showing an increment in the cells in S-phase confirming the proliferative effect. We observed a pressure-dependent single-cell detachment pattern on both cell lines when compared to controls. This migratory pattern was studied the cell-cell and cell-substrate adhesions molecules (E-cadherin and b-catenin, and b1integrin, respectively) which revealed decreased. It seems an amoeboid migration variant of the single-cell migration pat-tern.Besides that, cells submitted to pressure presented an increase in stress fibers, a cortical actin pattern, lamelipodia and filopodia. Conclusions: In this work, we demonstrated that the mechanical stimuli have a critical role in migration and invasion in the breast cancer and hepatocellular carcinoma cells. Our results open new perspectives on the role of mechanotransduction in cancer metastasis. The exploration of the signaling pathways involved could represent future possible therapeutic targets. Disclosure: All authors have declared no conflicts of interest.

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.

Recombinant trail: a synergistic effect in myeloid leukemias.

The tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL/Apo-2L) is a member of the TNF superfamily that trigger and activate 2 death receptors, DR4 and DR5, and 2 decoy receptors, DcR1 and DcR2. Several studies demonstrated that TRAIL in monotherapy can induces cancer cell death cells, but few have been done in leukemias in combination with conventional drugs. The aim of this work is to analyse the potential synergist effect of a recombinant TRAIL (rhTRAIL) in myeloid leukemias. For this, 2 myeloid leukemia cell lines, HL-60 (promyelocitic leukemia) and K-562 (Chronic Myeloid Leukemia) were treated with different concentrations of rhTRAIL as single agent and in combination with ATRA (all-transretinoic acid) and imatinib, respectively. The viability was measure using the trypan blue test and cell death by flow cytometry (FC) and Optical Microscopy. TRAIL and TRAIL-Rs were evaluated by FC. Our results show that rhTRAIL induced a decrease in cell viability inducing cell death, in a time, dose and cell type dependent manner. We observe an IC50 in HL-60 treated for 48h of 250 ng/mL, although in K562 cells, rhTRAIL wasn’t able to induce a significant effect. However, when we previously treated the cells with ATRA or IMATINIB a synergistic effect is observed, mainly in HL60 cells. These results may be correlated with the differential TRAIL receptors expression, namely the presence of the anti-apoptotic TRAIL receptors, in K562 cells. On the other hand, the higher percentage of pro-apoptotic TRAIL receptors may be related with the therapeutic efficacy of rhTRAIL in HL-60 cells. Our study suggests that rhTRAIL can be use as a new therapeutic aproach in APL, as single agent. However, it can potentiate the cytotoxic effect of conventional drugs.

New targeted therapies in myelodysplastic syndrome: the role of farnesyltransferase and proteasome inhibitors

One of the main mechanisms responsible for MDS molecular pathogenesis involves the activation of tyrosine-kinase receptors, such as FLT3, RAS proteins, and deregulation of apoptotic pathways. Regarding this, new drugs have been developed to target pathways involved in malignancy, such as Farnesyltransferase Inhibitors (IFTs) and proteasome inhibitors (PI). This work aims to clarify the role of IFTs and PI as potential therapeutic agents in Myelodysplastic Syndrome (MDS). For this, F-36P cells, were incubated with different concentrations of α-HFPA (IFT) and MG262 (PI), as single agents and in association with the conventional therapeutic drug, Cytosine Arabinoside (Ara-C). Cell growth and viability was evaluated by Trypan Blue test. Cell death was analyzed by optic microscopy and flow cytometry (FC). Expression of proteins involved in apoptosis and cell cycle regulation was evaluated by FC. The detection of RAS and FLT3 mutations was accessed by sequentiation and PCR, respectively. Our results show that α-HFPA and MG262, in monotherapy, induce a decrease in cell growth and viability in a time and dose-dependent manner (IC50, α-HFPA 125 μM; MG262 100 nM). The antiproliferative effect of α-HFPA could be related to RAS/MAPK pathway inhibition, as we observed a decrease in cyclin D1 levels, while the cytotoxicity induced by MG262 to an increase in BAX expression. Our results show that α-HFPA is effective independently of RAS mutations, once we didn’t identify mutations in none of the isoforms studied, but we observe ITD mutations in FLT3 gene. These results suggest that IFTs and PIs may constitute a potential therapeutic approach as single agents in MDS.

Metalloproteinase inhibitors as a potential therapeutic approach in multiple myeloma: a preliminary study

Multiple Myeloma (MM) is one of the B-cell malignancies with a poor prognosis, characterized by the clonal expansion of neoplastic plasma cells within the bone marrow, elevated serum immunoglobulin, and osteolytic bone disease. The first pathogenic step is a premalignant monoclonal gamopathy of undetermined significance (MGUS). With progression of MGUS to myeloma, complex genetic/epigenetic events occur in the neoplastic plasma cell, and in the bone marrow microenvironment. The resultant interactions of myeloma cells, bone marrow stromal cells, and microvessels contribute to persistence of the tumour and resistance to drugs. Matrix metalloproteinases (MMPs) play a critical rule in bone remodeling (osteolitic lesions) and tumor invasion, and could be a new therapeutic target in MM. The aim of this study is to evaluate the therapeutic potential of a metalloproteinase inhibitor, batimastat (BB-94), in a multiple myeloma cell line in culture. For this purpose a MM cell line, the NCI-H929 [H929] cells were cultured in absence and presence of different concentrations of the MMP inhibitor, BB-94, during different periods of time. Cell viability and death was determined by the alamar blue assay and by flow cytometry using the annexin V/propidium iodide incorporation. Our preliminary results show that BB-94 has, along a broad concentration range, an antiproliferative effect in MM cells that is accompanied by a cytotoxic effect as results of increased apoptosis levels as the concerntratio increases from 1 to 5µM . This study suggests that batimastat could be a new therapeutic approach in multiple myeloma in monotherapy.

Antitumoral effect of farnesiltransferase and mtor inhibitors in hepatocellular carcinoma: in vitro studies

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, often diagnosed at an advanced stage when the most potentially curative strategies are no longer effective. Advances in the understanding of tumor biology are opening new paths for the prevention and treatment of HCC, through the development of new targeted therapies. The design of drugs that block different growth-promoting pathways, activate apoptotic pathways may also open new horizons in the treatment of HCC. The aim of this study was to test the efficacy of new targeted drugs involved in signalling pathways, such as farnesiltransferase (L-744832) and mTOR inhibitors (everolimus) in a HCC cell line (HUH-7 cells). The HUH-7 cells were cultured in absence and presence of different concentrations of L-744832 and everolimus. The antiproliferative effect was assessed by the Alamar Blue assay and cell death by optic microscopy and flow cytometry. Our results showed that farnesiltransferase and mTOR inhibitors had an antiproliferative and cytotoxic effects in monotherapy in a dose and time dependent manner, inducing cell death preferentially by apoptosis. On, the other hand, the combination of L-744832 and everolimus with conventional anticarcinogenic drugs demonstrated a higher antiproliferative and cytotoxic effect for lower doses than the IC50 used in monotherapy (addition or potentiation synergism). These results suggested that farnesiltransferase and mTOR inhibitors may constitute a new potential therapeutic approach in HCC either in monotherapy or in association with conventional therapies.