Andréia Hanada Otake

Inhibition of angiotensin II receptor 1 limits tumor-associated angiogenesis and attenuates growth of murine melanoma

PurposeWe evaluated the involvement of angiotensin II (AngII)-dependent pathways in melanoma growth, through the pharmacological blockage of AT1 receptor by the anti-hypertensive drug losartan (LOS).ResultsWe showed immunolabeling for both AngII and the AT1 receptor within the human melanoma microenvironment. Like human melanomas, we showed that murine melanomas also express the AT1 receptor. Growth of murine melanoma, both locally and at distant sites, was limited in mice treated with LOS. The reduction in tumor growth was accompanied by a twofold decrease in tumor-associated microvessel density and by a decrease in CD31 mRNA levels. While no differences were found in the VEGF expression levels in tumors from treated animals, reduction in the expression of the VEGFR1 (Flt-1) at the mRNA and protein levels was observed. We also showed downregulation of mRNA levels of both Flt-4 and its ligand, VEGF-C.ConclusionsTogether, these results show that blockage of AT1 receptor signaling may be a promising anti-tumor strategy, interfering with angiogenesis by decreasing the expression of angiogenic factor receptors.

Platelet protease-activated receptor 1 and membrane expression of P-selectin in pulmonary arterial hypertension

INTRODUCTION Pulmonary arterial hypertension (PAH) is frequently associated with thrombotic events, particularly involving the pulmonary microcirculation at sites of vascular injury. We therefore decided to analyse protease-activated receptor 1 (PAR1), a key element in the activation of human platelets by thrombin, in PAH patients in stable clinical condition. METHODS Using flow cytometry, we analyzed platelet PAR1 density, PAR1-mediated exposure of P-selectin and the formation of platelet-leukocyte aggregates in 30 PAH patients aged 11 to 78 years (median 50.5 years). The control group consisted of 25 healthy subjects with the same age range as patients. RESULTS In patients, total platelet PAR1 density and uncleaved PAR1 density correlated negatively with platelet count (r(2)=0.33 and r(2)=0.34 respectively, p<0.0015). In patients with a low platelet count (<150x10(9) platelets/L), both densities were increased relative to controls (82% and 33% respectively, p<0.05). Thrombin peptide-induced platelet exposure of P-selectin was directly related to total and uncleaved PAR1 density (respectively, r(2)=0.33 and r(2)=0.29, p<0.0025) and increased in subjects with low platelet count (46% versus those with normal platelet count, p<0.05). Patients with low platelet count had decreased in vitro thrombin-induced formation of platelet-leukocyte aggregates (57% decrease versus controls, p<0.05). CONCLUSIONS There seems to be a subpopulation of PAH patients with increased propensity to thrombotic events as suggested by increased platelet PAR1 expression and PAR-mediated surface exposure of P-selectin associated with decreased platelet count.

Cationic technetium and rhenium complexes with pendant carbohydrates.

Three carbohydrate conjugated dipicolylamine chelators, 2-bis(2-pyridinylmethyl)amino)ethyl 1-deoxy-1-thio-beta-D-glucopyranoside (L(1)), 2-bis(2-pyridinylmethyl)amino)ethyl-beta-D-glucopyranoside (L(2)), and 2-bis(2-pyridinylmethyl)amino)carboxamide-N-(2-amino-2-deoxy-D-glucopyranose) (L(3)) were complexed to the [M(CO)(3)](+) core (M=Tc, Re) and the properties of the resulting complexes were investigated. Synthesis and characterization of the chelator 2-bis(2-pyridinylmethyl)amino)ethyl 1-deoxy-1-thio-beta-D-glucopyranoside (L(1)) and the corresponding Re complex are reported. All chelators were radiolabeled in high yield with [(99m)Tc(CO)(3)(H(2)O)(3)](+) (>98%) and [(186)Re(CO)(3)(H(2)O)(3)](+) (>80%). The chelators and Re-complexes were determined to not be substrates for the glucose metabolism enzyme hexokinase. However, the biodistribution of each of the (99m)Tc complexes demonstrated fast clearance from most background tissue, including >75% clearance of the activity in the kidneys and the liver within 2h post-injection.

Emerging targets for combination therapy in melanomas

Cutaneous melanomas are often difficult to treat when diagnosed in advanced stages. Melanoma cells adapt to survive in extreme environmental conditions and are among the tumors with larger genomic instability. Here we discuss some intrinsic and extrinsic mechanisms of resistance of melanoma cells to both conventional and target therapies, such as autophagy, adaptation to endoplasmic reticulum stress, metabolic reprogramming, mechanisms of tumor repopulation and the role of extracellular vesicles in this later phenomenon. These biological processes are potentially targetable and thus provide a platform for research and discovery of new drugs for combination therapy to manage melanoma patient treatment.

Mesenchymal stem cells do not prevent antibody responses against human α-L-iduronidase when used to treat mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPSI) is an autosomal recessive disease that leads to systemic lysosomal storage, which is caused by the absence of α-L-iduronidase (IDUA). Enzyme replacement therapy is recognized as the best therapeutic option for MPSI; however, high titers of anti-IDUA antibody have frequently been observed. Due to the immunosuppressant properties of MSC, we hypothesized that MSC modified with the IDUA gene would be able to produce IDUA for a long period of time. Sleeping Beauty transposon vectors were used to modify MSC because these are basically less-immunogenic plasmids. For cell transplantation, 4×106 MSC-KO-IDUA cells (MSC from KO mice modified with IDUA) were injected into the peritoneum of KO-mice three times over intervals of more than one month. The total IDUA activities from MSC-KO-IDUA before cell transplantation were 9.6, 120 and 179 U for the first, second and third injections, respectively. Only after the second cell transplantation, more than one unit of IDUA activity was detected in the blood of 3 mice for 2 days. After the third cell transplantation, a high titer of anti-IDUA antibody was detected in all of the treated mice. Anti-IDUA antibody response was also detected in C57Bl/6 mice treated with MSC-WT-IDUA. The antibody titers were high and comparable to mice that were immunized by electroporation. MSC-transplanted mice had high levels of TNF-alpha and infiltrates in the renal glomeruli. The spreading of the transplanted MSC into the peritoneum of other organs was confirmed after injection of 111In-labeled MSC. In conclusion, the antibody response against IDUA could not be avoided by MSC. On the contrary, these cells worked as an adjuvant that favored IDUA immunization. Therefore, the humoral immunosuppressant property of MSC is questionable and indicates the danger of using MSC as a source for the production of exogenous proteins to treat monogenic diseases.

Quantitative proteomic analysis and functional studies reveal that nucleophosmin is involved in cell death in glioblastoma cell line transfected with siRNA

Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1‐siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ‐treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.

Accumulation of prohibitin is a common cellular response to different stressing stimuli and protects melanoma cells from ER stress and chemotherapy-induced cell death

Melanoma is responsible for most deaths among skin cancers and conventional and palliative care chemotherapy are limited due to the development of chemoresistance. We used proteomic analysis to identify cellular responses that lead to chemoresistance of human melanoma cell lines to cisplatin. A systems approach to the proteomic data indicated the participation of specific cellular processes such as oxidative phosphorylation, mitochondrial organization and homeostasis, as well as the unfolded protein response (UPR) to be required for the survival of cells treated with cisplatin. Prohibitin (PHB) was among the proteins consistently accumulated, interacting with the functional clusters associated with resistance to cisplatin. We showed PHB accumulated at different levels in melanoma cell lines under stressing stimuli, such as (i) treatment with temozolomide (TMZ), dacarbazine (DTIC) and cisplatin; (ii) serum deprivation; (iii) tunicamycin, an UPR inducer. Prohibitin accumulated in the mitochondria of melanoma cells after cisplatin and tunicamycin treatment and its de novo accumulation led to chemoresistance melanoma cell lines. In contrast, PHB knock-down sensitized melanoma cells to cisplatin and tunicamycin treatment. We conclude that PHB participates in the survival of cells exposed to different stress stimuli, and can therefore serve as a target for the sensitization of melanoma cells to chemotherapy.

Morphological alterations and G0/G1 cell cycle arrest induced by curcumin in human SK-MEL-37 melanoma cells

The aim of this work was to study the effect of cur cumin on cell cycle in the human SK-MEL-37 melanoma cell line. In addition, morphological and structural analyses were also performed. Flow cytometric analysis showe d a G0/G1 arrest at 5 µM after 24 h exposure and a concentrat ion-dependent increase in the proportion of sub-G0 hypodiploid cells. Typical apoptotic events were al so observed by the fluorescence microscopy, transmi ssion and scanning electronic microscopy. Loss of mitochondri al membrane potential was not detected. Results su ggested that curcumin could arrest human melanoma cells at G0/G1 phase and induce a mitochondrial-independent apoptotic pathway .

Cell internalization of 7-ketocholesterol-containing nanoemulsion through LDL receptor reduces melanoma growth in vitro and in vivo : a preliminary report

Oxysterols are cholesterol oxygenated derivatives which possess several biological actions. Among oxysterols, 7-ketocholesterol (7KC) is known to induce cell death. Here, we hypothesized that 7KC cytotoxicity could be applied in cancer therapeutics. 7KC was incorporated into a lipid core nanoemulsion. As a cellular model the murine melanoma cell line B16F10 was used. The nanoparticle (7KCLDE) uptake into tumor cells was displaced by increasing amounts of low-density-lipoproteins (LDL) suggesting a LDL-receptor-mediated cell internalization. 7KCLDE was mainly cytostatic, which led to an accumulation of polyploid cells. Nevertheless, a single dose of 7KCLDE killed roughly 10% of melanoma cells. In addition, it was observed dissipation of the transmembrane potential, evidenced with flow cytometry; presence of autophagic vacuoles, visualized and quantified with flow cytometry and acridine orange; and presence of myelin figures, observed with ultrastructural microscopy. 7KCLDE impaired cytokenesis was accompanied by changes in cellular morphology into a fibroblastoid shape which is supported by cytoskeletal rearrangements, as shown by the increased actin polymerization. 7KCLDE was injected into B16 melanoma tumor-bearing mice. 7KCLDE accumulated in the liver and tumor. In melanoma tumor 7KCLDE promoted a >50% size reduction, enlarged the necrotic area, and reduced intratumoral vasculature. 7KCLDE increased the survival rates of animals, without hematologic or liver toxicity. Although more pre-clinical studies should be performed, our preliminary results suggested that 7KCLDE is a promising novel preparation for cancer chemotherapy.

7-Ketocholesterol loaded-phosphatidylserine liposome induces cell death, autophagy, and growth inhibition of melanoma and breast adenocarcinoma.

Background: The exposure of phosphatidylserine (PS) is one of the first steps of programmed cell death. Phagocytosis on cancer microenvironment is well described in tumors and is associated with malignancy and poor prognosis. Tumor associated macrophages (TAMs) act suppressing the anticancer immune response. The tumor parenchymal cells are also capable of phagocytosis cells in apoptosis. In a previous study we observed that 7-ketocholesterol is capable of inducing autophagy on melanoma cell. Aims: Evaluate the activities of a 7-ketocholesterol loaded-phosphatidylserine liposome on autophagy and phagocytosis of tumor microenvironment. Methods: Liposomes were constituted by 20 mg commercial Phosphatidylserine (PS) and PS associated with 5 mg of 7-ketocholesterol extracted with chloroform/methanol (10: 1), dried, resuspended in 10 mL phosphate buffer, homogenized and sonicated for 6 minutes. The size and Zeta Pontencial (ZP) of liposomes were evaluated. Antiinflammatory activity of liposomes was evaluated by paw edema induced by carrageenan. A dependent-dose effect of liposomes on J774 macrophages, B16F10 melanoma cells, and 4T1 breast cancer cells was assessed by MTT. Cell death evaluations, for the same cells, were performed by flow cytometry with propidium iodide (PI) staining. The presence of acid vacuoles related to autophagy was evaluated by flow cytomery by acridine orange staining. The effects of the liposomes in vivo were evaluated by B16F10 melanoma-bearing C57/bl6 mice and 4T1 breast cancer-bearing Balb c mice. Endocytosis efficiency of the liposomes was observed by labeling it with PKH26 fluorescent staining and evaluated in 4T1 cells after 12 h. Liposomes were radiolabeled by adding 1 to 30 mCi of 99mTc radiopharmaceuticals (99mTcO4-, 99mTc-dextran-70, 99mTc-MIBI, 99mTc-DISIDA) and 18FDG; the solution was homogenized and sonicated for 6 minutes. The samples were centrifuged and part of the supernatant was added to an Amicon® filter (10kD) and concentrated, the concentrated was diluted with 400 uL of PBS and concentrated again. Liposome incorporation was determined by quotient of the radioactivity in the Amicon® by sum of Amicon® and filtrated solutions. Furthermore, lipophilicity (L), hydrophilicity (H), and charge (-/0/+) of the radioactive material were considered in the final analysis. Results: PS liposomes presented 141,9nm + 9,101 size with a -25,2 ZP; PS-7-ketocholesterol (PS/7KC) liposomes presented 153,9 nm + 10,35 size with a -29,1 ZP. The paw edema was inhibited by both liposomes after 240 min of the carrageenan induction. The concentration of 26 uM/mL of PS and PS/7KC liposomes stimulated cell proliferation. PS/7KC at the concentrations above 84 uM/mL inhibited the cell proliferation. PS/7KC showed intense antiproliferative activity in melanoma cells and breast adenocarcinoma cells, assessed by the MTT method and by flow cytometry with PI. It was observed 10% more autophagic vacuoles on melanoma cells treated with PS/7KC than the control groups. Both in vivo tumor models had the same antiproliferative effect of the PS/7KC liposomes with daily doses. Daily doses of PS liposomes induced a high size of tumors. 99mTc-MIBI was efficiently and strongly incorporated to liposomes than the other proposed formulations. Conclusion: PS liposomes have effects in vivo and in vitro and must be related to phagocyte and autophagy activities. PS/7KC impairs J774 macrophage, B16F10 melanoma, and 4T1 breast adenocarcinoma cell growth. PS/7KC induces the presence of acid vacuoles corresponding to autophagy. The liposomes had a high endocytosis evaluated by PKH 26 labelled particles. PS keeps the tumor proliferation and PS/7KC inhibits tumor growth after ten days of daily doses. Supported by CNPq and Fundacao Araucaria. Citation Format: Giovani Marino Favero, Tharcisio Citrangulo Tortelli, Jr., Daniel Fernandes, Ana Paula Prestes, Louise N.B. Kmetiuk, Andreia Hanada Otake, Luciana N.S. Andrade, Daniele de Paula Faria, Camila de Godoi Carneiro, Alexandre Teles Garcez, Fabio L.N. Marques, Roger Chammas. 7-Ketocholesterol loaded-phosphatidylserine liposome induces cell death, autophagy, and growth inhibition of melanoma and breast adenocarcinoma [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; Sao Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A50.