Andrea Barretto Motoyama

SMYD2 is highly expressed in pediatric acute lymphoblastic leukemia and constitutes a bad prognostic factor.

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Although several clinical characteristics can be associated with worse prognosis, more robust biological markers still remains uncovered. SMYD2, a member of SMYD protein family, regulates the activity of several proteins through methylation. In this study, we performed quantitative real time PCR to compare the expression of SMYD2 in 83 pediatric ALL patients and non-neoplastic bone marrow samples (BMS). The study revealed that SMYD2 expression is altered in ALL BMS and its high expression was correlated with a bad prognosis. Moreover, we also revealed that SMYD2 expression level significantly decreases in patients that respond to chemotherapy treatment.

Altered expression of MLL methyltransferase family genes in breast cancer

The histone lysine methyltransferases contain a SET domain, which catalyzes the addition of methyl groups to specific lysine residues. The MLL family of genes encodes histone-modifying enzymes with histone 3-lysine 4 methyltransferase activity that can regulate gene transcription. The MLL family exists in multi-protein complexes and has been implicated in a variety of processes including normal development and cell growth. Although some of the MLL family members have already been described to be involved in cancer, a clear relationship of these genes with breast cancer is not determined to date. In the present study, we used quantitative PCR to investigate the expression profile of all five MLL genes [MLL (ALL-1), MLL2, MLL3, MLL4 and MLL5] in 7 breast cancer cell lines, 8 breast tumors and adjacent non-tumor tissues and in 12 normal tissues. We observed a diminished expression of all five genes in the breast cancer cell lines when compared to normal breast tissue. We found a significantly decreased expression of MLL2 in the tumor samples compared to the non-tumor controls. In tumor samples, MLL5 also showed a clear suppression tendency. Among the normal tissues analyzed, all genes showed a markedly higher expression in skeletal muscle and brain. Although further studies are required to determine the exact role of these methyltransferases in cancer development, our results indicate that the suppression of MLL genes, especially MLL2 and 5, take part in modulating breast carcinogenesis. Our assessment of the MLL family gene expression patterns in a diverse set of breast cancer cell lines and in a multitude of tissue types and breast tumors should lead to increasingly detailed information on the involvement of these genes in cancer progression.

Cytotoxic effect of tobacco extracts on human oral squamous cell carcinoma cell-line

Cancer is a public health problem worldwide. Incidences of oral carcinomas are increasing in the last decades, and the developed countries are the most affected. Current therapeutic options for this type of cancer are aggressive and/or invasive, including surgery, radiotherapy and chemotherapy. In addition, they have not yet translated into an improvement of life quality or expectancy to patients. In this scenario, new therapeutics are urgently needed and actively sought after. The goal of this study was to investigate the cytotoxic effects of tobacco crude extract (TCE) and two fractions thereof in the human lineage of oral squamous cell carcinoma, OSCC-3. Exposure of human oral cancer cells to TCE-induced cell death and decreased cell viability in a dose-dependent manner. Of the fractions tested, one was able to induce significant cell death (over 50%) after 48 h treatment. DNA fragmentation and caspase-3 activation indicated that the type of cell death induced by TCE and its fraction was apoptosis. Our results indicate that tobacco contains compounds that could be useful in inducing apoptosis in cancer cells. More specifically, because of the neutral chemical nature of the fraction capable of inducing apoptosis, we postulate that the putative compound responsible for the cell death is non-polar. Further investigation is needed to uncover its chemical nature and structure.

Cytotoxic effect of Pouteria torta leaf extracts on human oral and breast carcinomas cell lines

AIMS This study aimed at investigating the cytotoxic activity and the type of cell death induced by Pouteria torta (P. torta) leaf extracts on human oral squamous cell carcinoma and breast carcinoma cell lines. MATERIAL AND METHODS The effects of P. torta leaf hexanic (PTH), ethanolic (PTE) and aqueous (PTA) extracts at the concentration of 500 mg/mL were evaluated on OSCC-3 and MCF-7 cell lines, using crystal violet staining after 24 and 48 h of treatment. To obtain the dose-response curve, cells were treated with decreasing concentrations of the extracts (1000, 750, 500, 250, 125 mg/mL) for 24 h. To investigate the mechanism of cell death (apoptosis vs. necrosis), DNA fragmentation assay was performed. RESULTS All extracts were cytotoxic to both OSCC-3 and MCF-7, albeit at differing levels. PTH and PTE were effective at the concentration of 500 μg/mL, resulting in nearly 50% of cell death in both cancer cell lines. PTA was more effective at lower concentrations, with more significant cell death at 125 g/mL. Treatment with PTA and PTE caused apoptosis in MCF-7, whereas in OSCC-3 cells, the same effect could only be caused by PTH. On the other hand, PTA was able to induce necrosis in OSCC-3. CONCLUSIONS These findings demonstrated that P. torta leaf extracts may contain useful compounds to combat oral and breast cancer, and this study highlights the potential biological relevance of the Brazilian Cerrado Biome in cancer therapy.

Characterization of cytotoxic activity of compounds derived from anacardic acid, cardanol and cardol in oral squamous cell carcinoma

Background Cancer is the second leading cause of death worldwide, and oral cancer ranks tenth among all types [1]. Chemotherapy, radiotherapy and surgery are current therapeutic options; however these are not fully efficient. Permanent functional impairment and aesthetic scars are frequent [2]. In this scenario, it is crucial to find therapeutic alternatives, including those derived from the flora, which currently provides about 1/3 of all new medicines. Anacardic acids, cardanols and cardols are the main constituents of the cashew nut shell liquid (herein referred to as “LCC”) and together, account for 90% of its composition. The liquid is an industrial byproduct, with low economic value prior to processing. The nut, the proper fruit from the plant Anacardium occidentale, is edible. Anacardic acids, cardols and cardanols are made of a phenolic ring connected to a long side chain (usually C15H31-n) that can bear several to none insaturations. Additionally, a methyl group can be found in the phenolic ring [3]. Apart from current industrial uses, it has been demonstrated that some of these compounds may exert microbicide and antioxidative activities. Anacardic acid has been shown to be cytotoxic to lung, liver and gastric tumor cells through epigenetic mechanisms by inhibiting histone acetyltransferases (HATs) [4] and in a capase-independent manner [5]. However, given the possible molecular diversity obtained from LCC constituents, not all distinct LCC derivatives have yet been fully analyzed or characterized. Aim The aim of the present study was to screen for compounds with cytotoxic activity in oral cancer cells and characterize the observed effect.

Oropharynx HPV status and its relation to HIV infection

Background The number of oropharyngeal lesions caused by HPV (Human papillomavirus) has been increasing worldwide in the past years. In spite of the clinical relevance of HPV infection in the anogenital tract of HIV-positive patients, the relevance of oropharynx HPV infection in these patients is not clear. The aim of the present study was to detect HPV infection, and clinical and cytological changes in the oropharynx of HIV-positive patients. Methods Samples collected from the oropharynx of 100 HIV-positive patients were subjected to hybrid capture (HC), conventional and liquid-based cytology. Clinical data were also collected to investigate the relation with HPV status. Results High and low-risk types of HPV were present in 8% and 16.7% of the total sample. The mean ± sd (maximum-minimum) of the relative ratio light unit (RLU)/cutoff (CO) was 2.94 ± 2.58 (1.09–7.87) and 1.61 ± 0.65 (1.07–2.8) for high- and low-risk-HPV, respectively. By cytology, dysplasia was not detected, but atypical squamous cells of undetermined significance (ASC-US) were diagnosed in two samples. No clinical change, suggestive of dysplasia/cancer, was detected. Conclusion Our study was able to detect and characterize HPV infection by hybrid capture, which may represent a good tool for screening and follow-up of HPV in the studied population. The frequency and viral load of HPV were low. Neither clinical nor cytological changes suggestive of dysplasia/neoplasia were observed in oropharynx of HIV-positive patients.

Aerobic bacteria on cervical cytology

Dysbacteriosis is characterized by a shift in the microbiota, with a decrease in the amount of lactobacilli accompanied by an increase of anaerobic and/or aerobic bacteria. The infections of the genital tract by aerobic bacteria are associated with aerobic vaginitis (AV), pelvic inflammatory disease, infertility and pregnancy complications, such as chorioamnionitis, premature rupture of membranes and preterm delivery. Universal screening for group B streptococcus (GBS) at 35 to 37 weeks’ gestation is recommended in all pregnant women. Besides this, cervical colonization by aerobic bacteria is an independent and significant factor in the determination of success in assisted reproduction treatments. Dysbacteriosis may also serve to identify groups of patients with an increased risk for a smear with squamous abnormalities. Because of the poor recognition of AV, this condition is often misdiagnosed as bacterial vaginosis (BV). AV and BV may have similar symptoms, such as increased vaginal discharge, a deficiency in lactobacilli and an elevated pH. However, the abnormal microbiota in BV is anaerobic (Gardnerella vaginalis, Mobiluncus sp., Bacteroides sp., Prevotella sp., Peptostreptococcus sp., etc.), while in AV only aerobic enteric bacteria, such as Escherichia coli, Staphylococcus aureus, GBS and enterococci, are recovered. Samples for both conventional cervical cytology and culture of aerobic bacteria were collected from women who underwent annual routine screening. The bacterial microbiota on cervical cytology samples was analyzed in 47 samples with positive culture. For culture, an endocervical swab was taken and immediately placed in Stuart medium. Culture was performed on blood agar, chocolate agar, and MacConkey agar, and incubated at 35–378 for 24–48 hours. VITEK 2 grampositiveGP ID and gram-negative GN ID cards (bioM erieux, Marcy l’ Etoile, France), based on colorimetric detection, were used for identification of bacteria according to the manufacturer’s instructions. The samples for conventional cervical cytology were collected from ectocervix and endocervix with Ayre’s spatula and cytobrush, respectively. The smears were immediately fixed in 95% ethanol and stained by the Papanicolaou method. Individual squamous cells with a layer of coccobacilli along the margins of the cell membranes were considered “clue cells.” Coccobacilli, characterized by small bacilli and cocci organisms, were found both isolated and as microcolonies. Lactobacilli were characterized by the presence of elongated bacillary structures. The smears were analyzed by one pathologist who was blinded to the culture

Effects of Caffeine on Behavioural and Cognitive Deficits in Rats

There are many studies that have sought to find drug therapies to prevent harm arising from sepsis. Such studies have represented a progress in the support to septic patients and also in the development of new pharmacological alternatives. Our interest was to investigate the caffeine effect on sepsis behavioural and memory impairments. Male rats were anaesthetized and the surgery was made to allow exposure of the caecum, which was then squeezed to extrude a small amount of faeces from the perforation site, which was later placed back into the peritoneal cavity. This procedure, which served to generate experimental sepsis, is herein referred to as ceccum ligation and perforation (CLP). The caffeine (10 mg/kg) was administered by gavage route, once daily, during 7 or 14 consecutive days to investigate the effects of acute or subchronic caffeine treatment on long‐term behavioural and cognitive deficits induced by CLP. On the last day, 1 hr after caffeine administration, the animals were submitted to open‐field, elevated plus maze (EPM), forced swimming and step‐down inhibitory avoidance tests. The results showed that caffeine increased the percentage of open arm entries and open arm time in the EPM test, and reduced the immobility time when compared to the sham‐operated group. The caffeine also increased the latency in the inhibitory avoidance test platform. Our results demonstrated that the caffeine improved behavioural changes and improved the neurocognitive deficits of sepsis‐surviving animals. It is possible that blockage of the adenosine receptors may be responsible for the results here observed.

A panel of markers for identification of malignant and non-malignant cells in culture from effusions

The aim of the present study was to identify cell types in primary culture from malignant and non-malignant effusions. Effusion samples were subjected to cytology and culture. Immunocytochemistry was performed in cytological slides to evaluate malignancy (positivity for malignancy markers) and in culture slides for identification of cell types in growth. A total of 143 effusion samples (pleural n=76; peritoneal n=37; pericardial n=4; and peritoneal lavage n=26) were analyzed. Cell growth was observed in 34.9% of all samples and immunocytochemistry for identification of cell types in culture slides was conclusive in 90% of them. In non-malignant samples (n=28), growth of mesothelial cells, macrophages and of both cell types was identified in 82.14, 10.71 and 7.14%, respectively. In malignant samples (n=17, all carcinomas), growth of malignant epithelial cells and of both malignant epithelial and mesothelial cells was identified in 41.17 and 23.52%, respectively. In the remaining 35.29% of malignant samples, the only cells in growth were mesothelial and/or macrophages instead of malignant epithelial cells. In conclusion, in culture of malignant effusions, mesothelial cells may be simultaneously identified with malignant epithelial cells. Besides, mesothelial cells and macrophages may be the only cells identified in malignant effusion culture. Therefore, a broad panel of cell markers should be used for unmistakable identification of cells in studies of effusion primary culture. The ideal malignant effusion sample to obtain culture of neoplastic cells should be that without the presence of mesothelial cells and macrophages.

Abstract 3842: Expression analysis of SMYD5 in breast cancer and normal tissues

Lysine methyltransferases are a group of enzymes that carry a SET domain, which catalyzes the addition of methyl groups to specific lysine residues. Several lysine methyltransferases have been described to be involved in human carcinogenesis. The SMYD family of methyltransferases constitutes a group of five genes that encodes proteins bearing a SET and a MYND domain, whose activity have not yet been completely characterized. Although deregulation of SMYD family members has been reported to contribute to cancer progression through several different mechanisms, to date a clear relationship of SMYD5 with breast cancer, the second most frequent cancer worldwide and the most common among women, was not determined. In the present study we investigated the expression profile of SMYD5 in breast tumors and adjacent non-tumor samples, in breast cancer cell lines and in several normal tissues. qPCR revealed SMYD5 mRNA expression level was downregulated in 7 breast cancer cell lines when compared to normal breast tissues. Relative expression of SMYD5 in breast tumors and normal breast samples revealed a significant (p Note: This abstract was not presented at the meeting. Citation Format: Joao Nunes de Matos Neto, Maira de Azevedo Feitosa Araujo, Doralina do Amaral Rabello, Andrea Barretto Motoyama, Diego Madureira de Oliveira, Fabio Pittella Silva. Expression analysis of SMYD5 in breast cancer and normal tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3842. doi:10.1158/1538-7445.AM2015-3842