In this work, we report the synthesis of a new 1,3-thiazolium-5-thiolate derivative of a mesoionic compound (MIH 2.4Bl) and the characterization of its selective cytotoxicity on a panel of breast cancer cells lines. The cytotoxic effect of MIH 2.4Bl on breast cancer cell lines was determined by XTT and crystal violet assays, flow cytometry analysis, electron microscopy characterization, and terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) apoptosis assays. As determined using XTT cell growth and survival assays, MIH 2.4Bl exhibited growth inhibition activity on most breast cancer cell lines tested, compared with normal human mammary epithelial cells. Three breast cancer cell lines (MCF-7, T-47D, and ZR-75-1) showed a more potent sensitivity index to growth inhibition by MIH 2.4Bl than the other breast cancer cell lines. Interestingly, these 3 cell lines were derived from tumors of Luminal A origin and have ER (estrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2) positive expression. Additional analysis of cytotoxicity mediated by MIH 2.4Bl was performed using the MCF-7 cell line. MCF-7 cells displayed both time- and dose-dependent decreases in cell growth and survival, with a maximum cytotoxic effect observed at 72 and 96 hours. The MCF-7 cells were also characterized for cell cycle changes upon treatment with MIH 2.4Bl. Using flow cytometry analysis of cell cycle distribution, a treatment-dependent effect was observed; treatment of cells with MIH 2.4Bl increased the G2/M population to 34.2% compared with 0.1% in untreated (control) cells. Ultrastructural analysis of MFC-7 cells treated with MIH 2.4Bl at 2 different concentrations (37.5 and 75 μM) was performed by transmission electron microscopy. Cells treated with 37.5 μM MIH 2.4Bl showed morphologic changes beginning at 6 hours after treatment, while cells treated with 75 μM showed changes beginning at 3 hours after treatment. These changes were characterized by an alteration of nuclear morphology and mitochondrial degeneration consistent with apoptotic cell death. Results of a TUNEL assay performed on cells treated for 96 hours with MIH 2.4Bl supported the observation of apoptosis. Together, these results suggest that MIH 2.4Bl is a promising candidate for treating breast cancer and support further in vitro and in vivo investigation.

Breast cancer is the most commonly diagnosed cancer in women under 60. Localized breast cancer is easily treated, resulting in high survival rates. However, treatment for advanced disease is inadequate, with a five-year survival rate of less than 24%. Thus, there is a great need for new therapies capable of increasing therapeutic efficacy. Synthetic mesoionic compounds, belonging to the 1,3-thiazolium-5-thiolate group, are recognized for their broad spectrum of biological activities including antibiotic, antiparasitic, antiviral, anticonvulsant, antidepressant, antioxidant, analgesic, anti-inflammatory, and more recently for their potential antitumor activity. These compounds have the ability to cross cell membranes; the characteristic of mesoionic structures having distinct regions of positive and negative charge associated with a poly-heterocyclic aromatic ring system, indicates the capability of strong interactions with biomolecules such as DNA and proteins. In this study, the cytotoxic effects of mesoionic compound MI H 2.4 alone and in combination with zinc was examined in breast cancer cell lines (4TI, BT-20, BT-549, MCF7, MDA-MB-231, MDA-MB-436, MM2MT, T-47D, and ZR-75-1) and normal breast cell lineages (HuMEC, MCF-10A, and MCF-12A) were evaluated. The effect of this agent on cell cycle was also investigated. Different concentrations of mesoionic compound MI H 2.4 (MI H 2.4 free) and in combination with zinc (MI H 2.4 Zinc) were added to the cultured cells and incubated for 24, 48, 72 and 96 h. Cell survival and cytotoxicity were evaluated using crystal violet and MTT assays. Cell cycle analysis was performed using MCF7 cells that were stained with propidium iodide and analyzed by flow cytometry. The cytotoxic effects of mesoionic compounds (MI H 2.4 free and MI H 2.4 Zinc) were highest at72 and 96 h. The MI H 2.4 free and MI H 2.4 Zinc showed a similar inhibitory effect on breast cancer cell growth in the μM range. In contrast, the normal breast cell lineages showed low cytotoxicity to treatment with the mesoionic compounds. Treatment of MCF7 cells cultured with MI H 2.4 free blocked cell cycle progression at the G2 phase of the cell cycle after 24 h of treatment. Mitochondrial function of MCF7 cells was determined using a Seahorse XF-24 Extracellular Flux Analyzer. Treatment with MI H 2.4 free and MI H 2.4 Zinc for 24 h resulted in a decreased basal and maximal mitochondrial respiration. In summary, mesoionic compound MI H 2.4 may offer a novel therapeutic strategy in the treatment of breast cancer, considering that it has significant antitumoral activity in breast cancer cell lines and low cytotoxicity in normal cells. Citation Format: Luciana Amaral de Mascena Costa, Filipe Cássio Silva de Lima, Rodrigo da Silva Viana, Silvany de Sousa Araujo, Aurea Wischral, Helivaldo Diógenes da Silva Souza, Petrônio Filgueiras de Athayde-Filhoa, Leandro Araújo de Azevedo, Severino Alves Júnior, Manoel Adrião, J. Michael Mathis. Antitumor activity of the mesoionic compound MI H 2.4 on breast cancer cell lines [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 5877.

In the present study, the cytotoxic effects of a 1,3-thiazolium-5-thiolate derivative of a mesoionic compound, MIH 2.4Bl, were assessed in the MCF-7 breast cancer cell line. The cytotoxic effects of MIH 2.4Bl were determined using a crystal violet assay. Using a dose-response curve, the IC 50 value of MIH 2.4Bl was determined to be 45.8±0.8 µM. Additionally, the effects of MIH 2.4Bl on mitochondrial respiration were characterized using oxygen consumption rate analysis. Treating MCF-7 cells with increasing concentrations of MIH 2.4Bl resulted in a significant reduction in all mitochondrial respiratory parameters compared with the control cells, indicative of an overall decrease in mitochondrial membrane potential. The induction of autophagy by MIH 2.4Bl was also examined by measuring changes in the expression of protein markers of autophagy. As shown by western blot analysis, treatment of MCF-7 cells with MIH 2.4Bl resulted in increased protein expression levels of Beclin-1 and ATG5, as well as an increase in the microtubule-associated protein 1A/1B light chain 3B (LC3B)-II to LC3B-I ratio compared with the control cells. Microarray analysis of changes in gene expression following MIH 2.4Bl treatment demonstrated 3,659 genes exhibited a fold-change ≥2. Among these genes, 779 were up-regulated, and 2,880 were down-regulated in cells treated with MIH 2.4Bl compared with the control cells. Based on the identity of the transcripts and fold-change of expression, six genes were selected for verification by reverse transcription-quantitative (RT-q)PCR; activating transcription factor 3, acidic repeat-containing protein, heparin-binding EGF-like growth factor, regulator of G-protein signaling 2, Dickkopf WNT signaling pathway inhibitor 1 and adhesion molecule with Ig like domain 2. The results of RT-qPCR analysis of RNA isolated from control and MIH 2.4Bl treated cells were consistent with the expression changes identified by microarray analysis. Together, these results suggest that MIH 2.4Bl may be a promising candidate for treating breast cancer and warrants further in vitro and in vivo investigation.

Este artigo tem por objetivo propor uma reflexão em torno da contribuição do Coordenador Pedagógico (CP) enquanto agente de formação docente para uma Educação Ambiental Crítica. Buscamos conhecer o perfil desse profissional, suas atribuições e suas concepções em Educação Ambiental (EA). Realizou-se uma pesquisa do tipo revisão bibliográfica, de cunho sistemático, a partir de buscas em sites e fontes especializadas. Os dados foram analisados de forma qualitativa. O presente trabalho trouxe uma reflexão teórica sobre a questão ambiental, o papel do coordenador e a formação de cidadãos críticos.