Aptamer-Based Recognition of Breast Tumor Cells: A New Era for Breast Cancer Diagnosis.

Breast cancer is one of the leading causes of death among women worldwide and can be classified into four major distinct molecular subtypes based on the expression of specific receptors. Despite significant advances, the lack of biomarkers for detailed diagnosis and prognosis remains a major challenge in the field of oncology. This study aimed to identify short single-stranded oligonucleotides known as aptamers to improve breast cancer diagnosis. The Cell-SELEX technique was used to select aptamers specific to the MDA-MB-231 tumor cell line. After selection, five aptamers demonstrated specific recognition for tumor breast cell lines and no binding to non-tumor breast cells. Validation of aptamer specificity revealed recognition of primary and metastatic tumors of all subtypes. In particular, AptaB4 and AptaB5 showed greater recognition of primary tumors and metastatic tissue, respectively. Finally, a computational biology approach was used to identify potential aptamer targets, which indicated that CSKP could interact with AptaB4. These results suggest that aptamers are promising in breast cancer diagnosis and treatment due to their specificity and selectivity.

Next Generation of Ovarian Cancer Detection Using Aptamers.

Ovarian cancer is among the seven most common types of cancer in women, being the most fatal gynecological tumor, due to the difficulty of detection in early stages. Aptamers are important tools to improve tumor diagnosis through the recognition of specific molecules produced by tumors. Here, aptamers and their potential targets in ovarian cancer cells were analyzed by in silico approaches. Specific aptamers were selected by the Cell-SELEX method using Caov-3 and OvCar-3 cells. The five most frequent aptamers obtained from the last round of selection were computationally modeled. The potential targets for those aptamers in cells were proposed by analyzing proteomic data available for the Caov-3 and OvCar-3 cell lines. Overexpressed proteins for each cell were characterized as to their three-dimensional model, cell location, and electrostatic potential. As a result, four specific aptamers for ovarian tumors were selected: AptaC2, AptaC4, AptaO1, and AptaO2. Potential targets were identified for each aptamer through Molecular Docking, and the best complexes were AptaC2-FXYD3, AptaC4-ALPP, AptaO1-TSPAN15, and AptaO2-TSPAN15. In addition, AptaC2 and AptaO1 could detect different stages and subtypes of ovarian cancer tissue samples. The application of this technology makes it possible to propose new molecular biomarkers for the differential diagnosis of epithelial ovarian cancer.

Transforming growth factor-ß as a therapeutic target for the cardiac damage of Chagas disease.

Transforming growth factor beta (TGF-ß) is deeply involved on the pathogenesis of Chagas disease. Our group has been investigating the participation of this pleiotropic cytokine in different aspects of Chagas disease over the last 20 years. Important observations have been made, such as: (i) the ability of Trypanosoma cruzi in activating latent TGF-ß; (ii) the potential involvement of TGF-ß pathway on T. cruzi invasion of host cells; (iii) association of TGF-ß with parasite intracellular replication; (iv) cardiac fibrosis development and maintenance; (v) disruption of Connexin-43 plaque structures and (vi) inflammation and immune response. In this perspective article we intend to discuss the advances of the potential use of new therapies targeting TGF-ß to treat the cardiac alterations of Chagas disease-affected patients.

In Chagas disease, transforming growth factor beta neutralization reduces Trypanosoma cruzi infection and improves cardiac performance.

Chronic Chagasic cardiomyopathy (CCC), a progressive inflammatory and fibrosing disease, is the most prominent clinical form of Chagas disease, a neglected tropical disease caused by Trypanosoma cruzi infection. During CCC, the parasite remains inside the cardiac cells, leading to tissue damage, involving extensive inflammatory response and irregular fibrosis. Among the fibrogenic factors is transforming growth factor-ß (TGF-ß), a key cytokine controlling extracellular matrix synthesis and degradation. TGF-ß is involved in CCC onset and progression, with increased serum levels and activation of its signaling pathways in the cardiac tissue, which crucially contributes to fibrosis. Inhibition of the TGF-ß signaling pathway attenuates T. cruzi infection and prevents cardiac damage in an experimental model of acute Chagas disease. The aim of this study was to investigate the effect of TGF-ß neutralization on T. cruzi infection in both in vitro and in vivo pre-clinical models, using the 1D11 monoclonal antibody. To this end, primary cultures of cardiac cells were infected with T. cruzi trypomastigote forms and treated with 1D11. For in vivo studies, 1D11 was administered in different schemes for acute and chronic phase models (Swiss mice infected with 104 parasites from the Y strain and C57BL/6 mice infected with 102 parasites from the Colombian strain, respectively). Here we show that the addition of 1D11 to cardiac cells greatly reduces cardiomyocyte invasion by T. cruzi and the number of parasites per infected cell. In both acute and chronic experimental models, T. cruzi infection altered the electrical conduction, decreasing the heart rate, increasing the PR interval and the P wave duration. The treatment with 1D11 reduced cardiac fibrosis and reversed electrical abnormalities improving cardiac performance. Taken together, these data further support the major role of the TGF-ß signaling pathways in T. cruzi-infection and their biological consequences on parasite/host interactions. The therapeutic effects of the 1D11 antibody are promising and suggest a new possibility to treat cardiac fibrosis in the chronic phase of Chagas' heart disease by TGF-ß neutralization.

TGF-ß inhibitor therapy decreases fibrosis and stimulates cardiac improvement in a pre-clinical study of chronic Chagas' heart disease.

TGF-ß involvement in Chagas disease cardiomyopathy has been clearly demonstrated. The TGF-ß signaling pathway is activated in the cardiac tissue of chronic phase patients and is associated with an increase in extracellular matrix protein expression. The aim of this study was to investigate the effect of GW788388, a selective inhibitor of TßR1/ALK5, on cardiac function in an experimental model of chronic Chagas' heart disease. To this end, C57BL/6 mice were infected with Trypanosoma cruzi (102 parasites from the Colombian strain) and treated orally with 3mg/kg GW788388 starting at 120 days post-infection (dpi), when 100% of the infected mice show cardiac damage, and following three distinct treatment schedules: i) single dose; ii) one dose per week; or iii) three doses per week during 30 days. The treatment with GW788388 improved several cardiac parameters: reduced the prolonged PR and QTc intervals, increased heart rate, and reversed sinus arrhythmia, and atrial and atrioventricular conduction disorders. At 180 dpi, 30 days after treatment interruption, the GW3x-treated group remained in a better cardiac functional condition. Further, GW788388 treatment reversed the loss of connexin-43 enriched intercellular plaques and reduced fibrosis of the cardiac tissue. Inhibition of the TGF-ß signaling pathway reduced TGF-ß/pSmad2/3, increased MMP-9 and Sca-1, reduced TIMP-1/TIMP-2/TIMP-4, and partially restored GATA-6 and Tbox-5 transcription, supporting cardiac recovery. Moreover, GW788388 administration did not modify cardiac parasite load during the infection but reduced the migration of CD3+ cells to the heart tissue. Altogether, our data suggested that the single dose schedule was not as effective as the others and treatment three times per week during 30 days seems to be the most effective strategy. The therapeutic effects of GW788388 are promising and suggest a new possibility to treat cardiac fibrosis in the chronic phase of Chagas' heart disease by TGF-ß inhibitors.

Transforming growth factor-ß as a therapeutic target for the cardiac damage of Chagas disease

Transforming growth factor beta (TGF-β) is deeply involved on the pathogenesis of Chagas disease. Our group has been investigating the participation of this pleiotropic cytokine in different aspects of Chagas disease over the last 20 years. Important observations have been made, such as: (i) the ability of Trypanosoma cruzi in activating latent TGF-β; (ii) the potential involvement of TGF-β pathway on T. cruzi invasion of host cells; (iii) association of TGF-β with parasite intracellular replication; (iv) cardiac fibrosis development and maintenance; (v) disruption of Connexin-43 plaque structures and (vi) inflammation and immune response. In this perspective article we intend to discuss the advances of the potential use of new therapies targeting TGF-β to treat the cardiac alterations of Chagas disease-affected patients.

Avaliação do potencial de regeneração cardíaca após inibição da atividade de TGF-beta em modelo experimental de doença de Chagas crônica

Estudos publicados pelo grupo demonstram o envolvimento do TGF-\F062 na cardiopatia chagásica. A deposição excessiva de componentes de matriz extracelular (MEC) induz o processo fibrótico que leva ao comprometimento funcional do coração e o TGF-\F062 se destaca como uma das principais proteínas reguladoras desse processo. As metaloproteases atuam degradando a MEC, e estão diretamente envolvidas no remodelamento cardíaco, tendo sua atividade controlada por TIMPs. Dados paralelos do grupo indicam que animais infectados por T. cruzi durante a fase crônica experimental da doença de Chagas, apresentaram acometimento cardíaco, aumento da expressão e deposição de colágeno no coração e aumento dos níveis circulantes de TGF-\F062. O tratamento com o composto GW788388, inibidor da atividade de TGF-\F062, 120 dias pós-infecção (dpi), foi capaz de: melhorar a condução elétrica cardíaca; reverter a expressão e deposição de colágeno no tecido cardíaco; e diminuir os níveis circulantes do TGF-\F062. O objetivo deste estudo é compreender os mecanismos envolvidos na reversão da fibrose após o tratamento com GW788388, e avaliar possíveis candidatos envolvidos na regeneração cardíaca, durante a fase crônica da doença de Chagas experimental. Para isso, animais C57Bl/6 foram infectados com T. cruzi Cepa colombiana (102) e tratados com 3 mg/kg GW788388, após 120 dpi em dois diferentes esquemas de tratamento: i) 1x por semana ou ii) 3x por semana por via oral, durante 30 dias (até 150 dpi) O coração dos animais foi retirado e proteínas e RNA totais foram extraídos para investigação da expressão de MMP-2 e MMP-9 (RT-qPCR e Western blot), assim como suas atividades proteolíticas (Zimografia). Além disso, também foi investigada a expressão de TIMPs -1, -2 e -4 e troponina T por Western blot; e a expressão gênica de marcadores de regeneração cardíaca: Gata-4, Gata-6, T-box5, Nkx2-5, troponina T, desmina e titina por RT- qPCR. Técnicas histológicas foram utilizadas para avaliação de Cx43. Além disso, foram avaliados os níveis de TIMP-1 circulantes por ELISA. Nossos resultados demonstram que a infecção crônica leva à desorganização de Cx-43 e o tratamento recupera o padrão de Cx-43. Foi observada diminuição da atividade de MMP-2 e -9 com a infecção crônica e o tratamento com GW788388 aumentou suas atividades no coração dos animais. Os níveis de TIMP-1 circulantes aumentam em 120 dias após a infecção e caem 150 dias após a infecção, o tratamento com GW788388 aumenta esses níveis. Entretanto, a expressão cardíaca dos TIMPs -1, -2 e -4 aumentam em 150 dpi e o tratamento com GW788388 reverte esse aumento. A infecção crônica leva a diminuição da expressão de todos os marcadores de regeneração cardíaca avaliados, e o tratamento com GW788388 3x por semana aumentou a expressão de Gata-6 e T-box5. Nossos dados indicam que a reversão da fibrose cardíaca é mediada pela regulação da atividade de MMP-2, -9 por TIMPs. Além disso, o tratamento com GW788388 parece ser capaz de induzir regeneração cardíaca em animais cronicamente infectados, sendo promissor como uma nova possibilidade de tratar a fibrose cardíaca observada na fase crônica da doença de Chagas