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Triple-negative breast cancer has an aggressive clinical course and its treatment has been challenging due to high metastatic risk. Molecular targets have been sought to provide better strategies for this type of cancer. Integrins are cell adhesion receptors involved in tumor progression and α

Photodynamic Therapy (PDT) is a promising treatment for various types of cancer and other non-oncological diseases. This technique uses a photosensitizer (PS) that generates reactive oxygen species (ROS), which lead cells to death in the presence of light and molecular oxygen. In this study, we evaluated the photoactivity of four chlorins that presented an L-type structure conformation (6a, 6b, 8a, and 8b) in two tumoral (HEp-2 and HeLa) and one non-tumoral (Vero) cell line under PDT conditions. Intracellular accumulation and subcellular localization were determined by confocal microscopy, showing that these chlorins exhibited an excellent (120 min) internalization through the plasmatic membrane with mitochondria and lysosome localization. The chlorins 6a-b were two and three-fold more selective than the chlorins 8a-b to HEp-2 and HeLa compared to Vero at 3 and 6 J cm-2 doses, respectively. The fluorescence microscopy and flow cytometry analysis showed that the four chlorin derivatives lead tumor cells to death predominantly by apoptosis during the treatment with light. Considering that the chlorins 6a-b exhibited good water solubility and high phototoxicity to the tumor cells, we suggested that these tetrapyrrolic molecules have the potential for clinical treatment in cancer by PDT.

The 70 kDa heat shock proteins (Hsp70) are prone to self-assembly under thermal stress conditions, forming supramolecular assemblies (SMA), what may have detrimental consequences for cellular viability. In mitochondria, the cochaperone Hsp70-escort protein 1 (Hep1) maintains mitochondrial Hsp70 (mtHsp70) in a soluble and functional state, contributing to preserving proteostasis. Here we investigated the interaction between human Hep1 (hHep1) and HSPA9 (human mtHsp70) or HSPA1A (Hsp70-1A) in monomeric and thermic SMA states to unveil further information about the involved mechanisms. hHep1 was capable of blocking the formation of HSPA SMAs under a thermic treatment and stimulated HSPA ATPase activity in both monomeric and preformed SMA. The interaction of hHep1 with both monomeric and SMA HSPAs displayed a stoichiometric ratio close to 1, suggesting that hHep1 has access to most protomers within the SMA. Interestingly, hHep1 remodeled HSPA9 and HSPA1A SMAs into smaller forms. Furthermore, hHep1 was detected in the mitochondria and nucleus of cells transfected with the respective coding DNA and interacted with liposomes resembling mitochondrial membranes. Altogether, these new features reinforce that hHep1 act as a "chaperone for a chaperone", which may play a critical role in cellular proteostasis.

An amphiphilic chlorin derivative (CHL-T) was prepared from methylpheophorbide a (CHL) and 2-Amino-2-(hydroxymethyl)-1,3-propanediol (TRISMA®). The new chlorin was compared to other dyes (CHL and Hypericin) in relation to photophysical and photobiological activities in tumor and non-tumor cell lines. Cytotoxicity and cell death target were determined to evaluate the CHL-T efficiency, comparing to the precursor CHL and to the well-known dye hypericin (HY). All of the studied compounds exhibited absorption bands in the therapeutic window and presented a small fluorescence quantum yield compared to the reference dye (rhodamine B). CHL-T was about three times more efficient on singlet oxygen generation than the others photosensitizers. The lipophilicity order of the photosensitizers was CHL>HY>CHL-T. The tumoral HeLa cells presented improved accumulation for CHL and CHL-T compared to HY. The phototoxicity presented by the CHL-T was about ten times higher than by CHL, as demonstrated by the MTT assay. CHL-T showed more cytotoxicity to tumoral cell, comparing to non-tumoral cell in short incubation time. The cell death rises proportionally with increasing PSs concentrations, mainly by necrosis. These findings suggest that CHL-T is a potential new photosensitizer for PDT.

Background Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of α2β1 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an α2β1 integrin. Herein, we used ALT-C as a α2β1 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration. Methods ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The α2β1 integrin binding properties of ALT-C, its dissociation constant (Kd) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting. Results Our data demonstrate that ALT-C, after binding to α2β1 integrin, acts by two distinct mechanisms against tumor progression, depending on the cell type: in tumor cells, ALT-C decreases MMP-9 and MMP-2 contents and activity, but increases focal adhesion kinase phosphorylation and transmigration; and in endothelial cells, ALT-C inhibits MMP-2, which is necessary for tumor angiogenesis. ALT-C also upregulates c-Myc mRNA level, which is related to tumor suppression. Conclusion These results demonstrate that α2β1 integrin controls MMP expression and reveal this integrin as a target for the development of antiangiogenic and antimetastatic therapies. Electronic supplementary material The online version of this article (10.1186/s40409-018-0150-2) contains supplementary material, which is available to authorized users.

ADAMs are transmembrane multifunctional proteins that contain disintegrin and metalloprotease domains. ADAMs act in a diverse set of biological processes, including fertilization, inflammatory responses, myogenesis, cell migration, cell proliferation and ectodomain cleavage of membrane proteins. These proteins also have additional functions in pathological processes as cancer and metastasis development. ADAM9 is a member of ADAM protein family that is overexpressed in several types of human carcinomas. The aim of this study was to investigate the role of ADAM9 in hematogenous and lymphatic tumor cell dissemination assisting the development of new therapeutic tools. The role of ADAM9 in the interaction of breast tumor cells (MDA-MB-231) and endothelial cells was studied through RNA silencing. ADAM9 silencing in MDA-MB-231 cells had no influence in expression of several genes related to the metastatic process such as ADAM10, ADAM12, ADAM17, cMYC, MMP9, VEGF-A, VEGF-C, osteopontin and collagen XVII. However, there was a minor decrease in ADAM15 expression but an increase in that of MMP2. Moreover, ADAM9 silencing had no effect in the adhesion of MDA-MB-231 cells to vascular (HMEC-1 and HUVEC) and lymphatic cells (HMVEC-dLyNeo) under flow condition. Nevertheless, siADAM9 in MDA-MB-231 decreased transendothelial cell migration in vitro through HUVEC, HMEC-1 and HMVEC-dLyNeo (50%, 40% and 32% respectively). These results suggest a role for ADAM9 on the extravasation step of the metastatic cascade through both blood and lymph vessels.

A Terapia Fotodinâmica (PDT) é uma técnica utilizada para tratar vários tipos de tumores em que a luz estimula um fotossensibilizador (FS) a gerar espécies reativas de oxigênio (ROS) que levam à morte celular por apoptose ou necrose. A partir de uma clorina (CHL), cuja nomenclatura é metilfeoforbídio, visando torná-la mais anfifílica foi adicionado o grupo TRISMA (CHL-T). Os vários parâmetros usados na PDT (tipo de FS, concentração do FS, tempo de incubação do FS e dose de luz) podem desencadear diferentes vias para apoptose. Poucos estudos sobre o tipo de morte celular induzido com clorinas têm sido realizados. Diante disso, o objetivo deste estudo foi determinar a citotoxicidade dessa nova clorina, assim como identificar o tipo de morte celular envolvido na PDT e elucidar a participação da proteína apoptótica p53 nesse processo comparando-se com CHL e HY. A hipótese principal deste trabalho é que esta nova clorina modificada tem maior eficiência na indução de apoptose. Para testar esta hipótese, foi avaliada a indução de apoptose por microscopia de fluorecência e por citometria e a citotoxicidade pelo ensaio do MTT de três FSs: uma clorina (CHL), a clorina modificada (CHL-T) e a hipericina (HY). As clorinas apresentaram maior acumulação para as duas linhagens celulares quando comparada com a hipericina. A fototoxicidade apresentada pela nova clorina foi cerca de 10 a 20 vezes maior que a clorina de origem (CHL) nas duas linhagens celulares como demonstrado pelos resultados do ensaio com MTT. Os testes realizados por microscopia de fluorescência resultaram numa porcentagem de morte celular crescente com o aumento das concentrações diferenciando maior apoptose causada por PDT com CHL-T nas células HEp-2 e maior necrose nas células HeLa. A análise da apoptose por citometria também apresentou um efeito muito superior da CHL-T em relação aos demais FSs estudados para apoptose inicial (80,35%) para a concentração de 0,52 \'mü\'M, tempo de incubação de 2h e dose de luz 6 J/\'CM POT.2\'. Já a detecção de ROS por citometria não apresentou diferenças estatisticamente significativas para PDT nessas condições. Um discreto aumento na ativação de p53 com CHL-T e irradiação foi observado, porém não estatisticamente significativo. Os resultados sugerem que a indução da morte celular na PDT não depende dessa proteína e que a CHL-T tem excelente desempenho como FS em PDT. Photodynamic therapy (PDT) is a technique used in the treatment of various types of tumors in that light stimulates a photosensitizer (PS) to generate reactive oxygen species (ROS) that lead to cell death by apoptosis or necrosis. From a chlorine (CHL), whose nomenclature is methylfeoforbidio, hoping to make it over the amphiphilic the TRISMA group (CHL-T) has been added. Several parameters (type of PS, concentration of PS, incubation time and light dose) can trigger different apoptotic pathways. Few studies aiming the type of cell death induced by chlorines as a PS have been done. Thus, the objective of this project was to determine the cytotoxicity of this new chlorine, as well as identify the type of cell death involved in PDT as well as to elucidate the involvement of apoptotic protein p53 in this process comparing with CHL and HY. The main hypothesis of this study is that the modified chlorine has greater efficiency in the induction of apoptosis. To test this hypothesis, it was evaluated the induction of apoptosis by fluorescence microscopy and cytometry and the cytotoxicity by the MTT assay of three PSs: chlorine (CHL), the modified chlorine (CHL-T) and hypericin (HY).The chlorins accumulation was higher for the two cell lines compared with hypericin. Phototoxicity presented by the chlorin was about 10 to 20 times greater than by the chlorine source (CHL) for the two cell lines as demonstrated by the MTT assay. Tests conducted by fluorescence microscopy showed the percentage of cell death increased with increasing concentrations distinguishing higher apoptosis caused by PDT with CHL-T in HEp-2 cells and higher necrosis in HeLa cells. Analysis of apoptosis by flow cytometry also showed a superior response of CHL-T comparing to the others two studied PSs for initial apoptosis (80.35%) for concentration of 0.52 mM, incubation time of 2h and light dose 6 J/\'CM POT.2\'. However the detection of ROS by flow cytometry showed no statistically significant values for PDT in these conditions. A slight increase in the activation of p53 for CHL-T and irradiation was observed, but without being statistically significant. Thus, the results suggest that the induction of cell death in PDT does not depend on this protein and that CHL-T has excellent performance as a PS in PDT.