The involvement of heat shock proteins and related molecules in the resistance to therapies in breast and gynecologic cancer

The HSP response is implicated in conferring to breast and gynecologic malignancies different sensitivities to anticancer therapies including chemotherapy, endocrine therapy and immunotherapy (weare in the need of more studies about radiotherapy). The heat shock proteins are mainly implicated in cell death mechanisms, in cell differentiation including epithelial-mesenchymal transition, in tumordormancy, in angiogenesis, metastasis formation, and in the escape of immunosurveillance. Considering the ample functions where the HSPs are implicated and that the HSP response is quite complex it is not surprising that the HSP response affects the anticancer therapies. Several of the HSPs have different predominant roles according to the molecular partners with which they interact, thus it is difficult to dissect the molecular mechanisms to find the sensitivity to the therapies. In this review we present the implications of some the major HSPs (HSP27, HSP70 and HSP90) with drug resistance and present some of the main partners that are also implicated in drug resistance like p53, PTEN and MDR. We have given priority to the incorporation of clinical data where the HSPs have been studied using standard chemotherapies and new therapeutic strategies. It is clear that in order to have a significant understanding of the degree of drug resistance/sensitivity presented by a particular patient we need to examine the molecular status of several key molecular markers involved in the drug resistance pathways and that in this context the study of the HSP response should be incorporated. One of the other major problems in this field is that an inhibitor of one particular HSP will not be enough to achieve a significant anticancer response. Now that we know the complexity of this field we need to design strategies aiming to inhibit several molecular HSP pathways simultaneously without significantly affecting the normal cells, this is the principal challenge for the near future.Fil: Cuello Carrión, Fernando Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Fanelli, Mariel Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Castro, Gisela Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Cayado Gutiérrez, Niubys de Los Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Ciocca, Daniel Ramon. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

Differential expression and localization of beta-catenin and HSP27 after cisplatin/doxorubicin treatment in triple negative breast cancer cells

The treatment of triple-negative breast cancers involves the administration of the conventional chemotherapeutic drug doxorubicin, given the lack of specific targeted agents. Novel therapeutic strategies, such as cisplatin, are currently being tested for these patients. Many studies have demonstrated that aberrant Wnt/β-catenin signaling serves a role in the development of breast cancer, while others have concluded that abnormal regulation of Wnt pathway induces tumor cell chemoresistance. The small heat shock protein 27 (HSP27) is overexpressed in human breast cancer cells. As a result, cancer cells may suppress apoptosis and develop resistance to antineoplastic agents, such as doxorubicin. The present study sought to examine the role of the Wnt/β-catenin and HSP27 signaling pathway in response to cisplatin (CisPt)/doxorubicin (Doxo) treatment in human triple-negative (TN) breast cancer cell lines. Material and Methods: MDA-MB231 (TN) and MCF10A cell lines were used. Cell viability was measured using MTT assay and IC50 values were obtained after 48 h of CisPt or Doxo exposition. β-catenin and Hsp27 gene expression were measured by qPCR. Total and active β-catenin, phosphor ant total HSP27, phospho and GSK3β, phosphor and total p38 expressions were measured by western blot and immunofluorescence. 3D cell culture from MDA MB231 cells were treated with increasing concentrations of CisPt and Doxo for 48h. Results: MDA-MB231 cells showed higher IC50 values for CisPt and Doxo than the MCF10A cell line. In MDA-MB231 cells, the expression of β-catenin, active β-catenin, total and phospho-GSK3β and total HSP27 significantly decreased in the CisPt group (p<0.05). No changes were observed in Doxo-treated group. In MCF10A cells, the expression levels of total and active β-catenin did not modify with CisPt treatment, but in the Doxo group the proteins evaluated showed a tendency to increase. Also in MCF10A Doxo treatment significantly decreased the expression of GSK3β in comparison with control (p<0.05). In contrast, CisPt administration significantly increased phospho-GSK3β expression respect to the control group (p<0.05). Interestingly, in MDA-MB231 cells the nucleolus appeared disaggregated and active β-catenin increased at this subcellular localization after CisPt and Doxo treatment. In contrast, total β-catenin was preferentially localized in the Golgi. In the other hand 3D cell culture was more resistant to Doxo-treatment than 2D cell culture. CisPt induced a decrease in 3D cell culture growth. Conclusions: CisPt treatment was associated with decreased expression of β-catenin and HSP27. While in Doxo-treated cells, as related to stable levels of β-catenin and increased expression of HSP27. The differential expression and localizations of β-catenin and HSP27 could be related to a differential cellular response depending on the cytotoxicity mechanism of chemotherapeutic agent used., that in turns affect the cell fate decision. Our preliminary data indicate that β-catenin and HSP27 may be potential therapeutic targets in TNBC.Fil: Córdoba, María Evelyn. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Pennacchio, Gisela Erika. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Cayado Gutiérrez, Niubys de Los Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Cuello Carrión, Fernando Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Nadin, Silvina Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Vargas Roig, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Fanelli, Mariel Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaXXXVII Reunión Científica Anual de la Sociedad de Biología de CuyoSan LuisArgentinaSociedad de Biología de Cuy

In MMTV-Her-2/neu transgenic mammary tumors the absence of caveolin-1−/− alters PTEN and NHERF1 but not β-catenin expression

In a recent study, we have shown that in mammary tumors from mice lacking the Cav-1 gene, there are alterations in specific heat shock proteins as well as in tumor development. With this in mind, we have now investigated other proteins in the same mammary mouse tumor model (Her-2/neu expressing mammary tumors from Cav-1 wild type and Cav-1 null mice), to further comprehend the complex tumor-stroma mechanisms involved in regulating stress responses during tumor development. In this tumor model the cancer cells always lacked of Cav-1, so the KO influenced the Cav-1 in the stroma. By immunohistochemistry, we have found a striking co-expression of β-catenin and Her-2/neu in the tumor cells. The absence of Cav-1 in the tumor stroma had no effect on expression or localization of β-catenin and Her-2/neu. Both proteins appeared co-localized at the cell surface during tumor development and progression. Since Her-2/neu activation induces MTA1, we next evaluated MTA1 in the mouse tumors. Although this protein was found in numerous nuclei, the absence of Cav-1 did not alter its expression level. In contrast, significantly more PTEN protein was noted in the tumors lacking Cav-1 in the stroma, with the protein localized mainly in the nuclei. P-Akt levels were relatively low in tumors from both Cav-1 WT and Cav-1 KO mice. There was also an increase in nuclear NHERF1 expression levels in the tumors arising from Cav-1 KO mice. The data obtained in the MMTV-neu model are consistent with a role for Cav-1 in adjacent breast cancer stromal cells in modulating the expression and localization of important proteins implicated in tumor cell behavior.Fil: Cuello Carrión, Fernando Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Cayado Gutiérrez, Niubys de Los Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Natoli, Anthony L. . Peter MacCallum Cancer Centre. Metastasis Research Laboratory; AustraliaFil: Restall, Christina. Peter MacCallum Cancer Centre. Metastasis Research Laboratory; AustraliaFil: Anderson, Robin L.. Peter MacCallum Cancer Centre. Metastasis Research Laboratory; AustraliaFil: Nadin, Silvina Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Alvarez Olmedo, Daiana Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Castro, Gisela N.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Gago, Francisco E.. Facultad de Ciencias Médicas. Universidad Nacional de Cuyo; ArgentinaFil: Fanelli, Mariel Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Ciocca, Daniel Ramon. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

Heat shock proteins (HSPs) based anti-cancer vaccines

The importance of HSPs themselves in antigen presentation and cross-presentation remains controversial. Most studies agree that as part of their molecular chaperone function, HSPs can bind and present tumor associated antigens to professional antigen presenting cells through MHC class I and class II molecules, leading to the activation of anti-tumor CD8+ and CD4+ T cells. The regulation of the innate and adaptive immune responses by HSPs is still a matter of intense research. HSPs are seen as important anticancer vaccine adjuvants. They are used through different delivery systems: HSPs/antibodies, peptide/protein-HSP complexes, tumor antigen/HSP gene fusion, viral peptides/HSP complexes or gene fusion, viral proteins/bacterial HSP fusion. In preclinical models different administration routes, subcutaneous, intradermal, intramuscular or even peroral (under special conditions) can be used, and the animal toxicities are non-significant. The HSP-based vaccines can induce specific and non-specific cellular immune responses all of which are important to induce tumor rejection. In addition, the antibodies generated after vaccination are emerging as important protagonist in the antitumoral response. This response is significantly enhanced when the suppressive tumor microenvironment and the immune suppressing effector cells are blocked. Several clinical studies have been carried out and are ongoing, immunizing cancer patients with autologous tumor derived HSP-peptide complexes (HSPPCs). The most promising results have been observed in patients with melanoma and renal clear cell cancer without advanced disease. There are clinical trials with HSP-based anticancer vaccines other than with HSPPCs (including patients with non-Hodgkin lymphoma, high-grade transitional cell carcinoma of the bladder, high-grade cervical dysplasia, etc).Fil: Ciocca, Daniel Ramon. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Cayado Gutiérrez, Niubys de Los Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Maccioni, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Cuello Carrión, Fernando Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

Antitumoral activity of 3-isopropyl-4-methyl-5,7-dihydroxycoumarin and its action mechanism associated to HSPB1: Preliminary Results

Our laboratory has been involved in the study of HSPB1 (HSP27) in cancer; this protein participates in many events related with cancer progression and resistance to anticancer treatments. We are now exploring ways to interfere with the HSPB1 response testing phytochemicals selected from autochthonous plants grown in the semi desert area of Mendoza and San Luis. The study involves breast cancer, cervical cancer and glioma cells exposed to 3-isopropyl-4-methyl-5,7-dihydroxycoumarin (C13), a semi-synthetic coumarin previously characterized. The HSPB1 response has been examined by immunocytochemistry, the cytotoxic effect was studied by the MTT assay and the effect on the cell migration by the wound healing assay. Cell death was evaluated by flow cytometry and immunocytochemistry. C13 induced significant changes in HSPB1 total content and in phosphorylation protein status, with changes in cellular localization. The survival of the cancer cells was significantly decreased according to drug concentration and time of exposure. Cell migration decreased and cell death by apoptosis increased in treated cells. Aggresome structures were observed as a distinctive cytotoxic effect on Gli36 cells.Fil: Cayado Gutiérrez, Niubys de Los Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Cuello Carrión, Fernando Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: de Paola, Maria Matilde. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Garro, Hugo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; ArgentinaFil: Cifuente, Diego Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; ArgentinaFil: Pungitore, Carlos Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; ArgentinaFil: Ciocca, Daniel Ramon. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina24th Latin-American Symposium on Biotechnology, Biomedical, Biopharmaceutical, and Industrial Applications of Capillary Electrophoresis and Microchip Technology.MendozaArgentinaUniversidad Nacional de CuyoUniversidad de Buenos AiresUniversidad Nacional de La Plat

Down-regulation of Hsp27 (HSPB1) in MCF-7 human breast cancer cells induces up-regulation of PTEN.

Hsp27 (HSPB1) is usually overexpressed in breast cancers affecting the disease outcome and the sensitivity of tumors to chemotherapy and radiotherapy. Hsp27 interacts with other proteins such as β-catenin, histone deacetylase HDAC6, transcription factor STAT2 and procaspase-3. Phosphatase and tensin homologue (PTEN) is a tumor suppressor gene that is deleted in many human tumors. The PI3K/Akt signaling pathway is negatively regulated by PTEN. Hsp27 is described as a key component of the Akt signaling cascade: Akt, BAD, Forkhead transcription factors, Hsp27, mitogen-activated protein kinase kinase-3 and -6. Here, we have examined whether the downregulation of Hsp27 by siHsp27 affects the PTEN levels in the MCF-7 human breast cancer cell line. PTEN was detected with two different antibodies using western blots and immunocytochemistry. p-Akt was also evaluated by western blot. In addition, Hsp27 and PTEN were immunoprecipitated to know whether these proteins interact. Intracellular colocalization studies were carried out by confocal microscopy. A significant reduction in the Hsp27 levels was noted in the siHsp27 transfected cells. These Hsp27 downregulated cells showed a significant increased expression of PTEN. The MW 76 and 55 kDa PTEN forms were upregulated as revealed by two different antibodies. The phosphatase activity of PTEN seems to be active because p-Akt levels were reduced. Hsp27 immunoprecipitation was bringing PTEN and vice versa, these two proteins seem to interact at cytoplasmic level by FRET. Downregulation of Hsp27 stabilized PTEN protein levels. Chaperone-assisted E3 ligase C terminus of Hsc70-interacting protein (CHIP) levels were not significantly influenced by Hsp27 downregulation. In conclusion, we report a novel function of Hsp27 modulating the PTEN levels in human breast cancer cells suggesting an interaction between these two molecules.Fil: Cayado Gutiérrez, Niubys de Los Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Moncalero, Vera Lucia. Universidad Nacional de San Martín. Centro de Estudios de Salud y Medio Ambiente. Laboratorio de Neuro y Citogenética Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rosales, Eliana María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto Histología y Embriología de Mendoza; ArgentinaFil: Beron, Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto Histología y Embriología de Mendoza; ArgentinaFil: Salvatierra, Edgardo E.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires(i); Argentina. Fundación Instituto Leloir. Laboratorio de Terapia Molecular y Celular; ArgentinaFil: Alvarez Olmedo, Daiana Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Radrizzani, Martín. Universidad Nacional de San Martín. Centro de Estudios de Salud y Medio Ambiente. Laboratorio de Neuro y Citogenética Molecular; ArgentinaFil: Ciocca, Daniel Ramon. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

Molecular markers of DNA damage and repair in cervical cancer patients treated with cisplatin neoadjuvant chemotherapy: an exploratory study

Neoadjuvant (or induction) chemotherapy can be used for cervical cancer patients with locally advanced disease; this treatment is followed by radical surgery and/or radiation therapy. Cisplatin is considered to be the most active platinum agent drug for this cancer, with a response rate of 20%. In order to understand how the cisplatin treatment affects the stress response, in this work, we performed an exploratory study to analyze a number of stress proteins before and after cisplatin neoadjuvant chemotherapy. The study involved 14 patients; the pre- and post-chemotherapy paired biopsies were examined by hematoxylin and eosin staining and by immunohistochemistry. The proteins evaluated were p53, P16/INK4A, MSH2, nuclear protein transcriptional regulator 1 (NUPR1), and HSPB1 (total: HSPB1/t and phosphorylated: HSPB1/p). These proteins were selected because there is previous evidence of their relationship with drug resistance. The formation of platinum-DNA adducts was also studied. There was a great variation in the expression levels of the mentioned proteins in the pre-chemotherapy biopsies. After chemotherapy, p53 was not significantly affected by cisplatin, as well as P16/INK4A and MSH2 while nuclear NUPR1 content tended to decrease (p = 0.056). Cytoplasmic HSPB1/t expression levels decreased significantly following cisplatin therapy while nuclear HSPB1/t and HSPB1/p tended to increase. Since the most significant changes following chemotherapy appeared in the HSPB1 expression levels, the changes were confirmed by Western blot. The platinum-DNA adducts were observed in HeLa cell in apoptosis; however, in the tumor samples, the platinum-DNA adducts were observed in morphologically healthy tumor cells; these cells displayed nuclear HSPB1/p. Further mechanistic studies should be performed to reveal how HSPB1/p is related with drug resistance. When the correlations of the markers with the response to neoadjuvant chemotherapy were examined, only high pre-chemotherapy levels of cytoplasmic HSPB1/p correlated with a poor clinical and pathological response to neoadjuvant cisplatin chemotherapy (p = 0.056) suggesting that this marker could be useful opening its study in a larger number of cases.Fil: Real, Nilda E.. Hospital Diego Paroissien; ArgentinaFil: Castro, Gisela Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Cuello Carrión, Fernando Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Perinetti, Claudia. Hospital Diego Paroissien; ArgentinaFil: Röhrich, Hanna. Freie Universitaet Berlin; AlemaniaFil: Cayado Gutiérrez, Niubys de Los Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Guerrero Gimenez, Martin Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Ciocca, Daniel Ramon. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

Effects of temozolomide (TMZ) on the expression and interaction of heat shock proteins (HSPs) and DNA repair proteins in human malignant glioma cells

We previously reported the association of HSPA1A and HSPB1 with high-grade astrocytomas, suggesting that these proteins might be involved in disease outcome and response to treatment. With the aim to better understand the resistance/susceptibility processes associated to temozolomide (TMZ) treatment, the current study was performed in three human malignant glioma cell lines by focusing on several levels: (a) apoptotic index and senescence, (b) DNA damage, and (c) interaction of HSPB1 with players of the DNA damage response. Three human glioma cell lines, Gli36, U87, and DBTRG, were treated with TMZ evaluating cell viability and survival, apoptosis, senescence, and comets (comet assay). The expression of HSPA (HSPA1A and HSPA8), HSPB1, O6-methylguanine-DNA methyltransferase (MGMT), MLH1, and MSH2 was determined by immunocytochemistry, immunofluorescence, and Western blot. Immunoprecipitation was used to analyze protein interaction. The cell lines exhibited differences in viability, apoptosis, and senescence after TMZ administration. We then focused on Gli36 cells (relatively unstudied) which showed very low recovery capacity following TMZ treatment, and this was related to high DNA damage levels; however, the cells maintained their viability. In these cells, MGMT, MSH2, HSPA, and HSPB1 levels increased significantly after TMZ administration. In addition, MSH2 and HSPB1 proteins appeared co-localized by confocal microscopy. This co-localization increased after TMZ treatment, and in immunoprecipitation analysis, MSH2 and HSPB1 appeared interacting. In contrast, HSPB1 did not interact with MGMT. We show in glioma cells the biological effects of TMZ and how this drug affects the expression levels of heat shock proteins (HSPs), MGMT, MSH2, and MLH1. In Gli36 cells, the results suggest that interactions between HSPB1 and MSH2, including co-nuclear localization, may be important in determining cell sensitivity to TMZ.Fil: Castro, Gisela Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Cayado Gutiérrez, Niubys de Los Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Zoppino, Felipe Carlos Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Fanelli, Mariel Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Cuello Carrión, Fernando Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Sottile Fleury, Mayra Lis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Nadin, Silvina Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Ciocca, Daniel Ramon. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

Impact of high fat diet on the sterol regulatory element-binding protein 2 cholesterol pathway in the testicle

Male fertility has been shown to be dependent on cholesterol homeostasis. This lipid is essential for testosterone synthesis and spermatogenesis, but its levels must be maintained in an optimal range for proper testicular function. In particular, sperm cells' development is very sensitive to high cholesterol levels, noticeably during acrosomal formation. The aim of this work was to study whether the molecular pathway that regulates intracellular cholesterol, the sterol regulatory element-binding protein (SREBP) pathway, is affected in the testicles of animals under a fat diet. To investigate this, we took advantage of the non-obese hypercholesterolemia (HC) model in New Zealand rabbits that displays poor sperm and seminal quality. The testicular expression of SREBP isoform 2 (SREBP2) and its target molecules 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) and low-density lipoprotein receptor (LDLR) were studied under acute (6months) and chronic (more than 12months) fat intake by RT-PCR, western blot and immunofluorescence. Our findings showed that fat consumption promoted down-regulation of the SREBP2 pathway in the testicle at 6months, but upregulation after a chronic period. This was consistent with load of testicular cholesterol, assessed by filipin staining. In conclusion, the intracellular pathway that regulates cholesterol levels in the testicle is sensitive to dietary fats, and behaves differently depending on the duration of consumption: it has a short-term protective effect, but became deregulated in the long term, ultimately leading to a detrimental situation. These results will contribute to the understanding of the basic mechanisms of the effect of fat consumption in humans with idiopathic infertility.Fil: Funes, Abi Karenina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Simón, Layla Yamila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Colombo, Regina Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Avena Alvarez, María Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Monclus, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad del Aconcagua; ArgentinaFil: Crescitelli, Julieta. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología; Argentina. Universidad del Aconcagua; ArgentinaFil: Cabrillana, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Conte, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Cayado Gutiérrez, Niubys de Los Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad del Aconcagua; ArgentinaFil: Boarelli, Paola Vanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad "Juan Agustín Maza"; ArgentinaFil: Fornes, Miguel Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Saez Lancellotti, Tania Emilce Estefania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad del Aconcagua; Argentin

Pigment epithelium derived factor (PEDF) expression in the male tract of Wistar rats

Pigment epithelium derived factor (PEDF) expression has been described in many organs as showing neurotrophic, anti-angiogenic, anti-apoptotic, anti-inflammatory, anti-oxidant and pro-cell survival properties. However, references to its activity in the male reproductive system are scarce. We aimed to characterize the expression of PEDF in the male reproductive tract of Wistar rats by using RT-PCR, western blot and immunostaining and also evaluate the effect of flutamide in PEDF expression. We found that PEDF is expressed in the epididymis, prostate and seminal vesicles in Wistar rats, but notably not in the testes. Under the effect of flutamide PEDF expression decreased, recovering by suppressing the antiandrogen. The epididymis is an essential organ in sperm maturation-storages. The role of PEDF in this physiological process has not been fully elucidated yet, but considering that in other systems PEDF has anti-apoptotic, anti-oxidants and pro-cell survival properties, its expression along the epididymis could play a role in the protection of spermatozoa while they are stored.Fil: Conte, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Cabrillana, María Eugenia. Universidad de Mendoza; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Saez Lancellotti, Tania Emilce Estefania. Universidad de Mendoza; Argentina. Universidad del Aconcagua; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Simón, Layla Yamila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Funes, Abi Karenina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Cayado Gutiérrez, Niubys de Los Milagros. Universidad de Mendoza; Argentina. Universidad del Aconcagua; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Tagle-Delgado, Matias Gustavo. Universidad del Aconcagua; ArgentinaFil: Vincenti, Amanda Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Lopez, María Elis. Universidad de Mendoza; ArgentinaFil: Pietrobon, Elisa Olivia. Universidad de Mendoza; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Fornes, Miguel Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad del Aconcagua; Argentina. Universidad de Mendoza; ArgentinaFil: Monclus, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad del Aconcagua; Argentina. Universidad de Mendoza; Argentin