Tamoxifen alters lysosomes of breast cancer cells by a mechanism independent of its anti-estrogenic activity

Breast cancer is one of the most important causes of morbidity and mortality worldwide. It has been shown that the cells of some tumors have an increased lysosomal biogenesis in response to metabolic alterations, which also has an impact on the integrity and/or lysosomal functionality, showing increased levels of lysosomal proteases, such as cathepsin D (CatD). It has been demonstrated that this enzyme induces apoptosis when is released into the cytoplasm. Since the lysosomes could play a role either as initiators or executors of apoptotic processes when the membrane integrity is altered, this organelle could be taken as a potential therapeutic target against tumors. In breast cancer cell lines positive to estrogen receptor RE (REα), CatD is positively regulated by this hormone, while in cell lines negative for REα the enzyme is constitutively overexpressed. Tamoxifen (TAM) is one of the most common anti-estrogenic drugs used in breast cancer therapy. It interacts with ER and inhibits transcriptional activity in the mammary gland. The aim of this study was to evaluate the effect of estrogens and tamoxifen on lysosomal acidification and CatD processing in breast cancer cells. Mammary cell lines MCF-7 (tumorigenic expressing REα), MDA-MB231 (tumorigenic non-expressing REα) and MCF-10A (non-tumorigenic) were treated in the presence or absence of 17-β-estradiol and/or TAM. For quantification of acidic lysosomes, cells were treated with Lysotracker. Cultures were subjected to immunoblot analysis and fluorescence microscopy. As expected, TAM blocked the effect of estrogen on CatD processing in MCF-7 cells. However, TAM used alone, also altered CatD processing and decreased the number of acidic lysosomes in both cell lines. Neither effect of TAM was observed on MCF-10A cell line. In addition, a decreased level of another lysosomal protein, GM2AP (related to the development of tumors), was observed in the cells due to TAM. All these results suggest that TAM has additional effects independent of its anti-estrogenic activity, possibly due to lysosomotropic action.Fil: Pereyra, Laura Lucia. 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. Ministerio de Salud. Instituto Nacional del Cáncer; ArgentinaFil: Bannoud, Nadia. 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: Guarniolo, D.. 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: Chapana, Agostina 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: Sosa Escudero, Miguel Angel. 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 Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Carvelli, Flavia Lorena. 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 Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaXXXVI Reunión Científica Anual de la Sociedad de Biología de CuyoMendozaArgentinaSociedad de Biología de Cuy

Effect of botulinum neurotoxins from Mendoza of clostridium botulinum strains on cytoskeletal proteins of mammary tumor cells

The botulinum neurotoxin serotype A (BoNT A) produced by Clostridium botulinum, which causes botulism, is used for the treatment of multiple neurological diseases and its therapeutic action against cancer is currently being evaluated. In previous studies, we have shown that BoNT A from autochthonous soil strains (Su) have different properties than the reference A Hall strain. Among these, its molecular structure, its enzymatic activity against brain SNARE proteins and its greater specific toxic activity (AE) stand out. In cells from human mammary carcinoma (MCF-7) treated with BoNTs for 45 min, we found a marked effect on the expression of cytoskeletal proteins. Therefore, in this work, we delve into the study of the action of autochthonous BoNTs A and prototype A Hall on the distribution of actin and tubulin in these cells. Native forms of autochthonous BoNT (Su strains 1935 and 1891, Tupungato) and prototype A Hall were purified by saline precipitation. Their AE values (LD50 / mg protein) were established and their electrophoretic characteristics were evaluated under non-denaturing conditions. 250 LD50 of the BoNTs were incubated to MCF-7 cell cultures for 10 or 25 min. Later, the cells were fixed and processed for indirect immunofluorescence with the use of specific antibodies that recognize tubulin or actin. The samples were visualized by fluorescence microscopy. At the two times evaluated, the three types of BoNTs produced a marked redistribution of the actin cytoskeleton, patch form, on areas coinciding with the plasma membrane. Tubulin was redistributed to multiple areas with high signal density at 10 min of incubation only in the presence of BoNT 1891. At 25 min of incubation, the cells treated with BoNTs 1891 and 1935 showed this effect, while in those incubated with A Hall, the distribution of these proteins was not modified. The notable alterations in the distribution of components of the tumor cell cytoskeleton by BoNT from native strains of Mendoza soils open new perspectives for therapy against solid tumors.Fil: Chapana, Agostina Lucía. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Guarniolo, D.. Universidad Nacional de Cuyo. Facultad de Cs.médicas. Departamento de Patología. Area de Microbiología; ArgentinaFil: Carvelli, Flavia Lorena. 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 Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Sosa, E.. Universidad Nacional de Cuyo. Facultad de Cs.médicas. Departamento de Patología. Area de Microbiología; ArgentinaFil: Fernández, R. A.. Universidad Nacional de Cuyo. Facultad de Cs.médicas. Departamento de Patología. Area de Microbiología; ArgentinaFil: Sosa, M. A.. 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: Caballero, P. A.. Universidad Nacional de Cuyo. Facultad de Cs.médicas. Departamento de Patología. Area de Microbiología; ArgentinaIV Reunión Conjunta de Sociedades de Biología de la República ArgentinaArgentinaSociedad de Biología de CuyoSociedad Argentina de BiologíaSociedad de Biología de RosarioSociedad Chilena de Reproducción y DesarrolloAsociación de Biología de TucumánSociedad de Biología de Córdob

The neurotoxin of a autochthonous Clostridium botulinum affects the actin cytoskeleton in breast cancer cells

Botulism is a neuroparalitic disease caused by botulinum neurotoxins (NTBo, serotypes A-G) produced by Clostridium botulinum, whose main reservoir is the soil (Su). Infant botulism is a toxiinfection, caused by ingestion of spores, subsequent colonization and production of toxins in situ. The autochthonous NTBo would correspond to subtype A2, and have higher toxicity than A1 (Botox®), so in the future, they could be used as a therapeutic agent. The NTBos mechanism of action on certain pathologies is still to be clarified. Previous results from our laboratory showed that autochthonous NTBo 1935 from Su, degrades actin of rat brain homogenates, suggesting this protein could be an active target of NTBos. In this work, the action of this NTBo on the actin cytoskeleton in mammary tumor cells was evaluated. The NTBos of Su from strain 1935 and strain A Hall (both serotype A) were purified by saline precipitation. MCF7 cells (breast cancer cells) were cultured in petri dishes or coverslips with 250 LD50 of the NTBos for 25, 45 or 90 min. After incubations, cells were processed for western blot or immunofluorescence in order to evaluate the distribution and expression of actin. NTBo 1935 produced a higher actin degradation and an increased location of this protein at plasma membrane in comparison with A Hall in dependent-time manner. However, at 90 min of treatment, we observed 90% of cytotoxicity and further studies at this time were not evaluated. These results provide new insights about the NTBo mechanism of action and its possible use in the fight against breast cancer.Fil: Guarniolo, D. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Caballero, P. A.. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Chapana, A.. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Sosa, E.. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Fernández, R. A.. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Sosa, M. A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Histología y Embriología D/mend Dr.m.burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Carvelli, Flavia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Histología y Embriología D/mend Dr.m.burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaXXXVII Reunión Científica Anual de la Sociedad de Biología de CuyoSan LuisArgentinaSociedad de Biología de Cuy

Effect of botulinum neurotoxin of Mendoza clostridium botulinum atrain on tubulin in breast carcinoma cells (MCF 7)

Botulinum neurotoxin serotype A (BoNT A), produced by Clostridium botulinum, causes botulism and is used to treat multiple diseases. Its potential action in cancer therapy is currently being evaluated. In precedent studies, we have shown that BoNT A from native soil strains (Su), they have different characteristics to that of the prototype A Hall strain such as; greater specific toxic activity (AE) and differences in molecular structure and enzymatic activity against SNARE proteins. In this work, we study the action of an autochthonous BoNT A compared to the A Hall on tubulin in the MCF-7 cell line of breast carcinoma. The autochthonous BoNTs of the strain Su 1935 (Tupungato) and A Hall were used in their native form, purified by saline precipitation. The values of AE (LD50/mg protein) and electrophoretic characteristics under non-denaturing conditions were determined. MCF7 cells were cultured on coverslips and incubated with 250 and 500 LD50 of the BoNTs for 10, 25, 45, and 90 min. Later, the cells were fixed and processed for immunodetection. As primary antibodies were used anti-tubulin or anti-Golgina 97 and as secondary anti-mouse-Alexa 488. The preparations visualized by fluorescence microscopy. At 90 min incubation with 250 LD50, it was observed that ~90% of the cells were taken off and deformed by the action of the BoNT A 1935 and ~40% for the BoNT A Hall, while with 500 LD50 both toxins were deleterious to the cells. When the cells were incubated with the toxins for 25 min, a disruption of microtubules with both toxins was observed, the effect being greater with the BoNT A1935. This effect was accompanied by a redistribution of the Golgi apparatus. Western blotting showed the shape of new tubulin bands, possibly due to protein degradation. This effect was also greater in the BoNT 1935. These results show a cytotoxic action of BoNT A with disorganization of cell microtubules, being observed with greater intensity in the cells treated with the autochthonous BoNT A. The degradation of tubulin and its intracellular reorganization would be part of the deleterious action of this toxin on tumor cells, opening new perspectives for therapy against solid tumors.Fil: Chapana, Agostina Lucía. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Carvelli, Flavia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Histología y Embriología D/mend Dr.m.burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Guarniolo, D.. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Sosa, E.. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Fernandez, R. A.. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Barrera, Patricia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Histología y Embriología D/mend Dr.m.burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Sosa, Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Histología y Embriología D/mend Dr.m.burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Caballero, P. A.. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaXXXVII Reunión Científica Anual de la Sociedad de Biología de CuyoSan LuisArgentinaSociedad Biología de Cuy