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dc.date.available
2023-03-13T16:57:37Z  
dc.identifier.citation
Sasso, Corina Verónica; Pistone Creydt, Virginia; López Fontana, Constanza Matilde; Caron, Ruben Walter; (2023): Estradiol and progesterone regulate proliferation and apoptosis in colon cancer. Consejo Nacional de Investigaciones Científicas y Técnicas. (dataset). http://hdl.handle.net/11336/190356  
dc.identifier.uri
http://hdl.handle.net/11336/190356  
dc.description.abstract
Epidemiological studies describe estrogens as protectors in the development of colon cancer in postmenopausal women treated with hormone replacement therapy. However, the role of progesterone in colon cancer has been minimally studied and the results are controversial. For the above, the objective of this work was to determine the hormonal regulation exerted by natural ovarian steroids on proliferation and apoptosis in an experimental model of colon cancer in ovariectomized rats treated with 17-beta estradiol and progesterone. Sprague–Dawley rats were exposed to the carcinogen 1,2-dimethylhydrazine to induce colon tumors. Thirty days later, the rats were ovariectomized and treated with estradiol (60 μg/kg), progesterone (10 mg/kg), estradiol plus progesterone (60 μg/kg and 10 mg/kg) or vehicle. We observed no significant differences in colon cancer incidence and tumor multiplicity between the groups. Nevertheless, we observed a decrease in PCNA expression and a greater number of apoptotic index, higher expression of caspase 3, cleaved PARP and cleaved caspase 8 in tumors, confirming the activation of the extrinsic pathway of apoptosis by the combined treatment. In addition, we observed a higher expression of estrogen receptor beta in these tumors. We conclude that the action of both hormones, estradiol and progesterone, is necessary to reduce proliferation and increase apoptosis in colon tumors, probably through estrogen receptor beta activation.  
dc.rights
info:eu-repo/semantics/restrictedAccess  
dc.title
Estradiol and progesterone regulate proliferation and apoptosis in colon cancer  
dc.type
dataset  
dc.date.updated
2023-03-13T15:00:13Z  
dc.description.fil
Fil: Sasso, Corina Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina  
dc.description.fil
Fil: Pistone Creydt, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina  
dc.description.fil
Fil: López Fontana, Constanza Matilde. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina  
dc.description.fil
Fil: Caron, Ruben Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina  
dc.rights.license
Protección de datos personales (Ley 25.326)  
dc.datacite.PublicationYear
2023  
dc.datacite.Creator
Sasso, Corina Verónica  
dc.datacite.Creator
Pistone Creydt, Virginia  
dc.datacite.Creator
López Fontana, Constanza Matilde  
dc.datacite.Creator
Caron, Ruben Walter  
dc.datacite.affiliation
Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo  
dc.datacite.affiliation
Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo  
dc.datacite.affiliation
Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo  
dc.datacite.affiliation
Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo  
dc.datacite.affiliation
Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo  
dc.datacite.affiliation
Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo  
dc.datacite.affiliation
Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo  
dc.datacite.publisher
Consejo Nacional de Investigaciones Científicas y Técnicas  
dc.datacite.subject
Bioquímica y Biología Molecular  
dc.datacite.subject
Medicina Básica  
dc.datacite.subject
CIENCIAS MÉDICAS Y DE LA SALUD  
dc.datacite.ContributorType
DataCollector  
dc.datacite.ContributorType
DataCollector  
dc.datacite.ContributorType
DataCollector  
dc.datacite.ContributorName
Santiano, Flavia Eliana  
dc.datacite.ContributorName
Campo Verde Arbocco, Fiorella  
dc.datacite.ContributorName
Zyla, Leila Ester  
dc.datacite.date
01/04/2017-31/03/2019  
dc.datacite.DateType
Recolectado  
dc.datacite.language
eng  
dc.datacite.AlternateIdentifierType
info:eu-repo/semantics/altIdentifier/doi/10.1530/EC-18-0374.  
dc.datacite.version
1.0  
dc.datacite.description
Animals Virgin Sprague–Dawley female rats were kept in a light- (lights on 06.00–20.00 h) and temperature-controlled room (22–24°C) in our animal facility. Rat chow (Cargill, Córdoba, Argentina) and tap water were available ad libitum. Animal maintenance and handling were performed according to the NIH guide for the Care and Use of Laboratory Animals (NIH publication no. 86-23, revised 1991) and the UK requirements for ethics of animal experimentation (Animals Scientific Procedures, Act 1986). All the experimental procedures were approved by the Animal and Ethics Committee (CICUAL) of the School of Medicine of the National University of Cuyo, Mendoza, Argentina (0011463/2011). Experimental protocols To induce colon cancer, 45-day-old rats (approximately weighting 170 g) were treated subcutaneously once a week with 1,2-dimethylhydrazine (DMH, 21 mg/kg; Sigma), for 20 weeks as previously described (19, 20). Four weeks after the first DMH dose, the rats were anesthetized by an intraperitoneal injection of ketamine-xylazine (45 and 10 mg/kg) and were ovariectomized as previously described (21). In order to study the effects of the ovarian steroids on colon carcinogenesis, they started receiving subcutaneous injections twice a week with 17-beta estradiol (E2 group, 60 μg/kg, N = 13; Sigma), progesterone (P4 group, 10 mg/kg, N = 14; Sigma), E2 and P4 (E2 + P4 group, 60 μg/kg and 10 mg/kg, respectively, N = 13) or vehicle (V group, vegetal oil, N = 10) until they were killed. All the animals were periodically controlled for symptoms, irrespective of the treatment. The same observer checked weekly the rats in the same way, looking for loss of weight, diarrhea or any sign of distress. The incidence of colon cancer was calculated as the percentage of rats that presented tumors within the period studied. The rats were decapitated the day they were expected to receive the following hormonal dose. In consequence, they were killed 84 h after the last injection. Since the rats were killed when they exhibited symptoms of tumor presence, we compared the day of killing in order to have a parameter related to latency, and we expressed it as latency of appearance of evident symptoms. The animals without any symptom were killed at day 270 from the first DMH dose. Trunk blood samples were collected and allowed to clot at room temperature. Serum was separated and stored at −20°C until assayed for hormone determinations. Immediately after decapitation, a piece of the tumor was removed for histopathological, immunohistochemical and Western blot (WB) analysis. Hormone determinations To determine the serum levels of estradiol and progesterone, the specific commercial Coat-A-Count kits (TKE21 and TKPG1; Siemens Healthcare Diagnostics Inc.) were used according to the manufacturer’s instructions. A total of 100 µL of the calibrators or 100 µL of sera were added to the precoated tubes in duplicate. One milliliter of 125I estradiol or 125I progesterone was added to each tube and incubated for 3 h at room temperature. The content of the tubes was aspirated and counted for 1 min in a gamma counter. Assay sensitivity was 8 pg/mL for estradiol and 0.02 ng/mL for progesterone. The inter- and intra-assay coefficients of variation were <10% for both hormones. Tumor histology After decapitation, a small piece of tumor of each rat was fixed in 4% v/v formaldehyde for 24 h, dehydrated in ethanol and embedded in paraffin wax. Sections of 3–5 µm were cut in a HYRAX M 25 Rotary microtome (Zeiss) and stained with hematoxylin–eosin (H&E) for the analysis under the optic microscope. The tumor grade and type, the inflammation grade, fibrosis, necrosis and mitotic and apoptotic index were defined. The number of mitotic figures and apoptotic bodies present in the tumor cells in ten fields was counted under microscope at a magnification of 400×. The mitotic and apoptotic index was calculated dividing the number of mitotic figures by the number of apoptotic bodies for each tumor. Immunohistochemistry Sections of 3–5 μm from each tumor underwent an antigen retrieval protocol using heat (40 min in citrate buffer 0.01 M, pH 6.0). After two washes with distilled water, the endogen peroxidase was blocked with 0.1% w/v sodium azide for 30 min. The nonspecific binding sites were blocked with 10% w/v of skim milk. The primary antibodies used were PCNA (M0879, 1:600 dilution; Dako), caspase 3 (ab4051, 1:400 dilution; Abcam), ERA (ab32063, 1:200 dilution; Abcam), ERB (ab3577, 1:750 dilution; Abcam) and PR (sc-539, 1:100 dilution; Santa Cruz Biotechnology Inc.). The antibodies were incubated overnight at 4°C in humidity chambers. A commercial kit to detect mouse and rabbit antibodies was used (Dako EnVision Systems, horseradish peroxidase, diaminobenzidine; Dako). Slides were lightly counterstained with hematoxylin to reveal nuclei, examined and photographed. The immunostaining was evaluated considering the extent, intensity and localization of immunostaining independently by two experienced researchers blinded regarding the hormone treatments, and a few conflicting scores were resolved by consensus. The intensity score was measured as follows: 0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining; and a proportion score: 0 = no staining, 1 = staining less than 10% of the tumor cells, 2 = between 11 and 33%, 3 = between 34 and 65%, 4 = greater than 66%. The images were taken with a Nikon Eclipse E200 microscope (Nikon) equipped with a digital micrometrics SE High Quality camera (Accu-Scope, Commak, NY, USA) at a magnification of 400×. Protein isolation and WB Total proteins in 200 mg from each tumor were isolated by mechanical homogenization with two volumes of homogenization buffer (50 mM Tris, pH 7.5, 250 mM sucrose, 10 mM benzamidine, 10 mM NaF, 5 mM sodium pyrophosphate, 20 mM glycerophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, 10 mM p-nitrophenylphosphate, and aprotinin, leupeptin and pepstatin at 2 mg/L) in an ice bath. The homogenate was centrifuged at 12,500  g for 30 min and the supernatant was separated and frozen in several aliquots at −80°C until used. Proteins were quantified using the Micro BCA Protein Assay Kit (Thermo Scientific), and boiled for 5 min in loading buffer. Eighty micrograms of proteins were separated by SDS-PAGE and transferred to PVDF membranes (Immobilon-P, Merck Millipore). After rinsing and blocking with 2% w/v BSA (Sigma), the membranes were probed overnight at 4°C with antibodies targeting caspase 3 (ab4051, 1:500 dilution; Abcam), cleaved PARP (ab32064, 1:2000 dilution; Abcam), caspase 8 (ab25901, 1:1000 dilution; Abcam), ERA (ab32063, 1:2500 dilution; Abcam), ERB (ab3577, 1:3000 dilution; Abcam), PR (sc-539, 1:200 dilution; Santa Cruz Biotechnology Inc.) and B-actin (sc-47778, 1:3000 dilution; Santa Cruz Biotechnology Inc.). After a new rinsing, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies anti-rabbit (sc-2004, 1:2000 dilution; Santa Cruz Biotechnology Inc.) or anti-mouse (sc-2005, 1:2000 dilution; Santa Cruz Biotechnology Inc.) for 90 min at room temperature. The membranes were rinsed and the bands were detected by chemiluminescence (ECLTM; Amersham) using a ChemiDoc XRS + System with Image Lab Software from Bio-Rad and then quantified by densitometry using digital image processing by the NIH ImageJ 1.6 freeware program. Quantitative analysis of the different protein levels was performed by determining the ratio between the specific protein and B-actin levels by densitometry. Expression of the ESR1, ESR2 and PGR genes in human colon adenocarcinomas Tumors from The Cancer Genome Atlas (TCGA) colon cancer database (https://portal.gdc.cancer.gov accessed on November 26, 2018) were evaluated. Data was programmatically downloaded using R TCGAbiolinks package. Four hundred seventy-six primary tumors were obtained and patients were classified according to their gender (males N = 252 or females N = 224). Females were further divided according to their age: greater than 50 years (N = 191) or less than 50 years (N = 33) at the time of diagnosis. Raw RNA-Seq expression counts were used and normalized using Voom transformation from R Limma package (https://genomebiology.biomedcentral.com/articles/10.1186/gb-2014-15-2-r29 accessed on November 26, 2018). Transformed gene expression distribution was depicted using boxplots and expression correlation between the three genes was evaluated using Pearson’s correlation coefficient with its corresponding P value. The direction of the relation was calculated using simple linear regression and depicted as a straight line with a slope in a scatterplot.  
dc.datacite.DescriptionType
Métodos  
dc.subject.keyword
COLON CANCER  
dc.subject.keyword
17-BETA-ESTRADIOL  
dc.subject.keyword
PROGESTERONE  
dc.subject.keyword
APOPTOSIS  
dc.datacite.resourceTypeGeneral
dataset  
dc.conicet.datoinvestigacionid
5174  
dc.datacite.geolocation
Mendoza  
dc.datacite.formatedDate
2017-2019