The inhibitory and anticancer properties of Annona squamosa L. seed extracts / Propriedades inibitórias e anticancerígenas de extratos de sementes de Annona squamosa L

Although Annona squamosa Linn. (Annonaceae) has been used in traditional medicine and is known to have several pharmacological properties, its impact on EGFR kinase has not been fully investigated. An assay (biochemical) was used to govern the potential of different A. squamosa seed extracts to scavenge free radicals in petroleum ether, acetone, ethanol, and methanol. We also tested A. squamosa leaf extracts for their ability to inhibit the growth of HEK 293, MCF7, and HepG2 cell lines. The PSE, ASE, ESE, and MSE all contained anti-cancer substances like anethole, cyclopentane, 1,1,3-trimethyl, and phosphonate oxide tributyl, according to phytochemical analysis. ESE extracts from A. squamosa seeds have been selected based on free radical generation probabilities, cytotoxicity studies, and phytochemical analysis. Subsequent insilico studies have been conducted, and the results have shown that interactions between compounds present in ESE extracts and the EGFR kinase are what give these compounds their inhibitory effects. Preliminary phytochemical and pharmacological activities were studied and reported. A. squamosa ESE extracts inhibited the growth of MCF7 cells, and a pharmacokinetic study showed that the compounds anethole, cyclopentane, 1,1,3-trimethyl, and phosphonium oxide tributyl had few undesirable side effects. These substances can be used to both prevent and treat cancer diseases.

Embora a Annona squamosa Linn. (Annonaceae) tenha sido utilizada na medicina tradicional e seja conhecida por diversas propriedades farmacológicas, seu impacto na EGFR quinase ainda não foi totalmente investigado. Um ensaio bioquímico foi utilizado para controlar o potencial de diferentes extratos de sementes de A. squamosa para eliminar radicais livres em éter de petróleo, acetona, etanol e metanol. Extratos de folhas de A. squamosa também foram analisados em relação à sua capacidade de inibir o crescimento de linhagens celulares HEK 293, MCF7 e HepG2. O PSE, ASE, ESE e MSE continham substâncias anticancerígenas como anetol, ciclopentano, 1,1,3-trimetil e óxido de fosfonato tributil, de acordo com a análise fitoquímica. Extratos de ESE de sementes de A. squamosa foram selecionados com base em probabilidades de geração de radicais livres, estudos de citotoxicidade e análise fitoquímica. Estudos in silico subsequentes foram realizados e os resultados mostraram que as interações entre os compostos presentes nos extratos de ESE e a EGFR quinase são o que confere a esses compostos seus efeitos inibitórios. As atividades fitoquímicas e farmacológicas preliminares foram estudadas e relatadas. Os extratos de ESSE de A. squamosa inibiram o crescimento de células MCF7, e um estudo farmacocinético mostrou que os compostos anetol, ciclopentano, 1,1,3-trimetil e óxido de fosfônio tributil tiveram poucos efeitos colaterais indesejáveis. Essas substâncias podem ser usadas para prevenir e tratar doenças cancerígenas.

Changes in somatic cell count, composition and cytokine levels in milk from cows with mastitis due to mixed infections

Mastitis is a mammary gland inflammation that is very common worldwide, mostly caused by bacteria, and causes enormous economic losses. Many microorganisms cause this disease. The most common causes of mastitis by these microorganisms are Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Streptococcus agalactiae (S. agalactiae). The anti-inflammatory properties of transforming growth factor (TGF)-ß include: 1) limiting interferon (IFN)-γ production; 2) increasing the expression of the interleukine (IL)-1 receptor antagonist; 3) inhibiting macrophage production of chemokines, pro-inflammatory cytokines, nitric oxide, and reactive oxygen intermediates; and 4) increasing macrophage clearance of bacterial debris and damaged parenchymal cells. It is stated that cytokines and milk composition change in case of mastitis. In this study, it was aimed to reveal the changes in milk TGF-ß1 and Tumor necrosis factor (TNF)-α concentrations and milk composition in mixed infections caused by three pathogens causing mastitis. In this study, milk samples from 90 cows were divided into 5 groups. Tumor necrosis factor (TNF)-α and TGF-ß1 concentrations and milk composition were determined in these milk samples. The California Mastitis Test (CMT) was applied to the cows included in the study and scoring was done. According to the CMT results of the milk samples taken, CMT(-) cows were included in group 1 (n = 22). Those with the CMT(+) were sent to the microbiology laboratory for analysis within 2 h. After the bacteria was determined, combination groupings were formed. Group 2 (n = 17), in which S. aureus and E. coli grew together, group 3 (n = 21), in which S. aureus and S. agalactiae grew together, group 4 (n = 8), in which S. agalactiae and E. coli grew together in milk samples, and milk samples without any bacterial growth in CMT (+) formed group 5 (n = 22), respectively. Somatic cell count was measured with the DeLaval Cell Counter® (Cell Counter DCC) device. Mineral matter, fat, protein, lactose, electrical conductivity and specific gravity were measured in milk samples using Lactoscan Milk Analyzer (Milkotronic/EUROPE). Milk samples were then stored at -80°C to measure TGF-ß1 and TNF-α. Tumor necrosis factor-α and TGF-ß1 concentrations in milk samples were measured using ELISA kits (Sunred Biological Technology). Changes in milk TNF-α and TGF-ß1 concentration and milk composition were determined in milk samples with mastitis caused by mixed infection. The TNF-α concentration of group 4 was higher than the other groups. On the other hand, the highest concentration of TGF-ß1 was found in group 2. While the number of somatic cells in group 1 was lower than in groups 2, 3, and 4, there was no statistical difference between groups 1 and 5. The lowest milk fat ratio was found in group 1, and it was found to be statistically lower than groups 2, 3, and 4. While the rate of solid-non-fat of group 1 increased compared to groups 2 and 3, the highest protein ratio was found in groups 1 and 5. There was no difference between the 5 groups in terms of mineral matter ratios. While the specific gravity was highest in group 1, there was no statistical difference between the other 4 groups. Overall, it was concluded that there was an increase in TNF-α and TGF-ß1 concentrations and a change in milk composition in samples with bacterial growth.(AU)

Osteopontin expression and its relationship with prognostic biomarkers in canine mammary carcinomas

ABSTRACT: Osteopontin is a glycophosphoprotein implicated in different physiologic and pathologic processes and is known to be involved in progression and metastasis of various cancers in humans, but this relation is still little explored in the veterinary. The aim was to evaluate the expression of osteopontin in canine mammary carcinomas and its relation with well-established canine mammary tumor biomarkers. For that, expression of OPN, EGFR, HER2, and c-Kit were evaluated along with Ki67 rate in 43 mammary carcinomas. Osteopontin was demonstrated to be expressed by neoplastic epithelial cells in all carcinomas as well as in stromal cells from the tumor microenvironment. Relation between high osteopontin expression and EGFR positivity (P 0.001) and HER2 overexpression (P=0.012) was demonstrated. In conclusion, high OPN expression seems to be related to poor prognosis and MAPK pathway activation, given the association with EGFR and HER2, members of the MAPK signaling pathway.

RESUMO: A osteopontina é uma glicofosfoproteina implicada em diferentes processos fisiológicos e patológicos, sendo conhecida por estar envolvida na progressão e metástase de vários cânceres nos humanos, no entanto, essa relação é ainda pouco explorada na veterinária. O objetivo deste trabalho foi avaliar a expressão da osteopontina nos carcinomas mamários caninos e sua relação com biomarcadores bem estabelecidos para esta neoplasia. Para isto, foi avaliada a expressão de OPN, EGRH, HER2 e c-Kit juntamente com a taxa de Ki67 em 43 carcinomas mamários. A osteopontina foi expressa pelas células epiteliais neoplásicas em todos os carcinomas, assim como, nas células estromais do microambiente tumoral. Foi demonstrada uma relação entre uma alta expressão de osteopontina e positividade para EGFR (P 0.001) e superexpressão de HER2 (P=0.012). Em conclusão, alta expressão de OPN parece estar relacionada com mau prognóstico e ativação da via MAPK, devido a sua associação com EGRF e HER2, os quais são membros desta via de sinalização.

The interaction of Wnt signaling members with growth factors in cultured granulosa cells

Wnt family members have recently been distinguished in the adult ovary with potential roles in ovarian function. Though particular growth factors interact with Wnt signaling members in extraovarian cell types, it is unclear whether this interaction is applicable in the granulosa cells. Therefore, the current study aimed to determine the effect of insulin-like growth factor-1 (IGF-I), epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-β) on Wnt ligands WNT2 and WNT4 and Wnt receptor Frizzled-4 (FZD4) protein levels in cultured mouse granulosa cells. Granulosa cells were isolated from antral follicles of adult Balb/C mice and cultured for 24 hours in the presence of 100 ng/mL of IGF-I, or EGF or FGF-β. WNT2, WNT4 and FZD4 protein levels were evaluated through western blotting after the culture process. IGF-I treated granulosa cells had significantly the highest level of WNT2 and WNT4 as well as FZD4 when compared to FGF-β and EGF groups. FGF-β group had a significantly higher level of WNT2, WNT4 and FZD4 expression when compared to EGF group. FZD4 expression was at the highest level in the IGF-I group and this difference was statistically significant for all groups including uncultured cells and vehicle group. In addition, FGF-β was shown to positively affect the adhesion of granulosa cells. This study demonstrates that IGF-I, FGF-β and EGF have differential effects on the expressions of WNT2, WNT4, and FZD4 in cultured mouse granulosa cells, suggesting that particular growth factors related to ovarian function might conduct their roles in the ovary through Wnt signaling.

The interaction of Wnt signaling members with growth factors in cultured granulosa cells

Wnt family members have recently been distinguished in the adult ovary with potential roles in ovarian function. Though particular growth factors interact with Wnt signaling members in extraovarian cell types, it is unclear whether this interaction is applicable in the granulosa cells. Therefore, the current study aimed to determine the effect of insulin-like growth factor-1 (IGF-I), epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-β) on Wnt ligands WNT2 and WNT4 and Wnt receptor Frizzled-4 (FZD4) protein levels in cultured mouse granulosa cells. Granulosa cells were isolated from antral follicles of adult Balb/C mice and cultured for 24 hours in the presence of 100 ng/mL of IGF-I, or EGF or FGF-β. WNT2, WNT4 and FZD4 protein levels were evaluated through western blotting after the culture process. IGF-I treated granulosa cells had significantly the highest level of WNT2 and WNT4 as well as FZD4 when compared to FGF-β and EGF groups. FGF-β group had a significantly higher level of WNT2, WNT4 and FZD4 expression when compared to EGF group. FZD4 expression was at the highest level in the IGF-I group and this difference was statistically significant for all groups including uncultured cells and vehicle group. In addition, FGF-β was shown to positively affect the adhesion of granulosa cells. This study demonstrates that IGF-I, FGF-β and EGF have differential effects on the expressions of WNT2, WNT4, and FZD4 in cultured mouse granulosa cells, suggesting that particular growth factors related to ovarian function might conduct their roles in the ovary through Wnt signaling.(AU)

Osteopontin expression and its relationship with prognostic biomarkers in canine mammary carcinomas / Expressão de osteopontina e sua relação com biomarcadores prognóstico nos carcinomas mamários caninos

Osteopontin is a glycophosphoprotein implicated in different physiologic and pathologic processes and is known to be involved in progression and metastasis of various cancers in humans, but this relation is still little explored in the veterinary. The aim was to evaluate the expression of osteopontin in canine mammary carcinomas and its relation with well-established canine mammary tumor biomarkers. For that, expression of OPN, EGFR, HER2, and c-Kit were evaluated along with Ki67 rate in 43 mammary carcinomas. Osteopontin was demonstrated to be expressed by neoplastic epithelial cells in all carcinomas as well as in stromal cells from the tumor microenvironment. Relation between high osteopontin expression and EGFR positivity (P<0.001) and HER2 overexpression (P=0.012) was demonstrated. In conclusion, high OPN expression seems to be related to poor prognosis and MAPK pathway activation, given the association with EGFR and HER2, members of the MAPK signaling pathway.(AU)

A osteopontina é uma glicofosfoproteina implicada em diferentes processos fisiológicos e patológicos, sendo conhecida por estar envolvida na progressão e metástase de vários cânceres nos humanos, no entanto, essa relação é ainda pouco explorada na veterinária. O objetivo deste trabalho foi avaliar a expressão da osteopontina nos carcinomas mamários caninos e sua relação com biomarcadores bem estabelecidos para esta neoplasia. Para isto, foi avaliada a expressão de OPN, EGRH, HER2 e c-Kit juntamente com a taxa de Ki67 em 43 carcinomas mamários. A osteopontina foi expressa pelas células epiteliais neoplásicas em todos os carcinomas, assim como, nas células estromais do microambiente tumoral. Foi demonstrada uma relação entre uma alta expressão de osteopontina e positividade para EGFR (P<0.001) e superexpressão de HER2 (P=0.012). Em conclusão, alta expressão de OPN parece estar relacionada com mau prognóstico e ativação da via MAPK, devido a sua associação com EGRF e HER2, os quais são membros desta via de sinalização.(AU)

Osteopontin expression and its relationship with prognostic biomarkers in canine mammary carcinomas / Expressão de osteopontina e sua relação com biomarcadores prognóstico nos carcinomas mamários caninos

Osteopontin is a glycophosphoprotein implicated in different physiologic and pathologic processes and is known to be involved in progression and metastasis of various cancers in humans, but this relation is still little explored in the veterinary. The aim was to evaluate the expression of osteopontin in canine mammary carcinomas and its relation with well-established canine mammary tumor biomarkers. For that, expression of OPN, EGFR, HER2, and c-Kit were evaluated along with Ki67 rate in 43 mammary carcinomas. Osteopontin was demonstrated to be expressed by neoplastic epithelial cells in all carcinomas as well as in stromal cells from the tumor microenvironment. Relation between high osteopontin expression and EGFR positivity (P<0.001) and HER2 overexpression (P=0.012) was demonstrated. In conclusion, high OPN expression seems to be related to poor prognosis and MAPK pathway activation, given the association with EGFR and HER2, members of the MAPK signaling pathway.(AU)

A osteopontina é uma glicofosfoproteina implicada em diferentes processos fisiológicos e patológicos, sendo conhecida por estar envolvida na progressão e metástase de vários cânceres nos humanos, no entanto, essa relação é ainda pouco explorada na veterinária. O objetivo deste trabalho foi avaliar a expressão da osteopontina nos carcinomas mamários caninos e sua relação com biomarcadores bem estabelecidos para esta neoplasia. Para isto, foi avaliada a expressão de OPN, EGRH, HER2 e c-Kit juntamente com a taxa de Ki67 em 43 carcinomas mamários. A osteopontina foi expressa pelas células epiteliais neoplásicas em todos os carcinomas, assim como, nas células estromais do microambiente tumoral. Foi demonstrada uma relação entre uma alta expressão de osteopontina e positividade para EGFR (P<0.001) e superexpressão de HER2 (P=0.012). Em conclusão, alta expressão de OPN parece estar relacionada com mau prognóstico e ativação da via MAPK, devido a sua associação com EGRF e HER2, os quais são membros desta via de sinalização.(AU)

Clinicopathological and immunohistochemical features of pleomorphic lobular mammary carcinoma in a female dog

This report describes a pleomorphic lobular carcinoma (PLC) case, in an eight-year-old, female Shih Tzu dog, which attended the Veterinary Hospital of the University of Franca, Brazil. A single irregular, non-ulcerated, nonhyperaemic soft nodule, 2 cm in diameter, was observed during the clinical examination. The nodule was adhered within the deep tissues in the right caudal thoracic mammary gland. After removal of the nodule, the histopathologic examination showed that the neoplastic epithelial cells were scattered throughout the stroma in linear patterns, had abundant eosinophiliccytoplasm, eccentric nuclei and cytoplasmatic vacuoles, features compatible with the diagnosis of pleomorphic lobularcarcinoma. An immunohistochemical evaluation was performed in order to better characterise the tumour. Based on thenegative immunoreactivity for hormone receptors and human epidermal growth factor receptor-2 and positive expression for one of the basal markers, the basal-like triple-negative phenotype was characterized. The treatment indicated was chemotherapy with carboplatin; however, the disease progressed, and the patient had an overall survival of 47 days after surgery, confirming the aggressiveness of the disease.

Clinicopathological and immunohistochemical features of pleomorphic lobular mammary carcinoma in a female dog

This report describes a pleomorphic lobular carcinoma (PLC) case, in an eight-year-old, female Shih Tzu dog, which attended the Veterinary Hospital of the University of Franca, Brazil. A single irregular, non-ulcerated, nonhyperaemic soft nodule, 2 cm in diameter, was observed during the clinical examination. The nodule was adhered within the deep tissues in the right caudal thoracic mammary gland. After removal of the nodule, the histopathologic examination showed that the neoplastic epithelial cells were scattered throughout the stroma in linear patterns, had abundant eosinophiliccytoplasm, eccentric nuclei and cytoplasmatic vacuoles, features compatible with the diagnosis of pleomorphic lobularcarcinoma. An immunohistochemical evaluation was performed in order to better characterise the tumour. Based on thenegative immunoreactivity for hormone receptors and human epidermal growth factor receptor-2 and positive expression for one of the basal markers, the basal-like triple-negative phenotype was characterized. The treatment indicated was chemotherapy with carboplatin; however, the disease progressed, and the patient had an overall survival of 47 days after surgery, confirming the aggressiveness of the disease.(AU)

Expression of EGF receptors in canine prostate with proliferative inflammatory atrophy and carcinoma / Expressão de receptores EGF na próstata canina com atrofia inflamatória proliferativa e carcinoma

Gene expression of ErbB1 and ErbB2, and immunostaining of EGFR (Her1) and Her2 (c-erbB-2) were evaluated in this study to ascertain whether these receptors are involved in the evolution of canine premalignant and malignant prostatic lesions, as proliferative inflammatory atrophy (PIA) and prostatic carcinoma (PC). With regards to the intensity of EGFR immunostaining, there was no difference between normal prostatic tissue and tissues with PIA or PC. In relation to Her2 immunostaining, there were differences between normal prostatic tissue and those with PIA and PC, as also differences between prostates with PIA and PC. There was no correlation between EGFR and Her2 immunostaining. ErbB1 gene product was detected in two normal tissue samples, in one with PIA, and in all samples with PC. ErbB2 mRNA was recorded in two canine samples with PIA, in all with PC, but was not detected in normal prostatic tissue. It was concluded that EGFR and Her2 play roles in canine PIA and PC, suggesting that those receptors may be involved in canine prostatic carcinogenesis.

A expressão gênica de ErbB1 e ErbB2 e a imunomarcação de EGFR (Her1) e Her2 (c-erbB-2) foram avaliadas para verificar o envolvimento desses receptores em lesões pré-malignas e malignas da próstata canina, como a atrofia proliferativa inflamatória (PIA) e o carcinoma prostático (PC). Em relação à intensidade de imunomarcação para EGFR, não houve diferença entre o tecido prostático normal e com PIA e PC. Em relação a Her2, observou-se diferença de imunomarcação entre o tecido prostático normal e aqueles com PIA e PC e entre os com PIA e PC. Não houve correlação entre EGFR e Her2. O gene ErbB1 foi detectado em duas amostras normais, uma de PIA e em todas as amostras de PC. O gene ErbB2 foi detectado em duas amostras de PIA e em todas as amostras de PC, não sendo detectado no tecido prostático normal. Conclui-se que EGFR e Her2 atuam nas lesões de PIA e PC, sugerindo o envolvimento destes na carcinogênese da próstata canina.

Expression of EGF receptors in canine prostate with proliferative inflammatory atrophy and carcinoma / Expressão de receptores EGF na próstata canina com atrofia inflamatória proliferativa e carcinoma

Gene expression of ErbB1 and ErbB2, and immunostaining of EGFR (Her1) and Her2 (c-erbB-2) were evaluated in this study to ascertain whether these receptors are involved in the evolution of canine premalignant and malignant prostatic lesions, as proliferative inflammatory atrophy (PIA) and prostatic carcinoma (PC). With regards to the intensity of EGFR immunostaining, there was no difference between normal prostatic tissue and tissues with PIA or PC. In relation to Her2 immunostaining, there were differences between normal prostatic tissue and those with PIA and PC, as also differences between prostates with PIA and PC. There was no correlation between EGFR and Her2 immunostaining. ErbB1 gene product was detected in two normal tissue samples, in one with PIA, and in all samples with PC. ErbB2 mRNA was recorded in two canine samples with PIA, in all with PC, but was not detected in normal prostatic tissue. It was concluded that EGFR and Her2 play roles in canine PIA and PC, suggesting that those receptors may be involved in canine prostatic carcinogenesis.(AU)

A expressão gênica de ErbB1 e ErbB2 e a imunomarcação de EGFR (Her1) e Her2 (c-erbB-2) foram avaliadas para verificar o envolvimento desses receptores em lesões pré-malignas e malignas da próstata canina, como a atrofia proliferativa inflamatória (PIA) e o carcinoma prostático (PC). Em relação à intensidade de imunomarcação para EGFR, não houve diferença entre o tecido prostático normal e com PIA e PC. Em relação a Her2, observou-se diferença de imunomarcação entre o tecido prostático normal e aqueles com PIA e PC e entre os com PIA e PC. Não houve correlação entre EGFR e Her2. O gene ErbB1 foi detectado em duas amostras normais, uma de PIA e em todas as amostras de PC. O gene ErbB2 foi detectado em duas amostras de PIA e em todas as amostras de PC, não sendo detectado no tecido prostático normal. Conclui-se que EGFR e Her2 atuam nas lesões de PIA e PC, sugerindo o envolvimento destes na carcinogênese da próstata canina.(AU)

Prognostic factors in canine mammary carcinomas and HER2 expression relationship / Fatores de prognóstico em carcinomas mamários caninos e sua relação com expressão de HER2

Background: The human epidermal growth factor type 2 (HER2) receptor is a membrane glycoprotein tyrosine kinase. In woman, HER2 expression is diagnosed in 30% of breast carcinomas and it is associated with a worse prognosis, higher rate of recurrence and mortality. In the bitch, the HER2 overexpression in canine mammary tumors is still controversial and the prognostic value remains uncertain. Thus, we aimed to verify the HER2 expression in canine mammary carcinomas and relate it to the type and histological grade, lymph node metastasis and clinical staging. Materials, Methods & Results: Ninety bitches diagnosed with mammary carcinoma were included in this study. The inclusion criteria were bitches with complete clinical examination, thoracic radiographic examination and submitted unilateral or bilateral mastectomy. Ninety-nine samples of mammary carcinoma were used and the fragments of tumor and regional lymph nodes were fixed in 10% neutral formalin for histopathological and immunohistochemistry analysis. The lesions were evaluated by two pathologists and classified according to the type and histological grade. HER2 expression was performed by semi-quantitative analysis of the slides according to the HerceptTestTM (Dako) recommended score. Simple carcinomas were the most frequent (51.51%) followed by complex carcinomas (46.47%) and in situ carcinoma (2.02%). [...](AU)

Prognostic factors in canine mammary carcinomas and HER2 expression relationship / Fatores de prognóstico em carcinomas mamários caninos e sua relação com expressão de HER2

Background: The human epidermal growth factor type 2 (HER2) receptor is a membrane glycoprotein tyrosine kinase. In woman, HER2 expression is diagnosed in 30% of breast carcinomas and it is associated with a worse prognosis, higher rate of recurrence and mortality. In the bitch, the HER2 overexpression in canine mammary tumors is still controversial and the prognostic value remains uncertain. Thus, we aimed to verify the HER2 expression in canine mammary carcinomas and relate it to the type and histological grade, lymph node metastasis and clinical staging. Materials, Methods & Results: Ninety bitches diagnosed with mammary carcinoma were included in this study. The inclusion criteria were bitches with complete clinical examination, thoracic radiographic examination and submitted unilateral or bilateral mastectomy. Ninety-nine samples of mammary carcinoma were used and the fragments of tumor and regional lymph nodes were fixed in 10% neutral formalin for histopathological and immunohistochemistry analysis. The lesions were evaluated by two pathologists and classified according to the type and histological grade. HER2 expression was performed by semi-quantitative analysis of the slides according to the HerceptTestTM (Dako) recommended score. Simple carcinomas were the most frequent (51.51%) followed by complex carcinomas (46.47%) and in situ carcinoma (2.02%). [...]

Prognostic Factors in Canine Mammary Carcinomas and HER2 Expression Relationship

Background: The human epidermal growth factor type 2 (HER2) receptor is a membrane glycoprotein tyrosine kinase. In woman, HER2 expression is diagnosed in 30% of breast carcinomas and it is associated with a worse prognosis, higher rate of recurrence and mortality. In the bitch, the HER2 overexpression in canine mammary tumors is still controversial and the prognostic value remains uncertain. Thus, we aimed to verify the HER2 expression in canine mammary carcinomas and relate it to the type and histological grade, lymph node metastasis and clinical staging.Materials, Methods & Results: Ninety bitches diagnosed with mammary carcinoma were included in this study. The inclusion criteria were bitches with complete clinical examination, thoracic radiographic examination and submitted unilateral or bilateral mastectomy. Ninety-nine samples of mammary carcinoma were used and the fragments of tumor and regional lymph nodes were fixed in 10% neutral formalin for histopathological and immunohistochemistry analysis. The lesions were evaluated by two pathologists and classified according to the type and histological grade. HER2 expression was performed by semi-quantitative analysis of the slides according to the HerceptTestTM (Dako) recommended score. Simple carcinomas were the most frequent (51.51%) followed by complex carcinomas (46.47%) and in situ carcinoma (2.02%). The histologica

Analysis of gene expression EGFR and KRAS, microRNA-21 and microRNA-203 in patients with colon and rectal cancer and correlation with clinical outcome and prognostic factors

Purpose: To evaluate the expression of EGFR, KRAS genes, microRNAs-21 and 203 in colon and rectal cancer samples, correlated with their age at diagnosis, histological subtype, value of pretreatment CEA, TNM staging and clinical outcome. Methods: Expression of genes and microRNAs by real time PCR in tumor and non-tumor samples obtained from surgical treatment of 50 patients. Results: An increased expression of microRNAs-21 and 203 in tumor samples in relation to non-tumor samples was found. There was no statistically significant difference between the expression of these genes and microRNAs when compared to age at diagnosis and histological subtype. The EGFR gene showed higher expression in relation to the value of CEA diagnosis. The expression of microRNA-203 was progressively lower in relation to the TNM staging and was higher in the patient group in clinical remission. Conclusions: The therapy of colon and rectum tumors based on microRNAs remains under investigation reserving huge potential for future applications and clinical interventions in conjunction with existing therapies. We expect, based on the exposed data, to stimulate the development of new therapeutic possibilities, making the treatment of these tumors more effective.(AU)

AVALIAÇÃO DE FATORES ENVOLVIDOS NA FOLICULOGÊNESE DE BÚFALAS COM A UTILIZAÇÃO DE PRIMERS DELINEADOS PARA BOVINOS.

O conhecimento da fisiologia ovariana é importante durante a aplicação de biotécnicas assistidas de reprodução, tanto animal quanto humana. Durante a foliculogênese, os folículos apresentam aumento em número e diferenciação morfológica das células que circundam o oócito e, com a progressão do desenvolvimento, se tornam unidades funcionais no parênquima ovariano. Esses folículos podem ser considerados como pool de reserva e, na dependência do estímulo, podem permanecer quiescentes, sofrerem atresia ou chegar até a ovulação. Durante esse desenvolvimento, podemos ter a presença de fatores de crescimento produzidos no parênquima ovariano, que são modulados pela presença dos hormônios folículo estimulante (FSH) e luteinizante (LH). Além disso, o FSH e o LH também apresentam ação direta durante todo o curso da foliculogênese. Dentre os fatores de crescimento mais comuns, podemos citar o fibroblástico (FGF), insulínico (IGF), epidermal (EGF) e seus respectivos receptores que atuam na diferenciação e progressão do desenvolvimento folicular, desde as fases iniciais até a ovulação. Sendo assim, os objetivos do presente trabalho foram revisar a regulação parácrina dos fatores de crescimento durante a foliculogênese, atresia folicular, descrever e quantificar a expressão gênica desses fatores (FGF2, IGF1 e EGF), seus receptores (FSHR, FGFR2C e FGFR3C) e agentes envolvidos na apoptose (CASP3) presentes no ovário de búfalas vazias e gestantes. Em abatedouros, coletaram-se ovários de búfalas vazias (n=12) e gestantes (n=12), que se encontravam no terço final da gestação. Para a padronização das amostras, todas as búfalas vazias se encontravam cíclicas, com a presença de folículos em desenvolvimento e corpo lúteo evidente no parênquima. As amostras foram encaminhadas ao laboratório em tempo máximo de uma hora após a coleta. Todos os ovários foram dissecados, os folículos antrais aspirados e as amostras de tecido ovariano coletadas e armazenadas em nitrogênio líquido (-196°C) para posterior análise. No laboratório, foram utilizados primers delineados para bovinos na utilização do RT-qPCR na quantificação gênica das amostras. Após a padronização técnica, normalização das amostras e transformações dos valores em bases logarítmicas foi realizado a quantificação por RT-qPCR das amostras dos ovários de búfalas. Foram consideradas com diferença estatística, as amostras que apresentaram significância em 5% no pós-teste de Wilcoxon. Todos os primers foram expressos no RT-qPCR, entretanto, apenas o FSHR apresentou diferença significativa entre os grupos experimentais. O FSH atua na estimulação e regulação dos fatores parácrinos na regulação da foliculogênese, uma vez que houve variação da expressão de seu receptor (FSHR) entre os grupos experimentais. No grupo experimental de búfalas vazias, houve oscilação na expressão do FSHR decorrente das diferentes fases do ciclo estral que as fêmeas se encontravam, pois a modulação da secreção de esteroides pode afetar o desenvolvimento e progressão do desenvolvimento folicular. A presença do receptor FSHR no parênquima ovariano é um dos pontos iniciais para o início da foliculogênese e progressão do seu desenvolvimento, através da modulação de outros fatores de crescimento. Já nas búfalas gestantes, não houve essa variação, decorrente da atuação hormonal da progesterona. Sendo assim, conclui-se que houve expressão gênica dos fatores de crescimento testados estão presentes no tecido ovariano e são responsáveis pela modulação e progressão da foliculogênese em búfalas gestantes e vazias. Não foi possível visualizar a diferença na expressão do fator apoptótico pesquisado (CASP3) entre os grupos experimentais e, em adição, é viável a utilização de primers desenhados para bovinos na pesquisa de fatores de crescimento em ovário de búfalas.

Knowledge of ovarian physiology is important when applying assisted reproductive biotechniques, both animal and human. During folliculogenesis, the follicles show an increase in number and morphological differentiation of the cells that surround the oocyte and, with the progression of development, they become functional units in the ovarian parenchyma. These follicles can be considered as a reserve pool and, depending on the stimulus, can remain quiescent, suffer atresia or reach ovulation. During this development, we may have the presence of growth factors produced in the ovarian parenchyma, which are modulated by the presence of follicle-stimulating (FSH) and luteinizing (LH) hormones. Furthermore, FSH and LH also have a direct action throughout the course of folliculogenesis. Among the most common growth factors, we can mention fibroblastic (FGF), insulin (IGF), epidermal (EGF) and their respective receptors that act in the differentiation and progression of follicular development, from the initial stages to ovulation. Therefore, the objectives of this study were to review the paracrine regulation of growth factors during folliculogenesis, follicular atresia, describe and quantify the gene expression of these factors (FGF2, IGF1 and EGF), their receptors (FSHR, FGFR2C and FGFR3C) and agents involved in apoptosis (CASP3) present in the ovary of empty and pregnant buffaloes. In abattoirs, ovaries from empty buffaloes (n=12) and pregnant women (n=12), which were in the final third of gestation, were collected. For sample standardization, all empty buffaloes were cyclic, with the presence of developing follicles and corpus luteum evident in the parenchyma. The samples were sent to the laboratory within a maximum of one hour after collection. All ovaries were dissected, antral follicles aspirated and ovarian tissue samples collected and stored in liquid nitrogen (-196°C) for further analysis. In the laboratory, primers designed for cattle were used in the use of RT-qPCR for gene quantification of the samples. After technical standardization, normalization of samples and transformation of values into logarithmic bases, quantification by RT-qPCR of samples from buffalo ovaries was performed. The samples that presented significance in 5% in the Wilcoxon post-test were considered with statistical difference. All primers were expressed in RT-qPCR, however, only FSHR showed a significant difference between experimental groups. FSH acts in the stimulation and regulation of paracrine factors in the regulation of folliculogenesis, since there was variation in its receptor (FSHR) expression between the experimental groups. In the experimental group of empty buffaloes, there was an oscillation in the expression of the FSHR due to the different phases of the estrous cycle that the females were in, as the modulation of steroid secretion can affect the development and progression of follicular development. The presence of the FSHR receptor in the ovarian parenchyma is one of the starting points for the beginning of folliculogenesis and progression of its development, through the modulation of other growth factors. In pregnant buffaloes, however, there was no such variation, due to the hormonal action of progesterone. Thus, it is concluded that there was gene expression of the growth factors tested are present in ovarian tissue and are responsible for the modulation and progression of folliculogenesis in pregnant and empty buffaloes. It was not possible to visualize the difference in the expression of the investigated apoptotic factor (CASP3) between the experimental groups and, in addition, it is feasible to use primers designed for cattle in the research of growth factors in buffalo ovary.

Expression of epidermal growth factor (EGF) and its receptor in bovine endometrium throughout the luteal phase: effects of EGF on prostaglandin production in endometrial cells

Epidermal growth factor (EGF) is produced in bovine endometrium throughout the estrous cycle. However, little is known about the expression of the EGF receptor (EGFR) and the roles of EGF in bovine endometrium. To clarify whether EGF is involved in local regulation of bovine endometrial function, first we determined the EGF protein and the expression of EGFR mRNA in endometrial tissues throughout the luteal stages. EGF protein concentration was higher(P < 0.05) in the mid (days 8-12) luteal stage than in the other luteal stages. EGFR mRNA expression was higher (P < 0.05) in the mid and late (days 15-17) luteal stages than in the other luteal stages. To investigate the protein concentrations of EGF and EGFR mRNA expression in cultured bovine endometrial cells, epithelial and stromal cells were isolated between day 0 and day 4 post-ovulation from 22 uteri. Both EGF protein concentration and EGFR mRNA expression were higher (P < 0.05) in epithelial cells than in stromal cells. Then, to examine the possible role of EGF in the regulation of prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2), cultured endometrial epithelial and stromal cells were exposed to EGF (0, 1, 10 and 100 nm) for 24 h. In epithelial cells, EGF (10 and/or 100 nm) increased (P < 0.05) PGF2α and PGE2 secretion, but in stromal cells EGF (100 nm) increased (P < 0.05) PGF2α, but not PGE2 secretion. These results indicate that 1) the highest amount of EGF is produced by bovine endometrium at the mid-luteal stage, 2) endometrial EGFR mRNA expressions are higher at mid and late-luteal stages than other stages, 3) EGF is expressed mainly by uterine epithelial cells and 4) EGF has the ability to increase PGE2 and PGF2α production in both epithelial and stromal cells and therefore may play a role in local regulation of uterine function.

Expression of epidermal growth factor (EGF) and its receptor in bovine endometrium throughout the luteal phase: effects of EGF on prostaglandin production in endometrial cells

Epidermal growth factor (EGF) is produced in bovine endometrium throughout the estrous cycle. However, little is known about the expression of the EGF receptor (EGFR) and the roles of EGF in bovine endometrium. To clarify whether EGF is involved in local regulation of bovine endometrial function, first we determined the EGF protein and the expression of EGFR mRNA in endometrial tissues throughout the luteal stages. EGF protein concentration was higher(P < 0.05) in the mid (days 8-12) luteal stage than in the other luteal stages. EGFR mRNA expression was higher (P < 0.05) in the mid and late (days 15-17) luteal stages than in the other luteal stages. To investigate the protein concentrations of EGF and EGFR mRNA expression in cultured bovine endometrial cells, epithelial and stromal cells were isolated between day 0 and day 4 post-ovulation from 22 uteri. Both EGF protein concentration and EGFR mRNA expression were higher (P < 0.05) in epithelial cells than in stromal cells. Then, to examine the possible role of EGF in the regulation of prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2), cultured endometrial epithelial and stromal cells were exposed to EGF (0, 1, 10 and 100 nm) for 24 h. In epithelial cells, EGF (10 and/or 100 nm) increased (P < 0.05) PGF2α and PGE2 secretion, but in stromal cells EGF (100 nm) increased (P < 0.05) PGF2α, but not PGE2 secretion. These results indicate that 1) the highest amount of EGF is produced by bovine endometrium at the mid-luteal stage, 2) endometrial EGFR mRNA expressions are higher at mid and late-luteal stages than other stages, 3) EGF is expressed mainly by uterine epithelial cells and 4) EGF has the ability to increase PGE2 and PGF2α production in both epithelial and stromal cells and therefore may play a role in local regulation of uterine function.(AU)

Analysis of EGFR and HER-2 expressions in ductal carcinomas in situ in canine mammary glands / Análise da expressão de EGFR e HER-2 em carcinomas ductais in situ da glândula mamária canina

Biomolecular evidence has shown that ductal carcinoma in situ (DCIS) may develop into invasive carcinoma of the canine mammary gland, and mutations in proto-oncogenes HER2 and EGFR; two members of the family of epidermal growth factor receptors, may be involved in this process. The purpose of this study was the characterization of the immunohistochemical expression of the EGFR and HER2 proteins in the process of neoplastic transformation, supposedly present in ductal carcinomas in situ in canine mammary glands. Fifteen cases of DCIS were evaluated, with a higher expression of HER2 and EGFR being observed in low-grade carcinomas when compared with high-grade neoplasms, and with a high positive statistical correlation in the latter. Results suggest that aggressive tumors tend to lose the expression of EGFR and HER2 simultaneously. The loss of the expression of these markers may be related to the process of neoplastic progression in canine mammary tumors.(AU)

Evidências biomoleculares sugerem que o carcinoma ductal in situ (CDIS) pode progredir para carcinoma invasor na mama canina e que mutações nos proto-oncogenes HER-2 e EGFR, dois membros da família de receptores para fatores de crescimento epidérmicos, podem estar envolvidas neste processo. A partir disso, este trabalho teve por objetivo caracterizar a expressão imuno-histoquímica das proteínas EGFR e HER-2 no processo de transformação neoplásica supostamente presente em carcinomas ductais in situ da glândula mamária canina. Foram avaliados 15 casos de CDIS, sendo observada maior expressão de HER-2 e EGFR em carcinomas de baixo grau em comparação às neoplasias de alto grau, com correlação estatística positiva alta nestes últimos. Os resultados sugerem que tumores mais agressivos tendem a perder, simultaneamente, a expressão de EGFR e HER-2. A perda na expressão desses marcadores pode estar envolvida no processo de progressão neoplásica em tumores mamários caninos.(AU)

EFEITOS DE ALTAS DILUIÇÕES DE PHYTOLACCA DECANDRA NA VIABILIDADE E EXPRESSÃO DO FATOR HER-2 EM ADENOCARCINOMA MAMÁRIO MURINO (4T1) IN VITRO

Em estudo anterior, experimental, in vivo, observou-se que o tratamento de camundongos inoculados com células 4T1 com Phytolacca decandra em diferentes diluições homeopáticas apresentavam mudanças no crescimento tumoral e na expressão de HER-2. HER-2 (receptor de fator de crescimento epidermal, com atividade tirosina-quinase) é super-expressa em cerca de 30% dos carcinomas ductais invasivos humanos e, por isso, é alvo de tratamentos específicos. O objetivo desse estudo foi avaliar o efeito de diferentes diluições homeopáticas de Phytolacca decandra, preparadas segundo a Farmacopéia Homeopática Brasileira, na apoptose, morfologia e expressão de HER-2 em células 4T1 in vitro. Células 4T1 tratadas no momento do cultivo foram analisadas após 24 horas quanto à positividade para anexina V pelo método Countess® para a avaliação da apoptose. A expressão de HER-2 foi avaliada pelo método da imuno-citoquímica e a morfologia das células foi avaliada pelas colorações hematoxilina-eosina e Giemsa, a partir de esfregaços obtidos das células em suspensão. Células tratadas com Phytolacca decandra 200cH mostraram aumento no número de células positivas à anexina V (células em apoptose) e ocorrência de células bi ou multinucleadas. A expressão de HER-2 na superfície celular foi irregular nessas células, bem como nas células tratadas com a diluição 12cH, sugerindo infrarregulação desse receptor. Em suma, os resultados mostram alterações fenotípicas significativas em células 4T1 tratadas com Phytolacca decandra 200cH, as quais merecem estudos mais aprofundados.

In a previous study, experimental, the treatment of mice inoculated with 4T1 cells with Phytolacca decandra at different homeopathic dilutions produced changes in tumor growth and expression of HER-2. HER-2 protein (epidermal growth factor receptor, with tyrosine kinase activity) is superexpressed in about 30% of human invasive ductal carcinomas and, therefore, is targeted by specific treatments. The objective of this study was to evaluate the effect of different homeopathic dilutions of Phytolacca decandra, according to the Brazilian Homeopathic Pharmacopea, on apoptosis, morphology and expression of HER-2 in 4T1 cells in vitro. Treated 4T1 cells were analyzed after 24 hours of culture, for positivity to annexin V, using the Countess® method, for apoptosis evaluation. HER-2 expression was assessed by the immuno-cytochemistry method and cell morphology was evaluated by hematoxylin-eosin and Giemsa staining, in smears obtained from suspended cells. Cells treated with Phytolacca decandra 200cH showed increase in the number of annexin V positive cells (apoptosis cells) and occurrence of bi or multinucleated cells. HER-2 expression on the membrane was irregular in these cells, as well as in those treated with the 12cH dilution, suggesting downregulation of the receptor. In short, the results show significant phenotypic changes in 4T1 cells treated with Phytolacca decandra 200cH, which deserve further studies.