Effects of ß-glucan from S. cerevisiae on the expression of Casp9, Ccna2 and Sod1 genes in MCF-7 cells

ß-Glucans (ßG) are polysaccharides widely distributed in nature with chemopreventive properties. The aim of this study was to investigate the effects of ßG and the combined treatment with doxorubicin (Dox) on cell viability and mRNA levels of genes involved in cell cycle, apoptosis and antioxidant response. ßG was not cytotoxic. The mRNA levels of CCNA2of cells exposed to ß-glucan was upregulated and the exposure to Dox decreased the expression, while the combination led to an upregulation. Modulation of mRNA levels of CASP9suggest that ßG could inhibit promotion and progression steps of carcinogenesis, eliminatingneoplastic cells. The upregulation of CCNA2gene in combined treatment could be occurred due to ability of ßG in restoring the cell cycle distribution pattern after treatment with Dox. The upregulation of SOD1suggests that ßG can enhance the intracellular antioxidant defense, reducing the levels of superoxide dismutase induced by Dox. This response could reduce oxidative damage and attenuate tissue damage during chemotherapeutic treatment. Our data suggest that the drug combination may be less effective in killing tumor cells than the treatment with Dox alone. Thus, future studies should carefully consider this effect on indication of ßG during chemotherapy.Keywords:caspase-9; cyclin A2; superoxide dismutase 1; cell cycle; antioxidant.Received on July 2, 2020.Accepted on February 7, 2022.IntroductionGlucans are polysaccharides widely distributed in nature and oftenstudied due to chemopreventive properties. They are constituent of the cell wall of plants (oats and barley), algae, bacteria and fungi. ß-glucans (ßG)have a common structure comprising a main chain of ß-(1,3) and/or ß-(1,4) D-glucopyranosyl unit and they differ in length and branching structures. ßG of Saccharomyces cerevisiaehave 1→6 side branches while those of bacteria have 1→4 side branches (Chan, Chan, & Sze, 2009). ßGcan prevent DNA damage induced by chemical and physical agents (Ghavami,Goliaei, Taghizadeh, & Nikoofar, 2014). Some authors showed its significant efficacy in preventing mutagenic effects caused by doxorubicin, cyclophosphamide and cisplatin (Tohamy, El-Ghor, El-Nahas, & Noshy, 2003), methyl methanesulfonate (Oliveira et al., 2007)and hydrogen peroxide (Slamenová, 2003). Moreover, some studies have related the antioxidant ability of ßGagainst reactive free radicals formed by endogenous metabolic processes or exogenous chemicals (Tsiapali et al., 2001; Slamenová,2003; Sener, Eksioglu-Demiralp, Cetiner, Ercan, & Yegen, 2006; Guerra Dore et al., 2007; Kofuji et al., 2012; Lei et al., 2015). Yeast-derived ßGhave modulating action of humoral and cellular immune responses (Vetvicka et al., 2007).This activity provides protection to the organism against infections and cancer development (Samuelsen, Schrezenmeir, & Knutsen, 2014; Roudbary, Daneshmand, Hajimorad, Roudbarmohammadip, & Hassan, 2015). Despite postulated modes of action by which ß-glucan works are lacking information about the molecular mechanisms involved in the chemopreventive activity of this polysaccharide. In addition, compounds with chemopreventive properties can contribute to reduce side effects and toxicity during the chemotherapeutic treatment. Therefore, the aim of this study was to investigate the effects of ßG and the combined treatment with doxorubicin (Dox) on the expression of genes related with apoptosis (CASP9), cell cycle control (CCNA2)and antioxidant defense (SOD1)in human breast cancer MCF-7 cells. Doxorubicin (Dox) was chosen because it is one of the most used chemotherapeutic agent for cancer treatment. The limitation on the use of Dox in cancer treatment is the lack of selectivity against cancer cells and, consequently, its toxicity to patients.(AU)

Achados clínicos, histopatológicos e moleculares da mielopatia degenerativa canina: relato de caso / Clinical, histopathological and molecular findings of canine degenerative myelopathy: case report

Objetivou-se descrever os achados clínicos, histopatológicos e moleculares associados à MDC em um cão da raça Pastor-Suiço. O cão possuía uma paraparesia progressiva em membros pélvicos e foi submetido a avaliações clínicas, pelas quais se obteve, entre outros diferenciais, o diagnóstico presuntivo de MDC. Com a evolução dos sinais, o tutor optou pela eutanásia. Os achados histopatológicos da medula espinhal foram compatíveis com uma degeneração segmentar axonal e mielínica. O diagnóstico molecular foi realizado por meio da extração do DNA obtido por swab oral. Uma PCR foi otimizada utilizando-se primers descritos em literatura para amplificar a região do gene SOD1. A amostra foi, então, submetida a sequenciamento unidirecional, que revelou que o animal em questão era homozigoto para o alelo A para a mutação c.118G>A no éxon 2 do gene SOD1. O diagnóstico clínico presuntivo da MDC no presente caso foi esclarecido por meio dos achados histopatológicos, associados aos achados clínicos, e da sua caracterização molecular. Ressalta-se a contribuição deste relato, que traz aspectos clínicos, histopatológicos e moleculares associados à MDC na raça Pastor-Suíço, para a qual, até o presente momento, na literatura consultada, não há relato dessa enfermidade.(AU)

The objective of this study was to describe the clinical, histopathological and molecular findings associated with MDC in a Swiss Shepherd dog. The dog had a progressive paraparesis in pelvic limbs and was submitted to clinical evaluations where, among other differentials, the presumptive diagnosis of MDC was obtained. With the progression of the nervous deficits tutor opted for euthanasia. The histopathological findings of the spinal cord were compatible with axonal and myelinic segmental degeneration. Molecular diagnosis was performed by extracting the DNA obtained by oral swab. PCR was optimized using primers described in the literature to amplify the SOD1 gene region. The sample was then subjected to one-way sequencing which revealed that the animal in question was homozygous for the A allele for the c.118G>A mutation in exon 2 of the SOD1 gene. The presumptive diagnosis of MDC in the present case was clarified by histopathological findings, as well as by its molecular characterization. The contribution of this report brings clinical, histopathological and molecular aspects associated with canine degenerative myelopathy in the Swiss Shepherd breed, that until this moment, in the literature consulted, there is no report of this disease in the breed mentioned.(AU)

Achados clínicos, histopatológicos e moleculares da mielopatia degenerativa canina: relato de caso / Clinical, histopathological and molecular findings of canine degenerative myelopathy: case report

Objetivou-se descrever os achados clínicos, histopatológicos e moleculares associados à MDC em um cão da raça Pastor-Suiço. O cão possuía uma paraparesia progressiva em membros pélvicos e foi submetido a avaliações clínicas, pelas quais se obteve, entre outros diferenciais, o diagnóstico presuntivo de MDC. Com a evolução dos sinais, o tutor optou pela eutanásia. Os achados histopatológicos da medula espinhal foram compatíveis com uma degeneração segmentar axonal e mielínica. O diagnóstico molecular foi realizado por meio da extração do DNA obtido por swab oral. Uma PCR foi otimizada utilizando-se primers descritos em literatura para amplificar a região do gene SOD1. A amostra foi, então, submetida a sequenciamento unidirecional, que revelou que o animal em questão era homozigoto para o alelo A para a mutação c.118G>A no éxon 2 do gene SOD1. O diagnóstico clínico presuntivo da MDC no presente caso foi esclarecido por meio dos achados histopatológicos, associados aos achados clínicos, e da sua caracterização molecular. Ressalta-se a contribuição deste relato, que traz aspectos clínicos, histopatológicos e moleculares associados à MDC na raça Pastor-Suíço, para a qual, até o presente momento, na literatura consultada, não há relato dessa enfermidade.(AU)

The objective of this study was to describe the clinical, histopathological and molecular findings associated with MDC in a Swiss Shepherd dog. The dog had a progressive paraparesis in pelvic limbs and was submitted to clinical evaluations where, among other differentials, the presumptive diagnosis of MDC was obtained. With the progression of the nervous deficits tutor opted for euthanasia. The histopathological findings of the spinal cord were compatible with axonal and myelinic segmental degeneration. Molecular diagnosis was performed by extracting the DNA obtained by oral swab. PCR was optimized using primers described in the literature to amplify the SOD1 gene region. The sample was then subjected to one-way sequencing which revealed that the animal in question was homozygous for the A allele for the c.118G>A mutation in exon 2 of the SOD1 gene. The presumptive diagnosis of MDC in the present case was clarified by histopathological findings, as well as by its molecular characterization. The contribution of this report brings clinical, histopathological and molecular aspects associated with canine degenerative myelopathy in the Swiss Shepherd breed, that until this moment, in the literature consulted, there is no report of this disease in the breed mentioned.(AU)

Investigação da mutação no gene da superóxido dismutase 1 (SOD1: C.118 G>A) associado a mielopatia degenerativa canina na raça Pastor Alemão

A mielopatia degenerativa canina (MDC) é uma enfermidade progressiva e ascendente da medula espinal que ao longo dos anos vem sendo descrita em várias raças. Os cães desenvolvem uma paraparesia/plegia progressiva, e com a piora dos sinais clínicos aliado a falta de tratamento para a cura, muitos tutores optam pela eutanásia. Os componentes genéticos que envolvem a enfermidade são cada vez mais estudados tendo em vista sua similaridade com a esclerose lateral amiotrófica (ELA), e trata-se de uma doença genética autossômica recessiva associada a mutações no gene SOD1. O diagnóstico definitivo dá-se apenas no post mortem por meio dos achados histopatológicos na medula espinal que revelam uma axonopatia caracterizada por degeneração axonal e mielínica. Objetivou-se genotipar cães da raça Pastor Alemão para SOD1: c.118G> A a fim de conhecer como a mutação está distribuída nesta raça no Brasil. Por meio da reação em cdeia da polimerase-Polimorfismo do comprimento do fragmento de restrição (PCR-RFLP), 95 cães foram genotipados. Destes 23 foram identificados como heterozigotos AG e 72 homozigotos GG. A frequência observada do alelo A (associado à MDC) foi 12,1053%. Descreve-se também os achados clínicos, histopatológicos e moleculares associados à MDC em um cão da raça Pastor Suiço, o qual foi utilizado para a validação do teste. Nesse cão, os achados histopatológicos da medula espinal foram compatíveis com uma degeneração segmentar axonal e mielínica. Uma PCR foi otimizada e a amostra foi então submetida ao sequenciamento unidirecional que revelou que o animal em questão era homozigoto para o alelo A associado a mutação c.118G>A no exon 2 do gene SOD1. O diagnóstico presuntivo da MDC no presente caso foi esclarecido por meio dos achados histopatológicos, e também pela sua caracterização molecular, esta ultima ainda é pouco explorada em descrições da doença no Brasil. A investigação da mutação neste estudo permitiu o conhecimento sobre a presença do alelo A (SOD1:c.118G >A), em uma população de cães da raça Pastor Alemão no Brasil. Estes resultados têm valor para documentação, do ponto de vista epidemiológico, das frequências deste alelo na referida raça.

Canine degenerative myelopathy (MDC) is an ascending progressive disease of the spinal cord, which, through the years, has been described in various breeds. Dogs develop progressive paraparesis/plegia, and with evolution of clinical signs, several owners opt for euthanasia. Genetic components for this disease have been investigated due to its similarity with amyotrophic lateral sclerosis (ELA). Its a recessive autosomal disease caused by mutations in the SOD1 gene. A definitive diagnosis is obtained only on post mortem via histopathology findings for the spinal cord which show an axonopathy characterized by axonal and myelinic degeneration. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for genotyping dogs from German Shepherd breed to SOD1: c.118G> A, to see how the mutation in distributed in Brazil. Of the 95 dogs that underwent genotyping, 23 were identified as heterozygotes AG and 72 homozygotes GG. The frequency for the A allele (associated with MDC) was 12.1053%. The clinical, histopathologic, and molecular findings were associated with MDC in a Swiss Shepherd dog. Histopathology findings for the spinal cord were compatible with segmental axonal and myelinic degeneration. The molecular diagnosis was done via extraction of the DNA obtained from an oral swab. The PCR was optimized and the sample sent for unidirectional sequencing which revealed the animal in question was a homozygote for the A allele for the c.118G>A mutation on exon 2 of the SOD1 gene. A presumptive diagnosis of MDC in the present case was clarified via histopathology findings, and also due to its molecular characteristics. The research carried out allowed the identification of the distribution of the "A" allele (SOD1: c.118G> A) in a population of German Shepherd dogs in Brazil. These results have value for documentation, from the epidemiological point of view, of the frequencies of this allele in breed.