Efeito terapêutico do parecoxib: um estudo in vivo em Mus musculus
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2019-04-12
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As infeções pelo papilomavírus humano, como o HPV16, estão associadas ao desenvolvimento de vários cancros anogenitais como cancro do colo do útero, ânus, pénis, vagina e vulva. O cancro do colo do útero é o quarto tipo de cancro com maior incidência nas mulheres em todo o mundo. A ciclooxigenase-2 (COX-2) encontra-se sobreexpressa no cancro do colo do útero e está envolvida não só na génese da inflamação como também está associada à progressão maligna dos tumores. O parecoxib, pró-fármaco do valdecoxib, enquadra-se no grupo dos fármacos anti-inflamatórios não esteróides inibidores seletivos da COX-2. Este estudo teve como objetivo avaliar os efeitos terapêuticos do parecoxib num modelo animal transgénico do HPV16, que desenvolve lesões cutâneas semelhantes às causadas pelo HPV no cancro do colo do útero da mulher, com recurso à análise de parâmetros fisiológicos, histológicos, bioquímicos e genotóxicos. Utilizaram-se 43 murganhos fêmeas, com 20 semanas de idade, da estirpe FVB/n, sendo 21 wild-type e 22 transgénicas. O grupo I (10 animais HPV-/- ) foi constituído por animais tratados com parecoxib, o grupo II (11 animais HPV-/- ) foram usados como controlo, o grupo III foi tratado com parecoxib (11 animais HPV+/- ) e o grupo IV (11 animais HPV+/- ) constituído também como controlo. O parecoxib foi administrado por via intraperitoneal, na dose de 5 mg/kg, uma vez por dia, ao longo de 22 dias consecutivos. Os animais foram mantidos sobre condições de luz, temperatura e humidade controladas e alimentados em regime ad libitum. Ao longo do ensaio foram registados semanalmente o consumo de comida e de água, as massas corporais e avaliado o bem-estar de cada murganho. No dia seguinte à última administração do parecoxib, os animais foram eutanasiádos pela administração de uma overdose da associação de xilazina com ketamina e realizada uma necrópsia completa. Os órgãos dos animais foram recolhidos, registada a sua massa e fixados em formaldeído para posterior análise histológica. O sangue foi recolhido por punção cardíaca direta para análise bioquímica, para o ensaio do cometa e para o teste de micronúcleos. Em relação aos parâmetros fisiológicos foram observadas, de forma global, menor massa relativa do fígado e do baço do grupo III (HPV+/- ) tratado com parecoxib em relação ao grupo IV (HPV+/- ) controlo. A histologia demonstrou uma menor incidência de displasias da epiderme na pele e na orelha dos animais transgénicos (grupo III) tratados com parecoxib em relação ao seu grupo controlo (grupo IV). Os parâmetros bioquímicos não demonstraram qualquer diferença entre os vários grupos experimentais. O ensaio do cometa, realizado no sangue demonstrou ter efeitos positivos no grupo transgénico tratado com parecoxib (grupo III), em contrapartida observámos resultados opostos nos hepatócitos deste grupo. O teste de micronúcleos demonstrou haver uma menor instabilidade cromossómica nos eritrócitos do grupo III (HPV+/- ) em relação ao grupo IV (HPV+/- ). Em suma, o parecoxib teve efeitos benéficos no tratamento das lesões induzidas pelo HPV16 e não provocou alterações sistémicas bioquímicas. Através da análise do teste de micronúcleos o fármaco revelou não ser genotóxico para os animais sugerindo a hipótese que os danos detetados pelo ensaio do cometa sejam corrigidos pelos mecanismos de reparação de DNA.
The infections by human papillomavirus, such as HPV16, are associated with the development of several anogenital cancers, such as cervical cancer, anal cancer, penile cancer, vaginal cancer and vulvar cancer. Cervical cancer has the fourth highest incidence in women worldwide. Cycloxigenase-2 (COX2) is overexpressed in cervical cancer and it is involved not only on inflammation, but also in the progression of the malignant tumors. Parecoxib, which is a prodrug of valdecoxib, is a nonsteroidal anti-inflammatory drug and selective inhibitor of COX-2. The aim of this study is to evaluate the therapeutic effects of parecoxib in a transgenic animal model of HPV16-induced cancer, which develops skin lesions similar to the ones caused by HPV in cervical cancer, by analysing several physiological, histological, biochemical and genotoxic parameters. We have used 43 female mice of the FVB/n strain, by the age of 20 weeks, which 21 were wild-type and 22 transgenic. The group I (10 mice HPV-/- ) was treated with parecoxib; the group II (11 mice HPV-/- ) was used as experimental control group; the group III was treated with parecoxib (11 mice HPV+/- ) and the group IV (11 mice HPV+/- ) was used as control. Parecoxib was administered via intraperitoneal injection, at 5 mg/kg daily once, for 22 consecutive days. The animals were kept under controlled conditions of light, temperature and humidity and fed ad libitum. During this assay, the consumption of food and water, body weight and the welfare of each mice were registered weekly. The day after of last administration of parecoxib, the mice were euthanized with of xylazine and ketamine and a complete necropsy was performed. The organs were collected and their weight was recorded, being then fixed with formaldehyde for posterior histologic analysis. The blood was extracted through direct cardiac puncture for biochemical analysis, comet and micronucleus assay. Lower liver and spleen weights were observed in group III (HPV+/- ) treated with parecoxib compared with the control group IV (HPV+/- ). Histological analysis demonstrated a lower incidence of epidermal dysplasia on chest and ears skin of parecoxib-treated transgenic animals (group III), compared with to control group (group IV). Biochemical parameters did not demonstrated any difference between the experimental groups. The comet assays using white blood cells and showed conflicting results. The micronucleus test showed reduced to chromosomal instability in erythrocytes of group III (parecoxib-treated HPV+/- mice) compared with matched untreated controls from group IV. To conclude, parecoxib showed beneficial effects on treatment of HPV16-induced lesions and did not provoke systemic biochemical alterations. The micronucleus test indicates that the drug is not be genotoxic to mice, suggesting the hypothesis that the damage detected on the comet assay may be corrected by DNA repairing mechanisms.
The infections by human papillomavirus, such as HPV16, are associated with the development of several anogenital cancers, such as cervical cancer, anal cancer, penile cancer, vaginal cancer and vulvar cancer. Cervical cancer has the fourth highest incidence in women worldwide. Cycloxigenase-2 (COX2) is overexpressed in cervical cancer and it is involved not only on inflammation, but also in the progression of the malignant tumors. Parecoxib, which is a prodrug of valdecoxib, is a nonsteroidal anti-inflammatory drug and selective inhibitor of COX-2. The aim of this study is to evaluate the therapeutic effects of parecoxib in a transgenic animal model of HPV16-induced cancer, which develops skin lesions similar to the ones caused by HPV in cervical cancer, by analysing several physiological, histological, biochemical and genotoxic parameters. We have used 43 female mice of the FVB/n strain, by the age of 20 weeks, which 21 were wild-type and 22 transgenic. The group I (10 mice HPV-/- ) was treated with parecoxib; the group II (11 mice HPV-/- ) was used as experimental control group; the group III was treated with parecoxib (11 mice HPV+/- ) and the group IV (11 mice HPV+/- ) was used as control. Parecoxib was administered via intraperitoneal injection, at 5 mg/kg daily once, for 22 consecutive days. The animals were kept under controlled conditions of light, temperature and humidity and fed ad libitum. During this assay, the consumption of food and water, body weight and the welfare of each mice were registered weekly. The day after of last administration of parecoxib, the mice were euthanized with of xylazine and ketamine and a complete necropsy was performed. The organs were collected and their weight was recorded, being then fixed with formaldehyde for posterior histologic analysis. The blood was extracted through direct cardiac puncture for biochemical analysis, comet and micronucleus assay. Lower liver and spleen weights were observed in group III (HPV+/- ) treated with parecoxib compared with the control group IV (HPV+/- ). Histological analysis demonstrated a lower incidence of epidermal dysplasia on chest and ears skin of parecoxib-treated transgenic animals (group III), compared with to control group (group IV). Biochemical parameters did not demonstrated any difference between the experimental groups. The comet assays using white blood cells and showed conflicting results. The micronucleus test showed reduced to chromosomal instability in erythrocytes of group III (parecoxib-treated HPV+/- mice) compared with matched untreated controls from group IV. To conclude, parecoxib showed beneficial effects on treatment of HPV16-induced lesions and did not provoke systemic biochemical alterations. The micronucleus test indicates that the drug is not be genotoxic to mice, suggesting the hypothesis that the damage detected on the comet assay may be corrected by DNA repairing mechanisms.
Descrição
Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde
Palavras-chave
HPV16, K14-HPV16 , Ensaio do Cometa , Micronúcleos , AINES , Murganho