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Authors
Abstract(s)
O cancro continua a estar no topo das principais causas de morte em todo o mundo,
tendo ocorrido em 2020 aproximadamente 10 milhões de mortes devido a essa patologia.
Entre os cancros que mais afetam as mulheres em todo o mundo, o cancro do colo do
útero (CCU) é considerado o terceiro mais devastador, tendo provocado 341 831 mortes
em 2020. A infeção pelo vírus do papiloma humano (HPV) está intimamente associada
ao aparecimento de CCU, sendo por isso considerada como principal fator deste tipo de
carcinogénese, nomeadamente a infeção pelos HPV de alto risco. As infeções que podem
causar cancro, como é o caso das que são provocadas pelo HPV, são responsáveis por
cerca de 30% dos casos de cancro em países subdesenvolvidos. As oncoproteínas E6 e E7
produzidas pelos HPV de alto risco, ligam-se às proteínas supressoras de tumor p53 e
proteína do retinoblastoma (pRb), respetivamente, induzindo a sua degradação e
impedindo assim as funções de indução de apoptose e de controlo do ciclo celular
realizadas por estas proteínas supressoras de tumor. Estes dois processos acabam por
culminar na transformação de células normais em malignas e na imortalização destas
últimas. Tendo em consideração que as oncoproteínas E6 e E7 são continuamente
expressas em células cancerígenas infetadas por HPV, estas tornam-se alvos terapêuticos
interessantes para serem inibidos/bloqueados.
Embora já existam vacinas profiláticas contra a infeção por HPV (Gardasil ®, Cervarix
® e Gardasil ®-9), o seu caráter é exclusivamente preventivo, não existindo qualquer
efeito terapêutico em pessoas já infetadas. Tendo em consideração a falta de métodos
terapêuticos adequados e o facto dos agentes quimioterapêuticos existentes serem
inespecíficos e originarem efeitos secundários indesejados, torna-se fundamental
realizar estudos que se foquem no desenvolvimento de novos agentes anticancerígenos.
Recentemente, as moléculas esteroides têm sido amplamente estudadas no
desenvolvimento de novos agentes anticancerígenos, incluindo contra carcinomas
cervicais. Adicionalmente, estas moléculas apresentam características que as tornam
extremamente atrativas para esta área, nomeadamente elevada ação terapêutica e boa
biodisponibilidade. Assim, o presente trabalho tem como objetivo estudar pequenas
moléculas esteroides das seguintes famílias: 4-azaandrostenos (4a-4g), 4-azapregnenos
(7a-7g), derivados arilideno desidroepiandrosterona (DHEA) (8a-8m), epóxidos DHEA
(9a-9m) e trióis e trionas DHEA (10a-10f e 11a-11f), no sentido de verificar se podem ser
potenciais inibidores da oncoproteina E6 do HPV, impedindo a sua interação com o
supressor de tumor p53 nas células do CCU (HeLa HPV18 positivas e CaSki HPV16
positivas), sem afetar a viabilidade das células saudáveis, os fibroblastos normais da derme humana (NHDF). Para isso, realizaram-se primeiramente estudos de docking
molecular e estudos de previsão das propriedades de absorção, distribuição,
metabolismo, excreção e toxicidade (ADMET), para selecionar os compostos com maior
potencial para as avaliações biológicas in vitro. Realizou-se posteriormente o Thermal
Shift Assay (TSA), um ensaio que permite avaliar a estabilidade térmica da proteína E6
(de natureza recombinante) na presença e ausência dos compostos selecionados nos
estudos in silico, de forma a selecionar os que apresentam maior potencial de serem
testados em ensaios usando linhas celulares. Posteriormente, efetuaram-se estudos para
avaliar a viabilidade celular, pelo ensaio de brometo de 3-(4,5-dimetiltiazol-2-il) -2,5-
difeniltetrazólio (MTT), e determinar a metade da concentração inibitória máxima (IC50)
em células saudáveis (NHDF) e cancerígenas (HeLa HPV18, CaSki HPV16). Os
resultados mais promissores foram obtidos para os compostos 8h, 10c e 11c que
provocaram uma redução mais evidente da viabilidade das células cancerígenas. Estes
compostos esteroides reduziram especificamente a viabilidade em células HPV positivas
(IC50 38,63 µM (8h), 39,92 µM (10c), 22,01 µM (11c) para células HeLa e 34,30 µM (8h),
38,75 µM (10c) e 26,84 µM (11c) para células CaSki). Além disso, os referidos compostos
apresentam citotoxicidade claramente inferior nos NHDF.
Desta forma, os resultados obtidos indicam que os compostos 8h, 10c e 11c poderão vir a
ser considerados potenciais inibidores da oncoproteína E6 do HPV, representando um
possível avanço para terapia personalizada do CCU. No entanto, mais estudos têm de ser
realizados para corroborar os resultados já obtidos, nomeadamente a realização de
estudos de viabilidade em células HPV negativas, com o intuito de verificar se a
seletividade destes compostos se mantém apenas para células HPV positivais.
Cancer continues to be one of the leading causes of death worldwide, with approximately 10 million deaths from the disease in 2020. Among the cancers that most affect women worldwide, cervical cancer (CC) is considered the third most devastating, having caused 341 831 deaths in 2020. Human papilloma virus (HPV) infection is closely associated with the onset of CC and is therefore considered to be the main factor in this type of carcinogenesis, namely infection with high-risk HPV. Infections that can cause cancer, such as those caused by HPV, are responsible for around 30% of cancer cases in underdeveloped countries. The E6 and E7 oncoproteins produced by high-risk HPV bind to the tumor suppressor proteins p53 and retinoblastoma protein (pRb), respectively, inducing their degradation and thus preventing apoptosis-inducing and cell cyclecontrolling functions performed by these tumor suppressor proteins. These two processes eventually culminate in the transformation of normal cells into malignant ones and the immortalization of the latter. Considering that the E6 and E7 oncoproteins are continuously expressed in HPV infected cancer cells, they become interesting therapeutic targets to be inhibited/blocked. Although prophylactic vaccines against HPV infection already exist (Gardasil ®, Cervarix ® and Gardasil ®-9), their character is solely and exclusively preventive, with no therapeutic effect in people already infected. In view of the lack of appropriate therapeutic methods and the fact that existing chemotherapeutic agents are non-specific and give rise to unwanted side effects, it is essential to carry out studies focusing on the development of new anticancer agents. Steroid molecules have been widely studied in the development of new anticancer agents, including against cervical carcinomas. In addition, steroid molecules have characteristics that make them extremely attractive for this area, namely high therapeutic action and good bioavailability. Thus, the present work aims to study small steroid molecules from the following families: 4- azaandrostenes (4a-4g), 4-azapregnenes (7a-7g), arylidene dehydroepiandrosterone (DHEA) derivatives (8a-8m), DHEA epoxides (9a-9m) and DHEA triols and threonines (10a-10f and 11a-11f), in order to see if they could be potential inhibitors of HPV oncoprotein E6, preventing its interaction with the tumor suppressor p53 in CC cells (HeLa HPV18 positive and CaSki HPV16 positive), without affecting the viability of healthy cells, normal human dermal fibroblasts (NHDF). To this end, molecular docking studies and absorption, distribution, metabolism, excretion and toxicity (ADMET) prediction studies were first performed to select the compounds with the highest potential for in vitro biological evaluations. Subsequently, the Thermal Shift Assay (TSA), an assay that allows the evaluation of the thermal stability of the E6 protein (recombinant nature) in the presence and absence of the compounds selected in the in silico studies, was performed in order to select those with the greatest potential to be tested in assays using cell lines. To this end, studies were carried out to assess cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and to determine the half maximum inhibitory concentration (IC50) in nontumoral (NHDF) and tumoral cells (HeLa HPV18, CaSki HPV16). The most promising results were obtained for compounds 8h, 10c and 11c, which caused a more evident reduction in cancer cells viability. These steroid compounds specifically reduced viability in HPV positive cells (IC50 38.63 µM (8h), 39.92 µM (10c), 22.01 µM (11c) for HeLa cells and 34.30 µM (8h), 38.75 µM (10c) and 26.84 µM (11c) for CaSki cells). In addition, these compounds showed clearly lower cytotoxicity in NHDF. Thus, the results indicate that compounds 8h, 10c and 11c could be potential inhibitors of HPV oncoprotein E6, representing a possible advance for personalized therapy of CC. However, further studies need to be performed to corroborate the results already obtained, namely viability studies on HPV negative cells, in order to verify if the selectivity of these compounds is maintained only for HPV positive cells.
Cancer continues to be one of the leading causes of death worldwide, with approximately 10 million deaths from the disease in 2020. Among the cancers that most affect women worldwide, cervical cancer (CC) is considered the third most devastating, having caused 341 831 deaths in 2020. Human papilloma virus (HPV) infection is closely associated with the onset of CC and is therefore considered to be the main factor in this type of carcinogenesis, namely infection with high-risk HPV. Infections that can cause cancer, such as those caused by HPV, are responsible for around 30% of cancer cases in underdeveloped countries. The E6 and E7 oncoproteins produced by high-risk HPV bind to the tumor suppressor proteins p53 and retinoblastoma protein (pRb), respectively, inducing their degradation and thus preventing apoptosis-inducing and cell cyclecontrolling functions performed by these tumor suppressor proteins. These two processes eventually culminate in the transformation of normal cells into malignant ones and the immortalization of the latter. Considering that the E6 and E7 oncoproteins are continuously expressed in HPV infected cancer cells, they become interesting therapeutic targets to be inhibited/blocked. Although prophylactic vaccines against HPV infection already exist (Gardasil ®, Cervarix ® and Gardasil ®-9), their character is solely and exclusively preventive, with no therapeutic effect in people already infected. In view of the lack of appropriate therapeutic methods and the fact that existing chemotherapeutic agents are non-specific and give rise to unwanted side effects, it is essential to carry out studies focusing on the development of new anticancer agents. Steroid molecules have been widely studied in the development of new anticancer agents, including against cervical carcinomas. In addition, steroid molecules have characteristics that make them extremely attractive for this area, namely high therapeutic action and good bioavailability. Thus, the present work aims to study small steroid molecules from the following families: 4- azaandrostenes (4a-4g), 4-azapregnenes (7a-7g), arylidene dehydroepiandrosterone (DHEA) derivatives (8a-8m), DHEA epoxides (9a-9m) and DHEA triols and threonines (10a-10f and 11a-11f), in order to see if they could be potential inhibitors of HPV oncoprotein E6, preventing its interaction with the tumor suppressor p53 in CC cells (HeLa HPV18 positive and CaSki HPV16 positive), without affecting the viability of healthy cells, normal human dermal fibroblasts (NHDF). To this end, molecular docking studies and absorption, distribution, metabolism, excretion and toxicity (ADMET) prediction studies were first performed to select the compounds with the highest potential for in vitro biological evaluations. Subsequently, the Thermal Shift Assay (TSA), an assay that allows the evaluation of the thermal stability of the E6 protein (recombinant nature) in the presence and absence of the compounds selected in the in silico studies, was performed in order to select those with the greatest potential to be tested in assays using cell lines. To this end, studies were carried out to assess cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and to determine the half maximum inhibitory concentration (IC50) in nontumoral (NHDF) and tumoral cells (HeLa HPV18, CaSki HPV16). The most promising results were obtained for compounds 8h, 10c and 11c, which caused a more evident reduction in cancer cells viability. These steroid compounds specifically reduced viability in HPV positive cells (IC50 38.63 µM (8h), 39.92 µM (10c), 22.01 µM (11c) for HeLa cells and 34.30 µM (8h), 38.75 µM (10c) and 26.84 µM (11c) for CaSki cells). In addition, these compounds showed clearly lower cytotoxicity in NHDF. Thus, the results indicate that compounds 8h, 10c and 11c could be potential inhibitors of HPV oncoprotein E6, representing a possible advance for personalized therapy of CC. However, further studies need to be performed to corroborate the results already obtained, namely viability studies on HPV negative cells, in order to verify if the selectivity of these compounds is maintained only for HPV positive cells.
Description
Keywords
Hpv 4-Azaesteroides Cancro do Colo do Útero Citotoxicidade Derivados do Dhea E6-P53 Thermal Shift Assay