Copper(II) Complex Containing 4-Fluorophenoxyacetic Acid Hydrazide and 1,10-Phenanthroline: A Prostate Cancer Cell-Selective and Low-Toxic Copper(II) Compound.

Prostate Cancer (PCa) is the second leading cause of cancer-related deaths among men worldwide. The treatment of advanced cases is based on chemotherapy, which lacks specificity and efficacy, due to severe side effects and resistance to the traditional drugs. Copper complexes have shown antitumoral efficacy and low toxicity, being considered a promising class of metal-based drugs for the treatment of malignant neoplasms. Thus, the present study aimed to evaluate the cellular effects of a copper(II) complex with 4-fluorophenoxyacetic acid hydrazide and 1,10-phenanthroline (1) on PCa cell lines, as well as the mutagenic/recombinogenic and anticarcinogenic potential of 1 in Drosophila melanogaster. PNT-2 (non-tumorigenic), LNCaP (hormone-responsive PCa) and PC-3 (androgen-independent PCa) cells were cultured, and cytotoxicity was assessed using the MTT assay. The expression levels of the proliferation markers Ki-67 and Cyclin D1 were analyzed by flow cytometry. Furthermore, the Somatic Mutation and Recombination Test (SMART) and the Epithelial Tumor Test (ETT) were performed. Complex 1 was selective to LNCaP cells, significantly reducing Ki-67 and Cyclin D1 expression levels. Sub-toxic concentrations of complex 1 were defined by the toxicity test in D. melanogaster, and no mutagenic/recombinogenic/carcinogenic effects were observed. Anticarcinogenic potential was observed in D. melanogaster, suggesting modulating activity of the complex 1 against Doxorubicin, a drug used as control by its carcinogenic properties. Therefore, complex 1 is a possible starting point for the development of new antitumor agents for the treatment of PCa.

Avaliação do efeito carcinogênico do bromazepam em Drosophila melanogaster / Evaluation of carcinogen bromazepam effect in Drosophila melanogaster

Introdução: embora o câncer seja um dos maiores problemas de saúde pública enfrentados mundialmente, diversas substâncias presentes no meio, como os fármacos, não estão muito bem elucidadas sobre seu possível potencial carcinogênico. Entre eles, estão os benzodiazepínicos, fármacos que possuem crescente aumento do consumo desde o século XX e, principalmente, na segunda década do século XXI, por suas ações ansiolíticas, sedativas e anticonvulsivantes. Objetivo: avaliar o efeito carcinogênico do bromazepam por meio do teste para detecção de tumores epiteliais (ETT) em Drosophila melanogaster. Metodologia: para realização do ETT foram utilizadas duas linhagens mutantes de D. melanogaster: wts (fêmeas) e mwh (machos). As larvas descendentes desse cruzamento foram tratadas isoladamente com cinco concentrações de bromazepam, sendo elas: 0,0375; 0,075; 0,15; 0,30 e 0,60 mM. A Doxorrubicina foi utilizada como controle positivo e a água ultrapura como controle negativo. Após tratamento, coleta e armazenamento, as moscas foram analisadas, identificando-se as frequências tumorais, por região corporal, em cada concentração testada. Resultados: o bromazepam não apresentou efeito carcinogênico em nenhuma das concentrações experimentadas neste estudo, não havendo diferença estatisticamente significativa nas frequências tumorais observadas nos indivíduos tratados com bromazepam quando comparadas à frequência obtida nos indivíduos tratados com o controle negativo. Conclusão: Nas presentes condições experimentais, o bromazepam não apresentou atividade carcinogênica, no entanto, há a necessidade de novos estudos, com diferentes metodologias e diferentes organismos testes, para a maior compreensão da ação do bromazepam no organismo.

Introduction: although cancer is one of the biggest public health problems faced worldwide, several substances present in the environment, such as drugs are not very well understood about its possible carcinogenic potential. Among them are benzodiazepines, drugs that have increased their consumption since the 20th century and, mainly, in the second decade of the 21st century, due to their anxiolytic, sedative and anticonvulsant actions. Objective: Evaluate the carcinogenic effect of bromazepam through the test to detect epithelial tumor clones (ETT) in Drosophila melanogaster. Methodology: to perform the ETT, two mutant strains of D. melanogaster were used: wts (female) and mwh (male). The descending larves of this cross were treated separately with five concentrations of bromazepam, namely: 0.0375; 0.075; 0.15; 0.30 and 0.60 mM. Doxorubicin was used as a positive control and ultrapure water as a negative control. After treatment, collection and storage, the flies were analyzed, identifying the tumor frequencies, by body region, at each concentration tested. Results: bromazepam did not have a carcinogenic effect at any of the concentrations experienced in this study, with no statistically significant difference in tumor frequencies observed in individuals treated with bromazepam when compared to the frequency obtained in individuals treated with the negative control. Conclusion: In the present experimental conditions, bromazepam did not show carcinogenic activity, however, there is a need for further studies with different methodologies and different test organisms to better understand the action of bromazepam in the body.

A selective CuII complex with 4-fluorophenoxyacetic acid hydrazide and phenanthroline displays DNA-cleaving and pro-apoptotic properties in cancer cells.

The thin line between efficacy and toxicity has challenged cancer therapy. As copper is an essential micronutrient and is important to tumor biology, CuII complexes emerged as an alternative to chemotherapy; however, its biological properties need to be better understood. Thus, we report in vitro the antitumor effects of two CuII complexes named [Cu(4-fh)(phen)(ClO4)2] (complex 1) and [Cu(4-nh)(phen)(ClO4)2]·H2O (complex 2), in which 4-fh = 4-fluorophenoxyacetic acid hydrazide; 4-nh = 4-nitrobenzoic hydrazide and phen = 1,10-phenanthroline. Both complexes presented cytotoxic activity against tumor cells, but only complex 1 showed significant selectivity. Complex 1 also induced DNA-damage, led to G0/G1 arrest and triggered apoptosis, which was initiated by an autophagy dysfunction. The significant in vitro selectivity and the action mechanism of complex 1 are noteworthy and reveal this prodrug as promising for anticancer therapy.

Efeito modulador do Firocoxib (Previcox®) sobre a ação carcinogênica da doxorrubicina por meio do teste de tumores epiteliais (ETT) em Drosophila melanogaster / Modulatory effect of Firocoxib (Previcox®) on the carcinogenic action of doxorubicin through the epithelial tumor teste (TTE) in Drosophila melanogaster

Introdução: a superexpressão da COX-2 relaciona-se com o aumento da produção de fatores de crescimento vascular e como consequência, com o desenvolvimento tumoral. O Firocoxib é um anti-inflamatório não esteroidal utilizado para inflamação associada à osteoartrite em cães. É o inibidor mais seletivo da COX-2, reduzindo eficientemente a ação desta enzima. Estudos indicam os benefícios do Firocoxib na terapia antineoplásica. Objetivo: este trabalho objetivou avaliar o efeito modulador do Firocoxib sobre a ação da doxorrubicina (DXR) por meio do teste de tumores epiteliais (ETT) em Drosophila melanogaster. Metodologia: foram preparadas três concentrações de Firocoxib: 2,5; 5 e 10 mg/mL, utilizadas isoladamente e em associação à doxorrubicina. O tratamento ocorreu com larvas de D. melanogaster descendentes do cruzamento de fêmeas wts/TM3 com machos mwh/mwh. Resultados: os resultados sugerem que o Firocoxib possui atividade moduladora sobre a ação carcinogênica da DXR, pois houve redução significativa nas frequências tumorais dos indivíduos tratados com diferentes concentrações de Firocoxib em cotratamento com a doxorrubicina quando comparadas à frequência tumoral do controle positivo. Conclusão: conclui-se que, nas presentes condições experimentais, o Firocoxib reduziu a frequência de tumores induzidos pela doxorrubicina em D. melanogaster.

Introduction: COX-2 overexpression is related to increased production of vascular growth factors and, as a consequence, to tumor development. Firocoxib is a non-steroidal anti-inflammatory drug used for inflammation associated with osteoarthritis in dogs. It is the most selective inhibitor of COX-2, efficiently reducing the action of this enzyme. Studies indicate the benefits of Firocoxib in anticancer therapy. Objective: this study aimed to evaluate the modulatory effect of Firocoxib on the action of doxorubicin (DXR) by means of the epithelial tumor test (ETT) in Drosophila melanogaster. Methodology: three concentrations of Firocoxib were prepared: 2.5; 5 and 10 mg/mL, used alone and in association with doxorubicin. The treatment occurred with D. melanogaster larvae descended from the crossing of wts/TM3 females with mwh/mwh males. Results: the results suggest that Firocoxib has modulating activity on the carcinogenic action of DXR, as there was a significant reduction in tumor frequencies in individuals treated with different concentrations of Firocoxib in co-treatment with doxorubicin when compared to the tumor frequency of the positive control. Conclusion: it is concluded that, under the present experimental conditions, Firocoxib reduced the frequency of tumors induced by doxorubicin in D. melanogaster.

Modulating effect of vitamin D3 on the mutagenicity and carcinogenicity of doxorubicin in Drosophila melanogaster and in silico studies.

Vitamin D3 (VD3) deficiency increases DNA damage, while supplementation may exert a pro-oxidant activity, prevent viral infections and formation of tumors. The aim of this study was to investigate the mutagenicity and carcinogenicity of VD3 alone or in combination with doxorubicin (DXR) using the Somatic Mutation and Recombination Test and the Epithelial Tumor Test, both in Drosophila melanogaster. For better understanding of the molecular interactions of VD3 and receptors, in silico analysis were performed with molecular docking associated with molecular dynamics. Findings revealed that VD3 alone did not increase the frequency of mutant spots, but reduced the frequency of mutant spots when co-administered with DXR. In addition, VD3 did not alter the recombinogenic effect of DXR in both ST and HB crosses. VD3 alone did not increase the total frequency of tumor, but significantly reduced the total frequency of tumor when co-administered with DXR. Molecular modeling and molecular dynamics between calcitriol and Ecdysone Receptor (EcR) showed a stable interaction, indicating the possibility of signal transduction between VD3 and EcR. In conclusion, under these experimental conditions, VD3 has modulatory effects on the mutagenicity and carcinogenicity induced by DXR in somatic cells of D. melanogaster and exhibited satisfactory interactions with the EcR.

Betulinic acid modulates urethane-induced genotoxicity and mutagenicity in mice and Drosophila melanogaster.

Betulinic acid (BA) is a pentacyclic triterpenoid found in several plant species. Urethane (URE) is a known promutagen. Here, we examine the genotoxicity and mutagenicity of BA alone or in combination with URE using the bone marrow micronucleus assay in mice bone marrow cells and the Somatic Mutation and Recombination Test in Drosophila melanogaster. Findings revealed that BA alone was not genotoxic, but reduced the frequency of micronucleus when compared to the positive control. No significant differences were observed in the cytotoxicity. Biochemical analyzes showed no significant differences for liver (AST and ALT) or renal (creatinine and urea) function parameters, indicating the absence of hepatotoxic and nephrotoxic effects. BA alone did not increase the frequency of mutant spots, but reduced the total frequency of mutant spots when co-administered with URE in both ST and HB crosses. In addition, BA reduced the recombinogenic effect of URE at the highest concentrations of both crosses. In conclusion, under experimental conditions, BA has modulatory effects on the genotoxicity induced by URE in mice, as well as in somatic cells of D. melanogaster. We suggest that the modulatory effects of BA may be mainly due to its antioxidant and apoptotic properties.

Protective effect of aspirin against mitomycin C-induced carcinogenicity, assessed by the test for detection of epithelial tumor clones (warts) in Drosophila melanogaster.

The present study assessed the protective effect of aspirin against carcinogenicity induced by mitomycin C (MMC) by the test for detection of warts/epithelial tumor clones in Drosophila melanogaster. Larvae were treated with different concentrations of aspirin alone (10, 20 or 40 mg/mL) or aspirin in association with MMC. MMC and ultrapure water were employed as the positive and negative control, respectively. Antioxidant activity was determined using the DPPH method. For performing cytotoxicity assay on HeLa cells, the aspirin concentrations used ranged from 200 mmol/L to 3,125 mmol/L. For assessment of apoptosis and necrosis, cells were incubated for 24 h with complete medium in the absence (control group) or presence of aspirin (12.5 mmol/L and 25 mmol/L). The results obtained in the assessment of the possible carcinogenic effects of aspirin at the three concentrations tested indicate no statistically significant increase in tumor frequency compared to the negative control. The anticarcinogenic activity assessment, where the larvae of D. melanogaster were previously induced to tumor formation by MMC and later treated with aspirin, showed a statistically significant reduction in the number of tumors compared to the positive control. Antioxidant activity across the three aspirin concentrations (10, 20 or 40 mg/mL) ranged from 20.81% to 26.5%. It was observed that aspirin reduced growth viability of HeLa cells in a concentration-dependent manner in comparison with the control. These results indicate that aspirin did not induce tumors in Drosophila and reduced MMC-induced carcinogenicity. The antioxidant activity and apoptosis induction appear to be the main mechanisms involved in reducing the frequency of tumors.

Efeito redutor do Viagra (Citrato de Sildenafila) sobre a frequência de tumores epiteliais induzidos pela Doxorrubicina em Drosophila melanogaster / Viagra (Sildenafil citrate) reduced the frequency of epithelial tumors induced by doxorubicin in Drosophila melanogaster

O consumo de medicamentos está entre as principais causas de desenvolvimento do câncer. Faz-se, então, necessário investigar os efeitos de drogas amplamente consumidas no mundo, como é o caso do Viagra® (Citrato de Sildenafila - CS). O objetivo do presente estudo foi avaliar o efeito modulador do CS na carcinogênese induzida pela Doxorrubicina (DXR), por meio do Teste para Detecção de Clones de Tumores Epiteliais em Drosophila melanogaster. Foram utilizadas duas linhagens mutantes do organismo teste (wts e mwh) no intuito de obter larvas heterozigotas wts +/+ mwh, que foram tratadas com três concentrações de CS: 10 mM, 20 mM e 40 mM, isoladamente e em associação com a DXR. Constatou-se que o CS não exerceu efeito carcinogênico nas concentrações isoladamente avaliadas. Quando em associação com a DXR, propiciou efeito redutor sobre as frequências dos tumores induzidos por ela, conferindo papel anticarcinogênico ao CS. Acredita-se que este efeito seja devido a uma supra-regulação da via de apoptose dependente de caspase e ao impedimento do crescimento celular promovido pelo fármaco utilizado. As evidências obtidas comprovam o efeito protetor tumoral atribuído ao Viagra® nas condições experimentais propostas.(AU)

Drug consumption is among the leading causes of cancer development. Therefore, it is necessary to investigate the effects of drugs widely consumed in the world, as the case of Viagra® (Sildenafil Citrate - SC). The objective of the study was to evaluate the modulatory effect of SC on the carcinogenicity induced by Doxorubicin (DXR) by the Test for Detection of Epithelial Tumor Clones in Drosophila melanogaster. Two mutant lines (wts and mwh) were used in order to obtain heterozygous wts+/+mwh larvae, which were treated with three concentrations of CS: 10 mM, 20 mM and 40 mM, alone and in combination with doxorubicin. We verified that SC had no carcinogenic effect at the concentrations evaluated alone. In association with DXR, it was provided a reduced effect of tumor-induced, conferring anticarcinogenic role to SC. It is believed that this effect is due to up-regulation of the caspase-dependent apoptosis and the impediment of cell growth promoted by the drug used. Evidence obtained confirms the tumor protective effect attributed to Viagra® under experimental conditions. (AU)

Efeito modulador do óleo de copaíba (Copaifera officinalis L) sobre a carcinogenicidade da doxorrubicina, avaliado por meio do teste para detecção de clones de tumores em Drosophila melanogaster / Modulatory effect of the copaiba oil (Copaifera officinalis L) on the carcinogenicity of doxorubicin, evaluated through the test for detection of tumor clones in Drosophila melanogaster

Introdução: o óleo de copaíba é o composto extraído do tronco da copaibeira (Copaifera sp.) que tem sido utilizado na medicina popular desde a chegada dos portugueses ao Brasil. Objetivo: o objetivo deste estudo foi avaliar possíveis efeitos carcinogênicos e/ ou anticarcinogênicos do óleo de copaíba (Copaifera officinalis L.), por meio do teste para detecção de clones de tumores epiteliais (warts) em Drosophila melanogaster. Metodologia: foram preparadas três soluções de óleo de copaíba, nas proporções de 0,5%, 1% e 2%. Nessas soluções foram cultivadas Drosophilas melanogaster expostas simultaneamente à doxorrubicina na concentração de 0,4 mM, agente conhecidamente cancerígeno, também utilizado como controle positivo na presente pesquisa. Para controle negativo foi utilizado Tween 80 (1%). O tratamento foi realizado com todas as larvas descendentes do cruzamento de fêmeas wts/ TM3 com machos mwh/mwh. Resultados: o óleo de copaíba apresentou atividade carcinogênica quando utilizado isoladamente na concentração 2%, visto que houve aumento estatisticamente significativo (p ≤ 0,05) na frequência de tumores em comparação com o controle negativo. Além disso, evidenciou-se potencialização do efeito carcinogênico da doxorrubicina nas concentrações 0,5% e 1%, uma vez que houve um aumento estatisticamente significativo (p ≤ 0,05) na frequência de tumores nessas concentrações, quando associadas à DXR, em comparação com o controle positivo. Conclusão: os resultados evidenciaram o efeito carcinogênico isolado do óleo de copaíba, bem como seu efeito potencializador quando associado à doxorrubicina.

Introduction: the copaiba oil is a compound extracted from the trunk of the copaiba tree (Copaifera sp.) that has been used in popular medicine since the arrival of the Portuguese in Brazil. Purpose: this study aimed to evaluate the possible carcinogenic and/ or anticarcinogenic effects of the copaiba oil (Copaifera officinalis L.) through the test for detection of epithelial tumor clones (warts) in Drosophila melanogaster. Methodology: three solutions of copaiba oil were prepared in the proportions 0,5%, 1% and 2%. In these solutions were cultivated Drosophilas melanogaster previously exposed to doxorubicin at a 0.4 mM concentration, admittedly carcinogenic agent, which was also used for positive control in the present research. For negative control Tween 80 (1%) was used. The treatment was performed on all larvae descendant of the crossing of females wts/TM3 with males mwh/mwh. Results: the results showed that the copaiba oil presented carcinogenic activity when used in isolation at the concentration of 2%, seeing that there was a statistically significant increase (p ≤ 0,05) in tumor frequency in comparison to the negative control. Moreover, there was a potentiation of doxorubicin's carcinogenic effect at concentrations 0,5% and 1%, since there was a statistically significant increase (p ≤ 0,05) of tumor frequency in these concentrations, when associated to DXR, in comparison to the positive control. Conclusion: the results evidenced the isolated carcinogenic effect of copaiba oil, as well as its potentiating effect when associated with doxorubicin

Modulatory effects of metformin on mutagenicity and epithelial tumor incidence in doxorubicin-treated Drosophila melanogaster.

Metformin (MET) is an anti-diabetic drug used to prevent hepatic glucose release and increase tissue insulin sensitivity. Diabetic cancer patients are on additional therapy with anticancer drugs. Doxorubicin (DXR) is a cancer chemotherapeutic agent that interferes with the topoisomerase II enzyme and generates free radicals. MET (2.5, 5, 10, 25 or 50 mM) alone was examined for mutagenicity, recombinogenicity and carcinogenicity, and combined with DXR (0.4 mM) for antimutagenicity, antirecombinogenicity and anticarcinogenicity, using the Somatic Mutation and Recombination Test and the Test for Detecting Epithelial Tumor Clones in Drosophila melanogaster. MET alone did not induce mutation or recombination. Modulating effects of MET on DXR-induced DNA damage were observed at the highest concentrations. In the evaluation of carcinogenesis, MET alone did not induce tumors. When combined with DXR, MET also reduced the DXR-induced tumors at the highest concentrations. Therefore, in the present experimental conditions, MET alone did not present mutagenic/recombinogenic/carcinogenic effects, but it was able to modulate the effect of DXR in the induction of DNA damage and of tumors in D. melanogaster. It is believed that this modulating effect is mainly related to the antioxidant, anti-inflammatory and apoptotic effects of this drug, although such effects have not been directly evaluated.

Assessment of the carcinogenic potential of high intense-sweeteners through the test for detection of epithelial tumor clones (warts) in Drosophila melanogaster.

High intensity-sweeteners (HIS) are natural or synthetic substances, sweeter than sugar, providing sweetness without calories. Sweeteners are mainly used as an aid in losing weight, preventing obesity and controlling blood sugar levels for diabetics. The objective of this study was to evaluate the carcinogenic potential of the sweeteners aspartame, sucralose, sodium saccharin and steviol glycoside, using the test for detection of epithelial tumor clones in Drosophila melanogaster. Larvae of 72 ± 4h, obtained from wts/TM3 female mated with mwh/mwh males, were treated for approximately 48h with different concentrations of aspartame (0.85, 1.7, 3.4, 6.8 or 13.6 mM ); sucralose (0.5, 1.25, 2.5, 5.0 or 10 mM); sodium saccharin (25; 50; 100; 200 or 400 mM) and steviol glycoside (2.5; 5.0; 10; 20 or 40 mM). Water (Reverse Osmosis) and doxorubicin (DXR 0.4 mM) were used as negative and positive controls, respectively. No statistically significant differences were observed (p > 0.05) in tumor frequencies in individuals treated with all concentrations of these sweeteners when compared to negative control. It was therefore concluded that, in these experimental conditions, aspartame, sucralose, sodium saccharin and steviol glycoside have no carcinogenic effect in D. melanogaster.