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Glioblastoma multiforme (GBM) is a notably lethal brain tumor associated with high proliferation rate and therapeutic resistance, while currently effective treatment options are still lacking. Imidazo[1,2-a]pyridine derivatives and organoselenium compounds are largely used in medicinal chemistry and drug development. This study is aimed at further investigating the antitumor mechanism of IP-Se-06 (3-((2-methoxyphenyl)selanyl)-7-methyl-2-phenylimidazol[1,2-a]pyridine), a selenylated imidazo[1,2-a]pyridine derivative in glioblastoma cells. IP-Se-06 exhibited high cytotoxicity against A172 cells ( I C 50 = 1.8 μ M ) and selectivity for this glioblastoma cell. The IP-Se-06 compound has pharmacological properties verified in its ADMET profile, especially related to blood-brain barrier (BBB) permeability. At low concentration (1 μM), IP-Se-06 induced intracellular redox state modulation with depletion of TrxR and GSH levels as well as inhibition of NRF2 protein. IP-Se-06 also decreased mitochondrial membrane potential, induced cytochrome c release, and chromatin condensation. Furthermore, IP-Se-06 induced apoptosis by decreasing levels of Bcl-xL while increasing levels of γ-H2AX and p53 proteins. Treatment with IP-Se-06 induced cell cycle arrest and showed antiproliferative effect by inhibition of Akt/mTOR/HIF-1α and ERK 1/2 signaling pathways. In addition, IP-Se-06 displayed significant inhibition of p38 MAPK and p-p38, leading to inhibition of inflammasome complex proteins (NLRP3 and caspase-1) in glioblastoma cells. These collective findings demonstrated that IP-Se-06 is a bioactive molecule that can be considered a candidate for the development of a novel drug for glioblastoma treatment.

Glioblastoma multiforme (GBM) is a notably lethal brain tumor associated with high proliferation rate and therapeutic resistance, while currently effective treatment options are still lacking. Imidazo[1,2

MNT, a transcription factor of the MXD family, is an important modulator of the oncoprotein MYC. Both MNT and MYC are basic-helix-loop-helix proteins that heterodimerize with MAX in a mutually exclusive manner, and bind to E-boxes within regulatory regions of their target genes. While MYC generally activates transcription, MNT represses it. However, the molecular interactions involving MNT as a transcriptional regulator beyond the binding to MAX remain unexplored. Here we demonstrate a novel MAX-independent protein interaction between MNT and REL, the oncogenic member of the NF-κB family. REL participates in important biological processes and it is altered in a variety of tumors. REL is a transcription factor that remains inactive in the cytoplasm in an inhibitory complex with IκB and translocates to the nucleus when the NF-κB pathway is activated. In the present manuscript, we show that MNT knockdown triggers REL translocation into the nucleus and thus the activation of the NF-κB pathway. Meanwhile, MNT overexpression results in the repression of IκBα, a bona fide REL target. Both MNT and REL bind to the IκBα gene on the first exon, suggesting its regulation as an MNT-REL complex. Altogether our data indicate that MNT acts as a repressor of the NF-κB pathway by two mechanisms: (1) retention of REL in the cytoplasm by MNT interaction, and (2) MNT-driven repression of REL-target genes through an MNT-REL complex. These results widen our knowledge about MNT biological roles and reveal a novel connection between the MYC/MXD and NF-κB pathways, two of the most prominent pathways in cancer.

© The Author(s) 2021., MNT, a transcription factor of the MXD family, is an important modulator of the oncoprotein MYC. Both MNT and MYC are basic-helix–loop–helix proteins that heterodimerize with MAX in a mutually exclusive manner, and bind to E-boxes within regulatory regions of their target genes. While MYC generally activates transcription, MNT represses it. However, the molecular interactions involving MNT as a transcriptional regulator beyond the binding to MAX remain unexplored. Here we demonstrate a novel MAX-independent protein interaction between MNT and REL, the oncogenic member of the NF-κB family. REL participates in important biological processes and it is altered in a variety of tumors. REL is a transcription factor that remains inactive in the cytoplasm in an inhibitory complex with IκB and translocates to the nucleus when the NF-κB pathway is activated. In the present manuscript, we show that MNT knockdown triggers REL translocation into the nucleus and thus the activation of the NF-κB pathway. Meanwhile, MNT overexpression results in the repression of IκBα, a bona fide REL target. Both MNT and REL bind to the IκBα gene on the first exon, suggesting its regulation as an MNT–REL complex. Altogether our data indicate that MNT acts as a repressor of the NF-κB pathway by two mechanisms: (1) retention of REL in the cytoplasm by MNT interaction, and (2) MNT-driven repression of REL-target genes through an MNT–REL complex. These results widen our knowledge about MNT biological roles and reveal a novel connection between the MYC/MXD and NF-κB pathways, two of the most prominent pathways in cancer., The work was supported by grant SAF2017-88026-R from Agencia Estatal de Investigación, Spanish Government, to J.L. and M.D.D. J.L.-P. and M.C.L.-N. were recipients of F.P.U. fellowships and L.G.-G. of a F.P.I. fellowship from the Spanish Government.

This work evaluated the antitumor effects of albendazole (ABZ) and its relationship with modulation of oxidative stress and induction of DNA damage. The present results showed that ABZ causes oxidative cleavage on calf-thymus DNA suggesting that this compound can break DNA. ABZ treatment decreased MCF-7 cell viability (EC50=44.9 for 24 h) and inhibited MCF-7 colony formation (~67.5% at 5 μM). Intracellular ROS levels increased with ABZ treatment (~123%). The antioxidant NAC is able to revert the cytotoxic effects, ROS generation and loss of mitochondrial membrane potential of MCF-7 cells treated with ABZ. Ehrlich carcinoma growth was inhibited (~32%) and survival time was elongated (~50%) in animals treated with ABZ. Oxidative biomarkers (TBARS and protein carbonyl levels) and activity of antioxidant enzymes (CAT, SOD and GR) increased, and reduced glutathione (GSH) was depleted in animals treated with ABZ, indicating an oxidative stress condition, leading to a DNA damage causing phosphorylation of histone H2A variant, H2AX, and triggering apoptosis signaling, which was confirmed by increasing Bax/Bcl-xL rate, p53 and Bax expression. We propose that ABZ induces oxidative stress promoting DNA fragmentation and triggering apoptosis and inducing cell death, making this drug a promising leader molecule for development of new antitumor drugs., Graphical abstract fx1, Highlights • The ABZ redox signaling pathway was examined in cancer inhibition. • The oxidative stress can explain the ABZ antitumor mechanisms of action. • The ABZ oxidative stress modulation can be used for cancer therapeutics development. • ABZ can be used as a molecule prototype in possible drug repositioning.

Antiinflammatory and antitumor activity has been reported in Passiflora edulis (yellow passion fruit) nevertheless the intrinsic mechanisms of action are not fully elucidated. The present study aimeds to perform a comparison between the antitumor activity involving the crude extract (HCE) and the supercritical fluid extract with ethanol as co-solvent (SFEtOH) from P. edulis f. flavicarpa Deg. The in vitro cytotoxicity was evaluated in MCF-7 cells, while the in vivo antitumor activity was assessed in male Balb/c mice inoculated with Ehrlich carcinoma cells. SFEtOH exhibited higher antitumor activity compared to HCE. Wherein, SFEtOH showed an EC50 of 264.6 μg/mL against MCF-7 cells as well as an increased inhibition of tumor growth of 48.5% (p

Xyloglucan-block-polycaprolactone (XGO-PCL) copolymer nanoparticles have been proposed as nanocarriers for drug delivery. However, the possible harmful effects of exposure to nanoparticles still remain a concern. Therefore, the aim of this study is to evaluate the potential toxicity of XGO-PCL nanoparticles using in vitro and in vivo assays. Cytotoxicity and genotoxicity studies were conducted on MRC-5 human fetal lung fibroblast cells upon exposure to XGO-PCL nanoparticles. No significant reduction in the cell viability and no DNA damage were observed at the different concentrations tested. Erythrocyte toxicity was assessed by the incubation of nanoparticles with human blood. XGO-PCL nanoparticles induced a hemolytic ratio of less than 1%, indicating good blood compatibility. Finally, the subacute toxicity of XGO-PCL nanoparticles (10 mg/kg/day) was evaluated in BALB/c mice when administered orally or intraperitoneally for 14 days. Results of the in vivo toxicity study showed no clinical signs of toxicity, mortality, weight loss, or hematological and biochemical alterations after treatment with nanoparticles. Also, microscopic analysis of the major organs revealed no histopathological abnormalities, corroborating the previous results. Thus, it can be concluded that XGO-PCL nanoparticles induced no effect indicative of toxicity, indicating their potential use as drug delivery systems.

MNT is a transcription factor of the MXD family. MNT-MAX dimers down-regulate genes by binding to E-box sequences, which can also be bound by MYC-MAX to activate transcription. MNT has been described as a modulator of MYC activity but little is known about MNT regulation and whether MNT has MAX-independent functions. Using a MAX deficient cell line and siRNA-mediated silencing of MAX, we show that in the absence of MAX, the total MNT levels are elevated and that MNT localizes both in the cytoplasm and the nucleus. In contrast, MNT is predominantly nuclear when MAX is expressed. MNT is required for optimal cell proliferation even in the absence of MAX, being the first report of a MAX-independent function of MNT. Interestingly, MNT forms homodimers and autoregulates its expression by repressing its own promoter. The tight MNT regulation and its activity in absence of MAX suggest its importance on cell homeostasis.

Pharmacological doses of ascorbate were evaluated for its ability to potentiate the toxicity of sodium orthovanadate (Na(3)VO(4)) in tumor cells. Cytotoxicity, inhibition of cell proliferation, generation of ROS and DNA fragmentation were assessed in T24 cells. Na(3)VO(4) was cytotoxic against T24 cells (EC(50)=5.8 μM at 24 h), but in the presence of ascorbate (100 μM) the EC(50) fell to 3.3 μM. Na(3)VO(4) plus ascorbate caused a strong inhibition of cell proliferation (up to 20%) and increased the generation of ROS (4-fold). Na(3)VO(4) did not directly cleave plasmid DNA, at this aspect no synergism was found occurring between Na(3)VO(4) and ascorbate once the resulting action of the combination was no greater than that of both substances administered separately. Cells from Ehrlich ascites carcinoma-bearing mice were used to determine the activity of antioxidant enzymes, the extent of the oxidative damage and the type of cell death. Na(3)VO(4) alone, or combined with ascorbate, increased catalase activity, but only Na(3)VO(4) plus ascorbate increased superoxide dismutase activity (up to 4-fold). Oxidative damage on proteins and lipids was higher due to the treatment done with Na(3)VO(4) plus ascorbate (2-3-fold). Ascorbate potentiated apoptosis in tumor cells from mice treated with Na(3)VO(4). The results indicate that pharmacological doses of ascorbate enhance the generation of ROS induced by Na(3)VO(4) in tumor cells causing inhibition of proliferation and apoptosis. Apoptosis induced by orthovanadate and ascorbate is closer related to inhibition on Bcl-xL and activation of Bax. Our data apparently rule out a mechanism of cell demise p53-dependent or related to Cdk2 impairment.

The purpose of the study was to obtain further in vivo data of antitumor effects and mechanisms triggered by juglone and Q7 in combination with ascorbate. The study was done using Ehrlich ascites tumor-bearing mice. Treatments were intraperitoneal every 24 h for 9 days. Control group was treated with excipient. Previous tests selected the doses of juglone and Q7 plus ascorbate (1 and 100 mg/kg, respectively). Samples of ascitic fluid were collected to evaluate carbonyl proteins, GSH and activity of antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase. Hypoxia inducible factor HIF-1α, GLUT1, proteins driving cell cycle (p53, p16 and cyclin A) and apoptosis (poly-ADP-polymerase PARP, Bax and Bcl-xL) were assessed by western blot. Tumor cells were categorized by the phase of cell cycle using flow cytometry and type of cell death using acridine orange/ethidium bromide. A glucose uptake assessment was performed by liquid scintillation using Ehrlich tumor cells cultured with (14)C-deoxyglucose. Treatments caused increased protein carbonylation and activity of antioxidant enzymes and decreased levels of GSH, HIF-1α, GLUT1 and glucose uptake in tumor cells. They also caused increased number of tumor cells in G1, p53 and p16 activation and decreased cyclin A, but only when combined with ascorbate. Apoptosis was induced mostly when treatments were done with ascorbate, causing PARP and Bax cleavage, and increased Bax/Bcl-xL ratio. Juglone and Q7 in combination with ascorbate caused inhibition of tumor progress in vivo by triggering apoptosis and cell cycle arrest associated with oxidative stress, suppression of HIF-1 and uncoupling of glycolytic metabolism.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2016. Introdução. Evidências indicam um promissor efeito antitumoral das quinonas juglona, fenilaminonaftoquinona-7 (Q7) e fenilaminonaftoquinona-9 (Q9), especialmente quando associadas ao ascorbato. Objetivo. Dar continuidade aos estudos sobre o efeito antitumoral das naftoquinonas juglona, Q7 e Q9 em associação ao ascorbato de sódio, bem como avaliar a melhor associação para o tratamento do câncer a partir dos efeitos obtidos in vitro e in vivo. Metodologia. Foram analisados os efeitos das quinonas juglona, Q7 e Q9 associadas ou não ao ascorbato de sódio sobre o CT-DNA (Calf Thymus-DNA). Em células MCF-7 (carcinoma de mama) foram avaliados a viabilidade celular, os níveis de EROs intracelular, os danos ao DNA e a proliferação celular. Proteínas oriundas dos lisados de células MCF-7 foram utilizados para os ensaios de imunoeletroforese para verificar a integridade da proteína poli (ADP-ribose) polimerase (PARP), gama-H2AX (?H2AX) e pAkt. A atividade antitumoral in vivo das quinonas associadas ou não ao ascorbato de sódio foi inicialmente analisada através de parâmetros de sobrevida e crescimento tumoral de camundongos portadores do tumor ascítico de Ehrlich. Posteriormente, amostras do tumor foram coletadas e utilizadas para análise de marcadores de estresse oxidativo como: substâncias reativas ao ácido tiobarbitúrico (TBARS), proteína carbonilada, glutationa reduzida (GSH), superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx), glutationa redutase (GR) e glutationa-S-transferase (GST). Ainda em amostras do tumor ascítico de Ehrlich foram avaliados danos ao DNA, parada do ciclo celular, influência na captação de glicose e tipo de morte celular induzida pelos tratamentos. As mesmas amostras foram utilizadas para a análise de imunoeletroforese de proteínas envolvidas no dano ao DNA e proliferação celular (?H2AX e pAkt, respectivamente), na progressão do ciclo celular (p53, p16 e ciclina A), na captação de glicose (HIF-1a e GLUT1) e proteínas envolvidas na sinalização da morte celular (PARP, Bax, Bcl-xL). Resultados. As quinonas, quando associadas ao ascorbato, causaram um aumento na intercalação e clivagem oxidativa do CT-DNA. Em células MCF-7, a associação com ascorbato diminuiu significativamente a viabilidade celular. As associações juglona/ascorbato e, principalmente Q7/ascorbato, aumentaram os níveis de EROs intracelulares e os danos ao DNA. As três associações testadas causaram diminuição no número de colônias, porém, juglona/ascorbato e Q7/ascorbato foram capazes de inibir a proteína Akt fosforilada (pAkt). Os resultados in vitro foram reproduzidos in vivo. Juglona/ascorbato e Q7/ascorbato inibiram significativamente o desenvolvimento do tumor e aumentaram o tempo de sobrevida dos animais. Os marcadores de estresse oxidativo foram alterados, indicando uma indução na geração de EROs pelos tratamentos, aumentando assim a peroxidação lipídica e a carbonilação de proteínas, danificando o DNA das células do tumor ascítico de Ehrlich e promovendo também, a parada do ciclo celular em G1/S, redução da captação de glicose e morte celular por apoptose. Conclusão. Os resultados apresentados mostraram que o ascorbato de sódio potencializou o efeito antitumoral das quinonas juglona e, principalmente da Q7, também in vivo, evidenciando que estas associações são promissores agentes para o tratamento do câncer. Abstract : Introduction. Evidences point to the promising antitumor effect of quinones juglone, phenylaminonaphthoquinone-7 (Q7) and phenylaminonaphthoquinone-9 (Q9), especially when associated to ascorbate. Objective. To continue the studies on the antitumor effect of naphthoquinones juglone, Q7 and Q9 associated with sodium ascorbate, as well as to evaluate the best association for cancer treatment from the effects obtained in vivo and in vitro. Methodology. The effects of quinones juglone, Q7 and Q9 associated or not to sodium ascorbate on Calf Thymus-DNA were analyzed. Cell viability, levels of intracellular ROS, damages on DNA and cell proliferation were evaluated in MCF-7 cells (breast carcinoma). Proteins from MCF-7 cell lysate were used in immunoblotting assays to check the integrity of poly (ADP-ribose) polymerase (PARP), gamma-H2AX (?H2AX) and pAkt. The in vivo antitumor activity of quinones associated or not with sodium ascorbate was firstly analyzed by means of survival parameters and tumor growth of mice carrying Ehrlich ascitic tumor. After that, samples of the tumor were collected and analyzed for oxidative stress markers such as: thiobarbituric acid reactive substances (TBARS), carbonylation of protein, reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST). Also, of Ehrlich ascitic tumor, DNA damages, cellular cycle arrest, influence in glucose capture and type of cell death induced by the treatments were evaluated. The same samples were used in immunoblotting assays of proteins involved in damage to DNA and cell proliferation (?H2AX and pAkt, respectively), cell cycle progression (p53, p16 and cyclin A) and glucose capture (HIF-1a and GLUT1) and proteins involved in cell death signaling (PARP, Bax, Bcl-xL). Results. When associated to ascorbate, quinones promoted an increase in interleaving and oxidative cleavage of CT-DNA. In MCF-7 cells, the association with ascorbate declined significantly cell viability. The associations juglone/ascorbate and, mainly Q7/ascorbate increased levels of intracellular EROs and damage to DNA. The three assessed associations promoted the decrease in the number of colonies, however, julone/ascorbate and Q7/ascorbate were able to inhibit the protein pAkt. The in vitro results were in vivo reproduced. Juglone/ascorbate and Q7/ascorbate significantly inhibited the growth of the tumor and increased survival time of the animals. The oxidative stress markers were altered pointing to an induction in the generation of ROS by these treatments, thereby increasing lipid peroxidation and protein carbonylation, damaging DNA of cells of Ehrlich ascitic tumor also promoting, cellular cycle arrest in G1/S, reduction of glucose capture and cellular death by apoptosis. Conclusion. The results presented here showed that the sodium ascorbate potentialized the antitumor effect of juglone, especially Q7, also in vivo, evidencing that those associations are promising agents for cancer treatment.

Ethnopharmacological relevance Ayurvedic and Chinese traditional medicine and tribal people use herbal preparations containing Piper nigrum fruits for the treatment of many health disorders like inflammation, fever, asthma and cancer. In Brazil, traditional maroon culture associates the spice Piper nigrum to health recovery and inflammation attenuation. Aims of the study The aim of the current work was to evaluate the relationship between reactive oxygen species (ROS) overproduction, DNA fragmentation, cell cycle arrest and apoptosis induced by Piper nigrum ethanolic extract and its antitumor activity. Methods The plant was macerated in ethanol. Extract constitution was assessed by TLC, UV–vis and ESI-IT-MS/MS spectrometry. The cytotoxicity, proliferation and intracellular ROS generation was evaluated in MCF-7 cells. DNA damage effects were evaluated through intercalation into CT-DNA, plasmid DNA cleavage and oxidative damage in CT-DNA. Tumor growth inhibition, survival time increase, apoptosis, cell cycle arrest and oxidative stress were assessed in Ehrlich ascites carcinoma-bearing mice. Results Extraction yielded 64 mg/g (36% piperine and 4.2% piperyline). Treatments caused DNA damage and reduced cell viability (EC 50 =27.1±2.0 and 80.5±6.6 µg/ml in MCF-7 and HT-29 cells, respectively), inhibiting cell proliferation by 57% and increased ROS generation in MCF-7 cells (65%). Ehrlich carcinoma was inhibited by the extract, which caused reduction of tumor growth (60%), elevated survival time (76%), cell cycle arrest and induced apoptosis. The treatment with extract increased Bax and p53 and inhibited Bcl-x L and cyclin A expression. It also induced an oxidative stress in vivo verified as enhanced lipid peroxidation and carbonyl proteins content and increased activities of glutathione reductase, superoxide dismutase and catalase. GSH concentration was decreased in tumor tissue from mice. Conclusion The ethanolic extract has cytotoxic and antiproliferative effect on MCF-7 cells and antitumor effect in vivo probably due to ROS overproduction that induced oxidative stress affecting key proteins involved in cell cycle arrest at G1/S and triggering apoptosis. Finally , the overall data from this study are well in line with the traditional claims for the antitumor effect of Piper nigrum fruits.

The aim of this study was to enhance the understanding of the antitumor mechanism of 1,4-naphthoquinones and ascorbate. Juglone, phenylaminonaphthoquinone-7, and 9 (Q7/Q9) were evaluated for effects on CT-DNA and DNA of cancer cells. Evaluations in MCF-7 cells are DNA damage, ROS levels, viability, and proliferation. Proteins from MCF-7 lysates were immunoblotted for verifying PARP integrity,γH2AX, and pAkt. Antitumor activity was measured in Ehrlich ascites carcinoma-bearing mice. The same markers of molecular toxicity were assessedin vivo. The naphthoquinones intercalate into CT-DNA and caused oxidative cleavage, which is increased in the presence of ascorbate. Treatments caused DNA damage and reduced viability and proliferation of MCF-7 cells. Effects were potentiated by ascorbate. No PARP cleavage was observed. Naphthoquinones, combined with ascorbate, caused phosphorylation of H2AX and inhibited pAkt. ROS were enhanced in MCF-7 cells, particularly by the juglone and Q7 plus ascorbate. Ehrlich carcinoma was inhibited by juglone, Q7, or Q9, but the potentiating effect of ascorbate was reproducedin vivoonly in the cases of juglone and Q7, which caused up to 60% inhibition of tumor and the largest extension of survival. Juglone and Q7 plus ascorbate caused enhanced ROS and DNA damage and inhibited pAkt also in Ehrlich carcinoma cells.

Purpose: To evaluate the antitumor effect of a latex extract from Euphorbia tirucalli Linn. (Euphorbiaceae) and its toxicity. Methods: Aqueous ethanol and petroleum ether extracts were obtained through maceration. .Maximum tolerated dose was determined in healthy mice. Antitumor activity was measured in Ehrlich ascites tumor-bearing mice treated with the extract through intraperitoneal injection (62.5, 125 or 250 mg/kg) every 48 h (four doses). Efficacy was assessed by weight gain, abdominal circumference, volume of ascitic fluid and packed tumor cells, tumor cell viability and survival. Toxicity indicators were serum glucose, triglycerides, total proteins, activity of alanine and aspartate aminotransferases and mass of heart, spleen, kidney and liver. A hemolysis assay was also performed. Results: Doses of 62.5 and 125 mg/kg caused no antitumor activity, while 250 mg/kg dose reduced weight gain (3-fold), abdominal circumference and volume of ascitic fluid (> 50 %) and packed cells (50 %), but lowered tumor cell viability (40 %). However, mice treated with the extract survived for a shorter time than control mice. Furthermore, the 250 mg/kg dose caused cardiac atrophy, splenomegaly and fasting hyperglycemia. The extract caused hemolysis, and the half-maximal effective concentration (EC 50 ) was 1.6 (0.9 – 2.7) mg/mL. Conclusion: Euphorbia tirucalli extract inhibits Ehrlich ascites tumor in mice, but the therapeutic dose is also harmful to non-tumor tissues. Keywords: Euphorbia tirucalli , Ehrlich ascites tumor-bearing mice, Antitumor, Toxicity, Cardiac atrophy, Splenomegaly

Naphthoquinones interact with biological systems by generating reactive oxygen species (ROS) that can damage cancer cells. The cytotoxicity and the antitumor activity of 3‑acyl‑2‑phenylamino‑1,4‑naphthoquinones (DPB1‑DPB9) were evaluated in the MCF7 human breast cancer cell line and in male Ehrlich tumor‑bearing Balb/c mice. DPB4 was the most cytotoxic derivative against MCF7 cells (EC50 15 µM) and DPB6 was the least cytotoxic one (EC50 56 µM). The 1,4‑naphthoquinone derivatives were able to cause DNA damage and promote DNA fragmentation as shown by the plasmid DNA cleavage assay (FII form). In addition, 1,4‑naphthoquinone derivatives possibly interacted with DNA as intercalating agents, which was demonstrated by the changes caused in the fluorescence of the DNA‑ethidium bromide complexes. Cell death of MCF7 cells induced by 3‑acyl‑2‑phenylamino‑1,4‑naphthoquinones was mostly due to apoptosis. The DNA fragmentation and subsequent apoptosis may be correlated to the redox potential of the 1,4‑naphthoquinone derivatives that, once present in the cell nucleus, led to the increased generation of ROS. Finally, certain 1,4‑naphthoquinone derivatives and particularly DPB4 significantly inhibited the growth of Ehrlich ascites tumors in mice (73%).

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Nutrição, Florianópolis, 2010 Os carotenóides têm sido bastante investigados como agentes quimiopreventivos, funcionando como antioxidantes em sistemas biológicos. A astaxantina é um carotenóide do grupo das xantofilas, de ocorrência natural em algas, peixes e frutos do mar, sendo o camarão uma de suas principais fontes alimentares. Sua atividade antioxidante parece ser muito superior àquela dos demais carotenóides. No presente estudo foi avaliada a atividade antioxidante in vitro dos carotenóides totais (CT) extraídos do músculo de camarões Litopenaues vannamei cultivados em cativeiro, utilizando-se como parâmetros a medida de substâncias reativas ao ácido tiobarbitúrico (TBARS), o seqüestro do radical livre 2,2-difenil-1-picrilhidrazil (DPPH) e a co-oxidação do ?-caroteno/ácido linoléico, utilizando-se a astaxantina sintética como padrão. Foram utilizados dois grupos de camarões: camarão controle (CC) que recebeu ração controle não suplementada, com 3 ppm de carotenóides e camarão suplementado (CS) que recebeu ração contento 60 ppm de carotenóides provenientes da alga Haematococcus pluvialis. Após 30 dias de tratamento, os camarões foram capturados, extraídos os carotenóides totais e realizada a quantificação da astaxantina. Soro e homogenato de fígados extraídos de ratos Wistar adultos foram incubados com os agentes oxidantes CuCl2 0,1 mM e 2,2´-azobis(2-amidinopropano) dihidrocloreto (AAPH) 5 mM, na presença ou ausência dos carotenóides oriundos dos camarões, nas concentrações 0, 0,25, 1,25, 2,5 e 5 ?M. Posteriormente foram realizadas as medidas de TBARS. Para a análise de DPPH utilizaram-se as concentrações de 5 e 10 ?M de carotenóides totais, enquanto que para a co-oxidação foram utilizadas as concentrações de 10 e 20 ?M de carotenóides totais. Os resultados estão expressos como média ± erro padrão da média (E.P.M.). Os dados foram analisados pela análise de variância de duas vias (ANOVA) com medidas repetidas, seguido pelo teste post-hoc de Bonferroni. O nível de significância considerado foi de P < 0,05. Nas análises de TBARS, no soro e homogenato de fígado, após a indução da oxidação com CuCl2 e AAPH observa-se que a proteção contra a lipoperoxidação dos CT extraídos do CS foi semelhante àquela obtida com a astaxantina sintética, principalmente nas concentrações mais elevadas (2,5 e 5 ?M), indicando um perfil concentração-resposta. Nas análises de co-oxidação do ?-caroteno/ácido linoléico, os CT extraídos dos camarões do grupo CC não apresentaram atividade antioxidante nas concentrações de 10 e 20 ?M, enquanto que os CT extraídos do grupo CS mostraram uma atividade antioxidante estatisticamente semelhante àquela da astaxantina sintética para ambas as concentrações testadas. Para o seqüestro do radical livre DPPH, observou-se novamente um possível efeito concentração-resposta dos CT extraídos dos grupos CC e CS, onde os CT do grupo CS apresentaram uma capacidade de seqüestrar o radical DPPH semelhante àquela da astaxantina sintética (aproximadamente 75%), apesar da diferença estatística. Ainda, os resultados da cromatografia líquida de alta eficiência (CLAE) indicam não haver diferença nos níveis de astaxantina nos dois grupos de camarões (CC e CS), sendo este o carotenóide majoritário nos crustáceos de ambos os grupos. Foi observada uma maior atividade antioxidante in vitro dos carotenóides extraídos dos camarões que receberam uma suplementação de astaxantina na ração (grupo CS). Neste grupo, o efeito protetor da astaxantina pode ter evitado a oxidação de ácidos graxos poliinsaturados e colesterol nos crustáceos, evitando desta forma a presença de compostos oxidados nos extratos. No grupo CC, com menor teor de astaxantina, uma maior concentração de compostos oxidados presentes nos extratos poderia estar interferindo nas análises. Os carotenóides do grupo suplementado exerceram uma maior proteção contra a lipoperoxidação no soro e homogenato de fígado de ratos, proteção contra a oxidação de um ácido graxo poliinsaturado e maior atividade no seqüestro de radicais livres. A partir destes resultados observados in vitro novos estudos devem ser conduzidos com o intuito de verificar os possíveis efeitos benéficos in vivo do consumo de alimentos que possuam astaxantina como o carotenóide majoritário, como o camarão.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2018. Introdução. As seleno-diidropirimidinonas são compostos sintéticos com potenciais atividades biológicas, entre elas a antitumoral. Destacam-se por possuírem o selênio, elemento essencial ao funcionamento do organismo, que tem por característica reduzir a proliferação e induzir morte em células tumorais. Objetivo. Avaliar o potencial citotóxico, antiproliferativo e antitumoral in vitro de novos compostos de seleno-diidropirimidinona. Metodologia. Os compostos em estudo (concentrações de 1 µM - 1000 µM) foram testados quanto à sua citotoxicidade em linhagens tumorais MCF-7 (carcinoma de mama humano) e HeLa (adenocarcinoma cervical humano), bem como em linhagem normal McCoy (fibroblasto de camundongo), pelo ensaio MTT após 72 horas de incubação. A interação dos compostos com o DNA, foi realizada por espectrofotometria UV-Vis, utilizando concentração fixa de DNA (150 µM), variando as concentrações dos compostos (50 µM a 300 µM).Para ensaio clonogênico, foi avaliado tratamento com concentração inibitória de 30% (IC30) dos compostos por 72h. Para o ensaio de morte celular, o tratamento com IC30 dos compostos foi realizado por 72h, e as células foram coradas com uma solução de 10 µM de laranja acridina e iodeto de propídio. A fragmentação do DNA de células MCF-7 foi verificada pelo ensaio cometa (IC30 / 72h). A análise do ciclo celular foi realizada de acordo com o conteúdo de DNA celular medido por citometria de fluxo usando um kit de solução PI / RNAse da Immunostep®. Resultados. Os tratamentos com as Seleno- diidropirimidinonas indicaram efeitos citotóxicos e seletivos sobre as células tumorais cultivadas in vitro. As seleno-diidropirimidinonas 3j, 8a, 8b e 4a foram capazes de interagir e intercalar com o DNA plasmidial. Em doses sub tóxica IC30, foram capazes de inibir a proliferação, induzindo a morte celular tanto por necrose como por apoptose. Os compostos 8a e 8b promoveram fragmentação do DNA em células MCF-7 aumentando o número de células na fase sub/G1, indicativo de indução de apoptose, e reduziram o número de células na fase S, em tratamentos de 72h com doses sub tóxicas. Conclusão. Os resultados obtidos permitem sugerir que os compostos de Seleno-diidropirimidinonas 8a e 8b tratam-se de promissores protótipos na pesquisa de novos agentes na terapia contra o câncer, se fazendo necessários futuros ensaios para elucidar melhor suas ações e mecanismos. Abstract : Introduction. Selenium-dihydropyrimidinones are synthetic compounds with potential biological activities, including antitumor. It stands out for having selenium, an essential element to the functioning of the organism, which has the characteristic of reducing proliferation and inducing death in tumor cells. Objective. Evaluation of the in vitro cytotoxic, antiproliferative and antitumor potential of new selenide-dihydropyrimidinone compounds. Methodology. The compounds under study (concentrations of 1 µM - 1000 µM) were tested for their cytotoxicity to the MCF-7 (human breast carcinoma), HeLa (human cervical adenocarcinoma) and normal McCoy (mouse fibroblast) MTT assay in 72 hours of incubation. The interaction of the compounds with DNA was performed by UV-Vis spectrophotometry, with fixed concentration for the DNA of 150 µM and variations of the concentrations of the compounds of 50 µM to 300 µM. In the clonogenic assay, the IC30 treatment of the compounds was 72 h, which was incubated for 15 days until colony formation. For the cell death assay, the IC30 treatment of the compounds was 72 h, and the cells were stained with a solution of 10 µM orange acridine plus propidium iodide and categorized by microscopy as viable, apoptotic and necrotic. DNA fragmentation of MCF-7 cells was verified by the comet assay (IC30 / 72 h). And the cell cycle analysis was performed according to the cellular DNA content measured by flow cytometry using an Immunostep® PI / RNAse solution kit. Results. Treatments with Selenidipyrimidinones indicated cytotoxic and selective effects on tumor cells cultured in vitro. The selene-dihydropyrimidinones 3j, 8a, 8b and 4a led to the interaction and intercalation of the DNA. At sub-toxic IC30 doses, they were able to inhibit proliferation, inducing cell death both by necrosis and by apoptosis. Compounds 8a and 8b promoted DNA fragmentation in MCF-7 cells and caused an increase in the number of cells in the sub- G1 phase, indicating death by apoptosis and imprisoned the S-phase MCF-7 cells in 72 h treatments at sub-toxic doses. Conclusion. The results suggest that Selen-dihydropyrimidinone compounds 8a and 8b are promising agents in cancer therapy, and future trials are needed to better elucidate their actions and mechanisms.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2018. Tanto as imidazo[1,2-a]piridinas quanto os compostos organosselênicos demonstram alto potencial antitumoral contra diversas linhagens tumorais em estudos in vitro e in vivo, sendo propícia sua fusão em uma única molécula. Desta forma, onze novos derivados de imidazo[1,2-a]piridinas, sendo dez deles selenilados, foram testadas quanto ao seu potencial citotóxico, por meio do teste do MTT, contra duas linhagens tumorais (MCF-7 e HeLa) e uma linhagem de fibroblasto murinho (McCoy), definindo-se a CI50 para os onze compostos (0-1000 µM), selecionando-se os compostos com CI50 menores. Ensaios moleculares com espectrofotometria UV visível e com fluorescência (iodeto de propídio) foram realizados para avaliar a interação e a intercalação dos compostos selecionados (50-250 µM) com CT-DNA (150 µM). O teste do cometa para avaliar a fragmentação do DNA, o ensaio clonogênico e análise do ciclo celular para avaliar a atividade antiprolieferativa, a análise de morte celular (iodeto de propídio/laranja de acridina) para avaliar o tipo de morte induzido pelos compostos selecionados em células MCF-7 foram realizados após 72h de tratamento em concentrações subtóxicas, a CI30. A imunoeletroforese para avaliar a expressão de proteínas relacionadas ao dano de DNA, ciclo celular e apoptose foi realizada após o tratamento de 48h. Os compostos IP-Se-05 (CI50 = 26 µM/ MCF-7) e IP-Se-06 (CI50 = 12,5 µM/ MCF-7) obtiveram as menores CI50. Os testes de interação e intercalação com CT-DNA demonstraram a capacidade desses dois compostos de interagirem e intercalarem com o CT-DNA. O ensaio cometa detectou a fragmentação do DNA e a imunoeletroforese demonstrou o aumento da expressão de ?H2AX após o tratamento das células MCF-7 com IP-Se-05 e IP-Se-06. O ensaio clonogênico e a análise do ciclo celular comprovaram a atividade antiproliferativa desses dois compostos, ocorrendo diminuição estatisticamente significativa no número de colônias e a parada do ciclo celular na fase G2/M de células MCF-7, 66,22% para IP-Se-05 e 57,97% para IP-Se-06. Ainda, ambos compostos causaram diminuição da expressão da ciclina A e da pAkt. O ensaio de morte celular comprovou a morte apoptótica e necrótica das células tratadas com IP-Se-05 e morte apoptótica das células tratadas com IP-Se-06, aumentando a expressão de p53. De maneira geral, IP-Se-06 demonstrou maior atividade citotóxica e antiproliferativa em menor concentração, além de desencadear a indução da morte celular somente por apoptose, maior expressão de p53 e menor expressão da ciclina A, talvez pela presença do grupamento 2-metoxifenil. Por fim, supõe-se que IP-Se-05 e IP-Se-06 intercalem com o DNA, causando sua fragmentação na fase S e aumentando a expressão de ?H2AX. Isto ativa a parada do ciclo celular na fase G2/M, diminuindo a expressão da ciclina A e da pAkt, enquanto aumenta a expressão da p53, o que encaminha as células MCF-7 para morte celular apoptótica. Conclue-se que IP-Se-06 é o mais promissor dentre os onze compostos testados neste trabalho. Ele causou maior efeito citotóxico e antiproliferativo in vitro, provavelmente, pela presença do grupamento 2-metoxifenil em sua estrutura química, o que confere polaridade e eletrofilicidade a este composto, destacando-se dos demais. Abstract : Imidazo[1,2-a]pyridines and organoselenic compounds display a high antitumor potential against several tumor lines in in vitro and in vivo studies. In this way, their merge into a single molecule might bring promising results. In this study, eleven novel imidazo[1,2-a]pyridines were tested initially for their cytotoxic and antiproliferative potential in vitro. To verify cytotoxic and selective activity of these compounds, the MTT test was performed on a panel of tumor cells lines (MCF-7 e HeLa) and a fibroblast cell line (McCoy). The IC50 was determined for the eleven compounds (0-1000 µM) and used to select the compounds with the lowest concentrations. Molecular assays with visible UV spectrophotometry and fluorescence (propidium iodide) was performed to evaluate the compounds interaction and intercalation (50-250 µM) with CT-DNA (150 µM). Comet test was performed to evaluate DNA fragmentation in MCF-7 cells after a 72h treatment at subtoxic concentration, the IC30. Clonogenic assay and cell cycle analysis was performed to evaluate antiproliferative activity. Cell death (propidium iodide/acridine orange) analysis induced by the compounds were performed in MCF-7 cells after a 72h treatment with the IC30. Immunoelectrophoresis to evaluate the expression of proteins related to DNA damage, cell cycle and apoptosis was performed after a 48h treatment. Compounds IP-Se-05 (IC50 = 26 µM /MCF-7) and IP-Se-06 (IC50 = 12.5 µM /MCF-7) exhibited the lowest IC50. Interaction and CT-DNA intercalation tests demonstrated the ability of these compounds to interact and intercalate with DNA. The comet assay detected DNA fragmentation and immunoelectrophoresis demonstrated increased ?H2AX expression in MCF-7 cells after IP-Se-05 and IP-Se-06 treatments. The clonogenic assay and the analysis of the cell cycle evidenced the antiproliferative activity of these two compounds, as statistically significant decrease in the number of colonies and cell cycle gap in the G2/M phase of MCF-7 cells (66.22% for IP-Se-05 and 57.97% for IP-Se-06) were observed. In addition, decreased expression of cyclin A and pAkt was detected. The cell death analysis demonstrated the apoptotic and necrotic death of cells treated with IP-Se-05 and apoptotic death of cells treated with IP-Se-06, increasing p53 expression. In general, IP-Se-06 demonstrated higher cytotoxic and antiproliferative activity in a lower concentration, in addition to induction of cell death mostlyby apoptosis, higher expression of p53 and lower expression of cyclin A, perhaps linked to the presence of a 2-phenylmethoxy group. Finally, it is assumed that IP-Se-05 and IP-Se-06 intercalate with the DNA, causing its fragmentation and increasing the expression of ?H2AX. This activates cell cycle arrest in the G2/M phase, decreasing expression of cyclin A and pAkt, while increasing the expression of p53, which routes MCF-7 cells to apoptotic cell death. It is concluded that IP-Se-06 is the most promising among the eleven compounds evaluated in this work. It displayed greater cytotoxic and antiproliferative effect in vitro, probably, due to the presence of a 2-methoxyphenyl group in its chemical structure, which confers more polarity and electrophilicity to the compound, when comparing it to the others.