Evaluation of the effects of the association between sulforaphane and vitamin D on cytotoxicity, genotoxicity, cell migration and modulation of gene expression in human prostate tumor cells (DU145 e PC-3)

Atualmente, o câncer de próstata é o segundo mais incidente em homens no Brasil. Apesar das terapias disponíveis, não existe um tratamento que seja eficiente para os casos de câncer em estágio avançado ou metastático. Sulforafano e vitamina D separados têm apresentado atividade anticâncer promissora em estudos in vitro e in vivo, no entanto, quando utilizados em testes clínicos seus efeitos têm sido limitados devido à baixa biodisponibilidade. Deste modo, o objetivo deste trabalho foi avaliar os efeitos da associação de sulforafano e vitamina D sobre a viabilidade celular, citotoxicidade, indução de apoptose e danos no DNA, estresse oxidativo e autofagia, atividade clonogênica e migratória, modulação do ciclo celular, expressão de genes e proteínas em células humanas tumorais de próstata obtidas de metástases (DU145 e PC-3), a fim de verificar um possível efeito sinérgico entre eles. Em células DU145 após 72 horas de tratamento, vitamina D diminuiu a viabilidade celular em ensaio de vermelho neutro e sulforafano diminuiu a viabilidade celular em ensaio de MTT e vermelho neutro. Em células PC-3, vitamina D e sulforafano diminuíram a viabilidade celular em ensaio de MTT e vermelho neutro. O tratamento associado (sulforafano + vitamina D) diminuiu a viabilidade celular quando comparado aos tratamentos isolados após 24 horas, mas não houve alteração após 72 horas. Sulforafano induziu citotoxicidade e sua associação com vitamina D potencializou o efeito citotóxico do sulforafano em ambas as linhagens. Sulforafano induziu morte por apoptose e parada do ciclo celular em DU145 (fase S e G2) e PC-3 (fase G2). Vitamina D e sulforafano inibiram a atividade clonogênica em PC-3, mas somente sulforafano diminuiu a capacidade de formar colônias em células DU145. Após o tratamento associado, não foi observada qualquer diferença na indução de apoptose, modulação do ciclo celular ou capacidade clonogênica quando comparadas aos tratamentos isolados. Não houve alteração na migração celular em nenhuma das concentrações testadas, tanto em tratamento isolado como associado nas duas linhagens. Após o tratamento isolado, vitamina D e sulforafano não induziram formação de espécies reativas e danos no DNA. Entretanto, o tratamento associado foi genotóxico em DU145 e induziu estresse oxidativo em DU145 (3 e 24 horas) e PC-3 (3 horas). Sulforafano aumentou a autofagia e a associação do sulforafano com a vitamina D potencializou seu efeito autofágico em ambas as linhagens. Após o tratamento associado em células DU145, a expressão dos genes MAPK1, CCNB1, CDK1 e CTNNB1 aumentou. As proteínas BAX, CASP8, CASP3, NRF2 e JNK foram mais expressas, e BCL2 menos expressa em relação ao controle. Em células PC-3 foi observado aumento da expressão dos genes MAPK1 e CCNB1 e das proteínas BAX e NRF2, e diminuição da expressão do gene TNF e das proteínas BCL2, CASP8 e JNK. Após a predição das interações entre alvos, sulforafano e vitamina D, foi apresentada a via MAPK como possível via de sinalização responsável pelos efeitos encontrados com o tratamento associado. Esses resultados demonstram que a associação de sulforafano e vitamina D tem potencial para uso na terapia do câncer de próstata.Currently, prostate cancer is the second most incident in men in Brazil. Despite the available therapies, there is no treatment that is efficient for cases of advanced or metastatic cancer. Separated sulforaphane and vitamin D have shown promising anticancer activity in in vitro and in vivo studies, however, when used in clinical trials, their effects have been limited due to low bioavailability. Thus, the objective of this work was to evaluate the effects of the association of sulforaphane and vitamin D on cell viability, cytotoxicity, induction of apoptosis and DNA damage, oxidative stress and autophagy, clonogenic and migratory activity, cell cycle modulation, expression of genes and proteins in human prostate tumor cells obtained from metastases (DU145 and PC-3), in order to verify a possible synergistic effect between them. In DU145 cells after 72 hours of treatment, vitamin D decreased cell viability in neutral red assay and sulforaphane decreased cell viability in MTT and neutral red assay. In PC-3 cells, vitamin D and sulforaphane decreased cell viability in MTT and neutral red assay. The associated treatment (sulforaphane + vitamin D) decreased cell viability when compared to isolated treatments after 24 hours, but there was no change after 72 hours. Sulforaphane induced cytotoxicity and its association with vitamin D potentiated the cytotoxic effect of sulforaphane on both cell lines. Sulforaphane induced cell death by apoptosis and cell cycle arrest in DU145 (S and G2 phase) and PC-3 (G2 phase). Vitamin D and sulforaphane inhibited clonogenic activity in PC-3, but only sulforaphane decreased the ability to form colonies in DU145 cells. After the associated treatment, no difference was observed in the induction of apoptosis, cell cycle modulation or clonogenic capacity when compared to the isolated treatments. There was no change in cell migration at any of the concentrations tested, either in isolated or associated treatment in the two cell lines. Vitamin D and sulforaphane did not induce formation of reactive species and DNA damage. However, the associated treatment was genotoxic in DU145 and induced oxidative stress in DU145 (3 and 24 hours) and PC-3 (3 hours). Sulforaphane increased the autophagy and its association with vitamin D potentiated this effect in both cell lines. After the associated treatment in DU145 cells, the expression of MAPK1, CCNB1, CDK1 and CTNNB1 genes was increased. The proteins BAX, CASP8, CASP3, NRF2 and JNK were more expressed, and BCL2 less expressed in relation to the control. In PC-3 cells, there was an increase in the expression of the MAPK1 and CCNB1 genes and of the BAX and NRF2 proteins, and a decrease in the expression of the TNF gene and of the BCL2, CASP8 and JNK proteins. After predicting the interactions between targets, sulforaphane and vitamin D, the MAPK pathway was presented as a possible signaling pathway responsible for the effects found with the associated treatment. These results demonstrate that the association of sulforaphane and vitamin D has potential for use in prostate cancer therapy

Sulforaphane Combined with Vitamin D Induces Cytotoxicity Mediated by Oxidative Stress, DNA Damage, Autophagy, and JNK/MAPK Pathway Modulation in Human Prostate Tumor Cells

Prostate cancer ranks second in incidence worldwide. To date, there are no available therapies to effectively treat advanced and metastatic prostate cancer. Sulforaphane and vitamin D alone are promising anticancer agents in vitro and in vivo, but their low bioavailability has limited their effects in clinical trials. The present study examined whether sulforaphane combined with vitamin D at clinically relevant concentrations improved the cytotoxicity of the compounds alone towards DU145 and PC-3 human prostate tumor cells. To assess the anticancer activity of this combination, we analyzed cell viability (MTT assay), oxidative stress (CM-H2DCFDA), autophagy (fluorescence), DNA damage (comet assay), and protein expression (Western blot). The sulforaphane–vitamin D combination (i) decreased cell viability, induced oxidative stress, DNA damage, and autophagy, upregulated BAX, CASP8, CASP3, JNK, and NRF2 expression, and downregulated BCL2 expression in DU145 cells; and (ii) decreased cell viability, increased autophagy and oxidative stress, upregulated BAX and NRF2 expression, and downregulated JNK, CASP8, and BCL2 expression in PC-3 cells. Therefore, sulforaphane and vitamin D in combination have a potential application in prostate cancer therapy, and act to modulate the JNK/MAPK signaling pathway

Diallyl Disulfide Induces Chemosensitization to Sorafenib, Autophagy, and Cell Cycle Arrest and Inhibits Invasion in Hepatocellular Carcinoma

Hepatocellular carcinoma is the seventh most common type of cancer in the world, with limited treatment options. A promising strategy to treat cancer is to associate chemotherapeutics and plant bioactive compounds. Here, we examined whether diallyl disulfide (DADS; 50–200 μM) and sorafenib (SORA; 8 μM), either alone or in combination, were toxic to hepatocellular carcinoma cells (HepG2) in vitro. We assessed whether DADS and/or SORA induced cell death (LIVE/DEAD assay and autophagy) and cell cycle changes (flow cytometry), altered expression of key genes and proteins (RT-qPCR and Western blot), and modulated tumorigenesis signatures, such as proliferation (clonogenic assay), migration (wound healing), and invasion (inserts). The DADS + SORA combination elicited autophagic cell death by upregulating LC3 and NRF2 expression and downregulating FOS and TNF expression; induced the accumulation of cells in the G1 phase which thereby upregulated the CHEK2 expression; and inhibited invasion by downregulating the MMP2 expression. Predictive analysis indicated the participation of the MAPK pathway in the reported results. The DADS + SORA combination suppressed both cell invasion and clonogenic survival, which indicated that it dampened tumor growth, proliferation, invasion, and metastatic potential. Therefore, the DADS + SORA combination is a promising therapy to develop new clinical protocols

The Antitumoral/Antimetastatic Action of the Flavonoid Brachydin A in Metastatic Prostate Tumor Spheroids In Vitro Is Mediated by (Parthanatos) PARP-Related Cell Death

Metastatic prostate cancer (mPCa) is resistant to several chemotherapeutic agents. Brachydin A (BrA), a glycosylated flavonoid extracted from Fridericia platyphylla, displays a remarkable antitumoral effect against in vitro mPCa cells cultured as bidimensional (2D) monolayers. Considering that three-dimensional (3D) cell cultures provide a more accurate response to chemotherapeutic agents, this study investigated the antiproliferative/antimetastatic effects of BrA and the molecular mechanisms underlying its action in mPCa spheroids (DU145) in vitro. BrA at 60–100 μM was cytotoxic, altered spheroid morphology/volume, and suppressed cell migration and tumor invasiveness. High-content analysis revealed that BrA (60–100 µM) reduced mitochondrial membrane potential and increased apoptosis and necrosis markers, indicating that it triggered cell death mechanisms. Molecular analysis showed that (i) 24-h treatment with BrA (80–100 µM) increased the protein levels of DNA disruption markers (cleaved-PARP and p-γ-H2AX) as well as decreased the protein levels of anti/pro-apoptotic (BCL-2, BAD, and RIP3K) and cell survival markers (p-AKT1 and p-44/42 MAPK); (ii) 72-h treatment with BrA increased the protein levels of effector caspases (CASP3, CASP7, and CASP8) and inflammation markers (NF-kB and TNF-α). Altogether, our results suggest that PARP-mediated cell death (parthanatos) is a potential mechanism of action. In conclusion, BrA confirms its potential as a candidate drug for preclinical studies against mPCa

<i>Pouteria ramiflora</i> (Mart.) Radlk. extract: Flavonoids quantification and chemopreventive effect on HepG2 cells

<p><i>Pouteria ramiflora</i> (Mart.) Radlk., popularly known as curriola, is commonly used in Brazil as medicinal plant to treat worm infections, dysentery, pain, inflammation, hyperlipidemia, and obesity. At present the safety of this extract when used therapeutically in human remains to be determined. Thus, the aim of this study was to examine cytotoxicity, antiproliferative, and antimutagenic actions of this extract. The hydroalcoholic extract from <i>P. ramiflora</i> leaves consisted of flavonoids identified and quantified as myricetin-3-<i>O</i>-β-D-galactopyranoside (13.55 mg/g) and myricetin-3-<i>O</i>-α-L-rhamnopyranoside (9.61 mg/g). The extract exhibited cytotoxicity at concentrations higher than 1.5 µg/ml in human hepatocarcinoma (HepG2)and 2.5 µg/ml in non-tumoral primary gastric (GAS) cells using the MTT assay, and at concentrations higher than 3 µg/ml in HepG2 and 3.5 µg/ml in GAS cells by the neutral red assay. The extract did not show antiproliferative effect as evidenced by the nuclear division index (NDI). However, in the presence of benzo[a]pyrene (BaP) (positive control), an enhanced cytostatic effect in the NDI and flow cytometry was noted. It is of interest that when the extract was co-incubated with BaP a significant decrease in DNA damage was observed indicating an antimutagenic action. This protective effect might be attributed to myricetin and gallic acid found in <i>P. ramiflora</i> extract. The low cytotoxicity action and protective effect observed in the present study encourage further studies regarding other biological effects of <i>P. ramiflora</i>, as well as its potential use as a chemopreventive agent.</p