RESUMO
Gyrophoric acid (GA), a lichen secondary metabolite, has attracted more attention during the last years because of its potential biological effects. Until now, its effect in vivo has not yet been demonstrated. The aim of our study was to evaluate the basic physicochemical and pharmacokinetic properties of GA, which are directly associated with its biological activities. The stability of the GA in various pH was assessed by conducting repeated UV-VIS spectral measurements. Microsomal stability in rat liver microsomes was performed using Ultra-Performance LC/MS. Binding to human serum albumin (HSA) was assessed using synchronous fluorescence spectra, and molecular docking analysis was used to reveal the binding site of GA to HSA. In the in vivo experiment, 24 Sprague-Dawley rats (Velaz, Únetice, Czech Republic) were used. The animals were divided as follows. The first group (n = 6) included healthy males as control intact rats (âINT), and the second group (n = 6) included healthy females as controls (âINT). Groups three and four (âGA/n = 6 and âGA/n = 6) consisted of animals with daily administered GA (10 mg/kg body weight) in an ethanol-water solution per os for a one-month period. We found that GA remained stable under various pH and temperature conditions. It bonded to human serum albumin with the binding constant 1.788 × 106 dm3mol-1 to reach the target tissue via this mechanism. In vivo, GA did not influence body mass gain, food, or fluid intake during the experiment. No liver toxicity was observed. However, GA increased the rearing frequency in behavioral tests (p < 0.01) and center crossings in the elevated plus-maze (p < 0.01 and p < 0.001, respectively). In addition, the time spent in the open arm was prolonged (p < 0.01 and p < 0.001, respectively). Notably, GA was able to pass through the blood-brain barrier, indicating its ability to permeate into the brain and to stimulate neurogenesis in the hilus and subgranular zone of the hippocampus. These observations highlight the potential role of GA in influencing brain function and neurogenesis.
Assuntos
Simulação de Acoplamento Molecular , Ratos Sprague-Dawley , Animais , Ratos , Masculino , Feminino , Humanos , Microssomos Hepáticos/metabolismo , Concentração de Íons de Hidrogênio , Albumina Sérica Humana/metabolismo , Albumina Sérica Humana/química , Ligação ProteicaRESUMO
A combination of biochemical, biophysical and biological techniques was used to study calf thymus DNA interaction with newly synthesized 7-MEOTA-tacrine thiourea 12-17 and urea heterodimers 18-22, and to measure interference with type I and II topoisomerases. Their biological profile was also inspected in vitro on the HL-60 cell line using different flow cytometric techniques (cell cycle distribution, detection of mitochondrial membrane potential dissipation, and analysis of metabolic activity/viability). The compounds exhibited a profound inhibitory effect on topoisomerase activity (e.g. compound 22 inhibited type I topoisomerase at 1 µM concentration). The treatment of HL-60 cells with the studied compounds showed inhibition of cell growth especially with hybrids containing thiourea (14-17) and urea moieties (21 and 22). Moreover, treatment of human dermal fibroblasts with the studied compounds did not indicate significant cytotoxicity. The observed results suggest beneficial selectivity of the heterodimers as potential drugs to target cancer cells.
Assuntos
Acridinas/farmacologia , Antineoplásicos/farmacologia , Tacrina/farmacologia , Tioureia/farmacologia , Células A549 , Acridinas/síntese química , Acridinas/química , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Fibroblastos/efeitos dos fármacos , Células HL-60 , Humanos , Relação Estrutura-Atividade , Tacrina/química , Tioureia/químicaRESUMO
This study examines the binding properties of a series of newly synthetized tacrine derivatives 1-4 and their anticancer effects. Spectroscopic techniques (UV-Vis, fluorescence spectroscopy, thermal denaturation, and linear spectropolarimetry) and viscometry were used to study DNA binding properties and to determine the types of DNA interaction with the studied derivatives. The binding constants for the complexes with DNA were obtained using UV-Vis spectroscopic titrations (K = 1.6 × 10(4)-4.0 × 10(5) M(-1)) and electrophoretic methods were used to determine the effect of the derivatives on topoisomerase I and II activity. Monotacrine derivative 1 showed evidence of topoisomerase Irelaxation activity at a concentration of 30 × 10(-6) M, while bistacrine derivatives 2-4 produced a complete inhibition of topoisomerase Iat a concentration of 5 × 10(-6) M. The biological activities of the derivatives were studied using MTT-assay and flow cytometric methods (detection of mitochondrial membrane potential and measurement of cell viability) following incubation of 24 and 48 h with human leukemic cancer cell line HL60. The ability of the derivatives to impair cell proliferation was also tested through the analysis of cell cycle distribution.
Assuntos
Antineoplásicos/farmacologia , Tacrina/farmacologia , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase II/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , DNA Topoisomerases/metabolismo , Células HL-60 , Humanos , Substâncias Intercalantes/química , Substâncias Intercalantes/farmacologia , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Tacrina/química , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase II/químicaRESUMO
A novel series of trisubstituted acridines were synthesized with the aim of mimicking the effects of BRACO19. These compounds were synthesized by modifying the molecular structure of BRACO19 at positions 3 and 6 with heteroacyclic moieties. All of the derivatives presented in the study exhibited stabilizing effects on the human telomeric DNA quadruplex. UV-vis spectroscopy, circular dichroism, linear dichroism and viscosimetry were used in order to study the nature of the DNA binding in more detail. The results show that all of the novel derivatives were able to fold the single-stranded DNA sequences into antiparallel G-quadruplex structures, with derivative 15 exhibiting the highest stabilizing capability. Cell cycle analysis revealed that a primary trend of the "braco"-like derivatives was to arrest the cells in the S- and G2M-phases of the cell cycle within the first 72h, with derivative 13 and BRACO19 proving particularly effective in suppressing cell proliferation. All studies derivatives were less toxic to human fibroblast cell line in comparison with HT 29 cancer cell line.
Assuntos
Acridinas/química , Acridinas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , DNA/metabolismo , Quadruplex G/efeitos dos fármacos , Animais , Bovinos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/química , Humanos , Ligantes , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismoRESUMO
Preferential uptake of photosensitizer by tumour tissue is an elementary prerequisite of effective and successful photodynamic therapy (PDT). Therefore intracellular concentration of photosensitizer is one of the limiting factors affecting PDT efficiency. Hypericin (HY) has found applications in photodynamic diagnostics solely due to its high specificity for tumour cells and tissues. However, here we suggest that not only HY uptake, but importantly also the cell ability to manage oxidative stress induced by HY-PDT can be important decisive factors finally affecting the cell death response. We showed that despite the higher accumulation of HY in FHC human fetal colon epithelial cells compared to HT-29 colon adenocarcinoma cells, the cytotoxic effects of this photosensitizer were more pronounced in the latter cell line, and this was associated with enhanced accumulation of HY-PDT-induced reactive oxygen species (ROS).
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Células Epiteliais/efeitos dos fármacos , Perileno/análogos & derivados , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêutico , Antracenos , Linhagem Celular , Colo/citologia , Células Epiteliais/efeitos da radiação , Feto , Humanos , Microscopia de Fluorescência , Perileno/uso terapêutico , Perileno/toxicidade , Fármacos Fotossensibilizantes/toxicidade , Espécies Reativas de Oxigênio/metabolismoRESUMO
Since many studies have suggested the impact of dietary polyunsaturated fatty acids on cancer progression and prognosis, there is an assumption of possible pre-sensitizing effects of their application in combined treatment. The present work evaluates modulation of photodynamic therapy (PDT) with hypericin by pre-treatment with n-3 and n-6 fatty acids in HT-29 and HeLa tumour cells. We observed stimulation of cytotoxic effects by docosahexaenoic acid (n-3) and arachidonic acid (n-6) in several stages of action in both cell lines. Treatment with either fatty acids or photodynamic therapy alone induced apoptosis in a dose- and time-dependent manner; however the effect was even more striking in mutual combination applied as pre-treatment with fatty acids prior to photodynamic therapy. Moreover, the combination also induced changes in membrane lipid composition leading to alteration in cell membrane fluidity. Increased toxicity of combined treatment was also confirmed by the presence of oxidative stress demonstrated by ROS production, RNS accumulation and increased presence of lipoperoxides. In conclusion, we suggest that pre-treatment with polyunsaturated fatty acids may contribute to cytotoxic effects induced by photodynamic therapy with hypericin.
Assuntos
Antineoplásicos/uso terapêutico , Apoptose , Ácidos Graxos Insaturados/toxicidade , Estresse Oxidativo , Perileno/análogos & derivados , Antracenos , Ácido Araquidônico/toxicidade , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Ácidos Docosa-Hexaenoicos/toxicidade , Células HT29 , Humanos , Fluidez de Membrana , Perileno/uso terapêutico , FotoquimioterapiaRESUMO
Photodynamic therapy (PDT) is a flexible multi-target therapeutic approach. One of the main requirements of successful PDT is sufficient intracellular concentration of an applicable photosensitizer. Mechanisms of anticancer drug elimination by tumour cells are mostly linked to the elevated expression and activity of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), breast cancer resistance protein (BCRP) and P450 monooxygenases. The interaction of hypericin with this cell drug-defence system is still unclear. We report here for the first time increased activity of MRP1 and BCRP in HT-29 colon cancer cells treated with hypericin per se. On the contrary, pre-treatment with proadifen (SKF525A) affected the function of MRP1 and BCRP leading to increased hypericin content, which might indicate a possible link between proadifen and these ABC transporter proteins. Subsequent enhanced intracellular oxidative stress was accompanied by loss of mitochondrial membrane potential, activation of caspase-9 and -3, PARP cleavage and onset of apoptosis. In conclusion, our study suggests that drug efflux transporters MRP1 and BCRP affect the pharmacokinetics of hypericin in HT-29 colon adenocarcinoma cells, and the action of hypericin-mediated PDT (HY-PDT) should be modulated by pre-treatment with their specific inhibitors.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenocarcinoma/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Perileno/análogos & derivados , Fármacos Fotossensibilizantes/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Adenocarcinoma/secundário , Antracenos , Caspase 3/metabolismo , Caspase 9/metabolismo , Inibidores Enzimáticos/farmacologia , Células HT29 , Humanos , Luz , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Oxigenases de Função Mista/metabolismo , Perileno/farmacologia , Fotoquimioterapia , Proadifeno/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Photodynamic therapy with hypericin (HY-PDT) is known as an efficient modality for treatment of various cancerous and non-cancerous diseases. Although the role of p53 protein in cell death signaling is well established, relatively little is known of its impact on the efficiency of HY-PDT. Comparison of sensitivity and long-term survival of p53-null versus wt-p53-expressing HCT-116 cells is reported here. The lack of p53 function did not affect cell proliferation or attenuate the initial phases of programmed cell death. However, analyses of apoptosis in the final stages revealed suppression of its incidence and delayed activation of caspase-3 in p53-null cells. Significantly higher clonogenic ability, especially in hypoxia, was identified in the case of p53-null cells. Induction of Mcl-1 and Bax levels were more prominent in wt-pt53 cells. Interestingly, the level of Bcl-2 did not react to HY-PDT at all, in both cell lines. Bringing the evidence together, we prove that despite insignificant impact on overall toxicity, expression of p53 affects the clonogenic efficiency of HCT-116 cells. Since destruction of tumor tissue and its vascular system by PDT tends to lead to hypoxia, superior survival of p53-deficient tumor cells under given conditions might result in recurrence of cancer diseases.
Assuntos
Neoplasias do Colo/patologia , Perileno/análogos & derivados , Fotoquimioterapia , Proteína Supressora de Tumor p53/metabolismo , Antracenos , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ensaio de Unidades Formadoras de Colônias , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/efeitos da radiação , Perileno/farmacologia , Perileno/uso terapêutico , Fosfatidilserinas/metabolismo , Fatores de Tempo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Recombinant human erythropoietin is widely used to treat anemia associated with cancer and with the myelosuppressive effects of chemotherapy, particularly platinum-based regimens. Erythropoietin is the principal regulator of erythroid cell proliferation, differentiation, and apoptosis. Recently, the antiapoptotic and proliferative effects of erythropoietin on nonhematopoietic cells were also established. We now show the effect of erythropoietin treatment on the response of A2780 and SKOV3 ovarian carcinoma cell lines to photodynamic therapy (PDT) using hypericin. SKOV3 exhibited an increased resistance to hypericin when cells were treated with erythropoietin. This resistance was reversed by treatment of SKOV3 cells with the specific Janus kinase 2 kinase inhibitor AG490 or the tyrosine kinase inhibitor genistein. These results support a role for the specific erythropoietin-induced Janus kinase 2/STAT signal transduction pathway in PDT resistance. Evidence of erythropoietin signaling was obtained by the demonstration of Akt phosphorylation in both A2780 and SKOV3 cells. Erythropoietin-treated SKOV3 cells exhibited decreased apoptosis induced by hypericin, an effect that was blocked by the phosphoinositide 3-kinase/Akt inhibitor wortmannin. These results may have important implications for ovarian cancer patients undergoing PDT and receiving erythropoietin.
Assuntos
Apoptose/efeitos dos fármacos , Eritropoetina/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Fotoquimioterapia , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Feminino , Humanos , Neoplasias Ovarianas/patologia , Proteínas RecombinantesRESUMO
The synthesis of novel 1',1''-(acridin-3,6-diyl)-3',3''-dialkyldiureas was reported. Their biological activity to inhibit cell proliferation was assessed by a MTT assay on two cell lines, HeLa and HCT-116, at micromolar concentration. 1',1''-(Acridin-3,6-diyl)-3',3''-dihexyldiurea hydrochloride was active on a HCT-116 cell line with an IC(50) value of 3.1 microM. The interaction of these compounds with calf thymus DNA was investigated by a variety of spectroscopic techniques including UV-vis, fluorescence and CD spectroscopy. From spectrofluorimetric titrations, binding constants for the DNA-drug complexes were determined (K=0.9-4.2x10(5) M(-1)). Antiproliferative activity of synthesized derivatives might be related to their intercalation into DNA.
Assuntos
Acridinas/química , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , DNA/química , Proflavina/química , Ureia/síntese química , Ureia/toxicidade , Alquilação , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Bovinos , Linhagem Celular Tumoral , DNA/genética , Humanos , Estrutura Molecular , Fotoquímica , Espectrofotometria , Relação Estrutura-Atividade , Titulometria , Ureia/químicaRESUMO
One proposal to increase the efficiency of photodynamic therapy (PDT) is to accompany photosensitization with other treatment modalities, including modulation of arachidonic acid (AA) metabolism. The aim of this study was to evaluate the effectiveness of a combined modality approach employing 48 and 24 h pretreatment with various inhibitors of lipoxygenase (LOX; nordihydroguaiaretic acid, esculetin, AA-861, MK-886 and baicalein), cyclooxygenase (COX; diclofenac, flurbiprofen, ibuprofen, indomethacin, SC-560 and rofecoxib) and cytochrome P450-monooxygenase (proadifen) pathways, followed by hypericin-mediated PDT. Cytokinetic parameters like MTT assay, adherent and floating cell numbers, viability and cell cycle distribution analysis were examined 24 h after hypericin activation. Pretreatment of human colon cancer cells HT-29 prior to PDT with 5-LOX inhibitor MK-886 as well as 5, 12-LOX and 12-LOX inhibitors (esculetin and baicalein, respectively) resulted in significant and dose-dependent effects on all parameters tested. Pretreatment with diclofenac, flurbiprofen, ibuprofen and indomethacin, the nonspecific COX inhibitors, promoted hypericin-mediated PDT, but these effects were probably COX-independent. In contrast, application of SC-560 and rofecoxib, specific inhibitors of COX-1 and COX-2, respectively, attenuated PDT. Inhibition of P450 monooxygenase with proadifen implied also the significance of this metabolic pathway in cell survival and cell resistance to hypericin photocytotoxicity. In conclusion, our results testify that application of diverse inhibitors of AA metabolism may have different consequences on cellular response to hypericin-mediated PDT and that some of them could be considered for potentiation of PDT.
Assuntos
Adenocarcinoma/tratamento farmacológico , Ácido Araquidônico/antagonistas & inibidores , Neoplasias do Colo/tratamento farmacológico , Perileno/análogos & derivados , Fotoquimioterapia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Antracenos , Ácido Araquidônico/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Células HT29 , Humanos , Perileno/farmacologia , Perileno/uso terapêuticoRESUMO
BACKGROUND: Hypericin-mediated photodynamic therapy (HY-PDT) has recently captured increased attention as an alternative minimally invasive anticancer treatment, although cancer cells may acquire resistance. Therefore, combination treatments may be necessary to enhance HY-PDT efficacy. Histone deacetylase inhibitors (HDACis) are often used in combination treatments due to their non-genotoxic properties and epigenetic potential to sensitize cells to external stimuli. Therefore, this study attempts for the first time to investigate the therapeutic effects of HDACis in combination with visible light-mediated PDT against cancer. Specifically, the colorectal cancer cell model was used due to its known resistance to HY-PDT. RESULTS: Two chemical groups of HDACis were tested in combination with HY-PDT: the hydroxamic acids Saha and Trichostatin A, and the short-chain fatty acids valproic acid and sodium phenylbutyrate (NaPB), as inhibitors of all-class versus nuclear HDACs, respectively. The selected HDACis manifest a favorable clinical toxicity profile and showed similar potencies and mechanisms in intragroup comparisons but different biological effects in intergroup analyses. HDACi combination with HY-PDT significantly attenuated cancer cell resistance to treatment and caused the two HDACi groups to become similarly potent. However, the short-chain fatty acids, in combination with HY-PDT, showed increased selectivity towards inhibition of HDACs versus other key epigenetic enzymes, and NaPB induced the strongest expression of the otherwise silenced tumor suppressor CDKN1A, a hallmark gene for HDACi-mediated chromatin modulation. Epigenetic regulation of CDKN1A by NaPB was associated with histone acetylation at enhancer and promoter elements rather than histone or DNA methylation at those or other regulatory regions of this gene. Moreover, NaPB, compared to the other HDACis, caused milder effects on global histone acetylation, suggesting a more specific effect on CDKN1A chromatin architecture relative to global chromatin structure. The mechanism of NaPB + HY-PDT was P53-dependent and likely driven by the HY-PDT rather than the NaPB constituent. CONCLUSIONS: Our results show that HDACis potentiate the antitumor efficacy of HY-PDT in colorectal cancer cells, overcoming their resistance to this drug and epigenetically reactivating the expression of CDKN1A. Besides their therapeutic potential, hypericin and these HDACis are non-genotoxic constituents of dietary agents, hence, represent interesting targets for investigating mechanisms of dietary-based cancer prevention.
Assuntos
Antineoplásicos/farmacologia , Cromatina/efeitos dos fármacos , Neoplasias do Colo/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Fotoquimioterapia/métodos , Antracenos , Antineoplásicos/química , Linhagem Celular Tumoral , Cromatina/genética , Neoplasias do Colo/tratamento farmacológico , Metilação de DNA/efeitos dos fármacos , Sinergismo Farmacológico , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Perileno/análogos & derivados , Perileno/farmacologia , Fenilbutiratos/química , Fenilbutiratos/farmacologia , Ácido Valproico/química , Ácido Valproico/farmacologiaRESUMO
Proadifen (SKF-525A) is a P450 monooxygenase inhibitor with potential anti-proliferative activity and the ability to potentiate the toxicity of hypericin-mediated photodynamic therapy and mitoxantrone via alteration of ABC transport proteins. Elevated expression of some ABC transporters may also determine the efficacy of cisplatin-based chemotherapy. Thus, the purpose of this study was to investigate the ability of proadifen to sensitize A2780 and A2780cis ovarian cancer cells to cisplatin (CDDP). Herein, we show for the first time that proadifen sensitized resistant ovarian cancer cells to CDDP-induced cell death. The chemosensitizing effect of proadifen on CDDP action was also confirmed by MTT assays in multicellular spheroids. The possible mechanisms responsible for the enhanced cytotoxicity of proadifen/CDDP combined treatment may be attributed to a decrease of reduced relative glutathione levels, downregulation of multidrug resistance-associated proteins 1 and 2 (MRP1, MRP2) and attenuation of survivin expression. Taken together, our results indicate that proadifen is a promising compound for further in vivo experiments related to overcoming multidrug resistance and sensitization of resistant ovarian carcinoma to CDDP.
Assuntos
Cisplatino/farmacologia , Proadifeno/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Regulação para Baixo , Feminino , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Membranas Mitocondriais/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Survivina , Regulação para Cima , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Multidrug resistance caused by the overexpression of ABC transporter proteins in cancer cells remains a major obstacle limiting chemotherapy efficacy. Drugs inhibiting these transporters have been shown to increase the anti-proliferative properties of chemotherapeutics. As we previously described, proadifen, a P450 monooxygenase inhibitor, might also be able to inhibit some ABC transporters, including breast cancer resistance protein (BCRP). Because mitoxantrone (MTX) is a strong BCRP substrate and is often used in the treatment of leukemia, we investigated the effect of 24 h proadifen pre-treatment on the cytotoxicity of MTX in leukemic cell lines that are sensitive to MTX (HL-60) and MTX-resistant ABCG2-overexpressing subclone (cBCRP). We show for the first time that proadifen is able to enhance the cytotoxic properties of MTX in cBCRP cells, particularly through the inhibition of BCRP expression and activity. This proadifen-MTX synergism was also mediated by the inhibition of various cellular proteins engaged in apoptosis, including Mc-1, Bcl-xL, survivin and activation of procaspase-3. Proadifen also decreased the expression of γH2AX, which is involved in the recruitment of reparation proteins. Moreover, the inhibition of DNA damage repair proteins Ku86 and B23 after proadifen treatment indicate a possible role of proadifen in DNA repair blockage, thus suppressing the reparation rate of MTX-induced DSBs.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Promielocítica Aguda/patologia , Mitoxantrona/farmacologia , Proadifeno/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Pretreatment with 5-LOX pathway inhibitor MK-886 potentiates cytotoxic effects of photodynamic therapy mediated by natural photosensitizer, hypericin. In this study, we focused on elucidating mechanisms beyond the increased efficacy of combined treatment. METHODS: Metabolic activity/viability, caspase-3 activation/mitochondrial membrane potential dissipation, intracellular hypericin level, glutathione level and redox status (NAD(P)H/oxidized flavins ratio) analyses, as well as drug efflux assays, were performed by flow cytometry. Changes in protein expression of ATP-binding cassette transporters, GDF-15 and other selected proteins were evaluated by Western blotting. Silencing of gdf-15 was carried out to verify its role in response to treatment. RESULTS: MK-886 pretreatment led to a concentration-dependent increase in intracellular hypericin content, accompanied by changes in ATP-binding cassette transporters levels and efflux efficiency. Intracellular accumulation of cytokine GDF-15 correlated with increased cell death markers; however, the impact of gdf-15 silencing on the evaluated markers was negligible. A marked decrease in the glutathione level of a majority of cells was observed after more toxic combination treatment. CONCLUSION: The significant increase in cell death markers after combination treatment confirms the potentiating effect of MK-886 on hypericin-mediated photodynamic therapy in HT-29 and MCF-7 cells. Although BCRP downregulation was not confirmed as leading mechanism responsible for elevated levels of hypericin content, changes in expression and efflux activity of ABC transporters caused by MK-886 suggest its potential in combination treatment with drugs that are substrates of these transporters, predominantly MRP1. However, complex cellular response to MK-886 pretreatment needs to be considered and further elucidated.
Assuntos
Indóis/farmacologia , Neoplasias/tratamento farmacológico , Perileno/análogos & derivados , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Transportadores de Cassetes de Ligação de ATP/biossíntese , Antracenos , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Glutationa/biossíntese , Fator 15 de Diferenciação de Crescimento/antagonistas & inibidores , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Oxirredução , Perileno/farmacologia , RNA Interferente Pequeno/metabolismoRESUMO
HL-60 cancer cells were treated with a series of novel acridine derivatives (derivatives 1-4) in order to test the compounds' ability to inhibit both cancer cell growth and topoisomerase I and II activity. Binding studies of derivatives 1-4 with calf thymus DNA were also performed using a number of techniques (UV-Vis and fluorescence spectroscopy, thermal denaturation, linear dichroism and viscometry) to determine the nature of the interaction between the compounds and ctDNA. The binding constants for the complexes of the studied acridine derivatives with DNA were calculated from UV-Vis spectroscopic titrations (K=3.1×10(4)-2.0×10(3)M(-1)). Some of the compounds showed a strong inhibitory effect against Topo II at the relatively low concentration of 5µM. Topo I/II inhibition mode assays were also performed and verified that the novel compounds are topoisomerase suppressors rather than poisons. The biological activities of derivatives were studied using MTT assay and flow cytometric methods (detection of mitochondrial membrane potential, measurement of cell viability) after 24 and 48h incubation. The ability of derivatives to impair cell proliferation was tested by an analysis of cell cycle distribution.
Assuntos
Acridinas/farmacologia , Proliferação de Células/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Leucemia Promielocítica Aguda/tratamento farmacológico , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase II/farmacologia , Acridinas/síntese química , Acridinas/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Química Farmacêutica , DNA/química , DNA/metabolismo , Relação Dose-Resposta a Droga , Células HL-60 , Temperatura Alta , Humanos , Leucemia Promielocítica Aguda/enzimologia , Leucemia Promielocítica Aguda/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Tecnologia Farmacêutica/métodos , Fatores de Tempo , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/metabolismo , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/metabolismo , ViscosidadeRESUMO
St. John's wort (SJW, Hypericum perforatum L.) is a commonly used natural antidepressant responsible for the altered toxicity of some anticancer agents. These interactions have been primarily attributed to the hyperforin-mediated induction of some pharmacokinetic mechanisms. However, as previously demonstrated by our group, hypericin induces the expression of two ABC transporters: multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP). Because cisplatin (CDDP) and mitoxantrone (MTX) are potential substrates of ABC transporters, we investigated the effect of 24h hypericin pre-treatment on the cytotoxicity of CDDP and MTX in human cancer cell lines. CDDP-sensitive and -resistant ovarian adenocarcinoma cell lines A2780/A2780cis, together with HL-60 promyelocytic leukemia cells and ABCG2-over-expressing cBCRP subclone, were used in our experiments. We present CDDP cytotoxicity attenuated by hypericin pre-treatment in both A2780 and A2780cis cells and MTX cytotoxicity in HL-60 cells. In contrast, hypericin potentiated MTX-induced death in cBCRP cells. Interestingly, hypericin did not restore cell proliferation in rescued cells. Nevertheless, hypericin did increase the expression of MRP1 transporter in A2780 and A2780cis cells indicating the impact of hypericin on certain resistance mechanisms. Additionally, our results indicate that hypericin may be the potential substrate of BCRP transporter. In conclusion, for the first time, we report the ability of hypericin to affect the onset and/or progress of CDDP- and MTX-induced cell death, despite strong cell cycle arrest. Thus, hypericin represents another SJW metabolite that might be able to affect the effectiveness of anti-cancer drugs and that could interact with ABC transporters, particularly with BCRP.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Mitoxantrona/farmacologia , Perileno/análogos & derivados , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antracenos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Interações Medicamentosas , Humanos , Hypericum , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Perileno/farmacologia , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Photodynamic therapy (PDT) is a highly efficient approach for tumour therapy, though it also has its drawbacks, too. There are multiple mechanisms involved in cell death regulation that can be successfully targeted for improvement of PDT in particular cases. We assumed, however, that the potential to manage radical stress might be the primary factor responsible for resistance to hypericin-mediated PDT (HY-PDT). METHODS: We compared the sensitivity of six colon-derived cancer cell lines to HY-PDT at IC50 equitoxic doses acquired by formazan-based (MTT) assay. Intracellular hypericin content, cell survival/metabolic activity, caspase-3 activation/mitochondrial membrane potential dissipation, apoptosis, glutathione level, redox status (NAD(P)H/oxidized flavins ratio) and Western blot analyses of proteins relevant in apoptosis regulation were measured to demonstrate differences between tested cell lines. RESULTS: Analyses revealed a whole spectrum of responses from insignificant to high cytotoxicity, despite the MTT-based "equitoxicity". Further critical evaluation of multiple parameters linked to cell physiology and proteomics proved that intracellular hypericin content, glutathione level or redox status demonstrate partial but not direct correlation with resistance to HY-PDT, when considered separately. However, their logical conjunction did copy the trend of cellular resistance. CONCLUSIONS: We may conclude that intracellular level of hypericin and glutathione together with redox state of the target cell represent a potential combination of parameters responsible for the primary cytotoxicity of HY-PDT. We also present evidence that cytotoxic assays, such as the MTT, should be accompanied with other tests of cell survival/cytotoxicity in order to avoid incorrect conclusions.
Assuntos
3-Hidroxiesteroide Desidrogenases/metabolismo , Neoplasias do Colo/química , Neoplasias do Colo/tratamento farmacológico , Glutationa/metabolismo , Perileno/análogos & derivados , Fotoquimioterapia , Antracenos , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos da radiação , Humanos , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Perileno/análise , Perileno/uso terapêutico , Fármacos Fotossensibilizantes/análise , Fármacos Fotossensibilizantes/farmacocinética , Fármacos Fotossensibilizantes/uso terapêuticoRESUMO
Proadifen (SKF-525A) is a well-known inhibitor of cytochrome P450 monooxygenases. Besides the prevention of drug metabolism it affects the proliferation of cancer cells, although the mechanisms of possible anti-cancer activity of proadifen have not been fully understood yet. The aim of this study therefore was to evaluate the potential anti-proliferative effect of proadifen on HT-29 colon cancer cells. Our results show that proadifen inhibited the growth of HT-29 cells by the accumulation of cells in the G1 phase of the cell cycle, reduction of metabolic activity and colony formation and by the induction of apoptosis. Analyses of Western blots and flow cytometry revealed time- and dose-dependent phosphatidylserine externalization, caspase-3 activation and PARP cleavage. Intense upregulation of NAG-1 and ATF3 and downregulation of Mcl-1 and Egr-1 were also observed. Further investigation showed that NAG-1 gene silencing by siRNA had no effect on the pro-apoptotic action of proadifen. In contrast, we found that AR-A014418, the specific inhibitor of glycogen synthase kinase-3 ß (GSK-3ß), significantly decreased proadifen-induced apoptosis. Inactivation of GSK-3ß (phosphorylation at serine 9) resulted in changes in phosphatidylserine externalization and caspase-3 activation. These data suggest that GSK-3ß is an important factor in the induction of apoptosis in HT-29 colon cancer cells treated with proadifen.