RESUMO
Age-related hearing loss (AHL) is the most common sensory disorder of aged population. Currently, one of the most important sources of experimental medicine for AHL is medicinal plants. This study performed the first investigation of the effect of thymoquinone (TQ), a potent antioxidant, on AHL. Here, we used inbred C57BL/6J mice (B6 mice) as a successful experimental model of the early onset of AHL. The behavioral assessment of hearing revealed that the injection of a high dose of TQ (40 mg/kg; TQ40) significantly improved the auditory sensitivity of B6 mice at all tested frequencies (8, 16 and 22 kHz). Histological sections of cochlea from B6 mice injected with a low dose (20 mg/kg; TQ20) and high dose showed relatively less degenerative signs in the modiolus, hair cells and spiral ligaments, the main constituents of the cochlea. In addition, TQ40 completely restored the normal pattern of hair cells in B6 mice, as shown in scanning electron micrographs. Our data indicated that TQ20 and TQ40 reduced levels of Bak1-mediated apoptosis in the cochlea of B6 mice. Interestingly, the level of Sirt1, a positive regulator of autophagy, was significantly increased in B6 mice administered TQ40. In conclusion, TQ relieves the symptoms of AHL by downregulating Bak1 and activating Sirt1 in the cochlea of B6 mice.
Assuntos
Antioxidantes/farmacologia , Benzoquinonas/farmacologia , Cóclea/efeitos dos fármacos , Audição/efeitos dos fármacos , Presbiacusia/tratamento farmacológico , Sirtuína 1/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Limiar Auditivo/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cóclea/metabolismo , Cóclea/fisiopatologia , Cóclea/ultraestrutura , Modelos Animais de Doenças , Feminino , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/ultraestrutura , Camundongos Endogâmicos C57BL , Presbiacusia/metabolismo , Presbiacusia/patologia , Presbiacusia/fisiopatologia , Transdução de Sinais , Sirtuína 1/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genéticaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Shikonin, one of the main active ingredients of Chinese herbal medicine Lithospermum erythrorhizon, has been widely used to treat various disease including virus infection and inflammation in clinical. Its anti-tumor activity has been recorded in "Chinese herbal medicine". Recently, some studies about its anti-glioma effects have been reported. However, little is known about the molecular pharmacological activity of Shikonin in glioma. AIM: This study aimed to systematically uncover and validate the pharmacological mechanism of Shikonin against glioma. MATERIAL AND METHODS: Network pharmacology approach, survival analysis, and Pearson co-expression analysis were performed to uncover and test the pharmacological mechanisms of Shikonin in glioma. Apoptosis assay, Caspase-3 activity assay and immunoblot analysis were practiced to validate the mechanisms. RESULTS: Network pharmacology results suggested, anti-glioma effect of Shikonin by interfering endoplasmic reticulum (ER) stress-mediated tumor apoptosis targeting Caspase-3, and Bax/Bak-induced mitochondrial outer membrane permeabilization (MOMP) triggering cancer cell apoptosis. Survival analysis suggested the association of CASP3 with glioma (P < 0.05). Pearson correlation analysis indicated possible interaction of CASP3 with PERK through positive feedback regulation. Shikonin or in combination with 14G2a induced cell apoptosis in oligodendroglioma Hs683 cells in a dose-dependent manner with at a maximum apoptosis rate of 33%-37.5%, and 73%-77% respectively. Immunoblot analysis showed that Shikonin increased Caspase-3 activity to about 4.29 times, and increased 9 times when it combined with 14G2a. Shikonin increased also the expression levels of the proteins PERK and CHOP by about 4.4 and 5.6 folds, respectively, when it combined with 14G2a. CONCLUSIONS: This study highlights the pharmacological mechanisms of Shikonin in the induction of tumor apoptosis in glioma cells.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glioma/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Naftoquinonas/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/fisiologia , Humanos , Membranas Mitocondriais/fisiologiaRESUMO
BACKGROUND: Supplementation of the nicotinamide adenine dinucleotide (NAD) precursor nicotinamide riboside (NR) has recently been shown to increase life-span of cells, tissues, and entire organisms. [Correction added on 13 December 2019, after first online publication: In the preceding sentence, "adenine nicotinamide" was revised to "nicotinamide adenine."] The impact of NR on platelet longevity has not been tested. STUDY DESIGN AND METHODS: A pool-and-split design of buffy coat derived platelet concentrates (PCs) was used. One arm was treated with cumulative doses of NR-triflate, the control arm with sodium triflate. Storage lesion was monitored for 23 days. Platelet metabolic and functional parameters were tested. Clearance of human platelets was measured in a mouse model of transfusion. RESULTS: Total intracellular NAD levels in platelets decreased two-fold from 4.8 ± 0.5 fmol (mean ± SD, n = 6) to 2.1 ± 1.8 fmol per 103 control cells, but increased almost 10-fold to 41.5 ± 4.1 fmol per 103 NR treated platelets. This high intracellular NAD level had no significant impact on platelet count, mean platelet volume, swirling, nor on lactate and glucose levels. Platelet aggregation and integrin αIIb ß3 activation declined steadily and comparably in both conditions. GPIbα levels were slightly lower in NR-treated platelets compared to control, but this was not caused by reduced receptor shedding because glycocalicin increased similarly. Apoptotic markers cytochrome c, Bcl-xL, cleaved caspase-3, and Bak were not different throughout storage for both conditions. Platelet survival in a mouse model of transfusion was not different between NR-treated and control platelets. CONCLUSION: Platelets carry the cellular machinery to metabolize NR into NAD at rates comparable to other eukaryotic cells. Unlike those cells, platelet life-span cannot be prolonged using this strategy.
Assuntos
Plaquetas/metabolismo , Preservação de Sangue , NAD/metabolismo , Niacinamida/análogos & derivados , Agregação Plaquetária/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Plaquetas/citologia , Caspase 3/metabolismo , Citocromos c/metabolismo , Humanos , Niacinamida/farmacologia , Compostos de Piridínio , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
BACKGROUND: As an effective antimalarial medicine, Dihydroartemisinin (DHA) has therapeutic potential on human cervical cancer. However, its working mechanism has not been elucidated. PURPOSE: This study aimed to investigate the reversal effect of DHA on human cervical cancer HeLa cells, and explored its mechanism of action in vitro and in vivo. STUDY DESIGN/METHODS: The effect and mechanism of DHA on HeLa cells was examined by using CCK-8 assay, flow cytometry, transmission electron microscopy, immunofluorescence, and Western blot analysis in human hepatocellular carcinoma cells. RESULTS: In this study, it was confirmed that DHA had statistically equivalent anti-tumor efficiency in HeLa cells with a clinical chemotherapeutic agent of cisplatin. Meanwhile, DHA triggered autophagy, where LC3B-II expression was dose-dependently increased. Further, it was revealed that DHA promotes reactive oxygen species (ROS) generation, with DNA double-strand breaks (DSB) damage, as up-regulation of γH2AX protein and foci formation. Interestingly, we firstly demonstrated that DHA induced autophagy through promotion of the phosphorylation of Bcl-2 (Ser70), independent of the phosphorylated JNK1/2 (Thr183/Tyr185). Moreover, DHA-treated HeLa cells displayed an increase in the pro-autophagic protein Beclin-1 with downregulated the phospho-mTOR (Ser2448). Furthermore, upregulated pro-apoptotic protein Bak-1, but not Bax, suggesting Bak-1 is included in DHA-induced autophagy. CONCLUSION: Therefore, DHA upregulates the phosphorylation of Bcl-2 (Ser70) and mTOR (Ser2448) and induces autophagic cell death in Hela cells. This study provided a mechanism to support DHA, an autophagy inducer, as a potential therapeutic agent for human cervical cancer.
Assuntos
Antimaláricos/farmacologia , Antineoplásicos/farmacologia , Artemisininas/farmacologia , Autofagia/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/química , Apoptose , Proteína Beclina-1/metabolismo , Cisplatino/farmacologia , Quebras de DNA de Cadeia Dupla , Feminino , Células HeLa , Humanos , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima , Neoplasias do Colo do Útero , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
Cardiomyocyte apoptosis is the major risk factor for the development of heart failure (HF). The purpose of this study was to evaluate the effects of Gamma-aminobutyric acid (GABA) tea on hypertension-induced cardiac apoptotic pathways in spontaneously hypertensive rats (SHR). In order to reveal the mechanisms, 36 male SHR at eight weeks of age, 200 g were divided into six groups. One group was fed water as a control group. Other rats were administered one of the following treatments: GABA tea at dose 150 and 300 mg/kg/day as low GABA tea (LGT) and high GABA tea (HGT) groups, respectively, pure GABA at dose 150 and 300 mg/kg/day as LG and HG groups, respectively, green tea (GT) as control of LGT and HGT groups. After 12 weeks, cardiac tissues were analyzed by histological analysis, western blotting, and TUNEL assays. GABA tea, GT, and pure GABA decreased hypertension-induced cardiac abnormalities, including abnormal myocardial architecture. In addition, GABA tea, GT, and pure GABA dramatically increased anti-apoptotic protein, Bcl2. Furthermore, GABA tea, GT, and pure GABA also decreased activated-caspase 9 and activated-caspase 3. Additionally, the survival associated protein IGF-I and PI3K/Akt were enhanced in cardiac tissues upon treatment. Our results showed an optimistic anti-apoptotic and pro-survival effects of GABA tea treatment against hypertensive rat hearts.
Assuntos
Apoptose/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Chá/química , Ácido gama-Aminobutírico/farmacologia , Animais , Caspase 3/metabolismo , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Hipertensão/patologia , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Endogâmicos SHR , Receptores de Somatomedina/metabolismo , Chá/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Ácido gama-Aminobutírico/uso terapêuticoRESUMO
A Moringa oleifera seed lectin (MOSL) was purified by using chitin column with the molecular mass of 17±1kDa. The lectin agglutinated mouse, cow and human erythrocytes and the hemagglutination activity was inhibited by methyl-α-d-mannopyranoside, methyl-ß-d-galactopyranoside, lactose and glucose. The lectin exhibited 100% hemagglutination activity at the pH range from 8.0 to 9.0 and temperature range from 30 to 60°C. Additionally, the lectin gradually lost its activity in the presence of urea but the activity abolish completely when treated with EDTA. MOSL showed mild toxicity against brine shrimp nauplii with a LC50 value of 131.0µg/ml. Antiproliferative activity was studied against Ehrlich ascites carcinoma (EAC) cells and 71.08% cell growth inhibition was observed in vitro at 200µg/ml. The lectin was injected (i.p.) into EAC mice at the doses of 2.0 and 4.0mg/kg/day for five consecutive days and 25.38% and 55% of cell growth inhibition was observed, respectively. MOSL caused the cell cycle arrest at G2/M phase as determined by FACS flow cytometry. The cell growth inhibition was due to the induction of apoptosis in the EAC cells which was confirmed by cell morphological study, caspase-3 inhibitor and activation of Bak and suppression of Bcl-2 and NF-κB genes expression.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma de Ehrlich/tratamento farmacológico , Carcinoma de Ehrlich/patologia , Lectinas/uso terapêutico , Moringa oleifera/química , NF-kappa B/genética , Sementes/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Animais , Inibidores de Caspase/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hemaglutinação/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Lectinas/isolamento & purificação , Lectinas/farmacologia , Lectinas/toxicidade , Camundongos , NF-kappa B/metabolismo , Desnaturação Proteica/efeitos dos fármacos , Temperatura , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
STUDY QUESTION: Does coumestrol inhibit proliferation of human placental choriocarcinoma cells? SUMMARY ANSWER: Coumestrol promotes cell death in the choriocarcinoma cells by regulating ERK1/2 MAPK and JNK MAPK signaling pathways and through disruption of Ca2+ and ROS homeostasis. WHAT IS KNOWN ALREADY: A number of patients who suffer from choriocarcinomas fail to survive due to delayed diagnosis or a recurrent tumor and resistance to traditional chemotherapy using platinum-based agents and methotrexate. To overcome these limitations, it is important to discover novel compounds which have no adverse effects yet can inhibit the expression of a target molecule to develop, as a novel therapeutic for prevention and/or treatment of choriocarcinomas. STUDY DESIGN, SIZE, DURATION: Effects of coumestrol on human placental choriocarcinoma cell lines, JAR and JEG3, were assessed in diverse assays in a dose- and time-dependent manner. PARTICIPCANTS/MATERIALS, SETTING, METHODS: Effects of coumestrol on cell proliferation, apoptosis (annexin V expression, propidium iodide staining, TUNEL and invasion assays), mitochondria-mediated apoptosis, production of reactive oxygen species (ROS), lipid peroxidation, glutathione levels and endoplasmic reticulum (ER) stress proteins in JAR and JEG3 cells were determined. Signal transduction pathways in JAR and JEG3 cells in response to coumestrol were determined by western blot analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Results of the present study indicated that coumestrol suppressed proliferation and increased apoptosis in JAR and JEG3 cells by inducing pro-apoptotic proteins, Bax and Bak. In addition, coumestrol increased ROS production, as well as lipid peroxidation and glutathione levels in JAR and JEG3 cells. Moreover, coumestrol-induced depolarization of mitochondrial membrane potential (MMP) and increased cytosolic and mitochondrial Ca2+ levels in JAR and JEG3 cells. Consistent with those results, treatment of JAR and JEG3 cells with a Ca2+ chelator and an inhibitor of IP3 receptor decreased coumestrol-induced depolarization of MMP and increased proliferation in JAR and JEG3 cells. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: A lack of in vivo animal studies is a major limitation of this research. The effectiveness of coumestrol to induce apoptosis of human placental choriocarcinoma cells requires further investigation. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that coumestrol induces apoptotic effects on placental choriocarcinoma cells by regulating cell signaling and mitochondrial-mediated functions, with a potential to impair progression of the cancer. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by grants from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. HI15C0810 awarded to G.S. and HI17C0929 awarded to W.L.).
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Cumestrol/farmacologia , Células Epiteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Mitocôndrias/efeitos dos fármacos , Fitoestrógenos/farmacologia , Apoptose/genética , Cálcio/agonistas , Cálcio/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Córion/efeitos dos fármacos , Córion/metabolismo , Córion/patologia , Relação Dose-Resposta a Droga , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Glutationa/metabolismo , Humanos , Peroxidação de Lipídeos , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Proteína Killer-Antagonista Homóloga a bcl-2/agonistas , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/agonistas , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Malignant melanoma has exhibited a rising incidence in recent years worldwide. Although various molecular targeted drugs are being researched and developed for melanoma patients, their efficacy appears to be unsatisfactory. Over the past few years, several reports have demonstrated that Coptidis Rhizoma water extracts (CR) or its major active chemical component, berberine, has anticancer activities in various types of cancer, including melanoma. However, their underlying mechanisms have not been well understood. In the present study, we determined that CR suppressed melanoma cell viability, which was mainly mediated through apoptosis. In addition, the expression levels of anti-apoptotic proteins, BCL2A1, MCL1 and BCL-w, were strongly suppressed by CR treatment. Furthermore, multi-domain pro-apoptotic proteins BAX and BAK were activated by CR treatment and were also required for the CR-induced apoptosis. Collectively, CR or some formulations containing CR, may be effective safe treatment strategies for human melanoma.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Melanoma/tratamento farmacológico , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Antineoplásicos/uso terapêutico , Proteínas Reguladoras de Apoptose/metabolismo , Berberina/farmacologia , Berberina/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Coptis chinensis , Medicamentos de Ervas Chinesas/uso terapêutico , Humanos , Melanoma/patologia , Camundongos , Antígenos de Histocompatibilidade Menor/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Células NIH 3T3 , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Background: Cancer is one of the leading causes of death worldwide. Despite certain advances in cancer therapy, still there is considerable demand for developing efficient therapeutic agents. Nowadays, there is a rising interest in the use of natural-based anti-cancer drugs. In this study, the cytotoxicity of farnesiferol C and microlobin isolated from Ferula szowitsiana was investigated against MCF-7, HeLa and KYSE cancer cell lines. In addition, the mechanism of binding of these compounds to apoptotic proteins (Bax, Bak and Bcl-2) was analyzed by an in silico method. Materials and methods: We used MTT assay in order to assess the cytotoxicity of compounds against cancer cell lines. For in silico study, the AutoDock 4 was adopted. Results and discussion: According to the in vitro findings, in general, farnesiferol C showed significant cytotoxicity at higher concentrations (>50 µM) following 48 and 72 h incubation with the selected cancer cells; however, microlobin exhibited almost no activity at concentrations up to 100 µM. The in silico results revealed that both compounds could bind to Bax more efficiently rather than to Bcl-2 or Bak proteins. Conclusion: The results obtained by our preliminary in vitro and in silico studies suggest that these compounds might induce apoptosis through Bax activation; however further studies, either in vitro or in vivo are needed to clarify these activities.
Assuntos
Cumarínicos/farmacologia , Simulação de Acoplamento Molecular , Sesquiterpenos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cumarínicos/isolamento & purificação , Relação Dose-Resposta a Droga , Ferula/química , Humanos , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sesquiterpenos/isolamento & purificação , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Colon cancer, a common malignancy, can occur due to poor eating habits and increasing age. Consequently, careful regulation of eating habits may serve as a possible method for preventing the occurrence or progression of colon cancer. Extracts of the fruit of Cnidium monnieri (L.) Cusson are wellknown as an effective herbal medicine for the treatment of pain in female genitalia and carbuncle. However, there have been no studies on the apoptotic effects of Cnidium monnieri (L.) Cusson (CME). Adenosine monophosphateactivated protein kinase (AMPK), the major regulator of energy metabolism, is activated by metabolic stress, including hypoxia and glucose deprivation. Activation of AMPK inhibits cell proliferation and induces apoptosis through the inhibition of phosphorylated (p)Akt and control of Bcell lymphoma 2 (Bcl2) family members. The proapoptotic proteins Bcl2associated X protein (Bax) and Bcl2homologous antagonist killer (Bak), are activated by their translocation to mitochondria from the cytosol. Translocation of Bax/Bak induces outer membrane permeabilization and is likely to lead to apoptosis through cytochrome C release and caspase activity. In the present study, the apoptotic effects and influence on mitochondriamediated apoptotic proteins of CME in HCT116 cells were assessed. We hypothesized that CME may have an effect on the inhibition of pAkt in an AMPKindependent pathway. The present study demonstrated that CME induced the release of LDH and apoptosis through its inhibition of pAkt to control Bcl2 and activate Bax and Bak. Cotreatment with CME and AMPK inhibitors showed that CMEinduced apoptosis does not occurr through a AMPKdependent pathway. Therefore, the present study determined, for the first time, that CME induced apoptosis as a result of causing metabolic stresses due to directly regulation of the dephosphorylation of Akt, whereas it did not control the AMPK-dependent pathway in HCT116 colon cancer cells.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Cnidium/química , Neoplasias do Colo/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos Fitogênicos/química , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Células HCT116 , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2RESUMO
Annona reticulata Linn. (Common name: Bullock's-heart) (Annonaceae family) is a semi-evergreen and small deciduous tree. The extracts of various parts of Annona reticulata L. have been reported as cytotoxic to many cancer cells. Annona reticulata L. leaves' methanolic extract (ARME) was prepared and used against the breast cancer cells. The breast cancer cells (T-47D) viability and IC50 were evaluated by Vybrant® MTT Cell Proliferation Assay Kit. Detection of phosphatidylserine on membranes of apoptotic cells was done by Attune flow cytometer. RNA transcripts were quantified in ARME treated and untreated cells. Finally, the Vybrant® FAM Poly Caspases assay kit was used for analysis of polycaspases activity in T-47D cells. The IC50 (5 ± 0.5 µg/mL) of the ARME was found against breast cancer cells (T-47D). The Paclitaxel was used as a control standard drug for the study. The downregulation of Bcl-2 and upregulation of Bax and Bak, and caspases activation suggested induction of apoptosis in T-47D cells by ARME through mitochondrial pathway. The cell cycle halted at G2/M phase in the ARME treated cells. The ARME was found to be effective against Breast cancer cells (T-47D).
Assuntos
Annona/química , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Extratos Vegetais/farmacologia , Antineoplásicos Fitogênicos/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Humanos , Concentração Inibidora 50 , Metanol/química , Folhas de Planta/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
BCL2-antagonist/killer 1 (BAK1) and apoptotic peptidase activating factor 1 (APAF1) are significant genes in apoptosis signalling pathway of Kashin-Beck disease (KBD). We aimed to verify the protein expression levels of BAK1 and APAF1 in the cartilage and chondrocytes of patients with KBD. Additionally, we explored the relationship between the levels of these proteins and selenium concentration. Chondrocytes was cultured and treated with sodium selenite in vitro. Immunohistochemistry and Western blotting were used to verify the expression levels of BAK1 and APAF1. Compared with the control samples, APAF1 was upregulated and BAK1 was downregulated in the cartilage and chondrocytes of KBD patients. APAF1 expression was higher in the middle and deep zone in the KBD cartilage. APAF1 levels decreased gradually with the increasing selenium concentration (0.05, 0.10 and 0.25 mg/L). BAK1 expression in the 0.25 mg/L selenium group was lower than that of the control group. Different selenium concentrations had varying effects on BAK1 and APAF1 levels. APAF1 may play an important role in the pathogenesis of KBD. APAF1-related apoptosis was more pronounced in the middle and deep zones of the KBD cartilage. APAF may represent a potentially novel molecular target, which may be a biomarker of the role of selenium on the prevention and treatment of KBD. The role of BAK1 in the pathogenesis of KBD requires further study.
Assuntos
Fator Apoptótico 1 Ativador de Proteases/fisiologia , Doença de Kashin-Bek/patologia , Selênio/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/fisiologia , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Humanos , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
Chemoprevention of breast cancer is feasible with the use of non-toxic phytochemicals from edible and medicinal plants. Benzyl isothiocyanate (BITC) is one such plant compound that prevents mammary cancer development in a transgenic mouse model in association with tumor cell apoptosis. Prior studies from our laboratory have demonstrated a role for reactive oxygen species (ROS)-dependent Bax activation through the intermediary of c-Jun N-terminal kinases in BITC-induced apoptosis in human breast cancer cells. The present study demonstrates that truncated Recepteur d'Origine Nantais (sfRON) is a novel regulator of BITC-induced apoptosis in breast cancer cells. Overexpression of sfRON in MCF-7 and MDA-MB-361 cells resulted in augmentation of BITC-induced apoptosis when the apoptotic fraction was normalized against vehicle control for each cell type (untransfected and sfRON overexpressing cells). ROS generation and G2 /M phase cell cycle arrest resulting from BITC treatment were significantly attenuated in sfRON overexpressing cells after normalization with vehicle control for each cell type. Increased BITC-induced apoptosis by sfRON overexpression was independent of c-Jun N-terminal kinase or p38 mitogen-activated protein kinase hyperphosphorylation. On the other hand, activation of Bax and Bak following BITC exposure was markedly more pronounced in sfRON overexpressing cells than in controls. sfRON overexpression also augmented apoptosis induction by structurally diverse cancer chemopreventive phytochemicals including withaferin A, phenethyl isothiocyanate, and D,L-sulforaphane. In conclusion, the present study provides novel mechanistic insights into the role of sfRON in apoptosis regulation by BITC and other electrophilic phytochemicals.
Assuntos
Neoplasias da Mama/genética , Expressão Gênica , Isotiocianatos/farmacologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Apoptose , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Isotiocianatos/uso terapêutico , Células MCF-7 , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Malaria is a significant global health issue, with nearly 200 million cases in 2013 alone. Parasites obtain folate from the host or synthesize it de novo. Folate consumption has increased in many populations, prompting concerns regarding potential deleterious consequences of higher intake. The impact of high dietary folate on the host's immune function and response to malaria has not been examined. Our goal was to determine whether high dietary folate would affect response to malarial infection in a murine model of cerebral malaria. Mice were fed control diets (CD, recommended folate level for rodents) or folic acid-supplemented diets (FASD, 10x recommended level) for 5 weeks before infection with Plasmodium berghei ANKA. Survival, parasitemia, numbers of immune cells and other infection parameters were assessed. FASD mice had reduced survival (p<0.01, Cox proportional hazards) and higher parasitemia (p< 0.01, joint model of parasitemia and survival) compared with CD mice. FASD mice had lower numbers of splenocytes, total T cells, and lower numbers of specific T and NK cell sub-populations, compared with CD mice (p<0.05, linear mixed effects). Increased brain TNFα immunoreactive protein (p<0.01, t-test) and increased liver Abca1 mRNA (p<0.01, t-test), a modulator of TNFα, were observed in FASD mice; these variables correlated positively (rs = 0.63, p = 0.01). Bcl-xl/Bak mRNA was increased in liver of FASD mice (p<0.01, t-test), suggesting reduced apoptotic potential. We conclude that high dietary folate increases parasite replication, disturbs the immune response and reduces resistance to malaria in mice. These findings have relevance for malaria-endemic regions, when considering anti-folate anti-malarials, food fortification or vitamin supplementation programs.
Assuntos
Antimaláricos/uso terapêutico , Ácido Fólico/efeitos adversos , Malária/tratamento farmacológico , Plasmodium berghei/patogenicidade , Animais , Malária Cerebral/tratamento farmacológico , Camundongos , Parasitemia/tratamento farmacológico , Plasmodium berghei/efeitos dos fármacos , Modelos de Riscos Proporcionais , Linfócitos T/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Epi-reevesioside F, a new cardiac glycoside isolated from the root of Reevesia formosana, displayed potent activity against glioblastoma cells. Epi-reevesioside F was more potent than ouabain with IC50 values of 27.3±1.7 vs. 48.7±1.8 nM (P < 0.001) and 45.0±3.4 vs. 81.3±4.3 nM (P < 0.001) in glioblastoma T98 and U87 cells, respectively. However, both Epi-reevesioside F and ouabain were ineffective in A172 cells, a glioblastoma cell line with low Na+/K+-ATPase α3 subunit expression. Epi-reevesioside F induced cell cycle arrest at S and G2 phases and apoptosis. It also induced an increase of intracellular concentration of Na+ but not Ca2+, cleavage and exposure of N-terminus of Bak, loss of mitochondrial membrane potential, inhibition of Akt activity and induction of caspase cascades. Potassium supplements significantly inhibited Epi-reevesioside F-induced effects. Notably, Epi-reevesioside F caused cytosolic acidification that was highly correlated with the anti-proliferative activity. In summary, the data suggest that Epi-reevesioside F inhibits Na+/K+-ATPase, leading to overload of intracellular Na+ and cytosolic acidification, Bak activation and loss of mitochondrial membrane potential. The PI3-kinase/Akt pathway is inhibited and caspase-dependent apoptosis is ultimately triggered in Epi-reevesioside F-treated glioblastoma cells.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Ouabaína/química , Saponinas/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Antineoplásicos/química , Neoplasias Encefálicas/tratamento farmacológico , Cálcio/química , Linhagem Celular Tumoral , Proliferação de Células , Citosol/metabolismo , Citometria de Fluxo , Glioblastoma/tratamento farmacológico , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Potencial da Membrana Mitocondrial , Potássio/química , Estrutura Terciária de Proteína , Rodaminas/química , Sódio/químicaRESUMO
Chemoresistance in cancer has previously been attributed to gene mutations or deficiency. Caspase mutations or Bax deficiency can lead to resistance to cancer drugs. We recently demonstrated that Bak initiates a caspase/Bax-independent cell death pathway. We show that Plumbagin (PL) (5-hydroxy-2-methyl-1,4-napthoquinone), a medicinal plant-derived naphthoquinone that is known to have anti-tumor activity in a variety of models, induces caspase-independent cell death in HCT116 Bax knockout (KO) or MCF-7 Bax knockdown (KD) cells that express wild-type (WT) Bak. The re-expression of Bax in HCT116 Bax KO cells fails to enhance the PL-induced cell death. Additionally, Bak knockdown by shRNA efficiently attenuates PL-induced cell death. These results suggest that PL-induced cell death depends primarily on Bak, not Bax, in these cells. Further experimentation demonstrated that p53 Ser15 phosphorylation and mitochondrial translocation mediated Bak activation and subsequent cell death. Knockdown of p53 or a p53 Ser15 mutant significantly inhibited p53 mitochondrial translocation and cell death. Furthermore, we found that Akt mediated p53 phosphorylation and the subsequent mitochondrial accumulation. Taken together, our data elaborate the role of Bak in caspase/Bax-independent cell death and suggest that PL may be an effective agent for overcoming chemoresistance in cancer cells with dysfunctional caspases.
Assuntos
Apoptose , Resistencia a Medicamentos Antineoplásicos/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Caspases/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imunofluorescência , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Humanos , Mitocôndrias/metabolismo , Naftoquinonas/farmacologia , Fosforilação , RNA Interferente Pequeno , Transfecção , Proteína X Associada a bcl-2/metabolismoRESUMO
Melanoma is an aggressive skin cancer. Unfortunately, there is currently no chemotherapeutic agent available to significantly prolong the survival of the most patients with metastatic melanomas. Here we report that the Ginkgo biloba extract (EGb761), one of the most widely sold herbal supplements in the world, potently induces apoptosis in human melanoma cells by disturbing the balance between pro- and anti-apoptosis Bcl-2 family proteins. Treatment with EGb761 induced varying degrees of apoptosis in melanoma cell lines but not in melanocytes. Induction of apoptosis was caspase-dependent and appeared to be mediated by the mitochondrial pathway, in that it was associated with reduction in mitochondrial membrane potential and activation of Bax and Bak. Although EGb761 did not cause significant change in the expression levels of the BH3-only Bcl-2 family proteins Bim, Puma, Noxa, and Bad, it significantly downregulated Mcl-1 in sensitive but not resistant melanoma cells, suggesting a major role of Mcl-1 in regulating apoptosis of melanoma cells induced by EGb761. Indeed, siRNA knockdown of Mcl-1 enhanced EGb761-induced apoptosis, which was associated with increased activation of Bax and Bak. Taken together, these results demonstrate that EGb761 kills melanoma cells through the mitochondrial apoptotic pathway, and that Mcl-1 is a major regulator of sensitivity of melanoma cells to apoptosis induced by EGb761. Therefore, EGb761 with or without in combination with targeting Mcl-1 may be a useful strategy in the treatment of melanoma.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Ginkgo biloba , Melanoma/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Extratos Vegetais/farmacologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Técnicas de Silenciamento de Genes , Humanos , Melanoma/metabolismo , Melanoma/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
We investigated the role of VDAC2 in human epithelial thyroid tumours using proteomic 2D-DIGE analysis and qRT-PCR. We found a significant up-regulation of VDAC2 in thyroid tumours and in thyroid tumour cell lines (TPC-1 and CAL-62). We did not detect overexpression of VDAC2 in a normal thyroid cell line (Nthy-ori 3-1). Silico analysis revealed that two proteins, BAK1 and BAX, had a strong relationship with VDAC2. BAK1 gene expression showed down-regulation in thyroid tumours (follicular and papillary tumours) and in TPC-1 and CAL-62 cell lines. Transient knockdown of VDAC2 in TPC-1 and CAL-62 promoted upregulation of the BAK1 gene and protein expression, and increased susceptibility to sorafenib treatment. Overexpression of the BAK1 gene in CAL-62 showed lower sorafenib sensitivity than VDAC2 knockdown cells. We propose the VDAC2 gene as a novel therapeutic target in these tumours.
Assuntos
Neoplasias Epiteliais e Glandulares/metabolismo , Proteômica/métodos , Neoplasias da Glândula Tireoide/metabolismo , Eletroforese em Gel Diferencial Bidimensional/métodos , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/patologia , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Compostos de Fenilureia/farmacologia , Sorafenibe , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/patologia , Regulação para Cima , Canal de Ânion 2 Dependente de Voltagem/genética , Adulto Jovem , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Osteosarcoma (OS) is a relatively rare form of cancer, but OS is the most commonly diagnosed bone cancer in children and adolescents. Chemotherapy has side effects and induces drug resistance in OS. Since an effective adjuvant therapy was insufficient for treating OS, researching novel and adequate remedies is critical. Hyperthermia can induce cell death in various cancer cells, and thus, in this study, we investigated the anticancer method of hyperthermia in human OS (U-2 OS) cells. Treatment at 43 °C for 60 min induced apoptosis in human OS cell lines, but not in primary bone cells. Furthermore, hyperthermia was associated with increases of intracellular reactive oxygen species (ROS) and caspase-3 activation in U-2 OS cells. Mitochondrial dysfunction was followed by the release of cytochrome c from the mitochondria, and was accompanied by decreased anti-apoptotic Bcl-2 and Bcl-xL, and increased pro-apoptotic proteins Bak and Bax. Hyperthermia triggered endoplasmic reticulum (ER) stress, which was characterized by changes in cytosolic calcium levels, as well as increased calpain expression and activity. In addition, cells treated with calcium chelator (BAPTA-AM) blocked hyperthermia-induced cell apoptosis in U-2 OS cells. In conclusion, hyperthermia induced cell apoptosis substantially via the ROS, ER stress, mitochondria, and caspase pathways. Thus, hyperthermia may be a novel anticancer method for treating OS.
Assuntos
Apoptose , Retículo Endoplasmático/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Calpaína/antagonistas & inibidores , Calpaína/genética , Calpaína/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Regulação para Baixo/efeitos dos fármacos , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Mitocôndrias/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/metabolismo , Temperatura , Regulação para Cima/efeitos dos fármacos , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
Grape seed proanthocyanidins (GSPs), a biologically active component of grape seeds, have been reported to possess a wide array of pharmacological and biochemical properties. Recently, the inhibitory effects of GSPs on various cancers have been reported, but their effects on cervical cancer remain unclear. Here, we explored the effect of GSPs on cervical cancer using in vitro and in vivo models. In vitro, the treatment of HeLa and SiHa cells with GSPs resulted in a significant inhibition of cell viability. Further investigation indicated that GSPs led to the dose-dependent induction of apoptosis in cancer cells. The underlying mechanism was associated with increased expression of the pro-apoptotic protein Bak-1, decreased expression of the anti-apoptotic protein Bcl-2, the loss of mitochondrial membrane potential, and the activation of caspase-3, suggesting that GSPs induced cervical cancer cell apoptosis through the mitochondrial pathway. In addition, the administration of GSPs (0.1%, 0.2%, and 0.4%, w/v) as a supplement in drinking water significantly inhibited the tumor growth of HeLa and SiHa cells in athymic nude mice, and the number of apoptotic cells in those tumors was also increased significantly. Taken together, our studies demonstrated that GSPs could inhibit the growth of cervical cancer by inducing apoptosis through the mitochondrial pathway, which provides evidence indicating that GSPs may be a potential chemopreventive and/or chemotherapeutic agent for cervical cancer.