RESUMEN
Elevated autophagy is highly associated with cancer development and progression. Fruit extracts of several plants inhibit activity of autophagy-related protease ATG4B and autophagy activity in colorectal cancer cells. However, the effects of these plant extracts in oral cancer cells remain unclear. In this study, we found that the extracted Tribulus terrestris fruit (TT-(fr)) and Xanthium strumarium fruit had inhibitory effects on autophagy inhibition in both SAS and TW2.6 oral cancer cells. Moreover, the fruit extracts had differential effects on cell proliferation of oral cancer cells. In addition, the fruit extracts hampered cell migration and invasion of oral cancer cells, particularly in TT-(fr) extracts. Our results indicated that TT-(fr) extracts consistently inhibited autophagic flux, cell growth and metastatic characteristics of oral cancer cells, suggesting TT-(fr) might contain function ingredient to suppress oral cancer cells.
Asunto(s)
Neoplasias de la Boca , Tribulus , Autofagia , Proliferación Celular , Frutas , Humanos , Neoplasias de la Boca/tratamiento farmacológico , Extractos Vegetales/farmacologíaRESUMEN
BACKGROUND: We identified an autophagy-inducing areca nut (AN) ingredient (AIAI) in the 30-100 kDa fraction of AN extract (ANE 30-100K). This study was to analyze the role of endocytosis in ANE 30-100K-induced autophagy. METHODS: We used benzyl alcohol, dynasore, and shRNA of clathrin and dynamin to assess whether ANE 30-100K-induced cytotoxicity and accumulation of microtubule-associated protein 1 light chain 3 (LC3)-II were affected in oral (OECM-1) and esophageal (CE81T/VGH) carcinoma cells. RESULTS: Both benzyl alcohol and dynasore effectively reduced ANE 30-100K-induced cytotoxicity and LC3-II accumulation in OECM-1 and CE81T/VGH cells. Downregulated protein expression of both clathrin and dynamin by their shRNA also significantly attenuated ANE 30-100K-induced elevation of LC3-II levels in CE81T/VGH cells. CONCLUSIONS: These results indicate that AIAI may be engulfed by cells through clathrin-mediated endocytosis, which promotes the execution of the following autophagy program.
Asunto(s)
Areca/química , Autofagia/efectos de los fármacos , Clatrina/farmacología , Endocitosis/efectos de los fármacos , Neoplasias de la Boca/inducido químicamente , Extractos Vegetales/farmacología , Alcohol Bencilo/farmacología , Línea Celular Tumoral/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Humanos , Hidrazonas/farmacología , Proteínas Asociadas a Microtúbulos/metabolismo , Nueces/química , Extractos Vegetales/química , ARN Interferente Pequeño/metabolismoRESUMEN
BACKGROUND: Cryptocarya-derived crude extracts and their compounds have been reported to have an antiproliferation effect on several types of cancers but their impact on oral cancer is less well understood. METHODS: We examined the cell proliferation effect and mechanism of C. concinna-derived cryptocaryone (CPC) on oral cancer cells in terms of cell viability, apoptosis, reactive oxygen species (ROS), mitochondrial depolarization, and DNA damage. RESULTS: We found that CPC dose-responsively reduced cell viability of two types of oral cancer cells (Ca9-22 and CAL 27) in MTS assay. The CPC-induced dose-responsive apoptosis effects on Ca9-22 cells were confirmed by flow cytometry-based sub-G1 accumulation, annexin V staining, and pancaspase analyses. For oral cancer Ca9-22 cells, CPC also induced oxidative stress responses in terms of ROS generation and mitochondrial depolarization. Moreover, γH2AX flow cytometry showed DNA damage in CPC-treated Ca9-22 cells. CPC-induced cell responses in terms of cell viability, apoptosis, oxidative stress, and DNA damage were rescued by N-acetylcysteine pretreatment, suggesting that oxidative stress plays an important role in CPC-induced death of oral cancer cells. CONCLUSIONS: CPC is a potential ROS-mediated natural product for anti-oral cancer therapy.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Cryptocarya/química , Neoplasias de la Boca/tratamiento farmacológico , Fitoterapia , Extractos Vegetales/uso terapéutico , Pironas/uso terapéutico , Antineoplásicos Fitogénicos/farmacología , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Daño del ADN , Humanos , Estrés Oxidativo , Extractos Vegetales/farmacología , Pironas/farmacologíaRESUMEN
Purpose Radiation combined with natural products may improve the radiosensitivity of cancer cells. This study investigated the potential of a combined modality treatment with Ultraviolet C (UVC; wavelength range 200-280 nm) and our previously identified anti-oral cancer agent (methanolic extracts of Cryptocarya concinna roots; MECCrt) in oral cancer cells. Materials and methods The mechanism of the possible synergy of UVC and MECCrt was explored in terms of cell viability, cell cycle, apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MitoMP), and DNA damage analyses. Results In cell viability (%) at 24 h treatment, the low doses of UVC (14 J/m(2)) and MECCrt (10 µg/ml) resulted in slight damage to human oral cancer Ca9-22 cells (83.2 and 80.4) but was less harmful to human oral normal HGF-1 cells (93.4 and 91.8, respectively). The combined treatment of UVC and MECCrt (UVC/MECCrt) had a lower viability (54.5%) than UVC or MECCrt alone in Ca9-22 cells but no showed significant change in HGF-1 cells. In Ca9-22 cells, the expression of flow cytometry-based apoptosis (sub-G1 phase, annexin V, and pancaspase assays) was significantly higher in UVC/MECCrt than in UVC or MECCrt alone (p < 0.0001). Using flow cytometry, intracellular ROS levels of UVC/MECCrt and MECCrt alone were higher than for UVC alone. MitoMP change and H2A histone family member X (γH2AX; H2AFX)-based DNA damage were synergistically inhibited and induced by MECCrt/UVC compared to its single treatment in Ca9-22 cells, respectively. Conclusion UVC plus MECCrt treatment had selective killing and synergistic anti-proliferative effects against oral cancer cells involving apoptosis, oxidative stress, and DNA damage. This combination therapy appears to have a great clinical potential against oral cancer cells.
Asunto(s)
Cryptocarya/química , Daño del ADN , Neoplasias de la Boca/fisiopatología , Neoplasias de la Boca/terapia , Extractos Vegetales/química , Raíces de Plantas/química , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Quimioradioterapia/métodos , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Humanos , Metanol/química , Neoplasias de la Boca/patología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/efectos de la radiación , Extractos Vegetales/administración & dosificación , Tolerancia a Radiación/efectos de los fármacos , Dosificación Radioterapéutica , Especies Reactivas de Oxígeno/metabolismo , Resultado del Tratamiento , Hipoxia Tumoral/efectos de los fármacos , Hipoxia Tumoral/efectos de la radiación , Terapia Ultravioleta/métodosRESUMEN
Glycosaminoglycans (GAGs), in particular chondroitin sulfate, are an accepted marker of chondrogenic cells. In this study, a cell-based sulfated GAG assay for identifying the chondrogenesis of mesenchymal stem cells was developed. Based on fluorescent staining using safranin O and 4',6-diamidino-2-phenylindole (DAPI), this method was highly sensitive. The results were both qualitative and quantitative. The method is suitable for identifying the chondrogenic process and also for screening compounds. The method may be helpful for discovering novel bioactive compounds for cartilage regeneration.
Asunto(s)
Condrogénesis/efectos de los fármacos , Sulfatos de Condroitina/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Niño , Sulfatos de Condroitina/química , ADN/química , ADN/metabolismo , Humanos , Indoles/química , Límite de Detección , Fenazinas/químicaRESUMEN
Areca nut (AN) is a popular carcinogen used by about 0.6-1.2 billion people worldwide. Although AN contains apoptosis-inducing ingredients, we previously demonstrated that both AN extract (ANE) and its 30-100 kDa fraction (ANE 30-100K) predominantly induce autophagic cell death in both normal and malignant cells. In this study, we further explored the action mechanism of ANE 30-100K-induced autophagy (AIA) in Jurkat T lymphocytes and carcinoma cell lines including OECM-1 (mouth), CE81T/VGH (esophagus), SCC25 (tongue), and SCC-15 (tongue). The results showed that chemical- and small hairpin RNA (shRNA)-mediated inhibition of AMP-activated protein kinase (AMPK) resulted in the attenuation of AIA in Jurkat T but not in OECM-1 cells. Knockdown of Atg5 and Beclin 1 expressions ameliorated AIA in OECM-1/CE81T/VGH/Jurkat T and OECM-1/SCC25/SCC-15, respectively. Furthermore, ANE 30-100K could activate caspase-3 after inhibition of Beclin 1 expression in OECM-1/SCC25/SCC15 cells. Meanwhile, AMPK was demonstrated to be the upstream activator of the extracellular-regulated kinase (ERK) in Jurkat T cells, and inhibition of MEK attenuated AIA in Jurkat T/OECM-1/CE81T/VGH cells. Finally, we also found that multiple myeloma RPMI8226, lymphoma U937, and SCC15 cells survived from long-term non-cytotoxic ANE 30-100K treatment exhibited stronger resistance against serum deprivation through upregulated autophagy. Collectively, our studies indicate that Beclin-1 and Atg5 but not AMPK are commonly required for AIA, and MEK/ERK pathway is involved in AIA. Meanwhile, it is also suggested that long-term AN usage might increase the resistance of survived tumor cells against serum-limited conditions.
Asunto(s)
Areca/química , Autofagia/efectos de los fármacos , Neoplasias de la Boca/tratamiento farmacológico , Nueces/química , Extractos Vegetales/química , Extractos Vegetales/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 5 Relacionada con la Autofagia , Beclina-1 , Caspasa 3/metabolismo , Línea Celular Tumoral , Humanos , Células Jurkat , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Boca/efectos de los fármacos , Boca/metabolismo , Neoplasias de la Boca/metabolismo , Células U937 , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Grape seeds extract (GSE) is a famous health food supplement for its antioxidant property. Different concentrations of GSE may have different impacts on cellular oxidative/reduction homeostasis. Antiproliferative effect of GSE has been reported in many cancers but rarely in oral cancer. METHODS: The aim of this study is to examine the antioral cancer effects of different concentrations of GSE in terms of cell viability, apoptosis, reactive oxygen species (ROS), mitochondrial function, and DNA damage. RESULTS: High concentrations (50-400 µg/ml) of GSE dose-responsively inhibited proliferation of oral cancer Ca9-22 cells but low concentrations (1-10 µg/ml) of GSE showed a mild effect in a MTS assay. For apoptosis analyses, subG1 population and annexin V intensity in high concentrations of GSE-treated Ca9-22 cells was increased but less so at low concentrations. ROS generation and mitochondrial depolarization increased dose-responsively at high concentrations but showed minor changes at low concentrations of GSE in Ca9-22 cells. Additionally, high concentrations of GSE dose-responsively induced more γH2AX-based DNA damage than low concentrations. CONCLUSIONS: Differential concentrations of GSE may have a differentially antiproliferative function against oral cancer cells via differential apoptosis, oxidative stress and DNA damage.
Asunto(s)
Apoptosis/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Extracto de Semillas de Uva/uso terapéutico , Neoplasias de la Boca/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Fitoterapia , Vitis , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Antioxidantes/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Extracto de Semillas de Uva/farmacología , Humanos , Mitocondrias/efectos de los fármacos , Especies Reactivas de Oxígeno , SemillasRESUMEN
BACKGROUND: We previously demonstrated the autophagy-inducing activity in the crude extract of areca nut (ANE) and its 30-100 kDa fraction (ANE 30-100 K). This study aimed to analyze whether chronic ANE and ANE 30-100 K stimulations lead to higher stress resistance and autophagic activity in oral cells, and whether the resulting autophagic status in stimulated cells correlates with stress resistance. MATERIALS AND METHODS: Malignant cells from the mouth oral epidermoid carcinoma Meng-1 (OECM-1) and blood (Jurkat T) origins were stimulated with non-cytotoxic ANE and ANE 30-100 K for 3 months. Sensitivity to anticancer drugs of and autophagy status in stimulated cells, analyzed respectively by XTT assay and calculating microtubule-associated protein 1 light chain 3-II LC3-II/ß-actin ratios from Western blot, were compared to non-treated cells. Autophagy inhibitors, 3-methyladenine (3-MA) and chloroquine (CQ), were used to assess whether autophagy inhibition interferes the altered chemoresistance. RESULTS: Areca nut extract-stimulated (ANE-s) and ANE 30-100 K-stimulated (30-100 K-s) OECM-1 and Jurkat T cells generally exhibited higher cisplatin and 5-fluorouracil (5-FU) resistances, compared to non-stimulated cells. Most stimulated cells expressed significantly higher levels of LC3-II and Atg4B proteins. Interestingly, these cells also showed stronger tolerances against hypoxia environment and expressed higher LC3-II levels under glucose-deprived and hypoxia conditions. Finally, both 3-MA and CQ alleviated, albeit to different degrees, the increased chemoresistance in ANE-s and/or 30-100 K-s cells. CONCLUSIONS: Chronic stimulations of ANE or ANE 30-100 K may increase tolerance of oral cancer and leukemia T cells to anticancer drugs, as well as to glucose deprivation and hypoxia conditions, and cause an elevation of autophagy activity responsible for increased drug resistance.
Asunto(s)
Areca , Autofagia/efectos de los fármacos , Resistencia a Antineoplásicos , Extractos Vegetales/farmacología , Actinas/análisis , Actinas/efectos de los fármacos , Adenina/análogos & derivados , Adenina/farmacología , Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Proteínas Relacionadas con la Autofagia , Carcinoma de Células Escamosas/patología , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Cloroquina/farmacología , Cisplatino/farmacología , Cisteína Endopeptidasas/análisis , Cisteína Endopeptidasas/efectos de los fármacos , Fluorouracilo/farmacología , Glucosa/metabolismo , Humanos , Indicadores y Reactivos , Células Jurkat/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Asociadas a Microtúbulos/efectos de los fármacos , Neoplasias de la Boca/patología , Sales de Tetrazolio , Factores de TiempoRESUMEN
BACKGROUND: The crude extract of the fruit bearing plant, Physalis peruviana (golden berry), demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown. METHODS: Herein, we isolated the main pure compound, 4beta-Hydroxywithanolide (4betaHWE) derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299) using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE) assay was used to evaluate the DNA damage due to the drug. RESULTS: It was shown that DNA damage was significantly induced by 1, 5, and 10 microg/mL 4betaHWE for 2 h in a dose-dependent manner (p < 0.005). A trypan blue exclusion assay showed that the proliferation of cells was inhibited by 4betaHWE in both dose- and time-dependent manners (p < 0.05 and 0.001 for 24 and 48 h, respectively). The half maximal inhibitory concentrations (IC50) of 4betaHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 microg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4betaHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 microg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 microg/mL for 24 h. CONCLUSIONS: In this study, we demonstrated that golden berry-derived 4betaHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer.
Asunto(s)
Apoptosis , Daño del ADN , Neoplasias Pulmonares/tratamiento farmacológico , Physalis/metabolismo , Extractos Vegetales/farmacología , Witanólidos/farmacología , Antineoplásicos/farmacología , División Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Ensayo Cometa/métodos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Fase G2 , Humanos , Concentración 50 InhibidoraRESUMEN
BACKGROUND: Areca (named as betel) is an important etiological factor linked with the high prevalence of oral squamous cell carcinoma (OSCC) in South-Asian countries. This in vitro study investigated the cellular changes and signaling activation in oral keratinocytes in response to areca nut extract (ANE) treatment. METHODS: Normal human oral keratinocyte (NHOK) and oral epidermoid carcinoma cell, Meng-1 (OECM-1) OSCC cell line were treated with variable dosages of ripen ANE. The morphological and cytoskeletal changes, as well as the activation of GTPase proteins and signaling kinases, were analyzed. RESULTS: Most NHOK cells in culture were polygonal, with only <5% cells exhibiting fibroblastoid morphology. However, 10 microg/ml ANE elicited fibroblastoid morphological change, genesis of lamellipodia, loss of subcortical actin, and stress-fiber formation in approximately 25% cultivated NHOK cells. Similar morphological changes were observed in nearly all OECM-1 cells following the ANE treatment. The activation of Rac and Rho GTPase, together with the prominent phosphorylation of a stress-activated kinases, particularly JNK1, was identified in treated OECM-1 cells. CONCLUSION: The novel evidences from the study that ANE impairs the actin organization and activates the signals in oral keratinocytes might bestow further insight into the impacts of ANE in oral pathogenesis.