RESUMO
Cinnamomum cassia Blume has been widely reported as the anti-tumor agent. However, the precise mechanism underlying its pro-apoptotic action is still not clear. Restraining aerobic glycolysis through suppression of pyruvate dehydrogenase kinase (PDHK) is a promising strategy for cancer inhibition. In this study, we performed to investigate the anti-tumor action of C. cassia is mediated by PDHK inhibition. The inhibition of water-extracted branch of C. cassia (WBCC) on the activity of PDHK using both in vitro and cell-based kinase assay were examined in several lung cancer cells. WBCC reduced viabilities of several lung cancer cells with minimal cytotoxicity on normal bronchial epithelial cells. WBCC decreased lactate production through inhibiting activity of PDHK. In consequence of PDHK inhibition, WBCC increased ROS production, which damage mitochondria membrane stability. In addition, WBCC induced ROS- and mitochondria-dependent apoptotic cell death. Among the components of WBCC, cinnamic acid was founded as a major inhibitor on PDHK activity. This is first report that WBCC induces apoptosis of lung cancer cells through inhibiting PDHK activity. Our findings suggest that WBCC and cinnamic acid can be potential candidates for developing novel anti-cancer drugs through glycolysis metabolism.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Cinnamomum aromaticum/química , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Extratos Vegetais/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Água , Células Cultivadas , Glicólise/efeitos dos fármacos , Humanos , Lactatos/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Estimulação Química , Células Tumorais CultivadasRESUMO
Among the herbal ingredients of HangAmDan-B, a medicinal formula that redirects macrophages to become tumoricidal effectors, we found that Panax notoginseng (Burk.) F. H. Chen is the active component responsible for its macrophage-mediated antitumor activity. The water extracted roots of P. notoginseng (PN) did not affect the viability of RAW264.7 murine macrophage-like cells and murine Lewis lung carcinoma (LLC) cells up to a concentration of 100[Formula: see text][Formula: see text]g/mL. However, the transfer of culture media from PN-treated RAW264.7 cells suppressed the growth of LLC cells. The expression of classically activated (M1) markers, such as interleukin (IL)-1[Formula: see text], monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-[Formula: see text], and inducible nitric oxide synthase (iNOS), was increased by PN treatment. The expression of alternatively activated (M2) markers including CD206, IL-10, and [Formula: see text]-[Formula: see text]-acetylhexosaminidases (YM-1) was reduced by PN treatment in the presence of IL-4. Flow cytometry also revealed that PN drives M1 activation of RAW264.7 cells. The transfer of culture media from PN-treated RAW264.7 cells induced the apoptosis of LLC cells as measured by flow cytometry using Annexin-V staining and western blot analysis for caspase cascade-related proteins. In addition, the results from in vivo tumor allograft model demonstrated that PN reduced both tumor volume and weight. The activation of macrophages toward an M1 phenotype was confirmed in the tumor allograft tumor model. These results collectively show that PN can serve as a potent anticancer agent through reeducation of macrophages toward an M1 phenotype.
Assuntos
Antineoplásicos Fitogênicos , Carcinoma Pulmonar de Lewis/patologia , Ativação de Macrófagos/efeitos dos fármacos , Panax notoginseng/química , Extratos Vegetais/farmacologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Extratos Vegetais/isolamento & purificação , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The parallel artificial membrane permeability assay (PAMPA) is widely used in early-stage drug discovery to discriminate compounds by intestinal permeability. The purpose of the current study was to establish a cassette (n-in-1) PAMPA to enable permeability screening of lipophilic compounds. A double-sink PAMPA consisting of a pH gradient (i.e., pH 6.5 and 7.4 for the donor and receiver compartments, respectively) and a lipophilic sink (i.e., a surfactant in the receiver solution) was utilized with cassette incubation of 10 reference compounds. Sample analysis was conducted using selected reaction monitoring (SRM) with a triple quadrupole LC-MS/MS system. Correlation between PAMPA permeability and human intestinal absorption (HIA) of the reference compounds yielded two false negatives, namely propranolol (PPN) and verapamil (VER); these two compounds showed a substantially lower recovery (ca. 10%) than other reference compounds (>69%). This cassette PAMPA was repeated subsequently with polysorbate 80 added to the donor compartments, which resulted in a significant increase in both the recovery and the permeability of the false negatives. Accordingly, the permeability class of all reference compounds could be unambiguously differentiated using this cassette PAMPA. Also, a strong linear correlation (r=0.9845) was observed between the cassette and discrete permeability of all reference compounds.
Assuntos
Química Farmacêutica/métodos , Corantes Fluorescentes/metabolismo , Membranas Artificiais , Permeabilidade/efeitos dos fármacos , Polissorbatos/metabolismo , Células CACO-2 , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Avaliação Pré-Clínica de Medicamentos/métodos , Corantes Fluorescentes/farmacologia , Humanos , Absorção Intestinal/efeitos dos fármacos , Absorção Intestinal/fisiologia , Polissorbatos/farmacologia , Espectrometria de Massas em Tandem/métodosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Anemone rivularis Buch.-Ham. ex DC. (Ranunculaceae) have been used as a traditional remedy for treatment of inflammation and cancer. However, there is no report demonstrating experimental evidence on anti-tumor action of A. rivularis. AIM OF STUDY: The Warburg's effect, preference of aerobic glycolysis rather than oxidative phosphorylation (OXPHOS) even in oxygen rich condition, is focused as one of major characteristics of malignant tumor. Thus, we investigated the effect of A. rivularis on the Pyruvate dehydrogenase (PDH) kinases (PDHKs), a major molecular targets for reducing aerobic glycolysis. MATERIALS AND METHODS: The ethanol extract of whole plant of A. rivularis (ARE), fingerprinted by high performance liquid chromatography (HPLC), was applied to in vitro and cell-based PDHK activity assays. The effect of ARE on cell viabilities of several tumor cells was estimated by MTT assay. The expression of phosphor-PDH, PDH and PDHK1 were measured by Western blot analysis. The production of reactive oxygen species (ROS) and apoptosis was measured by fluorescence-activated cell sorting analysis, using 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) and Annexin V/propidium iodide (PI) staining, respectively. Mitochondrial membrane potential was examined by tetramethylrhodamine methyl ester (TMRM) staining. In vivo anti-tumor efficacy of ARE was estimated by means of tumor volume and weight using allograft injection of murine Lewis lung carcinoma (LLC) cells to dorsa of C57BL/6 mice. RESULTS: ARE inhibited the viabilities of several cancer cells, including MDA-MB321, K562, HT29, Hep3B, DLD-1, and LLC. ARE suppressed PDHK activity in in vitro kinase assay, and also inhibited aerobic glycolysis by reducing phosphorylation of PDHA in human DLD-1 colon cancer and murine LLC cells. The expression of PDHK1, a major isoform of PDHKs in cancer, was not affected by ARE treatment. Moreover, ARE increased the both ROS production and mitochondrial damage. In addition, ARE suppressed the in vitro tumor growth through mitochondria-mediated apoptosis. The growth rates of allograft LLC cells were also reduced by ARE treatment. CONCLUSIONS: Here, we firstly report that ARE inhibits PDHK activity and growth of tumor in both in vitro and in vivo experiments. Therefore, we suggest ARE as a potential candidate for developing anti-cancer drugs.
Assuntos
Anemone/química , Antineoplásicos Fitogênicos/farmacologia , Extratos Vegetais/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Glicólise/efeitos dos fármacos , Células HT29 , Humanos , Células K562 , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Piruvato Desidrogenase Quinase de Transferência de Acetil , Espécies Reativas de Oxigênio/metabolismoRESUMO
The leaves and stems of Perilla frutescens var. acuta Kudo (PF) have been used to prevent threatened abortion in traditional medicine in the East Asian countries. Because reduced receptivity of endometrium is a cause of abortion, we analyzed the action of PF on the endometrial receptivity. PF increased the level of leukemia inhibitory factor (LIF), a major cytokine regulating endometrial receptivity, and LIF receptor in human endometrial Ishikawa cells. The PF-induced LIF expression was mediated by c-jun N-terminal kinase (JNK) and p38 pathways. Adhesion between Ishikawa cells and trophoblastic JAr cells stimulated by PF treatment was abolished by knock down of LIF expression or antagonism of LIFR. In addition, the expressions of integrin ß3 and ß5 were increased by PF treatment in Ishikawa cells. The PF-induced expression of integrin ß3 and ß5 was reduced with an LIFR antagonist. Neutralization of both integrins successfully blocked PF-stimulated adhesion of JAr cells and Ishikawa cells. These results suggest that PF enhanced the adhesion between Ishikawa cells and JAr cells by increasing the expression of integrin ß3 and ß5 via an LIF-dependent pathway. Given the importance of endometrial receptivity in successful pregnancy, PF can be a novel and effective candidate for improving pregnancy rate.
Assuntos
Endométrio/efeitos dos fármacos , Cadeias beta de Integrinas/biossíntese , Fator Inibidor de Leucemia/metabolismo , Perilla frutescens/química , Extratos Vegetais/farmacologia , Antracenos/farmacologia , Butadienos/farmacologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Imidazóis/farmacologia , Cadeias beta de Integrinas/metabolismo , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/antagonistas & inibidores , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nitrilas/farmacologia , Folhas de Planta/química , Raízes de Plantas/química , Piridinas/farmacologia , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Most cancer cells predominantly produce ATP by maintaining a high rate of lactate fermentation, rather than by maintaining a comparatively low rate of tricarboxylic acid cycle, i.e., Warburg's effect. In the pathway, the pyruvate produced by glycolysis is converted to lactic acid by lactate dehydrogenase (LDH). Here, we demonstrated that water extracts from the seeds of Myristica fragrans Houtt. (MF) inhibit the in vitro enzymatic activity of LDH. MF effectively suppressed cell growth and the overall Warburg effect in HT29 human colon cancer cells. Although the expression of LDH-A was not changed by MF, both lactate production and LDH activity were decreased in MF-treated cells under both normoxic and hypoxic conditions. In addition, intracellular ATP levels were also decreased by MF treatment, and the uptake of glucose was also reduced by MF treatment. Furthermore, the experiment on tumor growth in the in vivo mice model revealed that MF effectively reduced the growth of allotransplanted Lewis lung carcinoma cells. Taken together, these results suggest that MF effectively inhibits cancer growth and metabolism by inhibiting the activity of LDH, a major enzyme responsible for regulating cancer metabolism. These results implicate MF as a potential candidate for development into a novel drug against cancer through inhibition of LDH activity.