RESUMEN
The mTOR pathway is a master regulator of cellular growth and metabolism. The biosynthetic and energetic demand of rapidly proliferating cells such as cancer cells is met by metabolic adaptations such as an increased glycolytic rate known as the Warburg effect. Herein, we characterize the anti-tumor effect of rapamycin in a mouse model of T-cell lymphoma and examine the metabolic effects in vitro. The murine T-cell lymphoma line, MBL2, and human cutaneous T-cell lymphoma (CTCL) lines, HH and Hut78, were used in syngeneic or standard NSG mouse models to demonstrate a marked suppression of tumor growth by rapamycin accompanied by inhibition of mTORC1/2. Analysis of the metabolic phenotype showed a substantial reduction in the glycolytic rate and glucose utilization in rapamycin-treated lymphoma cells. This was associated with reduced expression of glucose transporters and glycolytic enzymes in cultured cells and xenograft tumors. As a result of the decrease in glycolytic state, rapamycin-treated cells displayed reduced sensitivity to low-glucose conditions but continued to rely on mitochondrial oxidative phosphorylation (OXPHOS) with sensitivity to inhibition of OXPHOS. Taken together, we demonstrate that rapamycin suppresses growth of T-cell lymphoma tumors and leads to a reduction in aerobic glycolysis counteracting the Warburg effect of cancer cells.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Glucólisis/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/genética , Animales , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Xenoinjertos , Humanos , Linfoma de Células T/tratamiento farmacológico , Linfoma de Células T/genética , Ratones , Ratones Endogámicos C57BL , Terapia Molecular Dirigida/métodos , Fosforilación Oxidativa/efectos de los fármacos , Fenotipo , Sensibilidad y Especificidad , Serina-Treonina Quinasas TOR/efectos de los fármacosRESUMEN
3-Bromopyruvate (3-BrPA) is an alkylating agent and a well-known inhibitor of energy metabolism. Rapamycin is an inhibitor of the serine/threonine protein kinase mTOR. Both 3-BrPA and rapamycin show chemopreventive efficacy in mouse models of lung cancer. Aerosol delivery of therapeutic drugs for lung cancer has been reported to be an effective route of delivery with little systemic distribution in humans. In this study, 3-BrPA and rapamycin were evaluated in combination for their preventive effects against lung cancer in mice by aerosol treatment, revealing a synergistic ability as measured by tumor multiplicity and tumor load compared treatment with either single-agent alone. No evidence of liver toxicity was detected by monitoring serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. To understand the mechanism in vitro experiments were performed using human non-small cell lung cancer (NSCLC) cell lines. 3-BrPA and rapamycin also synergistically inhibited cell proliferation. Rapamycin alone blocked the mTOR signaling pathway, whereas 3-BrPA did not potentiate this effect. Given the known role of 3-BrPA as an inhibitor of glycolysis, we investigated mitochondrial bioenergetics changes in vitro in 3-BrPA-treated NSCLC cells. 3-BrPA significantly decreased glycolytic activity, which may be due to adenosine triphosphate (ATP) depletion and decreased expression of GAPDH. Our results demonstrate that rapamycin enhanced the antitumor efficacy of 3-BrPA, and that dual inhibition of mTOR signaling and glycolysis may be an effective therapeutic strategy for lung cancer chemoprevention.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Piruvatos/farmacología , Sirolimus/farmacología , Adenosina Trifosfato/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Metabolismo Energético/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Glucólisis/efectos de los fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos A , Mitocondrias/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Docosahexaenoic acid (DHA; C22:6n-3) depresses mammary carcinoma proliferation and growth in cell culture and in animal models. The current study explored the role of interrupting bioenergetic pathways in BT-474 and MDA-MB-231 breast cancer cell lines representing respiratory and glycolytic phenotypes, respectively and comparing the impacts of DHA with a non-transformed cell line, MCF-10A. Metabolic investigation revealed that DHA supplementation significantly diminished the bioenergetic profile of the malignant cell lines in a dose-dependent manner. DHA enrichment also resulted in decreases in hypoxia-inducible factor (HIF-1α) total protein level and transcriptional activity in the malignant cell lines but not in the non-transformed cell line. Downstream targets of HIF-1α, including glucose transporter 1 (GLUT 1) and lactate dehydrogenase (LDH), were decreased by DHA treatment in the BT-474 cell line, as well as decreases in LDH protein level in the MDA-MB-231 cell line. Glucose uptake, total glucose oxidation, glycolytic metabolism, and lactate production were significantly decreased in response to DHA supplementation; thereby enhancing metabolic injury and decreasing oxidative metabolism. The DHA-induced metabolic changes led to a marked decrease of intracellular ATP levels by 50% in both cancer cell lines, which mediated phosphorylation of metabolic stress marker, AMPK, at Thr172. These findings show that DHA contributes to impaired cancer cell growth and survival by altering cancer cell metabolism, increasing metabolic stress and altering HIF-1α-associated metabolism, while not affecting non-transformed MCF-10A cells. This study provides rationale for enhancement of current cancer prevention models and current therapies by combining them with dietary sources, like DHA.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Ácidos Docosahexaenoicos/farmacología , Metabolismo Energético/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Mama/efectos de los fármacos , Mama/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismoRESUMEN
Honokiol is an important bioactive compound found in the bark of Magnolia tree. It is a nonadipogenic PPARγ agonist and capable of inhibiting the growth of a variety of tumor types both in vitro and in xenograft models. However, to fully appreciate the potential chemopreventive activity of honokiol, a less artificial model system is required. To that end, this study examined the chemopreventive efficacy of honokiol in an initiation model of lung squamous cell carcinoma (SCC). This model system uses the carcinogen N-nitroso-trischloroethylurea (NTCU), which is applied topically, reliably triggering the development of SCC within 24 to 26 weeks. Administration of honokiol significantly reduced the percentage of bronchial that exhibit abnormal lung SCC histology from 24.4% bronchial in control to 11.0% bronchial in honokiol-treated group (P = 0.01) while protecting normal bronchial histology (present in 20.5% of bronchial in control group and 38.5% of bronchial in honokiol-treated group. P = 0.004). P63 staining at the SCC site confirmed the lung SCCs phenotype. In vitro studies revealed that honokiol inhibited lung SCC cells proliferation, arrested cells at the G1-S cell-cycle checkpoint, while also leading to increased apoptosis. Our study showed that interfering with mitochondrial respiration is a novel mechanism by which honokiol changed redox status in the mitochondria, triggered apoptosis, and finally leads to the inhibition of lung SCC. This novel mechanism of targeting mitochondrial suggests honokiol as a potential lung SCC chemopreventive agent.
Asunto(s)
Anticarcinógenos/farmacología , Compuestos de Bifenilo/farmacología , Lignanos/farmacología , Neoplasias Pulmonares/patología , Mitocondrias/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Bronquios/efectos de los fármacos , Carcinógenos/química , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Carmustina/análogos & derivados , Carmustina/química , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Mitocondrias/efectos de los fármacos , Trasplante de Neoplasias , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
ExoU is a 74-kDa, water-soluble toxin injected directly into mammalian cells through the type III secretion system of the opportunistic pathogen, Pseudomonas aeruginosa. Previous studies have shown that ExoU is a Ca(2+)-independent phospholipase that requires a eukaryotic protein cofactor. One protein capable of activating ExoU and serving as a required cofactor was identified by biochemical and proteomic methods as superoxide dismutase (SOD1). In these studies, we carried out site-directed spin-labeling electron paramagnetic resonance spectroscopy to examine the effects of SOD1 and substrate liposomes on the structure and dynamics of ExoU. Local conformational changes within the catalytic site were observed in the presence of substrate liposomes, and were enhanced by the addition of SOD1 in a concentration-dependent manner. Conformational changes in the C-terminal domain of ExoU were observed upon addition of cofactor, even in the absence of liposomes. Double electron-electron resonance experiments indicated that ExoU samples multiple conformations in the resting state. In contrast, addition of SOD1 induced ExoU to adopt a single, well-defined conformation. These studies provide, to our knowledge, the first direct evidence for cofactor- and membrane-induced conformational changes in the mechanism of activation of ExoU.