RESUMEN
Obesity induces numerous physiological changes that can impact cancer risk and patient response to therapy. Obese patients with cervical cancer have been reported to have superior outcomes following chemoradiotherapy, suggesting that free fatty acids (FFA) might enhance response to radiotherapy. Here, using preclinical models, we show that monounsaturated and diunsaturated FFAs (uFFA) radiosensitize cervical cancer through a novel p53-dependent mechanism. UFFAs signaled through PPARγ and p53 to promote lipid uptake, storage, and metabolism after radiotherapy. Stable isotope labeling confirmed that cervical cancer cells increase both catabolic and anabolic oleate metabolism in response to radiotherapy, with associated increases in dependence on mitochondrial fatty acid oxidation for survival. In vivo, supplementation with exogenous oleate suppressed tumor growth in xenografts after radiotherapy, an effect that could be partially mimicked in tumors from high fat diet-induced obese mice. These results suggest that supplementation with uFFAs may improve tumor responses to radiotherapy, particularly in p53 wild-type tumors. SIGNIFICANCE: Metabolism of monounsaturated and diunsaturated fatty acids improves the efficacy of radiotherapy in cancer through modulation of p53 activity. See related commentary by Jungles and Green, p. 4513.
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Ácidos Grasos , Neoplasias del Cuello Uterino , Ratones , Animales , Femenino , Humanos , Ácidos Grasos/metabolismo , Ácido Oléico , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/radioterapia , Proteína p53 Supresora de Tumor , Dieta Alta en Grasa/efectos adversos , Ácidos Grasos no Esterificados/metabolismo , Obesidad/patología , Ácidos Grasos Monoinsaturados/metabolismoRESUMEN
Persistent HPV infection is causative for the majority of cervical cancer cases; however, current guidelines do not require HPV testing for newly diagnosed cervical cancer. Using an institutional cohort of 88 patients with cervical cancer treated uniformly with standard-of-care chemoradiation treatment (CRT) with prospectively collected clinical outcome data, we observed that patients with cervical tumors containing HPV genotypes other than HPV 16 have worse survival outcomes after CRT compared with patients with HPV 16+ tumors, consistent with previously published studies. Using RNA sequencing analysis, we quantified viral transcription efficiency and found higher levels of E6 and the alternative transcript E6*I in cervical tumors with HPV genotypes other than HPV 16. These findings were validated using whole transcriptome data from The Cancer Genome Atlas (n = 304). For the first time to our knowledge, transcript expression level of HPV E6*I was identified as a predictive biomarker of CRT outcome in our complete institutional data set (n = 88) and within the HPV 16+ subset (n = 36). In vitro characterization of HPV E6*I and E6 overexpression revealed that both induce CRT resistance through distinct mechanisms dependent upon p53-p21. Our findings suggest that high expression of E6*I and E6 may represent novel biomarkers of CRT efficacy, and these patients may benefit from alternative treatment strategies.
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Alphapapillomavirus/genética , Regulación Viral de la Expresión Génica , Infecciones por Papillomavirus/radioterapia , Neoplasias del Cuello Uterino/radioterapia , Adulto , Anciano , Anciano de 80 o más Años , Alphapapillomavirus/aislamiento & purificación , Biopsia , Cuello del Útero/patología , Cuello del Útero/virología , Quimioradioterapia , ADN Viral/genética , ADN Viral/aislamiento & purificación , Femenino , Estudios de Seguimiento , Técnicas de Genotipaje , Humanos , Persona de Mediana Edad , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/sangre , Infecciones por Papillomavirus/mortalidad , Infecciones por Papillomavirus/virología , Pronóstico , Supervivencia sin Progresión , Estudios Prospectivos , RNA-Seq , Neoplasias del Cuello Uterino/sangre , Neoplasias del Cuello Uterino/mortalidad , Neoplasias del Cuello Uterino/virología , Transcripción ViralRESUMEN
PURPOSE: Chemoradiotherapy for locally advanced cervical cancer fails in over a third of patients. Biomarkers with therapeutic implications are therefore needed. We investigated the relationship between an established prognostic marker, maximum standardized uptake value (SUVmax) on 18F-fluorodeoxyglucose positron emission tomography, and the inflammatory and immune state of cervical cancers. EXPERIMENTAL DESIGN: An SUVmax most prognostic for freedom from progression (FFP) was identified and compared with known prognostic clinical variables in a cohort of 318 patients treated with definitive radiation with prospectively collected clinical data. Gene set enrichment analysis (GSEA) and CIBERSORT of whole-transcriptome data from 68 patients were used to identify biological pathways and immune cell subpopulations associated with high SUVmax. IHC using a tissue microarray (TMA, N = 82) was used to validate the CIBERSORT findings. The impact of macrophages on cervical cancer glucose metabolism was investigated in coculture experiments. RESULTS: SUVmax <11.4 was most prognostic for FFP (P = 0.001). The GSEA showed that high SUVmax is associated with increased gene expression of inflammatory pathways, including JAK/STAT3 signaling. CIBERSORT and CD68 staining of the TMA showed high SUVmax tumors are characterized by a monocyte-predominant immune infiltrate. Coculture of cervical cancer cells with macrophages or macrophage-conditioned media altered glucose uptake, and IL6 and JAK/STAT3 signaling contribute to this effect. CONCLUSIONS: SUVmax is a prognostic marker in cervical cancer that is associated with activation of inflammatory pathways and tumor infiltration of myeloid-derived immune cells, particularly macrophages. Macrophages contribute to changes in cervical cancer glucose metabolism.See related commentary by Williamson et al., p. 4136.
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Fluorodesoxiglucosa F18/farmacocinética , Tomografía de Emisión de Positrones , Radiofármacos/farmacocinética , Neoplasias del Cuello Uterino/diagnóstico por imagen , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor , Femenino , Humanos , Inflamación/diagnóstico por imagen , Inflamación/etiología , Inflamación/metabolismo , Persona de Mediana Edad , Tomografía de Emisión de Positrones/métodos , Estudios Prospectivos , Neoplasias del Cuello Uterino/complicaciones , Adulto JovenRESUMEN
Approaches using a single type of data have been applied to classify human tumors. Here we integrate imaging features and transcriptomic data using a prospectively collected tumor bank. We demonstrate that increased maximum standardized uptake value on pretreatment 18F-fluorodeoxyglucose-positron emission tomography correlates with epithelial-to-mesenchymal transition (EMT) gene expression. We derived and validated 3 major molecular groups, namely squamous epithelial, squamous mesenchymal, and adenocarcinoma, using prospectively collected institutional (n = 67) and publicly available (n = 304) data sets. Patients with tumors of the squamous mesenchymal subtype showed inferior survival outcomes compared with the other 2 molecular groups. High mesenchymal gene expression in cervical cancer cells positively correlated with the capacity to form spheroids and with resistance to radiation. CaSki organoids were radiation-resistant but sensitive to the glycolysis inhibitor, 2-DG. These experiments provide a strategy for response prediction by integrating large data sets, and highlight the potential for metabolic therapy to influence EMT phenotypes in cervical cancer.
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Bases de Datos de Ácidos Nucleicos , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , RNA-Seq , Neoplasias del Cuello Uterino , Línea Celular Tumoral , Supervivencia sin Enfermedad , Femenino , Humanos , Tasa de Supervivencia , Neoplasias del Cuello Uterino/diagnóstico por imagen , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/mortalidadRESUMEN
The purpose of this study was to determine if radiation (RT)-resistant cervical cancers are dependent upon glutamine metabolism driven by activation of the PI3K pathway and test whether PI3K pathway mutation predicts radiosensitization by inhibition of glutamine metabolism. Cervical cancer cell lines with and without PI3K pathway mutations, including SiHa and SiHa PTEN-/- cells engineered by CRISPR/Cas9, were used for mechanistic studies performed in vitro in the presence and absence of glutamine starvation and the glutaminase inhibitor, telaglenastat (CB-839). These studies included cell survival, proliferation, quantification of oxidative stress parameters, metabolic tracing with stable isotope-labeled substrates, metabolic rescue, and combination studies with L-buthionine sulfoximine (BSO), auranofin (AUR), and RT. In vivo studies of telaglenastat ± RT were performed using CaSki and SiHa xenografts grown in immune-compromised mice. PI3K-activated cervical cancer cells were selectively sensitive to glutamine deprivation through a mechanism that included thiol-mediated oxidative stress. Telaglenastat treatment decreased total glutathione pools, increased the percent glutathione disulfide, and caused clonogenic cell killing that was reversed by treatment with the thiol antioxidant, N-acetylcysteine. Telaglenastat also sensitized cells to killing by glutathione depletion with BSO, thioredoxin reductase inhibition with AUR, and RT. Glutamine-dependent PI3K-activated cervical cancer xenografts were sensitive to telaglenastat monotherapy, and telaglenastat selectively radiosensitized cervical cancer cells in vitro and in vivo These novel preclinical data support the utility of telaglenastat for glutamine-dependent radioresistant cervical cancers and demonstrate that PI3K pathway mutations may be used as a predictive biomarker for telaglenastat sensitivity.
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Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glutaminasa/antagonistas & inhibidores , Estrés Oxidativo/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Compuestos de Sulfhidrilo/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Modelos Animales de Enfermedad , Femenino , Glutamina/metabolismo , Glutatión/metabolismo , Glucólisis , Humanos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/terapia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Therapies targeting reductive/oxidative (redox) metabolism hold potential in cancers resistant to chemotherapy and radiation. A redox imaging marker would help identify cancers susceptible to redox-directed therapies. Copper(II)-diacetyl-bis(4-methylthiosemicarbazonato) (Cu-ATSM) is a PET tracer developed for hypoxia imaging that could potentially be used for this purpose. We aimed to demonstrate that Cu-ATSM signal is dependent on cellular redox state, irrespective of hypoxia. Methods: We investigated the relationship between 64Cu-ATSM signal and redox state in human cervical and colon cancer cells. We altered redox state using drug strategies and single-gene mutations in isocitrate dehydrogenases (IDH1/2). Concentrations of reducing molecules were determined by spectrophotometry and liquid chromatography-mass spectrometry and compared with 64Cu-ATSM signal in vitro. Mouse models of cervical cancer were used to evaluate the relationship between 64Cu-ATSM signal and levels of reducing molecules in vivo, as well as to evaluate the change in 64Cu-ATSM signal after redox-active drug treatment. Results: A correlation exists between baseline 64Cu-ATSM signal and cellular concentration of glutathione, nicotinamide adenine dinucleotide phosphate (NADPH), and nicotinamide adenine dinucleotide (NADH). Altering NADH and NADPH metabolism using drug strategies and IDH1 mutations resulted in significant changes in 64Cu-ATSM signal under normoxic conditions. Hypoxia likewise changed 64Cu-ATSM signal, but treatment of hypoxic cells with redox-active drugs resulted in a more dramatic change than hypoxia alone. A significant difference in NADPH was seen between cervical tumor orthotopic implants in vivo, without a corresponding difference in 64Cu-ATSM signal. After treatment with ß-lapachone, there was a change in 64Cu-ATSM signal in xenograft tumors smaller than 50 mg but not in larger tumors. Conclusion:64Cu-ATSM signal reflects redox state, and altering redox state impacts 64Cu-ATSM metabolism. Our animal data suggest there are other modulating factors in vivo. These findings have implications for the use of 64Cu-ATSM as a predictive marker for redox therapies, though further in vivo work is needed.
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Compuestos Organometálicos , Tomografía de Emisión de Positrones , Tiosemicarbazonas , Hipoxia Tumoral , Animales , Artefactos , Línea Celular Tumoral , Transformación Celular Neoplásica , Complejos de Coordinación , Femenino , Humanos , Isocitrato Deshidrogenasa/genética , Ratones , Ratones Desnudos , Mutación , Oxidación-Reducción , Neoplasias del Cuello Uterino/diagnóstico por imagen , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
The purpose of this study was to evaluate the effect of obesity and obesity-associated factors on the outcomes of patients with cervical cancer. Outcomes were evaluated in 591 patients with FIGO Ib to IV cervical cancer treated uniformly with definitive radiation. Patients were stratified into 3 groups based upon pretreatment Body Mass Index (BMI): A ≤ 18.5; B 18.6 - 34.9; and C ≥ 35. The 5-year freedom from failure rates were 58, 59, and 73% for BMI groups A, B, and C (p = 0.01). Overall survival rates were 50, 59, and 68%, respectively (p = 0.02). High expression of phosphorylated AKT (pAKT) was associated with poor outcomes only in non-obese patients. Obese patients with PI3K pathway mutant tumors had a trend toward favorable outcomes, while a similar effect was not observed in non-obese patients. Compared to similar tumors from non-obese hosts, PIK3CA and PTEN mutant tumors from obese patients failed to express high levels of phosphorylated AKT and its downstream targets. These results show that patients with obesity at the time of diagnosis of cervical cancer exhibit improved outcomes after radiation. PI3K/AKT pathway mutations are common in obese patients, but are not associated with activation of AKT signaling.
RESUMEN
Highly glycolytic cervical cancers largely resist treatment by cisplatin and coadministered pelvic irradiation as the present standard of care. In this study, we investigated the effects of inhibiting glycolysis and thiol redox metabolism to evaluate them as alternate treatment strategies in these cancers. In a panel of multiple cervical cancer cell lines, we evaluated sensitivity to inhibition of glycolysis (2-deoxyglucose, 2-DG) with or without simultaneous inhibition of glutathione and thioredoxin metabolism (BSO/AUR). Intracellular levels of total and oxidized glutathione, thioredoxin reductase activity, and indirect measures of intracellular reactive oxygen species were compared before and after treatment. Highly radioresistant cells were the most sensitive to 2-DG, whereas intermediate radioresistant cells were sensitive to 2-DG plus BSO/AUR. In response to 2-DG/BSO/AUR treatment, we observed increased levels of intracellular oxidized glutathione, redox-sensitive dye oxidation, and decreased glucose utilization via multiple metabolic pathways including the tricarboxylic acid cycle. 2-DG/BSO/AUR treatment delayed the growth of tumors composed of intermediate radioresistant cells and effectively radiosensitized these tumors at clinically relevant radiation doses both in vitro and in vivo Overall, our results support inhibition of glycolysis and intracellular redox metabolism as an effective alternative drug strategy for the treatment of highly glycolytic and radioresistant cervical cancers.Significance: This study suggests a simple metabolic approach to strike at an apparent Achilles' heel in highly glycolytic, radioresistant forms of cervical cancers, possibly with broader applications in cancer therapy. Cancer Res; 78(6); 1392-403. ©2018 AACR.
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Glucólisis/efectos de los fármacos , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/radioterapia , Animales , Auranofina/farmacología , Butionina Sulfoximina/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Ciclo del Ácido Cítrico/efectos de los fármacos , Desoxiglucosa/farmacología , Femenino , Glutatión/metabolismo , Humanos , Ácido Láctico/metabolismo , Ratones Desnudos , Oxidación-Reducción , Tolerancia a Radiación/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Tiorredoxinas/metabolismo , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: PI3K/AKT pathway alterations are associated with incomplete response to chemoradiation in human cervical cancer. This study was performed to test for mutations in the PI3K pathway and to evaluate the effects of AKT inhibitors on glucose uptake and cell viability. EXPERIMENTAL DESIGN: Mutational analysis of DNA from 140 pretreatment tumor biopsies and 8 human cervical cancer cell lines was performed. C33A cells (PIK3CAR88Q and PTENR233*) were treated with increasing concentrations of two allosteric AKT inhibitors (SC-66 and MK-2206) with or without the glucose analogue 2-deoxyglucose (2-DG). Cell viability and activation status of the AKT/mTOR pathway were determined in response to the treatment. Glucose uptake was evaluated by incubation with 18F-fluorodeoxyglucose (FDG). Cell migration was assessed by scratch assay. RESULTS: Activating PIK3CA (E545K, E542K) and inactivating PTEN (R233*) mutations were identified in human cervical cancer. SC-66 effectively inhibited AKT, mTOR and mTOR substrates in C33A cells. SC-66 inhibited glucose uptake via reduced delivery of Glut1 and Glut4 to the cell membrane. SC-66 (1 µg/ml-56%) and MK-2206 (30 µM-49%) treatment decreased cell viability through a non-apoptotic mechanism. Decreases in cell viability were enhanced when AKT inhibitors were combined with 2-DG. The scratch assay showed a substantial reduction in cell migration upon SC-66 treatment. CONCLUSIONS: The mutational spectrum of the PI3K/AKT pathway in cervical cancer is complex. AKT inhibitors effectively block mTORC1/2, decrease glucose uptake, glycolysis, and decrease cell viability in vitro. These results suggest that AKT inhibitors may improve response to chemoradiation in cervical cancer.
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Ciclohexanonas/farmacología , Glucosa/metabolismo , Compuestos Heterocíclicos con 3 Anillos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Piridinas/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias del Cuello Uterino/patología , Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I , Femenino , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Transducción de Señal/efectos de los fármacos , Neoplasias del Cuello Uterino/metabolismoRESUMEN
PURPOSE: Cervical tumor response on posttherapy 2[(18)F]fluoro-2-deoxy-d-glucose-positron emission tomography (FDG-PET) is predictive of survival outcome. The purpose of this study was to use gene expression profiling to identify pathways associated with tumor metabolic response. EXPERIMENTAL DESIGN: This was a prospective tissue collection study for gene expression profiling of 62 pretreatment biopsies from patients with advanced cervical cancer. Patients were treated with definitive radiation. Fifty-three patients received concurrent chemotherapy. All patients underwent a pretreatment and a 3-month posttherapy FDG-PET/computed tomography (CT). Tumor RNA was harvested from fresh frozen tissue and hybridized to Affymetrix U133Plus2 GeneChips. Gene set enrichment analysis (GSEA) was used to identify signaling pathways associated with tumor metabolic response. Immunohistochemistry and in vitro FDG uptake assays were used to confirm our results. RESULTS: There were 40 biopsies from patients with a complete metabolic response (PET-negative group) and 22 biopsies from patients with incomplete metabolic response (PET-positive group). The 3-year cause-specific survival estimates were 98% for the PET-negative group and 39% for the PET-positive group (P < 0.0001). GSEA identified alterations in expression of genes associated with the PI3K/Akt signaling pathway in patients with a positive follow-up PET. Immunohistochemistry using a tissue microarray of 174 pretreatment biopsies confirmed p-Akt as a biomarker for poor prognosis in cervical cancer. The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 inhibited FDG uptake in vitro in cervical cancer cell lines. CONCLUSIONS: Activation of the PI3K/Akt pathway is associated with incomplete metabolic response in cervical cancer. Targeted inhibition of PI3K/Akt may improve response to chemoradiation.
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Perfilación de la Expresión Génica , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Adulto , Anciano , Animales , Desoxiglucosa/metabolismo , Femenino , Humanos , Ratones , Persona de Mediana Edad , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Análisis de Supervivencia , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/mortalidadRESUMEN
Neonatal polymorphonuclear leukocytes (PMN) exhibit delayed apoptosis both constitutively and under inflammatory conditions, and evidence has linked PMN longevity to the presence of antiapoptotic proteins. Activation of the survival-associated transcription factor, nuclear factor kappa B (NF-κB), promotes the synthesis of several antiapoptotic proteins including Flice inhibitory protein (FLIP). Neonatal and adult PMN were compared in this study to test the hypothesis that FLIP modulates age-related apoptosis. Expression of the short isoform, FLIP-S, was prominent at baseline and persisted during spontaneous apoptosis in neonatal PMN, whereas basal expression was lower and decreased under the same conditions in adult PMN. Stable FLIP-S expression in neonatal PMN was associated with a relative resistance to apoptosis in response to the protein synthesis inhibitor, cycloheximide (CHX), or the NF-κB inhibitor, gliotoxin. In contrast, similar treatment of adult PMN promoted greater overall apoptosis accompanied by FLIP degradation. Nuclear levels of phosphorylated p65, a critical NF-κB dimer, were relatively robust in neonatal PMN under basal conditions or after stimulation with TNF-α, a cytokine that induces FLIP. In conclusion, persistent FLIP-S expression is involved in the longevity of neonatal PMN, and our data suggest a contribution of NF-κB signaling and related survival mechanisms.
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Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Supervivencia Celular/fisiología , Neutrófilos/fisiología , Isoformas de Proteínas/metabolismo , Adolescente , Adulto , Apoptosis/fisiología , Células Cultivadas , Cicloheximida/farmacología , Femenino , Sangre Fetal/citología , Humanos , Etiquetado Corte-Fin in Situ , Recién Nacido , Persona de Mediana Edad , FN-kappa B/metabolismo , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Embarazo , Inhibidores de la Síntesis de la Proteína/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Adulto JovenRESUMEN
BCL-2/E1B-19 kDa-interacting protein 3 (BNIP3) is a BH3-only mitochondrial protein. Expression of BNIP3 is strongly stimulated by hypoxia. Up-regulation of BNIP3 has been detected in several human carcinomas including carcinomas of the lung and breast. The significance of BNIP3 overexpression in these cancers is not known. To determine whether BNIP3 plays a role in tumor growth, we generated A549 lung carcinoma cells that overexpressed BNIP3 and examined their ability to form tumors in the mouse xenograft model. All cell lines that overexpressed BNIP3 formed larger tumors compared to the parental or vector-transformed A549 cells. Breast carcinoma cell lines that overexpressed BNIP3 also induced tumors in athymic mice in the absence of hormone administration, while the parental cell line did not. Stable shRNA-mediated knockdown of endogenous BNIP3 severely impaired the tumorigenic activity of A549 cells. The tumor growth-enhancing activity was reduced by deletion of the BH3 domain of BNIP3. Expression of a dominant-negative mutant of BNIP3 lacking the C-terminal transmembrane domain also inhibited the tumorigenic potential of A549 cells. These results suggest that BNIP3 plays a fundamental role in the development of certain solid tumors such as the lung and breast carcinomas.
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Neonatal PMN (polymorphonuclear neutrophils) exhibit altered inflammatory responsiveness and greater longevity compared with adult PMN; however, the involved mechanisms are incompletely defined. Receptors containing immunoreceptor tyrosine-based inhibitory motif (ITIM) domains promote apoptosis by activating inhibitory phosphatases, such as Src homology domain 2-containing tyrosine phosphatase-1 (SHP-1), that block survival signals. Sialic acid-binding immunoglobulin-like lectin (Siglec)-9, an immune inhibitory receptor with an ITIM domain, has been shown to induce cell death in adult PMN in association with SHP-1. To test our hypothesis that neonatal PMN inflammatory function may be modulated by unique Siglec-9 and SHP-1 interactions, we compared expression of these proteins in adult and neonatal PMN. Neonatal PMN exhibited diminished cellular expression of Siglec-9, which was phosphorylated in the basal state. Granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment decreased Siglec-9 phosphorylation levels in neonatal PMN but promoted its phosphorylation in adult PMN, observations associated with altered survival signaling. Although SHP-1 expression was also diminished in neonatal PMN, GM-CSF treatment had minimal effect on phosphorylation status. Further analysis revealed that Siglec-9 and SHP-1 physically interact, as has been observed in other immune cells. Our data suggest that age-specific interactions between Siglec-9 and SHP-1 may influence the altered inflammatory responsiveness and longevity of neonatal PMN.
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Antígenos CD/metabolismo , Lectinas/metabolismo , Neutrófilos/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Adulto , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Recién Nacido , Neutrófilos/citología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico , Proteína Letal Asociada a bcl/metabolismoRESUMEN
Polyphenol phytoalexin (resveratrol), found in grapes and red wine is a strong chemopreventive agent with promising safety records with human consumption and unique forms of cell death induction in a variety of tumor cells. However, the mechanism of resveratrol-induced apoptosis upstream of mitochondria is still not defined. The results from this study suggest that caspase-2 activation occurs upstream of mitochondria in resveratrol-treated cells. The upstream activation of caspase-2 is not dependent on its antioxidant property or NF-kappaB inhibition. The activated caspase-2 triggers mitochondrial apoptotic events by inducing conformational changes in Bax/Bak with subsequent release of cytochrome c, apoptosis-inducing factor, and endonuclease G. Caspase-8 activation seems to be independent of these events and does not appear to be mediated by classical death receptor processing or downstream caspases. Both caspase-2 and caspase-8 contribute toward the mitochondrial translocation of Bid, since neither caspase-8 inhibition nor caspase-2 inhibition could prevent translocation of Bid DsRed into mitochondria. Caspase-2 inhibitors or antisense silencing of caspase-2 prevented cell death induced by resveratrol and partially prevented processing of downstream caspases, including caspase-9, caspase-3, and caspase-8. Studies using mouse embryonic fibroblasts deficient for both Bax and Bak indicate the contribution of both Bax and Bak in mediating cell death induced by resveratrol and the existence of Bax/Bak-independent cell death possibly through caspase-8- or caspase-2-mediated mitochondria-independent downstream caspase processing.
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Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Estilbenos/farmacología , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Apoptosis/fisiología , Factor Inductor de la Apoptosis/metabolismo , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Caspasa 2 , Caspasa 3 , Caspasa 8 , Caspasa 9 , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Citocromos c/metabolismo , Citosol/metabolismo , Endodesoxirribonucleasas/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/genética , Células HCT116 , Humanos , Ratones , Mitocondrias/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Conformación Proteica/efectos de los fármacos , Resveratrol , Proteína Destructora del Antagonista Homólogo bcl-2/química , Proteína X Asociada a bcl-2/química , Proteína X Asociada a bcl-2/genéticaRESUMEN
Multiple apoptotic stimuli induce conformational changes in Bax, a proapoptotic protein from the Bcl-2 family and its deficiency is a frequent cause of chemoresistance in colon adenocarcinomas. Curcumin, a dietary compound from turmeric, is known to induce apoptosis in a variety of cancer cells. To understand the role of Bax in curcumin-induced apoptosis we used HCT116 human colon cancer cells with one allele of Bax gene (Bax+/-) and Bax knockout HCT116 (Bax-/-) cells in which Bax gene is inactivated by homologous recombination. Cell viability decreased in a concentration-dependent manner in Bax+/- cells treated with curcumin (0-50 microM) whereas only minimal changes in viability were observed in Bax-/- cells upon curcumin treatment. In Bax-/- cells curcumin-induced activation of caspases 9 and 3 was blocked and that of caspase 8 remained unaltered. Curcumin-induced release of cytochrome c, Second mitochondria derived activator of caspase (Smac) and apoptosis inducing factor (AIF) was also blocked in Bax-/- cells and reintroduction of Bax, downregulation of the antiapoptotic protein Bcl-XL by antisense DNA as well as the overexpression of Smac, highly sensitized the Bax-/- cells toward curcumin-induced apoptosis. There was no considerable difference in the percentage of apoptotic cells in Bak RNAi transfected Bax+/- or Bax-/- cells treated with curcumin when compared with their corresponding vector transfected cells treated with curcumin. The present study demonstrates the role of Bax but not Bak as a critical regulator of curcumin-induced apoptosis and implies the potential of targeting antiapoptotic proteins like Bcl-XL or overexpression of proapoptotic proteins like Smac as interventional approaches to deal with Bax-deficient chemo-resistant cancers for curcumin-based therapy.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias del Colon/metabolismo , Curcumina/farmacología , Glicoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Factor Inductor de la Apoptosis , Inhibidores de Caspasas , Caspasas/metabolismo , Neoplasias del Colon/patología , Complejo de Ataque a Membrana del Sistema Complemento , Proteínas del Sistema Complemento , Citocromos c/metabolismo , Regulación hacia Abajo , Resistencia a Antineoplásicos , Activación Enzimática/efectos de los fármacos , Flavoproteínas/antagonistas & inhibidores , Flavoproteínas/metabolismo , Glicoproteínas/antagonistas & inhibidores , Glicoproteínas/genética , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Oligonucleótidos Antisentido/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
Curcumin, the yellow pigment derived from Curcuma longa, is known to induce apoptosis of several cancer cells. However, many cancer cells protect themselves by over-expressing antiapoptotic proteins such as Bcl-XL or Ku70. To study their role in curcumin-induced apoptosis, human colon cancer cells (SW480) were made to over-express or under-express Bcl-XL (by stable transfection) and Ku70 (by transient transfection) using plasmid constructs that express their genes in sense or antisense orientation, respectively. Stable cells that express Bax [Bax-GFP (green fluorescent protein)], a proapoptotic member of the Bcl-2 family, were also established. Curcumin-induced cell death and nuclear condensation was more in AsBcl-XL and AsKu70 cells that under-express Bcl-XL and Ku70, respectively, compared with the vector-transfected cells. Bcl-XL and Ku70 protected the cells by inhibiting the release of cytochrome c, Smac (second mitochondria derived activator of caspase) and apoptosis inducing factor (AIF), and the activation of caspases 9, 8 and 3 triggered by curcumin. AsBcl-XL and AsKu70 cells were more sensitive to curcumin through enhanced activation of caspases 9 and 3 and release of cytochrome c, Smac and AIF. Curcumin-induced activation of caspase 8 was blocked by Ku70 but not by Bcl-XL. However, caspase 8 activation by curcumin was accelerated in both AsBcl-XL and AsKu70 cells suggesting a possible feedback activation of caspase 8 by caspase 3. Bax-GFP cells were highly sensitized when Ku70 was down-regulated supporting the reported role of Ku70 in the retention of Bax within the cytosol. The study reveals the potential of antisense inhibition of antiapoptotic proteins as an effective strategy to tackle chemoresistant cancers with curcumin.
Asunto(s)
Antígenos Nucleares/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias del Colon/patología , Curcumina/farmacología , Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor Inductor de la Apoptosis , Inhibidores de Caspasas , Caspasas/metabolismo , Neoplasias del Colon/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento , Proteínas del Sistema Complemento , Citocromos c/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Regulación hacia Abajo , Activación Enzimática/efectos de los fármacos , Flavoproteínas/metabolismo , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Autoantígeno Ku , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
We have shown earlier that heat shock renders human colon cancer cells resistant to curcumin-induced apoptosis, but the contribution of individual heat shock proteins (hsps) to this resistance has not been tested. High expression of hsp27 and hsp70 in breast, endometrial and gastric cancers has been associated with metastasis, poor prognosis and resistance to chemo- or radiotherapy. In this study, SW480 cells were transfected with hsp70 cDNA in either the sense or antisense orientation and stable clones were selected and tested for their sensitivity to curcumin. The cells were protected from curcumin-induced cell death by hsp70 while cells harboring antisense hsp70 (Ashsp70) were highly sensitive to curcumin. Curcumin-induced nuclear condensation was less in hsp70 but more in Ashsp70 cells when compared with control vector-transfected cells. Loss of mitochondrial transmembrane potential induced by curcumin was further accelerated by antisense hsp70 expression and hsp70 restored it partly. Ashsp70 cells released more cytochrome c, AIF and Smac from mitochondria upon curcumin treatment than control cells. hsp70 partly prevented the release of AIF but not the other proteins. Activation of caspases 3 and 9 induced by curcumin was also inhibited by hsp70, whereas more activation could be seen in Ashsp70 cells, although caspase 8 activation was unaffected by changes in hsp70 expression. Curcumin-induced cleavage of PARP and DFF45 was inhibited by hsp70 but enhanced in Ashsp70 cells. The present study demonstrates the potential of hsp70 in protecting SW480 cells from curcumin-induced apoptosis and highlights that silencing the expression of hsp70 is an effective approach to augment curcumin-based therapy in cancers that are resistant due to hsp70 expression.