RESUMEN
Dietary boron intake is associated with reduced prostate and lung cancer risk and increased bone mass. Boron is absorbed and circulated as boric acid (BA) and at physiological concentrations is a reversible competitive inhibitor of cyclic ADP ribose, the endogenous agonist of the ryanodine receptor calcium (Ca(+2)) channel, and lowers endoplasmic reticulum (ER) [Ca(2+)]. Low ER [Ca(2+)] has been reported to induce ER stress and activate the eIF2α/ATF4 pathway. Here we report that treatment of DU-145 prostate cells with physiological levels of BA induces ER stress with the formation of stress granules and mild activation of eIF2α, GRP78/BiP, and ATF4. Mild activation of eIF2α and its downstream transcription factor, ATF4, enables cells to reconfigure gene expression to manage stress conditions and mild activation of ATF4 is also required for the differentiation of osteoblast cells. Our results using physiological levels of boric acid identify the eIF2α/ATF pathway as a plausible mode of action that underpins the reported health effects of dietary boron.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Ácidos Bóricos/toxicidad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Factor 2 Eucariótico de Iniciación/metabolismo , Neoplasias de la Próstata/metabolismo , Línea Celular Tumoral , Chaperón BiP del Retículo Endoplásmico , Humanos , Immunoblotting , Masculino , Microscopía Fluorescente , Fosforilación/efectos de los fármacosRESUMEN
The ryanodine receptor (RyR) is a large, intracellular calcium (Ca(2+)) channel that is associated with several accessory proteins and is an important component of a cell's ability to respond to changes in the environment. Three isoforms of the RyR exist and are well documented for skeletal and cardiac muscle and the brain, but the isoforms in non-excitable cells are poorly understood. The aggressiveness of breast cancers in women has been positively correlated with the expression of the RyR in breast tumor tissue, but it is unknown if this is limited to specific isoforms. Identification and characterization of RyRs in cancer models is important in understanding the role of the RyR channel complex in cancer and as a potential therapeutic target. The objective of this report was to identify the RyR isoforms expressed in widely used prostate cancer cell lines, DU-145 and LNCaP, and the non-tumorigenic prostate cell line, PWR-1E. Oligonucleotide primers specific for each isoform were used in semi-quantitative and real-time PCR to determine the identification and expression levels of the RyR isoforms. RyR1 was expressed in the highest amount in DU-145 tumor cells, expression was 0.48-fold in the non-tumor cell line PWR-1E compared to DU-145 cells, and no expression was observed in LNCaP tumor cells. DU-145 cells had the lowest expression of RyR2. The expression was 26- and 15-fold higher in LNCaP and PWR-1E cells, respectively. RyR3 expression was not observed in any of the cell lines. All cell types released Ca(2+) in response to caffeine showing they had functional RyRs. Total cellular RyR-associated Ca(2+) release is determined by both the number of activated RyRs and its accessory proteins which modulate the receptor. Our results suggest that the correlation between the expression of the RyR and tumor aggression is not related to specific RyR isoforms, but may be related to the activity and number of receptors.
Asunto(s)
Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , Humanos , Masculino , Neoplasias de la Próstata/patología , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/biosíntesisRESUMEN
Fruits, nuts, legumes, and vegetables are rich sources of boron (B), an essential plant nutrient with chemopreventive properties. Blood boric acid (BA) levels reflect recent B intake, and men at the US mean intake have a reported non-fasting level of 10 µM. Treatment of DU-145 prostate cancer cells with physiological concentrations of BA inhibits cell proliferation without causing apoptosis and activates eukaryotic initiation factor 2 (eIF2α). EIF2α induces cell differentiation and protects cells by redirecting gene expression to manage endoplasmic reticulum stress. Our objective was to determine the temporal expression of endoplasmic reticulum (ER) stress-activated genes in DU-145 prostate cells treated with 10 µM BA. Immunoblots showed post-treatment increases in eIF2α protein at 30 min and ATF4 and ATF6 proteins at 1 h and 30 min, respectively. The increase in ATF4 was accompanied by an increase in the expression of its downstream genes growth arrest and DNA damage-induced protein 34 (GADD34) and homocysteine-induced ER protein (Herp), but a decrease in GADD153/CCAAT/enhancer-binding protein homologous protein (CHOP), a pro-apoptotic gene. The increase in ATF6 was accompanied by an increase in expression of its downstream genes GRP78/BiP, calreticulin, Grp94, and EDEM. BA did not activate IRE1 or induce cleavage of XBP1 mRNA, a target of IRE1. Low boron status has been associated with increased cancer risk, low bone mineralization, and retinal degeneration. ATF4 and BiP/GRP78 function in osteogenesis and bone remodeling, calreticulin is required for tumor suppressor p53 function and mineralization of teeth, and BiP/GRP78 and EDEM prevent the aggregation of misfolded opsins which leads to retinal degeneration. The identification of BA-activated genes that regulate its phenotypic effects provides a molecular underpinning for boron nutrition and biology.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Factor de Transcripción Activador 6/metabolismo , Ácidos Bóricos/administración & dosificación , Ácidos Bóricos/farmacología , Factor 2 Eucariótico de Iniciación/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Masculino , Células Tumorales Cultivadas , Estados UnidosRESUMEN
INTRODUCTION: Cardiac toxicity, manifested as diminished contractility, ischemic heart disease, and heart failure is a major issue in drug safety. Concerns revolve around targeted drugs (TKIs) where contractility effects were not anticipated. The ability to predict cardiac toxicity early would help to de-risk drugs in development and prepare physicians to manage risk in the clinic. Issues with current preclinical studies include insufficient testing with informative, translatable models, and predictive biomarkers. The isolated heart model is amenable to multiple assessments which can be combined with current technologies to assess toxicity on a multi-scale level. METHODS: Rat isolated heart model was used to assess changes in left ventricular (LV) contractility and protein biomarkers BNP, IL6, TNFα, and cardiac troponins T (TnT) and I (TnI). Responses were assessed during perfusion with modified Henseleit Krebs (MHK), and 20 min concentration escalations of verapamil, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), isoproterenol, or 20 min escalations bracketing clinical blood concentrations of sunitinib, sorafenib, and erlotinib. LV parameters and effluent for biomarkers were collected before and during escalating drug concentrations. RESULTS: Verapamil reduced inotropy with no change in biomarkers, FCCP and isoproterenol reduced and increased heart function respectively and increased TnT and TNFα. Erlotinib had no significant effects on function or biomarkers. Sunitinib diminished function, increased TNFα at 0.1 µM, and increased TnT at higher concentrations. Sorafenib dose dependently increased TNFα beginning at 0.1 µM, reducing contractility and flow rate at 0.6 µM. DISCUSSION: The ex-vivo assay is a sensitive and predictive model for assessing changes in heart function and biomarkers of toxicity and injury. This assay demonstrates the potential for sunitinib and sorafenib to cause cardiac toxicity in humans. Also, TNFα appears to be a biomarker in the heart prior to injury. Due to its versatility, the isolated heart assay has potential to fill gaps in cardiac safety testing early in drug development.