RESUMEN
The indenoisoquinoline NSC724998 is a novel topoisomerase I (Top1) inhibitor entering Phase I clinical trials at the National Cancer Institute, USA. In this study, 2-D PAGE analysis was performed on nuclear lysates prepared from HCT-116 and A375 cells treated with 1 microM NSC724998 for 24 h and the differentially regulated spots identified by LC-MS/MS. One-hundred fourteen protein spot differentials were identified, 66 from A375 cells and 48 from HCT-116 cells. Proteins related to apoptosis changed specifically in A375 cells, whereas proteins involved in the ubiquitin-proteasome system were highly enriched in treated HCT-116 cells. Importantly, 12 differentially expressed proteins (ETFA, HCC1, HNRCL, KAP1, NPM, NUCL, PRDX1, PRP19, PSB6, RAE1L, RU2A, and SFRS9) were common to both cell lines. Western blotting and immunocytochemistry confirmed significant nuclear upregulation of both the proteasome subunit PSB6 and the transcriptional repressor KAP1. Interestingly, increased KAP1 polypeptide was accompanied by enhanced phosphorylation at Ser824. Similar to gammaH2AX, KAP1 phosphorylation was consistently enhanced in a panel of 12 cell lines and in A375 xenografts following NSC 724998 treatment. In summary, these data enhance our understanding of protein dynamics in the nucleus following DNA damage and provide an alternate marker (pKAP1) with potential for monitoring clinical responses to Top1 poisons.
Asunto(s)
Benzodioxoles/farmacología , Núcleo Celular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Isoquinolinas/farmacología , Proteómica , Inhibidores de Topoisomerasa I/farmacología , Western Blotting , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cromatografía Liquida , Daño del ADN , Electroforesis en Gel Bidimensional , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Espectrometría de Masas , Proteínas Represoras/metabolismo , Proteína 28 que Contiene Motivos TripartitoRESUMEN
The "Warburg effect," also termed aerobic glycolysis, describes the increased reliance of cancer cells on glycolysis for ATP production, even in the presence of oxygen. Consequently, there is continued interest in inhibitors of glycolysis as cancer therapeutics. One example is dichloroacetate (DCA), a pyruvate mimetic that stimulates oxidative phosphorylation through inhibition of pyruvate dehydrogenase kinase. In this study, the mechanistic basis for DCA anti-cancer activity was re-evaluated in vitro using biochemical, cellular and proteomic approaches. Results demonstrated that DCA is relatively inactive (IC(50) ≥ 17 mM, 48 hr), induces apoptosis only at high concentrations (≥ 25 mM, 48 hr) and is not cancer cell selective. Subsequent 2D-PAGE proteomic analysis confirmed DCA-induced growth suppression without apoptosis induction. Furthermore, DCA depolarizes mitochondria and promotes reactive oxygen species (ROS) generation in all cell types. However, DCA was found to have selective activity against rho(0) cells [mitochondrial DNA (mtDNA) deficient] and to synergize with 2-deoxyglucose in complex IV deficient HCT116 p53(-/-) cells. DCA also synergized in vitro with cisplatin and topotecan, two antineoplastic agents known to damage mitochondrial DNA. These data suggest that in cells "hardwired" to selectively utilize glycolysis for ATP generation (e.g., through mtDNA mutations), the ability of DCA to force oxidative phosphorylation confers selective toxicity. In conclusion, although we provide a mechanism distinct from that reported previously, the ability of DCA to target cell lines with defects in the electron transport chain and to synergize with existing chemotherapeutics supports further preclinical development.
Asunto(s)
Ácido Dicloroacético/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Transporte de Electrón , Glucólisis , Células HCT116 , Células HL-60 , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteómica , Conejos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Analogs of the malaria therapeutic, artemisinin, possess in vitro and in vivo anticancer activity. In this study, two dimeric artemisinins (NSC724910 and 735847) were studied to determine their mechanism of action. Dimers were >1,000 fold more active than monomer and treatment was associated with increased reactive oxygen species (ROS) and apoptosis induction. Dimer activity was inhibited by the antioxidant L-NAC, the iron chelator desferroxamine and exogenous hemin. Similarly, induction of heme oxygenase (HMOX) with CoPPIX inhibited activity, whereas inhibition of HMOX with SnPPIX enhanced it. These results emphasize the importance of iron, heme and ROS in activity. Microarray analysis of dimer treated cells identified DNA damage, iron/heme and cysteine/methionine metabolism, antioxidant response, and endoplasmic reticulum (ER) stress as affected pathways. Detection of an ER-stress response was relevant because in malaria, artemisinin inhibits pfATP6, the plasmodium orthologue of mammalian sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases (SERCA). A comparative study of NSC735847 with thapsigargin, a specific SERCA inhibitor and ER-stress inducer showed similar behavior in terms of transcriptomic changes, induction of endogenous SERCA and ER calcium mobilization. However, thapsigargin had little effect on ROS production, modulated different ER-stress proteins and had greater potency against purified SERCA1. Furthermore, an inactive derivative of NSC735847 that lacked the endoperoxide had identical inhibitory activity against purified SERCA1, suggesting that direct inhibition of SERCA has little inference on overall cytotoxicity. In summary, these data implicate indirect ER-stress induction as a central mechanism of artemisinin dimer activity.
Asunto(s)
Antineoplásicos/farmacología , Artemisininas/farmacología , Retículo Endoplásmico/efectos de los fármacos , Hemo-Oxigenasa 1/metabolismo , Hemo/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Artemisia/química , Biomarcadores/metabolismo , Western Blotting , Calcio/metabolismo , Ciclo Celular/efectos de los fármacos , Dimerización , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Humanos , Lisina/análogos & derivados , Lisina/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tapsigargina/farmacologíaRESUMEN
BACKGROUND: The nucleoside analog, ARC (NSC 188491) is a recently characterized transcriptional inhibitor that selectively kills cancer cells and has the ability to perturb angiogenesis in vitro. In this study, the mechanism of action of ARC was further investigated by comparing in vitro and in vivo activity with other anti-neoplastic purines. METHODS: Structure-based homology searches were used to identify those compounds with similarity to ARC. Comparator compounds were then evaluated alongside ARC in the context of viability, cell cycle and apoptosis assays to establish any similarities. Following this, biological overlap was explored in detail using gene-expression analysis and kinase inhibition assays. RESULTS: Results demonstrated that sangivamycin, an extensively characterized pro-apoptotic nucleoside isolated from Streptomyces, had identical activity to ARC in terms of 1) cytotoxicity assays, 2) ability to induce a G2/M block, 3) inhibitory effects on RNA/DNA/protein synthesis, 4) transcriptomic response to treatment, 5) inhibition of protein kinase C, 6) inhibition of positive transcription elongation factor b (P-TEFb), 7) inhibition of VEGF secretion, and 8) activity within hollow fiber assays. Extending ARC activity to PKC inhibition provides a molecular basis for ARC cancer selectivity and anti-angiogenic effects. Furthermore, functional overlap between ARC and sangivamycin suggests that development of ARC may benefit from a retrospective of previous sangivamycin clinical trials. However, ARC was found to be inactive in several xenograft models, likely a consequence of rapid serum clearance. CONCLUSION: Overall, these data expand on the biological properties of ARC but suggest additional studies are required before it can be considered a clinical trials candidate.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Nucleósidos/farmacología , Factor B de Elongación Transcripcional Positiva/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Nucleósidos de Pirimidina/farmacología , Pirimidinas/farmacología , Animales , Antibióticos Antineoplásicos/química , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , ADN de Neoplasias/biosíntesis , Ensayos de Selección de Medicamentos Antitumorales , Células HL-60 , Humanos , Ratones , Proteínas de Neoplasias/biosíntesis , Nucleósidos/química , Fosforilación , Inhibidores de Proteínas Quinasas/química , Nucleósidos de Pirimidina/química , Pirimidinas/química , ARN Polimerasa II/metabolismo , ARN Neoplásico/biosíntesis , Relación Estructura-Actividad , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Activities distinct from inhibition of Bcr/abl have led to adaphostin (NSC 680410) being described as "a drug in search of a mechanism." In this study, proteomic analysis of adaphostin-treated myeloid leukemia cell lines was used to further elucidate a mechanism of action. EXPERIMENTAL DESIGN: HL60 and K562 cells treated with adaphostin for 6, 12, or 24 h were analyzed using two-dimensional PAGE. Differentially expressed spots were excised, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. The contribution of the redox-active hydroquinone group in adaphostin was also examined by carrying out proteomic analysis of HL60 cells treated with a simple hydroquinone (1,4-dihydroxybenzene) or H(2)O(2). RESULTS: Analysis of adaphostin-treated cells identified 49 differentially expressed proteins, the majority being implicated in the response to oxidative stress (e.g., CALM, ERP29, GSTP1, PDIA1) or induction of apoptosis (e.g., LAMA, FLNA, TPR, GDIS). Interestingly, modulation of these proteins was almost fully prevented by inclusion of an antioxidant, N-acetylcysteine. Validation of the proteomic data confirmed GSTP1 as an adaphostin resistance gene. Subsequent analysis of HL60 cells treated with 1,4-dihydroxybenzene or H(2)O(2) showed similar increases in intracellular peroxides and an almost identical proteomic profiles to that of adaphostin treatment. Western blotting of a panel of cell lines identified Cu/Zn superoxide dismutase (SOD) as correlating with adaphostin resistance. The role of SOD as a second adaphostin resistance gene was confirmed by demonstrating that inhibition of SOD using diethyldithiocarbamate increased adaphostin sensitivity, whereas transfection of SOD I attenuated toxicity. Importantly, treatment with 1,4-dihydroxybenzene or H(2)O(2) replicated adaphostin-induced Bcr/abl polypeptide degradation, suggesting that kinase inhibition is a ROS-dependent phenomenon. CONCLUSION: Adaphostin should be classified as a redox-active-substituted dihydroquinone.