RESUMEN

Terbinafine N-dealkylation pathways result in formation of 6,6-dimethyl-2-hepten-4-ynal (TBF-A), a reactive allylic aldehyde, that may initiate idiosyncratic drug-induced liver toxicity. Previously, we reported on the importance of CYP2C19 and 3A4 as major contributors to TBF-A formation. In this study, we expanded on those efforts to assess individual contributions of CYP1A2, 2B6, 2C8, 2C9, and 2D6 in terbinafine metabolism. The combined knowledge gained from these studies allowed us to scale the relative roles of the P450 isozymes in hepatic clearance of terbinafine including pathways leading to TBF-A, and hence, provide a foundation for assessing their significance in terbinafine-induced hepatotoxicity. We used in vitro terbinafine reactions with recombinant P450s to measure kinetics for multiple metabolic pathways and calculated contributions of all individual P450 isozymes to in vivo hepatic clearance for the average human adult. The findings confirmed that CYP3A4 was a major contributor (at least 30% total metabolism) to all three of the possible N-dealkylation pathways; however, CYP2C9, and not CYP2C19, played a critical role in terbinafine metabolism and even exceeded CYP3A4 contributions for terbinafine N-demethylation. A combination of their metabolic capacities accounted for at least 80% of the conversion of terbinafine to TBF-A, while CYP1A2, 2B6, 2C8, and 2D6 made minor contributions. Computational approaches provide a more rapid, less resource-intensive strategy for assessing metabolism, and thus, we additionally predicted terbinafine metabolism using deep neural network models for individual P450 isozymes. Cytochrome P450 isozyme models accurately predicted the likelihood for terbinafine N-demethylation, but overestimated the likelihood for a minor N-denaphthylation pathway. Moreover, the models were not able to differentiate the varying roles of the individual P450 isozymes for specific reactions with this particular drug. Taken together, the significance of CYP2C9 and 3A4 and to a lesser extent, CYP2C19, in terbinafine metabolism is consistent with reported drug interactions. This finding suggests that variations in individual P450 contributions due to other factors like polymorphisms may similarly contribute to terbinafine-related adverse health outcomes. Nevertheless, the impact of their metabolic capacities on formation of reactive TBF-A and consequent idiosyncratic hepatotoxicity will be mitigated by competing detoxification pathways, TBF-A decay, and TBF-A adduction to glutathione that remain understudied.

Asunto(s)

Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Microsomas Hepáticos/metabolismo , Terbinafina/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Tasa de Depuración Metabólica , Microsomas Hepáticos/efectos de los fármacos , Terbinafina/farmacocinética

RESUMEN

BACKGROUND: Declining liver function is a concerning side effect associated with radiation therapy. Biomarkers of liver toxicity would be useful in personalizing therapy. METHODS: As part of two prospective clinical trials examining adaptive radiation therapy, we collected serum samples from patients receiving liver radiation. We performed a screen of 22 cytokines using a multiplex assay then used ELISA to quantify the cytokines of greatest interest. Subjects were split into screening and validation cohorts. Toxicity was defined as an increase in Child-Pugh score of 2 points or greater within 6 months. Logistic regression models were used to estimate the relationship between our toxicity endpoint and serum cytokine concentrations. RESULTS: Our initial screen (46 subjects, 11 events) identified hepatocyte growth factor (HGF), CD40L (CD154), and eotaxin (CCL11) as potentially predictive of toxicity. We then tested these markers in an expanded patient cohort (104 subjects, 18 events) with a batch correction due to varying age of the samples which confirmed that high HGF and low CD40L were associated with a subsequent decline in liver function following radiation therapy. Multivariate analysis factoring in baseline Child-Pugh score and mean liver radiation dose demonstrated that HGF and CD40L were potentially predictive of toxicity (HGF OR 4.3, P = .009; CD40L OR 0.5 P = .06). Additionally, higher than median baseline HGF levels (1.4 ng/ml) were significantly associated with decreased survival following liver radiation (27.1 vs 14.5 months, P = .03). CONCLUSIONS: Our study identifies high HGF and low CD40L as potential markers of liver toxicity following radiation therapy.

RESUMEN

Lamisil (terbinafine) is an effective, widely prescribed antifungal drug that causes rare idiosyncratic hepatotoxicity. The proposed toxic mechanism involves a reactive metabolite, 6,6-dimethyl-2-hepten-4-ynal (TBF-A), formed through three N-dealkylation pathways. We were the first to characterize them using in vitro studies with human liver microsomes and modeling approaches, yet knowledge of the individual enzymes catalyzing reactions remained unknown. Herein, we employed experimental and computational tools to assess terbinafine metabolism by specific cytochrome P450 isozymes. In vitro inhibitor phenotyping studies revealed six isozymes were involved in one or more N-dealkylation pathways. CYP2C19 and 3A4 contributed to all pathways, and so, we targeted them for steady-state analyses with recombinant isozymes. N-Dealkylation yielding TBF-A directly was catalyzed by CYP2C19 and 3A4 similarly. Nevertheless, CYP2C19 was more efficient than CYP3A4 at N-demethylation and other steps leading to TBF-A. Unlike microsomal reactions, N-denaphthylation was surprisingly efficient for CYP2C19 and 3A4, which was validated by controls. CYP2C19 was the most efficient among all reactions. Nonetheless, CYP3A4 was more selective at steps leading to TBF-A, making it more effective in terbinafine bioactivation based on metabolic split ratios for competing pathways. Model predictions did not extrapolate to quantitative kinetic constants, yet some results for CYP3A4 and CYP2C19 agreed qualitatively with preferred reaction steps and pathways. Clinical data on drug interactions support the CYP3A4 role in terbinafine metabolism, while CYP2C19 remains understudied. Taken together, knowledge of P450s responsible for terbinafine metabolism and TBF-A formation provides a foundation for investigating and mitigating the impact of P450 variations in toxic risks posed to patients.

Asunto(s)

Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP3A/metabolismo , Inhibidores Enzimáticos/farmacología , Terbinafina/farmacología , Biocatálisis , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Cinética , Modelos Moleculares , Estructura Molecular , Terbinafina/química , Terbinafina/metabolismo

RESUMEN

PURPOSE: The purpose of this study was to translate our in vitro therapy approach to an in vivo model. Increased glutamine uptake is known to drive cancer cell proliferation, making tumor cells glutamine-dependent. Studying lymph-node aspirates containing malignant lung tumor cells showed a strong correlation between glutamine consumption and glutathione (GSH) excretion. Subsequent experiments with A549 and H460 lung tumor cell lines provided additional evidence for glutamine's role in driving synthesis and excretion of GSH. Using stable-isotope-labeled glutamine as a tracer metabolite, we demonstrated that the glutamate group in GSH is directly derived from glutamine, linking glutamine utilization intimately to GSH syntheses. MATERIALS AND METHODS: To understand the possible mechanistic link between glutamine consumption and GSH excretion, we studied GSH metabolism in more detail. Inhibition of glutaminase (GLS) with BPTES, a GLS-specific inhibitor, effectively abolished GSH synthesis and excretion. Since our previous work, several novel GLS inhibitors became available and we report herein effects of CB-839 in A427, H460 and A549 lung tumor cells and human lungtumor xenografts in mice. RESULTS: Inhibition of GLS markedly reduced cell viability, producing ED50 values for inhibition of colony formation of 9, 27 and 217 nM in A427, A549 and H460, respectively. Inhibition of GLS is accompanied by ∼30% increased response to radiation, suggesting an important role of glutamine-derived GSH in protecting tumor cells against radiation-induced injury. In subsequent mouse xenografts, short-term CB-839 treatments reduced serum GSH by >50% and increased response to radiotherapy of H460-derived tumor xenografts by 30%. CONCLUSION: The results support the proposed mechanistic link between GLS activity and GSH synthesis and suggest that GLS inhibitors are effective radiosensitizers.

Asunto(s)

Bencenoacetamidas/farmacología , Glutaminasa/antagonistas & inhibidores , Neoplasias Pulmonares/radioterapia , Tolerancia a Radiación/efectos de los fármacos , Tiadiazoles/farmacología , Animales , Línea Celular Tumoral , Femenino , Glutamina/metabolismo , Glutatión/metabolismo , Humanos , Masculino , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Lamisil (terbinafine) may cause idiosyncratic liver toxicity through a proposed toxicological mechanism involving the reactive metabolite 6,6-dimethyl-2-hepten-4-ynal (TBF-A). TBF-A toxicological relevance remains unclear due to a lack of identification of pathways leading to and competing with TBF-A formation. We resolved this knowledge gap by combining computational modeling and experimental kinetics of in vitro hepatic N-dealkylation of terbinafine. A deep learning model of N-dealkylation predicted a high probability for N-demethylation to yield desmethyl-terbinafine followed by N-dealkylation to TBF-A and marginal contributions from other possible pathways. We carried out steady-state kinetic experiments with pooled human liver microsomes that relied on development of labeling methods to expand metabolite characterization. Those efforts revealed high levels of TBF-A formation and first order decay during metabolic reactions; actual TBF-A levels would then reflect the balance between those processes as well as reflect the impact of stabilizing adduction with glutathione and other biological molecules. Modeling predictions and experimental studies agreed on the significance of N-demethylation and insignificance of N-denaphthylation in terbinafine metabolism, yet differed on importance of direct TBF-A formation. Under steady-state conditions, the direct pathway was the most important source of the reactive metabolite with a Vmax/Km of 4.0 pmol/min/mg protein/µM in contrast to model predictions. Nevertheless, previous studies show that therapeutic dosing leads to accumulation of desmethyl-terbinafine in plasma, which means that likely sources for TBF-A would draw from metabolism of both the major metabolite and parent drug based on our modeling and experimental studies. Through this combination of novel modeling and experimental approaches, we are the first to identify pathways leading to generation of TBF-A for assessing its role in idiosyncratic adverse drug interactions.

Asunto(s)

Simulación por Computador , Modelos Biológicos , Terbinafina/metabolismo , Terbinafina/toxicidad , Antifúngicos/química , Antifúngicos/metabolismo , Antifúngicos/toxicidad , Línea Celular , Humanos , Estructura Molecular , Relación Estructura-Actividad , Terbinafina/química

RESUMEN

PURPOSE: Wee1 kinase inhibitors are effective radiosensitizers in cells lacking a G1 checkpoint. In this study we examined the potential effect of Wee1 kinase inhibition on inducing replication stress in hepatocellular carcinoma (HCC). METHODS AND MATERIALS: Five independent datasets from the Oncomine database comparing gene expression in HCC compared to normal tissue were combined and specific markers associated with Wee1 sensitivity were analyzed. We then performed a series of in vitro experiments to study the effect of Wee1 inhibition on irradiated HCC cell lines with varying p53 mutational status. Clonogenic survival assays and flow cytometry using anti-γH2AX and phospho-histone H3 antibodies with propidium iodide were performed to study the effect of AZD1775 on survival, cell cycle, and DNA repair. Additionally, nucleoside enriched medium was used to examine the effect of altering nucleotide pools on Wee1 targeted radiation sensitization. RESULTS: Our analysis of the Oncomine database found high levels of CDK1 and other cell cycle regulators indicative of Wee1 sensitivity in HCC. In our in vitro experiments, treatment with AZD1775 radiosensitized and chemosensitized Hep3B, Huh7, and HepG2 cell lines and was associated with delayed resolution of γH2AX foci and the induction of pan-nuclear γH2AX staining. Wee1 inhibition attenuated radiation-induced G2 arrest in the Hep3B (TP53 null) and Huh7 (TP53 mutant) cell lines but not in the TP53 wild-type cell line HepG2. Supplementation with nucleosides reversed the radiation-sensitizing effect of AZD1775 and reduced the amount of cells with pan-nuclear γH2AX staining after radiation. CONCLUSIONS: Radiation sensitization with Wee1 inhibition occurs in cells regardless of their p53 mutational status. In this study we show for the first time that replication stress via the overconsumption of nucleotides plays an important role in AZD1775-induced radiation sensitization.

Asunto(s)

Carcinoma Hepatocelular/radioterapia , Proteínas de Ciclo Celular/antagonistas & inhibidores , Genes p53 , Neoplasias Hepáticas/radioterapia , Mutación , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Proteína Quinasa CDC2/análisis , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Daño del ADN , Replicación del ADN , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Células Hep G2 , Histonas/análisis , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Pirimidinonas , Estrés Fisiológico

RESUMEN

To improve the efficacy of chemoradiation therapy for locally advanced pancreatic cancer and begin to establish patient selection criteria, we investigated the combination of the WEE1 inhibitor AZD1775 with gemcitabine-radiation in homologous recombination (HR) repair proficient and deficient pancreatic cancers. Sensitization to gemcitabine-radiation by AZD1775 was assessed in pancreatic cancer cells by clonogenic survival and in patient-derived xenografts by tumor growth. The contributions of HR repair inhibition and G2 checkpoint abrogation to sensitization were assessed by γH2AX, BRCA2 manipulation, and RAD51 focus formation and pHistone H3 flow cytometry, respectively. We found that AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type but not BRCA2 mutant pancreatic cancer cells. In all cells, AZD1775 caused inhibition of CDK1 phosphorylation and G2 checkpoint abrogation. However, sensitization by AZD1775 was associated with persistent γH2AX and inhibition of RAD51 focus formation. In HR-proficient (BRCA2 wild-type) or -deficient (BRAC2 null) isogenic cells, AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type, but not in BRCA2 null cells, despite significant G2 checkpoint abrogation. In patient-derived pancreatic tumor xenografts, AZD1775 significantly inhibited tumor growth and impaired RAD51 focus formation in response to gemcitabine-radiation. In conclusion, WEE1 inhibition by AZD1775 is an effective strategy for sensitizing pancreatic cancers to gemcitabine chemoradiation. Although this sensitization is accompanied by inhibition of CDK1 phosphorylation and G2 checkpoint abrogation, this mechanism is not sufficient for sensitization. Our findings demonstrate that sensitization to chemoradiation by WEE1 inhibition results from inhibition of HR repair and suggest that patient tumors without underlying HR defects would benefit most from this therapy.

Asunto(s)

Proteínas de Ciclo Celular/antagonistas & inhibidores , Desoxicitidina/análogos & derivados , Proteínas Nucleares/antagonistas & inhibidores , Neoplasias Pancreáticas/terapia , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Fármacos Sensibilizantes a Radiaciones/farmacología , Reparación del ADN por Recombinación/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Quimioradioterapia , Daño del ADN/efectos de los fármacos , Desoxicitidina/farmacología , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Ratones , Ratones Desnudos , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Gemcitabina

RESUMEN

PURPOSE: While the addition of radiation to chemotherapy improves survival in patients with locally advanced pancreatic cancer, more effective therapies are urgently needed. Thus, we investigated the radiosensitizing efficacy of the novel drug combination of Wee1 and PARP1/2 inhibitors (AZD1775 and olaparib, respectively) in pancreatic cancer. EXPERIMENTAL DESIGN: Radiosensitization of AsPC-1 or MiaPaCa-2 human pancreatic cancer cells was assessed by clonogenic survival and tumor growth assays. Mechanistically, the effects of AZD1775, olaparib, and radiation on cell cycle, DNA damage (γH2AX), and homologous recombination repair (HRR) were determined. RESULTS: Treatment of AsPC-1 and MiaPaCa-2 cells with either AZD1775 or olaparib caused modest radiosensitization, whereas treatment with the combination significantly increased radiosensitization. Radiosensitization by the combination of AZD1775 and olaparib was associated with G2 checkpoint abrogation and persistent DNA damage. In addition, AZD1775 inhibited HRR activity and prevented radiation-induced Rad51 focus formation. Finally, in vivo, in MiaPaCa-2-derived xenografts, olaparib did not radiosensitize, whereas AZD1775 produced moderate, yet significant, radiosensitization (P < 0.05). Importantly, the combination of AZD1775 and olaparib produced highly significant radiosensitization (P < 0.0001) evidenced by a 13-day delay in tumor volume doubling (vs. radiation alone) and complete eradication of 20% of tumors. CONCLUSIONS: Taken together, these results demonstrate the efficacy of combined inhibition of Wee1 and PARP inhibitors for radiosensitizing pancreatic cancers and support the model that Wee1 inhibition sensitizes cells to PARP inhibitor-mediated radiosensitization through inhibition of HRR and abrogation of the G2 checkpoint, ultimately resulting in unrepaired, lethal DNA damage and radiosensitization. Clin Cancer Res; 20(19); 5085-96. ©2014 AACR.

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Tolerancia a Radiación , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Modelos Animales de Enfermedad , Femenino , Humanos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Radiación , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/genética , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Carga Tumoral/efectos de la radiación , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Transarterial radioembolization (TARE) with (90)Y microspheres delivers low dose rate radiation (LDR) to intrahepatic tumors. In the current study, we examined clonogenic survival, DNA damage, and cell cycle distribution in hepatocellular carcinoma (HCC) cell lines treated with LDR in combination with varying doses and schedules of 5-fluorouracil (5-FU), gemcitabine, and sorafenib. Radiosensitization was seen with 1 to 3 µM 5-FU (enhancement ratio 2.2-13.9) and 30 to 100 nM gemcitabine (enhancement ratio 1.9-2.9) administered 24 hours before LDR (0.26 Gy/h to 4.2 Gy). Sorafenib radiosensitized only at high concentrations (3-10 µM) when administered after LDR. For a given radiation dose, greater enhancement was seen with LDR compared to standard dose rate therapy. Summarizing our clinical experience with low dose rate radiosensitization, 13 patients (5 with HCC, 8 with liver metastases) were treated a total of 16 times with TARE and concurrent gemcitabine. Six partial responses and one complete response were observed with a median time to local failure of 7.1 months for all patients and 9.9 months for patients with HCC. In summary, HCC is sensitized to LDR with clinically achievable concentrations of gemcitabine and 5-FU in vitro. Encouraging responses were seen in a small cohort of patients treated with TARE and concurrent gemcitabine. Future studies are needed to validate the safety and efficacy of this approach.

RESUMEN

PURPOSE: The combination of radiation with chemotherapy is the most effective therapy for unresectable pancreatic cancer. To improve upon this regimen, we combined the selective Checkpoint kinase 1 (Chk1) inhibitor MK8776 with gemcitabine-based chemoradiation in preclinical pancreatic cancer models. EXPERIMENTAL DESIGN: We tested the ability of MK8776 to sensitize to gemcitabine-radiation in homologous recombination repair (HRR)-proficient and -deficient pancreatic cancer cells and assessed Rad51 focus formation. In vivo, we investigated the efficacy, tumor cell selectivity, and pharmacodynamic biomarkers of sensitization by MK8776. RESULTS: We found that MK8776 significantly sensitized HRR-proficient (AsPC-1, MiaPaCa-2, BxPC-3) but not -deficient (Capan-1) pancreatic cancer cells to gemcitabine-radiation and inhibited Rad51 focus formation in HRR-proficient cells. In vivo, MiaPaCa-2 xenografts were significantly sensitized to gemcitabine-radiation by MK8776 without significant weight loss or observable toxicity in the small intestine, the dose-limiting organ for chemoradiation therapy in pancreatic cancer. We also assessed pChk1 (S345), a pharmacodynamic biomarker of DNA damage in response to Chk1 inhibition in both tumor and small intestine and found that MK8776 combined with gemcitabine or gemcitabine-radiation produced a significantly greater increase in pChk1 (S345) in tumor relative to small intestine, suggesting greater DNA damage in tumor than in normal tissue. Furthermore, we demonstrated the utility of an ex vivo platform for assessment of pharmacodynamic biomarkers of Chk1 inhibition in pancreatic cancer. CONCLUSIONS: Together, our results suggest that MK8776 selectively sensitizes HRR-proficient pancreatic cancer cells and xenografts to gemcitabine-radiation and support the clinical investigation of MK8776 in combination with gemcitabine-radiation in locally advanced pancreatic cancer.

Asunto(s)

Neoplasias Pancreáticas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quimioradioterapia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Concentración 50 Inhibidora , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Inhibidores de Proteínas Quinasas/administración & dosificación , Fármacos Sensibilizantes a Radiaciones/farmacología , Reparación del ADN por Recombinación/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Gemcitabina

RESUMEN

PURPOSE: To identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer and thus improve survival, we conducted an siRNA library screen in pancreatic cancer cells. We investigated PPP2R1A, a scaffolding subunit of protein phosphatase 2A (PP2A) as a lead radiosensitizing target. EXPERIMENTAL DESIGN: We determined the effect of PP2A inhibition by genetic (PPP2R1A siRNA) and pharmacologic (LB100, a small molecule entering phase I clinical trials) approaches on radiosensitization of Panc-1 and MiaPaCa-2 pancreatic cancer cells both in vitro and in vivo. RESULTS: PPP2R1A depletion by siRNA radiosensitized Panc-1 and MiaPaCa-2 cells, with radiation enhancement ratios of 1.4 (P < 0.05). Likewise, LB100 produced similar radiosensitization in pancreatic cancer cells, but minimal radiosensitization in normal small intestinal cells. Mechanistically, PPP2R1A siRNA or LB100 caused aberrant CDK1 activation, likely resulting from accumulation of the active forms of PLK1 (pPLK1 T210) and CDC25C (pCDC25C T130). Furthermore, LB100 inhibited radiation-induced Rad51 focus formation and homologous recombination repair (HRR), ultimately leading to persistent radiation-induced DNA damage, as reflected by γ-H2AX expression. Finally, we identified CDC25C as a key PP2A substrate involved in LB100-mediated radiosensitization as depletion of CDC25C partially reversed LB100-mediated radiosensitization. In a mouse xenograft model of human pancreatic cancer, LB100 produced significant radiosensitization with minimal weight loss. CONCLUSIONS: Collectively, our data show that PP2A inhibition radiosensitizes pancreatic cancer both in vitro and in vivo via activation of CDC25C/CDK1 and inhibition of HRR, and provide proof-of-concept evidence that PP2A is a promising target for the improvement of local therapy in pancreatic cancer.

Asunto(s)

Proteína Quinasa CDC2/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteína Fosfatasa 2/antagonistas & inhibidores , Reparación del ADN por Recombinación/efectos de los fármacos , Fosfatasas cdc25/metabolismo , Animales , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Humanos , Ratones , Neoplasias Pancreáticas/radioterapia , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Interferencia de ARN , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/genética , Fármacos Sensibilizantes a Radiaciones/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

PURPOSE: To identify new plasma proteomic markers before radiotherapy start to predict later grade ≥2 radiation-induced lung toxicity (RILT2). METHODS: Fifty-seven patients with non-small cell lung cancer received radiotherapy (RT) were eligible. Forty-eight patients with minimum follow-up of 1 year, nine with RILT2 with tumor stage matched to 39 without RILT2, were enrolled for this analysis. Platelet-poor plasma was obtained within 2 weeks before radiotherapy. The plasma proteomes were compared using a multiplexed quantitative proteomics approach involving ExacTag labeling, reverse-phase high-performance liquid chromatography, and nano liquid chromatography electrospray ionization tandem mass spectrometry. Z scores and Bonferroni-adjusted p values for the two-sample mean comparison were used to identify the differential protein expression between patients with and without RILT2. RESULTS: More than 200 proteins were identified and quantified. After excluding proteins that were not detected in at least 40% of the 48 patient samples, C4b-binding protein alpha chain and vitronectin had significantly higher (p < 0.001 and p = 0.02) expression levels in patients with RILT2 compared with patients without RILT2. These two proteins were validated by Western blot. Ingenuity pathway analysis revealed that they both play important roles in the inflammatory response and are associated with the known pathways of radiation-induced lung damage. CONCLUSIONS: This proteomic approach demonstrates new plasma protein biomarkers before treatment for future studies on RILT2 prediction.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/sangre , Neoplasias Pulmonares/sangre , Pulmón/efectos de la radiación , Proteínas/análisis , Proteómica/métodos , Traumatismos por Radiación/sangre , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Proteína de Unión al Complemento C4b/análisis , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/radioterapia , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Estudios Prospectivos , Traumatismos por Radiación/diagnóstico , Reproducibilidad de los Resultados , Vitronectina/sangre

RESUMEN

Chemoradiation is the treatment of choice for locally advanced head and neck squamous cell carcinoma (HNSCC). However, radioresistance, which contributes to local recurrence, remains a significant therapeutic problem. In this study, we characterized SM-164, a small second mitochondria-derived activator of caspase -mimetic compound that promotes degradation of cellular inhibitor of apoptosis-1(cIAP-1; also known as baculoviral IAP repeat-containing protein 2, BIRC2) and releases active caspases from the X-linked inhibitor of apoptosis inhibitory binding as a radiosensitizing agent in HNSCC cells. We found that SM-164 at nanomolar concentrations induced radiosensitization in some HNSCC cell lines in a manner dependent on intrinsic sensitivity to caspase activation and apoptosis induction. Blockage of caspase activation via short interfering RNA knockdown or a pan-caspase inhibitor, z-VAD-fmk, largely abrogated SM-164 radiosensitization. On the other hand, the resistant lines with a high level of Bcl-2 that blocks caspase activation and apoptosis induction became sensitive to radiation on Bcl-2 knockdown. Mechanistic studies revealed that SM-164 radiosensitization in sensitive cells was associated with NF-κB activation and TNFα secretion, followed by activation of caspase-8 and -9, leading to enhanced apoptosis. Finally, SM-164 also radiosensitized human tumor xenograft while causing minimal toxicity. Thus, SM-164 is a potent radiosensitizer via a mechanism involving caspase activation and holds promise for future clinical development as a novel class of radiosensitizer for the treatment of a subset of head and neck cancer patients.

Asunto(s)

Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Carcinoma de Células Escamosas/terapia , Caspasas/metabolismo , Neoplasias de Cabeza y Cuello/terapia , Triazoles/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Western Blotting , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Inhibidores de Caspasas , Caspasas/genética , Línea Celular Tumoral , Terapia Combinada , Inhibidores de Cisteína Proteinasa/farmacología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Activación Enzimática/efectos de la radiación , Citometría de Flujo , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones , Ratones Desnudos , Estructura Molecular , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , Fármacos Sensibilizantes a Radiaciones/química , Fármacos Sensibilizantes a Radiaciones/farmacología , Radioterapia/métodos , Triazoles/química , Ubiquitina-Proteína Ligasas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

The median survival for patients with locally advanced pancreatic cancer treated with gemcitabine and radiation is approximately 1 year. To develop improved treatment, we have combined a Chk1/2-targeted agent, AZD7762, currently in phase I clinical trials, with gemcitabine and ionizing radiation in preclinical pancreatic tumor models. We found that in vitro AZD7762 alone or in combination with gemcitabine significantly sensitized MiaPaCa-2 cells to radiation. AZD7762 inhibited Chk1 autophosphorylation (S296 Chk1), stabilized Cdc25A, and increased ATR/ATM-mediated Chk1 phosphorylation (S345 Chk1). Radiosensitization by AZD7762 was associated with abrogation of the G(2) checkpoint as well as with inhibition of Rad51 focus formation, inhibition of homologous recombination repair, and persistent gamma-H2AX expression. AZD7762 was also a radiation sensitizer in multiple tumor xenograft models. In both MiaPaCa-2- and patient-derived xenografts, AZD7762 significantly prolonged the median time required for tumor volume doubling in response to gemcitabine and radiation. Together, our findings suggest that G(2) checkpoint abrogation and homologous recombination repair inhibition both contribute to sensitization by Chk1 inhibition. Furthermore, they support the clinical use of AZD7762 in combination with gemcitabine and radiation for patients with locally advanced pancreatic cancer.

Asunto(s)

Reparación del ADN/efectos de los fármacos , Fase G2/efectos de los fármacos , Neoplasias Pancreáticas/radioterapia , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Fármacos Sensibilizantes a Radiaciones/farmacología , Tiofenos/farmacología , Urea/análogos & derivados , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Western Blotting , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Quimioterapia Combinada , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Fase G2/efectos de la radiación , Rayos gamma , Humanos , Immunoblotting , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recombinasa Rad51/metabolismo , Recombinación Genética/efectos de los fármacos , Recombinación Genética/efectos de la radiación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Urea/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Gemcitabina

RESUMEN

Treatment of C57BL/6 mice with cyclophosphamide (100 mg/kg) and fludarabine (200 mg/kg) induced nonmyeloablative lymphodepletion without inhibiting D5 melanoma tumor growth. Using this model, we found that induction of lymphopenia before adoptive transfer of ex vivo anti-CD3/CD28 activated and interleukin-2 expanded D5-G6 tumor draining lymph node cells enhanced the antitumor efficacy of the infused cells in both pulmonary metastases and subcutaneous D5 bearing mice. However, induction of lymphopenia did not promote intratumoral or extratumoral proliferation or accumulation of the infused cells. We have previously shown that radiotherapy enhances the therapeutic efficacy of intratumoral unpulsed dendritic cell vaccination in subcutaneous murine tumor models by augmenting the induction of antitumor cellular immune responses. Here, we confirmed this finding in a murine metastatic melanoma liver tumor model. Furthermore, local tumor irradiation combined with intratumoral dendritic cell administration significantly enhanced the therapeutic efficacy of tumor-reactive T cell adoptive transfer in this lymphodepleted liver tumor model. This was evident by reduced liver tumor size, decreased incidence of spontaneous intra-abdominal metastasis, and prolonged survival, resulting in 46% of mice cured. This enhanced antitumor activity was associated with a selective increase in proliferation, accumulation, and function of CD4+ rather than CD8+ infused cells. This multimodality regimen may have translational applications for the treatment of human cancers.

Asunto(s)

Linfocitos T CD4-Positivos/trasplante , Linfocitos T CD8-positivos/trasplante , Células Dendríticas/trasplante , Inmunoterapia Adoptiva , Neoplasias Hepáticas/terapia , Neoplasias Pulmonares/terapia , Ganglios Linfáticos/inmunología , Melanoma Experimental/terapia , Animales , Antineoplásicos/farmacología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Terapia Combinada , Ciclofosfamida/farmacología , Células Dendríticas/inmunología , Femenino , Inmunosupresores/farmacología , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/secundario , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/metabolismo , Linfopenia/inducido químicamente , Linfopenia/inmunología , Melanoma Experimental/patología , Melanoma Experimental/radioterapia , Ratones , Ratones Endogámicos C57BL , Vidarabina/análogos & derivados

RESUMEN

Epidermal growth factor receptor (EGFR) inhibitors are increasingly used in combination with radiotherapy in the treatment of various EGFR-overexpressing cancers. However, little is known about the effects of cell cycle status on EGFR inhibitor-mediated radiosensitization. Using EGFR-overexpressing A431 and UMSCC-1 cells in culture, we found that radiation activated the EGFR and extracellular signal-regulated kinase pathways in quiescent cells, leading to progression of cells from G(1) to S, but this activation and progression did not occur in proliferating cells. Inhibition of this activation blocked S-phase progression and protected quiescent cells from radiation-induced death. To determine if these effects were caused by EGFR expression, we transfected Chinese hamster ovary (CHO) cells, which lack EGFR expression, with EGFR expression vector. EGFR expressed in CHO cells also became activated in quiescent cells but not in proliferating cells after irradiation. Moreover, quiescent cells expressing EGFR underwent increased radiation-induced clonogenic death compared with both proliferating CHO cells expressing EGFR and quiescent wild-type CHO cells. Our data show that radiation-induced enhancement of cell death in quiescent cells involves activation of the EGFR and extracellular signal-regulated kinase pathways. Furthermore, they suggest that EGFR inhibitors may protect quiescent tumor cells, whereas radiosensitization of proliferating cells may be caused by downstream effects such as cell cycle redistribution. These findings emphasize the need for careful scheduling of treatment with the combination of EGFR inhibitors and radiation and suggest that EGFR inhibitors might best be given after radiation in order to optimize clinical outcome.

Asunto(s)

Ciclo Celular , Receptores ErbB/antagonistas & inhibidores , Neoplasias/patología , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Células CHO , Línea Celular Tumoral , Proliferación Celular , Cricetinae , Cricetulus , Factor de Crecimiento Epidérmico/farmacología , Citometría de Flujo , Humanos , Neoplasias/metabolismo

RESUMEN

BACKGROUND: Recent research has supported that a variety of cytokines play important roles during radiation-induced lung toxicity. The present study is designed to investigate the differences in early cytokine induction after radiation in sensitive (C57BL/6) and resistant mice (C3H). RESULTS: Twenty-two cytokines in the lung tissue homogenates, bronchial lavage (BAL) fluids, and serum from 3, 6, 12, 24 hrs to 1 week after 12 Gy whole lung irradiation were profiled using a microsphere-based multiplexed cytokine assay. The majority of cytokines had similar baseline levels in C57BL/6 and C3H mice, but differed significantly after radiation. Many, including granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and keratinocyte-derived chemokine (KC) were elevated significantly in specimens from both strains. They usually peaked at about 3-6 hrs in C57BL/6 and 6-12 hrs in C3H. At 6 hrs in lung tissue, G-CSF, IL-6, and KC increased 6, 8, and 11 fold in C57BL/6 mice, 4, 3, and 3 fold in the C3H mice, respectively. IL-6 was 10-fold higher at 6 hrs in the C57BL/6 BAL fluid than the C3H BAL fluid. MCP-1, IP-10, and IL-1alpha also showed some differences between strains in the lung tissue and/or serum. For the same cytokine and within the same strain of mice, there were significant linear correlations between lung tissue and BAL fluid levels (R2 ranged 0.46-0.99) and between serum and tissue (R2 ranged 0.56-0.98). CONCLUSION: Radiation induced earlier and greater temporal changes in multiple cytokines in the pulmonary fibrosis sensitive mice. Positive correlation between serum and tissue levels suggests that blood may be used as a surrogate marker for tissue.

Asunto(s)

Citocinas/sangre , Citocinas/metabolismo , Inmunidad Innata , Neumonitis por Radiación/sangre , Neumonitis por Radiación/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/química , Citocinas/análisis , Inmunidad Innata/fisiología , Inmunidad Innata/efectos de la radiación , Pulmón/química , Pulmón/metabolismo , Pulmón/patología , Masculino , Metaboloma , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Radiación , Traumatismos Experimentales por Radiación/sangre , Traumatismos Experimentales por Radiación/inmunología , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/patología , Neumonitis por Radiación/inmunología , Neumonitis por Radiación/patología , Extractos de Tejidos/análisis , Extractos de Tejidos/metabolismo

RESUMEN

We compared viability, phenotype, in vitro function and therapeutic efficacy of murine unpulsed-dendritic cells (-DC), DC pulsed with keyhole limpet hemocyanin (KLH-DC) and cryopreserved KLH-DC (C-KLH-DC). Mean viability (%+/-SE) of unpulsed-DC, KLH-DC and C-KLH-DC was 93.6+/-0.9, 93.9+/-0.8 and 87.4+/-1.6, respectively. Pulsing DC with KLH did not induce maturation or affect in vitro function. Cryopreservation of KLH-DC reduced MHC I, CD80 and CD86 expression, endocytic capacity and allogeneic splenocyte stimulatory capacity. Intratumoral (i.t.) vaccination of mice bearing s.c. D5 melanoma with unpulsed-DC, KLH-DC or C-KLH-DC elicited comparable anti-tumor immune responses and inhibited tumor growth to the same extent. Combining radiotherapy with i.t. unpulsed-DC, KLH-DC or C-KLH-DC administration enhanced induction of anti-tumor immune responses and inhibition of tumor growth to a similar degree. Cryopreservation of KLH-DC slightly reduces viability, expression of co-stimulatory cell surface markers and in vitro function; however, in vivo anti-tumor activity is fully maintained with or without radiotherapy.

Asunto(s)

Adyuvantes Inmunológicos/farmacología , Criopreservación/métodos , Células Dendríticas/inmunología , Hemocianinas/farmacología , Melanoma Experimental/terapia , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Endocitosis/inmunología , Femenino , Citometría de Flujo , Hemocianinas/inmunología , Inmunofenotipificación , Inmunoterapia Adoptiva/métodos , Prueba de Cultivo Mixto de Linfocitos , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Organismos Libres de Patógenos Específicos

RESUMEN

It has recently been shown that fixed-dose-rate (gemcitabine) infusion may be superior to bolus gemcitabine in the treatment of metastatic pancreas cancer. We wished to compare the radiosensitizing effects of fixed-dose-rate gemcitabine infusion to standard bolus injection. We measured weight loss and mouse intestinal crypt survival to determine equally toxic concentrations of gemcitabine administered through a 3-hour fixed-dose-rate infusion versus bolus injection in combination with fractionated radiation. To measure the effect of fixed-dose-rate gemcitabine infusion or bolus injection on radiosensitization, we treated mice bearing Panc-1 xenografts with equally toxic concentrations of gemcitabine (100 mg/kg fixed-dose-rate infusion or 500 mg/kg bolus injection) and fractionated radiation and monitored tumor growth. We found that 100 mg/kg gemcitabine through fixed-dose-rate infusion produced the same weight loss and intestinal crypt toxicity as the 500 mg/kg bolus injection. In nude mice bearing Panc-1 xenografts, fixed-dose-rate gemcitabine infusion produced greater radiosensitization than bolus injection with tumor doubling times of 44 +/- 5 versus 29 +/- 3 days, respectively (*P < .05). Fixed-dose-rate gemcitabine infusion produced enhanced radiosensitization without additional normal tissue toxicity compared to bolus gemcitabine injection. These data support an ongoing clinical trial using fixed-dose-rate gemcitabine infusion combined with conformal radiation in the treatment of locally advanced pancreatic cancer.

RESUMEN

Clinical evidence suggests that gemcitabine (Gem) plus oxaliplatin (Ox) is superior to gemcitabine alone in advanced pancreatic carcinoma. The addition of radiation to gemcitabine improves response and is a standard treatment for locally advanced disease. We investigated the effect of oxaliplatin on gemcitabine-based chemoradiation by determining whether gemcitabine and oxaliplatin produced synergistic cytotoxicity using median effect analysis and radiosensitization using clonogenic survival assays. We analyzed the effects of gemcitabine and oxaliplatin on cell cycle distribution by DNA content and on radiation-induced DNA damage repair by phosphorylated H2AX (gamma-H2AX). Gemcitabine and oxaliplatin produced schedule-dependent synergistic cytotoxicity in BxPC-3 and Panc-1 cells (combination indices: 0.76 +/- 0.05, 0.61 +/- 0.11). In BxPC-3 cells, oxaliplatin did not affect gemcitabine-mediated radiosensitization (Gem 1.99 +/- 0.27; Gem + Ox 2.38 +/- 0.30). In Panc-1 cells, oxaliplatin significantly enhanced gemcitabine-mediated radiosensitization (Gem 1.31 +/- 0.05; Gem + Ox 2.90 +/- 0.31). Radiosensitization by gemcitabine was accompanied by early S-phase arrest and induction/persistence of gamma-H2AX protein, which were unaltered by oxaliplatin. Addition of oxaliplatin to gemcitabine produces radiosensitization equal to or greater than gemcitabine alone, supporting our clinical investigation of oxaliplatin with gemcitabine-radiation in pancreatic cancer aimed at improving systemic disease control while maintaining local tumor radiosensitization.