The effect of radiotherapy dose on survival in stage III non-small-cell lung cancer patients undergoing definitive chemoradiotherapy.

BACKGROUND: In this study, we examined trends in the radiotherapy dose prescribed and the effect of dose escalation on survival in patients with stage III lung cancer. MATERIALS AND METHODS: Radiation dose prescription patterns were analyzed for 38,848 patients in the National Cancer Database with clinical stage III disease who underwent concurrent chemoradiation between 2004 and 2011 to a dose between 57 and 80 Gy. Survival information was available for patients diagnosed from 2004 to 2006 (n = 12,024). Overall survival (OS) was estimated using Kaplan-Meier methods. Cox proportional hazard regression was used to estimate hazard ratios (HRs). RESULTS: The percentage of patients treated to ≥ 64 Gy increased from 50% in 2004 to 62% in 2011 (P < .001). The 5-year OS was 12% for patients treated between 57 and 59.3 Gy, 14% for patients treated at 59.4 to 62.9 Gy, 16% for patients treated at 63 to 66 Gy and 66.1 to 73.9 Gy, and 13% for patients treated at 74 to 80 Gy (P < .0001). In multivariate analysis, the estimated HR (95% confidence interval) was 1.3 (1.1-1.6) for 57 to 59.3 Gy, 1.0 (0.9-1.2) for 59.4 to 62.9 Gy, 0.9 (0.9-1.2) for 63 to 66 Gy, 0.9 (0.8-1.1) for 66.1 to 73.9 Gy, and 1.0 (referent) for the 74 to 80 Gy cohort. There was no significant difference in the HR for the dose groups > 59.4 Gy compared with the 74 to 80 Gy cohort. CONCLUSION: There was no improvement in OS with radiotherapy dose escalation beyond 59.4 Gy for patients with unresectable clinical stage III lung cancer treated with chemoradiation.

RBx10080307, a dual EGFR/IGF-1R inhibitor for anticancer therapy.

Pharmacological intervention of epidermal growth factor receptor (EGFR) family members by antibodies or small molecule inhibitors has been one of the most successful approaches for anticancer therapy. However this therapy has its own limitations due to the development of resistance, over a period of time. One of the possible causes of the development of resistance to the therapy with EGFR inhibitors could be the simultaneous activation of parallel pathways. Both EGFR and insulin like growth factor-1 receptor (IGF-1R) pathways are reported to act reciprocal to each other and converge into the mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. Inhibiting one pathway alone may therefore not be sufficient and could be a cause of development of resistance. The other cause could be mutations of EGFR which would be less sensitive to the inhibitors. We, therefore, suggest that co-targeting IGF-1R and EGFR kinases by dual inhibitors can lead to improved efficacy and address the problems of resistance. In the present manuscript, we report the identification of a novel, small molecule dual EGFR/IGF-1R inhibitor, RBx10080307 which displayed in vitro activity at the molecular level and oral efficacy in mouse xenograft model. The compound also showed in vitro activity in an EGFR mutant cell line and may thus have the potential to show activity in resistant conditions. Additional efficacy studies are needed in EGFR resistant mouse cancer model and if found efficacious, this can be a major advantage over standalone erlotinib and other existing therapies.

Improved survival associated with neoadjuvant chemoradiation in patients with clinical stage IIIA(N2) non-small-cell lung cancer.

INTRODUCTION: Optimal management of clinical stage IIIA-N2 non-small-cell lung cancer (NSCLC) is controversial. This study examines whether neoadjuvant chemoradiation plus surgery improves survival rates when compared with other recommended treatment strategies. METHODS: Adult patients from the National Cancer Database, with clinical stage IIIA-N2 disease definitively treated between 1998 and 2004 at American College of Surgeons Commission on Cancer accredited facilities, were included in the study. Treatment was defined as neoadjuvant chemoradiation plus either lobectomy (NeoCRT+L) or pneumonectomy (NeoCRT+P), lobectomy plus adjuvant therapy (L+AT), pneumonectomy plus adjuvant therapy (P+AT), and concurrent chemoradiation (CRT). Median follow-up and overall survival (OS) were defined from date of diagnosis to last contact. Five-year OS was estimated using Kaplan-Meier methods. Cox proportional hazard regression was used to estimate hazard ratios and 95% confidence intervals (CIs), adjusting for sociodemographic, clinical, and facility characteristics. RESULTS: Median follow-up was 11.8 months for 11,242 eligible patients. Five-year OS was 33.5%, 20.7%, 20.3%, 13.35%, and 10.9% for NeoCRT+L, NeoCRT+P, L+AT, P+AT, and CRT, respectively (p < 0.0001). On multivariable analysis, the estimated hazard ratio was 0.51 (CI: 0.45-0.58) for NeoCRT+L; 0.77 (0.63-0.95) for NeoCRT+P; 0.66 (0.59-0.75) for L+AT; 0.69 (0.54-0.88) for P+AT; and 1.0 (reference) for the CRT group. Comorbidity did not attenuate the relationship between treatment and survival. CONCLUSION: This large study demonstrates that patients with clinical stage IIIA-N2 NSCLC, who underwent neoadjuvant chemoradiation followed by lobectomy, were associated with an improved survival.

Comparative deacetylase activity of wild type and mutants of SIRT1.

SIRT1, human ortholog of yeast SIR2 protein, deacetylates histones and several other transcription factors. Recently, SIRT1 has emerged as a drug target for treating age related diseases, type II diabetes, neurodegeneration, inflammation and cancer. Here, we have optimized production of functionally active wild type full-length SIRT1 protein and its N-terminal deleted mutants. In a comparative study, we found that the region containing 192-208 amino acids towards the N-terminus is critical for right conformational folding of the protein to retain its deacetylase activity. The EC(50) and IC(50) values obtained with standard modulators showed that the SRT(748) & SRT(556) can deacetylate substrate and are activated by resveratrol, whereas, deacetylase activity of all the other deletion mutants (SRT(540), SRT(532), SRT(507) and SRT(503)) was lost. We further report that the peptide substrate K(m) for SRT(748) (70+/-5.2 microM) was comparable to SRT(556) (93+/-5.4 microM). The K(m) for NAD(+) substrate was 176 & 274 microM for SRT(748) and SRT(556), respectively. Similar substrate affinity studies demonstrate that either of the protein (SRT(748) or SRT(556)) can be utilized for screening SIRT1 modulators. We have also examined critical regions in SIRT1 required for deacetylase activity as well as kinetic analyses of SIRT1 proteins.

Purification of recombinant human phosphodiesterase 7A expressed in Dictyostelium discoideum.

Phosphodiesterase plays an important role in regulating inflammatory pathways and T cell function. The development of phosphodiesterase 7 inhibitor may give better efficacy profile over phosphodiesterase 4 inhibitors. However, the recombinant phosphodiesterase 7 is required in large quantity for high-throughput screening of new drugs by in vitro enzymatic assays. In the present study, recombinant human PDE7A1 was expressed in Dictyostelium discoideum under the control of constitutively active actin-15 promoter. The cytosolic localization of the expressed protein was confirmed by immunofluorescence studies. Upto 2 mg of recombinant protein was purified using His-Tag affinity column chromatography followed by ion-exchange Resource Q column purification. The recombinant protein expressed in D. discoideum followed Michaelis-Menten kinetics similar to the protein expressed in mammalian system and showed no major changes in affinity to substrate or inhibitors. Thus, our study clearly demonstrates a robust expression system for successful bulk production of pharmacologically active isoform of human PDE7A1 required for high-throughput assays.

Differential gene expression analysis of a known hepatotoxin, N-acetyl-p-amino-phenol (APAP) as compared to its non-toxic analog, N-acetyl-m-amino-phenol (AMAP) in mouse liver.

Drug-induced hepatotoxicity is one of the most common adverse events associated with drug withdrawal from the market. Elucidating the molecular mechanism of hepatotoxicity is essential to predict the safety of a new molecule. To examine genes involved in hepatotoxicity, we have used oligonucleotide CodeLink Bioarrays and determined the transcriptional profile of mice liver treated with hepatotoxic drug N-acetyl-p-amino-phenol (APAP) as well as its non-toxic analog N-acetyl-m-amino-phenol (AMAP). Out of 20,000 genes analyzed, 896 showed differential expression of > or = 2-fold (648 upregulated and 248 downregulated) within the liver of APAP treated mice as compared to control. In comparison to AMAP treated mice, 62 genes were upregulated and 70 genes were downregulated in mice liver after APAP treatment. Functional classification of these differentially expressed genes identified genes associated with stress response, cell cycle, growth inhibition, cell death, structural components, cell signaling and inflammation. Gene expression profile was further correlated with biochemical analysis and histopathological lesions. These data show that gene expression profiling would help in better understanding the molecular basis of drug-induced hepatotoxicity that will lead to rational development of safer drugs, particularly in pre-clinical stages.

Development of a cell-based assay for screening of phosphodiesterase 10A (PDE10A) inhibitors using a stable recombinant HEK-293 cell line expressing high levels of PDE10A.

The cDNA encoding PDE10A (phosphodiesterase 10A) was cloned and a stable recombinant HEK-293 (human embryonic kidney-293) cell line expressing high levels of PDE10A was generated. Transient transfection of pCRE-Luc plasmid, harbouring the luciferase reporter gene under the control of CRE (cAMP-response element)-binding sequence, into the stable recombinant cell line, followed by treatment with PDE10 inhibitor, resulted in a dose-dependent increase in luciferase activity. This method provides a simple and sensitive cell-based assay for screening of PDE10 inhibitors for development of novel therapeutics for the treatment of neurological disorders.

Cloning, stable expression of human phosphodiesterase 7A and development of an assay for screening of PDE7 selective inhibitors.

Phosphodiesterases (PDEs) constitute a superfamily of enzymes that plays an important role in signal transduction by catalysing the hydrolysis of cAMP and cGMP. cDNA encoding PDE7A1 subtype was cloned and a stable recombinant HEK 293 cell line expressing high levels of PDE7A1 was generated. Transient transfection of pCRE-Luc plasmid, harboring luciferase reporter gene into the stable recombinant cell line and subsequent treatment with PDE7 inhibitor, resulted in a dose-dependent increase in luciferase activity. This method provides a simple and sensitive cell-based assay for screening of PDE7 selective inhibitors for the treatment of T cell mediated diseases.

A reporter gene assay for screening of PDE4 subtype selective inhibitors.

Phosphodiesterase (PDE) constitutes a superfamily of enzymes that catalyze the hydrolysis of cAMP and cGMP into their corresponding monophosphates and play an important role in diverse physiological functions. The present study provides a process for identifying PDE4 subtypes selective inhibitors using a reporter gene assay. Stable recombinant HEK-293 cell lines expressing high levels of PDE4A4B, PDE4B2A, and PDE4D3 subtypes individually were generated. Transient transfection of pCRE-Luc plasmid, harboring luciferase reporter gene under the control of cAMP response element (CRE)-binding sequence, into these stable recombinant cell lines followed by treatment with PDE4 inhibitor, resulted in a dose dependent increase in luciferase activity. This methods provide a novel, simple and sensitive assay for high throughput screening of PDE4 subtype selective inhibitors for treatment of asthma and COPD.

Assessment of Aegilops tauschii for resistance to biotypes of wheat curl mite (Acari: Eriophyidae).

Aegilops tauschii, the wild diploid D-genome progenitor of wheat, Triticum aestivum L., is an important source of resistance to several arthropod pests and pathogens. A total of 108 Ae. tauschii accessions from different geographic regions were evaluated for resistance to biotypes of the wheat curl mite, Aceria tosichella Keifer, from Kansas, Nebraska, and Montana. The wheat curl mite is the only vector known to transmit wheat streak mosaic virus. Wheat curl mite resistance was detected in germplasm from all the geographic locations represented. The highest percentage of resistant accessions originated from Turkey, followed by Afghanistan and the Caspian Sea region of Iran. Sixty-seven percent of the accessions exhibited resistance to at least one wheat curl mite biotype and 19% were resistant to all the three biotyopes. Resistance to the accessions tested occurred more frequently in the Nebraska and Kansas biotypes (69% and 64%, respectively) than did resistance to the Montana biotype (42%), although the frequency of resistance was not significant. The differential reactions of accessions to the different wheat curl mite biotypes suggests that Ae. tauschii has at least five different genes for resistance to mite colonization. Ae. tauschii continues to be a very useful source for wheat curl mite resistance genes for bread wheat improvement.