[Frontiers in Bioscience E3, 1349-1364, June 1, 2011]

Antitumor activity of NPB001-05, an orally active inhibitor of Bcr-Abl tyrosine kinase

Vilas Wagh1, Prabha Mishra2, Arvind Thakkar2, Vaibhav Shinde1, Somesh Sharma1, Muralidhara Padigaru2, Kalpana Joshi1

1Department of Pharmacology, Piramal Life Sciences Limited, 1-Nirlon complex, Goregaon, 400063 Mumbai, India,2Biomarker Discovery Group, Piramal Life Sciences Limited, 1-Nirlon complex, Goregaon, 400063 Mumbai, India


1. Abstract
2. Introduction
3. Materials and methods
3.1. Cell culture and drug treatments
3.2. Animals
3.3. Drug preparation and administration
3.4. In-vivo experiments and mouse xenograft models
3.5. RNA extraction and microarray analysis
3.6. Gene expression studies using RT-qPCR
3.7. Expression of protein markers
4. Results
4.1. Efficacy/toxicity studies in-vivo
4.1.1. Per-oral toxicity studies
4.1.2. Effect of NPB001-05 on human chronic myelogenous leukemia cells in-vivo.
4.1.3. Effect of NPB001-05 on P210Bcr-Abl cells derived tumors
4.2. Bcr-Abl expression and phosphorylation in tumor samples
4.3. Transcript profiling of NPB001-05 treated K562 cells in-vitro and in-vivo
4.4. Transcriptional profiling of NPB001-05 and imatinib treated K562 cells and analysis of differentially regulated genes.
4.4.1. Analysis of similarly expressed genes in NPB001-05 and imatinib treatment
4.4.2. Unique differentially expressed gene in NPB001-05
4.4.3. Gene expression validation by RT-qPCR and immunoblotting
5. Discussion
6. Acknowledgements
7. References


Scientists are constantly searching for phytochemical compounds with anti-cancer activity. In this study, activity of plant extract NPB001-05 from Piper betle was tested on human chronic myelogenous leukemia (CML) xenograft models. NPB001-05 was active when dosed orally (500 mg/kg) once or twice a day in xenograft tumor models. NPB001-05 showed activity to T315I tumor xenograft, where imatinib failed to show antitumor activity. NPB001-05 showed no relevant toxicity in animal models during 2 weeks exposure to drug. Responsive tumor showed inhibition of tyrosine kinase activity with lowered Bcr-Abl protein levels and increased apoptosis. Microarray based transcription profiling studies demonstrated that both imatinib and NPB001-05 dysregulated imatinib- responsive genes. NPB001-05 showed additional genes selectively dysregulated from ER stress, PI3K/AKT, MAPK pathways. Additionally, we tested gene expression of PI3K, AKT1, JUN, CASP3 and DDIT3 in K562, BaF3P210BCR-ABL and BaF3 P210BCR-ABLT315I cell line treated for 6- and 12- hours with NPB001-05 and imatinib. The data indicates that NPB001-05 mediated cell death in K562 affects the function of ER stress. NPB001-05 shows antitumor activity with favorable toxicity profile.