[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

TABLE OF CONTENTS

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

1. ABSTRACT

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.