Ornithine Decarboxylase activity, a clinical biomarker for evaluating cancer chemopreventive efficacy of phytomolecules Nusrat Masood , Vijaya Dubey , Suaib Luqman* CSIR-CIMAP CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow , India Email : [email protected] INTRODUCTION

 Biomarkers of cancer has made a strong traipse in Biomarker patent filings Classification of predicting the disease pattern and contributed according to type of cancer Biomarkers significantly to the understanding of tumour state, progression, characteristics and response to therapies.

 Ornithine decarboxylase (EC 4.1.1.17) catalyzes the Lung[6%] pyridoxal 5-phosphate (PLP)-dependent Liver[6%] General [47%] decarboxylation of ornithine to putrescine, the first Prostate [13%] critical step in biosynthetic pathway of polyamines. Breast  Expression of this enzyme is transiently increased [13%] upon stimulation by growth factors, but becomes constitutively activated during cell transformation induced by carcinogens, viruses or oncogenes.  Lots of research says that the phytomolecules having antioxioxidant property may have antimutagenic or anticarcinogenic activity . Therefore a need is there to REFERENCE find their interactive mode on known signature Analytical Biochemistry (1987)160:290-293 markers of cancer like ornithine decarboxylase Biochem.Biophys.Res.Comm.(1994)201:748-755 enzyme. J.Agric.Food Chem.(2002)50:2454-2458

Reactions inside the cell METHODOLOGY Structures of positive leads Exogenous Arginine L-Arginine Urea L-Ornithine HCl Arginase Reaction β– mercaptoethanol EDTA L-Ornithine Mixture Pyridoxal Phosphate

L-Citrulline Sample

Putrescine 37º C / 30 min Centrifugation Enhanced constitutive Spermidine Termination of enzymaticCenttri reaction expression Centrifugation of ODC Inhibition Spermine Polyamines of Pentanol extraction of supernatant Apoptosis Centrifugation

Nature Precedings : doi:10.1038/npre.2011.6643.1 Posted 23 Nov 2011 Enhanced polyamine uptake Final hydrophobic extraction of TNP-Putrescine Cell- Cell Promotion of Cell

interactions Proliferation

NO DPPH ODC AIM OF THE STUDY 100 60 NO DPPH ODC 40 1. To develop an improved, easy, reliable and cost-effective method for measuring the activity of 50 20 ornithine decarboxylase utilizing small quantity of chemicals.

2. Exploration of the phytochemicals as inhibitors of ornithine decarboxylase activity. 0 0 Percent Inhibition Percent A Inhibition Percent E 3. Finding the correlation between free radical scavenging potential with NO inhibition capacity B C F G Flavonoids D Terpenes H of phytomolecules. 4. Understanding the specificity of ODC enzyme to pH, time and substrate concentration. ODC, a highly inducible FUTURE PROSPECTS enzyme specific to pH, time RESULTS & substrate concentration.  A great no. of hurdles still 50µg 10µg 2µg 0.4µg 50µg 10µg 2µg 0.4µg 50µg 10µg 2µg 0.4µg 20 abound in the use of ODC

60 100

70 15 inhibitors, like DFMO as endpoints 50 80 60 for chemoprevention clinical trials.

10 40 50 Simply, ODC inhibitors may be 60 40 5 30 used to prevent or slow the rate of 40 30 0 20 Putrescine of µM pH5 pH6 pH7 pH8 pH9 20 tumour re-growth between doses of 10 20 therapy with radiation or other

Percent Inhibition Percent 10

Percent Inhibition Percent

Percent Inhibition Percent 0 0 0 40 more toxic chemotherapeutic A B C D E F G H A B C D E F G H A B C D E F G H 30 agents. Flavonoids Terpenes Flavonoids Terpenes Flavonoids Terpenes 20 Continuous progress in cancer Putrescine 10 drug development will move from

ODC inhibition assay DPPH scavenging assay NO scavenging assay 0

µM of µM

5 traditional cancer clinical trial to

10 15 20 30 60 90

DISCUSSION Time (minutes) 120 biomarker-driven and hypothesis-

40 testing trials .  ODC transform L-ornithine hydrochloride substrate to yellow coloured product putrescine 30 Also in near future some gaps in soluble in pentanol. our knowledge of phytomolecules

Putrescine 20  Some phytochemicals were found to inhibit enzyme activity in a concentration dependent 10 targeted cancer chemoprevention manner (0.4-50µg/mL). will close. µM of of µM 0  Molecules having phenolic group and lactone rings in their structure are better inhibitors. 0mM 1mM 2mM 3mM 5mM Comparatively flavonoids are better free radical scavengers. L-Ornithine HCl ACKNOWLEDGEMENT  A positive correlation was observed among the nitric oxide and DPPH inhibition (p<0.01) Council of Scientific and Industrial Research, New Delhi and  To act as inhibitor of ODC enzyme , phytochemical should interact in some way with the enzyme Council of Science and Technology (CST), Government of to prevent it from working in the normal manner. Uttar Pradesh.