Objective

Introduction

Cancer is currently one of the biggest health problems facing modern medicine. Cancers result from changes in the expression or activation of proteins fundamental to the normal functioning of cells within the body. Epidermal growth factor receptor (EGFR) is a crucial player in cell signalling, responsible for many cellular processes, such as DNA replication, cellular division, differentiation and apoptosis.

Aberrant signalling in EGFR has been linked to the development and progression of many cancer types, including breast, lung, gastric, pancreatic and colorectal cancers, amongst many others. Increased activity (phosphorylation) through EGFR is often linked to rapid cancer growth and progression, leading to poor patient prognosis. Current targeted treatments for EGFR include small molecule tyrosine kinase inhibitors (SM-TKIs) and antibody-based therapies. Whilst these therapies prove effective, patient relapse can be common due to cancer cells developing resistance to these therapies mechanistic actions.

With this in mind, the ongoing search for new drugs with novel mechanism of action against cancer is as important now as it ever was. Venoms could hold the key to a new class of EGFR-targeted molecules. Due to the complex nature of their biological make-up, venoms have been shown through previous research to display a range of anti-microbial, analgesic, anti-coagulant and anti-cancer properties. It has also been shown that venom-derived compounds have the potential to become FDA-licenced drugs for the treatment of human ailments.

Method

Using 2 EGFR-overexpressing cell lines as a model (A431 and MDA-MB-468) the effectiveness of a panel of 18 crude snake venoms, from a diverse range of species, were tested for their ability to effectively block the phosphorylation of EGFR. In-cell ELISAs and western blotting were used as methods to assess the effectiveness of each venom’s ability to reduce EGFR activity. Subsequent ‘hit’ venoms were fractionated via reverse-phase HPLC and the resultant fractions also tested in the ELISA to determine which venom component displayed the activity.

Results and Discussion

Crude snake venom ELISAs and western blots identified a range of venoms, predominantly pit vipers, capable of causing reduction in the phosphorylation of EGF receptors in both A431 and MDA-MB-468 cancer cells. At least one snake venom from each of the diverse groups (Cobra, Mamba, Viper, Pit Viper) showed capacity for causing a reduction in EGFR activity. Similar patterns of percentage reduction were seen across both cell lines. Cobra venoms showed the least reduction in EGFR phosphorylation, with some showing increases. Greater percentage reductions were seen in MDA-MB-468 cells then in A431 cells, likely to be dose-dependent limitations based on A431 cells expressing a greater level of surface EGFR receptors then MDA-MB-468.

Snake venom 12 showed fractions which had similar inhibitory effects on EGFR phosphorylation in both the tested cell lines. Fractions 7, 8 and 13 showed similar percentage reductions in both MDA-MB-468 and A431 cells, with inhibition of phosphorylation of greater than 50% in both.

2^nd Dimension Size exclusion HPLC showed fractions 7 and 13 contained one large detectible peak and so samples were sent off for mass spec analysis.