Mechanisms of Ergothioneine-Mediated Cytoprotection against Oxaliplatin-Induced Cytotoxicity

Abstract

Oxaliplatin is a third-generation platinum drug used to primarily treat metastatic colorectal cancer. However, its clinical effectiveness is hindered by dose-limiting peripheral neuropathy, presented in either its acute or chronic form. Previous work showed oxaliplatin to be a substrate for the electroneutral organic cation transporter protein, OCTN1, expressed in the dorsal root ganglion neurons. OCTN1 was thought to potentiate the development of oxaliplatin-induced neuropathy via a mechanism of oxaliplatin transport. Ergothioneine, the native OCTN1 substrate, is a known antioxidant and metal chelator and was shown to protect against oxaliplatin-induced neurotoxicity in vitro. The aims of this thesis were to investigate the cytoprotective effects of ergothioneine mediated by competitive inhibition of OCTN1 associated oxaliplatin transport and the chelation mediated inactivation of oxaliplatin. This was achieved through exposure of HEK-293 cells overexpressing mouse OCTN1 transporter and empty transfected cells to oxaliplatin in the absence or presence of ergothioneine. Platinum accumulation was measured by ICP-MS and normalized to cellular protein quantified through a bovine serum albumin standard curve. MTT cell viability assays were used to measure oxaliplatin-induced growth inhibition. An in vitro stability study was conducted with solutions containing oxaliplatin with or without ergothioneine incubated at 37°C, pH 7.4. The in vitro stability data was then extrapolated through in vivo scaling factors to predict effects on oxaliplatin clearance in human patients. An exploratory study utilizing ICP-MS to quantify platinum in HPLC fractions was conducted to further define the reaction mechanisms between the two. The findings of this thesis demonstrated that the level of ergothioneine-mediated cytoprotective effects against oxaliplatin-induced growth inhibition was dependent on ergothioneine concentration and OCTN1 expression. Additionally, ergothioneine degraded oxaliplatin in vitro in a time and concentration-dependent manner and was predicted to generate large effects on in vivo oxaliplatin clearance. It was also shown that ergothioneine markedly reduced the generation of Pt(DACH)Cl2. This reaction was speculated to be independent of the oxalate ring opening process and the generation of an ergothioneine-platinum chelate although that species was not identified. In conclusion, ergothioneine protected against oxaliplatin-induced growth inhibition via inhibition of OCTN1 mediated uptake and mediating the degradation of oxaliplatin. The large predicted ergothioneine-mediated effect on in vivo oxaliplatin clearance would be expected to alter both oxaliplatin toxicity and antitumor activity in vivo.