Abstract No. 428 Parthenolide induces thiol oxidation-mediated cytotoxicity in human and rat hepatocellular carcinoma cell lines

Abstract

Parthenolide (PTL) is a naturally occurring sesquiterpene lactone that has anti-tumor activity. The redox environment of hepatocellular carcinoma (HCC) becomes an important target due to the differences between cancer and normal tissue metabolism. We hypothesize that in vitro PTL will induce HCC cell death through increased thiol oxidation. Half maximal inhibitory concentrations (IC50) response to PTL was determined in human (HepG2, SNU-423) and rat (McARH7777) hepatoma cell lines using MTT assays for 24 h. Cellular respiration response to PTL was measured over 24 h using Seahorse Flux Analyzer. Intracellular glutathione (GSH) and oxidized glutathione (GSSG) levels were measured using spectrophotometry at 2 h, 4 h and 24 h. The overall oxidation status of cell lines was also determined using thiol redox-sensitive ratiometric sensor roGFP. PTL had over 90% cytotoxicity at high concentrations 20 μM for SNU-423, 54 μM for HepG2, and 40 μM for rat HCC after 24 h. IC50 concentrations of 6.6 μM for SNU-423, 18 μM for HepG2 and 13 μM for rat HCC were determined after 24 h. Cytotoxic doses of PTL induced a decline in cellular respiration of the HCC cell lines after 2 h. Furthermore, 2 h PTL treatment at IC50 or high concentration caused cyto-roGFP sensor to be oxidized 2.5- to 4-fold in human and rat HCC. PTL at IC50 doses resulted in increased percent GSSG as early as 2 h in human and rat cells. This increase was compensated by upregulation of total GSH levels to minimize cell toxicity in HepG2 cells, which appeared to be offset at high doses of PTL. In HepG2 cells increased percent GSSG levels also demonstrated a similar trend in both IC50 and high PTL doses. In vitro PTL decreased cellular respiration, increased overall oxidation status and increased glutathione oxidation in HCC as early as 2 h after treatment. In HepG2 cells, IC50 dose caused a compensatory upregulation of GSH levels to ostensibly overcome thiol oxidation induced cytotoxicity. However, higher concentrations of PTL overwhelmed this mechanism and caused significant cytotoxicity in all 3 cells lines suggesting PTL can potentially be delivered at high concentrations as locoregional therapy for HCC in vivo.