Modulation of Cell Surface Protein Free Thiols; A Potential Novel Mechanism of Action of the Sesquiterpene Lactone Parthenolide in Non-Hodgkin's Lymphoma.

Abstract 3774

Poster Board III-710

Recent data supports the concept that redox regulation of cell surface protein thiols (i.e., exofacial thiols) effects critical cellular functions. We therefore hypothesized that redox-active anti-cancer therapeutics would modulate exofacial thiols, and that such modulation could be related to cell death mechanisms. To test this hypothesis, we used the sesquiterpene lactone parthenolide, a known anti-cancer agent. Parthenolide treatment results in a dose dependent decrease in mantle cell (MCL) and diffuse large cell lymphoma cell viability. Indeed, parthenolide decreases the level of free (ie. reduced) exofacial thiols on Granta MCL cells as assessed by flow cytometry. Parthenolide specifically modifies free thiols of surface proteins having molecular weights of ∼12 kd and ∼22 kd, as determined using a biotinylated thiol reactive reagent N-(biotinoyl)-N-(iodoacetyl) ethylendiamine (BIAM), which binds to free thiol groups and is detected by streptavidin-peroxidase staining of western blots. We further show that the central redox regulator thioredoxin is one of the surface protein thiol targets modified by parthenolide; specifically; (a) thioredoxin is detected on the Granta cell surface; (b) parthenolide directly interacts with free thiol groups on purified human thioredoxin, and; (c) parthenolide directly modifies Granta membrane associated thioredoxin, as determined with BIAM and neutravidin pull-down. To examine the functional consequences of parthenolide induced surface protein thiol modification, Granta cells were pretreated with the cell impermeable thiol antioxidant glutathione (GSH). Pretreatment with GSH inhibits the parthenolide induced; (a) decrease in exofacial free thiols; (b) modification of surface thioredoxin and; (c) Granta cell death. Pretreatment of Granta cells with GSH also inhibits parthenolide-mediated activation of JNK and inhibition of NFkB, two activities previously associated with the drug's anti-cancer activity. Based on these data, we postulate that at least one component of parthenolide's anti-lymphoma activity derives from its ability to modify the redox state of critical exofacial thiols. Indeed, to our knowledge, our data is the first to suggest that cancer cell exofacial thiols may be novel and important targets for cancer therapy.

Supported by an NCI SPORE grant in lymphoma 1P50 CA130805.