Bacille-Calmette Guerin induces caspase-independent cell death in urothelial carcinoma cells together with release of the necrosis-associated chemokine high molecular group box protein 1

To evaluate the ability of bacille-Calmette Guerin (BCG) to induce caspase-independent cell death and release the necrosis-associated chemokine high molecular group box protein 1 (HMGB1) from urothelial carcinoma (UC) cells; a correlative clinical trial determined if BCG treatment resulted in increased urinary levels of HMGB1. The human UC cell lines 253 J and T24 were pretreated with apoptosis inhibitors, exposed to BCG, and cell viability and ultrastructural changes measured. HMGB1 levels were assessed in cell culture supernatant after BCG treatment. The expression/function of HMGB1 receptors on the UC cell lines was determined by reverse transcription-polymerse chain reaction and the ability of exogenous HMGB1 to activate nuclear factor (NF)-kappa B signalling assessed. An HMGB1 enzyme-linked immunosorbent assay was used to measure HMGB1 levels in urine obtained from BCG-treated patients. Inhibition of apoptotic pathways failed to inhibit BCG-induced cell death in UC cells. Electron microscopy showed BCG-dependent ultrastructural changes consistent with cellular necrosis. BCG exposure resulted in a binary increase in cell culture supernatant levels of HMGB1. UCs expressed multiple HMGB1 receptors. Treatment of UCs with HMGB1 activated NF-kappa B. In the clinical setting, six of seven patients had increased urinary levels of HMGB1 at 24 h after BCG treatment. BCG causes direct cytotoxicity in a subpopulation of UC cells. This cytotoxicity is caspase-independent and associated with ultrastructural changes and cellular protein release (HMGB1), characteristic of necrosis. Urinary levels of HMGB1 can be elevated in patients after BCG treatment. The expression and function of HMGB1 receptors in UC cells, coupled with the known role of HMGB1 on the host immune response, suggest a role for necrosis and HMGB1 release in the antitumour effect of BCG.