W1960 Reactive Oxygen Species Induced By the Compound Bisphenol-a-Diglycidyl-Ether Cause Senescence and Apoptosis in Colorectal Cancer Cell Lines Regardless of MDR1 or p53 Status

Abstract

Background: Reactive oxygen species (ROS) can be toxic. Tumors have elevated ROS, and the ability of tumor cells to accommodate additional ROS may be limited compared to normal cells, suggesting the possibility of “redox therapy”. ROS are critical to the mechanism of many cancer drugs, and experimental agents that affect ROS metabolism are being tested. However, this strategy has been difficult in colorectal cancer (CRC) due to high level expression of drug efflux pumps and loss of the stress-activated p53 pathway that normally controls expression of ROS scavenging enzymes. We show that ROS induced by the compound bisphenol-A-diglycidyl-ether (BADGE) cause senescence and apoptosis in CRC cell lines, including those with high levels of MDR1 efflux pump activity and loss of p53 function. Methods: ROS, Jnk kinase signaling, apoptosis, and senescence (irreversible cell cycle arrest) were measured in HT-29, SW620, HCT-116, and HCT-15 cells. ROS were measured using the redox-sensitive dye DCFDA. Jnk kinase activation was measured by Western blot, and apoptosis by annexinV staining. Markers of senescence included large cells with vacuolated cytoplasm, ability to maintain constant viable cell numbers over three weeks, and appearance of senescence-associated beta-galactosidase. Results: BADGE treatment caused immediate (<1h) dose-dependent increases in ROS that lasted for at least 12h. At later times, a further increase in ROS corresponding to apoptosis was observed. The consequences of BADGE treatment were proportional to the amount of ROS generated. 50μM BADGE caused senescence but not apoptosis, while higher doses (75-100μM) triggered ROS and Jnk kinasedependent apoptosis. Increasing glutathione-dependent ROS scavenging with N-acetylcysteine allowed greater doses of BADGE to be tolerated, while reducing glutathione with buthionine sulfoximine sensitized the cells to BADGE. BADGE triggered sustained Jnk kinase activation, and Jnk inhibitors decreased the pro-apoptotic effects of BADGE. The concentration of BADGE resulting in half-maximal apoptosis (IC50) was 75μM in all cell lines, regardless of MDR1 or p53 status. The MDR1 inhibitor verapamil, while reducing efflux of rhodamine 123, did not increase the IC50 of BADGE in HCT-15 cells. BADGE concentrations that caused ROS and apoptosis did not suppress PPARγ-dependent transcription, and PPARγ knockdown did not replicate or interfere with the effects of BADGE, suggesting that the effects of BADGE are PPARγ-independent. Conclusions: Depending on dose, ROS lead to senescence or apoptosis even in MDR1 high p53 mutant CRC cells. BADGE is a possible candidate for CRC redox therapy.