Xanthohumol Induces ROS through NADPH Oxidase, Causes Cell Cycle Arrest and Apoptosis.

Reactive oxygen species (ROS) are either toxic in excess or essential for redox signalling at the physiological level, which is closely related to the site of generation. Xanthohumol (XN) is an important natural product of hops (Humulus lupulus L.) and was reported to induce ROS in mitochondria. While in the present study, our data indicate that NADPH oxidase (NOX) is another site. In human acute myeloid leukemia HL-60 cells, we first identified that cell proliferation was inhibited by XN without affecting viability, and this could be alleviated by the antioxidant N-acetyl-L-cysteine (NAC); cell cycles were blocked at G1 phase, apoptosis was induced in a dose-dependent manner, and malondialdehyde (MDA) content was upregulated. XN-induced ROS generation was detected by flow cytometry, which can be inhibited by diphenyleneiodonium chloride (DPI, a NOX inhibitor), while not by NG-methyl-L-arginine acetate (L-NMMA, a nitric oxide synthase inhibitor). The involvement of NOX in XN-induced ROS generation was further evaluated: immunofluorescence assay indicated subunits assembled in the membrane, and gp91phox knockdown with siRNA decreased XN-induced ROS. Human red blood cells (with NOX, without mitochondria) were further selected as a cell model, and the XN-induced ROS and DPI inhibiting effects were found again. In conclusion, our results indicate that XN exhibits antiproliferation effects through ROS-related mechanisms, and NOX is a source of XN-induced ROS. As NOX-sourced ROS are critical for phagocytosis, our findings may contribute to the anti-infection and anti-inflammatory effect of XN.

An acute lytic cell death induced by xanthohumol obstructed ROS detecting in HL-60 cells.

Serum is an important component in cell culture medium. It also possesses potent antioxidant properties. Therefore, the conventional protocols for detecting reactive oxygen species (ROS) in cultured cells with fluorescent probes include washing and suspending cells with serum-free buffers, such as PBS. This transient serum deprivation is essential for the ROS detecting. Unfortunately, it may also cause unexpected results, which push us to choose more optimal experiment conditions. In the present study, we found an acute lytic cell death induced by xanthohumol (XN), which obstructed ROS detecting in human leukemia cell line HL-60 cells. XN induced ROS burst, caused cell swelling, membrane permeability increase, LDH release, and ultimately an acute lytic cell death and cell rupture. These effects could be alleviated by the antioxidant N-Acetyl-L-cysteine (NAC). Apoptosis, pyroptosis or necroptosis were not observed in this process. Results also indicated that 2% serum addition had already completely scavenged ROS induced by 10 µM XN. Taken together, it is strongly suggested to detecting ROS in a serum-free medium when studying where and how ROS generated in cells. The concentration at the ROS maximum point (10 µM XN in this study) can be selected as the optimal concentration.