Flavokawains A and B from kava (Piper methysticum) activate heat shock and antioxidant responses and protect against hydrogen peroxide-induced cell death in HepG2 hepatocytes

Context Flavokawains are secondary metabolites from the kava plant (Piper methysticum Forst. f., Piperaceae) that have anticancer properties and demonstrated oral efficacy in murine cancer models. However, flavokawains also have suspected roles in rare cases of kava-induced hepatotoxicity.Objective To compare the toxicity flavokawains A and B (FKA, FKB) and monitor the resulting transcriptional responses and cellular adaptation in the human hepatocyte cell line, HepG2.Materials and methods HepG2 were treated with 2-100M FKA or FKB for 24-48h. Cellular viability was measured with calcein-AM and changes in signalling and gene expression were monitored by luciferase reporter assay, real-time PCR and Western blot of both total and nuclear protein extracts. To test for subsequent resistance to oxidative stress, cells were pretreated with 50M FKA, 10M FKB or 10M sulphoraphane (SFN) for 24h, followed by 0.4-2.8mM H2O2 for 48h, and then viability was assessed.Results FKA (100M) was not toxic to HepG2, whereas FKB caused significant cell death (IC50=23.20.8M). Both flavokawains activated Nrf2, increasing HMOX1 and GCLC expression and enhancing total glutathione levels over 2-fold (p<0.05). FKA and FKB also activated HSF1, increasing HSPA1A and DNAJA4 expression. Also, flavokawain pretreatment mitigated cell death after a subsequent challenge with H2O2, with FKA being more effective than FKB, and similar to SFN.Conclusions Flavokawains promote an adaptive cellular response that protects hepatocytes against oxidative stress. We propose that FKA has potential as a chemopreventative or chemotherapeutic agent.