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

Abstract

<div class="section"> <a class="named-anchor" id="S1"> <!-- named anchor --> </a> <h5 class="section-title" id="d6653044e156">Context</h5> <p id="P1">Flavokawains are secondary metabolites from the kava plant ( <i>Piper methysticum</i> 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. </p> </div><div class="section"> <a class="named-anchor" id="S2"> <!-- named anchor --> </a> <h5 class="section-title" id="d6653044e164">Objective</h5> <p id="P2">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. </p> </div><div class="section"> <a class="named-anchor" id="S3"> <!-- named anchor --> </a> <h5 class="section-title" id="d6653044e169">Materials and methods</h5> <p id="P3">HepG2 were treated with 2–100 μM FKA or FKB for 24–48 h. Cellular viability was measured with calcein-AM and changes in signaling 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 pre-treated with 50 μM FKA, 10 μM FKB or 10 μM sulforaphane (SFN) for 24 h, followed by 0.4–2.8 mM H ₂O ₂ for 48 h, and then viability was assessed. </p> </div><div class="section"> <a class="named-anchor" id="S4"> <!-- named anchor --> </a> <h5 class="section-title" id="d6653044e180">Results</h5> <p id="P4">FKA (≤ 100 μM) was not toxic to HepG2, whereas FKB caused significant cell death (IC ₅₀ = 23.2 ± 0.8 μM). Both flavokawains activated Nrf2, increasing <i>HMOX1</i> and <i>GCLC</i> expression and enhancing total glutathione levels over 2-fold (p &lt; 0.05). FKA and FKB also activated HSF1, increasing <i>HSPA1A</i> and <i>DNAJA4</i> expression. Also, flavokawain pretreatment mitigated cell death after a subsequent challenge with H ₂O ₂, with FKA being more effective than FKB, and similar to SFN. </p> </div><div class="section"> <a class="named-anchor" id="S5"> <!-- named anchor --> </a> <h5 class="section-title" id="d6653044e207">Conclusions</h5> <p id="P5">Flavokawains promote an adaptive cellular response that protect hepatocytes against oxidative stress. We propose that FKA has potential as a chemopreventative or chemotherapeutic agent. </p> </div>