INTRODUCTION: Although prognosis of multiple myeloma has recently been improved by novel therapeutic regimens, all currently available therapuetics target symptomatic myeloma only. We saw an MGUS patient, experiencing a gradual and continuous decrease of M-protein (IgA-k) from 2,080 mg/dl to 878 mg/dl over 3 years (Fig. 1). History examination revealed that the decrease of M-protein initiated when the patient started taking a dietary supplement containing hesperetin-7-glucoside, which is a glycosylated form of hesperetin designated to achive an approximately 104-fold increase of bioavailability comparing to aglycon structure (Hayashibara Biochemical Laboratories, Inc.). Because this compound is eventually metabolized to hesperetin, a possible anti-myeloma effect of hesperetin was examined in vitro.
METHODS: Myeloma cells were obtained from myeloma patients and purified with CD138 magnetic beads. Anti-myeloma effects of test compounds on purified myeloma cells or myeloma cell lines were evaluated by WST-8 assay. Apoptosis of myeloma cells was quantified either by annexin V staining or the propidium iodide method, followed by flowcytomerty analysis or by morphological analysis of cytospin slides. Mitochondrial membrane potential was quantified using JC-1 staining Kit (Cayman Chemical Co.) and flow cytometry. Caspase activation and total ubiqutinated protein were analysed by western blotting. Proteasomal chymotrypsin-like activity was measured using 20S Proteasome Assay Kit (Enzo Life Sciences).
RESULTS: Hesperetin showed inhibitory effects in a dose-dependent manner on the growth of 4 myeloma cell lines and freshly isolated myeloma cells. Two myeloma cell ines, RPMI8226 and 12PE, were utilized as representative cell lines for further analysis. Hesperetin induced annexin V/PI positive cells, morphological fragmentation of the nucleus of myeloma cells, and activation of caspase-3, 8 and 9, at a concentration around 500 microM, which is clinically achievable with peroral administration of glycosylated hesperetin, suggesting that the observed anti-myeloma effects by hesperitine through apoptotic pathways. Further analysis revealed that hesperetin disrupted mitochondrial membrane potential which leads to release of cytochrome c from mitochondria to cytoplasm as assessed by western blot analysis. Caspase-8 and 9 inhibitors(Z-IETD-fmk and Z-LEHD-fmk, respectively)did not inhibit the hesperetine-induced apoptosis, although they completely inhibited anti-Fas antibody-induced apoptosis, suggesting that the hesperitine-induced apoptosis is not dependent on death receptor signaling. A pan-caspase inhibitor, Z-VAD-fmk, completely blocked the hesperetin-induced apoptosis in 12PE cells, but only partially in RPMI8226 cells, suggesting that hesperetin also mediated caspase-independent apoptosis in RPMI8226 cells. Moreover, western blot showed that hesperetin treatment induced an accumlation of poly-ubiquitine proteins. Analysis of proteasome activity revealed that hesperetin at 500 microM exerted a moderate inhibition of proteasome activity with 72.6% of the inhibition exerted by bortezomib at 40 nM.
CONCLUTIONS: This is the first report showing anti-myeloma effect of hesperetin. It showed anti-myeloma effects via caspase-dependent and independent apoptotic pathways and proteasome inhibition activity. Given a fact that hesperitin has been approved by Japanese Goverment as a safe supplementary compound, and the present MGUS case had been taking hesperitin over >3 years without any adverse events, this compound should be well torelated over a long period. If a delay of conversion from either MGUS or asymptomatic myeloma to symptomatic myeloma could be achieved, prognosis of MM could be improve. Based on these findings, an open-label, pilot clinical trial to test the efficacy of hespertin, enroling asymptomatic myeloma patients, has currently been underway (Approved by IRB of Kumamoto Univerisity Hospital).