Induction of differentiation of myelogenous leukemia cells by humulone, a bitter in the hop

doi:10.1016/S0145-2126(98)00046-0

Leukemia Research

Volume 22, Issue 7, July 1998, Pages 605-610

https://doi.org/10.1016/S0145-2126(98)00046-0 Get rights and content

Abstract

The active form of vitamin D, 1α,25-dihydroxyvitamin D₃ (VD₃), inhibits proliferation and induces differentiation of myelomonocytic leukemia cells, but its clinical use is limited by the adverse effect of hypercalcemia. VD₃ mobilizes calcium stores from bone by inducing the dissolution of bone mineral and matrix. We have recently found that humulone, a bitter in the hop extract for beer brewing, effectively inhibits bone resorption. In this study we examined the effect of humulone on the differentiation of human myelogenous leukemia cells. Humulone alone inhibited the growth of monoblastic leukemia U937 cells while only slightly increasing differentiation markers such as nitroblue tetrazolium (NBT)-reducing and lysozyme activities. Humulone effectively enhanced the differentiation-inducing action of VD₃. Other myelomonocytic leukemia cells were induced to differentiate by VD₃ and this was also enhanced by humulone. Since humulone is a less-toxic inhibitor of bone resorption, the combination of humulone and VD₃ may be useful in differentiation therapy of myelomonocytic leukemia.

Introduction

Human myelogenous leukemia cells are induced to undergo cell differentiation in vitro into mature neutrophils, macrophages, megakaryocytes, and hemoglobin-producing cells by various differentiation inducers 1, 2, 3. However, most inducers may be difficult to apply in clinical trials of differentiation therapy, since some agents are very unstable in vivo or require unusually high concentrations, while others exhibit serious adverse effects on normal host cells. Therefore, it is worthwhile to search for non-toxic differentiation inducers that have been used in clinical trials for other diseases or have been taken ordinarily as foods and beverages.

We found and isolated a strong bone resorption inhibitor from hop (Humulus lupulus L.) extract for beer brewing [4].

Humulone was the most active component in inhibiting bone resorption when assessed by counting the number of bone pits, and the concentration required 50% inhibition (IC₅₀) was 5.9 nM [4].

The active form of vitamin D, 1α,25-dihydroxyvitamin D₃ (VD₃) can induce monocytic differentiation of leukemia cells, and it has been used to treat myelodysplastic syndrome and smoldering leukemia, but it is scarcely effective [5], because it causes hypercalcemia and stimulates bone resorption at therapeutic dosages 5, 6. Examination of combined effects of VD₃ and agents preventing these adverse effects should be useful in development of differentiation therapy with VD₃. The present study was undertaken to examine the response of several kinds of human myelogenous leukemia cells to humulone.

Section snippets

Materials

Humulone [3,5,6-trihydroxy-4,6-bis(3-methyl-2-butenyl)-2-(3methyl-1-oxobutyl)-2,4-cyclohexadien-1-one, Fig. 1] was prepared from hop extract as previously described [4], and dissolved in ethanol. The stock solution was stored at −20°C and protected from light. The final concentration of ethanol did not exceed 0.4%. 12-O-tetradecanoylphorbol-13-acetate (TPA), nitroblue tetrazolium (NBT) and all-trans retinoic acid (ATRA) were purchased from Sigma (St. Louis, MO). VD₃ was obtained from Wako Pure

The effect of humulone on differentiation of U937 cells

Humulone inhibited the proliferation of human monoblastic leukemia U937 cells with an IC₅₀ of 3.4 μM, and slightly induced the NBT-reducing and lysozyme activities, which are typical markers of myelomonocytic differentiation (Fig. 2; data not shown). Humulone did not induce morphological changes of U937 cells. Thus humulone itself is a weak differentiation inducer.

VD₃ induces the monocytic differentiation of human leukemia cells-including U937 cells, but cannot be applied to clinical treatment

Discussion

The dried female flowers of hop are a Chinese crude drug used for the treatment of leprosy and tuberculosis [11]. Humulone has been reported as an antibiotic [12]and antifungal agent [13]. Humulone inhibited arachidonic acid-induced inflammatory ear edema and tumor promotion by TPA in mouse skin [11]. Humulone has a potent antioxidative activity [14], and IC₅₀ of humulone was 28 μM in hydrogen peroxide hemolysis test [15]. The antioxidative activity of humulone might be associated with

Acknowledgements

This work was supported by Grants-in-Aid for Cancer Research from the Ministry of Education, Science, Sports and Culture, and a grant from the Ministry of Health and Welfare, Japan.

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Humulus lupulus (Hop) contains numerous metabolites with anticancer potential. Despite this, their immunomodulatory activity, and in particular of bitter acids, is unknown. In this study, we demonstrated that a Hop pellet extract fraction containing bitter acids possesses immunomodulatory activity by enhancing Natural Killer (NK) cells function. After fractionation by semi-preparative Liquid Chromatography, three different fractions were obtained. The phytocomplex and the fractions were tested to verify the ability to modulate the NK compartment. Cytofluorimetric analysis revealed that a fraction containing bitter acids was able to up-regulate of NKG2D and NKp44 activating receptors. A further simplification yield a fraction mainly composed by Humulinones and Cohulupone derivatives, that at the concentration of 0.1 µg/mL induced selective activation of Nkp44 receptor and enhanced the cytolytic activity of NK cells against leukemia cell line K562. Those results point out the possible use of Hop bitter acids as natural immunostimulants and adjuvants in chemotherapy protocols.
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As referred we have found IC50 value of 16.16 ± 3.11 µg/mL for Caco-2 cells, using a mixture of both bitter acids, however one cannot completely parallel this values as exposure time was the double in our work. The pure constituent compounds of bitter acids humulone, isohumulone, lupulone and colupulone as well as extracts containing only α-acids or β-acids, have been studied during the last 20 years concerning their activity in leukemia, prostate, colon, breast and lung cancer cells (Farag & Wessjohann, 2013; Honma, Tobe, Makishima, Yokoyama, & Okabe-Kado, 1998; Lamy et al., 2007; Lamy et al., 2008; Liu et al., 2011; Tobe, Kubota, Yamaguchi, Kocha, & Aoyagi, 1997; Tyrrell et al., 2010) and a few mechanism of actions were proposed, mainly based on apoptosis via extrinsic pathways either via caspases activation or involving the Fas or TNF paths. Despite the paramount findings achieved in the study of individual compounds in the last years, in the present work our goal was also to study the beer bioactivity in a “shotgun” approach as opposed to the “silver bullet” method of studying the substances individually.
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The present review describes the morphological, phytochemical and ethnopharmacological aspects of Humulus lupulus L. (Cannabinaceae) and summarizes the most interesting findings obtained in the preclinical and clinical research related to the plant. The female inflorescences of Humulus lupulus (hops), well-known as bittering agent in brewing industry, have long been used in traditional medicine mainly to treat sleep disturbances. However the sedative activity is still under investigation in order to recognize the active principles responsible for the neuropharmacological effects observed in laboratory animals, and their mechanism of action. Here we report the data from our experiments as well as those obtained from other researchers, focusing on the variability of the results. Other traditional applications of hops as stomachic, antibacterial and antifungal remedy have been supported by in vivo and/or in vitro investigations. In recent years some prenylated chalcones present in hops have received much attention for their biological effects: in particular, xanthohumol has been shown to exert cancer chemopreventive activity in in vitro experiments, while 8-prenylnaringenin has been characterized as one of the most potent phytoestrogens isolated until now. Nevertheless much additional work is needed to open up new biomedical application of these compounds.
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Induction of differentiation of myelogenous leukemia cells by humulone, a bitter in the hop

Abstract

Introduction

Section snippets

Materials

The effect of humulone on differentiation of U937 cells

Discussion

Acknowledgements

Adv Cancer Res

Blood

Biochim Biophys Acta

Biochim Biophys Acta

Leuk Res

Biochim Biophys Acta

AML1a but not AML1b inhibits erythroid differentiation induced by sodium butyrate and enhances the megakaryocytic differentiation of K562 leukemia cells

Cell Growth Differ

Bone resorption inhibitors from hop extract

Biosci Biotechnol Biochem

1,25-Dihydroxyvitamin D3: in vivo and in vitro effects on human preleukemic and leukemic cells

Cancer Treat Rep

1,25-Dihydroxycholecalciferol: a potent stimulator of bone resorption in tissue culture

Science

1,25-Dihydroxyvitamin D₃: in vivo and in vitro effects on human preleukemic and leukemic cells