Cancer Letters

Cancer Letters

Volume 270, Issue 2, 8 November 2008, Pages 342-353
Cancer Letters

Mitochondria-cytochrome C-caspase-9 cascade mediates isorhamnetin-induced apoptosis

https://doi.org/10.1016/j.canlet.2008.05.040Get rights and content

Abstract

Isorhamnetin is a flavanoid present in plants of the Polygonaceae family and is also an immediate metabolite of quercetin in mammals. Since the plasma level of isorhamnetin is maintained longer than quercetin, isorhamnetin may be a key metabolite to mediate the anti-tumor effect of quercetin. In the present study, we investigated the apoptotic mechanism of isorhamnetin in Lewis lung cancer (LLC) cells in vitro and established its in vivo anti-cancer efficacy. In cell culture, isorhamnetin significantly increased DNA fragmentation, and TUNEL positive apoptotic bodies and sub-G1 apoptotic population in time- and dose-dependent manners. Western blot analyses revealed increased cleavage of caspase-3, and caspase-9 and PARP and increased cytosolic cytochrome C in isorhamnetin-treated cells. These events were accompanied by a reduced mitochondrial potential. Apoptosis was blocked by a general caspase inhibitor or the specific inhibitor of caspase-3 or -9. These in vitro results support mitochondria-dependent caspase activation to mediate isorhamnetin-induced apoptosis. Furthermore, an animal study revealed for the first time that isorhamnetin given by i.p. injection at a dose that is at least one order of magnitude lower than quercetin significantly suppressed the weights of tumors excised from LLC bearing mice. The in vivo anti-tumor efficacy was accompanied by increased TUNEL-positive and cleaved-caspase-3-positive tumor cells. Our data therefore support isorhamnetin as an active anti-cancer metabolite of quercetin in part through caspase-mediated apoptosis.

Introduction

The flavonoid quercetin has been evaluated for its anti-cancer potential in some models [4]. Isorhamnetin (3′-O-methyl quercetin) is a flavonol present in some plants such as Ginkgo biloba Linne and Persicaria thunbergii H. Gross. Both herbs are used for the treatment of rheumatism, hemorrhage and cancer in oriental medicine [7]. Structurally, isorhamnetin is one of the metabolites of quercetin [5], [6]. In addition to quercetin, isorhamnetin shares similar chemical structure with kaempferol.

Pharmacokinetic studies have shown that quercetin is absorbed quickly in mice and reaches the maximum plasma concentration 1 h after an intraperitoneal injection, and declines sharply 4 h after treatment [8], [9]. By i.v. injection, the life time of quercetin in plasma is 5 min [10]. On the other hand, the plasma concentration of isorhamnetin reaches a peak 2 h after injection and remains elevated longer than that of quercetin [8], [9]. Studies have revealed that isorhamnetin cannot be transformed back into quercetin or kaempferol in the Wistar rats. These observations suggest that the anti-cancer effect of quercetin may in large part be mediated through isorhamnetin. In fact, isorhamnetin has been known to induce apoptosis in human promyelocytic leukemia HL-60 cells more effectively than quercetin or kaempherol in vitro[11], [12]. Isorhamnetin was known to have anti-oxidant effect [13], anti-tumor activity against HeLa cells [11] and apoptotic activity against HL 60 cells [12].

However, to our knowledge, no in vivo study has been reported to establish the superior efficacy of isorhamnetin over quercetin, nor were the underlying mechanisms of isorhamnetin-induced apoptosis fully elucidated. Here, we evaluated the in vivo efficacy of isorhamnetin against the growth of Lewis lung cancer (LLC) cells and focused on elucidating the apoptotic signaling events.

We chose the LLC cell model and apoptosis mechanisms for several reasons. The first is that lung cancer is the leading cause of cancer mortality in the US [1] and worldwide [2], [3]. The survival benefit of current cytotoxic cancer therapeutic drugs is very dismal and they have serious drug side effects. Compounds that selectively induce apoptosis of cancer cells can lead to the elimination of neoplastic cells, affording a permanent chemoprevention against carcinogenesis [7], [14]. Most anti-cancer therapy drugs induce apoptosis to achieve therapeutic efficacy. Second, quercetin has been recently reported to inhibit the growth of LLC in vivo[10]. Identifying agents more effective than quercetin will be desirable for the prevention and treatment of lung cancer. In addition, LLC cells grow rapidly in syngenic mice and the tumors are strongly dependent on angiogenesis. We have extensive experience using this model to study the in vitro and in vivo effects of novel anti-cancer agents from Oriental herb extracts and compounds [11], [13], [15].

Section snippets

Materials

RPMI 1640 medium, fetal bovine serum, antibiotic–antimycotic were purchased from GIBCO (Grand island, NY). HEPES, sodium bicarbonate, 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide XTT (XTT), propidium iodide (PI), N-methyldibenzo-pyrazine methyl sulfate (PMS) and poly-l-lysine were from Sigma chemical Co. (St. Louis, MO). Dead End™ fluorometric TUNEL (TdT-mediated-dUTP Nick-End Labeling) assay kit was from Promega (Madison, WI), protease inhibitor cocktail from

Isorhamnetin inhibited the growth of LLC cells in association with apoptosis

To define an effective concentration for isorhamnetin to inhibit LLC growth and survival, we first carried out a dose–response experiment. As shown in Fig. 1A, exposure of LLC cells to isorhamnetin for 24 h decreased the number of metabolically active LLC cells in a concentration-dependent manner with IC50 of 40 μM. Next, we examined how rapid the overall growth inhibition effect could be detected given the exposure concentration at the IC50. The impact of isorhamnetin on the number of viable

Discussion

Our in vitro and in vivo studies described above significantly advanced the mechanistic understanding of the signaling events for apoptosis induction by isorhamnetin and their relevance to the in vivo tumor inhibitory efficacy. Mechanistically, our in vitro data support the induction of apoptosis by isorhamnetin through an activation of the mitochondria pathway: loss of transmembrane potential (Fig. 5B), increased mitochondria Bax (Fig. 5C), release of cytochrome C into cytosolic fraction (Fig.

Conflict of interest statement

All authors have nothing to disclose any financial and personal relationships with other people or organizations that could inappropriately influence (bias) their work.

Acknowledgements

This study was supported by MRC grant (R13-2007-019-00000-0) from MEST.

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