Relationship between the methylation status of dietary flavonoids and their growth-inhibitory and apoptosis-inducing activities in human cancer cells

Interest in the potential cancer chemopreventive and therapeutic properties of diet-derived compounds such as plant polyphenols has increased in recent years. A subgroup of polyphenols, flavonoids, contains a flavone backbone with hydroxylations at various positions. These compounds can be found in a number of fruits and vegetables including celery, apples, onions, parsley, capsicum pepper, etc. [Ramos, 2007], and have been shown to possess anti-cancer activities. The mechanisms by which flavonoids impart their anti-cancer effects are varied and may include action through anti-inflammation [Choi et al., 2004], free radical scavenging [Sim et al., 2007], modulation of survival and proliferation pathways [Lee et al., 2005; Lee et al., 2004], and inhibition of the ubiquitin-proteasome pathway [Chen et al., 2007; Chen et al., 2005]. Although numerous studies indicate positive anti-cancer effects, the bioavailability and therefore efficacy of ingested flavonoids may be limited due to conjugation reactions (methylation, sulfonation, and glucuronidation) to the free hydroxyl groups surrounding the molecules [Manach and Donovan, 2004]. Exogenously derived methylated flavonoids have been shown to promote oral bioavailability [Wen and Walle, 2006a; Wen and Walle, 2006b] but direct evidence to indicate whether these compounds promote apoptotic cell death in cancer cells remains unclear. Furthermore, the effects of methylated flavonoids on proteasome activity have not been examined. The eukaryotic proteasome is a large multi-catalytic, multi-subunit protease complex possessing at least three distinct activities, which are associated with three different β subunits, respectively: chymotrypsin-like (with β 5 subunit), trypsin-like (with β 2 subunit), and peptidyl-glutamyl peptide-hydrolyzing-like (PGPH- or caspase-like; with β 1 subunit) [Groll et al., 1997]. Inhibition of the chymotrypsin-like, but not the trypsin-like, activity of the proteasome has been found to be associated with induction of tumor cell apoptosis [An et al., 1998; Lopes et al., 1997]. By examining a broad range of cell culture models, it has been found that proteasome inhibitors rapidly induce tumor cell apoptosis, selectively activate the cell death program in cancer or oncogene-transformed, but not normal or untransformed cells, and are able to trigger apoptotic death in human cancer cells that are resistant to various anticancer agents [Adams, 2003; Almond and Cohen, 2002; Dou and Li, 1999; Dou et al., 2003; Goldberg, 1995]. We have previously observed cytotoxicity associated with proteasome inhibition by the hydroxylated flavonoids apigenin, luteolin, quercetin, and chrysin in leukemia cells but not in non-transformed cells [Chen et al., 2007; Chen et al., 2005]. Proteasome inhibition leads to accumulation of proteasome target proteins (such as IκBα) and subsequent induction of apoptosis in human cancer cell lines, apparent by activation of caspases and cleavage of poly(ADP-ribose) polymerase (PARP) [Chen et al., 2007; Chen et al., 2005]. In this work, we analyzed fully methylated flavonoids for their ability to impart cytotoxicity to human leukemia HL60 cells and whether cell death could be correlated to proteasomal-inhibitory activity. We found that methylated flavonoids inhibit tumor cell proliferation, but have little to no effect on proteasomal inhibition or apoptosis induction. These findings correlate with the notion that methylated flavonoids could increase bioavailability. However, due to their limited ability to induce apoptosis, the usefulness of methylated flavonoids as cancer therapeutic or preventative agents remains to be further studied.