Marine bromophenol bis(2,3-dibromo-4,5-dihydroxybenzyl) ether, induces mitochondrial apoptosis in K562 cells and inhibits topoisomerase I in vitro
Highlights
► BDDE exhibits apoptotic activity in K562 cells via mitochondrial pathway. ► BDDE inhibits the activity of topoisomerase I. ► BDDE does not stimulate the formation of topoisomerase I–DNA complex. ► BDDE mainly targets DNA and binds to DNA minor groove.
Introduction
DNA topoisomerase I (topo I) plays a pivotal role in the dynamic nature of DNA secondary and higher order structures and carries out vital cellular processes (Champoux, 2001). Topo I has recently emerged as a well-known anticancer target and topo I inhibitors have become one kind of the most effective and clinically used anticancer drugs (Salerno et al., 2010). Topo I inhibitors are classified as either topo I poisons or topo I catalytic inhibitors. Topo I poisons act by stabilizing the topo I–DNA covalent complex, such as campthotecin (CPT) and its derivatives (Hsiang et al., 1985), while topo I catalytic inhibitors act at any other stage in its catalytic cycle. However, current topo I inhibitors such as CPT and its derivatives often elicit life-threatening toxicities and induce tumor multidrug resistance, which limit their clinical usage (Seiter, 2005). To overcome these defects, continuous effort in searching for novel topo I inhibitors remains pressing.
Due to their multiple bioactivities, marine bromophenols, which usually exist in the marine sponges and algae, have attracted much attention in the field of functional food and pharmaceutical agents (Liu et al., 2011a). Previous studies have reported that some marine bromophenols, together with their synthetic derivatives analogs, could inhibit the proliferation of a number of cancer cell lines in vitro (Han et al., 2005, Ma et al., 2006, Popplewell and Northcote, 2009, Shi et al., 2009, Shoeib et al., 2004). Bromophenols isolated form Polysiphonia lanosa, as well as some synthesized isomers, have been reported to be cytotoxic against DLD-1 and HCT-116 cell lines (Shoeib et al., 2004). The Leathesia nana extract containing large amount of bromophenol derivatives, could inhibit the growth of Sarcoma 180 tumors in mice (Shi et al., 2009). The accumulated results both in vitro and in vivo indicate that marine bromophenols could be a promising family of anticancer compounds. However, the underlying anticancer mechanism of these compounds is unclear. Bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (BDDE, Fig. 1A), isolated from marine algae L. nana and Rhodomela confervoides, possesses various bioactivities, such as antimicrobials (Xu et al., 2003), protein tyrosine phosphatase 1B inhibition (Shi et al., 2008), and α-gulcosidase inhibition (Kim et al., 2010, Kurihara et al., 1999a, Kurihara et al., 1999b). BDDE also exhibits cytotoxicity against several cancer cell lines based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Xu et al., 2004). However, the apoptosis activity and the related molecular mechanism remain elusive.
In this study, we demonstrated that BDDE inhibited the proliferation of several tumor cells lines and induced apoptosis in K562 cells via mitochondrial pathway. We also found that BDDE inhibited the activity of topo I, but did not stimulate the formation of topo I–DNA complex nor intercalate into DNA. Ethidium bromide displacement fluorescence assay and molecular modeling results showed that BDDE acted as a novel topo I catalytic inhibitor by binding to DNA minor groove.
Section snippets
Drugs and reagents
BDDE was synthesized as described previously (Liu et al., 2011b). Antibodies against caspase-3, -9, Bcl-2, Bax, Survivin, and cytochrome C were purchased from Santa Cruz Biotechnology, Inc. Anti-mouse or anti-rabbit antibodies were purchased from Beijing Solarbio Science & Technology Co., Ltd. Annexin V-FITC Apoptosis Detection Kit was provided by Nanjing KeyGEN BioTECH. Co., Ltd, China. Calf thymus DNA topo I and pBR322 plasmid DNA were purchased from TAKARA, China. Other reagents and kits
BDDE inhibits cancer cell proliferation
To evaluate the cytotoxicity of BDDE in vitro, we examined its proliferative inhibition on several human tumor cell lines as well as normal MCF-10A cell line. As shown in Fig. 1B, BDDE displayed broad and potent cytotoxicity on the tested human cancer cell lines but relatively minor cytotoxicity to normal MCF-10A cells. K562 cells were relatively more sensitive to BDDE (the IC50 value of BDDE against K562 cell lines was 13.9 μg/mL) and were therefore selected for further analysis. Morphological
Discussion
BDDE is a bromophenol compound with multiple bioactivities (Liu et al., 2011a). Previous studies have shown that BDDE exhibits cytotoxicity to several human cancer cells (Xu et al., 2004). However, the mechanism of its cytotoxicity remains unclear. Here, we illustrated that BDDE induces apoptosis via mitochondrial pathway in K562 cells. This finding is consistent with the widely accepted conception that most chemotherapeutic agents induce apoptosis via mitochondrial pathway (Fulda and Debatin,
Conclusion
In conclusion, the present study showed that bromophenol compound BDDE displays broad-spectrum in vitro anticancer capabilities and exhibits potent apoptotic activity in K562 cells via mitochondrial pathway. BDDE interacts with the minor groove of DNA and inhibits the activity of topo I. With unique chemical structure different from the current topo I inhibitors, BDDE could serve as a lead template for rational drug design and for future anticancer agent development.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (81001396) and State Innovative Drugs Development Program of China (2009ZX09103-661 and 2009ZX09102).
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