(-)-Xanthatin selectively induces GADD45γ and stimulates caspase-independent cell death in human breast cancer MDA-MB-231 cells

exo-Methylene lactone group-containing compounds, such as (−)-xanthatin, are present in a large variety of biologically active natural products, including extracts of Xanthium strumarium (Cocklebur). These substances are reported to possess diverse functional activities, exhibiting anti-inflammatory, antimalarial, and anticancer potential. In this study, we synthesized six structurally related xanthanolides containing exo-methylene lactone moieties, including (−)-xanthatin and (+)-8-epi-xanthatin, and examined the effects of these chemically defined substances on the highly aggressive and farnesyltransferase inhibitor (FTI)-resistant MDA-MB-231 cancer cell line. The results obtained demonstrate that (−)-xanthatin was a highly effective inhibitor of MDA-MB-231 cell growth, inducing caspase-independent cell death, and that these effects were independent of FTase inhibition. Further, our results show that among the GADD45 isoforms, GADD45γ was selectively induced by (−)-xanthatin and that GADD45γ-primed JNK and p38 signaling pathways are, at least in part, involved in mediating the growth inhibition and potential anticancer activities of this agent. Given that GADD45γ is becoming increasingly recognized for its tumor suppressor function, the results presented here suggest the novel possibility that (−)-xanthatin may have therapeutic value as a selective inducer of GADD45γ in human cancer cells, in particular in FTI-resistant aggressive breast cancers. INTRODUCTION exo-Methylene lactone group-containing compounds are present ubiquitously in a large variety of biologically active natural products,1 including extracts of Xanthium strumarium (Cocklebur), and possess diverse activities, including anti-inflammatory, antimalarial, and cytotoxic activities for cancer cells.1 Although the xanthanolides are potentially attractive anticancer candidates, X. strumarium contains only trace amounts of the xanthanolide sesquiterpene lactones. It has been reported that natural product extracts that contain principally (+)-8-epi-xanthatin and (−)-xanthatin (see Figure 1) are effective growth inhibitors of several human tumor cell lines, with IC50 values ranging from 0.8 to 6.1 µM, and that farnesyltransferase (FTase) is a potential target of (+)-8-epi-xanthatin, exhibiting an IC50 value of 64 µM.2 FTase catalyzes post-translational prenylation reactions that are required for the oncogenic properties of certain GDP/GTP-binding GTPases, including Ras and Rho. 3,4 Although there is an apparent inconsistency in the reported IC50 values for (+)-8-epi-xanthatin-mediated inhibition of FTase activity versus its effect on cancer cell growth rates, FTase inhibition may be a mechanistic target of this agent’s growth suppression. It is noteworthy that (+)-8-epi-xanthatin has never been evaluated for its effects in human breast cancer-derived cells. Nor is there detailed experimental information investigating the effects of pure, chemically synthesized derivatives of (+)-8-epi-xanthatin or (−)-xanthatin on any human cancer cell lines, as previous studies have relied upon the use of cruder biological extracts. Figure 1 Chemical structures of six synthesized xanthanolide sesquiterpene lactones and β-caryophyllene and IC50 values for MDA-MB-231 cell growth inhibition. The exo-methylene lactone moiety, indicated with a gray inclusion, but not the dienone moiety ... The molecular mechanisms of breast cancer progression and metastasis are not fully understood.5−13 Among experimental human breast cancer cell lines, MDA-MB-231 cells have been established as a valuable model for preclinical studies as they are highly aggressive, both in vitro and in vivo. 7,14 Further, MDA-MB-231 cells have been characterized as farnesyltransferase-inhibitor (FTI) “resistant” cells.15,16 However, there is no information regarding the sensitivity of these cells to xanthanolide sesquiterpene lactone-mediated growth inhibition, such as for (+)-8-epi-xanthatin and (−)-xanthatin. Another increasingly recognized target in cancer biology is the growth arrest and DNA damage-inducible gene 45 (GADD45) pathway, inducible by a variety of DNA damaging agents, such as UV irradiation and methylmethane sulfonate.17 While GADD45 was initially identified as a gene rapidly induced by stimuli and assigned a role as a stress sensor,17 more recent attention has focused on GADD45 as a likely tumor suppressor gene that is inactivated in multiple tumor types.18 GADD45 stimulation results in the activation of mitogen-activated protein kinase (MAPK) signaling, including the p38 MAPK and c-Jun N-terminal kinase (JNK) pathways.19–21 In these respects, it is note-worthy that compounds containing a sesquiterpene lactone moiety can modulate MAPK pathways through the induction of cellular stress,1 although the details of this mechanism are not well established. To date, at least three major MAPK pathways, extracellular signal-regulated kinase 1 and 2 (ERK1/2), JNK, and p38, are recognized as physiologically important,19,21 undergoing activation by various stimuli, including UV irradiation, oxidation, and treatments with anticancer chemicals.21,22 The JNK and p38 signaling pathways appear particularly responsive to stress stimuli.19,20 Although an interplay in the apoptotic process has been established between the induction of GADD45 and the activation of stress stimuli-mediated p38/JNK signaling pathways,19 GADD45 pathways themselves have not been investigated as potential mediators of the cancer cell growth inhibitory effects exhibited by the xanthanolide sesquiterpene lactones. In the current study, we chemically synthesized six structurally related xanthanolides-containing exo-methylene lactones, including (−)-xanthatin and (+)-8-epi-xanthatin, and examined their activities on MDA-MB-231 cancer cells. The results revealed that (i) (−)-xanthatin possesses the most efficacious antiproliferation potential, demonstrated by the induction of caspase-independent death in MDA-MB-231 cells, and that (ii) the effect of (−) -xanthatin appears independent of FTase inhibition. Further, our results show that among the GADD45 isoforms, GADD45γ is selectively induced by (−) -xanthatin through cellular stress pathways including oxidative stress and that GADD45γ-primed JNK and p38 signaling pathways are, at least in part, involved in mediating (−)-xanthatin’s growth inhibition and potential anticancer activities.