(–)-Xanthatin induces the prolonged expression of c-Fos through an <i>N</i>-acetyl-L-cysteine (NAC)-sensitive mechanism in human breast cancer MDA-MB-231 cells

Abstract

-We reported that (-)-xanthatin, a xanthanolide sesquiterpene lactone present in the Cocklebur plant, exhibited potent anti-proliferative effects on human breast cancer cells, in which GADD45γ, a novel tumor suppressor gene, was induced. Mechanistically, topoisomerase IIα (Topo IIα) inhibition by (-)-xanthatin was shown to be the upstream trigger that stimulated the expression of GADD45γ mRNA and concomitantly produced reactive oxygen species (ROS) to maintain this expression. Since the anticancer drug etoposide, a selective Topo IIα inhibitor, has also been shown to induce intracellular ROS, (-)-xanthatin may exert its anti-proliferative effects on cancer cells in a similar manner to those of etoposide. In the present study, to generalize its applicability to cancer therapy, we further investigated the biological activities of (-)-xanthatin by comparing its activities to those of the established anti-cancer drug etoposide. After the exposure of breast cancer cells to (-)-xanthatin or etoposide, a prolonged and marked up-regulation in the expression of c-fos, a proapoptotic molecule, was detected together with GADD45γ; and the expression of these molecules was stabilized by ROS and abrogated by the pretreatment with N-acetyl-L-cysteine (NAC), a potent ROS scavenger. (-)-Xanthatin in particular exhibited stronger antiproliferative potential than that of etoposide, which underlies the marked induction of c-fos/GADD45γ and ROS production.

(–)-Xanthatin-mediated marked up-regulation of RhoB, a sensor for damaged DNA

“…β-Actin was used as a housekeeping gene for RT-PCR. (B) A working model of (-)-xanthatin-produced cell death signaling was described in combination with previous findings (Takeda et al, 2011(Takeda et al, , 2013a(Takeda et al, , 2013b. As shown in the Figure, (-)-xanthatin inhibits the catalytic activity of Topo IIα, which is coupled with the concomitant production of ROS; GADD45γ, which is up-regulated by DNA damage, may be an execution factor in cell death.…”

“…The results obtained showed that among members of the Rho family comprising RhoA, RhoB, and RhoC (Wheeler and Ridley, 2004), (-)-xanthatin strongly up-regulated the expression of the RhoB isoform in a NAC-sensitive manner. We summarized the anti-proliferative effects of (-)-xanthatin in combination with our previous findings (Takeda et al, 2011(Takeda et al, , 2013a(Takeda et al, , 2013b) (See Fig. 3B).…”

“…Although we and others (using extracts) demonstrated the anti-proliferative effects of (-)-xanthatin on human MDA-MB-231 breast cancer cells (Ramírez-Erosa et al, 2007;Takeda et al, 2011Takeda et al, , 2013aTakeda et al, , 2013b, the molecular mechanisms underlying (-)-xanthatin-induced anti-proliferative activity have not yet been elucidated in detail. We recently obtained evidence to show that i) (-)-xanthatin selectively induces growth arrest and the DNA damage-inducible gene 45 (GADD45) γ isoform, known as a stress sensor (Fornace et al, 1989), and ii) (-)-xanthatin inhibits DNA topoisomerase IIα (Topo IIα); inhibition of the Topo IIα enzyme, (possibly) accompanied by DNA damage, leads to deficient DNA replication (Mamouni et al, 2014).…”

“…The results of GADD45α/β/γ in the Figure were taken from a previous study (Takeda et al, 2011) (Takeda et al, 2011(Takeda et al, , 2013a(Takeda et al, , 2013b. These findings have been implicated in the (-)-xanthatin-mediated cell death response observed in MDA-MD-231 breast cancer cells (Takeda et al, 2011(Takeda et al, , 2013a(Takeda et al, , 2013b. However, further experimental evidence is needed for DNA damage primed by (-)-xanthatin.…”

Possible involvement of FosB in (–)-xanthatin-mediated anti-proliferative effects in human cancer MDA-MB-231 cells

“…1A, upper panel). To clarify the involvement of (-)-xanthatin in the extracts (Roussakis et al, 1994;Ramírez-Erosa et al, 2007) and to understand the SL's action mechanism, we have established a method for the complete synthesis of (-)-xanthatin in order to obtain "pure" (-)-xanthatin (Matsuo et al, 2010;Matsumoto et al, 2013) and applied this SL to biochemical analyses focusing on its anti-proliferative effects on the MDA-MB-231 cell line (Takeda et al, 2011(Takeda et al, , 2013a(Takeda et al, , 2013b(Takeda et al, , 2015, a model of basal-like triple negative (i.e., estrogen receptor α, progesterone receptor, and HER2/ ErbB2 negative) highly aggressive breast cancer (Roche-fort et al, 2003): the findings of that study indicated that the inhibition of (-)-xanthatin-mediated DNA topoisomerase IIα (Topo IIα) and production of reactive oxygen species (ROS) are involved in its anti-proliferative effects through the up-regulation of GADD45γ, an tumor suppressor gene (Ying et al, 2005;Zerbini and Liebermann, 2005). Although we and others have demonstrated the anti-proliferative effects of (-)-xanthatin on MDA-MB-231 cells (Ramírez-Erosa et al, 2007;Takeda et al, 2011, 2013a, 2013b: Yu et al, 2015, the molecular mechanisms underlying (-)-xanthatin-induced anti-proliferative activity have not yet been elucidated in detail.…”