In the current study, we investigated whether suppression of the antioxidant glutathione by BSO has the ability to sensitize antihormone resistant MCF-7:2A breast cancer cells to estradiol-induced apoptosis. Our results showed that glutathione levels and the enzymes involved in its synthesis, glutathione synthetase and glutathione peroxidase, were significantly elevated in MCF-7:2A cells compared to parental MCF-7 cells and that suppression of glutathione by BSO sensitized these cells to estrogen-induced apoptosis in vitro
and in vivo
. The BSO-mediated estradiol-induced apoptosis was associated with a marked decrease in the expression of antiapoptotic Bcl-2 and Bcl-xL proteins and a significant increase in proapoptotic Bax protein. It is worth noting that high-dose estrogen was generally considered the endocrine therapy of choice for postmenopausal women with breast cancer prior to the introduction of tamoxifen, however, due to undesirable side effects, the use of high-dose estrogen was largely abandoned [29
]. Here, we show that the killing effect of estradiol in antihormone resistant cells can be achieved at physiological concentrations when it is combined with non-toxic concentrations of BSO. Our present findings are consistent with previous studies which have shown that the cytotoxicity of a number of chemotherapeutic drugs, including melphalan [30
], doxorubicin [31
], and bleomycin [32
], are significantly enhanced when glutathione is depleted by BSO.
An important target of BSO plus estradiol-induced apoptosis appears to be Bcl-2, whose protein expression was dramatically decreased in MCF-7:2A cells following glutathione depletion. Previous studies have shown that Bcl-2 functions as an antioxidant to block apoptosis and that Bcl-2 protein levels and glutathione intracellular concentration is coordinately regulated with a decrease in either favoring cell death [23
]. It is believed that one mechanism by which Bcl-2 may function as an antioxidant is through upregulation of glutathione, leading to rapid detoxification of reactive oxygen species and inhibition of free radical-mediated mitochondrial damage. Bcl-2 also has the ability to shift the entire cellular redox potential to a more reduced state, which is independent of its effect on glutathione levels [33
]. It is worth noting that glutathione levels and Bcl-2 protein expression were significantly elevated in MCF-7:2A cells compared to parental MCF-7 cells. In phase I trials [34
], the concentration of BSO in blood has been shown to reach 0.5 to 1 mM, whereas, in mice [36
] the concentration has been estimated to be 5 to 6 mM following an in vivo
treatment of 4 mmol/kg. In our study, we showed that 100 μM BSO decreased glutathione concentrations by approximately 60% after 24 h and that BSO enhanced the apoptotic effect of estradiol in MCF-7:2A breast cancer cells as early as 48 h after treatment. Interestingly, treatment with BSO alone did not cause apoptosis in MCF-7:2A cells, indicating that glutathione depletion alone may not trigger apoptosis in these cells. This finding is consistent with previous studies by Mirkovic et al
] which showed that inhibition of glutathione by BSO did not increase susceptibility of mouse lymphoma cells to radiation-induced apoptosis even under conditions where glutathione levels were lowered by 50%. Other groups have made similar observations using BSO [39
]. One possible explanation for this apparent contradiction might be the fact that BSO does not lower glutathione levels in mitochondria as effectively as it does in the cytoplasm [40
]. Mitochondrial glutathione concentrations are regulated and have been implicated in apoptotic cell death [41
], hence, it would be of interest to evaluate relative glutathione concentrations in the mitochondrial matrix of MCF-7:2A cells following treatment with BSO either alone or in combination with estradiol. Another possibility could be that cellular thiols other than glutathione may play important roles in regulating apoptosis [39
]. The flavoprotein thioredoxin has been shown to be upregulated in several human tumors and is implicated in both cancer cell growth and apoptotic resistance [42
]. However, it is not known whether Bcl-2 or other apoptotic regulators can influence the levels of thioredoxin or whether such modulation may contribute to resistance in human tumor cells.
Apart from Bcl-2, we also found that proapoptotic Bax protein was markedly increased in MCF-7:2A cells by the combination of BSO plus estradiol and this induction coincided with a loss of mitochondrial membrane integrity and cytochrome c
release. Bax is normally found as a monomer in the cytosol of non-apoptotic cells and it oligomerizes and translocates to the outer mitochondrial membrane in response to apoptotic stimuli and induces mitochondrial membrane permeabilization and cytochrome c
]. In MCF-7:2A cells, Bax protein was induced as early as 24 h after BSO plus estradiol treatment (Figure ) and suppression of Bax expression using siRNA was able to partially reverse the apoptotic effect of the combination treatment (data not shown). The induction of Bax coincided with cytochrome c
release from the mitochondria into the cytosol, which was followed by activation of caspase 7, and PARP cleavage. It is worth noting that pretreatment of cells with the universal caspase inhibitor z-VAD almost completely blocked the apoptotic effect of BSO plus estradiol. It is also worth noting that antiapoptotic Bcl-2 and Bcl-xL proteins were also markedly decreased in MCF-7:2A cells following the combination treatment of BSO plus estradiol (Figure ) and overexpression of Bcl-xL partially blocked the apoptotic effect of BSO plus estradiol (data not shown). This finding is important because there is evidence that suggests that the ratio rather than the amount of antiapoptotic vs proapoptotic proteins determines whether apoptosis will proceed [43
]. Thus, it is reasonable to suggest that the apoptotic effect of BSO plus estradiol is mediated, in part, by the mitochondrial pathway through their ability to alter the ratio between proapoptotic and antiapoptotic proteins in target cells.
In addition to the mitochondrial pathway, BSO plus estradiol appears to induce apoptosis, in part, through activation of the JNK signaling pathway. JNKs are a group of mitogen-activated protein kinases (MAPKs) that bind the N-terminal activation domain of the transcription factor c-Jun and phosphorylate c-Jun on amino acid residues Ser63 and Ser73 [44
]. JNKs are stimulated by multiple factors including cytokines, DNA-damaging agents, and environmental stresses and are important in controlling programmed cell death or apoptosis. The inhibition of JNKs has been shown to enhance chemotherapy-induced inhibition of tumor cell growth, suggesting that JNKs may provide a molecular target for the treatment of cancer [44
]. We found that JNK activation (as measured by the increased levels of phospho-JNK1/2 and the JNK substrate phospho-c-Jun) correlated well with BSO plus estradiol-induced apoptosis in MCF-7:2A cells and pharmacologic disruption of this pathway using the JNK inhibitor SP600125 significantly attenuated this effect. Previously, Chen and coworkers [45
] reported that BSO enhanced the apoptotic effect of arsenic (As2
) in leukemia and lymphoma cells through activation of JNK and upregulation of death receptor (DR)5 and that inhibition of JNK by SP600125 decreased DR5 upregulation and apoptotic induction in U937 leukemia cells treated with arsenic plus BSO. While the exact mechanism by which JNK promotes apoptosis is not currently known, the phosphorylation of transcription factors such as c-Jun and p53, as well as pro- and antiapoptotic Bcl-2 family members [46
] has been suggested to be of importance. It is worth noting that treatment with BSO plus estradiol markedly increased phosphorylated c-Jun in MCF-7:2A cells and decreased phosphorylated Bcl-2 in these cells. These findings thus suggest that BSO plus estradiol might mediate their apoptotic effect, in part, through activation of JNK.