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 were significantly elevated in antihormone-resistant MCF-7:2A breast cancer cells compared to wild-type MCF-7 cells and that the combination treatment of BSO and estradiol caused a dramatic increase in apoptosis whereas the individual treatments had no effect on growth. Noteworthy, the killing effect of BSO and estradiol occurred at clinically achievable concentrations and was observed as early as 48 hours. These findings are consistent with previous studies which have shown that the cytotoxicity of a number of chemotherapeutic drugs, including melphalan [22
], doxorubicin [23
], and bleomycin [24
], are significantly enhanced when glutathione is depleted by BSO.
Our laboratory has previously demonstrated that when estrogen receptor positive breast cancer cells are grown and maintained in long term estrogen deprived (LTED) environments, they can ultimately develop enhanced responsiveness to greatly diminished levels of estrogen [7
]. These pre-clinical animal models show that initially, estrogen receptor expressing tumors are stimulated by estrogen and respond appropriately to tamoxifen with tumor regression. However, with continued exposure to tamoxifen, the tumors become resistant and re-grow [9
]. Additionally, treatment of these LTED tumors with post-menopausal levels of estrogen inhibits tumor growth as well as causes regression of established tamoxifen resistant tumors [7
] (). Mechanistic studies indicate that the apoptotic action of estrogen is due to its ability to either activate the fasR/FasL death receptor pathway [11
] or to disrupt mitochondrial function through activation of the bcl-2 family proteins [7
]. The paradoxical action of estrogen in these resistant cells is hypothesized to be due to increased sensitivity to estrogen due to adaptation to estrogen deprivation caused either by tamoxifen or an aromatase inhibitor [26
]. It is believed that this “estrogen hypersensitivity” helps to explain the effectiveness of high-dose estrogen in patients with extensive prior endocrine therapy [14
Interestingly, our present findings indicate that the ability of estradiol to induce apoptosis in antihormone resistant cells is influenced by the level of glutathione present in the cells. Glutathione levels were elevated ~ 1.4- to 1.6-fold in antihormone-resistant MCF-7:2A cells compared to wild-type MCF-7 cells and these cells failed to undergo apoptosis following 1 week of treatment with physiological concentrations of estradiol alone. In the presence of BSO, however, which depleted intracellular glutathione by ~60-70%, the combination treatment of BSO and estradiol caused a dramatic increase in apoptosis which was observed as early as 48 hours with maximum induction observed at day 7. Previous studies have shown that glutathione is an important component of tumor drug resistance [21
] and that depletion of intracellular glutathione by BSO significantly enhances the cytotoxicity of many cytotoxic agents, principally alkylating agents [15
] and platinating compounds [16
] but also irradiation [28
] and anthracyclines [29
]. The concentration of BSO used in our study was within the range of 10 μM to 1 mM, which is similar to what has previously been reported in the literature. However, we did observe some toxicity at higher concentrations of BSO (> 1 mM) in wild-type MCF-7 and antihormone-resistant MCF-7:2A cells (). It should be noted that BSO, at a clinically achievable concentration of 100 μM, was used for all of our combination experiments with estradiol since this concentration, as an individual treatment, did not significantly alter the growth of MCF-7:2A cells.
Glutathione, a sulfhydryl containing tripeptide, is involved in detoxifying cells from various toxins including chemotherapeutic agents [30
]. Several lines of evidence have shown that in tissue culture studies of cancer cell line made resistant to selected chemotherapy agents, glutathione levels correlated with increasing chemotherapy resistance [32
]. This resistance was not limited to the particular chemotherapy agent used to induce resistance, but was also evident when other chemotherapeutic agents were tested for cross-resistance [32
]. Additionally, translational studies of in vitro
cell lines derived from patients with chemorefractory disease were found to have elevated glutathione levels [33
]. BSO inhibits γ-glutamylcysteine synthetase (γ-GCS), the rate limiting enzyme in the production of glutathione, thus depleting glutathione levels within the cell [34
]. Both, GSH as well as resultant increase in γ-GCS levels as a result of BSO treatment can be monitored peripherally in patients by analysis of peripheral mononuclear cells (PMNs) [35
]. BSO also exhibits selectivity in that in vitro
studies have demonstrated greater depletion of glutathione levels in tumor tissues than sampled normal tissues [30
]. Based on its ability to target intracellular glutathione and reverse therapeutic resistance in refractory cancers, BSO is thought to be a potential antineoplastic agent and/or “therapeutic sensitizer” worthy of clinical evaluation.
Early phase clinical trials of BSO at doses resulting in both peripheral and tumor GSH depletion show that BSO can be safely administered to patients with refractory disease. BSO was administered intravenously twice daily either alone or together with chemotherapy to cancer patients whose disease who disease had progressed despite multiple lines of previous chemotherapy [35
]. In these patients treated with escalating doses of BSO, nausea and vomiting amenable to anti-emetic therapy were the main toxicities. Bone marrow suppression correlating with extent of previous chemotherapy exposure was found to be the rate limiting toxicity in the combination studies. No other significant toxicities were noted. Intracellular glutathione levels measured in PMNs decreased in a linear manner with repeated doses of BSO to a maximum of approximately 10-40% of baseline values [35
]. When tested in sequential tumor biopsies, glutathione was also found to be depleted to a variable extent in a similarly predictable pattern [36
]. Additionally, BSO administration resulted in an initial rapid inhibition of γ–GCS activity followed by γ–GCS recovery during the intervening time between dosings. In fact, γ–GCS levels mirrored peripheral BSO concentrations in patients thus demonstrating targeted delivery of BSO. Clinically, responses to treatment, including complete responses, have been achieved [27
In this present study, we demonstrated that glutathione depletion by BSO sensitized antihormone-resistant MCF-7:2A human breast cancer cells to estradiol-induced apoptosis in vitro. Taken together, it would be reasonable to incorporate this data into our working translational model for clinical evaluation (). We therefore propose utilizing BSO together with estrogen in patients for a defined therapeutic course in patients with hormonally sensitive metastatic breast cancer whose disease has progressed on prior antihormonal therapies to significantly reduce their disease burden, while potentially reversing resistance to antihormonal therapies. This would then be followed by continuing treatment with an aromatase inhibitor for maintenance of additional clinical benefit for these patients (). Our future goal will be to address this hypothesis in the context of a clinical trial based on these new pre-clinical findings.
Figure 6 Clinical protocol to investigate the efficacy of estradiol plus BSO combination treatment to induce apoptosis in long-term endocrine refractory breast cancer. An anticipated treatment plan for third-line endocrine therapy. Patients must have responded (more ...)