br Increases in GSH and MT require
Increases in GSH and MT require adequate maintenance of in-tracellular cysteine levels. The intimate connection between GSH/MT and intracellular cysteine is highlighted by the ability of extracellular treatment of cisplatin-resistant ovarian cancer 6-Hydroxydopamine with NAC. Treatment of OVcisR and SKVcisR with 3 mM NAC treatments increased both GSH and nuclear MT (Fig. S6 and S7, Supporting information). These results strongly suggest that regulation of sulfur metabolism in cisplatin-resistant ovarian cancer cells by CBS consists of two processes: flux through the transsulfuration pathway and uptake of extracellular cystine. Previous studies have shown that some cancer cell types maintain intracellular cysteine levels by importing cystine via the glu-tamate/cystine antiporter, xCT [32,33]. Elevated expression and ac-tivity of xCT in response to oxidative stress has been reported in breast cancer cells, alluding to a role for xCT toward protecting cancer cells
Fig. 8. Carbon monoxide (CO) inhibits CBS activity in cisplatin-resistant ovarian cancer cell lines OVcisR and SKVcisR. (a) CBS activity as measured by intracellular cystathionine (CTH). Levels of (b) cysteine, (d) γ-Glu-Cys and (f) GSH as measured by HPLC-MS in cisplatin-resistant ovarian cancer cell lines (normalized to total μg protein). Cells treated with either 30 μM photoCORM or 30 μM of inactivated photoCORM (iCORM) for 24 h. Data presented as averages ± SEM of n = 3 in-dependent experiments. For GSH, data presented as average pmol GSH/μg protein ± SEM of n = 3 independent experiments (*p < 0.05). (c) Effect of 30 μM photoCORM treatment on D4-cystine (D4-CC) uptake in OVcisR and SKVcisR. Cells grown for 48 h in media supplemented with D4-CC. Data presented as average fold difference from “iCORM” control ± SEM of n = 3 independent experiments. (e) Immunoblot of MT expression in 15 μg nuclear enriched fractions of cisplatin-resistant cell lines. Cell treated with 30 μM photoCORM or respective iCORM controls for 24 h prior to nuclear fraction isolation.
against oxidative stress induced loss of cell viability (a mechanism often exploited by chemotherapeutic drugs) . Overexpression of xCT has been shown to increase intracellular GSH and increase resistance to cisplatin . In addition, loss of xCT from cancer cells resulted in suppressed tumor growth of gastric cancer in pre-clinical models . In cisplatin-resistant ovarian cancer cells, we observed increased xCT activity, but not increased expression per se. We have provided strong evidence for the regulation of xCT activity by CBS via H2S, as lentiviral-mediated silencing of CBS in OVcisR(shCBS) and SKVcisR(shCBS) re-duced the activity of xCT, which was partially reversed by the addition of exogenous H2S, known to allosterically increase xCT activity (Fig. 6b) . To the best of our knowledge, this is the first report that demonstrated that CBS can upregulate GSH synthesis independent of
the transsulfuration pathway in cancer cells by inducing the upregu-lation of cystine uptake in cancer cells. This study further emphasized that CO mediated inhibition of CBS could exploit this mechanism to compromise the anti-oxidant potential of cancer cells resulting in their increased sensitivity to cisplatin.
The importance of overexpression of CBS in imparting cisplatin resistance was observed in SKV cells, which despite being sensitive to cisplatin, express detectable levels of CBS and CGL. This is in contrast to OV cells, where the expression of CBS and CGL is nearly undetectable. Interestingly, regression analysis of the effects of cisplatin on ovarian cancer cell lines in this study revealed that the ED50 of cisplatin for SKV cells is higher than that of OV cells (5.5 μM versus 2.3 μM, respectively, data not shown). The lower sensitivity of SKV cells to cisplatin versus
B. Kawahara et al.
OV (Fig. 1a) could possibly be attributed to the higher protein ex-pression of CBS and CGL in the former (Fig. 2a).
Finally, it is important to note that overexpression of CBS is ob-served in select malignancies, namely breast and ovarian cancers while the corresponding normal tissues in all such cases exhibit very low le-vels of expression or none at all . For this reason, CBS could be an important target in case of these two types of cancer where modulation of CBS activity by exogenous CO could thwart resistance to conven-tional chemotherapy. The effect of CO on non-transformed ovarian tissues in the context of resistance to cisplatin is intriguing, as the effect (s) of CO in non-cancerous cells are likely independent of CBS. Eluci-dation of other binding partners of CO in non-transformed ovarian cell lines could be an interesting follow-up study.