LY379268 br Fig Glutathione GSH and metallothionein MT
Fig. 4. Glutathione (GSH) and metallothionein (MT) are up regulated in cis-platin-resistant ovarian cancer cell lines. (a–b) Steady state levels of γ-Glu-Cys and GSH as measured by HPLC-MS in cisplatin-resistant cell lines, OVcisR and SKVcisR, and cisplatin-sensitive cell lines, OV and SKV. Data are presented as average pmol metabolite per total μg protein ± SEM of n = 3 independent experiments. (*p < 0.05). (c) Immunoblot of MT in 15 μg nuclear-enriched fractions of ovarian cancer cells. Lamin A was probed for determining equal loading of nuclear fractions. Blot is representative of n = 3 independent ex-periments.
cisplatin-resistant and cisplatin-sensitive LY379268 (Fig. 3c). This suggested that xCT activity per se, and not its expression, was important for the differential uptake of D4-CC.
B. Kawahara et al.
3.4. GSH and MT were found at increased levels in cisplatin-resistant ovarian cancer cell lines compared with cisplatin-sensitive ovarian cancer cell lines
Next, we wanted to determine whether increased cysteine levels observed in cisplatin-resistant cell lines was correlated with increased biosynthesis of GSH and MT, cysteine-containing species that bind and inactivate cisplatin . γ-Glu-Cys, the product of the rate-limiting step in the biosynthesis of GSH, was present at higher steady state levels, ~1.6-fold higher in OVcisR and ~1.4-fold higher in SKVcisR, versus OV and SKV respectively (Fig. 4a). Further connecting the dependence of γ-Glu-Cys with intracellular cysteine, 3 mM NAC significantly increased steady state levels of γ-Glu-Cys: > 2-fold in both OVcisR and SKVcisR (Supporting information, Fig. S5). Such increased levels of γ-Glu-Cys in OVcisR and SKVcisR were supported by concomitant increases in the steady-state levels of GSH. Intracellular levels of GSH were ~2.4-fold higher in OVcisR and ~1.4-fold higher in SKVcisR when compared with OV and SKV, their respective, cisplatin-sensitive, parent cell lines (Fig. 4b). NAC-treated OVcisR and SKVcisR cells both exhibited ~3.4-fold and ~2.4-fold higher steady state levels of GSH compared with their respective, vehicle treated controls (Supporting information, Fig. S6), demonstrating intracellular cysteine levels can influence GSH le-vels in cisplatin-resistant ovarian cancer cells.
In addition to GSH, MT, a cysteine-rich protein, is known to bind and inactivate cisplatin specifically when localized in the nucleus [14,15]. Qualitative measurement of nuclear MT indicated that OVcisR and SKVcisR cells exhibited considerably increased expression of nu-clear MT when compared with OV and SKV cells (Fig. 4c). Addition of 3 mM NAC, a donor of cysteine, also resulted in modest increased nu-clear MT expression as determined by Western analysis (Supporting information, Fig. S7). Together, these findings demonstrate the im-portance of elevated, intracellular cysteine levels toward maintaining
higher levels of GSH and nuclear MT, thiols known to bind and in-activate cisplatin.
3.5. Stable silencing of CBS expression in cisplatin-resistant ovarian cancer cell lines sensitized those cells to cisplatin
We next wanted to assess if and, if so, how CBS expression/activity was imparting cisplatin resistance. Toward this end, we prepared stable, lentiviral-mediated CBS-silenced cisplatin-resistant ovarian cancer cell lines [OVcisR(shCBS) and SKVcisR(shCBS)]. Western ana-lysis on whole cell lysates of OVcisR(shCBS) and SKVcisR(shCBS) re-vealed decreased CBS protein expression in those lysates compared to control shRNA-transfected cells OVcisR(scram) and SKVcisR(scram) respectively (Fig. 5a). Reduced expression of CBS also resulted in re-duced bioactivity, as measured by its enzymatic product CTH. Steady state levels of CTH in OVcisR(shCBS) were ~51% lower compared to transfection control OVcisR(scram) (Fig. 5b, left). Similarly, SKVcisR (shCBS) exhibited ~31% lower levels of intracellular CTH when com-pared to SKVcisR(scram) (Fig. 5b, right). To determine whether CBS over-expression in cisplatin-resistant cell lines was at least in part mediating cisplatin resistance, we measured the effects of cisplatin on cell viability in CBS-silenced cells versus scrambled controls. OVcisR (shCBS) and SKVcisR(shCBS), over a range of concentrations of cis-platin, exhibited significantly reduced cell viability compared to OV-cisR(scram) and SKVcisR(shCBS) respectively (Fig. 5c). Calculated ED50 values for cisplatin in OVcisR(shCBS) an SKVcisR(shCBS) were ~2.5 μM and ~3.6 μM respectively. These values were lower than the ED50 values for OVcisR(scram) and SKVcisR(scram), ~11 μM and ~30 μM respectively, reflecting increased sensitivity to cisplatin due to silencing CBS (Fig. 5c). r>