Fig Silencing cystathionine synthase CBS protein
Fig. 5. Silencing cystathionine β-synthase (CBS) protein expression attenuates cisplatin resistance in cisplatin-resistant ovarian cancer cell lines OVcisR and SKVcisR.
(a) Immunoblot for CBS of 20 μg whole cell lysate to measure efficacy of lentiviral-mediated gene silencing of CBS. Blots are representative of n = 3 independent experiments. (b) Intracellular cystathionine (CTH), as measured by HPLC-MS, to determine relative CBS enzymatic activity in CBS-silenced cell lines versus scrambled controls. Data presented as average pmol CTH per total μg protein ± SEM of n = 3 independent experiments. (c) Graph depicting the dose-dependency of cisplatin on cell viability 24 h post-treatment in cisplatin-resistant cell lines silenced for CBS (shCBS) versus those treated with scrambled controls (scram). ED50 values calculated from modeling of data shown. Data presented as average percentage of 0 μM cisplatin treatment ± SEM of n = 3 independent experiments. (*p < 0.05).
B. Kawahara et al.
3.6. CBS-silencing in cisplatin-resistant ovarian cancer cell lines decreased steady state levels of intracellular cysteine
Intracellular cysteine levels were ~60% lower in OVcisR(shCBS) and ~77% in SKVcisR(shCBS) compared to their respective scrambled lentiviral controls OVcisR(scram) and SKVcisR(scram) (Fig. 6a). CBS-silenced cell lines exhibited ≥50% reduced D4-CC uptake compared with their respective controls (Fig. 6b) strongly implicating a role for CBS in regulating cystine uptake. The uptake of cystine, as addressed in Results Section 3.3, may be dependent on the activity and/or expres-sion of xCT. Expression of xCT was however not noticeably different compared with respective scrambled controls (Fig. 6c). To partially elucidate the connection between CBS and cystine uptake, we turned our attention to H2S. H2S is a possible enzymatic product of CBS, whereby CBS catalyzes the condensation of homocysteine with cy-steine, rather than serine . It has been shown that H2S can allos-terically upregulate xCT activity and cystine uptake in neurons , though it had not yet been demonstrated in ovarian cancer cells. We therefore wanted to determine if H2S could at least partially restore the attenuated uptake of D4-CC. CBS-silenced Oxidopamine treated with 40 μM GYY 4137, a slow releaser of H2S, exhibited significant yet highly variable increases in D4-CC uptake versus those cells treated with vehicle con-trol: > 600% in OVcisR(shCBS) and > 33% in SKVcisR(shCBS) (Fig. 6b).
3.7. Cystathionine β-synthase (CBS) in cisplatin-resistant ovarian cancer cell lines regulated GSH biosynthesis and expression of nuclear MT In our next step we sought to determine whether the observed re-duction in steady state levels of cysteine, caused by CBS-silencing (Fig. 6a), affected the biosynthesis of GSH. Steady state levels of γ-Glu-Cys, the metabolic precursor to GSH, were significantly lower, ~30% less in OVcisR(shCBS) and ~60% less in SKVcisR(shCBS) versus OVcisR (scram) and SKVcisR(scram) respectively (Fig. 7a). Additionally, we observed steady state levels of GSH in CBS-silenced cell lines, 63 pmol/ μg in OVcisR(shCBS) and 58 pmol/μg SKVcisR(shCBS), were > 50% lower than that observed in scrambled controls, 130 pmol/μg in OVcisR (scram) and 150 pmol/μg in SKVcisR(scram) (Fig. 7b). CBS-silenced cell lines also expressed relatively less nuclear MT compared with scram-bled controls (Fig. 7c).
3.8. CO inhibited CBS, thereby decreasing nuclear metallothionein and GSH
Following confirmation of a substantial connection between CBS and cisplatin-resistance in OVcisR and SKVcisR, we investigated whe-ther CO-induced sensitization of cisplatin-resistant cells to cisplatin (Fig. 1b–c) was in part mediated by inhibition of CBS and the sub-sequent suppression of nuclear MT and GSH. CO, delivered by 30 μM photoCORM, significantly lowered CBS bioactivity, as measured by decreased steady state levels of intracellular CTH. CTH decreased ~2.7-fold in OVcisR and ~4.5-fold in SKVcisR (Fig. 8a). In a similar manner to lentiviral-mediated silencing of CBS (Fig. 6a), CO treatment reduced steady state levels of cysteine > 55% in the both cisplatin-resistant cell lines, OVcisR and SKVcisR (Fig. 8b). Concomitantly, CO reduced D4-CC uptake, ~73% in OVcisR and ~56% in SKVcisR (Fig. 8c). Steady state levels of γ-Glu-Cys, the product of the rate-limiting step of GSH synthesis, was also significantly lower in cisplatin-resistant cells treated with CO, ~63% lower in OVcisR and ~66% lower in SKVcisR, versus respective controls (Fig. 8d). Expression of nuclear MT, the thiol-con-taining peptide that is known to bind and inactivate cisplatin, was also lower in OVcisR and SKVcisR cells following treatment with CO (Fig. 8e). Treatment of cells with CO significantly lowered steady state levels of GSH in cisplatin-resistant cells, by ~40% in OVcisR and 65% SKVcisR (Fig. 8f). Journal of Inorganic Biochemistry 191 (2019) 29–39