br Discussion br The results reported above
The results reported above require critical evaluation. In previous studies, investigators have shown that silencing of CBS (by lentiviral transduction) enhances drug sensitivity in cisplatin-resistant ovarian cancer Oxidopamine . Also, CO delivered by tricarbonyldichlororuthenium (II) dimer, has been shown to promote global methylation in U937
Fig. 6. Cystathionine β-synthase (CBS) expression in cisplatin-resistant ovarian cancer cells correlates to the bioavailability of cysteine. (a) Steady state levels of intracellular cysteine, as measured by HPLC-MS, normalized to μg protein. Data presented as averages ± SEM of n = 3 independent experiments. (b) Relative D4-cystine (D4-CC) uptake from extracellular media in CBS-silenced cells. Cells treated with either 40 μM GYY or DMSO vehicle control, then in-cubated for 48 h. Data are presented as average fold difference from scrambled controls ± SEM of n = 3 independent experiments. (*p < 0.05). (c) Expression of xCT in 20 μg whole cell lysates of CBS-silenced ovarian cancer cells versus respective scrambled controls. GAPDH was used as a loading con-trol. Blot representative of n = 3 independent experiments.
B. Kawahara et al.
Fig. 7. Cisplatin resistance and cystathionine β-synthase (CBS) expression correlate to glutathione (GSH) biosynthesis and expression of nuclear me-tallothionein (MT) in cisplatin-resistant ovarian cancer cells. (a-b) Steady state levels of γ-Glu-Cys and GSH, normalized to total μg protein, in CBS-silenced cell lines (shCBS) compared with respective scrambled controls (scram), measured by HPLC-MS. Data presented as average pmol of metabolite per total μg protein ± SEM of n = 3 independent experiments. (c) Immunoblot of MT in 15 μg nuclear extracts of CBS-silenced cells (shCBS) versus transduction con-trols (scram). Lamin A is used as a nuclear fraction loading control. Blots re-presentative of n = 3 independent experiments. Journal of Inorganic Biochemistry 191 (2019) 29–39
human monoblastic leukemia cell, presumably by CO-mediated in-hibition of CBS . However, direct demonstration of a viable phar-macological inhibition of CBS to alleviate drug resistance in ovarian cancer has not been achieved. The present study for the first time provides evidence that CO, delivered from a photoCORM, inhibits CBS and such delivery could be a viable option to circumvent chemo-re-sistance in ovarian cancer. Specific small molecule inhibitors of CBS, such as benserazide, have shown promise in eradicating cancer cells . However, if further developed, these could only be used as a systemic therapy that will not spare normal cells. Such systemic therapy will result in serious adverse side effects, as CBS in the liver is the primary source of GSH, the major antioxidant in physiology . On the other hand, light-activated CO delivery via appropriate catheters, prototypes for which have already been developed in our laboratory , could be used to inhibit CBS locally in accessible cancer tissues. This would bypass systemic delivery and increase the exposure of af-fected tissues to effective concentrations of the inhibitor and at the same time minimize adverse side effects. In fact the feasibility of CO-releasing molecules for cancer treatment has recently been reviewed .
Platinum-containing anti-cancer drugs react readily with the sulfur-containing amino acids in proteins namely methionine and cysteine. Unlike methionine, whose reaction with platinum is readily reversible by replacement with thiols or nucleotide bases, the cysteine‑platinum complex is more stable . Therefore, cysteine-rich proteins such as GSH and MT, both of which are present at high concentrations in the cancer cell, readily bind and inactivate platinum drugs. Indeed, for-mation of a cisplatin-(GS)2 complex has been characterized (by NMR spectroscopy and HPLC-atomic absorption spectroscopy) in vitro and in vivo (in murine L1210 cells) in addition to efflux of the complex across the cell membrane . Along the same line, formation of a ternary complex between MT and Platinum-DNA adduct followed by release of platinum from the DNA (and formation of [(NH3)2Pt(S2-MT)] species) has been suggested to modulate DNA-repair and gene transcription leading to drug resistance . Immunohistochemical MT expression in various human tumours has been associated either with processes re-lated to carcinogenesis or with resistance against radiation and che-motherapy . In ovarian tumours an increasing percentage of MT expression has been observed during the progression of malignancy . Results shown in Fig. 8 now clearly indicate that CO-mediated CBS inhibition leads to the reduction of both GSH and MT (implicated in cisplatin inactivation) in ovarian cancer cells. Our observations are supported by earlier findings that CBS positively regulates GSH levels in breast cancer cells . The fact that exogenous CO could interfere with MT expression as well in refractory ovarian cancer cells, is in itself a novel finding in this study. The data that CBS silencing could lower levels of nuclear MT underlines the important role of CBS in regulating two major thiol moieties (GSH and MT) implicated in chemotherapeutic drug resistance [28–31]. Nuclear MT expression is induced by cisplatin and seems to protect DNA in cells from toxic effects of the drug. The proportion of the individual contributions of GSH and MT in in-activating cisplatin is however not explored in this work.