br Cisplatin resistance in ovarian cancer cells was
3.2. Cisplatin resistance in ovarian cancer cells was associated with increased expression and activity of CBS
Cisplatin resistance is attributed to increased drug inactivation by sulfur-containing nucleophilic species GSH and nuclear metallothionein (MT) binding and inactivating the drug [14,15]. Increased generation of GSH and MT places a significant demand for cysteine, as both GSH and MT require the same sulfur-containing amino acid. One of the major sources of cysteine can be from the enzymatic actions of CBS and cystathionine γ-lyase (CGL), transsulfuration pathway enzymes that convert homocysteine to cysteine . Immunoblot analysis of 20 μg whole cell lysates revealed dramatically increased expression of both CBS and CGL, transsulfuration pathway enzymes, in cisplatin-resistant versus the corresponding sensitive cells (Fig. 2a). Additionally, steady state levels of intracellular CTH, the enzymatic product of CBS, was present ~3.1-fold more in OVcisR and ~7.5-fold more in SKVcisR versus cisplatin-sensitive cell lines OV and SKV respectively (Fig. 2b). Journal of Inorganic Biochemistry 191 (2019) 29–39
3.3. Cisplatin-resistant ovarian cancer cell lines exhibited increased uptake of cystine and steady state levels of cysteine versus corresponding cisplatin-sensitive cell lines
In addition to overexpressing CBS, cisplatin-resistant ovarian cancer cell lines OVcisR and SKVcisR also exhibited overexpression of CGL (Fig. 2a), an enzyme downstream of CBS in the transsulfuration pathway. CGL breaks down CTH into the amino GSK J4 cysteine, which, like CTH, was found at higher steady state levels in cisplatin-resistant cell lines, ~2.7-fold more in OVcisR and ~1.4-fold compared to cis-platin-sensitive cells (Fig. 3a). CGL, however, is only one of several processes by which cysteine levels are regulated in the cell. In addition to the generation of cysteine through the transsulfuration pathway, uptake of cystine, an oxidized disulfide of two cysteine molecules, by the glutamate/cystine antiporter (xCT) is known to be an important source of intracellular cysteine for cancer cells, including ovarian cancer . Furthermore, certain cisplatin-resistant ovarian cancer cells are reported to have increased cystine-uptake via xCT . In consideration of this alternate source of cysteine, we measured relative uptake of extracellular deuterium-labeled cystine (D4-CC) by cisplatin-resistant ovarian cancer cells compared to their respective cisplatin-sensitive counterparts. The relative uptake of D4-CC was quantified by measuring intracellular levels of heavy labeled cystine and cysteine
Fig. 2. Cystathionine β-synthase (CBS) expression and activity correlates with cisplatin-resistance in ovarian cancer cells. (a) Immunoblot for CBS and cy-stathionine γ-lyase (CGL) of 20 μg whole cell lysate of ovarian cancer cells, both cisplatin-sensitive and cisplatin-resistant cells. GAPDH expression was used as a loading control. Blots are representative of n = 3 independent experiments. (b) Steady state levels of cystathionine (CTH) in ovarian cancer cell lines, as de-termined by HPLC-MS. Data presented as average pmol CTH per μg protein ± SEM of n = 3 independent experiments. (*p < 0.05).
Fig. 3. Cisplatin-resistant ovarian cancer cell lines, OVcisR and SKVcisR, maintain higher levels of intracellular cysteine compared with cisplatin-sensi-tive cell lines. (a) Steady state levels of cysteine in OVcisR and SKVcisR, cis-platin-resistant ovarian cancer cell lines, compared with OV and SKV, cisplatin-sensitive ovarian cancer cell lines. Data are presented as average pmol cysteine/ μg protein ± SEM of n = 3 independent experiments. (*p < 0.05) (b) Relative uptake of D4-cystine (D4-CC) by OVcisR and SKVcisR compared to OV and SKV respectively. Data expressed as average fold difference from OV and SKV ± SEM of n = 3 independent experiments. (*p < 0.05). (e) Representative immunoblot of three independent experiments, showing the relative expression of xCT in 20 μg whole cell lysate of cisplatin-sensitive and cisplatin-resistant cell lines. GAPDH was used as a loading control.
(D4-CC and D2-cystine respectively). We observed cisplatin-resistant cells, OVcisR and SKVcisR, contained ~2.1 and ~3.4 times more in-tracellular D2-cysteine (the reduced form of D4-CC) than OV and SKV, their respective cisplatin-sensitive cells, indicating a greater capacity to uptake cystine (Fig. 3b). Intracellular D4-CC was not detected at levels above background (data not shown). This is in accordance with pre-vious observations because intracellular cystine is quickly reduced to two cysteine residues, due to the reducing environment of the cytosol . Despite the increased uptake of cystine in cisplatin-resistant cells, xCT protein expression was not remarkably different between the