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  • Gene silencing experiments br Stable silencing of CBS

    2020-08-28

    2.9. Gene silencing experiments
    Stable silencing of CBS expression in OVcisR and SKVcisR was achieved utilizing short hairpin RNA (shRNA) packaged in lentiviral particles purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cells were transfected with shRNA complimentary to CBS mRNA (sc-60335-V) and selected for in the presence of 1 μg/mL puromycin to generate CBS-silenced cell lines OVcisR(shCBS) and SKVcisR(shCBS). Cells were transfected with scrambled shRNA (sc-108080) and selected for in the presence of 1 μg/mL puromycin to generate transfection control cell lines OVcisR(scram) and SKVcisR(scram).
    2.10. Statistical analysis
    Data are presented as means ± SEM of n = 3 independent experi-ments. Statistical comparison between two groups was performed using a two-tailed, paired Student's t-test, while comparisons between more than two groups was performed using ANOVA with post hoc Tukey's test. p values < 0.05 were considered statistically significant (*p < 0.05). Regression analysis and IC50/ED50 estimations were determined using GraphPad Prism purchased from GraphPad Software (La Jolla, CA).
    3. Results cancer 68181-17-9 to chemotherapeutics [7,9,10], though cisplatin resistance
    3.1. Cisplatin-resistant ovarian cancer cell lines were re-sensitized to and ovarian cancer had yet to be addressed. In this study, we wanted to
    assess the ability of CO, delivered by 30 μM photoCORM, to sensitize
    cisplatin upon co-treatment with CO established cisplatin-resistant ovarian cancer cells. 30 μM of photo-
    Previous studies have demonstrated the ability of CO to sensitize CORM was used in this study because CO concentrations higher than
    that delivered from > 30 μM photoCORM, were cytotoxic to the cells
    Fig. 1. Cisplatin-resistant ovarian cancer cell lines are sensitized to cisplatin upon co-treatment with carbon monoxide (CO), delivered from a photo-activated CO-releasing molecule (photoCORM). (a) Dose-dependency of cisplatin on cell viability in cisplatin-sensitive and resistant cell lines. Cells were treated with 0–40 μM cisplatin and/or DMSO vehicle control. Cell viability was then measured 24 h post-treatment via Trypan blue exclusion. Data are presented as average % of 0 μM cisplatin ± SEM of n = 3 independent experiments. (*p < 0.05). (b) Cell viability 24 h post-treatment, as measured by trypan blue exclusion, of cisplatin-resistant ovarian cancer cell lines, assessing the ability of CO to sensitize cisplatin-resistant ovarian cancer cell lines cells to cisplatin. iCORM, photo-inactivated photoCORM, was used as control for the non-CO scaffolding of the photoCORM. In lieu of cisplatin treatment, cells were treated with DMSO as vehicle control. Data are presented as average percentages of “Control” ± SEM of n = 3 independent experiments. (*p < 0.05). (c) Immunoblot of 20 μg whole cell lysate for cleaved PARP-1 in cisplatin-resistant ovarian cancer cell lines treated with CO and/or cisplatin. GAPDH was probed to assess equal protein loading. Blots presented are representative blots of n = 3 independent experiments.
    B. Kawahara et al.
    used in this study (Supporting information, Fig. S1). Cisplatin-resistant versions of ovarian cancer cell lines OVCAR-5 and SKOV-3 (abbreviated OVcisR and SKVcisR respectively) were assessed for their resistance to therapeutically relevant concentrations of cisplatin compared to their respective parent cancer cell lines, OVCAR-5 and SKOV-3 (abbreviated OV and SKV respectively). Dose-response experiments revealed that OVcisR, and SKVcisR exhibited significant cisplatin resistance, with > 2-fold increased ED50 values for cisplatin compared with OV and SKV respectively (Fig. 1a). Because CO has been shown to sensitize certain cancer cells to chemotherapeutics, we wanted to assess whether CO, delivered from a photoCORM, could work similarly in an ovarian cancer model, attenuating drug resistance for platinum-based che-motherapies in ovarian cancer cell lines. To determine this, we treated cisplatin-resistant cell lines, OVcisR and SKVcisR, with 30 μM photo-CORM and 20 μM cisplatin and compared these cell viability to cells treated with cisplatin alone. We observed that CO significantly en-hanced the ability of cisplatin to reduce cell viability of cisplatin-re-sistant cell lines compared with cisplatin treatment alone (Fig. 1b). OVcisR and SKVcisR cells treated with cisplatin alone exhibited ~40% and ~29% decreases in cell viability, which was enhanced > 2-fold in both cell lines by CO, delivered from 30 μM photoCORM (Fig. 1b). Light-inactivated photoCORM (iCORM) neither significantly alter cell viability itself nor enhanced the cytotoxicity of cisplatin toward OVcisR and SKVcisR (Fig. 1b), demonstrating the negligible effect of the mo-lecular scaffolding of the photoCORM toward increasing cisplatin sen-sitivity in OVcisR and SKVcisR. Lower doses of photoCORM also sen-sitized cisplatin-resistant cells to cisplatin in a concentration-dependent manner (Supporting information, Fig. S2). To determine whether the actions of CO and cisplatin together were apoptotic in nature, we as-sessed PARP-1 cleavage. Indeed, the sensitization of OVcisR and SKVcisR to cisplatin by co-treatment with 30 μM CO corresponded to increased PARP-1 cleavage in whole cell lysates, indicating an apoptotic process (Fig. 1c). Interestingly, we found 24 h treatment with that co-addition of 3 mM N-acetylcysteine (NAC), an antioxidant and efficient donor of cysteine, for 24 h was able to largely reverse CO's ability to re-sensitize of OVcisR and SKVcisR to cisplatin (Supporting information, Fig. S3). This finding indicated that NAC, and its effect on OVcisR and SKVcisR, disrupted the cellular processes by which CO sensitizes these cells to cisplatin. Since NAC is an efficient donor of cysteine, we mea-sured steady state levels of intracellular cysteine by HPLC-MS. Indeed, 3 mM NAC treatment for 24 h increased intracellular levels of cysteine in ~4.4-fold in OVcisR and ~3.8-fold in SKVcisR compared with re-spective vehicle controls (Supporting information, Fig. S4). Together, these findings regarding NAC suggested that intracellular levels of cy-steine, a sulfur-containing amino acid, might be mechanistically im-portant for the cisplatin-resistance phenotype in OVcisR and SKVcisR.