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  • Latrunculin A br Exogenous carbon monoxide CO has


    Exogenous carbon monoxide (CO) has recently been shown to de-crease chemotherapeutic resistance and proliferation in various cancer cell types [7–9]. Our group has recently reported that CO increased the sensitivity of human breast cancer Latrunculin A to doxorubicin mediated cell
    Corresponding authors.
    E-mail addresses: [email protected] (S. Sen), [email protected] (P.K. Mascharak).
    Available online 10 November 2018
    B. Kawahara et al.
    Abbreviations MT metallothionein
    CBS cystathionine β-synthase NEM N-ethylmalemide
    OV OVCAR-5
    CGL cystathionine γ-lyase OVcisR cisplatin-resistant OVCAR-5
    CO carbon monoxide PARP poly (ADP-ribose) polymerase
    CTH cystathionine PMSF phenylmethane sulfonyl fluoride
    FBS fetal bovine serum SKV SKOV-3
    GSH glutathione SKVcisR cisplatin-resistant SKOV-3
    death by > 40% via the inhibition of endogenous cystathionine β-syn-thase (CBS) enzymatic activity [10]. CBS is overexpressed selectively in human breast cancer tissues and not in normal human breast tissues making it a potential therapeutic target [11]. Interestingly, CBS is overexpressed in only a few other neoplasms, one of which is ovarian cancer, where CBS has been implicated in resistance to cisplatin [12]. However, the mitigation of chemotherapeutic drug resistance, using a pharmacological inhibitor of CBS has not yet been demonstrated in ovarian cancer cells. This study for the first time assessed the phar-macological inhibition of CBS by a light-induced CO delivery modality to counter chemotherapeutic drug resistance in human ovarian cancer cells. The results underscore the important role of the transsulfuration pathway in the development of chemotherapeutic drug resistance in ovarian cancer.
    The noxious nature of gaseous CO often poses challenging delivery issues in hospital settings. To avoid this problem, a designed metal car-bonyl complex namely [Mn(CO)3(phen)(PTA)]CF3SO3 (phen = 1,10-phenanthroline; PTA = 1,3,5-triza-7-phosphaadamantane; abbreviated “photoCORM” hereafter) has been employed as the exogenous CO source in this study (Scheme 1). This designed manganese carbonyl complex is water-soluble and rapidly releases CO only when exposed to low-power (10 mW/cm2) broadband visible light. This photoactive CO-releasing molecule (photoCORM) has been a convenient source of CO in delivery under controlled conditions [10,13].
    2. Materials and methods
    2.1. Materials and reagents
    Ovarian cancer cell lines OVCAR-5 (OV), cisplatin-resistant OVCAR-5 (OVcisR), SKOV-3 (SKV), and cisplatin-resistant SKOV-3 (SKVcisR) were a gift from Dr. Sivakumar Ramadoss and Dr. Gautam Chaudhuri of the David Geffen School of Medicine at University of California at Los Angeles. Cells were grown in 1× RPMI media supplemented with 10% fetal bovine serum, 100 I.U./mL penicillin and 100 μg/mL streptomycin at 37 °C + 5% CO2. Mycoplasma detection was performed regularly to confirm its absence with MycoAlert™ Mycoplasma Detection Kit (LT07-218) from Lonza (Basel, Switzerland). Cells were passaged no more  than five times after being received. Unless otherwise indicated, cells were allowed to seed overnight prior to treatments, then assayed 24 h post-treatment.
    2.3. CO and cisplatin treatments of cells
    Cells were seeded overnight, 100 × 103/well of 6-well tissue culture dishes or 1 × 106/well of 100 mm tissue culture dishes depending on the experiment being performed, and incubated at 37 °C + 5% CO2 24 h prior to cell culture treatments. Cells treated with CO were exposed to 30 μM photoCORM in the dark. To control for the effects of the non-CO backbone of the CO-releasing molecule, corresponding control cells were treated with 30 μM light-inactivated photoCORM (iCORM) in the dark. Upon addition of photoCORM or iCORM, cells were exposed to visible light for 30 min at room temperature, then returned back to 37 °C + 5% CO2. In experiments involving cisplatin, 20 μM cisplatin or DMSO vehicle control was administered either by itself or together with 30 μM photoCORM or iCORM. In experiments utilizing NAC, 3 mM NAC or vehicle control were added to cells along with photoCORM and cisplatin. Subsequent assays were performed 24 h post-treatment.
    2.4. Cell viability (Trypan blue exclusion and MTT) assay
    When assessing cell viability via Trypan blue exclusion method,
    100 × 103 cells/well of a 6-well tissue culture plate were allowed to seed overnight in a 37 °C incubator + 5% CO2. The next day, cells were treated as indicated and allowed to incubate for 24 h. Following treatment, cells were harvested with 1 mL 0.05% Trypsin-EDTA, after which trypsin was neutralized with 1 mL cell culture media
    Scheme 1. Structure of [Mn(CO)3(phen)(PTA)]CF3SO3, the photoCORM used in this study.
    B. Kawahara et al.
    supplemented with 10% FBS. This cell suspension was then visualized and quantified using a Vi-Cell XR cell viability analyzer from Beckman Coulter (Brea, CA). Cell viability was measured 24 h post-treatment and presented as the mean % of control or comparison group ± SEM of three independent experiments.