On the Question of CO's Ability to Induce HO-1 Expression in Cell Culture: A Comparative Study Using Different CO Sources

Abstract

With the recognition of the endogenous signaling roles and pharmacological functions of carbon monoxide (CO), there is an increasing need to understand CO's mechanism of actions. Along this line, chemical donors have been introduced as CO surrogates for ease of delivery, dosage control, and sometimes the ability to target. Among all of the donors, two ruthenium-carbonyl complexes, CORM-2 and -3, are arguably the most commonly used tools for about 20 years in studying the mechanism of actions of CO. Largely based on data using these two CORMs, there has been a widely accepted inference that the upregulation of heme oxygenase-1 (HO-1) expression is one of the key mechanisms for CO's actions. However, recent years have seen reports of very pronounced chemical reactivities and CO-independent activities of these CORMs. We are interested in examining this question by conducting comparative studies using CO gas, CORM-2/-3, and organic CO donors in RAW264.7, HeLa, and HepG2 cell cultures. CORM-2 and CORM-3 treatment showed significant dose-dependent induction of HO-1 compared to "controls," while incubation for 6 h with 250-500 ppm CO gas did not increase the HO-1 protein expression and mRNA transcription level. A further increase of the CO concentration to 5% did not lead to HO-1 expression either. Additionally, we demonstrate that CORM-2/-3 releases minimal amounts of CO under the experimental conditions. These results indicate that the HO-1 induction effects of CORM-2/-3 are not attributable to CO. We also assessed two organic CO prodrugs, BW-CO-103 and BW-CO-111. BW-CO-111 but not BW-CO-103 dose-dependently increased HO-1 levels in RAW264.7 and HeLa cells. We subsequently studied the mechanism of induction with an Nrf2-luciferase reporter assay, showing that the HO-1 induction activity is likely due to the activation of Nrf2 by the CO donors. Overall, CO alone is unable to induce HO-1 or activate Nrf2 under various conditions in vitro. As such, there is no evidence to support attributing the HO-1 induction effect of the CO donors such as CORM-2/-3 and BW-CO-111 in cell culture to CO. This comparative study demonstrates the critical need to consider possible CO-independent effects of a chemical CO donor before attributing the observed biological effects to CO. It is also important to note that such in vitro results cannot be directly extrapolated to in vivo studies because of the increased level of complexity and the likelihood of secondary and/or synergistic effects in the latter.

Scheme 1. CO Release Chemistry of CORM-2 upon Reacting with DMSO and Water-Gas Shift Reaction of CORM-3 Leading to CO₂ Generation (Inset Shows the Structure of Complex D Used as iCORM-2 in Some Studies)^,,,

Figure 1

CO release yield of CORMs and CO prodrugs in cell culture medium tested with methanizer-FID GC. Complete DMEM culture medium was supplemented with 10% FBS; concentrations: CORM-2 and CORM-3:50 μM; BW-CO-103 and BW-CO-111:10 μM; incubation time: 6 h. CO release yield was calculated using an external calibration standard curve (Figure S1).

Figure 2

Effects of CORM/iCORM-2 and CORM/iCORM-3 on RAW264.7 cells (a, c) and HeLa cells (b, d). (a) Western blot of RAW264.7 cells incubated with CORM/iCORM-2/-3 for 6 h; (b) Western blot of HeLa cells incubated with CORM/iCORM-2/-3 for 6 h. (c, d) Densitometry results of the Western blot for RAW264.7 cells (c) and HeLa cells (d). (Data show the fold changes compared to the vehicle control group after normalization with band optical density of β-Actin, n = 3, ^####P < 0.0001, ^###P < 0.001, ^##P < 0.01, ^#P < 0.05, t-test vs Veh group; ****P < 0.0001, one-way ANOVA vs Veh group.).

Figure 3

Effect of CO gas on RAW264.7, HeLa, and HepG2 cells: (a) RAW264.7 cells incubated with 250–500 ppm CO gas for 6 h; (b) HeLa cells incubated with 250–500 ppm CO for 6 h; (c) RAW264.7 cells incubated with 250 ppm CO for 18 h or 5% CO for 6 h; (d) HeLa cells incubated with 250 ppm CO for 18 h; (e) HepG2 cells incubated with CO gas dissolved in MEM culture medium (20 and 10 μM) in a normal cell incubator and 250 ppm CO gas in the CO chamber for 6 h; (f) RAW264.7 cells incubated in different conditions (described in the Results and Discussion section) for 6 h. Statistical significance, compared to the vehicle group: ns: not significant, **P < 0.01, one-way ANOVA.

Figure 4

Effect of CO prodrugs on RAW264.7 and HeLa cells. (a) CO release chemistry of BW-CO-103 and BW-CO-111; (b) Western blot of RAW264.7 cells incubated with BW-CO/CP-103 or BW-CO/CP-111 for 6 h; (c) Western blot of HeLa cells incubated with BW-CO/CP-103 for 6 h. (d) Dose-dependence test of HO-1 induction activity of BW-CO/CP-111 in RAW264.7 cells at 6 h time point; (e) densitometry analysis of the Western blot of BW-CO-111 (Data show the folds change comparing to the vehicle control group (0.5% DMSO) after normalization with the optical density of the β-Actin band (n = 3, ****P < 0.0001, ***P < 0.001 vs Veh group, one-way ANOVA)).

Figure 5

Nrf2 activation level tested with the Nrf2/ARE-luciferase reporter assay in transgenic HEK293 cells (0.5% DMSO in cell culture medium was used as the vehicle for all groups); CO gas concentration was 250 ppm, n = 3, data show the adjusted ratio by dividing chemiluminescent signal of the tested sample with the chemiluminescent signal of the naive Nrf2-luciferase cells (Compared to the vehicle group: ***P < 0.001, ****P < 0.0001, one-way ANOVA).