Pharmacological versus genetic inhibition of heme oxygenase-1 – the comparison of metalloporphyrins, shRNA and CRISPR/Cas9 system

Abstract

Inhibition of heme oxygenase-1 (HO-1, encoded by HMOX1), a cytoprotective, anti-apoptotic and anti-inflammatory enzyme, may serve as a valuable therapy in various pathophysiological processes, including tumorigenesis. We compared the effect of chemical inhibitors - metalloporphyrins, with genetic tools - shRNA and CRISPR/Cas9 systems, to knock-down (KD)/knock-out (KO) HO-1 expression/activity. 293T cells were incubated with metalloporphyrins, tin and zinc protoporphyrins (SnPPIX and ZnPPIX, respectively) or were either transduced with lentiviral vectors encoding different shRNA sequences against HO-1 or were modified by CRISPR/Cas9 system targeting HMOX1. Metalloporphyrins decreased HO activity but concomitantly strongly induced HO-1 mRNA and protein in 293T cells. On the other hand, only slight basal HO-1 inhibition in shRNA KD 293T cell lines was confirmed on mRNA and protein level with no significant effect on enzyme activity. Nevertheless, silencing effect was much stronger when CRISPR/Cas9-mediated knock-out was performed. Most of the clones harboring mutations within HMOX1 locus did not express HO-1 protein and failed to increase bilirubin concentration after hemin stimulation. Furthermore, CRISPR/Cas9-mediated HO-1 depletion decreased 293T viability, growth, clonogenic potential and increased sensitivity to HO treatment. In summary, we have shown that not all technologies can be used for inhibition of HO activity in vitro with the same efficiency. In our hands, the most potent and comprehensible results can be obtained using genetic tools, especially CRISPR/Cas9 approach.

Synthetically Lethal Interactions of Heme Oxygenase-1 and Fumarate Hydratase Genes

“…Such effect is generally considered as a disadvantage of the shRNA approach [18] and it underlines the importance of using several shRNA sequences and not to restrict to only one. Another alternative for future experiments could be the CRISPR/Cas9-mediated gene silencing approach, which, in comparison to shRNA, is known to be less susceptible to off-target effects than RNA interference [35] and may give better results in terms of inhibition of HO activity [19]. Although we did not investigate this very carefully, we cannot exclude the possibility that a certain extent of HO-1 inhibition is also required to cause decreased cell viability.…”

“…shRNA constructs (shHO-1) against human HMOX1 and non-targeting shRNA (scrambled shRNA) in pGFP-C-shLenti vectors were purchased from OriGene (Rockville, MD, USA) and were used for the production of lentiviral vectors in HEK293 cells according to the protocol described elsewhere [19]. After lentiviral vectors titration, 20 MOI (multiplicity of infection) of vectors encoding shRNA sequences against HMOX1 and scrambled shRNA were used.…”

“…We observed not only decreased viability upon SLV-11199 treatment ( Figure 5A) but also Clonogenic potential ( Figure 5B) of UOK 262 cells upon after stimulation with 10 and 25 µM SnPPIX was not affected; however, ZnPPIX diminished the colony formation potential at 10 µM concentration. We and others previously demonstrated that metalloporphyrins may significantly induce HMOX1 mRNA level [15,19], thus, we evaluated if such undesired effects are present in our models. In UOK 262 cells, both ZnPPIX and 10 µM SnPPIX demonstrated the stimulatory effect on HMOX1 expression ( Figure 5C).…”

“…The comprehensive computational study emphasized these off-target effects which may lead to misinterpretation of obtained results [18]. On the other hand, the disadvantages of commercially available metalloporphyrin-based inhibitors of heme oxygenase activity, such as inducing HMOX1 expression and the lack of selectivity towards HO isoforms, are well-known [15,19]. It is especially important as HO-2 isoform, on the contrary to the inducible HO-1 is constitutively expressed and is responsible for the maintenance of cellular homeostasis and for example the viability of endothelial cells [20][21][22].…”

Kidney injury by cyclosporine A is aggravated in heme oxygenase-1 deficient mice and involves regulation of microRNAs

“…Western blotting. Western blotting was performed as described previously (Mucha et al, 2018). Membranes were probed with primary antibodies against HO-1 (ADI-SPA-894, Enzo Life Sciences) and α-tubulin (CP06, Calbiochem), overnight at 4°C.…”