PMID- 29694447
OWN - NLM
STAT- MEDLINE
DCOM- 20181019
LR  - 20181029
IS  - 1734-154X (Electronic)
IS  - 0001-527X (Linking)
VI  - 65
IP  - 2
DP  - 2018
TI  - Pharmacological versus genetic inhibition of heme oxygenase-1 - the comparison of 
      metalloporphyrins, shRNA and CRISPR/Cas9 system.
PG  - 277-286
LID - 10.18388/abp.2017_2542 [doi]
AB  - 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 H(2)O(2) 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.
FAU - Mucha, Olga
AU  - Mucha O
AD  - Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and 
      Biotechnology, Jagiellonian University, Krakow, Poland.
FAU - Podkalicka, Paulina
AU  - Podkalicka P
AD  - Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and 
      Biotechnology, Jagiellonian University, Krakow, Poland.
FAU - Czarnek, Maria
AU  - Czarnek M
AD  - Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and 
      Biotechnology, Jagiellonian University, Krakow, Poland.
FAU - Biela, Anna
AU  - Biela A
AD  - Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and 
      Biotechnology, Jagiellonian University, Krakow, Poland.
FAU - Mieczkowski, Mateusz
AU  - Mieczkowski M
AD  - Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and 
      Biotechnology, Jagiellonian University, Krakow, Poland.
FAU - Kachamakova-Trojanowska, Neli
AU  - Kachamakova-Trojanowska N
AD  - Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and 
      Biotechnology, Jagiellonian University, Krakow, Poland.
FAU - Stepniewski, Jacek
AU  - Stepniewski J
AD  - Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and 
      Biotechnology, Jagiellonian University, Krakow, Poland.
FAU - Jozkowicz, Alicja
AU  - Jozkowicz A
AD  - Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and 
      Biotechnology, Jagiellonian University, Krakow, Poland.
FAU - Dulak, Jozef
AU  - Dulak J
AD  - Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and 
      Biotechnology, Jagiellonian University, Krakow, Poland.
AD  - Kardio-Med Silesia, Zabrze, Poland.
FAU - Loboda, Agnieszka
AU  - Loboda A
AD  - Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and 
      Biotechnology, Jagiellonian University, Krakow, Poland.
LA  - eng
PT  - Comparative Study
PT  - Journal Article
DEP - 20180425
PL  - Switzerland
TA  - Acta Biochim Pol
JT  - Acta biochimica Polonica
JID - 14520300R
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (Metalloporphyrins)
RN  - 0 (RNA, Small Interfering)
RN  - EC 1.14.14.18 (Heme Oxygenase-1)
SB  - IM
MH  - *CRISPR-Cas Systems/genetics
MH  - Enzyme Inhibitors
MH  - Gene Silencing
MH  - Genetic Techniques/standards
MH  - HEK293 Cells
MH  - Heme Oxygenase-1/*antagonists & inhibitors
MH  - Humans
MH  - Metalloporphyrins/pharmacology
MH  - Methods
MH  - RNA, Small Interfering
OTO - NOTNLM
OT  - HO-1
OT  - CRISPR/Cas9
OT  - heme oxygenase-1
OT  - inhibitors
OT  - off-target
OT  - shRNA
EDAT- 2018/04/26 06:00
MHDA- 2018/10/20 06:00
CRDT- 2018/04/26 06:00
PHST- 2017/12/08 00:00 [received]
PHST- 2018/01/19 00:00 [revised]
PHST- 2018/03/07 00:00 [accepted]
PHST- 2018/04/26 06:00 [pubmed]
PHST- 2018/10/20 06:00 [medline]
PHST- 2018/04/26 06:00 [entrez]
AID - 2542 [pii]
AID - 10.18388/abp.2017_2542 [doi]
PST - ppublish
SO  - Acta Biochim Pol. 2018;65(2):277-286. doi: 10.18388/abp.2017_2542. Epub 2018 Apr 
      25.