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Retinoic acid synergizes ATO-mediated cytotoxicity by precluding Nrf2 activity in AML cells.

Valenzuela M, Glorieux C, Stockis J, Sid B, Sandoval JM, Felipe KB, Kviecinski MR, Verrax J, Buc Calderon P - Br. J. Cancer (2014)

Bottom Line: Although co-administration of arsenic trioxide (ATO) with ATRA has emerged as an effective option to treat APL, the molecular basis of this effect remains unclear.The inhibitory effects of ATRA on ATO-mediated responses were not observed in either the ATRA-resistant NB4-R2 cells or in NB4 cells pre-incubated with the RARα antagonist Ro-41-52-53.The augmented cytotoxicity observed in leukaemia cells following combined ATO-ATRA treatment is likely due to inhibition of Nrf2 activity, thus explaining the efficacy of combined ATO-ATRA treatment in the APL therapy.

View Article: PubMed Central - PubMed

Affiliation: Toxicology and Cancer Biology Research Group (GTOX), Louvain Drug Research Institute, Université catholique de Louvain, Avenue Mounier, 73 bte B1.73.09, Brussels 1200, Belgium.

ABSTRACT

Background: Standard therapy for acute promyelocytic leukaemia (APL) includes retinoic acid (all-trans retinoic acid (ATRA)), which promotes differentiation of promyelocytic blasts. Although co-administration of arsenic trioxide (ATO) with ATRA has emerged as an effective option to treat APL, the molecular basis of this effect remains unclear.

Methods: Four leukaemia cancer human models (HL60, THP-1, NBR4 and NBR4-R2 cells) were treated either with ATO alone or ATO plus ATRA. Cancer cell survival was monitored by trypan blue exclusion and DEVDase activity assays. Gene and protein expression changes were assessed by RT-PCR and western blot.

Results: ATO induced an antioxidant response characterised by Nrf2 nuclear translocation and enhanced transcription of downstream target genes (that is, HO-1, NQO1, GCLM, ferritin). In cells exposed to ATO plus ATRA, the Nrf2 nuclear translocation was prevented and cytotoxicity was enhanced. HO-1 overexpression reversed partially the cytotoxicity by ATRA-ATO in HL60 cells. The inhibitory effects of ATRA on ATO-mediated responses were not observed in either the ATRA-resistant NB4-R2 cells or in NB4 cells pre-incubated with the RARα antagonist Ro-41-52-53.

Conclusions: The augmented cytotoxicity observed in leukaemia cells following combined ATO-ATRA treatment is likely due to inhibition of Nrf2 activity, thus explaining the efficacy of combined ATO-ATRA treatment in the APL therapy.

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Ectopic expression of HO-1 in HL60 cells precluded ATRA-promoted cell death. (A) HL60 cells, stably transfected with either pSFFV-neo or pSFFV-HO-1, were incubated for 12 h with ATO (6.25 μM) with or without ATRA (1 μM). Total protein extracts were prepared and HO-1 protein levels were evaluated by immunoblotting. Illustration of a typical western blot is shown. Cytotoxicity following ATO and/or ATRA treatment was assessed by either (B) trypan blue exclusion or (C) DEVDase activity assays after 24 or 48 h of treatment, respectively. Statistically significant differences with respect to the control condition are indicated (means±s.e.m.; n=3; *P⩽0.05). Ctl, control.
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fig4: Ectopic expression of HO-1 in HL60 cells precluded ATRA-promoted cell death. (A) HL60 cells, stably transfected with either pSFFV-neo or pSFFV-HO-1, were incubated for 12 h with ATO (6.25 μM) with or without ATRA (1 μM). Total protein extracts were prepared and HO-1 protein levels were evaluated by immunoblotting. Illustration of a typical western blot is shown. Cytotoxicity following ATO and/or ATRA treatment was assessed by either (B) trypan blue exclusion or (C) DEVDase activity assays after 24 or 48 h of treatment, respectively. Statistically significant differences with respect to the control condition are indicated (means±s.e.m.; n=3; *P⩽0.05). Ctl, control.

Mentions: Previous results show that HO-1 expression – via Nrf2 activation – would have a major protective role in the survival of cells exposed to both ATRA and ATO. To explore this, HL60 cells were stably transfected with a plasmid expressing the human HO-1 gene (pSFFV-HO-1). As shown in Figure 4A, the SFFV promoter was activated in these cells and ectopic HO-1 expression was maintained even in the presence of ATRA-ATO combination. Such forced HO-1 expression was sufficient to reduce ATO-mediated cytotoxicity, but partially reverted (not significant) the enhanced cytotoxicity triggered by ATRA during ATO challenge, as determined by the trypan blue exclusion (Figure 4B) and DEVDase activity (Figure 4C) assays.


Retinoic acid synergizes ATO-mediated cytotoxicity by precluding Nrf2 activity in AML cells.

Valenzuela M, Glorieux C, Stockis J, Sid B, Sandoval JM, Felipe KB, Kviecinski MR, Verrax J, Buc Calderon P - Br. J. Cancer (2014)

Ectopic expression of HO-1 in HL60 cells precluded ATRA-promoted cell death. (A) HL60 cells, stably transfected with either pSFFV-neo or pSFFV-HO-1, were incubated for 12 h with ATO (6.25 μM) with or without ATRA (1 μM). Total protein extracts were prepared and HO-1 protein levels were evaluated by immunoblotting. Illustration of a typical western blot is shown. Cytotoxicity following ATO and/or ATRA treatment was assessed by either (B) trypan blue exclusion or (C) DEVDase activity assays after 24 or 48 h of treatment, respectively. Statistically significant differences with respect to the control condition are indicated (means±s.e.m.; n=3; *P⩽0.05). Ctl, control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150280&req=5

fig4: Ectopic expression of HO-1 in HL60 cells precluded ATRA-promoted cell death. (A) HL60 cells, stably transfected with either pSFFV-neo or pSFFV-HO-1, were incubated for 12 h with ATO (6.25 μM) with or without ATRA (1 μM). Total protein extracts were prepared and HO-1 protein levels were evaluated by immunoblotting. Illustration of a typical western blot is shown. Cytotoxicity following ATO and/or ATRA treatment was assessed by either (B) trypan blue exclusion or (C) DEVDase activity assays after 24 or 48 h of treatment, respectively. Statistically significant differences with respect to the control condition are indicated (means±s.e.m.; n=3; *P⩽0.05). Ctl, control.
Mentions: Previous results show that HO-1 expression – via Nrf2 activation – would have a major protective role in the survival of cells exposed to both ATRA and ATO. To explore this, HL60 cells were stably transfected with a plasmid expressing the human HO-1 gene (pSFFV-HO-1). As shown in Figure 4A, the SFFV promoter was activated in these cells and ectopic HO-1 expression was maintained even in the presence of ATRA-ATO combination. Such forced HO-1 expression was sufficient to reduce ATO-mediated cytotoxicity, but partially reverted (not significant) the enhanced cytotoxicity triggered by ATRA during ATO challenge, as determined by the trypan blue exclusion (Figure 4B) and DEVDase activity (Figure 4C) assays.

Bottom Line: Although co-administration of arsenic trioxide (ATO) with ATRA has emerged as an effective option to treat APL, the molecular basis of this effect remains unclear.The inhibitory effects of ATRA on ATO-mediated responses were not observed in either the ATRA-resistant NB4-R2 cells or in NB4 cells pre-incubated with the RARα antagonist Ro-41-52-53.The augmented cytotoxicity observed in leukaemia cells following combined ATO-ATRA treatment is likely due to inhibition of Nrf2 activity, thus explaining the efficacy of combined ATO-ATRA treatment in the APL therapy.

View Article: PubMed Central - PubMed

Affiliation: Toxicology and Cancer Biology Research Group (GTOX), Louvain Drug Research Institute, Université catholique de Louvain, Avenue Mounier, 73 bte B1.73.09, Brussels 1200, Belgium.

ABSTRACT

Background: Standard therapy for acute promyelocytic leukaemia (APL) includes retinoic acid (all-trans retinoic acid (ATRA)), which promotes differentiation of promyelocytic blasts. Although co-administration of arsenic trioxide (ATO) with ATRA has emerged as an effective option to treat APL, the molecular basis of this effect remains unclear.

Methods: Four leukaemia cancer human models (HL60, THP-1, NBR4 and NBR4-R2 cells) were treated either with ATO alone or ATO plus ATRA. Cancer cell survival was monitored by trypan blue exclusion and DEVDase activity assays. Gene and protein expression changes were assessed by RT-PCR and western blot.

Results: ATO induced an antioxidant response characterised by Nrf2 nuclear translocation and enhanced transcription of downstream target genes (that is, HO-1, NQO1, GCLM, ferritin). In cells exposed to ATO plus ATRA, the Nrf2 nuclear translocation was prevented and cytotoxicity was enhanced. HO-1 overexpression reversed partially the cytotoxicity by ATRA-ATO in HL60 cells. The inhibitory effects of ATRA on ATO-mediated responses were not observed in either the ATRA-resistant NB4-R2 cells or in NB4 cells pre-incubated with the RARα antagonist Ro-41-52-53.

Conclusions: The augmented cytotoxicity observed in leukaemia cells following combined ATO-ATRA treatment is likely due to inhibition of Nrf2 activity, thus explaining the efficacy of combined ATO-ATRA treatment in the APL therapy.

Show MeSH
Related in: MedlinePlus