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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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ATO induced Nrf2 targets in AML cells. (A) HL60 and THP-1 nuclear (N) or total (T) protein levels of Nrf2, TBP1, HO-1, NQO1 and β-actin. Cells were incubated for 24 h with 6.25 μM ATO. (B) Reverse-transcription PCR (upper panels) analysis of GAPDH, HO-1, NQO1, GCLM and ferritin targets mRNA levels and nuclear Nrf2 translocation (bottom panels) in HL60 and THP-1 cells following ATO (6.25 μM) or t-BHQ (25 μM) treatments for 24 h. (C) HL60 cells were electroporated either with the pRS-control or pRS-shNrf2 plasmids and treated with ATO (6.25 μM) for 12 h. Total protein extracts were analysed for the presence of Nrf2, NQO1, HO-1 and β-actin by immunoblotting. (D) HL60 and THP-1 cells were treated with different concentrations of ATO, Ara-C or daunorubicin for 24 h, as indicated. Following treatment, total protein extracts were prepared and NQO1, HO-1 and β-actin protein levels were evaluated by immunoblotting. A typical western blot out of three experiments is shown. (E) GSH content was determined in HL60 and THP-1 cells treated with ATO (6.25 μM), Ara-C (5 μM), daunorubicin (0.5 μM) or t-BHQ (25 μM) for 24 h. Statistically significant differences with respect to the control condition are indicated (means±s.e.m.; n=3; *P⩽0.05, **P⩽0.01, ***P⩽0.001). Ctl, control.
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fig1: ATO induced Nrf2 targets in AML cells. (A) HL60 and THP-1 nuclear (N) or total (T) protein levels of Nrf2, TBP1, HO-1, NQO1 and β-actin. Cells were incubated for 24 h with 6.25 μM ATO. (B) Reverse-transcription PCR (upper panels) analysis of GAPDH, HO-1, NQO1, GCLM and ferritin targets mRNA levels and nuclear Nrf2 translocation (bottom panels) in HL60 and THP-1 cells following ATO (6.25 μM) or t-BHQ (25 μM) treatments for 24 h. (C) HL60 cells were electroporated either with the pRS-control or pRS-shNrf2 plasmids and treated with ATO (6.25 μM) for 12 h. Total protein extracts were analysed for the presence of Nrf2, NQO1, HO-1 and β-actin by immunoblotting. (D) HL60 and THP-1 cells were treated with different concentrations of ATO, Ara-C or daunorubicin for 24 h, as indicated. Following treatment, total protein extracts were prepared and NQO1, HO-1 and β-actin protein levels were evaluated by immunoblotting. A typical western blot out of three experiments is shown. (E) GSH content was determined in HL60 and THP-1 cells treated with ATO (6.25 μM), Ara-C (5 μM), daunorubicin (0.5 μM) or t-BHQ (25 μM) for 24 h. Statistically significant differences with respect to the control condition are indicated (means±s.e.m.; n=3; *P⩽0.05, **P⩽0.01, ***P⩽0.001). Ctl, control.

Mentions: In both HL60 and THP-1 cells, a rapid Nrf2 nuclear accumulation was observed, which remained stable from 3 to 12 h and it decreased after 24 h incubation in the presence of 6.25 μM ATO (Figure 1A). The concomitant increase in the expression of two downstream Nrf2 targets (NQO1 and HO-1) remained fairly stable up to 24 h incubation with ATO (6.25 μM). No changes in Nrf2 expression were observed in untreated cells (basal conditions) during the 24 h incubation time (data not shown). This enhanced protein expression was associated with transcriptional activation of Nrf2 targets by ATO and by t-BHQ, a well-known electrophilic Nrf2 activator, as shown by RT-PCR analysis of HO-1, NQO1, GCLM and ferritin mRNA levels (Figure 1B, upper panels). Interestingly, nuclear accumulation of Nrf2 was more pronounced when cells were treated with ATO as compared with t-BHQ (Figure 1B, lower panels). To confirm that the observed induction of downstream targets was really dependent on Nrf2 activation, its expression was silenced by using shRNA (shNrf2 cells). In shNrf2 cells, induction of both HO-1 and NQO1 by ATO was reduced (Figure 1C).


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)

ATO induced Nrf2 targets in AML cells. (A) HL60 and THP-1 nuclear (N) or total (T) protein levels of Nrf2, TBP1, HO-1, NQO1 and β-actin. Cells were incubated for 24 h with 6.25 μM ATO. (B) Reverse-transcription PCR (upper panels) analysis of GAPDH, HO-1, NQO1, GCLM and ferritin targets mRNA levels and nuclear Nrf2 translocation (bottom panels) in HL60 and THP-1 cells following ATO (6.25 μM) or t-BHQ (25 μM) treatments for 24 h. (C) HL60 cells were electroporated either with the pRS-control or pRS-shNrf2 plasmids and treated with ATO (6.25 μM) for 12 h. Total protein extracts were analysed for the presence of Nrf2, NQO1, HO-1 and β-actin by immunoblotting. (D) HL60 and THP-1 cells were treated with different concentrations of ATO, Ara-C or daunorubicin for 24 h, as indicated. Following treatment, total protein extracts were prepared and NQO1, HO-1 and β-actin protein levels were evaluated by immunoblotting. A typical western blot out of three experiments is shown. (E) GSH content was determined in HL60 and THP-1 cells treated with ATO (6.25 μM), Ara-C (5 μM), daunorubicin (0.5 μM) or t-BHQ (25 μM) for 24 h. Statistically significant differences with respect to the control condition are indicated (means±s.e.m.; n=3; *P⩽0.05, **P⩽0.01, ***P⩽0.001). Ctl, control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4150280&req=5

fig1: ATO induced Nrf2 targets in AML cells. (A) HL60 and THP-1 nuclear (N) or total (T) protein levels of Nrf2, TBP1, HO-1, NQO1 and β-actin. Cells were incubated for 24 h with 6.25 μM ATO. (B) Reverse-transcription PCR (upper panels) analysis of GAPDH, HO-1, NQO1, GCLM and ferritin targets mRNA levels and nuclear Nrf2 translocation (bottom panels) in HL60 and THP-1 cells following ATO (6.25 μM) or t-BHQ (25 μM) treatments for 24 h. (C) HL60 cells were electroporated either with the pRS-control or pRS-shNrf2 plasmids and treated with ATO (6.25 μM) for 12 h. Total protein extracts were analysed for the presence of Nrf2, NQO1, HO-1 and β-actin by immunoblotting. (D) HL60 and THP-1 cells were treated with different concentrations of ATO, Ara-C or daunorubicin for 24 h, as indicated. Following treatment, total protein extracts were prepared and NQO1, HO-1 and β-actin protein levels were evaluated by immunoblotting. A typical western blot out of three experiments is shown. (E) GSH content was determined in HL60 and THP-1 cells treated with ATO (6.25 μM), Ara-C (5 μM), daunorubicin (0.5 μM) or t-BHQ (25 μM) for 24 h. Statistically significant differences with respect to the control condition are indicated (means±s.e.m.; n=3; *P⩽0.05, **P⩽0.01, ***P⩽0.001). Ctl, control.
Mentions: In both HL60 and THP-1 cells, a rapid Nrf2 nuclear accumulation was observed, which remained stable from 3 to 12 h and it decreased after 24 h incubation in the presence of 6.25 μM ATO (Figure 1A). The concomitant increase in the expression of two downstream Nrf2 targets (NQO1 and HO-1) remained fairly stable up to 24 h incubation with ATO (6.25 μM). No changes in Nrf2 expression were observed in untreated cells (basal conditions) during the 24 h incubation time (data not shown). This enhanced protein expression was associated with transcriptional activation of Nrf2 targets by ATO and by t-BHQ, a well-known electrophilic Nrf2 activator, as shown by RT-PCR analysis of HO-1, NQO1, GCLM and ferritin mRNA levels (Figure 1B, upper panels). Interestingly, nuclear accumulation of Nrf2 was more pronounced when cells were treated with ATO as compared with t-BHQ (Figure 1B, lower panels). To confirm that the observed induction of downstream targets was really dependent on Nrf2 activation, its expression was silenced by using shRNA (shNrf2 cells). In shNrf2 cells, induction of both HO-1 and NQO1 by ATO was reduced (Figure 1C).

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