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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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Loss of ATRA-mediated Nrf2 inhibitory mechanism in the NB4-R2 retinoic acid-resistant cell line. (A) HL60, NB4 and NB4-R2 cells were treated for 3 days with ATRA 1 μM. Differentiation was determined by the NTB reduction assay as indicated in Materials and Methods. (B) NB4-R2 and NB4 cells were treated with ATO (0.75 μM) in the presence of ATRA (1 μM) for 24 h. After treatment, total protein extracts were prepared and HO-1 and β-actin protein levels were assessed by immunoblotting. Illustration of a typical western blot is shown. (C) GSH content was measured in NB4-R2 cells treated with ATO (0.75 μM) in the presence of ATRA (1 μM) for 24 h. Cell survival was assessed in NB4-R2 cells treated with ATO (0.75 μM) in the presence of ATRA (1 μM) for 24 h using (D) DEVDase activity or (E) the trypan blue exclusion assays. (F) NB4 cells were incubated with Ro-41-5351 (0.5 μM) for 24 h and then treated with ATRA (1 μM) and ATO (0.75 μM) for additional 24 h. Cell extracts were collected and cell survival was assessed by using the trypan blue exclusion assay. 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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fig5: Loss of ATRA-mediated Nrf2 inhibitory mechanism in the NB4-R2 retinoic acid-resistant cell line. (A) HL60, NB4 and NB4-R2 cells were treated for 3 days with ATRA 1 μM. Differentiation was determined by the NTB reduction assay as indicated in Materials and Methods. (B) NB4-R2 and NB4 cells were treated with ATO (0.75 μM) in the presence of ATRA (1 μM) for 24 h. After treatment, total protein extracts were prepared and HO-1 and β-actin protein levels were assessed by immunoblotting. Illustration of a typical western blot is shown. (C) GSH content was measured in NB4-R2 cells treated with ATO (0.75 μM) in the presence of ATRA (1 μM) for 24 h. Cell survival was assessed in NB4-R2 cells treated with ATO (0.75 μM) in the presence of ATRA (1 μM) for 24 h using (D) DEVDase activity or (E) the trypan blue exclusion assays. (F) NB4 cells were incubated with Ro-41-5351 (0.5 μM) for 24 h and then treated with ATRA (1 μM) and ATO (0.75 μM) for additional 24 h. Cell extracts were collected and cell survival was assessed by using the trypan blue exclusion assay. 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: Previous results showed that ATRA enhances ATO-mediated cytotoxicity by inhibiting the Nrf2 pathway in AML cells. This led us to explore the potential role of RARα in the ATRA-mediated effects on Nrf2 pathway in response to ATO. To this end, in addition to NB4 cells, we included in our study the ATRA-resistant NB4-R2 cells, a cellular subline bearing a missense mutation in the PML fragment of PML–RARα that inhibits RARα transcriptional function (Duprez et al, 2000). These cells do not differentiate even following 3 days of exposure to 1 μM ATRA (Figure 5A). Conversely, HL60 and NB4 were able to differentiate under such conditions. Subsequently, NB4-R2 and NB4 cells were incubated with ATO-ATRA. Western blot analysis revealed that HO-1 induction was not reduced in NB4-R2 cells treated with ATO in the presence of ATRA, as was the case for NB4 parental cells (Figure 5B). Similar results were observed when GSH content was determined (Figure 5C).


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)

Loss of ATRA-mediated Nrf2 inhibitory mechanism in the NB4-R2 retinoic acid-resistant cell line. (A) HL60, NB4 and NB4-R2 cells were treated for 3 days with ATRA 1 μM. Differentiation was determined by the NTB reduction assay as indicated in Materials and Methods. (B) NB4-R2 and NB4 cells were treated with ATO (0.75 μM) in the presence of ATRA (1 μM) for 24 h. After treatment, total protein extracts were prepared and HO-1 and β-actin protein levels were assessed by immunoblotting. Illustration of a typical western blot is shown. (C) GSH content was measured in NB4-R2 cells treated with ATO (0.75 μM) in the presence of ATRA (1 μM) for 24 h. Cell survival was assessed in NB4-R2 cells treated with ATO (0.75 μM) in the presence of ATRA (1 μM) for 24 h using (D) DEVDase activity or (E) the trypan blue exclusion assays. (F) NB4 cells were incubated with Ro-41-5351 (0.5 μM) for 24 h and then treated with ATRA (1 μM) and ATO (0.75 μM) for additional 24 h. Cell extracts were collected and cell survival was assessed by using the trypan blue exclusion assay. 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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Show All Figures
getmorefigures.php?uid=PMC4150280&req=5

fig5: Loss of ATRA-mediated Nrf2 inhibitory mechanism in the NB4-R2 retinoic acid-resistant cell line. (A) HL60, NB4 and NB4-R2 cells were treated for 3 days with ATRA 1 μM. Differentiation was determined by the NTB reduction assay as indicated in Materials and Methods. (B) NB4-R2 and NB4 cells were treated with ATO (0.75 μM) in the presence of ATRA (1 μM) for 24 h. After treatment, total protein extracts were prepared and HO-1 and β-actin protein levels were assessed by immunoblotting. Illustration of a typical western blot is shown. (C) GSH content was measured in NB4-R2 cells treated with ATO (0.75 μM) in the presence of ATRA (1 μM) for 24 h. Cell survival was assessed in NB4-R2 cells treated with ATO (0.75 μM) in the presence of ATRA (1 μM) for 24 h using (D) DEVDase activity or (E) the trypan blue exclusion assays. (F) NB4 cells were incubated with Ro-41-5351 (0.5 μM) for 24 h and then treated with ATRA (1 μM) and ATO (0.75 μM) for additional 24 h. Cell extracts were collected and cell survival was assessed by using the trypan blue exclusion assay. 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: Previous results showed that ATRA enhances ATO-mediated cytotoxicity by inhibiting the Nrf2 pathway in AML cells. This led us to explore the potential role of RARα in the ATRA-mediated effects on Nrf2 pathway in response to ATO. To this end, in addition to NB4 cells, we included in our study the ATRA-resistant NB4-R2 cells, a cellular subline bearing a missense mutation in the PML fragment of PML–RARα that inhibits RARα transcriptional function (Duprez et al, 2000). These cells do not differentiate even following 3 days of exposure to 1 μM ATRA (Figure 5A). Conversely, HL60 and NB4 were able to differentiate under such conditions. Subsequently, NB4-R2 and NB4 cells were incubated with ATO-ATRA. Western blot analysis revealed that HO-1 induction was not reduced in NB4-R2 cells treated with ATO in the presence of ATRA, as was the case for NB4 parental cells (Figure 5B). Similar results were observed when GSH content was determined (Figure 5C).

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