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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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Pharmacologic inhibition of Nrf2 targets (HO-1 and GSH) sensitises AML and APL cells to ATO. (A) Cell viability was determined in HL60, THP-1 and NB4 cells exposed for 48 h to different concentrations of ATO (0–25 μM) in the presence (triangles) or absence (squares) of ZnPP (5 μM) using the trypan blue exclusion assay. (B) HL60 cells treated with ATO (6.25 μM) in the presence of ZnPP (5 μM) for 24 h. Cell extracts were prepared and DEVDase activity was assessed as described in the Materials and Methods section. The pan-caspase inhibitor 10 μM Q-VD-OPh (QVD) was included as a negative control (data not shown). (C–D) HL60 and NB4 cells were pre-incubated in the presence or the absence of BSO (10 μM) for 24 h (+BSO) and then treated with ATO (6.25 and 0.75 μM, respectively) for an additional 12 h. Cytotoxicity was determined either by (C) trypan blue exclusion assay or (D) DEVDase activity. 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).
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fig2: Pharmacologic inhibition of Nrf2 targets (HO-1 and GSH) sensitises AML and APL cells to ATO. (A) Cell viability was determined in HL60, THP-1 and NB4 cells exposed for 48 h to different concentrations of ATO (0–25 μM) in the presence (triangles) or absence (squares) of ZnPP (5 μM) using the trypan blue exclusion assay. (B) HL60 cells treated with ATO (6.25 μM) in the presence of ZnPP (5 μM) for 24 h. Cell extracts were prepared and DEVDase activity was assessed as described in the Materials and Methods section. The pan-caspase inhibitor 10 μM Q-VD-OPh (QVD) was included as a negative control (data not shown). (C–D) HL60 and NB4 cells were pre-incubated in the presence or the absence of BSO (10 μM) for 24 h (+BSO) and then treated with ATO (6.25 and 0.75 μM, respectively) for an additional 12 h. Cytotoxicity was determined either by (C) trypan blue exclusion assay or (D) DEVDase activity. 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).

Mentions: Antioxidant elements induced during the activation of Nrf2 pathway (that is, HO-1 and GSH) may have a critical role in a putative resistance against ATO-mediated cytotoxicity in both AML and APL cells. To explore this likelihood, HL60, THP-1 and the NB4 (PML–RARα/RARα) cells were first incubated with different concentrations of ATO in the presence or absence of either the active HO-1 inhibitor ZnPP or CuPP, its inactive form. As shown in Figure 2A, ZnPP (5 μM) augmented cell cytotoxicity in HL60 and THP-1 exposed to ATO for 48 h, as determined by the trypan blue exclusion assay. This effect was not observed when CuPP (5 μM) was used as a control (data not shown). Interestingly, NB4 cells were sensitive to lower ATO concentrations and the effect of ZnPP was limited to concentrations varying between 0.35 and 0.75 μM ATO. To discard unspecific effects of ZnPP, K562 cells (which do not induce HO-1 in response to either ATO or t-BHQ) were subjected to similar conditions. ATO-mediated cytotoxicity was not affected by the presence of ZnPP in these cells (Supplementary Figure 2). Regarding the apoptosis-like induced cell death, the DEVDase assay indicated that caspase activity was increased by three-fold when HL60 cells were treated with 6.25 μM ATO in the presence of 5 μM ZnPP as compared with ATO alone (Figure 2B). Moreover, the incubation of HL60 cells in the presence of 5 μM ZnPP did not modify the caspase activation induced by methotrexate, daunorubicin or Ara-C (Supplementary Figure 3).


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)

Pharmacologic inhibition of Nrf2 targets (HO-1 and GSH) sensitises AML and APL cells to ATO. (A) Cell viability was determined in HL60, THP-1 and NB4 cells exposed for 48 h to different concentrations of ATO (0–25 μM) in the presence (triangles) or absence (squares) of ZnPP (5 μM) using the trypan blue exclusion assay. (B) HL60 cells treated with ATO (6.25 μM) in the presence of ZnPP (5 μM) for 24 h. Cell extracts were prepared and DEVDase activity was assessed as described in the Materials and Methods section. The pan-caspase inhibitor 10 μM Q-VD-OPh (QVD) was included as a negative control (data not shown). (C–D) HL60 and NB4 cells were pre-incubated in the presence or the absence of BSO (10 μM) for 24 h (+BSO) and then treated with ATO (6.25 and 0.75 μM, respectively) for an additional 12 h. Cytotoxicity was determined either by (C) trypan blue exclusion assay or (D) DEVDase activity. 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).
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Related In: Results  -  Collection

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fig2: Pharmacologic inhibition of Nrf2 targets (HO-1 and GSH) sensitises AML and APL cells to ATO. (A) Cell viability was determined in HL60, THP-1 and NB4 cells exposed for 48 h to different concentrations of ATO (0–25 μM) in the presence (triangles) or absence (squares) of ZnPP (5 μM) using the trypan blue exclusion assay. (B) HL60 cells treated with ATO (6.25 μM) in the presence of ZnPP (5 μM) for 24 h. Cell extracts were prepared and DEVDase activity was assessed as described in the Materials and Methods section. The pan-caspase inhibitor 10 μM Q-VD-OPh (QVD) was included as a negative control (data not shown). (C–D) HL60 and NB4 cells were pre-incubated in the presence or the absence of BSO (10 μM) for 24 h (+BSO) and then treated with ATO (6.25 and 0.75 μM, respectively) for an additional 12 h. Cytotoxicity was determined either by (C) trypan blue exclusion assay or (D) DEVDase activity. 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).
Mentions: Antioxidant elements induced during the activation of Nrf2 pathway (that is, HO-1 and GSH) may have a critical role in a putative resistance against ATO-mediated cytotoxicity in both AML and APL cells. To explore this likelihood, HL60, THP-1 and the NB4 (PML–RARα/RARα) cells were first incubated with different concentrations of ATO in the presence or absence of either the active HO-1 inhibitor ZnPP or CuPP, its inactive form. As shown in Figure 2A, ZnPP (5 μM) augmented cell cytotoxicity in HL60 and THP-1 exposed to ATO for 48 h, as determined by the trypan blue exclusion assay. This effect was not observed when CuPP (5 μM) was used as a control (data not shown). Interestingly, NB4 cells were sensitive to lower ATO concentrations and the effect of ZnPP was limited to concentrations varying between 0.35 and 0.75 μM ATO. To discard unspecific effects of ZnPP, K562 cells (which do not induce HO-1 in response to either ATO or t-BHQ) were subjected to similar conditions. ATO-mediated cytotoxicity was not affected by the presence of ZnPP in these cells (Supplementary Figure 2). Regarding the apoptosis-like induced cell death, the DEVDase assay indicated that caspase activity was increased by three-fold when HL60 cells were treated with 6.25 μM ATO in the presence of 5 μM ZnPP as compared with ATO alone (Figure 2B). Moreover, the incubation of HL60 cells in the presence of 5 μM ZnPP did not modify the caspase activation induced by methotrexate, daunorubicin or Ara-C (Supplementary Figure 3).

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