Limits...
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.

Show MeSH

Related in: MedlinePlus

The association of ATRA and ATO inhibited Nrf2 nuclear translocation promoting cell death in both AML and APL cells. HL60 and NB4 cells were pretreated with ATRA (1 μM) for 2 h and then exposed to ATO (3.1–6.25 μM; 12 h and 0.75–1.5 μM; 24 h), respectively. (A) After treatment, total and nuclear protein extracts were prepared and HO-1, NQO1, Nrf2 and β-actin protein levels were assessed by immunoblotting. Illustrations of typical western blots are shown. (B) GSH content in both HL60 and NB4 cells, under the same conditions described above, was determined as described in the Materials and Methods section. Cytotoxicity was assessed by using (C) trypan exclusion assay and (D) DEVDase activity. HL60 and NB4 cells were exposed to ATO (6.25 and 0.75 μM) for 24 or 48 h, respectively. 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

License
getmorefigures.php?uid=PMC4150280&req=5

fig3: The association of ATRA and ATO inhibited Nrf2 nuclear translocation promoting cell death in both AML and APL cells. HL60 and NB4 cells were pretreated with ATRA (1 μM) for 2 h and then exposed to ATO (3.1–6.25 μM; 12 h and 0.75–1.5 μM; 24 h), respectively. (A) After treatment, total and nuclear protein extracts were prepared and HO-1, NQO1, Nrf2 and β-actin protein levels were assessed by immunoblotting. Illustrations of typical western blots are shown. (B) GSH content in both HL60 and NB4 cells, under the same conditions described above, was determined as described in the Materials and Methods section. Cytotoxicity was assessed by using (C) trypan exclusion assay and (D) DEVDase activity. HL60 and NB4 cells were exposed to ATO (6.25 and 0.75 μM) for 24 or 48 h, respectively. 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: As ATRA inhibited the Nrf2 transcriptional activity in breast cancer cells (Wang et al, 2007), we explored whether retinoic acid may modulate the characterised Nrf2 activation in response to ATO in both AML and APL cells. Rather strikingly, when HL60 and NB4 cells were exposed to ATO in the presence of 1 μM ATRA, Nrf2 nuclear translocation, induction of HO-1 and NQO1 expression (Figure 3A), as well as the increase in GSH content (Figure 3B) were reduced.


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)

The association of ATRA and ATO inhibited Nrf2 nuclear translocation promoting cell death in both AML and APL cells. HL60 and NB4 cells were pretreated with ATRA (1 μM) for 2 h and then exposed to ATO (3.1–6.25 μM; 12 h and 0.75–1.5 μM; 24 h), respectively. (A) After treatment, total and nuclear protein extracts were prepared and HO-1, NQO1, Nrf2 and β-actin protein levels were assessed by immunoblotting. Illustrations of typical western blots are shown. (B) GSH content in both HL60 and NB4 cells, under the same conditions described above, was determined as described in the Materials and Methods section. Cytotoxicity was assessed by using (C) trypan exclusion assay and (D) DEVDase activity. HL60 and NB4 cells were exposed to ATO (6.25 and 0.75 μM) for 24 or 48 h, respectively. 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

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

fig3: The association of ATRA and ATO inhibited Nrf2 nuclear translocation promoting cell death in both AML and APL cells. HL60 and NB4 cells were pretreated with ATRA (1 μM) for 2 h and then exposed to ATO (3.1–6.25 μM; 12 h and 0.75–1.5 μM; 24 h), respectively. (A) After treatment, total and nuclear protein extracts were prepared and HO-1, NQO1, Nrf2 and β-actin protein levels were assessed by immunoblotting. Illustrations of typical western blots are shown. (B) GSH content in both HL60 and NB4 cells, under the same conditions described above, was determined as described in the Materials and Methods section. Cytotoxicity was assessed by using (C) trypan exclusion assay and (D) DEVDase activity. HL60 and NB4 cells were exposed to ATO (6.25 and 0.75 μM) for 24 or 48 h, respectively. 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: As ATRA inhibited the Nrf2 transcriptional activity in breast cancer cells (Wang et al, 2007), we explored whether retinoic acid may modulate the characterised Nrf2 activation in response to ATO in both AML and APL cells. Rather strikingly, when HL60 and NB4 cells were exposed to ATO in the presence of 1 μM ATRA, Nrf2 nuclear translocation, induction of HO-1 and NQO1 expression (Figure 3A), as well as the increase in GSH content (Figure 3B) were reduced.

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