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Elevation of cAMP Levels Inhibits Doxorubicin-Induced Apoptosis in Pre- B ALL NALM- 6 Cells Through Induction of BAD Phosphorylation and Inhibition of P53 Accumulation.

Fatemi A, Kazemi A, Kashiri M, Safa M - Int J Mol Cell Med (2015)

Bottom Line: Western blot results revealed the reduced expression of p53 protein in cells treated with combination of cAMP-elevating agents and doxorubicin in contrast to cells treated with doxorubicin alone.Expression of total BAD protein was not affected by doxorubicin and cAMP-elevating agents.However, phosphorylation of BAD protein was induced in the presence of cAMP-elevating agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, School of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.

ABSTRACT
Recognition of the molecular mechanisms of cAMP action against DNA damage-induced apoptosis can be useful to improve the efficacy of DNA damaging therapeutic agents. Considering the critical role of bcl-2-associated death promoter (BAD) and p53 proteins in DNA damage -induced apoptosis, the aim of this study was to assess the effect of cAMP-elevating agents on these proteins in doxorubicin-treated pre-B acute lymphoblastic leukemia (pre-B ALL) NALM-6 cells.The pre-B ALL cell line NALM-6 was cultured and treated with doxorubicin in combination with or without cAMP-elevating agents forskolin and 3-isobutyl-1-methylxanthine (IBMX). Cell viability was measured by trypan blue staining and MTT assay. For evaluation of apoptosis, annexin-V staining by flow cytometry and caspase-3 activity assay were used. Protein expression of p53, BAD and phoshorylated BAD was detected by western blotting analysis.cAMP-increasing agents diminished the doxorubicin-mediated cytotoxicity in NALM-6 cells as indicated by the viability assays. Annexin-V apoptosis assay showed that the cAMP-elevating agents decreased doxorubicin-induced apoptosis. Moreover, doxorubicin-induced caspase-3 activity was attenuated in the presence of cAMP-increasing agents. Western blot results revealed the reduced expression of p53 protein in cells treated with combination of cAMP-elevating agents and doxorubicin in contrast to cells treated with doxorubicin alone. Expression of total BAD protein was not affected by doxorubicin and cAMP-elevating agents. However, phosphorylation of BAD protein was induced in the presence of cAMP-elevating agents. Our study suggests that elevated cAMP levels inhibit doxorubicin-induced apoptosis in pre-B ALL cells through induction of BAD phosphorylation and abrogation of p53 accumulation.

No MeSH data available.


Related in: MedlinePlus

Doxorubicin-induced caspase activation was diminished by cAMP-increasing agents. NALM-6 cells were treated with forskolin and IBMX (FI). After 30 min, the cells were incubated with doxorubicin (250 nM) for 8 and 24 h. The cells were then assayed for caspase-3 protease activity (* P<0.05
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Figure 4: Doxorubicin-induced caspase activation was diminished by cAMP-increasing agents. NALM-6 cells were treated with forskolin and IBMX (FI). After 30 min, the cells were incubated with doxorubicin (250 nM) for 8 and 24 h. The cells were then assayed for caspase-3 protease activity (* P<0.05

Mentions: Cytotoxicity of doxorubicin in the presence or absence of cAMP-increasing agents (forskolin and IBMX) was determined by trypan blue exclusion and MTT assay. Forskolin activates adenylyl cyclase, and IBMX is an inhibitor of the phosphodiesterase which converts cAMP to AMP, resulting in elevated cAMP levels within the cell (20). Viability of NALM-6 cells after exposure to doxorubicin was reduced in a dose dependent manner with 50% reduction in viability at 500 nM concentration of the drug. As shown in Fig. 1 (trypan blue exclusion assay) and Fig. 2 (MTT assay), cAMP-increasing agents attenuated the doxorubicin-mediated cytotoxicity in NALM-6 cells. To investigate the effect of elevated level of cAMP on doxorubicin-induced apoptosis, NALM-6 cells were assayed for annexin V staining and caspase-3 protease activity. As shown in Fig. 3, pretreatment of cells with cAMP-increasing agents was associated with considerable lower percentages of annexin-V positive cells (apoptotic cells) in comparison to the cells treated with doxorubicin only. Moreover, cotreatment of cells with doxorubicin and cAMP-increasing agents resulted in significant attenuation of the doxorubicin-induced caspase-3 activation (Fig. 4). These findings indicate the inhibitory effect of cAMP levels on doxorubicin- induced apoptosis in NALM- 6 cells.


Elevation of cAMP Levels Inhibits Doxorubicin-Induced Apoptosis in Pre- B ALL NALM- 6 Cells Through Induction of BAD Phosphorylation and Inhibition of P53 Accumulation.

Fatemi A, Kazemi A, Kashiri M, Safa M - Int J Mol Cell Med (2015)

Doxorubicin-induced caspase activation was diminished by cAMP-increasing agents. NALM-6 cells were treated with forskolin and IBMX (FI). After 30 min, the cells were incubated with doxorubicin (250 nM) for 8 and 24 h. The cells were then assayed for caspase-3 protease activity (* P<0.05
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499571&req=5

Figure 4: Doxorubicin-induced caspase activation was diminished by cAMP-increasing agents. NALM-6 cells were treated with forskolin and IBMX (FI). After 30 min, the cells were incubated with doxorubicin (250 nM) for 8 and 24 h. The cells were then assayed for caspase-3 protease activity (* P<0.05
Mentions: Cytotoxicity of doxorubicin in the presence or absence of cAMP-increasing agents (forskolin and IBMX) was determined by trypan blue exclusion and MTT assay. Forskolin activates adenylyl cyclase, and IBMX is an inhibitor of the phosphodiesterase which converts cAMP to AMP, resulting in elevated cAMP levels within the cell (20). Viability of NALM-6 cells after exposure to doxorubicin was reduced in a dose dependent manner with 50% reduction in viability at 500 nM concentration of the drug. As shown in Fig. 1 (trypan blue exclusion assay) and Fig. 2 (MTT assay), cAMP-increasing agents attenuated the doxorubicin-mediated cytotoxicity in NALM-6 cells. To investigate the effect of elevated level of cAMP on doxorubicin-induced apoptosis, NALM-6 cells were assayed for annexin V staining and caspase-3 protease activity. As shown in Fig. 3, pretreatment of cells with cAMP-increasing agents was associated with considerable lower percentages of annexin-V positive cells (apoptotic cells) in comparison to the cells treated with doxorubicin only. Moreover, cotreatment of cells with doxorubicin and cAMP-increasing agents resulted in significant attenuation of the doxorubicin-induced caspase-3 activation (Fig. 4). These findings indicate the inhibitory effect of cAMP levels on doxorubicin- induced apoptosis in NALM- 6 cells.

Bottom Line: Western blot results revealed the reduced expression of p53 protein in cells treated with combination of cAMP-elevating agents and doxorubicin in contrast to cells treated with doxorubicin alone.Expression of total BAD protein was not affected by doxorubicin and cAMP-elevating agents.However, phosphorylation of BAD protein was induced in the presence of cAMP-elevating agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, School of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.

ABSTRACT
Recognition of the molecular mechanisms of cAMP action against DNA damage-induced apoptosis can be useful to improve the efficacy of DNA damaging therapeutic agents. Considering the critical role of bcl-2-associated death promoter (BAD) and p53 proteins in DNA damage -induced apoptosis, the aim of this study was to assess the effect of cAMP-elevating agents on these proteins in doxorubicin-treated pre-B acute lymphoblastic leukemia (pre-B ALL) NALM-6 cells.The pre-B ALL cell line NALM-6 was cultured and treated with doxorubicin in combination with or without cAMP-elevating agents forskolin and 3-isobutyl-1-methylxanthine (IBMX). Cell viability was measured by trypan blue staining and MTT assay. For evaluation of apoptosis, annexin-V staining by flow cytometry and caspase-3 activity assay were used. Protein expression of p53, BAD and phoshorylated BAD was detected by western blotting analysis.cAMP-increasing agents diminished the doxorubicin-mediated cytotoxicity in NALM-6 cells as indicated by the viability assays. Annexin-V apoptosis assay showed that the cAMP-elevating agents decreased doxorubicin-induced apoptosis. Moreover, doxorubicin-induced caspase-3 activity was attenuated in the presence of cAMP-increasing agents. Western blot results revealed the reduced expression of p53 protein in cells treated with combination of cAMP-elevating agents and doxorubicin in contrast to cells treated with doxorubicin alone. Expression of total BAD protein was not affected by doxorubicin and cAMP-elevating agents. However, phosphorylation of BAD protein was induced in the presence of cAMP-elevating agents. Our study suggests that elevated cAMP levels inhibit doxorubicin-induced apoptosis in pre-B ALL cells through induction of BAD phosphorylation and abrogation of p53 accumulation.

No MeSH data available.


Related in: MedlinePlus