European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Q1 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 Q2 60 61 62 63 64 65 66

Contents lists available at ScienceDirect

European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

cAMP protects acute promyelocytic leukemia cells from arsenic trioxide-induced caspase-3 activation and apoptosis Majid Safa a,b,n, Kazem Mousavizadeh c,nn, Shekoofeh Noori d, Arefeh Pourfathollah e, Hamid Zand f a

Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran Department of Hematology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran,Iran c Oncopathology Research Center, and Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran d Department of Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran e Department of Medical Laboratory Sciences, Tehran University of Medical Sciences, Tehran, Iran f National Institute and Faculty of Nutrition and Food Technology, Department of Molecular Nutrition, Shahid Beheshti University of Medical Sciences, Tehran, Iran b

art ic l e i nf o

a b s t r a c t

Article history: Received 13 December 2013 Received in revised form 19 April 2014 Accepted 23 April 2014

More recently, arsenic trioxide (ATO), was integrated into acute promyelocytic leukemia (APL) treatment, showing high efficacy and tolerability in patients with both ATRA-sensitive and ATRA-resistant APL. ATO could induce apoptosis at relatively high concentrations (0.5 to 2.0 micromol/L) and partial differentiation at low concentrations (0.1 to 0.5 micromol/L) in leukemic promyelocytes. It is known that cAMP agonists enhance low-dose ATO-induced APL cells differentiation. Less well appreciated was the possible interaction between relatively high-doses of ATO and enhanced levels of cAMP in APL cells. Here, we show that elevation of cAMP levels by forskolin inhibited ATO-mediated apoptosis in APL-derived NB4 cells, and this inhibition could be averted by cell permeable cAMP-dependent protein kinase inhibitor (14–22) amide. Inactivating phosphorylation of the proapoptotic protein Bad at Ser118 and phosphorylation of the CREB proto-oncogene at Ser133 were observed upon elevation of cAMP levels in NB4 cells. Phosphorylation of these PKA target proteins is known to promote cell survival in AML cells. The ability of cAMP to endow the APL cells with survival advantage is of particular importance when cAMP agonists may be considered as adjuncts to APL therapy. & 2014 Published by Elsevier B.V.

Keywords: Arsenic trioxide NB4 cells cAMP Apoptosis

1. Introduction Acute promyelocytic leukemia (APL), is a distinct subtype of acute myeloid leukemia characterized by differentiation block of granulopoiesis at the promyelocytic stage (Grignani et al., 1994). It is well known that in the leukemic blasts of great majority of APL patients, RAR(alpha) is fused to the PML gene as a result of the t(15;17) translocation leading to PML-RAR oncoprotein expression. This specific fusion protein blocks differentiation and promotes survival of myeloid precursor cells (de The et al., 1991). Several developments have paved the way to change APL from a fatal

n Corresponding author at: Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran. Tel.: þ 98 21 8670 4667; fax: þ98 21 8862 2576. nn Corresponding author. Tel.: þ 98 21 8670 4653; fax: þ98 21 8862 2578. E-mail addresses: [email protected] (M. Safa), [email protected] (K. Mousavizadeh).

disease to one that is now highly curable (Sanz, 2006). Early treatment regimens for APL relied mainly on anthracycline-based chemotherapy with daunorubicin or idarubicin as single agents (Sanz et al., 2003). The addition of all-trans-retinoic acid (ATRA), a non-chemotherapeutic differentiating agent to the anthracyclinebased chemotherapy has improved dramatically the management and survival of those with APL and made the disease curable in most cases (Huang et al., 1988). More recently, ATO has been included into APL treatment, showing high efficacy in the treatment of newly diagnosed patients with APL as well as in patients with relapses or refractory disease after ATRA or chemotherapy or both (Ghavamzadeh et al., 2011; Shen et al., 1997). Arsenic trioxide targets PML gene product and restores the cell death program (de The and Chen, 2010). In-vitro studies revealed that ATO exerts a dose-dependent dual effect on APL blasts. Higher concentrations (0.5–2 mM) primarily trigger apoptosis, whereas lower concentrations (0.1–0.25 mM) induce partial differentiation (Chen et al., 1997). in vivo, both differentiation and apoptosis were observed among patients with APL or in APL animal models

http://dx.doi.org/10.1016/j.ejphar.2014.04.040 0014-2999/& 2014 Published by Elsevier B.V.

Please cite this article as: Safa, M., et al., cAMP protects acute promyelocytic leukemia cells from arsenic trioxide-induced caspase-3 activation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.040i

M. Safa et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

(Chen et al., 1997; Lallemand-Breitenbach et al., 1999). The second messenger molecule cyclic adenosine monophosphate (cAMP) is an important intracellular signaling mediator. cAMP-dependent protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC) are the major cAMP mediators (Cheng et al., 2008). The cAMP signaling pathway contributes to the regulation of a wide array of biologically distinct cellular processes including metabolism, cell differentiation, and apoptosis (14). A striking effect of cAMP is enhanced ATRA and ATO-induced maturation of APL cells which is in favor of using cAMP signaling stimulators in current APL therapy (Guillemin et al., 2002; Zhu et al., 2002). In spite of the fact that cAMP sensitizes APL cells to differentiation, little is known about the impact of cAMP on APL cell survival. While some reports indicated that cAMP potentiates apoptosis in leukemia cells, other studies showed that cAMP prevents apoptosis (Dong et al., 2010; Insel et al., 2012; Naderi et al., 2009). This modulatory effect of cAMP is of particular concern when cAMP inducers are associated as adjuncts with APL treatment protocols. Our study showed that elevation of cAMP levels protected NB4 cells against ATO-induced apoptosis. The protection is accompanied by phosphorylation of two known PKA-target sites at Ser133 CREB and Ser118 Bad proteins. Moreover, we found that inhibition of PKA by the PKA inhibitory peptide Myr-PKI, reversed the protective effect of cAMP on ATO-induced apoptosis.

2. Materials and methods 2.1. Chemicals and reagents The following substances were purchased from the following manufacturers: forskolin (PubChem CID: 47936), propidium iodide (PubChem CID: 104981) and caspase 3 colorimetric assay kit were purchased from Sigma; PKI 14–22 amide myristoylated was from Tocris Bioscience; the cAMP analog 6-MB-cAMP was from Biolog; Annexin-V-Flous staining kit was from Roche Applied Science. 2.2. Cell culture NB4 (human acute promyelocytic leukemia cell line) and NALM-6 (human B-cell precursor acute lymphoblastic leukemia cell line) cells were grown in suspension in RPMI medium supplemented with 2 mM L-glutamine, 10% FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin in a humidified 5% CO2 incubator at 37 1C under standard cell culture conditions. 2.3. Metabolic activity measured by MTT assay The effect of various concentrations of ATO on cell proliferation was assayed in the presence or absence of the cAMP analog 6-MBcAMP and forskolin by the MTT colorimetric method. Briefly, exponentially growing cancer cells were seeded into a 96-well culture plate at a density of 5  103 cells/well and incubated with various concentrations of ATO in the presence or absence of cAMP and cAMP-increasing agents for 24 h. After removing the medium, cells were incubated with MTT solution (5 mg/ml in PBS) for 4 h and the resulting formazan was solubilized with DMSO (100 ml). The absorbance of each well was measured at 570 nm in an ELISA reader.

24 h. NB4 cells were then harvested and washed twice with PBS and fixed with 70% ethanol. Then cells were treated with 0.5 μg/ml RNase in PBS, and incubated at 37 1C for 30 min before staining with 50 μg/ml PI for 30 min. The cells were analyzed using a FACScan flow cytometer (Becton Dickinson). 2.5. Phosphatidylserine (PS) externalization (annexin-V assay) NB4 cells were treated with ATO in the presence or absence of forskolin for 24 h and were then washed with PBS after the incubation time. A total of 5  105 cells per sample were resuspended in a total volume of 100 ml of the incubation buffer. Annexin-V-Flous (0.5 ml per sample) and propidium iodide (final concentration 20 mg/ml) were added, and cell suspension was incubated for 20 min in the dark. Fluorescence was then measured using flow cytometery. The data were evaluated using CellQuest Software (Becton Dickinson) and expressed as percentage of the cells positive for annexin-V and negative for propidium iodide (early-apoptotic phase). 2.6. Caspase-3 activity assay To evaluate caspase-3 activity, cell lysates were prepared after their respective treatment with ATO in combination with or without forskolin. Briefly, the cells were treated with the indicated agents, and the cell lysates were prepared. The reaction mixture (total volume, 100 ml) contained 20 mg of cell lysate and 10 ml of the caspase-3 substrate acetyl-Asp-Glu-Val-Asp-p-nitroanilide (AcDEVD-pNA) was incubated in a 96-well plate at 37 1C for 2 h. The absorbance of samples was then read at 405 nm in an ELISA reader. 2.7. Western blot analysis Cells were centrifuged at different time points after various treatments, and cellular pellets were washed with cold PBS and lysed (5  106 cells/aliquots) in 0.2 ml of RIPA buffer (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate, and 0.5% sodium deoxycholate) containing protease and phosphatase inhibitor cocktails (Sigma). After centrifugation at 13,000g for 20 min at 4 1C, the supernatant was collected. Protein concentrations were determined by Bradford protein assay, and equivalent amounts of total cellular proteins were separated by 10% SDS-PAGE, according to the method of Laemmli. The gels were then electroblotted onto nitrocellulose membranes (Hybond-ECL, Amersham Corp.). Subsequently, membranes were blocked with 5% nonfat dry milk in TBS containing 0.1% (v/v) Tween-20, 1 h at room temperature or overnight at 4 1C, and probed with specific primary antibodies (Cell Signaling Technology, UK) against cleaved caspase-3 (9664), caspase-9 (9502), caspase-7 (9492), phospho-CREB (9198), Bad (9239), P-Ser155Bad (9297), cleaved PARP (5625), p21 (2947), p53 (2527) and pSer15-p53 (9284). After 5 washes in TBS-T, membranes were incubated with HRP-conjugated secondary antibodies. Proteins were then visualized with a chemiluminescence detection system (Amersham ECL Advance Kit, GE Healthcare). Densitometric quantification was done using Image J Software.

2.4. Sub-G1 DNA content analysis 2.8. Statistical analysis Apoptotic cells were detected using PI staining of ATO-treated cells followed by flow cytometry to detect the so-called sub-G1 peak. Briefly, NB4 cells were seeded into 6 well plates at the concentration of 0.8  106 cells/ml and incubated with different concentrations of ATO in the presence or absence of forskolin for

The significance of differences between experimental variables was determined by the use of the paired Student's t test. A probability level of po 0.05 was considered statistically significant.

Please cite this article as: Safa, M., et al., cAMP protects acute promyelocytic leukemia cells from arsenic trioxide-induced caspase-3 activation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.040i

M. Safa et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

3. Results 3.1. Elevation of cAMP levels inhibits ATO-induced apoptosis in acute promyelocytic leukemia NB4 cells Viability of NB4 cells was assessed by sub-G1 apoptosis assay after treatment of cells with 1 mM and 2 mM concentrations of ATO in the presence or absence of various concentrations of forskolin or 200 mM concentration of cAMP analog 6-MB-cAMP. Forskolin is an activator of adenylyl cyclase, resulting in increased cAMP levels within the cell (Insel and Ostrom, 2003). As shown in Fig. 1A, elevation of cAMP levels significantly attenuated the cytotoxic effects of different concentrations of ATO in NB4 cells. Metabolic activity of NB4 cells was assessed by MTT assay after treatment of cells with 1 μM and 2 μM concentrations of ATO in the presence or absence of the cAMP analog 6-MB-cAMP and forskolin. As presented (Fig. 1B), cell metabolic activity in cells treated with ATO was significantly decreased in comparison to the groups treated with ATO in combination with forskolin or cAMP which is related to the number of viable cells. To address whether the apoptosis induced by ATO in NB4 cells can be inhibited by elevation of cAMP, the cells were also analyzed for Annexin-V binding, and Annexin-V combined with propidium iodide (PI) by Annexin V-FLOUSStaining Kit. As presented in Fig. 1C, the presence of cAMPelevating agent forskolin substantially decreased percentages of Annexin-V and Annexin-V/PI double positive cells in comparison to the cells treated with ATO alone which indicates that cAMP protects NB4 cells from ATO-induced apoptosis. In addition we found that cAMP was able to profoundly inhibits the doxorubicininduced cell death in acute promyelocytic leukemia cells (data not shown) which represents a general action of cAMP against apoptosis induced by chemotherapeutic drugs in APL cells. 3.2. Increased levels of cAMP leads to G1 arrest in acute promyelocytic leukemia cells The effects of ATO and forskolin or their combination on cell cycle distribution of NB4 cells were examined. As shown in Fig. 2A, the cell population in sub-G1 region was decreased in the presence of forskolin which confirms the anti-apoptotic effect of cAMP on NB4 cells. Analysis of the cell cycle distribution showed that NB4 cells accumulated at the G1 phase under forskolin treatment alone or when ATO and forskolin treatments were combined, but not in the presence of ATO alone (Fig. 2A). It has been well known that p21, an important cyclin kinase inhibitor, negatively regulates the progression from G1 into S phase (Abbas and Dutta, 2009). Here we showed that NB4 cells express high levels of p21 expression in the presence of forskolin (Fig. 2B) which indicates that cAMP induces G1 arrest through upregulation of p21 protein in NB4 cells. 3.3. cAMP attenuates ATO-induced caspase activation in acute promyelocytic leukemia NB4 cells It has been shown that arsenic trioxide induces apoptosis of myeloid leukemia cells through caspase activation (Huang et al., 1999). Therefore, to ascertain whether enhanced levels of cAMP could influence the arsenic trioxide-induced caspase activation, NB4 cells were treated with 2 mM ATO in the presence or absence of 50 mM forskolin. The cells were harvested at different time points and caspase-3 protease activity was assessed in cell lysates by measuring hydrolysis of colorimetric caspase-3 substrate. Cotreatment of cells with forskolin and ATO resulted in significant attenuation of the ATOinduced increase in caspase-3 activities (Fig. 3A). In the next experiment, activation of caspase-3, caspase-9, caspase-7, and cleavage of poly (ADP-ribose) polymerase (PARP) was confirmed by western blot analysis. As shown in Fig. 3B, cleaved products of caspase-3, caspase-7,

3

caspase-9, and PARP were observed considerably within 24 h in response to arsenic trioxide treatment and this effect was reduced in the presence of forskolin. Our results indicate that caspase-mediated apoptosis was attenuated upon treatment of cells with ATO in the presence of forskolin, providing compelling evidence in support of a potent anti-apoptotic action of elevated cAMP levels in APL-derived NB4 cells. 3.4. Increased cAMP levels do not affect p53 protein in NB4 cells We have previously shown that elevation of cAMP levels inhibits doxorubicin-induced apoptosis in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells through dephosphorylation of p53 serine residues and prevention of p53 accumulation (Safa et al., 2010a). To this end, we wished to assess the effect of cAMP levels on p53 protein and its phosphorylation at Ser15 residue in NB4 cells. We first treated BCP-ALL cell line NALM-6 with doxorubicin in the presence or absence of forskolin to show the inhibitory effect of cAMP on p53 protein levels. The result shows that cAMP elevated levels dramatically reduced p53 protein accumulation (Fig. 4A). p53 protein expression was then evaluated at different times after treatment of NB4 cells with ATO in the presence or absence of cAMP-increasing agent forskolin. We found that NB4 cells constitutively expressed p53 protein which was not affected by cAMP levels at different time points. Furthermore, as shown in Fig. 4B, phosphorylation of p53 was also reduced upon elevation of cAMP levels in NALM-6 cells which indicates that inhibition of p53 is a mechanism of anti-apoptotic action of cAMP in these cells. To evaluate the influence of cAMP on p53 phosphorylation in NB4 cells, the cells were treated with DNAdamaging drug daunorubicin (1 mM) and ATO (2 mM) in the presence or absence of forskolin (50 mM). Although daunorubicin augmented phosphorylation of Ser15 p53 in comparison to untreated cells, the amount of phosphorylation was not changed in the presence of forskolin (Fig. 4B). These results suggest that cAMP-mediated inhibition of apoptosis is not related to p53 protein levels alteration in acute promyelocytic leukemia NB4 cells. 3.5. Inhibition of PKA reversed the protective effect of cAMP against ATO-induced apoptosis It is believed that PKA is the main effector for cAMP signal in eukaryotic cells (Beebe, 1994). To substantiate whether the inhibitory effect of cAMP on ATO-induced apoptosis is PKA dependent, NB4 cells were treated with PKI-Myr-(14–22)-amide (30 mM), a cell-permeable selective PKA C subunit inhibitor peptide, for 60 min prior to the addition of cAMP- increasing agent forskolin and ATO. After 24 h, cells were harvested and subjected to the apoptosis assay. As shown in Fig. 5A, inhibition of PKA by PKI-Myr(14–22)-amide diminished the protective effect of elevated cAMP levels against ATO-induced apoptosis. We further examined the phosphorylation of cAMP response element binding protein (CREB) at Ser-133, which serves as an indicator of PKA activation (Gonzalez and Montminy, 1989). We observed that elevation of cAMP levels by forskolin enhanced the phosphorylation of CREB at Ser133 whereas forskolin–induced CREB phosphorylation was totally inhibited by the PKA inhibitor PKI-Myr-(14–22)-amide (Fig. 5B). These findings suggest a major role for PKA in the cAMP-mediated inhibition of ATO-induced apoptosis 3.6. Elevation of cAMP levels increased phosphorylation of Bcl-2-associated death promoter (BAD) protein at Ser118 Phosphorylation of Ser118 Bad (Ser155 in murine Bad) by PKA results in the liberation of anti-apoptotic proteins Bcl-2/Bcl-XL sequestered by Bad and the consequent promotion of cell survival (Datta et al., 2000; Virdee et al., 2000). Therefore, to ascertain

Please cite this article as: Safa, M., et al., cAMP protects acute promyelocytic leukemia cells from arsenic trioxide-induced caspase-3 activation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.040i

M. Safa et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

35

30

30

%Sub-G1 cells

35

25 20 15 10

15 10

+

+

+

20

40

60

80

+ +

+

ATO (2 μM) Forskolin 6-MB-cAMP (200 μM)

100

+

+

+

+

+

20

40

60

80

+ +

+

Solvent

+

Solvent

100

+

Solvent

ATO (1 μM) Forskolin

20

0

0

6-MB-cAMP (200 μM)

25

5

5

Solvent

100 90

% Metabolic activity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

% Sub-G1 cells

4

80 70 60 50 40 30 20 10 0

Fig. 1. ATO induces dose-dependent cell death in NB4 cells that can be inhibited by increased cAMP levels. (A) NB4 cells were treated with the indicated doses of ATO with various concentrations of forskolin or 200 mM 6-MB-cAMP and cultured for 24 h before cell death analysis by Sub-G1 assay (n ¼3; n Po 0.05, relative to cells treated with ATO only). (B) Using MTT, cellular metabolic activity was decreased in NB4 cells exposed to ATO alone. (C) NB4 cells were treated with the indicated doses of ATO with forskolin (50 mM) for 24 h. Cells were analyzed for Annexin-V and Annexin-V plus PI uptake by flow cytometry. One representative experiment of at least three performed is presented.

Please cite this article as: Safa, M., et al., cAMP protects acute promyelocytic leukemia cells from arsenic trioxide-induced caspase-3 activation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.040i

M. Safa et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

+

ATO

+

+

+

+

Forskolin Time (hr)

+

+

+

+

+

+

12

8

+

16

24

p21 Actin

Relative expression of p21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

5

1.2

1.03

ATO

1

ATO+Fsk 0.73

1.8

0.62

1.6 0.31

1.4 1.2

0.1 0.0

0.0

0.0

0

8

12 16 Incubation time (hrs)

24

Fig. 2. Elevation of cAMP levels induces p21 protein expression and cell cycle arrest at the G1-S boundary. (A) NB4 cells were treated with the indicated concentrations of ATO in the presence or absence of forskolin (50 mM) for 36 h. The changes in cell cycle phase distribution were assessed by cell cycle analysis. One representative experiment among three independent assays is shown. (B) NB4 cells were treated with ATO (2 mM) in the presence or absence of forskolin (50 mM). Cells were harvested at the indicated times and subjected to western blot analysis of p21 protein expression levels. Actin serves as loading control. The figure shows one representative blot of 3 experiments. The relative expression of p21 was calculated by dividing the intensity of each band, quantified using ImageJ, by the respective intensity of actin (n¼ 3; n Po 0.05, relative to cells treated with ATO only).

Please cite this article as: Safa, M., et al., cAMP protects acute promyelocytic leukemia cells from arsenic trioxide-induced caspase-3 activation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.040i

M. Safa et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

6

Relative caspase-3 activity (405 nm)

0.25

NALM-6

Con

Doxorubicin Fsk

ATO

0.2

ATO + Fsk 0.15

25 µM 50 µM

p53

*

Fsk

Actin

Dox 0.1

NB4 ATO

0.05

Fsk (50 µM) Time (hr)

0 8 24 Incubation time (hrs)

8

12

16

24

p53 Actin

ATO (2 µM) Fsk (50 µM) Dox(200 nM)

+

+ +

NALM-6

+ +

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

Doxorubicin Full Length Caspase-9 (47 KDa)

Fsk Caspase-9

Caspase-3

Cleaved caspase-9 (37 KDa) Cleaved caspase-9 (35 KDa) *

pS15 p53

Cleaved caspase-3 (19 KDa) Cleaved caspase-3 (17 KDa)

Actin

25 µM

50 µM

Full Length Caspase-7 (35 KDa)

NB4

Caspase-7 Cleaved p20 kDa PARP-1

DNR

Cleaved p85 kDa

ATO Actin

Fsk (50 µM) Fig. 3. Effects of cAMP on ATO-induced caspase-3 activation. (A) NB4 cells were treated with ATO (2 mM) or forskolin (50 mM) or combination of ATO and forskolin for indicated times and harvested in lysis buffer. Doxorubicin-treated NB4 cells were used as positive control for caspase-3 activity. Enzymatic activities of caspase-3 were determined by incubation of 20 mg total protein with chromogenic substrate (DEVDpN) in a 100 ml assay buffer for 2 h at 37 1C. The release of chromogenic p-nitroaniline (pNA) was monitored spectrophotometrically at 405 nm (n¼ 3; n Po0.05). (B) NB4 cells were incubated with ATO (2 mM) in the presence or absence of forskolin (50 mM) for 24 h. Equal amounts of cell lysates (60 mg) were subjected to electrophoresis and analyzed by western blot for cleavage of caspase 3, caspase-9, caspase7, and PARP. The asterisk represents a nonspecific band seen using the caspase-9 antibody. Doxorubicin-treated NB4 cells were used as positive control for apoptosis. The immunoblot is representative of 3 independent experiments.

whether enhanced levels of cAMP could influence the level of Bad phosphorylation at Ser118, NB4 cells were treated with ATO (2 mM) in the presence or absence of forskolin (50 mM). Cells were harvested after 6 h to examine phosphorylation of Bad protein. As presented in Fig. 6A, elevation of cAMP levels increased Bad phosphorylation at Ser118 in NB4 cells. This finding indicates that cAMP-mediated Bad phosphorylation may be a one mechanism for protection of NB4 cells against ATO-induced apoptosis.

4. Discussion Arsenic trioxide is an effective treatment of acute promyelocytic leukemia even for APL patients who have been resistant to

pS15 p53

Actin Fig. 4. cAMP does not affect expression of p53 protein and its phosphorylation at Ser 15 residue in NB4 cells. (A) NALM-6 cells were used as positive control for the inhibitory action of cAMP on p53 protein. NALM-6 cells were treated with doxorubicin 0.5 mM in the presence or absence of forskolin 25 or 50 mM for 6 h and p53 protein levels were determined by immunoblotting. NB4 cells were treated with ATO (2 mM) alone or in combination with in forskolin (50 mM) for the times shown, and then subjected to Western blot analysis with p53 and actin antibodies. The figure shows one representative blot of 3 experiments. (B) NALM-6 cells were used as positive control to show the phosphorylation status of p53 upon elevation of cAMP levels. NALM-6 cells were treated with doxorubicin 0.5 mM in the presence or absence of forskolin 25 or 50 mM for 6 h and phospho-Ser15 p53 protein levels were determined by immunoblotting. NB4 cells were treated with ATO (2 mM) or daunorubicin (1 mM) in the presence or absence of forskolin for 4 h. Cells were harvested and processed for immunoblot analysis with anti-Ser15-phosphorylated p53 antibody.

all-trans retinoic acid. After the promising results obtained with ATO in APL, research activities have been conducted to investigate its mechanisms of action. The clinical response to arsenic trioxide is associated with incomplete cytodifferentiation and the induction of apoptosis with caspase-3 activation in leukemic cells

Please cite this article as: Safa, M., et al., cAMP protects acute promyelocytic leukemia cells from arsenic trioxide-induced caspase-3 activation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.040i

M. Safa et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

ATO Fsk

+

+ +

+

Bad P-S118 Bad Actin

ATO Fsk Myr-PKI

Ratio of p-Bad/Bad protein

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

7

3.5 3 2.5 2 1.5 1 0.5 0

P-S133 CREB Actin Fig. 5. Inhibition of PKA reversed cAMP-mediated protection of NB4 cells. (A) NB4 cells were pretreated with PKI-Myr-(14–22)-amide (30 mM) for 60 min. Cells were then incubated with or without forskolin (50 mM) and ATO (2 mM) for 24 h. PKA inhibitor was included throughout the experiment. Cells were then analyzed for Annexin-V staining by flow cytometry (n¼ 3; n Po 0.05). (B) NB4 cells were pretreated with PKI-Myr-(14–22)-amide for 60 min before exposure to ATO (2 mM) and forskolin (50 mM) for a further 6 h. Whole cell extracts were then prepared, and western blot analysis was performed using phospho-CREB and actin antibodies.

(Soignet et al., 1998). Cell biology studies indicated that ATO within the concentration range found in the plasma of APL patients exerts dual effects on APL cells. Apoptosis is observed when cells are treated with 0.5–2.0 mM of ATO while incomplete differentiation is evident using low concentrations (0.1–0.5 mM) of the drug (Shao et al., 1998; Zhang et al., 2001). Identification of novel approaches to induce leukemic cell differentiation and apoptosis is a milestone on the road of successful treatment of acute promyelocytic leukemia. It has well been established that cAMP in combination with both ATO and ATRA stimulates differentiation of APL blasts (Guillemin et al., 2002; Zhu et al., 2002). This means that cAMP agonists might be suitable candidates as adjuncts to the treatment of acute promyelocytic leukemia. However, there is evidence that cAMP may interfere with intracellular signaling pathways in leukemia cells leading to undesirable consequences (Naderi et al., 2011; Safa et al., 2010b). Whether the antiapoptotic function of cAMP agonists can impede arsenic trioxide-mediated apoptosis that has not been elucidated yet. We show here that elevation of cAMP levels protect NB4 acute promyelocytic leukemia cells against apoptosis induced by clinically relevant concentrations of arsenic trioxide. In our recent study it was shown that cAMPincreasing agents decrease p53 protein levels in pre-B acute lymphoblastic leukemia cells both with and without exposure to doxorubicin (Safa et al., 2011). Although elevation of cAMP levels had no effect on p53 protein in NB4 cells, inhibition of caspase-3 activation was seen in ATO-treated cells in the presence of cAMPincreasing agent forskolin (Fig. 3). The cyclin-dependent kinase inhibitor p21 is induced by both p53-dependent and -independent

Fig. 6. The phosphorylation status of Bad in forskolin- and ATO-exposed leukemic cells. (A) NB4 cells were treated with ATO (2 mM) in the presence or absence of forskolin (50 mM) for 4 h, and whole cell extracts analyzed by immunoblotting for Bad, P-Ser118Bad, with actin as loading control. (B) The levels of phosphorylated Ser118 normalized to total Bad protein levels that were obtained by densitometric quantification of three independent experiments using ImageJ. n P o0.05, relative to cells that were not treated with forskolin.

mechanisms in response to many stimuli and is involved in regulation of fundamental cellular processes such as cell proliferation, differentiation, senescence, and apoptosis (Gartel and Tyner, 2002; Romanov et al., 2012). Since its discovery, it has become increasingly clear that p21 expression can both promote and inhibit tumorigenic processes, depending on the cellular context (Warfel and El-Deiry, 2013). Of particular interest, several recent studies have pointed out that p21 confers resistance to apoptosis induced by multiple chemotherapeutic drugs and other stimuli (Ferrandiz et al., 2010; Gorospe et al., 1996; Koster et al., 2010; Ruan et al., 1998; Xaus et al., 1999). One of the most straight forward mechanisms by which p21 might protect cells from apoptosis is the direct binding and inhibition of pro-caspase-3 that is mediated by PKA (Suzuki et al., 1998; Suzuki et al., 1999). In addition, p21 can protect cells from IR-induced apoptosis by suppression of CDK activity that seems to be required for activation of the caspase cascade downstream of the mitochondria (Sohn et al., 2006). Based on the above mentioned studies, it is postulated that activation of cAMP-PKA signaling pathway and concomitant upregulation of p21 protein might be a mechanism that protects NB4 cells from caspase-3 mediated apoptosis. In our experimental system, the inhibition of PKA in the presence of forskolin increased arsenic trioxide-induced apoptosis indicating that the inhibitory effect of cAMP is mediated by PKA. PKA activation in the cAMP-protected NB4 cells may provide prosurvival effects through phosphorylation of specific target proteins. Our results demonstrate increased phosphorylation of S118Bad and S133CREB which are recognized PKA substrate sites (Gonzalez and Montminy, 1989; Virdee et al., 2000). PKA can thereby suppress the pro-apoptotic function of Bad by disruption of its ability to heterodimerize with the pro-survival proteins Bcl-2 and Bcl-Xl (Burlacu, 2003). CREB is overexpressed and constitutively phosphorylated in the majority of primary AML samples which is

Please cite this article as: Safa, M., et al., cAMP protects acute promyelocytic leukemia cells from arsenic trioxide-induced caspase-3 activation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.040i

M. Safa et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

Arsenic trioxide (0.1 ~ 0.5 μM)

Arsenic trioxide (1.0 ~ 2.0 μM)

cAMP

Partial differentiation

Apoptosis

Fig. 7. Model depicting pro-differentiating and pro-survival effects of cAMP on ATO-exposed NB4 cells.

associated with poor outcome in these patients (Sakamoto and Frank, 2009). Given the fact that AML cells are more dependent on CREB than normal myeloid progenitor cells (Sandoval et al., 2009), over-activation of CREB by cAMP agonists can provide prosurvival signal in acute promyelocytic leukemia cells. Our results are consistent with the recent study by Gausdal et al. reporting that cAMP-increasing agents promote APL progression in NSG mice carrying the NB4 APL cell line and protect APL cells against anthracycline-induced death in vitro (Gausdal et al., 2013). Based on these findings together with previous studies (Chelbi-alix et al., 2003; Miller, 2002; Zhao et al., 2004; Zhu et al., 2002), we propose a model in which activation of cAMP signaling exerts differential modulatory effects in ATO-treated APL cells, depending on the concentration of the drug (Fig. 7). In conclusion, our study reveals a cAMP/PKA dependent mechanism of protection against ATO-induced apoptosis in APLderived NB4 cells. In this regard, under inflammatory conditions increased levels of cAMP elevating prostaglandins acting via adenyl cyclase may efficiently prevent apoptotic initiating pathways in acute promyelocytic leukemia cells. Understanding transducing mechanisms that confer inducible resistance toward chemotherapeutic agents in APL cells may increase our knowledge in current strategies for the treatment of APL.

Declaration of interest The authors declare that they have no conflict of interest.

Acknowledgments This study was supported by the Grant 13073 from Iran University of Medical Sciences. References Abbas, T., Dutta, A., 2009. p21 in cancer: intricate networks and multiple activities. Nat. Rev. Cancer 9, 400–414. Beebe, S.J., 1994. The cAMP-dependent protein kinases and cAMP signal transduction. Semin. Cancer Biol. 5, 285–294. Burlacu, A., 2003. Regulation of apoptosis by Bcl-2 family proteins. J. Cell Mol. Med. 7, 249–257. Chelbi-alix, M.K., Bobe, P., Benoit, G., Canova, A., Pine, R., 2003. Arsenic enhances the activation of Stat1 by interferon gamma leading to synergistic expression of IRF-1. Oncogene 22, 9121–9130.

Chen, G.Q., Shi, X.G., Tang, W., Xiong, S.M., Zhu, J., Cai, X., Han, Z.G., Ni, J.H., Shi, G.Y., Jia, P.M., Liu, M.M., He, K.L., Niu, C., Ma, J., Zhang, P., Zhang, T.D., Paul, P., Naoe, T., Kitamura, K., Miller, W., Waxman, S., Wang, Z.Y., de The, H., Chen, S.J., Chen, Z., 1997. Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): I. As2O3 exerts dose-dependent dual effects on APL cells. Blood 89, 3345–3353. Cheng, X., Ji, Z., Tsalkova, T., Mei, F., 2008. Epac and PKA: a tale of two intracellular cAMP receptors. Acta Biochim. Biophys. Sin. (Shanghai) 40, 651–662. Datta, S.R., Katsov, A., Hu, L., Petros, A., Fesik, S.W., Yaffe, M.B., Greenberg, M.E., 2000. 14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol. Cell 6, 41–51. de The, H., Chen, Z., 2010. Acute promyelocytic leukaemia: novel insights into the mechanisms of cure. Nat. Rev. Cancer 10, 775–783. de The, H., Lavau, C., Marchio, A., Chomienne, C., Degos, L., Dejean, A., 1991. The PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR. Cell 66, 675–684. Dong, H., Zitt, C., Auriga, C., Hatzelmann, A., Epstein, P.M., 2010. Inhibition of PDE3, PDE4 and PDE7 potentiates glucocorticoid-induced apoptosis and overcomes glucocorticoid resistance in CEM T leukemic cells. Biochem. Pharmacol. 79, 321–329. Ferrandiz, N., Caraballo, J.M., Albajar, M., Gomez-Casares, M.T., Lopez-Jorge, C.E., Blanco, R., Delgado, M.D., Leon, J., 2010. p21(Cip1) confers resistance to imatinib in human chronic myeloid leukemia cells. Cancer Lett. 292, 133–139. Gartel, A.L., Tyner, A.L., 2002. The role of the cyclin-dependent kinase inhibitor p21 in apoptosis. Mol. Cancer Ther. 1, 639–649. Gausdal, G., Wergeland, A., Skavland, J., Nguyen, E., Pendino, F., Rouhee, N., McCormack, E., Herfindal, L., Kleppe, R., Havemann, U., Schwede, F., Bruserud, O., Gjertsen, B.T., Lanotte, M., Segal-Bendirdjian, E., Doskeland, S.O., 2013. Cyclic AMP can promote APL progression and protect myeloid leukemia cells against anthracycline-induced apoptosis. Cell Death Dis. 4, e516. Ghavamzadeh, A., Alimoghaddam, K., Rostami, S., Ghaffari, S.H., Jahani, M., Iravani, M., Mousavi, S.A., Bahar, B., Jalili, M., 2011. Phase II study of single-agent arsenic trioxide for the front-line therapy of acute promyelocytic leukemia. J. Clin. Oncol. 29, 2753–2757. Gonzalez, G.A., Montminy, M.R., 1989. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59, 675–680. Gorospe, M., Wang, X., Guyton, K.Z., Holbrook, N.J., 1996. Protective role of p21 (Waf1/Cip1) against prostaglandin A2-mediated apoptosis of human colorectal carcinoma cells. Mol. Cell Biol. 16, 6654–6660. Grignani, F., Fagioli, M., Alcalay, M., Longo, L., Pandolfi, P.P., Donti, E., Biondi, A., Lo Coco, F., Pelicci, P.G., 1994. Acute promyelocytic leukemia: from genetics to treatment. Blood 83, 10–25. Guillemin, M.C., Raffoux, E., Vitoux, D., Kogan, S., Soilihi, H., Lallemand-Breitenbach, V., Zhu, J., Janin, A., Daniel, M.T., Gourmel, B., Degos, L., Dombret, H., Lanotte, M., De The, H., 2002. in vivo activation of cAMP signaling induces growth arrest and differentiation in acute promyelocytic leukemia. J. Exp. Med. 196, 1373–1380. Huang, M.E., Ye, Y.C., Chen, S.R., Chai, J.R., Lu, J.X., Zhoa, L., Gu, L.J., Wang, Z.Y., 1988. Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood 72, 567–572. Huang, X.J., Wiernik, P.H., Klein, R.S., Gallagher, R.E., 1999. Arsenic trioxide induces apoptosis of myeloid leukemia cells by activation of caspases. Med. Oncol. 16, 58–64. Insel, P.A., Ostrom, R.S., 2003. Forskolin as a tool for examining adenylyl cyclase expression, regulation, and G protein signaling. Cell Mol. Neurobiol. 23, 305–314. Insel, P.A., Zhang, L., Murray, F., Yokouchi, H., Zambon, A.C., 2012. Cyclic AMP is both a pro-apoptotic and anti-apoptotic second messenger. Acta Physiol. (Oxf) 204, 277–287. Koster, R., di Pietro, A., Timmer-Bosscha, H., Gibcus, J.H., van den Berg, A., Suurmeijer, A.J., Bischoff, R., Gietema, J.A., de Jong, S., 2010. Cytoplasmic p21 expression levels determine cisplatin resistance in human testicular cancer. J. Clin. Invest. 120, 3594–3605. Lallemand-Breitenbach, V., Guillemin, M.C., Janin, A., Daniel, M.T., Degos, L., Kogan, S.C., Bishop, J.M., de The, H., 1999. Retinoic acid and arsenic synergize to eradicate leukemic cells in a mouse model of acute promyelocytic leukemia. J. Exp. Med. 189, 1043–1052. Miller Jr., W.H., 2002. Molecular targets of arsenic trioxide in malignant cells. Oncologist 7 (Suppl 1), 14–19. Naderi, E.H., Findley, H.W., Ruud, E., Blomhoff, H.K., Naderi, S., 2009. Activation of cAMP signaling inhibits DNA damage-induced apoptosis in BCP-ALL cells through abrogation of p53 accumulation. Blood 114, 608–618. Naderi, E.H., Jochemsen, A.G., Blomhoff, H.K., Naderi, S., 2011. Activation of cAMP signaling interferes with stress-induced p53 accumulation in all-derived cells by promoting the interaction between p53 and HDM2. Neoplasia 13, 653–663. Romanov, V.S., Pospelov, V.A., Pospelova, T.V., 2012. Cyclin-dependent kinase inhibitor p21(Waf1): contemporary view on its role in senescence and oncogenesis. Biochemistry (Mosc) 77, 575–584. Ruan, S., Okcu, M.F., Ren, J.P., Chiao, P., Andreeff, M., Levin, V., Zhang, W., 1998. Overexpressed WAF1/Cip1 renders glioblastoma cells resistant to chemotherapy agents 1,3-bis(2-chloroethyl)-1-nitrosourea and cisplatin. Cancer Res. 58, 1538–1543. Safa, M., Kazemi, A., Zaker, F., Razmkhah, F., 2011. Cyclic AMP-induced p53 destabilization is independent of EPAC in pre-B acute lymphoblastic leukemia cells in vitro. J. Recept Signal Transduct. Res. 31, 256–263. Safa, M., Kazemi, A., Zand, H., Azarkeivan, A., Zaker, F., Hayat, P., 2010a. Inhibitory role of cAMP on doxorubicin-induced apoptosis in pre-B ALL cells through dephosphorylation of p53 serine residues. Apoptosis 15, 196–203.

Please cite this article as: Safa, M., et al., cAMP protects acute promyelocytic leukemia cells from arsenic trioxide-induced caspase-3 activation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.040i

M. Safa et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Safa, M., Zand, H., Mousavizadeh, K., Kazemi, A., Bakhshayesh, M., Hayat, P., 2010b. Elevation of cyclic AMP causes an imbalance between NF-kappaB and p53 in NALM-6 cells treated by doxorubicin. FEBS Lett. 584, 3492–3498. Sakamoto, K.M., Frank, D.A., 2009. CREB in the pathophysiology of cancer: implications for targeting transcription factors for cancer therapy. Clin. Cancer Res. 15, 2583–2587. Sandoval, S., Pigazzi, M., Sakamoto, K.M., 2009. CREB: a key regulator of normal and neoplastic hematopoiesis. Adv. Hematol. 2009, 634292. Sanz, M.A., 2006. Treatment of acute promyelocytic leukemia. Hematol. Am. Soc. Hematol. Educ. Program, 147–155. Sanz, M.A., Martin, G., Lo Coco, F., 2003. Choice of chemotherapy in induction, consolidation and maintenance in acute promyelocytic leukaemia. Best Pract. Res. Clin. Haematol. 16, 433–451. Shao, W., Fanelli, M., Ferrara, F.F., Riccioni, R., Rosenauer, A., Davison, K., Lamph, W.W., Waxman, S., Pelicci, P.G., Lo Coco, F., Avvisati, G., Testa, U., Peschle, C., GambacortiPasserini, C., Nervi, C., Miller Jr., W.H., 1998. Arsenic trioxide as an inducer of apoptosis and loss of PML/RAR alpha protein in acute promyelocytic leukemia cells. J. Natl. Cancer Inst. 90, 124–133. Shen, Z.X., Chen, G.Q., Ni, J.H., Li, X.S., Xiong, S.M., Qiu, Q.Y., Zhu, J., Tang, W., Sun, G.L., Yang, K.Q., Chen, Y., Zhou, L., Fang, Z.W., Wang, Y.T., Ma, J., Zhang, P., Zhang, T.D., Chen, S.J., Chen, Z., Wang, Z.Y., 1997. Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): II. clinical efficacy and pharmacokinetics in relapsed patients. Blood 89, 3354–3360. Sohn, D., Essmann, F., Schulze-Osthoff, K., Janicke, R.U., 2006. p21 blocks irradiation-induced apoptosis downstream of mitochondria by inhibition of cyclin-dependent kinase-mediated caspase-9 activation. Cancer Res. 66, 11254–11262. Soignet, S.L., Maslak, P., Wang, Z.G., Jhanwar, S., Calleja, E., Dardashti, L.J., Corso, D., DeBlasio, A., Gabrilove, J., Scheinberg, D.A., Pandolfi, P.P., Warrell Jr., R.P., 1998.

9

Complete remission after treatment of acute promyelocytic leukemia with arsenic trioxide. N. Engl. J. Med. 339, 1341–1348. Suzuki, A., Tsutomi, Y., Akahane, K., Araki, T., Miura, M., 1998. Resistance to Fasmediated apoptosis: activation of caspase 3 is regulated by cell cycle regulator p21WAF1 and IAP gene family ILP. Oncogene 17, 931–939. Suzuki, A., Tsutomi, Y., Yamamoto, N., Shibutani, T., Akahane, K., 1999. Mitochondrial regulation of cell death: mitochondria are essential for procaspase 3-p21 complex formation to resist Fas-mediated cell death. Mol. Cell Biol. 19, 3842–3847. Virdee, K., Parone, P.A., Tolkovsky, A.M., 2000. Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novel site, contributes to cell survival. Curr. Biol. 10, R883. Warfel, N.A., El-Deiry, W.S., 2013. p21WAF1 and tumourigenesis: 20 years after. Curr. Opin. Oncol. 25, 52–58. Xaus, J., Cardo, M., Valledor, A.F., Soler, C., Lloberas, J., Celada, A., 1999. Interferon gamma induces the expression of p21waf-1 and arrests macrophage cell cycle, preventing induction of apoptosis. Immunity 11, 103–113. Zhang, T.D., Chen, G.Q., Wang, Z.G., Wang, Z.Y., Chen, S.J., Chen, Z., 2001. Arsenic trioxide, a therapeutic agent for APL. Oncogene 20, 7146–7153. Zhao, Q., Tao, J., Zhu, Q., Jia, P.M., Dou, A.X., Li, X., Cheng, F., Waxman, S., Chen, G.Q., Chen, S.J., Lanotte, M., Chen, Z., Tong, J.H., 2004. Rapid induction of cAMP/PKA pathway during retinoic acid-induced acute promyelocytic leukemia cell differentiation. Leukemia 18, 285–292. Zhu, Q., Zhang, J.W., Zhu, H.Q., Shen, Y.L., Jia, P.M., Yu, Y., Cai, X., Waxman, S., Lanotte, M., Chen, S.J., Chen, Z., Tong, J.H., 2002. Synergic effects of arsenic trioxide and cAMP during acute promyelocytic leukemia cell maturation subtends a novel signaling cross-talk. Blood 99, 1014–1022.

Please cite this article as: Safa, M., et al., cAMP protects acute promyelocytic leukemia cells from arsenic trioxide-induced caspase-3 activation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.040i

cAMP protects acute promyelocytic leukemia cells from arsenic trioxide-induced caspase-3 activation and apoptosis.

More recently, arsenic trioxide (ATO), was integrated into acute promyelocytic leukemia (APL) treatment, showing high efficacy and tolerability in pat...
2MB Sizes 0 Downloads 4 Views