SB202190 | ATPases Signal

The p38 pathway inhibitor SB202190 activates MEK/MAPK to stimulate the growth of leukemia cells
Megumi Hirosawaa,1, Masako Nakaharaa,b,1, Risa Otosakab, Akemi Imotoa,
Toshio Okazakia,b, Shinichiro Takahashia,b,∗
a Division of Molecular Hematology, Kitasato University Graduate School of Medical Sciences, Japan
b Division of Hematology, Kitasato University School of Allied Health Sciences, Japan

A R T I C L E I N F O A B S T R A C T

Article history:
Received 22 February 2008
Received in revised form 14 August 2008 Accepted 25 September 2008
Available online 7 November 2008

Keywords:
SB202190
Leukemia p38
ERK
Cell growth THP-1
MV4-11

In this study, the biological effects of signal transduction inhibitors on leukemia cells were examined. We found that the p38 inhibitor SB202190 enhanced the growth of THP-1 and MV4-11 cells. To deter- mine the pathway affected by SB202190, we examined the 50% effective dose (ED50) values for THP-1 cell growth in combination with several inhibitors. In the presence of SB202190, the ED50 values for the far- nesyltransferase inhibitor FPT inhibitor II and MEK inhibitor U0126 were signiﬁcantly decreased. Western blot analysis revealed that SB202190 increased the phosphorylation of C-Raf and extracellular regulated kinase (ERK), suggesting that Ras–Raf–MEK–mitogen-activated protein kinase (MAPK) pathway activation is involved in the leukemia cell growth induced by SB202190.
© 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Exploitation of knowledge gained from studying cell sig- naling pathways can be extremely attractive when considering chemotherapeutic options for treating a variety of neoplasias. The mitogen-activated protein kinase (MAPK) pathways are key regulators of cell proliferation, differentiation and survival [1]. Thus, the constituents of these pathways are ideal candidates for novel anti-neoplastic chemotherapies [2]. Although genetic alter- ations affecting the functions of transcription factors that regulate myeloid maturation play important roles in leukemogenesis [3], inappropriate MAPK activation may also play a role in leukemic transformation. The Ras–MAPK and phosphoinositide 3-kinase (PI3K)–Akt pathways are frequently activated in hematological malignancies [4,5]. Upregulation of the MAPK network in acute myeloid leukemia (AML) has been reported to arise through sev- eral mechanisms, including the Fms-like tyrosine 3 kinase receptor, c-KIT and Ras mutations frequently detected in AML [6–9].

∗ Corresponding author at: Division of Molecular Hematology, Kitasato University Graduate School of Medical Sciences, Japan. Tel.: +81 42 778 8216;
fax: +81 42 778 8216.
E-mail address: [email protected] (S. Takahashi).
1 These authors contributed equally to this work.

Small molecules designed to selectively target key components of these signal transduction cascades induce apoptosis and/or markedly increase the conventional drug sensitivities of AML blasts in vitro. To further understand the roles of abnormal kinase path- ways in leukemia cells, we initially examined the effects of various inhibitors, including the receptor tyrosine kinase inhibitor AG1296, MAPK/extracellular regulated kinase (ERK) kinase (MEK) inhibitor U0126, farnesyltransferase inhibitor L-744832, FPT inhibitor II, PI3K/Akt inhibitor LY294002 and p38 inhibitor SB202190, on the growth of leukemia cells.
SB202190, a pyridinyl imidazole compound, acts as a speciﬁc inhibitor of p38α and p38β [10,11], but not p38μ and p386 [12], through competition with ATP for the same binding site on p38 [13]. Crystal structure and mutagenesis analyses revealed that a single residue difference between p38 and other MAPKs, such as Jun N-terminal kinase and ERK, determines the speciﬁcity of this pyridinyl imidazole compound [13,14]. This speciﬁc inhibitor has been widely used in investigations of the physiological functions of p38 [15,16].
In the present study, we found that SB202190 potently activated the growth of THP-1 and MV4-11 leukemia cells. Furthermore, we found that C-Raf and ERK were activated by SB202190, suggesting that activation of the Raf–MEK–MAPK pathway was involved in the aberrant cell growth. To the best of our knowledge, this is the ﬁrst report demonstrating aberrant leukemia cell growth induced by

Fig. 1. The p38 inhibitor SB202190 increases THP-1 and MV4-11 cell survival. (A–E) Inhibitors (A: AG1296; B: U0126; C: L-744832; D: LY294002; E: SB202190) were added to the indicated cells. The survival rates of the cells were measured by cell growth assays at 3 days after the reagent addition. Concentration 0 refers to solvent (DMSO)-treated cells. The numbers presented in the bars are the reagent concentrations (µM) added to the cells. Data are the means of triplicate samples and the standard deviations are shown as error bars. The data shown are representative of at least three independent experiments.

the p38 inhibitor SB202190. These ﬁndings may warrant caution regarding the use of pharmacological inhibitors of p38 for a certain portion of leukemia patients.

2. Materials and methods

2.1. Cell culture and reagents

MV4-11, RS4-11, PL-21, NB4, THP-1 and KG-1a cells were grown in RPMI medium (GIBCO BRL, Rockville, MD) containing 10% heat-inactivated fetal bovine serum (Biowest, Miami, FL), 100 U/ml penicillin and 100 µg/ml streptomycin (GIBCO BRL) as described previously [17]. Cells were incubated in the presence or absence of the kinase inhibitors AG1296 (Calbiochem, San Diego, CA), L-744832 (Calbiochem), FPT inhibitor II (Calbiochem), U0126 (Calbiochem), LY294002 (Sigma, St. Louis, MO) or SB202190 (Sigma) as indicated and assayed for cell growth and signaling.

2.2. Cell growth assays

Cell growth was determined by a dye reduction assay involving a tetrazolium salt, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H- tetrazolium, monosodium salt (Dojindo, Tokyo, Japan), which is a modiﬁcation of the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolim (MTT) assay. The 50% effective dose (ED50 ) values were calculated from the data obtained by the cell growth assays. We selected seven different drug doses, with one solvent dose as a control, and performed the assays at 3 days after addition of the drugs. We calculated the drug effects as the ratio of the absorbance (490 nm) of drug-treated cells to the absorbance of solvent-treated cells in the cell growth assays. The calculated ratios were analyzed using a website (http://chiryo.phar.nagoya- cu.ac.jp/javastat/Graded50-j.htm) and the ED50 values were obtained. Viability was measured by the Trypan blue dye exclusion method as previously described [18].

2.3. Immunoblotting

Anti-phospho-ERK rabbit polyclonal, anti-phospho-Akt rabbit monoclonal, anti-phospho-C-Raf (Ser289/296/301) rabbit polyclonal and anti-total-Akt rabbit monoclonal antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-total-ERK mouse monoclonal and anti-total-C-Raf mouse monoclonal antibodies were obtained from BD Biosciences (San Jose, CA). Immunoblotting was performed as previously described [17].

2.4. Sequencing of Ras mutations

The following primers were used to identify mutations in codons 12, 13 and 61 of N-Ras and K-Ras: N-Ras: forward, 5∗-CTGTCCAAAGCAGAGGCAGTG- 3∗ and reverse, 5∗ -AGGCTTCCTCTGTGTATTTGCC-3∗ [19]; K-Ras: forward, 5∗- CCTGCTGAAAATGACTGAAT-3∗ and reverse, 5∗ -ATACACAAAGAAAGCCCTCC-3∗ [20].
cDNAs were prepared from each cell line using reverse transcriptase (Superscript II; Invitrogen, Carlsbad, CA). The sequencing procedures used were described previously [21]. Brieﬂy, the ampliﬁed cDNAs were puriﬁed by 2% agarose gel elec- trophoresis, recovered with a Wizard SV Gel and PCR Clean-up System (Promega, Madison, WI) and cloned into the pGEMT easy vector (Promega). At least ﬁve inde- pendent clones were employed for each sequence analysis. The T7 primer was used for sequencing with a Big Dye Sequencing Kit (Applied Biosystems, Foster City, CA). The products were analyzed using an ABI Prism DNA Sequencer 3130 (Applied Biosystems).

3. Results

3.1. p38 inhibitor SB202190 potently enhances the growth of THP-1 and MV4-11 cells

First, we examined the effects of treatment with the inhibitors AG1296, U0126, L-744832, LY294002 and SB202190 on the growth
of leukemia cell lines. The characteristics of the cells used in this study are summarized in Table 1. Among the inhibitors examined, we found that addition of SB202190 was not cytotoxic and rather increased the survival of several cell lines, including THP-1 cells (Fig. 1). To further examine the cell growth, Trypan blue dye exclu- sion assays were performed (Fig. 2). As a result, we found that the growth rates of THP-1 cells were signiﬁcantly increased in the presence of 1.3–5 µM SB202190. Although the difference was mod- est, MV4-11 cell growth was also reproducibly increased by 1.3 µM SB202190.

Table 1
Characteristics of the cells used in this study.

Cell line Type Ras mutation
THP-1 AML M4 N-Ras/12a
MV4-11 AML M5 K-Ras/12, 18a
KG1a AML M6 Nonea
NB4 AML M3 Nonea
PL21 AML M3 None
RS4-11 ALL None
a Ref. [44].

3.2. ED50 values for U0126 and FPT inhibitor II are signiﬁcantly decreased by SB202190 in THP-1 cells

Since we found that leukemia cell growth was stimulated by SB202190, we examined which pathway was responsible for the aberrant cell growth. To achieve this, cells were cultured in the presence or absence of SB202190 together with various amounts of inhibitors. The ED50 for each inhibitor was calculated (http://chiryo. phar.nagoya-cu.ac.jp/javastat/Graded50-j.htm) and the change in ED50 after the addition of SB202190 was evaluated. We selected THP-1 cells for these experiments because their growth stimu- lation by SB202190 was most evident (Figs. 1 and 2). As shown in Fig. 3, the ED50 values of U0126 and FPT inhibitor II were signiﬁcantly decreased in the presence of SB202190 (U0126: ED50 average [av.] 22.95 µM, standard deviation [S.D.] ± 4.42; SB202190 + U0126: ED50 av. 11.17 µM, S.D. ± 4.28; FPT inhibitor II: ED50 av. 25.69 µM, S.D. ± 5.20; SB202190 + FPT inhibitor II: ED50
av. 14.39 µM, S.D. ± 3.09). Although not signiﬁcant, the ED50 of L- 744832 also tended to be reduced by SB202190 (L-744832: ED50
av. 10.26 µM, S.D. 2.892; SB202190 + L-744832: ED50 av. 9.33 µM,
S.D. 0.898). Since inhibitors are more effective toward activated kinases, these ﬁndings suggest that the Ras–MEK–MAPK path- way was activated by the addition of SB202190. In contrast, the ED50 of the PI3K–Akt inhibitor LY294002 was increased in the presence of SB202190 (LY294002: ED50 av. 2.57 µM, S.D. ± 0.45; SB202190 + LY294002: ED50 av. 4.71, S.D. 0.92), indicating sup- pression of the PI3K–Akt pathway.

3.3. Aberrant activation of ERK and C-Raf by SB202190

Next, we veriﬁed whether the changes in ED50 induced by SB202190 reﬂected its impacts on kinase pathways. We exam- ined ERK and Akt phosphorylation because the ED50 values of the

Fig. 2. SB202190 stimulates the growth of THP-1 and MV4-11 cells. The indicated amounts of SB202190 were added to THP-1, MV4-11, KG1a, NB4, PL21 and RS4- 11 cells. Trypan blue dye exclusion assays were performed every 2 days and live cells were plotted. The data shown are representative of at least three independent experiments and reproducibility was conﬁrmed.

Fig. 3. Combined effects of inhibitors on the growth of THP-1 cells. (A) Survival rates of cells in the presence of combinations of inhibitors. Cells were cultured in the presence (5 µM) or absence of SB202190 with various amounts of the indicated inhibitors for 3 days and subjected to cell growth assays. The data shown are representative of a series of experiments. (B) Changes in ED50 values. The ED50 for each inhibitor was calculated (http://chiryo.phar.nagoya-cu.ac.jp/javastat/Graded50-j.htm) from the above experiments and the change in ED50 after the addition of SB202190 was evaluated. SB denotes SB202190. The data shown were calculated from three to four independent experiments.

farnesyltransferase–MEK–MAPK inhibitors and Akt inhibitor were affected by SB202190 addition to THP-1 cells (Fig. 3B). Western blot analyses revealed that ERK was constitutively phosphorylated in THP-1, MV4-11, KG-1a, NB4 and PL21 cells (Fig. 4A). As expected, ERK phosphorylation was signiﬁcantly increased by the addition of SB202190 to these cells. In SB202190-insensitive RS4-11 cells, neither basal phosphorylation of ERK nor induction of ERK phos- phorylation was observed after addition of SB202190. Furthermore, consistent with the changes in the ED50 values, reduced levels of Akt phosphorylation were observed after addition of SB202190 to THP-1, MV4-11 and KG1a cells. To further clarify the detailed bio- chemical mechanisms of the observed biological effects on THP-1 cells, we performed time-dependent analyses of MAPK and Akt sig- naling. As shown in Fig. 4B, ERK phosphorylation was observed after only 15 min of stimulation with SB202190, whereas Akt suppres- sion was hardly seen after 6 h. These ﬁndings demonstrate that ERK phosphorylation precedes the effects of SB202190 on the PI3K–Akt pathway and indicate that activation of ERK is a pivotal effect of SB202190. ERK phosphorylation was even observed in the pres- ence of 0.62 µM SB202190 (Fig. 4C). Next, we examined the effects of SB202190 on C-Raf phosphorylation, since C-Raf is one of the major upstream components of the MEK–MAPK signaling pathway [22]. As shown in Fig. 5, phosphorylation of C-Raf was augmented

after the addition of SB202190, suggesting that C-Raf is involved in the MEK–MAPK pathway activation induced by this reagent.

4. Discussion

Over the years, it has been established that the p38 pathway is activated in response to stress, heat shock and radiation [10,23,24]. In addition, the p38 pathway is required for interferon α-dependent gene transcription [25] and the generation of growth inhibitory responses [26]. Furthermore, p38 exhibits regulatory effects on the induction of apoptosis [27,28]. There are strong indications that this signaling cascade acts as a convergence point for signaling path- ways activated by different cytokines to mediate myelosuppressive signals, since addition of another pyridinyl imidazole compound inhibitor of p38, namely SB203580, to bone marrow samples from aplastic anemia patients was found to stimulate hematopoietic pro- genitor colony formation in vitro [29]. Parmar et al. [30] recently found that pharmacological inhibition of p38 reverses the suppres- sive effects of STI571 on primary leukemic CFU-GM progenitors, indicating that activation of this signaling cascade is essential for the antileukemic effects of imatinib mesylate. These studies sup- port our ﬁndings of increased leukemia cell growth after inhibition of p38.

Fig. 4. SB202190 modulates ERK and Akt phosphorylation. (A) The indicated cells were cultured in the presence (5 µM) or absence of SB202190 for 6 h and analyzed for their ERK and Akt activities by western blotting. The membranes were ﬁrst probed with an anti-phospho-ERK rabbit polyclonal antibody or anti-phospho-Akt rabbit monoclonal antibody. Next, the membranes were stripped and reprobed with an anti-total-ERK mouse monoclonal antibody or anti-total-Akt rabbit monoclonal antibody to verify equal protein loading. (B) Time-dependent analyses of ERK and Akt phosphorylation. The phosphorylation levels of ERK and Akt were examined by western blotting at 15 min, 60 min and 6 h after the addition of 5 µM SB202190 to THP-1 cells. (C) Concentration-dependent analyses of ERK phosphorylation. THP-1 cells were incubated with the indicated concentrations of SB202190 for 15 min and analyzed for their levels of ERK phosphorylation by western blotting. The expression of total ERK was also examined (lower panel).

Fig. 5. SB202190 induces phosphorylation of C-Raf. THP-1 cells were incubated with the indicated concentrations of SB202190 for 15 min and analyzed for their lev- els of C-Raf phosphorylation (Ser289/296/301) by western blotting. The amount of total C-Raf was also examined (lower panel). The membrane was ﬁrst probed with an anti-phospho-C-Raf (Ser289/296/301) rabbit polyclonal antibody to detect phosphorylated C-Raf and then left for 4 days until the signals disappeared. The membrane was then reprobed with an anti-total-C-Raf mouse monoclonal antibody to verify equal protein loading.

Most of the ED50 values obtained for the kinase inhibitors in the present study were comparable with previously reported values in the literature. For example, the ED50 of L-744832 was reported to inhibit the growth of more than 70% of tumor cell lines in vitro at concentrations of 2–20 µM [31]. Furthermore, the 50% inhibitory concentration (IC50) for ERK phosphorylation of U0126 is 14 µM [32], while that of the PI3 kinase activity of LY294002 is 1.4 µM
[33] and those of the geranylgeranyltransferase I and II activities of FPT inhibitor II are 24 µM, respectively [34]. These literature values are comparable to our ED50 for cell growth in the present study. In contrast, the IC50 values for kinase inhibition of SB202190 are 50 and 100 nM for p38α/SAPK2α and p38β2/SAPK2β, respectively [35], and far lower than the concentrations of SB202190 ( 1.3 µM) observed to affect cell growth in the present study. Therefore, SB202190-induced abnormal cell growth may not only depend on inhibition of p38, but also on its effects on other pathways. Hall- Jackson et al. [36] and Kalmes et al. [37] found that SB203580, a similar compound to SB202190, markedly caused Raf activation in cells by an unknown mechanism. Therefore, we hypothesize that inhibition of p38 activity also results in compensatory activation of an upstream regulator of these kinase pathways. This is clearly supported by the ﬁnding that C-Raf was dramatically phosphory- lated after the addition of SB202190. To clarify the mechanisms of C-Raf activation, we examined the activation of Ras by Ras pull down assay. Western blotting with antibody against pan-Ras was employed to detect the binding of GTP-Ras with GST-Ras-binding domain of C-Raf. We found that the proportion of GTP-Ras did not signiﬁcantly increased after the exposure of SB202190 (data not shown). Although we cannot exclude the involvement of the activation of certain isoforms of Ras, this result may indicate the direct effect of SB202190 to C-Raf. As SB compounds target threo- nine 106 at SAPKα/p38 and Raf is one of the protein kinase that possesses threonine at this position [36], SB202190 may inhibit C-Raf directly. SB203580 was previously shown to inhibit C-Raf at micromolar concentrations [36], consistent with the concentra- tions obtained in the present study. Therefore, regarding activation of C-Raf, a mechanism may exist whereby C-Raf suppresses its own activation and SB202190 may directly modulate the activ- ity of C-Raf. However, other mechanisms for C-Raf activation by SB202190 are also possible. For example, at a higher concentration of SB202190, its interaction with C-Raf may cause C-Raf to oligomer- ize [38,39] or become targeted to the plasma membrane [40,41], which are well-established mechanisms for inducing activation of C-Raf.
SB203580 has also been shown to enhance the phosphoryla-
tion of ERK in HL-60 and ML-1 cells [42,43]. Further studies may be required to understand the biological effects of SB202190 on the Ras–Raf–MEK–MAPK pathway. In the present study, we exam- ined the presence of several common activating mutations of N-Ras and K-Ras. We did not ﬁnd such mutations in SB202190-insensitive RS4-11 cells. In contrast, we detected several Ras mutations in SB202190-sensitive THP-1 and MV4-11 cells, consistent with a pre- vious report [44]. Therefore, taken together with the ED50 changes in the farnesyltransferase inhibitors in the presence of SB202190, we speculate that Ras mutations may be related to the aberrant Ras–Raf–MEK–MAPK pathway activation by SB202190.
Ishii et al. [45] reported that SB203580 activates ERK and induces the differentiation of human promyelocytic HL-60 cells and myeloid HT93 and ML-1 cells. However, they found no effect on the differentiation of THP-1 cells. Consistent with that study, we investigated the effects of SB202190 on the morphological differen- tiation of THP-1 and MV4-11 cells and found no remarkable changes (data not shown). Therefore, SB202190-mediated activation of the MEK–MAPK pathway predominantly affects growth stimulation, rather induction of differentiation, in these cells.

We observed that Akt phosphorylation was somewhat decreased after the addition of SB202190. p38 inhibitors have some non-speciﬁc effects on PKB (Akt) and 1 µM SB202190 represses approximately 10% of PKB activity [46]. Since activation of the PI3K–Akt signaling pathway is crucial for many aspects of cell growth, survival and apoptosis [6], we speculate that activation of the MEK–MAPK pathway, rather than suppression of Akt, may be more responsible for the aberrant cell growth observed in the present study.
It should be pointed out that pharmacological inhibitors of p38 are currently under development for the treatment of rheuma- toid arthritis, bronchial asthma and other inﬂammatory diseases [47–50], based on their well-documented abilities to decrease the production of proinﬂammatory cytokines. Our ﬁndings raise the possibility that concomitant use of kinase inhibitors for molecular targeted therapy against ERK-activated leukemia cells with such pharmacological inhibitors for p38 may induce the expansion of leukemic clones. It is possible that pharmacological inhibitors of the p38 pathway should be avoided in the design and development of future trials for ERK-activated leukemias or even for leukemia patients in remission.

Conﬂict of interest statement

None declared.

Acknowledgements

We thank Prof. Kenzo Ohtsuki, Department of Genetical Bio- chemistry and Signal Biology, Kitasato University Graduate School of Medical Sciences, for constructive comments. We also thank for Dr. Naohito Ishii and Prof. Masato Katagiri, Department of Clini- cal Physiology, Kitasato University School of Allied Health Sciences, for generous supports. This work is supported in part by a grant from Nakayama Foundation for Human Science, Mitsubishi Pharma Research Foundation, Japan Leukaemia Research Foundation, Foun- dation from Kitasato University School of Allied Health Sciences (Grant-in-Aid for Research Project, No. 2007-1003, No. 2008-1001) and Foundation from Kitasato University Graduate School of Med- ical Sciences Research Project.
Contributions. Dr. S. Takahashi contributed to acquired data the concept and design, interpreted and analyzed the data, provided drafting of the article, provided critical revision of the article for important intellectual content, gave ﬁnal approval of the article, provided study materials, supplied statistical expertise, obtained a funding source, provided administrative, technical or logistical sup- port, and collected and assembled data. Ms. M. Hirosawa acquired data and supplied statistical expertise. Dr. M. Nakahara acquired data. Ms. R. Otosaka acquired data and supplied statistical expertise. Ms. A. Imoto acquired data. Dr. T. Okazaki provided study materials.

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