Valproic acid induces apoptosis in human leukemia cells by stimulating both caspase-dependent and -independent apoptotic signaling pathways

Leukemia Research 26 (2002) 495–502

Valproic acid induces apoptosis in human leukemia cells by stimulating both caspase-dependent and -independent apoptotic signaling pathways Rika Kawagoe, Hiroyuki Kawagoe, Kimihiko Sano∗ Department of Pediatrics, Kobe University School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan Received 21 May 2001; accepted 13 August 2001

Abstract We investigated the effects of valproic acid (VPA) on the growth and survival of human leukemia cell lines. VPA induced cell death in all of the nine cell lines tested in a dose dependent manner. VPA-treatment induced apoptotic changes in MV411 cells including DNA fragmentation, phosphatidylserine externalization, cytochrome c release from mitochondria, and activation of caspases-3, -8, and -9. A caspase inhibitor, zVAD-FMK, inhibited the DNA fragmentation induced by VPA but not cell death. These findings suggest that VPA exerts an anti-leukemic effect by both caspase-dependent and -independent apoptotic signaling pathways. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Valproic acid; Apoptosis; Leukemia; Caspase-dependent and -independent pathways

1. Introduction Accumulating evidence indicates that some members of short chain fatty acids (SCFAs) affect the proliferation and/or the differentiation of a wide variety of tumor cells [1–6]. For instance, aromatic SCFAs such as butyrate, phenylacetate, and phenylbutyrate, promote differentiation or induce apoptosis in many types of tumor cells including colon cancer [1,2], head and neck squamous carcinoma [3], renal cell carcinoma [4], neuroblastoma [5], and leukemia [6,7]. Valproic acid (2-propylpentanoic acid, VPA) is a member of branched SCFAs and has been widely used in the management of various types of epilepsy for decades. As similar to the other members of SCFAs, VPA affects the growth and differentiation of some types of cells. It is reported that VPA induces differentiation in neuroblastoma cells and inhibits their growth [8,9]. Furthermore, VPA affects the growth of hematopoietic cells. VPA inhibited the formation of normal granulocyte and macrophage colonies at the higher concentration [10]. Tittle et al. showed that VPA inhibited the growth of murine B-lymphoid leukemia cell lines and a human T-lymphoblastic leukemia cell line, Jurkat [11]. The Abbreviations: VPA, valproic acid; SCFA, short chain fatty acid; FCS, fetal calf serum; PBS, phosphate-buffered saline; FITC, fluorescence isothiocyanate; SDS, sodium dodecyl sulfate; PI, propidium iodide; PS, phosphatidylserine; HMBA, hexamethylene bisacetamide; AIF, apoptosis inducing factor; MLL, mixed lineage leukemia ∗ Corresponding author. Tel.: +81-78-382-6090; fax: +81-78-382-6099. E-mail address: [email protected] (K. Sano).

mechanism underlying these effects of VPA on normal and malignant hematopoietic cells is unknown at present. In this study, we firstly aimed to clarify the effect of VPA on the growth of various human leukemia cell lines and the mechanism underlying it. We demonstrated that VPA induces apoptosis in a variety of human leukemia cell lines in a dose dependent manner. LD50 of the four of nine cell lines tested were within the range that did not inhibit the growth of normal hematopoietic progenitors. It is proposed that there are some apoptotic signaling pathways and with a few exceptions [12–16], they all activate the family of cysteine proteases known as caspases. Though many anti-cancer drugs are known to induce apoptosis in tumor cells, few are reported to be independent to caspases. If a drug dose induces apoptosis independently to caspases, it might be applicable as an option for the cancer treatment. We show in this study that VPA led the cells to death by stimulating both caspase-dependent and -independent apoptotic signaling pathways. Our findings suggest that VPA may be useful as an anti-leukemic agent without serious toxicity to normal hematopoiesis.

2. Materials and methods 2.1. Cell lines RS4-11, a human B-cell precursor leukemia cell line [17] and a human biphenotypic (B-myeloid) cell line, MV4-11

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[18] were obtained from American Type Culture Collection (Manassas, VA). KOCL-33, KOCL-44 and KOPB-26 (B-cell precursor) [19] were kindly gifted by Drs. K. Sugita and S. Nakazawa (Department of Pediatrics, Yamanashi Medical College, Japan). THP-1, a human monocytic leukemia cell line [20], was purchased from Japanese Collection of Research Bioresources (Tokyo, Japan). MOLT-4 [21] and Jurkat, human T-lymphoblastic leukemia cell lines, and KG-1, a human myeloid leukemia cell line [22], were obtained from Health Science Research Resources Bank (Osaka, Japan).

Osaka, Japan), and Ac-LEHD-MCA for caspase-9 (Peptide Institute). MV4-11 cells cultured in the presence or absence of VPA were suspended in chilled cell lysis buffer (Clontech). After a centrifugation, the supernatant was incubated with caspase substrates at 37 ◦ C for 1 h. The fluorescence was measured at 505 nm for DEVD-AFC or 460 nm for Ac-IETD-MCA and Ac-LEHD-MCA using a fluorometer (F-4010, Hitachi, Tokyo) [25,26].

2.2. Cell culture and viability assay

After culturing the MV4-11 cells in the presence or absence of VPA for 24 h, the mitochondria were stained with Mito Tracker Red CMTRos (Molecular Probes, Leiden, The Netherlands) according to the manufacturer’s direction. Cells were fixed, incubated with anti-cytochrome c mouse monoclonal antibody (1 ␮g/ml) (Promega, Madison, WI) at 4 ◦ C for 1 h, and then with FITC-conjugated anti-mouse IgG goat F(ab )2 fragments at 4 ◦ C for 30 min. The stained cells were immediately observed under a confocal laser microscope (MRC-1024, Bio Rad, Hercules, CA) [14].

Cells were cultured in RPMI1640 medium containing 10% fetal calf serum (FCS). For viability assays, cells were cultured in the presence of 0–300 ␮g/ml VPA (Wako, Osaka, Japan) for 5 days, and then trypan blue exclusion assay was performed. In all experiments, except for cell viability assays, cells were cultured with 200 ␮g/ml VPA for 24–72 h. 2.3. Morphological and flow cytometric analyses of apoptotic cells MV4-11 cells cultured in the presence or absence of VPA were fixed in a 1% para-formaldehyde solution, washed with phosphate-buffered saline (PBS), and then incubated with 0.2 mM Hoechst 33258 (Wako). The stained cells were observed under a fluorescence microscope (Zeiss, Germany). For flow cytometric analysis, cells were washed with PBS, and then suspended in the binding buffer (Immunotech, Marseille, France). Cells were stained with fluorescence isothiocyanate (FITC)-conjugated annexin-V and propidium iodide (PI) (Immunotech) according to the manufacturer’s direction. The stained cells were immediately analyzed on flow cytometer (Epics Elite, Beckman Coulter, Marseille, France) as previously described [23].

2.6. Immunohistochemical analysis of cytochrome c release

3. Results 3.1. VPA induces cell death in human leukemia cell lines To examine the effects of VPA on the growth and survival of human leukemic cells, nine human leukemia cell lines (four B-cell precursor, two T-lymphoid, one myeloid, one monocytic and one biphenotypic cell lines) were cultured with various concentrations of VPA (0–300 ␮g/ml) for 5 days. As shown in Fig. 1, VPA decreased the number of

2.4. DNA fragmentation analysis MV4-11 cells cultured in the presence or absence of VPA for 72 h were lysed in a solution containing 500 ␮g/ml of proteinase K, 500 ␮g/ml of RNase A, and 0.5% sodium dodecyl sulfate (SDS) at 37 ◦ C for 30 min. A 300 ␮l of a solution containing 6 M NaI/13 mM EDTA/0.5% sodium N-lauroilsarcosine/10 mg glycogen/26 mM Tris–HCl, pH8.0 was added to the tube and incubated at 60 ◦ C for 15 min. DNA was precipitated with 2-propanol, run on a 2% agarose gel, and stained with ethidium bromide [24]. 2.5. Caspase activity assay The following fluorescence-conjugated caspase substrates were used: DEVD-AFC for caspase-3 (Clontech, Palo Alto, CA), Ac-IETD-MCA for caspase-8 (Peptide Institute,

Fig. 1. Viability of the VPA-treated cells nine human leukemia cell lines were cultured for 5 days with various concentrations of VPA (0–300 ␮g/ml) and the cell viability was assessed. The viability of the VPA treated cells were expressed as relative values to those of the untreated control. Means of the data obtained from three independent experiments were plotted.

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Fig. 2. Detection of the apoptotic changes induced by VPA cells were treated with or without 200 ␮g/ml of VPA and analyzed. (A) MV4-11 cells cultured for 24 h were stained with Hoechst 33258 to assess the nuclear morphological changes. Left panel, VPA(−); and right panel, VPA(+). (B) MV4-11 cells cultured for 24 h were stained with PI and annexin V-FITC and analyzed on flow cytometer to assess the PS externalization on the surface of the cell membrane. The percentage of the cells in each quadrant indicates the mean ± S.D. of three independent experiments. Left panel, VPA(−); middle panel, VPA(+); right panel, pretreated with 100 ␮M zVAD-FMK before cultured with VPA. (C) MV4-11 cells were treated for 72 h and DNA was extracted to detect its fragmentation. DNA was run on 2% agarose gel. Lane 1, 123 bp ladder marker; lane 2, VPA(+); and lane 3, VPA(−).

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viable cells in all the cell lines tested in a dose-dependent manner. The sensitivity to VPA was different among the cell lines, and there was no correlation between the sensitivity and the cell phenotype. The growth of two cell lines, MV4-11 and KOCL-44, were completely inhibited by VPA at the clinically used concentration (100–150 ␮g/ml). LD50 of the four of the nine cell lines (MV4-11, KOCL-44, KOCL-33, and KOPB-26) were less than the level that

inhibited the growth of normal hematopoietic progenitors in vitro (200 ␮g/ml) [10]. 3.2. VPA induces apoptosis in a human leukemia cell line, MV4-11 To elucidate the mechanism of anti-leukemic effect of VPA, we used MV4-11, which was the most sensitive cell

Fig. 3. (A) Detection of the cytochrome c release from the mitochondria MV4-11 cells cultured with or without 200 ␮g/ml VPA for 24 h were stained with Mito Tracker Red CMTRos and anti-cytochrome c mouse monoclonal antibody. Upper panels, VPA(−); and lower panels, VPA(+). (B) Detection of caspase activation MV4-11 cells were cultured with or without 200 ␮g/ml VPA for 24 h and the caspase activity was measured as described under Section 2. Each caspase activity of untreated cells was set as 1 and that of VPA-treated cells was given as relative value to the control. The data presented were the mean ± S.D. of three independent experiments.

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line to VPA, for a further study. Firstly, the nuclear morphological changes of the cells were assessed by staining the cells with Hoechst 33258. After a 24-h culture with VPA, the cells exhibited nuclear shrinkage and chromatin condensation (Fig. 2A). Such morphological changes were not apparent in the control cells. For a further assessment of apoptosis, we examined the exposition of phosphatidylserine (PS) on the cell surface by using annexin V-FITC and PI. After a 24-h culture with VPA, the number of the annexin V+ /PI− cells, which were apoptotic but not necrotic, became 10-fold higher than that of the control (Fig. 2B, left and middle). Agarose gel electrophoresis showed that the DNA extracted from the cells treated with VPA for 72 h was fragmented (Fig. 2C, lane 2). These results indicate that VPA induces apoptosis in MV4-11 cells.

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3.3. VPA induces the cytochrome c release from mitochondria and activates caspases in MV4-11 cells To clarify the pathways through which VPA induces apoptosis, we analyzed for cytochrome c release. As shown in Fig. 3A, the mitochondria and cytochrome c were colocalized in control MV4-11 cells. In contrast, the distributions of them were different in the VPA-treated cells, suggesting that VPA induces the release of cytochrome c from the mitochondria. Next, we tested whether or not VPA activates caspases in MV4-11 cells. As shown in Fig. 3B, the activities of caspases-3, -8 and -9 in the VPA-treated cells were 12-, 5-, and 7-fold higher than those in the control, respectively, indicating that VPA activates these caspases in MV4-11 cells.

Fig. 4. Effects of general caspase inhibitor, zVAD-FMK, on VPA-induced nuclear apoptotic changes. (A) Inhibition of nuclear morphological changes by zVAD-FMK MV4-11 cells were pretreated with 100 ␮M zVAD-FMK for 1 h and then cultured with 200 ␮g/ml VPA for 24 h. Nuclear changes were assessed by Hoechst 33258 staining. Left panel, zVAD-FMK(−)/VPA(+); and right panel, zVAD-FMK(+)/VPA(+). (B) Inhibition of DNA fragmentation by zVAD-FMK. One million MV4-11 cells were pretreated with or without 100 ␮M zVAD-FMK for 1 h and then cultured with or without 200 ␮g/ml VPA for 24 h. Extracted DNA was run on a 2% agarose gel. Lane 1, 123 bp ladder marker; lane 2, treated VPA alone; lane 3, treated both zVAD-FMK and VPA; and lane 4, untreated control.

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3.4. VPA stimulates both caspase-dependent and -independent pathways In a next set of experiments, we elucidated the role of caspases in the VPA-induced apoptosis by using a general caspase inhibitor, zVAD-FMK. MV4-11 cells were pretreated with 100 ␮M zVAD-FMK for 1 h, and then cultured with 200 ␮g/ml VPA for 24 h. The morphological analysis by Hoechst 33258 staining revealed that the pre-treatment of the cells with zVAD-FMK prevented nuclear changes induced by VPA (Fig. 4A). As shown in Fig. 4B, DNA fragmentation induced by VPA was also inhibited by zVAD-FMK. These findings suggest that the activation of caspases by VPA is indispensable to promote the apoptotic changes of cell nuclei. We, then, investigated the effect of caspase inhibitor on the exposition of PS, an apoptotic change of a cell membrane, induced by VPA. After a 24-h culture with 200 ␮g/ml VPA, the number of annexin V+ /PI− cells in zVAD-FMK-pretreated cells was four-fold lower than that of the control without zVAD-FMK-pretreatment though it was not completely inhibited (Fig. 2B, right). At 48 h, the number of annexin V+ /PI− cells in these cells increased to the level comparable to that of the cells treated VPA alone for 24 h (data not shown). These results indicate that zVAD-FMK can delay, but not completely inhibit the apoptotic change of a membrane induced by VPA in MV4-11 cells. Finally, we tested the effect of zVAD-FMK on the viabilities of the cells treated with VPA, and found that zVAD-FMK failed to protect the cells from VPA-induced cell death (Fig. 5). Taken together, these results indicate that VPA induces cell death by stimulating both caspase-dependent and -independent apoptotic pathways.

4. Discussion In the present study, we demonstrated that VPA, a member of SCFAs, induces cell death in a variety of human leukemia cell lines including pre-B and T-lymphoid, and myeloid lineages. These results are consistent with the previous report showing that VPA inhibits the growth of murine B-lymphoid cell lines [11]. Among the cell lines tested, two cell lines, MV4-11 and KOCL-44, were completely killed by the clinical dose (100–150 ␮g/ml) of VPA (Fig. 1). Importantly these two cell lines carry the mixed lineage leukemia (MLL) gene abnormality, which is often observed in infantile leukemia showing the resistance to the chemotherapy. In addition, LD50 of the four of the nine cell lines were less than 200 ␮g/ml. This level of VPA does not inhibit the growth of normal hematopoietic progenitors [10]. Therefore, VPA has a potential as being used as an anti-leukemic agent without serious adverse effects. In the previous studies, the mechanism underlying the growth inhibitory effect of VPA on hematopoietic cells was not elucidated. We aimed to clarify the mechanism of the anti-leukemic effect of VPA by using a human biphenotypic (B-myeloid) cell line, MV4-11, which was the most sensitive cells to VPA. VPA caused chromatin condensation, DNA fragmentation, and PS externalization in the MV4-11 cells (Fig. 2). These results indicate that VPA induces apoptosis in leukemic cells as other members of SCFAs do in a variety of tumor cells. It is known that in the execution of apoptosis, cytochrome c is often released from the mitochondria. This event subsequently causes the activation of caspase-9, which finally activates caspase-3. In consistent with this proposed pathway, VPA

Fig. 5. The effect of general caspase inhibitor on VPA-induced cell death MV4-11 cells were cultured in the presence or absence of 100 ␮M zVAD-FMK for 1 h and then cultured with or without 200 ␮g/ml of VPA for 0, 36, 72, and 108 h. The viability of the control cells was given as 1 and the viability of the treated cells was expressed as relative value. The data represent the mean ± S.D. of the data from three independent experiments.

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causes the release of cytochrome c from the mitochondria as well as the activation of caspases-3 and -9 in MV4-11 cells (Fig. 3). This pathway is likely to be important in the VPA-induced apoptosis. VPA activated caspase-8 in addition to caspases-9 and -3 in MV4-11 cells (Fig. 3B). Classically, caspase-8 is thought to be activated by the signal through the death domain of cell surface receptors. However, it was recently reported that caspase-8 was also activated in the downstream of the cytochrome c release and subsequent caspase-3 activation [27]. A similar mechanism may cause the activation of caspase-8 by VPA in MV4-11 cells. In addition to the apoptotic pathway depending on the activation of caspases, increasing evidence has suggested that there are caspase-independent apoptotic pathways [12–16]. The exact mechanism of the caspase-independent apoptotic death is not clear, but a mitochondrial protein, apoptosis inducing factor (AIF), seems to be related to this pathway [14]. It was reported that anti-CD2 and staurosporine induced apoptosis in peripheral T-lymphocytes in both caspase-dependent and -independent pathways. In this case, a general caspase inhibitor zVAD-FMK inhibited nuclear apoptotic changes, but did not completely inhibit PS externalization on cell surface [15]. Similar to this observation, pre-treatment of the MV4-11 cells with zVAD-FMK inhibited the VPA-induced apoptotic nuclear events including chromatin condensation and DNA fragmentation but failed to inhibit the VPA-induced cell death as well as the exposition of PS in the cell surface. These findings suggest that VPA activates two different apoptotic signaling pathways: (1) a caspase-dependent pathways that mediates nuclear apoptotic changes and (2) a caspaseindependent pathways that mediates the events on a cell membrane. Many drugs are reported to induce apoptosis in variety of tumor cells, but there are few that are known to induce apoptosis through caspase-independent pathway [12]. Our result suggests that VPA might be useful as an option for the treatment of leukemia that are resistant to usual chemotherapy, though we must identify the feature of the tumors that are sensitive to VPA, as in some reports VPA was described to inhibit apoptosis in some cell lines [28,29]. We also have to evaluate the apoptosis-inducing effect of VPA in vivo. VPA can be a useful tool in clinical scene, as we know from the long clinical experience that it has minimal side effects, and at the same time it can be useful in elucidating the unknown apoptotic-signaling pathways.

Acknowledgements R. Kawagoe collected, assembled and analyzed the data and drafted the manuscript. H. Kawagoe provided the concept and design. K. Sano obtained the funding provided study materials, gave critical input into the revision, and gave final approval.

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