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FK228 inhibits Hsp90 chaperone function in K562 cells via hyperacetylation of Hsp70
Biochemical and Biophysical Research Communications 356 (2007) 998–1003 www.elsevier.com/locate/ybbrc
FK228 inhibits Hsp90 chaperone function in K562 cells via hyperacetylation of Hsp70 q Ying Wang 1, Sheng-Yu Wang 1, Xu-Hui Zhang, Ming Zhao, Chun-Mei Hou, Yuan-Ji Xu, Zhi-Yan Du, Xiao-Dan Yu * Department of Pathology, Beijing Institute of Basic Medical Sciences, 27 Taiping Road, Beijing 100850, China Received 14 March 2007 Available online 22 March 2007
Abstract Some pan-histone-deacetylase (HDAC) inhibitors have recently been reported to exert their anti-leukemia effect by inhibiting the activity of class IIB HDAC6, which is the deacetylase of Hsp90 and a-tubulin, thereby leading to hyperacetylation of Hsp90, disruption of its chaperone function and apoptosis. In this study, we compared the effect of a class I HDAC inhibitor FK228 with the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on the Hsp90 chaperone function of K562 cells. We demonstrated that, although having a weaker inhibitory effect on HDAC6, FK228 mediated a similar disruption of Hsp90 chaperone function compared to SAHA. Unlike SAHA, FK228 did not mediate hyperacetylation of Hsp90, instead the acetylation of Hsp70 was increased and Bcr-Abl was increasingly associated with Hsp70 rather than Hsp90, forming an unstable complex that promotes Bcr-Abl degradation. These results indicated that FK228 may disrupt the function of Hsp90 indirectly through acetylation of Hsp70 and inhibition of its function. 2007 Elsevier Inc. All rights reserved. Keywords: FK228; SAHA; HDAC inhibitor; Hsp90; Hsp70; Acetylation; Chaperone function; Leukemia; Apoptosis
The acetylation and deacetylation of histones have important roles in regulating gene expression [1]. Histone acetylation is generally associated with activation of gene transcription, whereas deacetylation is often accompanied by repression of transcription. Levels of histone acetylation in cells are determined by the balance between histone acetyltransferases (HATs) and histone deacetylases (HDACs) [2]. In mammalian cells, there are 18 different HDACs, which can be further divided into four types. HDAC 1, 2, 3, and 8 are class I deacetylases that localize almost exclusively to the nucleus and are ubiquitously expressed in various human cell lines and tissues. HDAC 4, 5, 6, 7, 9, and 10 are class II deacetylases, which shuttle between the q
This work was supported by the National Natural Science Foundation of China (Grant Nos. 30330620 and 30470898). * Corresponding author. Fax: +86 10 68213039. E-mail address: [email protected] (X.D. Yu). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.03.076
nucleus and cytoplasm with certain cellular signals. Class III comprises the conserved nicotinamide adenine dinucleotide-dependent Sir2 family of deacetylases. HDAC11 is the only member of the class IV HDACs. Generally, panHDAC inhibitors inhibit class I, II, and IV but not class III HDACs [3]. Altered expression or mutation of genes that encode HATs will lead to aberrant transcription, and the abnormal activity of HDACs or their binding partners also have a causative role in the onset and progression of cancer [4,5]. Consistent with this, several HDAC inhibitors have anticancer activity against many types of tumors, including leukemia [2,3,6]. A proposed mechanism for the antitumor effects of HDAC inhibitors is that histone acetylation leads to transcriptional activation of a select number of genes, the expression of which causes inhibition of tumor cell growth [2]. However, despite tremendous efforts and advances in microarray technology, these genes have not yet been identified. Meanwhile, several studies have found
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that inhibition of HDAC increases acetylation levels of the core histones as well as some non-histone proteins [7,8], raising the possibility that transcription-independent effects of HDAC inhibitors are also important for their anticancer activity [3]. It has recently been reported that pan-HDAC inhibitors such as LAQ824, LBH589, and SAHA exert their antitumor activity by inhibition of HDAC6, which is the deacetylase of a-tubulin and Hsp90 [8–10]. The inhibition of HDAC6 results in acetylation of Hsp90 and disruption of its chaperone function [10,11,12]. As Hsp90 controls the intracellular trafficking and folding of diverse cellular proteins, disruption of Hsp90 chaperone function will lead to the destabilization and eventual degradation of Hsp90 client proteins and induces apoptosis [13,14]. FK228 (also known as depsipeptide) is produced by Chromobacterium violaceum and has potent antitumor activity [15]. Previously, FK228 has been reported to kill chronic myeloid leukemia (CML) K562 cells at nanomolar concentrations and to increase imatinib-mesylate-induced apoptosis [16]. Currently, it is undergoing clinical trials in patients with chronic lymphocytic leukemia, acute myeloid leukemia, and T-cell lymphoma [17,18]. The mechanisms by which FK228 exerts its effects are not fully understood, although it has been reported that FK228 treatment led to upregulation of TNF-a, activation of caspase-8, and downregulation of c-FLIP protein [19,20]. As a class I HDAC inhibitor, FK228 has only a weak effect on HDAC6 [21,22], but it has been reported to disrupt the chaperone function of Hsp90 and induce apoptosis in human nonsmall-cell lung cancer cells [7]. However, it is unknown whether FK228 can influence the Hsp90 chaperone function in K562 CML cells and, if so, by which mechanism it can disturb Hsp90 chaperone function without inhibiting HDAC6 activity. To answer this question, we compared the anti-leukemia effect of FK228 with that of SAHA on CML K562 cells, and investigated the influence of FK228 on Hsp90 function. We found that, similar to SAHA, FK228 reduced the levels of Hsp90 client proteins including Bcr-Abl, Raf-1, Cdk4, Akt, survivin, and XIAP via a proteasome-dependent mechanism. Treatment with FK228 also resulted in inactivation of the Raf/Mek/Erk and PI3K/Akt survival-signaling pathways and apoptotic cell death. However, unlike with SAHA, the inhibition effect of FK228 on Hsp90 chaperone function seems to involve increased acetylation of Hsp70 rather than of Hsp90. As Hsp70 is required for the assembly of client protein–Hsp90 complexes, it seems that FK228 may disrupt the function of Hsp90 indirectly through acetylation of Hsp70. Materials and methods Cell line and reagents. The K562 cell line was available in our laboratory and cultured in RPMI-1640 medium containing 10% fetal bovine serum and antibiotics. FK228 was kindly provided by Dr. David S. Schrump (Thoracic Oncology, Surgery Branch, NIH/NCI, USA) and suberoylanilide hydroxamic acid (SAHA) was provided by AstraZeneca Company (Macclesfield, UK), Acetylated tubulin antibody
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was purchased from Sigma–Aldrich (St. Louis, MO, USA). Antibodies for Raf-1, phospho-Akt, Akt, phospho-Erk, Erk, acetylated-histone H3, histone H3, acetylated lysine, survivin, and XIAP were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-cAbl and Cdk4 were purchased from BD Biosciences (San Diego, CA, USA). AntiHsp90 and anti-Hsp70 were obtained from Stressgen Biotechnologies (Victoria, BC, Canada). Cytotoxicity assays. For cell-viability assays, K562 cells were seeded on 96-well plates. After 24 h, cells were treated with FK228 (5–150 ng/mL) or SAHA (0.5–5 lM) for 12–48 h. At appropriate time points, the percentages of viable cells after treatment were measured using the MTT assay. Each experiment was performed three times. Western blot and immunoprecipitation analyses. For Western blot analysis, control or drug-treated K562 cells were lysed in Laemmli’s buffer (Bio-Rad Laboratories, CA, USA), and approximately 60 lg of total proteins were resolved on SDS–polyacrylamide gels and an immunoblot was performed as previously described [7]. For the immunoprecipitation experiments, cellular extracts from approximately 1 · 107 cells were prepared in radio-immunoprecipitation (RIPA) buffer, and approximately 400 lg of total proteins were used for immunoprecipitation analyses according to a previously described procedure [7]. Statistical analyses. Data were expressed as means ± SEM. Statistical analyses were performed with the Student’s t test and p values less than 0.05 were considered significant.
Results and discussion The cytotoxic effect of FK228 and SAHA on K562 K562 cells were treated with FK228 or SAHA for 0–48 h. The MTT assay found that both reagents mediated significant dose- and time-dependent reductions in cell viability; the effect of FK228 is stronger than that of SAHA (Fig. 1A). Treatment with 25 ng/mL (which is equivalent to 0.05 lM) of FK228 or 2 lM SAHA caused maximal cytotoxicity after 48 h and increasing the doses did not further increase the drug-induced cytotoxicity. According to this result, 25 ng/mL FK228 and 2 lM SAHA were selected for subsequent experiments. The effects of FK228 and SAHA on histone deacetylases To investigate the possible mechanism of FK228induced cytotoxicity, we compared the effects of FK228 and SAHA treatment on the acetylation of histone H3, which is the substrate of class I HDACs. Western blot analyses showed that K562 cells had low levels of acetylated histone H3 at 0 h, Treatment with HDAC inhibitors increased the acetylation of histone H3, but unlike the cytotoxicity effects, this increased acetylation did not increase in a time-dependent manner (Fig. 1B). This indicates that increased histone acetylation alone is not sufficient to account for HDAC-inhibitor-induced apoptosis. We also compared the effects of FK228 and SAHA treatment on the acetylation of a-tubulin, the substrate of HDAC6, and found that both SAHA and FK228 induced marked tubulin acetylation (Fig. 1B), but the effect of FK228 was not as strong as that of SAHA. The effect of FK228 on a-tubulin hyperacetylation was also confirmed in several other cell lines, including prostate cancer
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Fig. 1. Comparison of the effects of FK228 and SAHA on cell growth and inhibition of histone deacetylase of K562 cells. (A) K562 cells were treated with FK228 (5–100 ng/mL) or SAHA (0.5–5 lM) for indicated times and the cytotoxicity was evaluated by the MTT assay. All experiments were performed in triplicate. (B) Western blot analyses of acetylated histone H3 and acetylated tubulin in the cell lysates from K562 cells treated with FK228 or SAHA for 12–48 h. The levels of histone H3 and b-actin were used as the loading control.
LNCaP, DU145, PC-3, non-small cell lung cancer A549 and H322 cells (unpublished data). Considering that HDAC6 is the only known deacetylase of a-tubulin, the increased acetylation of a-tubulin indicates that FK228 can partially inhibit HDAC6 activity. Although this result is in direct contrast with a previous report [22], the dose used by the authors of the previous report was 10 ng/mL, which is lower than the dose in the present study (25 ng/ mL). However, it is unknown whether the increased acetylation of a-tubulin is due to direct inhibition of HDAC6 by FK228 or due to FK228-induced mitotic arrest with increased microtubule stability, which affects the affinity of HDAC6 for microtubules, as shown for the anticancer reagent paclitaxel [23]. Immunoprecipitation experiments were then performed to assess the effect of FK228 and SAHA on acetylation of Hsp90, another HDAC6 substrate. As expected for a pan-HDAC inhibitor, SAHA mediated hypercetylation of both Hsp90 and a-tubulin. However, in FK228-treated cells, increased Hsp90 acetylation could not be detected with either immunoprecipitation with anti-acetylated lysine antibody and blotting with anti-Hsp90 or immunoprecipitation with anti-Hsp90 and blotting with anti-acetylated lysine antibody (Fig. 2). This is in contrast to the observation in SKBR3 breast cancer cells [7], LNCaP prostate cancer cells and Src-transformed NIH-3T3 cells (unpublished data), in which FK228 (25 ng/mL) increases Hsp90 acetylation after 3–12 h treatment. Further experiments are needed to investigate whether FK228-induced increase in Hsp90 acetylation is cell-type-dependent. Interestingly, we found that levels of acetylated Hsp70 were markedly increased in both FK228- and SAHA-treated cells
(Fig. 2). It is known that Hsp70 is required for the assembly of the signaling protein–Hsp90 heterocomplex. Hsp90 is involved in two multi-chaperone complexes and promotes correct folding or degradation of client proteins, depending on its conformation. When adenosine triphosphate (ATP) is bound to the amino-terminal nucleotidebinding pocket, Hsp90 is associated with co-chaperone proteins p23 and p50Cdc37 and contacts the client protein directly to stabilize the interactions. When adenosine diphosphate (ADP) is bound, Hsp90 is assembled into the complex with co-chaperone proteins Hsp70 and p60Hop, and in this complex it is Hsp70 that directly contacts the client protein to promote client protein ubiquitination and degradation [13,14]. Therefore, disruption of Hsp70 function may indirectly affect the chaperone function of Hsp90. Effect of FK228 and SAHA on Hsp90 chaperone function As a molecular chaperone, Hsp90 controls the intracellular trafficking and folding of diverse cellular proteins; particularly those involved in signal transduction, cell-cycle regulation, and survival [13,14]. Inhibition of the Hsp90 chaperone function causes degradation of its client proteins via the ubiquitin–proteasome pathway. To investigate the possible effect of hyperacetylation of Hsp70 on Hsp90 chaperone function, we evaluated the impact of increased acetylation of Hsp70 on the associations of Hsp90 and Hsp70 with the client protein Bcr-Abl. Bcr-Abl was immunoprecipitated from cell lysates of untreated and FK228treated K562 cells. The binding of Bcr-Abl to Hsp90 was compared with binding to Hsp70 using antibodies against
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Fig. 2. Comparison of the effects of FK228 and SAHA on Hsp90/Hsp70 acetylation. SAHA increases Hsp90 and Hsp70 acetylation, but FK228 only increases Hsp70 acetylation. The figure shows immunoprecipitates with anti-acetylated lysine antibody that were immunoblotted with anti-Hsp90 and anti-Hsp70 antibodies or immunoprecipitates with anti-Hsp90 or anti-Hsp70 antibodies that were immunoblotted with anti-acetylated-lysine antibody in the RIPA lysates from K562 cells treated with FK228 or SAHA for the times indicated. The levels of Hsp90 and Hsp70 in the non-immunoprecipitated cell lysates or immunoprecipitated pellet were used as the loading controls.
Fig. 3. Hsp90 chaperone function was inhibited by FK228 and Hsp70 inhibitor. (A) FK228 shifts the chaperone association of Bcr-Abl from Hsp90 to Hsp70. Immunoprecipitation was performed with anti-c-Abl antibody and immunoblotted separately with anti-c-Abl, anti-Hsp90 or anti-Hsp70 antibodies in the RIPA lysates from K562 cells treated with FK228 for 6–24 h. The levels of c-Abl, Hsp90 and Hsp70 in the non-immunoprecipitated cell lysates were used as the loading control. (B) Western blot analyses of Bcr-Abl, Raf-1, Akt, Cdk4, survivin, and XIAP in the cell lysates from K562 cells treated with FK228 or SAHA for 12–48 h. The levels of b-actin were used as the loading control. (C) Western blot analyses of Bcr-Abl, XIAP, Cdk4, and cleaved PARP in the cell lysates from K562 cells treated with 25–50 lM quercetin alone or in combination with FK228 for 24 h. The levels of b-actin were used as the loading control.
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Hsp90 and Hsp70. Fig. 3A shows that, although there was no increased acetylation of Hsp90, FK228 treatment shifted the chaperone association of Bcr-Abl from Hsp90 to Hsp70, indicating that Bcr-Abl is associated with the unstable multi-chaperone complexes necessary for polyubiquitination and proteasomal degradation [24]. We then evaluated the effect of FK228 on the chaperone function of Hsp90, and found that, similar to SAHA, FK228 mediated marked reductions in Bcr-Abl, Raf-1, Cdk4, Akt, survivin, and XIAP levels. As Bcr-Abl, Raf-1, Cdk4, Akt, and survivin [13,25] have previously been shown to be Hsp90 client proteins, which normally require interaction with Hsp90 to maintain a mature, stable, and functional conformation, the degradation of these proteins indicates that the Hsp90 chaperone function was disrupted by SAHA and FK228 treatment (Fig. 3B). In addition, pretreatment of cells with the proteasome inhibitor MG132 prevents FK228-induced reduction of Bcr-Abl (data not shown), indicating that the reduction of this fusion protein is mediated by proteasomal degradation, as has previously been shown for SAHA- and LAQ824-treated K562 cells [8,10]. A previous study has reported that class I HDACs are co-immunoprecipitated with Hsp70 [26] and that FK228 and SAHA treatment can increase acetylation of Hsp70, indicating that one of the class I HDACs is the deacetylase of Hsp70. We propose that hyperacetylation may impair Hsp90 as well as Hsp70 function. A previous report has provided circumstantial evidence that a HDAC inhibitor can disrupt Hsp70 function, because HDAC inhibitor trichostatin A-induced cell death was found to be prevented via increasing the Hsp70 levels [27]. To further confirm that inhibition of Hsp70 function can indirectly affect Hsp90 chaperone function, K562 cells were treated with quercetin, a well-known Hsp70 inhibitor, alone or in combination with FK228, and its effects on reducing levels of Hsp90 client proteins and inducing apoptosis was assessed by Western-blot analysis. As shown in Fig. 3C, quercetin alone could partially deplete Bcr-Abl levels, but when combined with FK228, there were greater reductions in multiple Hsp90 clients and increased apoptotic cell death. This result indicates that Hsp70 function is important for maintaining Hsp90 chaperone function. Of the SAHA- and FK228-depleted proteins, survivin and XIAP are members of the inhibitor of apoptosis gene family. Recently, survivin has been shown to be a new client protein for Hsp90 [25]. Although it has not been directly proved that XIAP is a client protein of Hsp90, it has been shown that the conserved IAP repeat in survivin contains a Hsp90-binding site. Therefore, it is likely that Hsp90 is also involved in folding and maturation of XIAP [25]. We also found that the number of multinucleated cells increased concurrently with survivin depletion, which is consistent with a dual role of survivin in mitotic control and inhibition of apoptosis [28].
The effect of FK228 and SAHA on cell-survival signaling pathways As Bcr-Abl kinase activity initiates multiple intracellular signaling cascades that lead to leukemogenesis [29], additional experiments were performed to determine the effect of depletion of Bcr-Abl, Raf-1, and Akt protein levels on survival signaling pathways in K562 cells. As shown in Fig. 4, treatment with HDAC inhibitors reduced the levels of phosphorylated Erk and phosphorylated Akt in a time-dependent manner, indicating that Erk and Akt activities were inhibited in these cells. Concurrent with the inhibition of Erk and Akt activities, the level of cleaved poly(ADP)ribosome polymerase (PARP) was increased, indicating that inhibition of the Raf-1/MEK/ ERK and PI3K/Akt survival signaling pathways is involved in HDAC-inhibitor-induced apoptosis. Although both SAHA and FK228 have similar inhibitory effects on Hsp90 chaperone function, FK228 had greater cytotoxic effects than SAHA. We propose that HDAC-inhibitorinduced inhibition of Hsp90 chaperone function has a role in initiating apoptotic signal cascades, whereas other factors may be involved in amplifying the apoptotic process. For example, FK228 has been reported to specifically induce caspase-1 expression and activate caspase-1, which can cleave Bid to form truncated Bid and promote apoptosis, whereas SAHA has no effect on caspase-1 regulation [30]. In addition, the expression of other small subsets of genes have been shown to be selectively altered by SAHA or FK228 treatment [30], and these changes may also be involved in determining the reagent’s cytotoxicity. Taken together, these results demonstrate that the cytotoxic activity of FK228 in CML K562 cells is associated with the disruption of Hsp90 chaperone function by hyperacetylation of Hsp70. To our knowledge, this is the first study to show that Hsp70 acetylation affects the chaperone function of Hsp90, but the possible mechanisms remain to be determined.
Fig. 4. Inactivation of ERK and AKT signaling pathways coincide with increased apoptosis in K562 cells. Western blot analyses were performed on p-Erk, Erk, p-Akt, Akt, PARP, and cleaved PARP in the cell lysates from K562 cells treated with FK228 or SAHA for 12–48 h.
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FK228 inhibits Hsp90 chaperone function in K562 cells via hyperacetylation of Hsp70 q Ying Wang 1, Sheng-Yu Wang 1, Xu-Hui Zhang, Ming Zhao, Chun-Mei Hou, Yuan-Ji Xu, Zhi-Yan Du, Xiao-Dan Yu * Department of Pathology, Beijing Institute of Basic Medical Sciences, 27 Taiping Road, Beijing 100850, China Received 14 March 2007 Available online 22 March 2007
Abstract Some pan-histone-deacetylase (HDAC) inhibitors have recently been reported to exert their anti-leukemia effect by inhibiting the activity of class IIB HDAC6, which is the deacetylase of Hsp90 and a-tubulin, thereby leading to hyperacetylation of Hsp90, disruption of its chaperone function and apoptosis. In this study, we compared the effect of a class I HDAC inhibitor FK228 with the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on the Hsp90 chaperone function of K562 cells. We demonstrated that, although having a weaker inhibitory effect on HDAC6, FK228 mediated a similar disruption of Hsp90 chaperone function compared to SAHA. Unlike SAHA, FK228 did not mediate hyperacetylation of Hsp90, instead the acetylation of Hsp70 was increased and Bcr-Abl was increasingly associated with Hsp70 rather than Hsp90, forming an unstable complex that promotes Bcr-Abl degradation. These results indicated that FK228 may disrupt the function of Hsp90 indirectly through acetylation of Hsp70 and inhibition of its function. 2007 Elsevier Inc. All rights reserved. Keywords: FK228; SAHA; HDAC inhibitor; Hsp90; Hsp70; Acetylation; Chaperone function; Leukemia; Apoptosis
The acetylation and deacetylation of histones have important roles in regulating gene expression [1]. Histone acetylation is generally associated with activation of gene transcription, whereas deacetylation is often accompanied by repression of transcription. Levels of histone acetylation in cells are determined by the balance between histone acetyltransferases (HATs) and histone deacetylases (HDACs) [2]. In mammalian cells, there are 18 different HDACs, which can be further divided into four types. HDAC 1, 2, 3, and 8 are class I deacetylases that localize almost exclusively to the nucleus and are ubiquitously expressed in various human cell lines and tissues. HDAC 4, 5, 6, 7, 9, and 10 are class II deacetylases, which shuttle between the q
This work was supported by the National Natural Science Foundation of China (Grant Nos. 30330620 and 30470898). * Corresponding author. Fax: +86 10 68213039. E-mail address: [email protected] (X.D. Yu). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.03.076
nucleus and cytoplasm with certain cellular signals. Class III comprises the conserved nicotinamide adenine dinucleotide-dependent Sir2 family of deacetylases. HDAC11 is the only member of the class IV HDACs. Generally, panHDAC inhibitors inhibit class I, II, and IV but not class III HDACs [3]. Altered expression or mutation of genes that encode HATs will lead to aberrant transcription, and the abnormal activity of HDACs or their binding partners also have a causative role in the onset and progression of cancer [4,5]. Consistent with this, several HDAC inhibitors have anticancer activity against many types of tumors, including leukemia [2,3,6]. A proposed mechanism for the antitumor effects of HDAC inhibitors is that histone acetylation leads to transcriptional activation of a select number of genes, the expression of which causes inhibition of tumor cell growth [2]. However, despite tremendous efforts and advances in microarray technology, these genes have not yet been identified. Meanwhile, several studies have found
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that inhibition of HDAC increases acetylation levels of the core histones as well as some non-histone proteins [7,8], raising the possibility that transcription-independent effects of HDAC inhibitors are also important for their anticancer activity [3]. It has recently been reported that pan-HDAC inhibitors such as LAQ824, LBH589, and SAHA exert their antitumor activity by inhibition of HDAC6, which is the deacetylase of a-tubulin and Hsp90 [8–10]. The inhibition of HDAC6 results in acetylation of Hsp90 and disruption of its chaperone function [10,11,12]. As Hsp90 controls the intracellular trafficking and folding of diverse cellular proteins, disruption of Hsp90 chaperone function will lead to the destabilization and eventual degradation of Hsp90 client proteins and induces apoptosis [13,14]. FK228 (also known as depsipeptide) is produced by Chromobacterium violaceum and has potent antitumor activity [15]. Previously, FK228 has been reported to kill chronic myeloid leukemia (CML) K562 cells at nanomolar concentrations and to increase imatinib-mesylate-induced apoptosis [16]. Currently, it is undergoing clinical trials in patients with chronic lymphocytic leukemia, acute myeloid leukemia, and T-cell lymphoma [17,18]. The mechanisms by which FK228 exerts its effects are not fully understood, although it has been reported that FK228 treatment led to upregulation of TNF-a, activation of caspase-8, and downregulation of c-FLIP protein [19,20]. As a class I HDAC inhibitor, FK228 has only a weak effect on HDAC6 [21,22], but it has been reported to disrupt the chaperone function of Hsp90 and induce apoptosis in human nonsmall-cell lung cancer cells [7]. However, it is unknown whether FK228 can influence the Hsp90 chaperone function in K562 CML cells and, if so, by which mechanism it can disturb Hsp90 chaperone function without inhibiting HDAC6 activity. To answer this question, we compared the anti-leukemia effect of FK228 with that of SAHA on CML K562 cells, and investigated the influence of FK228 on Hsp90 function. We found that, similar to SAHA, FK228 reduced the levels of Hsp90 client proteins including Bcr-Abl, Raf-1, Cdk4, Akt, survivin, and XIAP via a proteasome-dependent mechanism. Treatment with FK228 also resulted in inactivation of the Raf/Mek/Erk and PI3K/Akt survival-signaling pathways and apoptotic cell death. However, unlike with SAHA, the inhibition effect of FK228 on Hsp90 chaperone function seems to involve increased acetylation of Hsp70 rather than of Hsp90. As Hsp70 is required for the assembly of client protein–Hsp90 complexes, it seems that FK228 may disrupt the function of Hsp90 indirectly through acetylation of Hsp70. Materials and methods Cell line and reagents. The K562 cell line was available in our laboratory and cultured in RPMI-1640 medium containing 10% fetal bovine serum and antibiotics. FK228 was kindly provided by Dr. David S. Schrump (Thoracic Oncology, Surgery Branch, NIH/NCI, USA) and suberoylanilide hydroxamic acid (SAHA) was provided by AstraZeneca Company (Macclesfield, UK), Acetylated tubulin antibody
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was purchased from Sigma–Aldrich (St. Louis, MO, USA). Antibodies for Raf-1, phospho-Akt, Akt, phospho-Erk, Erk, acetylated-histone H3, histone H3, acetylated lysine, survivin, and XIAP were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-cAbl and Cdk4 were purchased from BD Biosciences (San Diego, CA, USA). AntiHsp90 and anti-Hsp70 were obtained from Stressgen Biotechnologies (Victoria, BC, Canada). Cytotoxicity assays. For cell-viability assays, K562 cells were seeded on 96-well plates. After 24 h, cells were treated with FK228 (5–150 ng/mL) or SAHA (0.5–5 lM) for 12–48 h. At appropriate time points, the percentages of viable cells after treatment were measured using the MTT assay. Each experiment was performed three times. Western blot and immunoprecipitation analyses. For Western blot analysis, control or drug-treated K562 cells were lysed in Laemmli’s buffer (Bio-Rad Laboratories, CA, USA), and approximately 60 lg of total proteins were resolved on SDS–polyacrylamide gels and an immunoblot was performed as previously described [7]. For the immunoprecipitation experiments, cellular extracts from approximately 1 · 107 cells were prepared in radio-immunoprecipitation (RIPA) buffer, and approximately 400 lg of total proteins were used for immunoprecipitation analyses according to a previously described procedure [7]. Statistical analyses. Data were expressed as means ± SEM. Statistical analyses were performed with the Student’s t test and p values less than 0.05 were considered significant.
Results and discussion The cytotoxic effect of FK228 and SAHA on K562 K562 cells were treated with FK228 or SAHA for 0–48 h. The MTT assay found that both reagents mediated significant dose- and time-dependent reductions in cell viability; the effect of FK228 is stronger than that of SAHA (Fig. 1A). Treatment with 25 ng/mL (which is equivalent to 0.05 lM) of FK228 or 2 lM SAHA caused maximal cytotoxicity after 48 h and increasing the doses did not further increase the drug-induced cytotoxicity. According to this result, 25 ng/mL FK228 and 2 lM SAHA were selected for subsequent experiments. The effects of FK228 and SAHA on histone deacetylases To investigate the possible mechanism of FK228induced cytotoxicity, we compared the effects of FK228 and SAHA treatment on the acetylation of histone H3, which is the substrate of class I HDACs. Western blot analyses showed that K562 cells had low levels of acetylated histone H3 at 0 h, Treatment with HDAC inhibitors increased the acetylation of histone H3, but unlike the cytotoxicity effects, this increased acetylation did not increase in a time-dependent manner (Fig. 1B). This indicates that increased histone acetylation alone is not sufficient to account for HDAC-inhibitor-induced apoptosis. We also compared the effects of FK228 and SAHA treatment on the acetylation of a-tubulin, the substrate of HDAC6, and found that both SAHA and FK228 induced marked tubulin acetylation (Fig. 1B), but the effect of FK228 was not as strong as that of SAHA. The effect of FK228 on a-tubulin hyperacetylation was also confirmed in several other cell lines, including prostate cancer
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Fig. 1. Comparison of the effects of FK228 and SAHA on cell growth and inhibition of histone deacetylase of K562 cells. (A) K562 cells were treated with FK228 (5–100 ng/mL) or SAHA (0.5–5 lM) for indicated times and the cytotoxicity was evaluated by the MTT assay. All experiments were performed in triplicate. (B) Western blot analyses of acetylated histone H3 and acetylated tubulin in the cell lysates from K562 cells treated with FK228 or SAHA for 12–48 h. The levels of histone H3 and b-actin were used as the loading control.
LNCaP, DU145, PC-3, non-small cell lung cancer A549 and H322 cells (unpublished data). Considering that HDAC6 is the only known deacetylase of a-tubulin, the increased acetylation of a-tubulin indicates that FK228 can partially inhibit HDAC6 activity. Although this result is in direct contrast with a previous report [22], the dose used by the authors of the previous report was 10 ng/mL, which is lower than the dose in the present study (25 ng/ mL). However, it is unknown whether the increased acetylation of a-tubulin is due to direct inhibition of HDAC6 by FK228 or due to FK228-induced mitotic arrest with increased microtubule stability, which affects the affinity of HDAC6 for microtubules, as shown for the anticancer reagent paclitaxel [23]. Immunoprecipitation experiments were then performed to assess the effect of FK228 and SAHA on acetylation of Hsp90, another HDAC6 substrate. As expected for a pan-HDAC inhibitor, SAHA mediated hypercetylation of both Hsp90 and a-tubulin. However, in FK228-treated cells, increased Hsp90 acetylation could not be detected with either immunoprecipitation with anti-acetylated lysine antibody and blotting with anti-Hsp90 or immunoprecipitation with anti-Hsp90 and blotting with anti-acetylated lysine antibody (Fig. 2). This is in contrast to the observation in SKBR3 breast cancer cells [7], LNCaP prostate cancer cells and Src-transformed NIH-3T3 cells (unpublished data), in which FK228 (25 ng/mL) increases Hsp90 acetylation after 3–12 h treatment. Further experiments are needed to investigate whether FK228-induced increase in Hsp90 acetylation is cell-type-dependent. Interestingly, we found that levels of acetylated Hsp70 were markedly increased in both FK228- and SAHA-treated cells
(Fig. 2). It is known that Hsp70 is required for the assembly of the signaling protein–Hsp90 heterocomplex. Hsp90 is involved in two multi-chaperone complexes and promotes correct folding or degradation of client proteins, depending on its conformation. When adenosine triphosphate (ATP) is bound to the amino-terminal nucleotidebinding pocket, Hsp90 is associated with co-chaperone proteins p23 and p50Cdc37 and contacts the client protein directly to stabilize the interactions. When adenosine diphosphate (ADP) is bound, Hsp90 is assembled into the complex with co-chaperone proteins Hsp70 and p60Hop, and in this complex it is Hsp70 that directly contacts the client protein to promote client protein ubiquitination and degradation [13,14]. Therefore, disruption of Hsp70 function may indirectly affect the chaperone function of Hsp90. Effect of FK228 and SAHA on Hsp90 chaperone function As a molecular chaperone, Hsp90 controls the intracellular trafficking and folding of diverse cellular proteins; particularly those involved in signal transduction, cell-cycle regulation, and survival [13,14]. Inhibition of the Hsp90 chaperone function causes degradation of its client proteins via the ubiquitin–proteasome pathway. To investigate the possible effect of hyperacetylation of Hsp70 on Hsp90 chaperone function, we evaluated the impact of increased acetylation of Hsp70 on the associations of Hsp90 and Hsp70 with the client protein Bcr-Abl. Bcr-Abl was immunoprecipitated from cell lysates of untreated and FK228treated K562 cells. The binding of Bcr-Abl to Hsp90 was compared with binding to Hsp70 using antibodies against
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Fig. 2. Comparison of the effects of FK228 and SAHA on Hsp90/Hsp70 acetylation. SAHA increases Hsp90 and Hsp70 acetylation, but FK228 only increases Hsp70 acetylation. The figure shows immunoprecipitates with anti-acetylated lysine antibody that were immunoblotted with anti-Hsp90 and anti-Hsp70 antibodies or immunoprecipitates with anti-Hsp90 or anti-Hsp70 antibodies that were immunoblotted with anti-acetylated-lysine antibody in the RIPA lysates from K562 cells treated with FK228 or SAHA for the times indicated. The levels of Hsp90 and Hsp70 in the non-immunoprecipitated cell lysates or immunoprecipitated pellet were used as the loading controls.
Fig. 3. Hsp90 chaperone function was inhibited by FK228 and Hsp70 inhibitor. (A) FK228 shifts the chaperone association of Bcr-Abl from Hsp90 to Hsp70. Immunoprecipitation was performed with anti-c-Abl antibody and immunoblotted separately with anti-c-Abl, anti-Hsp90 or anti-Hsp70 antibodies in the RIPA lysates from K562 cells treated with FK228 for 6–24 h. The levels of c-Abl, Hsp90 and Hsp70 in the non-immunoprecipitated cell lysates were used as the loading control. (B) Western blot analyses of Bcr-Abl, Raf-1, Akt, Cdk4, survivin, and XIAP in the cell lysates from K562 cells treated with FK228 or SAHA for 12–48 h. The levels of b-actin were used as the loading control. (C) Western blot analyses of Bcr-Abl, XIAP, Cdk4, and cleaved PARP in the cell lysates from K562 cells treated with 25–50 lM quercetin alone or in combination with FK228 for 24 h. The levels of b-actin were used as the loading control.
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Hsp90 and Hsp70. Fig. 3A shows that, although there was no increased acetylation of Hsp90, FK228 treatment shifted the chaperone association of Bcr-Abl from Hsp90 to Hsp70, indicating that Bcr-Abl is associated with the unstable multi-chaperone complexes necessary for polyubiquitination and proteasomal degradation [24]. We then evaluated the effect of FK228 on the chaperone function of Hsp90, and found that, similar to SAHA, FK228 mediated marked reductions in Bcr-Abl, Raf-1, Cdk4, Akt, survivin, and XIAP levels. As Bcr-Abl, Raf-1, Cdk4, Akt, and survivin [13,25] have previously been shown to be Hsp90 client proteins, which normally require interaction with Hsp90 to maintain a mature, stable, and functional conformation, the degradation of these proteins indicates that the Hsp90 chaperone function was disrupted by SAHA and FK228 treatment (Fig. 3B). In addition, pretreatment of cells with the proteasome inhibitor MG132 prevents FK228-induced reduction of Bcr-Abl (data not shown), indicating that the reduction of this fusion protein is mediated by proteasomal degradation, as has previously been shown for SAHA- and LAQ824-treated K562 cells [8,10]. A previous study has reported that class I HDACs are co-immunoprecipitated with Hsp70 [26] and that FK228 and SAHA treatment can increase acetylation of Hsp70, indicating that one of the class I HDACs is the deacetylase of Hsp70. We propose that hyperacetylation may impair Hsp90 as well as Hsp70 function. A previous report has provided circumstantial evidence that a HDAC inhibitor can disrupt Hsp70 function, because HDAC inhibitor trichostatin A-induced cell death was found to be prevented via increasing the Hsp70 levels [27]. To further confirm that inhibition of Hsp70 function can indirectly affect Hsp90 chaperone function, K562 cells were treated with quercetin, a well-known Hsp70 inhibitor, alone or in combination with FK228, and its effects on reducing levels of Hsp90 client proteins and inducing apoptosis was assessed by Western-blot analysis. As shown in Fig. 3C, quercetin alone could partially deplete Bcr-Abl levels, but when combined with FK228, there were greater reductions in multiple Hsp90 clients and increased apoptotic cell death. This result indicates that Hsp70 function is important for maintaining Hsp90 chaperone function. Of the SAHA- and FK228-depleted proteins, survivin and XIAP are members of the inhibitor of apoptosis gene family. Recently, survivin has been shown to be a new client protein for Hsp90 [25]. Although it has not been directly proved that XIAP is a client protein of Hsp90, it has been shown that the conserved IAP repeat in survivin contains a Hsp90-binding site. Therefore, it is likely that Hsp90 is also involved in folding and maturation of XIAP [25]. We also found that the number of multinucleated cells increased concurrently with survivin depletion, which is consistent with a dual role of survivin in mitotic control and inhibition of apoptosis [28].
The effect of FK228 and SAHA on cell-survival signaling pathways As Bcr-Abl kinase activity initiates multiple intracellular signaling cascades that lead to leukemogenesis [29], additional experiments were performed to determine the effect of depletion of Bcr-Abl, Raf-1, and Akt protein levels on survival signaling pathways in K562 cells. As shown in Fig. 4, treatment with HDAC inhibitors reduced the levels of phosphorylated Erk and phosphorylated Akt in a time-dependent manner, indicating that Erk and Akt activities were inhibited in these cells. Concurrent with the inhibition of Erk and Akt activities, the level of cleaved poly(ADP)ribosome polymerase (PARP) was increased, indicating that inhibition of the Raf-1/MEK/ ERK and PI3K/Akt survival signaling pathways is involved in HDAC-inhibitor-induced apoptosis. Although both SAHA and FK228 have similar inhibitory effects on Hsp90 chaperone function, FK228 had greater cytotoxic effects than SAHA. We propose that HDAC-inhibitorinduced inhibition of Hsp90 chaperone function has a role in initiating apoptotic signal cascades, whereas other factors may be involved in amplifying the apoptotic process. For example, FK228 has been reported to specifically induce caspase-1 expression and activate caspase-1, which can cleave Bid to form truncated Bid and promote apoptosis, whereas SAHA has no effect on caspase-1 regulation [30]. In addition, the expression of other small subsets of genes have been shown to be selectively altered by SAHA or FK228 treatment [30], and these changes may also be involved in determining the reagent’s cytotoxicity. Taken together, these results demonstrate that the cytotoxic activity of FK228 in CML K562 cells is associated with the disruption of Hsp90 chaperone function by hyperacetylation of Hsp70. To our knowledge, this is the first study to show that Hsp70 acetylation affects the chaperone function of Hsp90, but the possible mechanisms remain to be determined.
Fig. 4. Inactivation of ERK and AKT signaling pathways coincide with increased apoptosis in K562 cells. Western blot analyses were performed on p-Erk, Erk, p-Akt, Akt, PARP, and cleaved PARP in the cell lysates from K562 cells treated with FK228 or SAHA for 12–48 h.
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