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Introduction
Molecular chaperones are proteins that are responsible for maintaining the correct folding, function and stability of client
proteins. Of these, heat shock protein 90 (Hsp90) has recently emerged as a focus of interest because of its role in regulating
proteins that are responsible for malignant transformation. Approximately 50 proteins have been identified as clients of
Hsp90[1]. Most of these proteins play important roles in the control of cell cycle, growth and apoptosis and their dysregulated
function might lead to transformation. Examples include bcr-abl, Her2, Raf-1, Akt, Cdk4, mutant p53, estrogen and androgen
receptors. In the absence of Hsp90, or when the function of Hsp90 is disrupted, multiple client proteins are targeted for
ubiquitination and proteasomal degradation. This, in turn, leads to growth arrest and apoptosis in cancer cells
in vitro, and to inhibition or regression of tumor growth in
animals[2].
Hsp90 is present in cells in equilibrium between a low chaperoning activity `latent state¡¯ and an `activated state¡¯, with
increased chaperoning efficiency. The shift in equilibrium might be dictated by the amount of `stress¡¯ on the system, such
as mutated and dysregulated proteins[3]. Thus, the effects of inhibiting Hsp90 function could depend more on the `activity¡¯
and degree of involvement of the co-chaperone-protein-Hsp90 complexes and less on its cellular levels. Hsp90 participates
in at least two multimolecular chaperone complexes that then associate with client proteins. One such complex comprises
Hsp90, p23, and p50 (immunophilin in the case of steroid receptors), and the other consists of Hsp90, Hsp70, and
p60Hop. Association of client proteins with a P23-containing Hsp90 complex correlates with functionality; however, association with
a complex containing Hsp70 and p60Hop may
not[4]. Collectively, Hsp90 inhibitors will disrupt crucial chaperone functions in
a transformed cell, which might not be toxic to normal cells. These studies confirm that Hsp90 is a promising target for novel
cancer therapeutics and pave the road for the introduction of Hsp90 inhibitors in the treatment of cancers. Recently,
attention has been directed at the development of pharmacological Hsp90 inhibitors as chemotherapeutic agents. Such
efforts have focused on the ansamycin antibiotics, including geldanamycin (GA) and its closely related analogue,
17-AAG[5].
Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by a well-defined genetic abnormality
involving the bcr-abl translocation, which occurs in the Philadelphia (Ph) chromosome. This genetic alteration results from
a chromosome 9:22 translocation that leads to expression of a chimeric fusion protein, bcr-abl, with deregulated tyrosine
kinase activity[6]. The bcr-abl fusion protein and its constitutively-activated tyrosine kinase activity are essential for
malignant progression in CML. Because of this characteristic feature of CML, the bcr-abl kinase is a good candidate for
molecular-targeted chemotherapy. Earlier studies have demonstrated that multiple signal transduction pathways are involved in
abnormal growth signaling by bcr-abl, including Ras, Stat5, and phosphatidylinositol-3
kinase[7].
Because p210bcr/abl is one of the client proteins of Hsp90, disruption of the chaperone functions of Hsp90 may reduce the
protein level of p210bcr/abl, and therefore potentially
retard several signal transduction pathways initiated by
p210bcr/abl.
Curcumin (Cur), a natural compound present in turmeric,
possessing both anti-inflammatory and antioxidant effects, has
been studied vigorously as a chemopreventative agent in several cancer
models[8]. Because curcumin has already been
shown to have low systemic toxicity in animal and human
studies[9], we explored the effect
of curcumin on K562, a human CML cell line that expresses
p210bcr/abl. Our previously published work was the first to show that curcumin inhibited the
proliferation of K562 cells and the inhibition effect was correlated with down-regulation of
p210bcr/abl/Ras/Raf/MEK-1/ERK/Elk-1 and
p210bcr/abl /Ras/MEKK/SEK/JNK/c-Jun signal transduction
pathway[10]. But why was curcumin able to block several
signal pathways at the same time? How dose curcumin down-regulate
the p210bcr/abl protein level? By decreasing its synthesis
or by increasing its degradation? Will curcumin be able to influence the chaperone function of Hsp90? With these questions
in mind, we have now examined the effects of curcumin on
p210bcr/abl and the functions of its molecular chaperone, Hsp90, in
more detail.
In present study, we have demonstrated that curcumin time-dependently
depleted p210bcr/abl by disrupting
its binding with the molecular chaperone,
Hsp90. The present study suggests that curcumin could be worthy of being evaluated as a
potential chemotherapeutic agent to CML.
Materials and methods
Drugs and antibodies Curcumin was extracted and purified
from Curcuma longa Lgrowing in Jianyang County, Fujian
province; its purity was 97%.
Anti-p210bcr/abl, anti-Hsp90, and anti-Hsp70 monoclonal antibodies were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). Anti-actin monoclonal antibody was purchased from NeoMarkers (Fremant, CA),
anti-p60Hop and anti-p23 monoclonal antibodies were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Protein
A-Sepharose was purchased from BOEHRINGER MANNHEIM (Gmbh,
Germany).
Cell culture The CML cell line K562 was maintained in RPMI-1640 medium supplemented with 10%
(v/v) fetal calf serum, streptomycin 100 mg/L, penicillin 100 IU/L at 37
oC in humidified 5% CO2. After incubating for 24 h, exponentially growing
cells (1×109/L) were treated with curcumin of different concentrations (13.6 or 27.2
µmol/L) or GA 5 µmol/L for the indicated
length of time.
Flow cytometry Cells
(1×107/aliquot) treated with curcumin for different lengths of time were collected, washed with
phosphate buffered saline (PBS, dibasic sodium phosphate 9.1 mmol/L, monobasic sodium phosphate 1.7 mmol/L, and NaCl
150 mmol/L. pH was adjusted to 7.4 with NaOH.) 3
times, and resuspended in a final volume of 100 µL of ice-PBS. One milliliter
of 70% (v/v) ethanol in PBS was added to the resuspended cells and was mixed vigorously. Cells were then fixed
overnight. Fixed cells were incubated with primary Abs
(antip210bcr/abl, 1:400 dilution) for 1 h before being washed with PBS 3
times, and then incubated with Fluorescein (mistakenly abbreviated by its commonly-used reactive isothiocyanate form,
FITC) -labeled secondary IgG antibodies for 30 min before flow cytometry analysis. K562 cells without curcumin treatment were used as
p210bcr/abl positive control, and K562 cells incubated with secondary IgG antibodies directly but without primary Abs as
negative control. Flow cytometry measurements were made on a FACSCalibur machine, and the data was analyzed with
WinBryte software (Becton Dickinson).
Western blot analysis Protein was extracted from
curcumin-treated cells with a lysis buffer (Tris-HCl 50
mmol/L, pH 8.0, NaCl 150 mmol/L, dithiothreitol 1 mmol/L, edetic acid 0.5 mmol/L, nonidet P40 0.1%, sodium dodecylsulfate 0.1%,
phenylmethylsulfonyl fluoride 100 mg/L) supplemented with proteinase inhibitors: aprotinin 1 mg/L, leupeptin 2 mg/L, and
sodium orthovanadate 100 µmol/L. Appropriate protein amounts (20 µg) were subjected to sodium dodecylsulfate-
polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred to nitrocellulose membrane (150
mA; 4 oC) for 1.5 h. The blots were blocked in blocking-buffer (1% BSA, Tris-HCl 20 mmol/L, pH 7.5, NaCl 150 mmol/L, 0.05%
Tween-20) for 1 h at room temperature, which was followed by incubation with primary Abs
(anti-p210bcr/abl, anti-Hsp90,
anti-Hsp70, anti-p60Hop, anti-p23 or anti-Actin mAb, 1:1000 dilution) for 1 h at room temperature and then with antimouse
peroxidase-conjugated secondary IgG antibodies and developed with substrate, resulting in a visible color reaction on the membrane.
Co-immunoprecipitation[5]and Western
blot Curcumin-treating cells
(1×107/aliquot) and control cells were washed twice
with ice-cold PBS and resuspended in 1 mL of ice-cold lysis buffer. All further steps were performed at 4
oC. After a 15-min incubation, samples were sedimented
at12 000×g for 5 min. Supernatants were left to react overnight with 5 µg of monoclonal
anti-p210bcr/abl antibody, diluted with 30 µl of pre-swollen protein A-Sepharose beads, and incubated for an additional 2 h with
gentle agitation. The beads were sedimented at
3200×g for 2 min, washed 4 times with 1 mL aliquots of wash buffer [150
mmol/L NaCl, 20 mmol/L HEPES(pH 7.5), 1 mmol/L sodium orthovanadate, 10%
(v/v) glycerol, 0.1%
(w/v) Triton X-100, and 1%
(w/v) thiodiglycol], and eluted by heating for 20 min at 65
oC with 50 µL of SDS sample buffer consisting of 2%
(w/v) SDS, 62.5 mmol/L Tris-HCl (pH 6.8), 1mmol/L EDTA, and 5%
(v/v) b-mercapto-ethanol. The immunoprecipitate was subjected to
electrophoresis and immunoblotting as described
previously.
p210bcr/abl mRNA
expression[11] Total cellular RNA was isolated from untreated and curcumin-treated k562 cells using
TRIzol according to the manufacturer¡¯s instruction. First strand cDNA synthesis was performed using random hexamers.
The sequence of primers are as follows: bcr-abl sense
5¡¯-CTCCAGAC TGTCCACAGCATTCCG-3¡¯; anti sense 5¡¯-TCAGACCCTGSGGCTCAAAGTC-3¡¯;
b-actin sense 5¡¯-TACCTCATGAAGATCCTCA-3¡¯; antisense
5¡¯-TTCGTGGA-TGCCACAGGAC- 3¡¯. The reactions were denatured for 1 min at 95
oC, annealed for 2 min at 55
oC, and extended at 72 oC for
1.5 min in a Perkin-Elmer Thermal Cycler 480 (Branchburg, NJ, USA). PCR products were separated on 2% agarose gels
containing ethidium bromide and were photographed under UV light.
Results
Curcumin time-dependently depleted
P210bcr/abl Treatment with Cur inhibited the proliferation of K562 cells in a
concentration- and time-dependent manner, Cur 27.2
µmol/L for 24 h inhibited the proliferation of K562 by
54.5%[10]. When K562 cells were treated with Cur 27.2 µmol/L, down-regulation
of p210bcr/abl (Figure 1) was observed from 1 to 24 h. The inhibition rate of
1 h, 6 h, 12 h, and 24 h was 31.2%, 63.7%,
81.3%, and 94.5%, respectively. This result demonstrated that curcumin was able to deplete the abundance of
p210bcr/abl quickly. The half-life of
p210bcr/abl in K562 cells after curcumin treatment was
estimated to be 5 h.
Effects of curcumin on
p210bcr/abl mRNA expression levels To further elucidate the mechanism responsible for the
changes in amounts of p210bcr/abl protein, the levels of
p210bcr/abl mRNA were determined. In contrast to the protein levels, the
p210bcr/abl mRNA expression levels had no apparent change after treatment with Cur 27.2 µmol/L for 1 h, 6 h, 12 h, 24 h, or 48
h (Figure 2).
Effects of curcumin on the
Hsp90/p210bcr/abl complex
When K562 cells were treated with Cur for 24 h, total lysate was prepared, resolved by SDS-PAGE, and analyzed by Western
blotting. Although Cur treatment induced obvious down-regulation of
p210bcr/abl, the drug induced no clear decrease in
Hsp90, p23, and p60Hop; while the abundance of
Hsp70 protein increased significantly (Figure 3). Next
p210bcr/abl was immunoprecipitated from untreated cells or Cur 27.2
µmol/L for 24 h to examine the effects of drug exposure on the association of
p210bcr/abl with Hsp90 and other associated
chaperones (Figure 4). After immunoprecipitation with
anti-p210bcr/abl and blotting with anti-Hsp90, anti-p23 and anti-Hsp70,
the abundance of Hsp90 or p23 protein binding with
p210bcr/abl decreased significantly, in contrast, Hsp70 binding with
p210bcr/abl increased dramatically, while the amounts of
p60Hop protein binding with
p210bcr/abl had no apparent change. These
data show that the exposure of K562 cells to Cur 27.2
µmol/L for 24 h dissociated
p210bcr/abl from Hsp90/p23 complexes, but
drug treatment increased the association of the protein with Hsp70/
p60Hop complexes.
Discussion
Curcumin (diferuloylmethane) is a polyphenol derived from the plant
Curcuma longa, which is commonly used as a yellow coloring and flavoring agent in foods. Extensive published research over the last 50 years has indicated
thatthis polyphenol can both prevent and treat
cancer[8]. Recently, curcumin has been considered by oncologists as a potential third
generation cancer chemopreventive agent, and clinical trials have been carried out in several laboratories. It has been shown
to be a potent inhibitor of protein kinase C, EGF-receptor tyrosine kinase, c-Jun N-terminal kinase, protein tyrosine kinases,
protein serine/threonine kinases and IkappaB
kinase[12]. In addition, curcumin inhibits the activation of NFkappaB and the
expression of c-jun, c-fos, c-myc, cyclin D1, NIK, MAPK, ERK, ELK, PI3K, Akt, CDK, HER2 and
iNOS[13]. Curcumin inhibited
p185neu in vitro and depleted
p185neu protein in vivo by
disrupting its binding with the molecular chaperone GRP94
(glucose-regulated protein)[14].
Results in this study showed that curcumin reduced the protein level of
p210bcr/abl in a time-dependent manner. After
curcumin treatment for 6 h, the inhibition rate of
p210bcr/abl was 63.7%. It has been reported that the normal half-life of
p210bcr/abl protein is in excess of 24
h[15]; our data showed that curcumin treatment significantly decreased the half-life of
p210bcr/abl to about 5 h. Additionally, the
p210bcr/abl mRNA expression levels had no apparent change after treatment with Cur 27.2
µmol/L. Collectively, these data showed that the ability of curcumin to deplete
p210bcr/abl within 24 h might not be a result of the
inhibition of bcr/abl mRNA synthesis but is, at least in part, a result of an increase in protein
degradation.
Why was curcumin able to down-regulate a variety of transcription factors and signaling protein kinases? Moreover,
many of these proteins are the client proteins of Hsp90. Our data in the present study is the first to demonstrate that curcumin
is able to disrupt the molecular chaperone functions of Hsp90. This is a newly
discovered mechanism of the anti-cancer effect
of curcumin. The result of co-immunoprecipitation analysis showed that the
p210bcr/abl protein formed a stable complex with
Hsp90 and p23 in the absence of curcumin (Figure 4). This demonstrates that most of the
p210bcr/abl protein in untreated K562 cells exist in the mature Hsp90 complex. More importantly, after the addition of curcumin,
p210bcr/abl protein was dissociated from an Hsp90/p23 chaperone complex and associated instead with
Hsp70/p60Hop (Figure 4). Disruption of Hsp90/p23/
p210bcr/abl complexes may be followed rapidly by the proteolytic degradation of
p210bcr/abl. An ubiquitin-dependent proteasome-
mediated pathway may be implicated with this phenomenon because most client proteins have been reported to be
degrad-ed by the proteasome system[5].
Further study is required to examine this phenomenon and to
systematically test this possibility. Another possibility, that some
p210bcr/abl may be degraded in a Hsps-independent pathway, such as the
disruption of post-transcriptional activity of bcr/abl mRNA, also requires further study. Our results were similar to the
research of Blagosklonny et
al[16], who showed that composition of the multichaperone complexes associated with
p210bcr/abl was altered by geldanamycin before destabilization of these kinases, so that p23 association was lost and
Hsp70/p60Hop association either increased or was unaffected. All of these data suggest that curcumin inhibits the transition of
p210bcr/abl from an immature
p60Hop-Hsp70 complex to a complex including Hsp90-p23 that allows the chaperoned protein to acquire a
functional and perhaps stable conformation. Although the function of the
p60Hop-Hsp70 complex remains vague, it has been
proposed to mediate proteolytic degradation of its associated client protein. The study of
Schneider et al showed that Hsp90, in cooperation with Hsp70, p60 and other factors, functioned as a quality control system in the refolding or
degradation of client proteins[17]. These specific Hsp90 substrates may have an intrinsic structural instability under normal cellular
conditions, and this may render them especially sensitive to Hsp90 inhibitors. Normal dissociation of Hsp90 from client
proteins depends on the ATPase activity of Hsp70 and is mediated by
p60[17].
In conclusion, we offered evidence that curcumin inhibited
p210bcr/abl, dissociated the binding of
p210bcr/abl with Hsp90/p23 complex. In contrast, association of
p210bcr/abl with
Hsp70/p60Hop complex increased. Pharmacologically, curcumin has been
found to be safe. Human clinical trials indicate no dose-limiting toxicity when administered at doses of up to 10
g/d[8]. All of these studies suggest that curcumin has enormous potential in the therapy of CML and other cancers.
Acknowledgements
We thank Dr Da-li Zheng and Dr Qing-ling Huang for their valuable technical assistance and discussions.
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