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Histone deacetylase inhibitor- and proteasome inhibitor-mediated cleavage of HSP90β
induces cell apoptosis and suppresses cell growth

Sangkyu Park , Jae-Hyung Jeon , Jeong-A Park ,
1
2
1
Jun-Kyu Choi , Ye-Ram Choi , Ha-Eun Shin and Younghee Lee 1, 2*
2
2
2
1 Biotechnology Research Institute, Chungbuk National University, Cheongju, Chungbuk 28644, Republic of Korea
2 Department of Biochemistry, College of Natural Sciences, Chungbuk National University, Cheongju, Chungbuk 28644, Republic of Korea
.
ABSTRACT
HSP90 is one of the molecular chaperones which contributes to
protein stability in most living organisms. Previously, we
found that HSP90 cleavage occurred when histone deacetylase
inhibitor or proteasome inhibitor were treated in leukemia cells.
In this study, we found that HSP90 cleavage of HSP90 was
induced by treatment of SAHA and MG132 in 6 out of 16 solid
tumor cell lines. To investigate the effects of the cleavage of
HSP90 on cells, we predicted the potential cleavage site of
HSP90 and established mutant constructs. Through in vitro
cleavage assay using recombinant proteins, we found that the
294 th aspartic acid residue of the HSP90β was mainly cleaved Figure 2. Cleavage of HSP90β occurs mainly at the 294th
by caspase 10. We then established K562 and Mia-PaCa-2 aspartic acid residue by active caspase 10. (A) The cleavage
mutant cell lines expressing HSP90β D294A using retroviral site was predicted by the CaspDB database. Thereafter, four
system. The cells showed reduced cleavage of HSP90 by sites (252nd, 259th, 278th, and 294th aspartic acids) were Figure 4. Inhibition of HSP90 cleavage reduces cell growth
treatment of SAHA and MG132 compared with the K562 and selected for potential cleavage sites based on the size of the and increases apoptosis. (A) The K562-HSP90β WT and
Mia-PaCa-2 cell lines expressing HSP90β WT. Furthermore, cleaved HSP90 fragment. (B) Alignment for the predicted K562-HSP90β D294A cells were treated with the DMSO
cell growth was increased and cell apoptosis was reduced by cleavage sites of HSP90α and HSP90β. (C) Four mutant control or indicated doses of MG132 for 24 h. The cell lysates
HSP90β D294A expression in SAHA- and MG132-treated were subjected to Western blot analysis. The relative intensities
condition. Therefore, we suggest that the HSP90 cleavage constructs encoding recombinant human HSP90β were of Akt and GSK3β bands are shown as a graph after
widely occurs in several cell lines, and the cleavage of HSP90 established (aspartic acid to alanine; D252A, D259A, D278A, normalization with the levels of GAPDH. P values were
evaluated using t test. Values are the mean ± SD. *P < 0.05 vs
may be one of the mechanisms involved in the anti-tumor D294A) by site-directed mutagenesis. The rhHSP90β mutant DMSO control. (B) The cell viability of the K562-HSP90β WT
effects of anti-cancer drugs. proteins produced in E. coli were incubated with 2 U of active and K562-HSP90β D294A cells treated with the indicated doses
recombinant caspase 10 for 4 h. The mixtures were subjected to of MG132 was measured using the MTS assay. P values were
Western blot analysis. evaluated using a ratio paired t test. Values are the means ± SD.
RESULTS *P < 0.05, **P < 0.01 vs. K562-HSP90β WT. (C) The
percentages of apoptotic cells were detected by flow cytometry
using annexin V-FITC and PI staining. The cells were obtained
after treatment with DMSO or indicated doses of MG132 for 24
h. Values are the means ± SD. *P < 0.05 vs. K562-HSP90β WT.

CONCLUSION
1. Proteasome inhibitor and HDAC inhibitor induce the cleavage
of HSP90 in various tissue-derived cancer cell lines.
2. The 294 th aspartic acid residue is the major site of active
caspase 10-mediated HSP90 cleavage in the cells treated with
MG132 or SAHA.
3. The cleavage of HSP90 induces down-regulation of client
proteins, reduces cell viability, and increases apoptosis.
Figure 3. The mutation at the 294th aspartic acid residue
4. The cleavage of HSP90 may be a novel action mechanism of
decreases MG132-mediated cleavage of HSP90. (A) The various anti-cancer drugs and has a potential to be used as a
expression of exogenous HSP90β mRNA and protein in cells marker for finding anti-cancer drug candidates.
was determined using RT-PCR (left) and Western blot analysis
(right). Exogenous HSP90β was tagged with the Myc-tag. REFERENCES
GAPDH was used as a loading control. (B) The K562-HSP90β
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