Betulinic Acid ubat Kanser Prostat
Betulinic Acid Selectively Increases Protein Degradation and Enhances
Prostate Cancer-Specific Apoptosis: Possible Role for Inhibition of
Deubiquitinase Activity
Abstract
Inhibition of the ubiquitin-proteasome system (UPS) of
protein degradation is a valid anti-cancer strategy and has led to the approval
of bortezomib for the treatment of multiple myeloma. However, the alternative
approach of enhancing the degradation of oncoproteins that are frequently
overexpressed in cancers is less developed. Betulinic acid (BA) is a
plant-derived small molecule that can increase apoptosis specifically in cancer
but not in normal cells, making it an attractive anti-cancer agent. Our results
in prostate cancer suggested that BA inhibited multiple deubiquitinases (DUBs),
which resulted in the accumulation of poly-ubiquitinated proteins, decreased
levels of oncoproteins, and increased apoptotic cell death. In normal
fibroblasts, however, BA did not inhibit DUB activity nor increased total
poly-ubiquitinated proteins, which was associated with a lack of effect on cell
death. In the TRAMP transgenic mouse model of prostate cancer, treatment with
BA (10 mg/kg) inhibited primary tumors, increased apoptosis, decreased
angiogenesis and proliferation, and lowered androgen receptor and cyclin D1
protein. BA treatment also inhibited DUB activity and increased ubiquitinated
proteins in TRAMP prostate cancer but had no effect on apoptosis or
ubiquitination in normal mouse tissues. Overall, our data suggests that
BA-mediated inhibition of DUBs and induction of apoptotic cell death
specifically in prostate cancer but not in normal cells and tissues may provide
an effective non-toxic and clinically selective agent for chemotherapy.
By virtue of their high proliferative capacity, cancer
cells frequently respond to the accumulation of unfolded proteins or
proteotoxic stress by enhancing the ubiquitin-proteasome system (UPS) in order
to resist apoptotic cell death [1].
The UPS is the major cellular pathway that degrades unfolded proteins and
controls the expression levels of specific proteins important in cell cycle,
proliferation, and apoptosis [2].
Proteins are targeted for UPS-mediated degradation by the addition of multiple
ubiquitin units (poly-Ub), which facilitates recognition and degradation by the
UPS complex. Inhibition of the UPS and subsequent increase in multiple proteins
is a valid anti-cancer strategy that has led to the development of bortezomib,
an FDA approved drug for the treatment of multiple myeloma [3].
Clinically, however, bortezomib alone does not display substantial activity in
castration-resistant prostate cancer (CRPC) and is often associated with dose
limiting side effects such as neuropathy [4].
An alternative but less developed therapeutic strategy is
to exploit the UPS by enhancing its activity and specificity in order to
increase the degradation of proliferation and pro-survival proteins that are
frequently overexpressed in cancers, i.e., oncoproteins. A feasible and
clinically relevant method is to pursue the identification of small molecules
that can activate UPS-mediated degradation of proteins such as androgen
receptor (AR) in prostate cancer (PC). Betulinic acid (BA) is a plant-derived
small molecule that can increase apoptosis in cancer cells, thus making it an
attractive anti-cancer agent [5].
At present, BA is one of only two small molecules reported to directly activate
chymotrypsin-like UPS activity in
vitro [6],
[7].
Another report demonstrates that BA inhibits the growth of LNCaP PC cells by
selectively activating UPS-dependent degradation of AR as well as specificity
protein (Sp) transcription factors that regulate VEGF expression [8].
However, the mechanisms by which BA specifically activates UPS-dependent
degradation of AR and other factors are unknown.
In addition to stimulating UPS activity, another possible
way BA can increase the degradation of specific proteins is by inhibiting
deubiquitinases (DUBs). Reversible ubiquitination is a crucial mechanism in the
regulation of the UPS and in the maintenance of many cell cycle and
pro-survival proteins [9]–[11].
Recent findings indicate that DUBs play critical regulatory roles in most
pathways involving Ub [9]–[11].
Approximately one hundred human DUBs fall into five classes, the best
characterized being ubiquitin-specific proteases (USP) and ubiquitin C-terminal
hydrolases (UCH). DUB-mediated removal of poly-Ub from key proteins renders
them less susceptible to degradation by the UPS and therefore increases their
levels. In fact, several DUBs are overexpressed in cancer and are considered to
be oncogenes [9]–[11].
Because DUBs have a role in oncogenic transformation,
recent attention has focused on the identification of small molecule inhibitors
of DUBs. The idea is that inhibiting DUBs will elevate poly-Ub on oncoproteins
and increase their recognition and degradation by the UPS pathway, resulting in
greater apoptosis and improved drug efficacy [12].
Several small molecule inhibitors of DUB activity have been identified to
increase the accumulation of poly-Ub proteins and enhance apoptosis in cancer
cells, suggesting that DUB inhibitors are promising anti-cancer agents [13]–[18].
In this report, we showed that the ability of BA to increase the degradation of
multiple proliferation and pro-survival proteins in PC cells was correlated to
inhibition of DUBs. In contrast to PC cells, BA had no effect on DUB activity
and degradation of proteins in normal cells, resulting in no toxicity. Our
results suggested that the PC-specific effect provided by BA therapy was due to
its ability to inhibit DUBs in cancer but not in non-cancer cells.
Ethics Statement
All animal
studies were carried out with the approval of the Institutional Animal Care and
Use Committee (protocol #6996.06 MR) of the Miami Veterans Affairs Medical
Center (Association for Assessment and Accreditation of Laboratory Animal Care
accredited) and conducted in accordance with the NIH Guidelines for the Care
and Use of Laboratory Animals.
Reagents
BA for
cell culture experiments was purchased from A.G. Scientific; digitonin and
polyvinyl-pyrrolidone (PVP) from Sigma-Aldrich; MG132, doxorubicin, and
Coomassie blue from EMD Biosciences; N-ethylmaleimide (NEM) from Sigma; and
trypan blue (0.4%) from Invitrogen.
Treatment of TRAMP
mice with BA
TRAMP
transgenic mice (Jackson Laboratories) were identified by tail biopsy and PCR
using Wizard SV Genomic DNA purification (Promega) and DNA primers PB-1 forward
5′-CCGGTCGACCGGAAGCTTCCACAAGTGCATTTA-3′ and Tag reverse
5′-CTCCTTT CAAGACCTAGAAGGTCCA-3′. BA was obtained from
Ze-Qi Xu at Advanced Life Sciences and prepared as previously described [19].
Mice with palpable prostate tumors were randomly divided into experimental and
control groups and injected i.p. 11 times over 14 days with BA (5 [BA5] or 10
[BA10] mg/kg body weight; n = 10 each dose) or vehicle control (n = 12). On day
15, primary prostate tumors were removed and their weights determined. An outer
portion of primary prostate tumor was fixed in formalin for
immunohistochemistry. TRAMP males without palpable prostate tumors were similarly
treated with BA10 or vehicle control (n = 3, each group), prostates, spleen,
thymus removed, and analyzed by immunohistochemistry.
Immunohistochemistry
Immunostaining
for apoptotic (cleaved caspase-3, Cell Signaling Technology; ApopTag Peroxidase
In Situ Apoptosis Detection, Millipore) and proliferating (Ki67, NCL-Ki67p,
Leica Microsystems; PCNA [PC10], Santa Cruz Biotechnology) cells was performed
using rabbit polyclonal, mouse monoclonal, and biotinylated goat
anti-rabbit/mouse secondary antibodies (Vector Laboratories), as previously
described [20].
Blood vessel density was determined by immunostaining for CD-31 using a goat
polyclonal antibody (M20; Santa Cruz Biotechnology) and a biotinylated rabbit
anti-goat secondary antibody or for CD-34 using a rat polyclonal (RAM34, BD
Biosciences) and goat anti-rat secondary antibody. The number of cleaved
caspase-3 or Ki67 positive cells and CD-31 positive vessels were determined for
BA10 and vehicle controls (n = 5 each group), as previously described [20].
Similarly, AR (N-20), cyclin D1 (DCS-6), and ubiquitin (P4D1) from Santa Cruz
Biotechnology were immunostained; the number of AR positive cells was
determined for BA10 and vehicle control prostate tumors.
Cell lines
Human PC
cell lines LNCaP, DU145, and PC3 [21]
were obtained from the American Type Culture Collection (ATCC) and used within
6 months of resuscitation of original cultures. All PC cells were maintained in
RPMI 1640 medium (Invitrogen) with 5% fetal bovine serum (Hyclone), 100 U/ml
penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin (Invitrogen).
PrEC normal human prostate epithelial cells were obtained from Lonza and
maintained in PrEGM media. RWPE-1 normal prostate epithelial cells (obtained
from Dr. Bal Lokeshwar, University of Miami and originally from ATCC) were
maintained in Keratinocyte-SFM media (Invitrogen). Human foreskin BJ fibroblast
cells (passage 24) and fetal lung fibroblasts (IMR-90, MRC-5) were obtained
from Dr. Priyamvada Rai (University of Miami) and originally obtained from ATCC
(CRL-2522, CCL-186, and CCL-171). BJ, IMR-90, and MRC-5 cells were maintained
in DMEM medium (Invitrogen) with 10% fetal bovine serum, 100 U/ml penicillin,
and 100 µg/ml streptomycin.
BA cell proliferation
assay
The
CellTiter Aqueous cell proliferation colorimetric method from Promega was used
to determine cell viability of PC cells in media containing BA (2.5, 5, 7.5,
and 10 µM) or control (0.5% DMSO). Cell viability was normalized against the
vehicle control and the data expressed as a percentage of control from three
independent experiments done in triplicate.
Drug treatments
PC cells
were cultured in media containing BA (10 µM), MG132 (1 µM), docetaxel (1 nM),
or DMSO control for varying times (24–72 h). BJ, IMR-90, MRC-5, PrEC, and
RWPE-1 cells were cultured in media containing BA, doxorubicin (1 µM), or DMSO
control for varying times (24–72 h). In all the experiments, floating and trypsinized
attached cells were pooled for further analysis.
Western blot analysis
Preparation
of total protein lysates and western blot analysis was done as previously
described [22].
The following antibodies were used: cleaved PARP (9541), CoxIV (4844), AKT
(9272), and survivin (71G4B7) from Cell Signaling Technology; cytochrome c
(7H8.2C12), Smac (612245), Bcl-xL (610211) and Rb (544144) from BD Biosciences;
AIF (E-1), actin (C-11), cyclin A (H432), cyclin B1 (GNS1), cyclin D1 (DCS-6),
Cdk1 (17), Cdk2 (D-12), Cdk4 (M2), p21 (C-19), p27 (C-19), E2F1 (KH95), AR
(N-20), Mcl-1 (S-19), Bcl-2 (N-19), HA (Y-11), Ub (P4D1), Rb (C-15), and
horseradish peroxidase-conjugated secondary antibody from Santa Cruz
Biotechnology. Our preference was to use Coomassie blue staining of total
protein as loading controls because drug treatments often affect the levels of
typical housekeeping proteins such as actin or tubulin.
Trypan blue exclusion
assay
Treated
and control PC, BJ, IRM-90, MRC-5, PrEC, or RWPE-1 cells were harvested,
resuspended in PBS, diluted 1:1 in 0.4% trypan blue, dead blue and live
non-blue cells immediately counted using a hemacytometer, and the % dead blue
cells determined from at least three independent experiments done in duplicate.
Annexin-FITC/propidium
iodide (PI) flow cytometry
Treated
and control PC cells were resuspended in binding buffer followed by the
addition of annexin V-FITC and PI (Annexin V Kit sc-4252 AK, Santa Cruz
Biotechnology). After 20 min., cells were analyzed by flow cytometry using a
Coulter XL flow cytometer and the percentage of annexin+ cells determined using
WinMDI version 2.8 from two independent experiments done in triplicate.
Mitochondrial protein
release assay
Treated
and control PC cells were resuspended in a buffer containing 100–200 µM
digitonin, 20 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl, 1 mM EGTA, 1 mM EDTA, 1
mM DTT, 250 mM sucrose, and protease inhibitors (Roche) at 50 µl/1×106
cells. After 5 min. on ice, cells were centrifuged 5 min and the supernatant
used for western blot analysis. Digitonin is a detergent that preferentially
permeabilizes plasma membrane compared to mitochondrial membrane [23].
Flow cytometric cell
cycle analysis
Propidium/hypotonic
citrate method was used to study cell cycle distribution of BA treated PC cells
[24].
Six to 8 samples were analyzed from at least three independent experiments and
DNA distribution histograms generated as previously described [22],
[25].
Transient transfection
of AR, cyclin D1 wild type and T286A mutant
CMV/AR
expression plasmid was transfected into PC3 cells for 24 h using FuGene-HD
(Roche) followed by BA (24, 48, 72 h) or control (24 h) treatment and AR
protein analyzed by western blot. Cyclin D1 expression plasmids pBABE/cyclin D1
wild type (9050) and pcDNA/cyclin D1 T286A mutant (11182; cannot be degraded by
UPS; [26])
were obtained from AddGene. These plasmids were transfected into LNCaP cells
for 24 h followed by BA or control treatment for 24 h. Protein levels of
transfected cyclin D1 protein was determined by western blot analysis using
anti-HA and cyclin D1.
Proteasome assay
The
Proteasome-Glo Chymotrypsin-like Cell-Based Assay (Promega) was used to
determine the effect of BA on proteasome activity in PC cells. Cells were
treated with BA, BA+MG132, or control for 8, 24, 48, and 72 h, cell numbers
determined, and proteasome activity measured with a luminometer (TD-20/20,
Turner Designs). Light units were normalized to cell number (control treatment
= 1) and 6–8 samples were analyzed from at least four independent experiments.
DUB assay
The
DUB-Glo Protease Assay (Promega) was used to determine the effect of BA on DUB
activity in PC, BJ, IRM-90, and RWPE-1 cells. Cells were treated with BA, 1 nM
docetaxel, or control for 8, 24, 48, and 72 h, lysed in DUB buffer (50 mM
Tris-HCl, pH 7.5, 0.1% NP-40, 5 mM MgCl2, 250 mM sucrose, 1 mM DTT,
1 mM PMSF), centrifuged 10 min., and 10 µg protein used to determine DUB
activity. Control lysates were pre-incubated with 4 mM NEM, a known DUB
inhibitor, for 1 h before addition of substrate. Light units (control treatment
= 1) from 6–8 samples were determined from at least three independent
experiments. DUB activity was also measured in vehicle control (n = 4) and BA10
(n = 5) TRAMP prostate tumors.
DUB labeling assay
Cell
lysates were prepared as described above for the DUB assay, 20 µg of protein incubated
with 500 ng HA-Ub vinyl sulfone (VS), an irreversible specific inhibitor of
most DUBs (Boston Biochem) for 1.5 h at room temperature, and samples analyzed
by western blot using anti-HA antibody to detect DUB labeling.
Statistical analysis
Statistical
differences between drug-treated and controls were determined by two-tailed
Student's t-test
(unequal variance) with P<0.05
considered significant.
Results
BA inhibits the growth
of TRAMP prostate tumors by increasing apoptosis and decreasing angiogenesis
and proliferation
We
previously utilized BA (Fig.
1) as an agent that can enhance the sensitivity of PC cell lines to cell
death when combined with antimitotic agents by increasing NF-κB activity, in
part due to enhanced degradation of IκBα [27],
[28].
To evaluate the in vivo
therapeutic efficacy of BA, we utilized the TRAMP transgenic mouse model of PC [29],
[30].
TRAMP mice contain the prostate-specific probasin promoter linked to the SV40 T
antigen oncogene, which results in the development of aggressive metastatic PC.
Our results indicated that BA (5 and 10 mg/kg) significantly reduced the final
weights of primary prostate tumors compared to vehicle control tumors (Fig.
2A). There were no differences in the final body weights between BA treated
and control mice (data not shown). Immunohistochemistry (IHC) of cleaved
(active) caspase-3, a marker for apoptotic cells, showed a significant increase
in BA10 compared to vehicle control tumors (Fig.
2B and Supplementary Fig.
S1A). IHC of CD31, a marker for blood vessels, and Ki67, a marker for
proliferating cells, showed a significant decrease in BA10 compared to vehicle
control tumors. Further confirmation using TUNEL for apoptosis, CD34 for
angiogenesis, and PCNA for proliferation is shown in Supplementary Fig.
S1B. These results indicated that BA induced apoptosis and inhibited
angiogenesis and proliferation in TRAMP prostate tumors
BA decreases the
levels of AR in TRAMP prostate tumors but not in normal prostate
We then
sought to determine whether BA can decrease the expression levels of AR and
cyclin D1 proteins in TRAMP prostate tumors. IHC and counting of AR+ cells
showed a significant decrease in BA10 compared to vehicle control tumors (Figs.
2C, D). IHC of cyclin D1 also showed a significant decrease in BA10
compared to vehicle control tumors, correlating with the decrease in the Ki67
and PCNA proliferation markers (Figs.
2B, C). We next sought to determine if the BA-mediated decrease in AR also
occurred in non-cancerous prostate tissue. Unlike the results in prostate
tumors, the levels of AR in normal prostate were similar in BA10 compared to
vehicle control, suggesting that the BA-mediated degradation of AR occurred
only in tumor cells (Fig.
2C).
BA inhibits the
proliferation and increases apoptosis of PC cells
To address
the mechanisms of BA as a single agent in PC, we used androgen-dependent LNCaP
and castration-resistant DU145 and PC3 cells. Using a three-day cell
proliferation assay, we found that 10 µM BA inhibited the growth of all PC
cells, including the more chemotherapy resistant DU145 and PC3 cells (Fig.
3A). All subsequent experiments were done using 10 µM BA. The BA anti-PC
cell effect was due to increased apoptotic cell death, as determined by trypan
blue exclusion assay, western blot analysis of cleaved-PARP (substrate for
activated caspases), and annexin V-FITC/PI flow cytometry (Figs.
3B, C). BA is reported to target mitochondria to initiate the intrinsic pathway
of apoptosis by increasing the release of mitochondrial proteins such as
cytochrome c, which activates the caspase cascade [5].
Our results in PC3 cells showed that BA increased the release of cytochrome c,
Smac (blocks inhibitor of apoptosis [IAP] family; [31]),
and apoptosis-inducing factor (AIF; translocates to nucleus to increase DNA
fragmentation; [32])
from the mitochondria (Fig.
3D). Similar results were obtained in BA treated LNCaP and DU145 cells
(Supplementary Fig.
S2). Thus, the BA-mediated release of pro-apoptotic proteins from the
mitochondria coincided with the observed increase in apoptosis in PC cells
BA increases G1/S cell
cycle arrest in PC cells
To
investigate the cell cycle effects of BA on PC cells, we used flow cytometry
analysis. Treatment of PC cells with BA for 24 h resulted in a significantly
increased G1 and decreased S phase of the cell cycle, indicating a major block
in G1/S (Supplementary Fig.
S3A). After longer times of BA treatment, there was a significant increase
in the sub-G1 cell cycle phase in all PC cells, which was reflective of greater
DNA degradation that occurred in apoptotic cells (Supplementary Fig.
S3B). In DU145 and PC3 but not in LNCaP there was a significant increase in
G2/M after 72 h of BA treatment. These results indicated that BA induced
significant disruptions in the normal cell cycle of PC cells.
BA increases the
degradation of multiple cell cycle and pro-survival proteins in PC cells
Since BA
is reported to increase the degradation of multiple proteins, we investigated
the effect of BA on cell cycle and pro-survival proteins in PC cells by western
blot analysis. Our results indicated that the protein levels of multiple cell
cycle proteins including cyclins A, B1, D1; Cdks1, 2, 4; E2F1, and Rb decreased
after BA treatment starting at 24 h in LNCaP and PC3 cells (Fig.
4A). In LNCaP cells, the Cdk inhibitor p21 also decreased after BA
treatment but in PC3, p21 protein increased at 24 h and returned to basal
levels by 48 and 72 h. In contrast, the Cdk inhibitor p27 increased after BA
treatment, suggesting a role in the G1/S cell cycle block. Similar results were
obtained in DU145 cells (Supplementary Fig.
S4).
BA
treatment increased the levels of cleaved-PARP with time in PC cells,
indicating elevated levels of activated caspases and apoptosis (Fig.
4B; Supplementary Fig.
S4). Interestingly, BA treatment dramatically decreased the protein levels
of AR in LNCaP and in AR transfected PC3/DU145 cells. Furthermore, BA treatment
decreased the levels of pro-survival proteins AKT, survivin, and Mcl-1. In
contrast, BA treatment did not reduce the levels of anti-apoptotic proteins
Bcl-2 and Bcl-xL (Fig.
4B; Supplementary Fig.
S4). Overall, these results indicated that BA selectively increased the
degradation of several proliferation and pro-survival proteins.
BA-mediated protein
degradation is dependent on the UPS
Previous
studies suggest that BA-mediated increase in UPS activity is a reason for
enhanced protein degradation [6],
[8].
Our results confirmed that in LNCaP cells, the UPS inhibitor MG132 blocked the
BA-mediated decrease in AR, AKT, and Mcl-1 proteins. In addition, MG132
antagonized the BA-mediated increase in apoptotic cell death, as determined by
trypan exclusion and western blot analysis of cleaved-PARP (Fig.
5A). A cyclin D1 mutant that cannot be degraded by the UPS was resistant to
BA-mediated degradation, further suggesting a role for the UPS (Fig.
5B). However, our proteasome assay results showed that BA had no direct
effect on UPS activity in LNCaP cells (Fig.
5C). In contrast to LNCaP cells, BA treatment of DU145 and PC3 cells
resulted in significantly increased UPS activity (Supplementary Fig.
S5). Overall, these results indicated that BA variably enhanced UPS
activity in some (DU145 and PC3) but not all (LNCaP) PC cells.
BA inhibits multiple
DUBs leading to increased total poly-Ub proteins in PC cells
In LNCaP cells,
a possible way BA can increase the selective degradation of proteins without
directly activating the UPS is by inhibiting DUBs. In this case, inhibition of
DUBs should result in an accumulation of total poly-Ub proteins and increase
their degradation by the UPS. Our western blot results showed that BA treatment
of LNCaP cells increased total poly-Ub proteins, similar to MG132 treatment (Fig.
6A). Similar results were also observed in DU145 and PC3 cells (not shown).
Despite the increase in poly-Ub, it is not clear why BA treatment has no effect
on UPS activity in LNCaP cells (Fig.
5C). In TRAMP prostate tumors, BA treatment also increased Ub proteins as
determined by IHC and decreased DUB activity (Supplementary Fig.
S6). Our DUB assay results showed that BA treatment of LNCaP cells reduced
DUB activity starting at 24 h. In contrast, treatment of LNCaP with 1 nM
docetaxel, a dose that increased apoptotic cell death [28],
had less effect on DUB activity (Fig.
6B). Similar results were obtained with BA treatment of DU145 and PC3 cells
with the exception that BA inhibited DUB activity starting at a later time (48
h) and docetaxel had a stronger DUB inhibitory effect compared to LNCaP cells
(Supplementary Fig.
S7). Overall, the ability of 10 µM BA to inhibit DUB activity correlated
with inhibition of cell proliferation (not shown).
To further
determine if BA can inhibit DUBs in LNCaP cells, we used a DUB labeling assay
with HA-UbVS, a potent, irreversible, and specific inhibitor of most DUBs [33].
Since HA-UbVS only binds to active DUBs, the HA tag allows for the labeling of
active DUBs present in LNCaP cells after BA treatment and analysis by western
blot. Our results showed that BA treatment of LNCaP cells decreased multiple
DUBs at 24 and 48 h compared to control cells (Fig.
6C). In contrast, treatment of LNCaP cells with docetaxel had no effect on
DUB activity. Similar results were obtained in DU145 and PC3 cells
(Supplementary Figs.
S8A, B). Overall, these results suggested that BA inhibited multiple DUBs,
resulting in increased poly-Ub proteins, which were likely rapidly degraded by
the UPS pathway.
BA has no effect on
DUB activity in non-cancer cells
BA is
reported to be a more selective agent against cancer compared to normal cells
but the mechanisms of how this occurs have not been determined [34],
[35].
Because our results in TRAMP mice showed that BA treatment did not reduce AR
protein levels in non-cancerous prostate, we sought to determine the effect of
BA on non-cancer cells by utilizing BJ human foreskin fibroblast cells. BJ
cells treated with BA for 72 h did not increase cell death or cleaved-PARP
compared to control treated cells (Fig.
7A). In contrast, treatment of BJ cells with 1 µM doxorubicin (a DNA
damaging drug) resulted in substantial cell death and cleaved-PARP. Our results
further showed that BA treatment did not decrease the levels of AR or increase
total poly-Ub proteins in BJ cells as in LNCaP cells (Figs.
7B, C). Finally, we showed that BA had no effect on DUB activity in BJ
cells, unlike what was observed in PC cells (Fig.
7D and Supplementary Fig.
S8C). Similar results were obtained using human IMR-90 and MRC-5 fetal lung
fibroblast cells (Supplementary Fig.
S9).
Unlike in fibroblast cells, BA inhibited DUB activity and
increased poly-Ub accumulation and cell death in proliferating PrEC and RWPE-1
normal prostate epithelial cells (Fig.
8). However, when RWPE-1 cells were allowed to reach confluency (high cell
density) before starting BA treatment (cRWPE-1), there was no effect on DUB
activity, poly-Ub accumulation, or cell death. In contrast, BA treatment of
confluent PC3 cells inhibited DUB activity and increased poly-Ub accumulation
greater than in proliferating PC3 cells (Fig.
8). IHC results in normal mouse tissues further demonstrated that BA
treatment did not increase apoptosis or ubiquitin accumulation (Supplementary Fig.
S10). Overall, these results suggested that the selectivity of BA to
increase apoptotic cell death in PC cells may be due to its ability to inhibit
DUBs specifically in cancer but not in non-cancer cells.
BA is an effective anti-cancer agent without toxicity to
normal cells and tissues making it ideal for testing in PC therapy.
Significantly, our results showed that BA treatment of TRAMP transgenic mice
containing advanced PC inhibited tumor growth, increased apoptosis, and
decreased proliferation and angiogenesis without toxicity to normal tissues. A
likely mechanism for this tumor-specific effect is that BA inhibited DUB
activity specifically in PC but not in normal cells, resulting in increased
poly-Ub proteins and enhanced degradation of proliferation and pro-survival proteins
such as cyclin D1 and AR by the UPS. Overall, these results provide evidence
that BA can be an effective anti-PC agent by inhibiting multiple DUBs, which
are new targets for PC therapy.
BA has potent anti-proliferation effects against the
commonly used PC cell lines due to a strong induction of apoptosis (Fig.
3). Numerous other carcinoma cell lines including lung, colon, liver,
pancreatic, breast, ovarian, head and neck, and renal are also inhibited by BA
mainly due to induction of mitochondrial targeted apoptosis [5],
[35],
[36].
Our results are similar to these reports because BA increased the release of
mitochondrial proteins such as cytochrome c, Smac, and AIF in PC cells (Fig.
3D). A recent report demonstrates that ectopic overexpression of Sp1
transcription factor renders pancreatic cancer cells more resistant to
BA-mediated toxicity [37].
Therefore, it is likely that that the degradation of specific proliferation and
pro-survival proteins individually or in combination are required for
BA-mediated apoptosis, although the precise mechanisms are not clear.
Alternatively, the BA-mediated pro-apoptotic death signal may originate from
the accumulation of poly-Ub proteins resulting from the inhibition of multiple
DUBs [38].
In LNCaP cells, the dependence of BA on the UPS pathway
for increased protein degradation was supported by results demonstrating that
1) a cyclin D1 mutant that cannot be degraded by the UPS was resistant to
BA-mediated degradation; and 2) the UPS inhibitor MG132 blocked the BA-mediated
decrease in AR, AKT, and Mcl-1. Since BA is reported to increase the
degradation of multiple members of the Sp transcription factor family in PC
cells, transcription of proliferation and pro-survival genes dependent on Sp
may also be further reduced [8].
Therefore, the BA-mediated degradation of Sp transcription factors likely
amplifies the UPS-mediated decrease in the expression of proliferation and
pro-survival genes. Similar to our in
vitro results in PC cell lines, BA treatment decreased AR and
cyclin D1 protein levels and increased total Ub proteins in TRAMP tumors. A
chemotherapy agent such as BA that can specifically degrade AR and cyclin D1 is
especially important in PC therapy due to the importance of these proteins in
tumor progression. AR is the most important factor for the emergence of CRPC
and cyclin D1 has a role in PC progression, regulation of AR activity, and may
be a significant prognostic marker for aggressive metastatic PC [39]–[42].
The mechanism why BA increased the degradation of cell
cycle and pro-survival proteins was likely by the inhibition of multiple DUBs,
which resulted in increased levels of total poly-Ub proteins that are
recognized by the UPS and degraded. Our preliminary data suggests that BA
specifically inhibits USP7, 9x, and 10 in PC3 cells (data not shown). USP7,
also known as herpesvirus-associated ubiquitin-specific protease or HAUSP, is
overexpressed in PC, deubiquitinates PTEN protein to block its function, and
regulates p53 levels [43],
[44].
USP9x regulates the levels of the anti-apoptotic protein Mcl-1 and has a role
in TGFβ signaling by controlling Smad4 mono-Ub [45],
[46].
At present, the DUB that regulates AR protein levels is not known. USP10 is an
AR cofactor important for activation of AR regulated genes [47],
[48].
However, it is not clear if inhibition or shRNA knockdown of USP10 lowers AR
protein levels. Once the DUB(s) that regulate AR protein levels are identified,
this should provide new high impact drug target(s) for the identification of
other small molecule inhibitors in addition to BA that could have therapeutic
efficacy in PC.
An alternative strategy to target the UPS without the
toxic side effects to normal cells may be by inhibiting upstream DUB activity.
One possible mechanism for BA's lack of toxicity to non-cancer cells is that BA
had no effect on DUB activity in normal cells such as BJ, IMR-90, MRC-5
fibroblasts and confluent RWPE-1 prostate epithelial cells compared to PC cells
(Figs.
6–8;
supplemental Fig.
S9). The reason for this difference is not clear but may reflect that DUBs
are often overexpressed in cancer cells [9]–[11].
Interestingly, the DUB labeling assay revealed only two DUBs present in PC but
not in BJ cells (bands 2 and 3, Supplementary Fig.
S8D); other DUB labeled bands that appeared more highly expressed in PC
compared to BJ cells include bands 1, 4, and 5. We are currently investigating
the identities of the DUB family members inhibited by BA and play a role in
sensitizing PC cells to BA treatment without harming normal cells.
Alternatively, the reason BA inhibits DUB activity in PC but not in non-cancer
cells may be due to an indirect mechanism.
Inhibition of the UPS pathway for anti-cancer
chemotherapy has led to the successful development of bortezomib [3].
This report addressed the utilization of a small molecule such as BA to
increase the degradation of proliferation and pro-survival proteins via the UPS
by inhibiting upstream DUB activity in PC cells. Further investigation will be
required to determine whether BA directly or indirectly inhibits DUB activity
in PC cells. In contrast, BA had no effect on DUB activity in normal fibroblast
and confluent prostate epithelial cells, perhaps explaining why BA was
non-toxic to normal cells and tissues. Since DUBs are overexpressed in PC
cells, they provide a new target that will result in an increase in therapeutic
efficacy by reducing pro-survival proteins such as AR. Our data suggests that
BA-mediated inhibition of DUBs and induction of apoptotic cell death
specifically in PC but not in non-cancer cells will provide an effective
non-toxic and clinically selective agent for chemotherapy.
BA treatment of TRAMP mice with prostate tumors increases
apoptosis and decreases angiogenesis and proliferation. Representative immunostaining for cleaved (cl)-caspase-3
and TUNEL (apoptosis), CD31 and CD34 (angiogenesis), and Ki67 and PCNA
(proliferation) in prostate tumors from TRAMP mice treated with vehicle control
or BA10 (×200).
doi:10.1371/journal.pone.0056234.s001
(TIF)
BA increases the release of mitochondrial proteins in
LNCaP and DU145 cells.
Mitochondrial protein release assay and western blot analysis showed increased
levels of cytochrome c, Smac, and AIF in LNCaP and DU145 cells treated with BA
compared to control (0 hrs) cells. Cox IV protein was negative or weak
indicating no or minimal mitochondrial contamination whereas actin was the
positive control. Coomassie blue stain of total protein was loading control. +C
was lysate prepared using the standard method for total proteins.
doi:10.1371/journal.pone.0056234.s002
(TIF)
(A) BA increased G1/S cell cycle block in PC cells. Flow
cytometric analysis of LNCaP,
DU145, and PC3 treated with BA or control for 24 h resulted in increased cells
in G1 and decreased cells in S phase. Numbers in parenthesis are the percentage
of cells in each cell cycle phase from three independent experiments done in
duplicate. There was no change in G2/M and increased sub (s)-G1. (B) BA increased cells
in the sub-G1 cell cycle phase at later time points. Flow cytometric analysis
of LNCaP, DU145, and PC3 treated with BA for 48 (LN) or 72 h (DU/PC) showed
increased cells in sub-G1, indicating DNA breakage. In DU145 and PC3 but not in
LNCaP cells, there was significantly increased cells in G2/M. Numbers in
parenthesis were the percentage of cells in each cell cycle phase from three
independent experiments done in duplicate.
doi:10.1371/journal.pone.0056234.s003
(TIF)
BA increases the degradation of multiple cell cycle and
pro-survival proteins in DU145 cells. Western blot analysis showed that BA treatment resulted
in lower protein levels of cyclins, Cdks, E2F1, Rb, AR (transfected), AKT, and
survivin and higher levels of p27 and cl-PARP in DU145 cells, similar to
results in LNCaP and PC3 cells. BA treatment also decreased the levels of
mutant p53 protein. Unlike in LNCaP and PC3 cells, BA treatment of DU145 cells
did not decrease Mcl-1 protein.
doi:10.1371/journal.pone.0056234.s004
(TIF)
UPS assay showed significantly increased proteasome
activity in DU145 and PC3 cells treated with BA for 24, 48, and 72 h (*, P<0.03;
**, P<0.003; ***, P<3×10−5). Addition of MG132 (MG) to BA resulted in decreased
proteasome activity.
doi:10.1371/journal.pone.0056234.s005
(TIF)
(A) BA treatment of TRAMP mice with prostate tumors
increased immunostaining for ubiquitin compared to vehicle control (×200). (B)
DUB assay showed that BA10 (n = 5) significantly decreased DUB activity in
TRAMP prostate tumors relative to vehicle control (n = 4) (*, P<6×10−5).
Control lysates pre-treated with 4 mM NEM for 1 h resulted in decreased DUB
activity.
doi:10.1371/journal.pone.0056234.s006
DUB assay showed that BA treatment of DU145 and PC3 cells
significantly decreased DUB activity at 48 and 72 h relative to control treated
cells ( = 1). Unlike in LNCaP,
treatment of DU145 and PC3 with 1 nM Doc also reduced DUB activity, although
not as great as in BA treated cells (*, P<0.05;
**, P<7×10−3).
Control lysates pre-treated with 4 mM NEM for 1 h resulted in decreased DUB
activity.
doi:10.1371/journal.pone.0056234.s007
(TIF)
DUB activity labeling with HA-UbVS showed that BA but not
Doc inhibited multiple DUBs in DU145 (A) and PC3 (B) cells. Protein bands 1–5 showed the strongest decrease in
activity with BA treatment. In contrast, BA does not inhibit DUB activity in BJ
(C)
cells. Control lysates without addition of HA-UbVS or pre-incubated with NEM
for 1 h were the controls. (D)
Control LNCaP (LN), LN-AI (AI, androgen-independent variant of LNCaP), DU
(DU145), PC (PC3) cells labeled with HA-UbVS are compared with BJ cells.
Molecular weight markers (kDa) are shown to the left. Coomassie blue stain of
total protein were loading controls.
doi:10.1371/journal.pone.0056234.s008
(TIF)
BA had no effect on non-cancer IRM-90 and MRC-5 fetal
lung fibroblasts. (A) Trypan blue
exclusion assay showed that BA did not increase cell death in IRM-90 and MRC-5
after 72 h. In contrast, doxorubicin (Dox) increased cell death (n = 7–11,
three experiments). (B)
Western blot analysis showed that Dox but not BA increased cl-PARP. +C is LNCaP
BA 24 h. (C)
In contrast to LNCaP (+C), BA did not increase poly-Ub accumulation in MRC-5
and IRM-90. (D)
DUB assay showed that BA treatment of IRM-90 cells had no effect on DUB
activity. Control lysates pre-treated with NEM for 1 h resulted in decreased
DUB activity (n = 6, two experiments).
doi:10.1371/journal.pone.0056234.s009
(TIF)
BA did not increase apoptosis or ubiquitin in normal
mouse tissue. IHC (×200) results
showed little difference between BA10 and vehicle control TRAMP spleen and
thymus for cleaved caspase-3 (apoptosis) and ubiquitin.
doi:10.1371/journal.pone.0056234.s010
(TIF)
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