Mitochondrial apoptosis and FAK signaling disruption by a novel histone deacetylase inhibitor, HTPB, in antitumor and antimetastatic mouse models.
ABSTRACT Compound targeting histone deacetylase (HDAC) represents a new era in molecular cancer therapeutics. However, effective HDAC inhibitors for the treatment of solid tumors remain to be developed.
Here, we propose a novel HDAC inhibitor, N-Hydroxy-4-(4-phenylbutyryl-amino) benzamide (HTPB), as a potential chemotherapeutic drug for solid tumors. The HDAC inhibition of HTPB was confirmed using HDAC activity assay. The antiproliferative and anti-migratory mechanisms of HTPB were investigated by cell proliferation, flow cytometry, DNA ladder, caspase activity, Rho activity, F-actin polymerization, and gelatin-zymography for matrix metalloproteinases (MMPs). Mice with tumor xenograft and experimental metastasis model were used to evaluate effects on tumor growth and metastasis. Our results indicated that HTPB was a pan-HDAC inhibitor in suppressing cell viability specifically of lung cancer cells but not of the normal lung cells. Upon HTPB treatment, cell cycle arrest was induced and subsequently led to mitochondria-mediated apoptosis. HTPB disrupted F-actin dynamics via downregulating RhoA activity. Moreover, HTPB inhibited activity of MMP2 and MMP9, reduced integrin-β1/focal adhesion complex formation and decreased pericellular poly-fibronectin assemblies. Finally, intraperitoneal injection or oral administration of HTPB efficiently inhibited A549 xenograft tumor growth in vivo without side effects. HTPB delayed lung metastasis of 4T1 mouse breast cancer cells. Acetylation of histone and non-histone proteins, induction of apoptotic-related proteins and de-phosphorylation of focal adhesion kinase were confirmed in treated mice.
These results suggested that intrinsic apoptotic pathway may involve in anti-tumor growth effects of HTPB in lung cancer cells. HTPB significantly suppresses tumor metastasis partly through inhibition of integrin-β1/FAK/MMP/RhoA/F-actin pathways. We have provided convincing preclinical evidence that HTPB is a potent HDAC targeted inhibitor and is thus a promising candidate for lung cancer chemotherapy.
- [Show abstract] [Hide abstract]
ABSTRACT: Deoxyribonucleic acid is wrapped around an octamer of core histone proteins to form a nucleosome, the basic structure of chromatin. Two main families of enzymes maintain the equilibrium of acetyl groups added to or removed from lysine residues. Histone deacetylases (HDACs) catalyze the removal of acetyl groups from lysine residues in histone amino termini and non-histone proteins also, leading to chromatin condensation and transcriptional repression. HDAC overexpression, resulting in tumor suppressor genes silencing, has been found in several human cancer tissues, indicating that aberrant epigenetic activity is associated with cancer development. Therefore, inhibitors of these enzymes are emerging anticancer agents and there is evidence supporting their role in hematological malignancies. The minimal efficacy of conventional chemotherapy has prompted a renewed focus on targeted therapy based on pathways altered during the pathogenesis of lung cancer. We identify the pleiotropic antitumor effects of HDAC inhibitors in lung cancer, focusing on the result caused by their use individually, as well as in combination with other chemotherapeutic agents, in lung cancer cell lines and in clinical trials. We searched reviews and original papers in Pubmed over the last 10 years. We identified 76 original papers on this topic. Numerous preclinical studies have shown that HDAC inhibitors exhibit impressive antitumor activity in lung cancer cell lines. Nevertheless, Phase III randomized studies do not support HDAC inhibitors use in lung cancer patients in everyday practice. Ongoing and future studies would help determine their role in lung cancer treatment.Cancer Chemotherapy and Pharmacology 09/2013; · 2.80 Impact Factor
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ABSTRACT: Normal cellular function is dependent on a number of highly regulated homeostatic mechanisms, which act in concert to maintain conditions suitable for life. During periods of nutritional deficit, cells initiate a number of recycling programs which break down complex intracellular structures, thus allowing them to utilize the energy stored within. These recycling systems, broadly named “autophagy”, enable the cell to maintain the flow of nutritional substrates until they can be replenished from external sources. Recent research has shown that a number of regulatory components of the autophagy program are controlled by lysine acetylation. Lysine acetylation is a reversible post-translational modification that can alter the activity of enzymes in a number of cellular compartments. Strikingly, the main substrate for this modification is a product of cellular energy metabolism: acetyl-CoA. This suggests a direct and intricate link between fuel metabolites and the systems which regulate nutritional homeostasis. In this review, we examine how acetylation regulates the systems that control cellular autophagy, and how global protein acetylation status may act as a trigger for recycling of cellular components in a nutrient-dependent fashion. In particular, we focus on how acetylation may control the degradation and turnover of mitochondria, the major source of fuel-derived acetyl-CoA.Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 01/2014; · 4.13 Impact Factor
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ABSTRACT: Many histone deacetylase (HDAC) inhibitors show limited therapeutic effects for solid tumors. Here, we develop a novel HDAC inhibitor YCW1 and verify the combination effect of YCW1 and cisplatin in lung cancer pre-clinical models. YCW1 exerted cancer-specific cytotoxicity via mitochondria-mediated apoptosis. YCW1 and cisplatin showed synergistic anti-tumor effects through impairment of DNA damage repair. YCW1 inhibited tumor growth in lung orthotopic and subcutaneously implanted xenograft models. YCW1 significantly suppressed lung metastases via inhibition of focal adhesion complex. Our findings suggested that YCW1 is a potential HDAC inhibitor for lung cancer treatment as single and in combination regimens with cisplatin.Cancer letters 12/2013; · 5.02 Impact Factor
Mitochondrial Apoptosis and FAK Signaling Disruption
by a Novel Histone Deacetylase Inhibitor, HTPB, in
Antitumor and Antimetastatic Mouse Models
Jiunn-Min Shieh1, Tzu-Tang Wei2, Yen-An Tang3, Sin-Ming Huang4, Wei-Ling Wen4, Mei-Yu Chen2, Hung-
Chi Cheng3,5, Santosh B. Salunke6, Ching-Shih Chen7, Pinpin Lin8, Chien-Tien Chen6*, Yi-Ching Wang2,3*
1Department of Internal Medicine, Chi Mei Medical Center, Tainan, Taiwan, 2Department of Pharmacology, National Cheng Kung University, Tainan, Taiwan, 3Institute of
Basic Medical Science, National Cheng Kung University, Tainan, Taiwan, 4Department of Life Science, National Taiwan Normal University, Taipei, Taiwan, 5Institute of
Biochemistry, National Cheng Kung University, Tainan, Taiwan, 6Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan, 7Division of Medicinal
Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio, United States of America, 8Division of Environmental Health and
Occupational Medicine, National Health Research Institutes, Zhunan, Taiwan
Background: Compound targeting histone deacetylase (HDAC) represents a new era in molecular cancer therapeutics.
However, effective HDAC inhibitors for the treatment of solid tumors remain to be developed.
Methodology/Principal Findings: Here, we propose a novel HDAC inhibitor, N-Hydroxy-4-(4-phenylbutyryl-amino)
benzamide (HTPB), as a potential chemotherapeutic drug for solid tumors. The HDAC inhibition of HTPB was confirmed
using HDAC activity assay. The antiproliferative and anti-migratory mechanisms of HTPB were investigated by cell
proliferation, flow cytometry, DNA ladder, caspase activity, Rho activity, F-actin polymerization, and gelatin-zymography for
matrix metalloproteinases (MMPs). Mice with tumor xenograft and experimental metastasis model were used to evaluate
effects on tumor growth and metastasis. Our results indicated that HTPB was a pan-HDAC inhibitor in suppressing cell
viability specifically of lung cancer cells but not of the normal lung cells. Upon HTPB treatment, cell cycle arrest was induced
and subsequently led to mitochondria-mediated apoptosis. HTPB disrupted F-actin dynamics via downregulating RhoA
activity. Moreover, HTPB inhibited activity of MMP2 and MMP9, reduced integrin-b1/focal adhesion complex formation and
decreased pericellular poly-fibronectin assemblies. Finally, intraperitoneal injection or oral administration of HTPB efficiently
inhibited A549 xenograft tumor growth in vivo without side effects. HTPB delayed lung metastasis of 4T1 mouse breast
cancer cells. Acetylation of histone and non-histone proteins, induction of apoptotic-related proteins and de-
phosphorylation of focal adhesion kinase were confirmed in treated mice.
Conclusions/Significance: These results suggested that intrinsic apoptotic pathway may involve in anti-tumor growth
effects of HTPB in lung cancer cells. HTPB significantly suppresses tumor metastasis partly through inhibition of integrin-b1/
FAK/MMP/RhoA/F-actin pathways. We have provided convincing preclinical evidence that HTPB is a potent HDAC targeted
inhibitor and is thus a promising candidate for lung cancer chemotherapy.
Citation: Shieh J-M, Wei T-T, Tang Y-A, Huang S-M, Wen W-L, et al. (2012) Mitochondrial Apoptosis and FAK Signaling Disruption by a Novel Histone Deacetylase
Inhibitor, HTPB, in Antitumor and Antimetastatic Mouse Models. PLoS ONE 7(1): e30240. doi:10.1371/journal.pone.0030240
Editor: Jean-Marc Vanacker, Institut de Ge ´nomique Fonctionnelle de Lyon, France
Received May 4, 2011; Accepted December 16, 2011; Published January 18, 2012
Copyright: ? 2012 Shieh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by grants CMNCKU9906 from the Chi Mei Medical Center, Taiwan (JMS) and NSC100-2325-B-400-012 (PL) and NSC 100-
2325-B-006-009 from the National Science Council, Taiwan (YCW). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (Y-CW); email@example.com (C-TC)
The development of molecular-targeted therapies represents a
new era in cancer treatment . Molecular-targeted drugs
specifically against cancer cells without affecting normal cells are
being developed [2–4]. Many of the molecular-targeted drugs are
inhibitors of proteins involved in signaling transduction, such as
growth factors, growth factor receptors or kinases [2,5].
Recent findings of overexpression and/or increased activity of
histone deacetylases (HDACs) in cancer cells and low basal level in
normal cells make HDACs potential therapeutic targets for cancer
treatment [6–8]. HDACs catalyze the removal of acetyl-groups
from lysine residues in the N-terminal tails of histones, leading to
chromatin condensation and transcriptional repression. In addition
to histones, HDACs have many other substrates involved in the
regulation of cellular function, such as p53, p21, HSP90, tubulin,
and of various transcription factors . It has been demonstrated
that inhibition of HDACs reverses aberrant epigenetic status and
exhibits potent antitumor activities by inducing cell cycle arrest,
differentiation and/or apoptosis in diverse cancer cells [10,11].
To date, more than 15 HDAC inhibitors have been tested in
clinical trials in several hematological malignancies and solid
tumors . These HDAC inhibitors include the short chain fatty
acids such as phenylbutyrate, butyrate, and valproic acid; the
PLoS ONE | www.plosone.org1 January 2012 | Volume 7 | Issue 1 | e30240
benzamides such as MS-275 and CI-994 [13,14]; the hydroxamic
acids such as Trichostatin A (TSA), LAQ-824, and pyroxamide;
the cyclic peptides such as FK-228. Specifically, the U.S. Food and
Drug Administration has approved two HDAC inhibitors,
vorinostat (SAHA, suberoylanilide hydroxamic acid, ZolinzaH)
and romidepsin (FK228, depsipeptide, IstodaxH), for the treatment
of cutaneous manifestations of cutaneous T-cell lymphoma .
However, some adverse events occurred in patients treated with
vorinostat or other HDAC inhibitors, which may have resulted
from the high dose of inhibitors used during the treatment for solid
tumors in clinical trials [8,16].
The structures of HDAC inhibitors such as TSA and SAHA
could be divided into three motifs: a zinc-chelating motif
(hydroxamate), a linker consisting an aliphatic chain, and a polar
cap group. We have previously developed an HDAC inhibitor, N-
has been optimized for HDAC inhibition by structure-based
analyses . In the present study, the antitumor and antimeta-
static activities of HTPB and the underlying mechanisms were
studied in lung cancer cell and animal models. The goal was to
develop an HDAC inhibitor with low IC50to treat solid tumor
without significant side effects in preclinical models.
HTPB is a pan-HDAC inhibitor and exhibits cancer cell-
specific cytotoxicity by promoting acetylation of various
The structure of HTPB and SAHA are shown in Fig. 1A (upper
panel). The cytotoxicity of HTPB was assessed in the IMR90
normal lung cell line and two human lung cancer cell lines including
A549 and H1299. SAHA was included as a positive control HDAC
inhibitor. HTPB induced significant cytotoxicity in A549 and
H1299 lung cancer cell lines with extrapolated IC50 value of
1.59 mM for A549 and 2.19 mM for H1299 (1.2–2.1 times more
potency than SAHA; IC50: A549=1.89 mM, H1299=4.59 mM),
without showing apparent cytotoxicity towards IMR90 normal lung
cell line (Fig. 1A, lower panel).
To examine the target specificity of HTPB, in vitro HDAC
inhibition assay was performed with class I, II, and IV HDACs. As
Figure 1. Effect of HTPB on cell viability and on the biomarkers associated with broad inhibition on numerous HDACs. (A) Chemical
structure of HTPB (upper left). Dose-dependent effects of HTPB on cell viability in IMR90, H1299, and A549 cells (lower left). Cells were treated with
0.5–10 mM of HTPB for 48 hours, and cell viability was assessed by MTT assay. A known HDAC inhibitor, SAHA, was used for comparison. (B) HTPB
suppressed activities of class I (HDAC1 and HDAC8), class II (HDAC4 and HDAC6), and class IV (HDAC11) HDACs in A549 cells. Data represent mean 6
SEM from three independent experiments. *** P,0.001. Dose-dependent effects (C) and time-dependent effects (D) of HTPB on the histone and non-
histone proteins. SAHA was included for comparison. (E) HTPB induced acetylation of histone H3 and H4 without affecting the total protein levels of
HDAC1 and HDAC 6. In addition, HTPB induced p21 protein expression in both A549 (p53 wild-type) and H1299 (p53 null) cells. The immunoblots
shown are representatives of three independent experiments.
Antitumor and Antimetastatic Activity of HTPB
PLoS ONE | www.plosone.org2January 2012 | Volume 7 | Issue 1 | e30240
shown in Fig. 1B, the deacetylase activities of different HDAC
isotypes including class I (HDAC1 and HDAC8), class II (HDAC4
and HDAC6), and class IV (HDAC11) were significantly inhibited
by HTPB. The biomarkers of HDAC inhibition are acetylation of
histone and non-histone proteins [11,18]. Exposure to HTPB
induced acetylation of histone H3, histone H4, p53 and tubulin in
a time- and dose-dependent manner (Fig. 1C & 1D), while it did
not affect the HDAC1 and HDAC6 protein levels (Fig. 1E).
Notably, HTPB was more potent than SAHA for induction of
tubulin acetylation (Fig. 1D). Despite the p53 status, HTPB
induced the expression of p21Cip1protein in A549 (p53 wild-type)
and H1299 (p53 null) cells (Fig. 1E), suggesting that activation of
p21Cip1involved changes in promoter-associated proteins, includ-
ing HDACs, not via p53-dependent transcriptional activation.
These results suggested that HTPB is a pan-HDAC inhibitor and
induces acetylation of histone and non-histone proteins.
HTPB induces cell cycle arrest and mitochondrial-
To investigate the underlying mechanism of cell growth
repression by HTPB, the effects of HTPB on cell cycle progression
in A549 and H1299 cells were assessed by flow cytometry.
Treatment with 5 mM HTPB caused cell accumulation at G2/M
phase and apoptosis (sub-G1) in both cells and an additional G1
arrest in H1299 cells at 48 hours treatment (Fig. 2A), indicating
that HTPB exerted a cell cycle deregulation effect.
To further elucidate the HTPB-induced apoptosis, we
performed a DNA ladder analysis and found that ladders
appeared in A549 and H1299 cells after HTPB treatments
(Fig. 2B). Moreover, treatment with 5 mM HTPB caused a time-
dependent increase in pro-apoptotic protein, Bad and Bak, while
it decreased the anti-apoptotic protein Bcl-2 and Bcl-XL(Fig. 2C).
HTPB treatment significantly stimulated caspase-3 and caspase-
9 (an indicator of the intrinsic mitochondrial pathway) activities
after 24 hours treatment whereas the activity of caspase-8 (an
indicator of the extrinsic membrane receptor pathway) remained
unaffected in both cells lines tested (Fig. 2D). The cleavage of
pro-caspase-9 and -3 was also seen after HTPB treatment (Fig.
S1). These results suggested that intrinsic apoptotic pathway
may play a role in HTPB-induced cytotoxicity in lung cancer
HTPB at non-cytotoxic doses suppresses migration ability
in lung cancer cell lines via inhibiting activity of integrin-
b1/FAK/MMP/RhoA/F-actin motility control
To investigate whether HTPB inhibited cell migration in A549
and H1299 lung cancer cell lines, trans-well migration assay and
wound-healing assay were performed at non-cytotoxic doses. As
shown in Fig. 3A and 3B, the percentage and distance of migrated
cells were significantly reduced after HTPB treatment. These
results suggested that HTPB significantly inhibited lung cancer cell
migration at non-cytotoxic doses.
Figure 2. HTPB induces cell cycle arrest and apoptosis. (A) The effects of HTPB on cell cycle distribution in A549 and H1299 cells. Cells were
treated with 5 mM HTPB for indicated times and assessed by flow cytometry. The percentage of G2/M and sub-G1 fraction population is plotted in the
histogram. G2/M arrest and sub-G1 induction are indicated by arrows. (B) HTPB caused apoptotic DNA ladders in A549 and H1299 cells treated with
5 mM HTPB for 48 hours. HTPB induced intrinsic apoptosis. Cells were treated with 5 mM HTPB for indicated times and cell lysates were subjected to
Western blot analyses (C) and caspase activity assay (D). Pro-apoptotic proteins Bad and Bak were up-regulated and anti-apoptotic proteins Bcl-2 and
Bcl-XL were down-regulated. Caspases-3 and -9 were up-regulated in both A549 and H1299 cells. Data represent mean 6 SEM from three
independent experiments. * P,0.05; ** P,0.01.
Antitumor and Antimetastatic Activity of HTPB
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To delineate the mechanism of HTPB-induced migration
inhibition, enzyme activities of matrix metalloproteinases MMP-2
and MMP-9 were examined bysubstrate-specific gelatin-zymography
assay. The data indicated that MMP-2 and MMP-9 enzyme activities
were significantly decreased in A549 and H1299 cells treated with
RhoA activity in lung cancer cells (Fig. 3C). To determine whether
focal adhesion complex played a role in HTPB-inhibited cancer cell
migration, we examined the activities of focal adhesion kinase (FAK)
actin dynamics as detected by confocal immunofluorescence
microscopy illustrated that the polymerization of F-actin was
dramatically inhibited after HTPB treatment in H1299 and A549
cells (Fig. 3E). These results suggested that HTPB decreased
migratory activities of lung cancer cells partly through inhibiting the
activities of MMPs and RhoA protein and disrupting focal adhesion
complex and F-actin cytoskeleton arrangement.
HTPB inhibits lung tumor xenograft growth in vivo
without significant side effects
To further evaluate the antitumoractivity of HTPB, Balb/c nude
mice bearing A549 lung tumor xenograft were injected intraper-
itoneally or orally with 25–100 mg/kg of HTPB, 3 days/week for
three weeks. As shown in Fig. 4A and Fig. S2A, intraperitoneal
treatment with 25 and 50 mg/kg HTPB significantly inhibited
tumor growth by 80% and 94%, compared with DMSO control,
while 50 mg/kg SAHA inhibited tumor growth only by 65% (left
panel). Oral administration with 50 and 100 mg/kg HTPB
significantly inhibited tumor growth by 39% and 79% (right panel)
respectively. In addition, significantly less tumor weight was
observed in mice treated by HTPB intraperitoneally (Fig. 4B, left
panel)ororally(rightpanel)than incontrolmice. Note that the anti-
tumor growth effect of HTPB was 2–4 times more potent than
SAHA as assessed by tumor weight (Fig. 4B, left panel). Treatment
with HTPB did not adversely affect body weight (Fig. 4C and Fig.
S2B) and caused no detectable toxicity as examined by hematolog-
ical biochemistry examinations (Fig. 4D).
Figure 3. HTPB inhibits cancer cell migration via reduced activities of matrix metalloproteinases, RhoA, and focal adhesion
complex. (A) The image from trans-well migration assay and (B) wound-healing assay indicated that after 48 hours treatment at non-cytotoxic
doses, HTPB inhibited migratory activity in a dose-dependent manner. * P,0.05; ** P,0.01; scale bars: 400 mm. (C) Gelatin-zymography assay and
RhoA-GTP GST pull-down assay showed that MMP-2 and MMP-9 enzyme activities were suppressed and RhoA-GTP expression was reduced in A549
and H1299 cells after 2.5 mM HTPB treatment for 48 hours. (D) Expression of integrin-b1 and phosphorylation of FAK at Tyr-397 were down-regulated
in H1299 and A549 cells after HTPB treatment for 48 hours at the indicated doses. (E) HTPB led to F-actin dysregulation by immunofluorescence
analyses. Cells were treated with 5 mM HTPB for 48 hours, and then fixed and stained with phalloidin (F-actin). Scale bars: 40 mm.
Antitumor and Antimetastatic Activity of HTPB
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HTPB significantly inhibits cancer cell metastasis in vivo
To explore the anti-metastasis activity of HTPB, highly
metastatic 4T1-luc breast cancer cells were treated with
1.92 mM HTPB for 48 hours. Such a treatment did not change
cell viability (Fig. 5A and Fig. S3A) or cell cycle distribution (Fig.
S3B) of 4T1-luc breast cancer cells but it decreased transwell
migration capacities to 50% compared to the un-treated control
(Fig. S3C). Interestingly, pericellular poly-fibronectin assemblies
were decreased in 4T1-luc cells after HTPB treatment (Fig. 5B).
The treated cells were then injected intravenously via tail vein into
Balb/c mice and photographed by IVIS-50 imaging system at
day-1, 4, 7, and 13 to observe in vivo cancer cell metastasis after
Figure 4. HTPB effectively inhibits A549 xenograft growth without significant side effects. (A) Balb/c nude mice bearing the established
A549 tumors (,50 mm3) were treated with HTPB via intraperitoneal (left panel) or oral administration (right panel) for three weeks (3 days/week). A
known HDAC inhibitor, SAHA, was used for comparison in intraperitoneal experiments. The tumor volumes of mice were measured twice weekly.
Points, mean; bars, 6SEM. Three mice per group for intraperitoneal injection and five mice per group for oral treatment were used in the xenograft
experiment. (B) The tumor weights of mice were measured. P values were for comparisons with DMSO or vehicle control (* P,0.05, ** P,0.01).
(C) HTPB treatments did not cause significant body weight loss of tested animals. (D) Hematological biochemistry tests including GOT, GPT, albumin
and creatinine were examined and the results showed no significant differences between HTPB treatment and DMSO or solvent control.
Antitumor and Antimetastatic Activity of HTPB
PLoS ONE | www.plosone.org5 January 2012 | Volume 7 | Issue 1 | e30240
drug treatment. As shown in Fig. 5C and Fig. S4, HTPB
significantly delayed lung metastasis of 4T1-luc cells (right panel),
compared to DMSO control (left panel). These results suggested
that HTPB inhibited metastasis of 4T1-luc cells in vivo.
HTPB induces protein acetylation, apoptosis and FAK
inhibition in vivo
To confirm that HTPB suppressed tumor growth and tumor
metastasis via targeting the HDACs, inducing apoptosis and
inhibiting FAK in vivo, mice bearing established A549 tumors were
treated with a single dose of HTPB at 50 mg/kg. After treatment,
tumors were dissected and cell lysates were subjected to Western blot
orimmunohistochemistryanalysis. Acetylation ofhistone H3,histone
H4 and p53 were profoundly increased after 2 hours treatment in
tumor xenograftcollected. The protein levels of anti-apoptoticBcl-XL
started to decreaseafter 2 hourstreatment (Fig. 6A),whilethe level of
cleaved caspase-3 protein was increased in tumor xenograft collected
on day 25 (Fig. 6B, upper panel). Activated phosopho-FAK and
phosopho-AKT were also decreased in HTPB-treated tumor
xenograft (Fig. 6B, middle and lower panels). These results
demonstrated that HTPB could induce apoptosis and down-regulate
migration regulators, FAK and AKT, in vivo. In addition, increase of
HDAC inhibition biomarkers such as acetylation of histone H3,
histone H4 and p53 was evident in tumors of treated mice.
Figure 5. HTPB delays lung metastasis of 4T1-luc breast cancer cell in animal models. (A) 4T1-luc mouse breast cancer cells were treated
with 1.92 mM HTPB for 48 hours. HTPB did not significantly affect cell growth of 4T1-luc cells during the indicated treatments. (B) Fibronectin
assembly on the surface of 4T1-luc cells measured by immunofluorescence analyses showed that HTPB treatment reduced pericellular poly-
fibronectin assemblies. (C) The treated 4T1-luc cells were injected intravenously via tail vein into Balb/c mice and observed for the luciferase signals
and photographed using IVIS50 for 13 days after drug treatment. HTPB significantly delayed lung metastasis.
Antitumor and Antimetastatic Activity of HTPB
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Since HDACs are promising targets for cancer therapy, a
number of HDAC inhibitors are in clinical trials as single agent
and/or in combination with other anticancer drugs . However,
none have yet been demonstrated to be effective as treatment for
solid tumors. Here, we provide compelling evidence from cell and
animal studies that HTPB, a phenylbutyrate-based compound, is a
potential HDAC inhibitor for lung cancer treatment. HTPB
targeted numerous members within three classes of HDACs in vitro
and efficiently stimulated protein acetylation in cell and animal
models (Fig. 1 and 6). HTPB repressed cell viability and induced
apoptosis in lung cancer cell lines (Fig. 1 and 2). In addition, HTPB
reduced cell migration at non-cytotoxic dose via inhibition of Rho/
F-actin and integrin-b1/FAK/MMP pathways (Fig. 3). The
xenograft experiments further confirmed that HTPB induced cell
apoptosis and thereby inhibited tumor growth in vivo without
adversely affecting body weight and hematological parameters
(Fig. 4 and 6). In addition, HTPB significantly inhibited lung
metastasis in vivo through inhibition of pericellular poly-fibronectin
assemblies at non-cytotoxic concentrations (Fig. 5 and 6).
Collectively, these results suggested that HTPB is a promising
candidate HDAC inhibitor for lung cancer treatment.
We provide the first evidence that HTPB significantly inhibited
tumor growth by both intraperitoneal and oral administrations in
animal model (Fig. 4). The anti-tumor growth effect could be
optimized if a better solvent was used during oral administration.
Upon HTPB treatment, G2/M arrest were induced and
subsequently led to mitochondria-mediated apoptosis (Fig. 2).
The G1 arrest observed in H1299 cells could be due to the
induced expression of p21Cip1after HTPB treatment (Fig. 1E).
p21Cip1is a cyclin-dependent kinase inhibitor and has been shown
to mediate G1 cell cycle arrest . Our results (Fig. 2 and 6)
showed that treatment of HTPB resulted in a time-dependent
reduction in the levels of the anti-apoptotic proteins Bcl-2 and
Figure 6. HTPB effectively induced protein acetylation, apoptosis and pFAK/pAKT inactivation in vivo. (A) Mice bearing established
(about 100,200 mm3) A549 tumors were injected intraperitoneally with a single dose of HTPB at 50 mg/kg. After treatment for the indicated time,
tumors from two representative mice of each time point (a–f) were harvested and subjected to Western blot using anti-actyl-histone H3, H4 and p53
and Bcl-XLantibodies. (B) Immunohistochemistry analyses were performed using antibody against cleaved-form of caspase-3, p-FAK and p-AKT
(brownish color). Original magnification6200.
Antitumor and Antimetastatic Activity of HTPB
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Bcl-XL. Concomitantly, the level of pro-apoptotic proteins Bad and
Bak was increased. The Bcl-2 family of proteins constitutes a
critical mediator in the mitochondrial pathway of apoptosis .
Furthermore, the progression of apoptosis involves the activation
of a cascade of proteases called caspases. Theoretically, the
extrinsic pathway is related to the activation of caspase-8 and the
intrinsic pathway is associated with activation of caspase-9. Both
pathways converge to a common pathway involving the activation
of caspase-3 [20,21]. As shown in our data, HTPB apparently
stimulated caspases-3, caspase-9 and to a lesser extent caspase-8
activities. Importantly, induced caspase-3 and reduced Bcl-XL
were confirmed in HTPB-treated tumor xenograft (Fig. 6). It is
noteworthy that we also found that HTPB caused induction of the
acetylated-p53 protein, which are highly expressed and correlated
with apoptosis induction . Together, these results suggested
that HTPB induced the execution of apoptosis partly through the
activation of the intrinsic mitochondrial pathway. Experiments
using specific caspase inhibitors to test whether intrinsic apoptosis
accounted for HTPB-induced cell death are worthy of further
We have also showed for the first time that HTPB significantly
delayed lung metastases in animal model (Fig. 5). Our cellular
data indicated that HTPB inhibited cancer cell migration
through inhibiting activity of matrix metalloproteinases, RhoA,
integrin-b1 and focal adhesion complex (Fig. 3) and disrupting F-
actin arrangement (Fig. 3). Activated integrins control down-
stream signaling pathway through non-receptor tyrosine kinase
FAK [23,24] which correlates with cancer metastasis .
Integrin-b1 has been genomically identified and shown to
clinically promote cancer metastasis . In addition, the Rho/
Rac/CDC42 GTPase proteins, which are downstream effectors
of FAK, control cell motility through WASP and ARP2/3
complex signaling pathway to regulate the extension and
branching of actin filament and cell protrusion . We
confirmed that HTPB treatment decreased integrin-b1, p-
FAK(Y397) and decreased the activities of RhoA and matrix
metalloproteases, MMP-2 and MMP-9. Importantly, reduced p-
FAK(Y397) and its downstream effector p-AKT were confirmed
in HTPB-treated tumor xenograft (Fig. 6). Note that integrin-b1
downstream signaling players RhoA is known to regulate the
actin stress fiber-coordinated fibronectin matrix assembly ,
which is associated with cancer colonization and metastasis in the
lungs . Our study also confirmed the inhibition of pericellular
poly-fibronectin assembly by HTPB treatment (Fig. 5). The
question remained is whether the anti-motility effect of HTPB is
due to HDAC inhibition. Our previous study on another HDAC
inhibitor, OSU-HDAC-44 showed that OSU-HDAC-44 de-
creased the activity of RhoA via induction of srGAP1 and
contributed to dysregulation of F-actin dynamics . The
srGAP1 gene, which encodes a GTPase activating protein known
to regulate axon guidance , was confirmed to be in the open
chromatin structure and increased in expression level in our
previous study . HTPB may inhibit cancer cell motility partly
through reactivation of srGAP1 via promoting histone acetylation
of srGAP1 promoter and further attenuation of downstream Rho/
FAK/MMP signaling pathway. Note that TSA has been shown
to up-regulate RECK via transcriptional activation to inhibit
MMP activity in human lung cancer cells . Whether other
Rho family of GTPases, such as Rac and Cdc42 and other
metastasis-related proteins such as RECK, WASP and ARP2/3
complex are involved in HTPB-induced migration inhibition is
worth further investigation.
In conclusion, our findings show that HTPB is a novel pan-
HDAC inhibitor that exhibits antitumor and antimetastatic
activities in lung cancer cells but not in normal lung cells in cell
and xenograft models, which involves not only histone acetylation-
dependent activation of gene transcription, but also activation of
intrinsic apoptotic pathways and down-regulation of integrin-b1/
FAK/MMP/RhoA/F-actin motility control pathway. Note that
HTPB induced stronger cytotoxicity in lung cancer cells and had
greater inhibitory effect on tumor growth in lung tumor xenografts
than SAHA. In addition, HTPB inhibited the invasion of lung
cancer cells at a lower dose than SAHA did . A better efficacy
in vivo of HTPB over SAHA may be due to longer retention time of
HTPB than SAHA in animal. Pharmacokinetics and pharmaco-
dynamics studies for both SAHA and HTPB are under the
investigations. Furthermore, HTPB showed significant inhibition
of in vitro HDAC activity compared to MS275 (Fig. S5), a class I
HDAC inhibitor with preference for HDAC1. In comparison with
our previous HDAC inhibitor, OSU-HDAC-44, the synthesis of
HTPB is easier (only 2 steps, but 3 steps for HDAC-44) and with
higher yield. Methyl groups at a-position of HDAC-44 were
removed to form the HTPB in order to minimize the bulkiness and
steric hindrance of amide linkage. The IC50 values of HTPB in
various lung cancer cells were close to OSU-HDAC-44 . In
addition, HTPB significantly inhibited lung metastasis in vivo at
non-cytotoxic concentrations. These anti-metastasis data in vitro
and in vivo were not shown for OSU-HDAC-44. It is worthy to
investigate whether there is selective chromatin change in a
fraction of gene loci by genome-wide chromatin immunoprecip-
itation-on-chip assay. Collectively, our data provide compelling
evidence that HTPB is an HDAC inhibitor and could be tested for
lung cancer treatment and combination chemotherapy.
Materials and Methods
All animals were obtained from the National Laboratory
Animal Center (Republic of China, Taiwan) with the approval
of Institutional Animal Care and Use Committee (IACUC),
National Cheng Kung University (IACUC Approval No. 99131)
and were maintained in pathogen free conditions. The study
approval by the review board institution and ethics committee was
confirmed by National Cheng Kung University.
Cell lines and culture conditions
Human normal lung cell line IMR90 and human lung cancer
cell lines, A549 and H1299, were obtained from the American
Type Culture Collection (ATCC, Manassas, VA). Luciferase
expressing murine breast cancer cell line, 4T1-Luc, was obtained
from Dr. M.L.Kuo (Institute of Toxicology, National Taiwan
University, Taipei, Taiwan). All cell lines were cultured in
Dulbecco’s Modified Eagle’s Medium (GIBCO, Grand Island,
NY) containing 10% fetal bovine serum (FBS) (BIOCHROM AG,
Leonorenstr, Berlin, Germany) and 1% penicillin-streptomycin
(GIBCO), and incubated at 37uC in 5% CO2atmosphere.
Preparation of HTPB
Synthesis of N-hydroxy-4-(4-phenylbutanamido)-benzamide 2
(HTPB) was successfully accomplished in two steps as shown in
Fig. S6. 4-(4-phenylbutanamido) benzoic acid 1 was prepared by
treatment of 4-phenylbutanoic acid with oxalyl chloride followed
by 4-amino benzoic acid. Standard peptide coupling reaction
between the resulting acid 1 and hydroxyl amine hydrochloride,
using PyBop as a coupling reagent and Et3N provided the
hydroxamte 2 (HTPB) as evidenced by1H,13CNMR and high-
resolution mass spectroscopy.
Antitumor and Antimetastatic Activity of HTPB
PLoS ONE | www.plosone.org8January 2012 | Volume 7 | Issue 1 | e30240
4-(4-phenylbutanamido)benzoic acid (1)
To a stirred solution of 4-phenylbutanoic acid (328 mg,
2 mmol, 1 equiv) in anhydrous CH2Cl2(5 ml) was added oxalyl
chloride (504 mg, 336 ml, 4 mmol, 2 equiv) at 0uC under N2. The
resulting mixture was warmed to ambient temperature. After
having been stirred for 4 hours, the reaction mixture was
concentrated in vacuo. The crude residue was co-evaporated with
anhydrous CH2Cl2(265 ml) and dried in vacuo. The resulting acid
chloride residue was dissolved in anhydrous CH2Cl2(5 ml) and 4-
amino benzoic acid (329 mg, 2.4 mmol, 1.2 equiv) was added at
0uC followed by Et3N (417 ml, 303 mg, 3 mmol, 1.5 equiv) under
N2. After having been stirred for 10 hours at ambient tempera-
ture, the reaction mixture was quenched with water (10 ml) and
then extracted with CH2Cl2(2610 ml). The combined organic
layers were dried (MgSO4), filtered and concentrated under
reduced pressure. The crude residue was purified by column
chromatography (EtOAc/hexanes, 9/1) on silica gel to afford
498 mg of 4-(4-phenylbutanamido)benzoic acid 1 as a white solid:
1H NMR (DMSO-d6, 400 MHz) d12.65 (br s, 1H, COOH), 10.18
(s, 1H, NH), 7.87 (d, J=8.8 Hz, 2H), 7.70 (d, J=8.4 Hz, 2H),
7.31-7.16 (m, 5H), 2.63 (t, J=7.6 Hz, 2H), 2.36 (t, J=7.4 Hz,
2H), 1.90 (quin, J=7.6 Hz, 2H);
100 MHz) d 171.9, 167.2, 143.4, 141.8, 130.5, 128.5, 126.0,
125.1, 118.5, 36.0, 34.7, 26.8; MS (ESI) Calculated for
C17H16NO3: 283, Found: 282 (M-H+, 100); High-Resolution
MS (TOF -ESI) Calculated for C17H16NO3(M-H+): 282.1130,
Found: 282.1123; Rf0.32 (EtOAc/hexanes, 9/1).
13C NMR (DMSO-d6,
To a stirred solution of acid 1 (283 mg, 1 mmol, 1 equiv) in
DMF (1 mL) was added triethyl amine (121 mg, 167 mL,
1.2 mmol, 1.2 equiv) followed by PyBOP (624 mg, 1.2 mmol,
1.2 equiv) at 0uC. The Resulting reaction mixture was warmed at
ambient temperature. After having been stirred for 4 hours at
ambient temperature, the reaction mixture was cooled to 0uC and
hydroxylamine hydrochloride (138 mg, 2 mmol, 2 equiv) was
added followed by triethyl amine (151 mg, 209 ml, 1.5 mmol, 1.5
equiv) and the resulting mixture was stirred at ambient
temperature for overnight. The reaction mixture was then
quenched with water (5 ml) and extracted with EtOAc (365 ml).
The combined organic layers were washed with brine (10 ml),
dried (MgSO4), filtered and concentrated under reduced pressure.
The crude residue was purified by column chromatography
(EtOAc/hexanes, 9/1) on silica gel to afford 259 mg of N-
hydroxy-4-(4-phenylbutanamido)benzamide 2 as a white solid:1H
NMR (DMSO-d6, 400 MHz) d 11.08 (s, 1H), 10.08 (s, 1H), 8.92 (s,
1H), 7.70 (d, J=8.8 Hz, 2H), 6.64 (d, J=8.4 Hz, 2H), 7.31-7.16
(m, 5H), 2.62 (t, J=7.4 Hz, 2H), 2.34 (t, J=7.4 Hz, 2H), 1.90
(quin, J=7.6 Hz, 2H);13C NMR (DMSO-d6, 100 MHz) d 171.3,
163.9, 141.8, 141.6, 128.3, 128.3, 127.6, 126.9, 125.8, 118.3, 35.8,
34.6, 26.6; MS (ESI) Calculated for C17H18N2O3: 298, Found:
282 (M-H+, 100); High-Resolution MS (TOF -ESI) Calculated for
C17H17N2O3 (M-H+): 297.1239, Found: 297.1236; Rf 0.35
Cell cytotoxicity/MTT assay
Cells were seeded at 56104cells/well in 12-well plates and
treated with various concentrations of HTPB or SAHA for
48 hours, followed by 0.5 mg/ml of 3-(4.5-dimethylthiazol-2-ly)-
2,5-diphenyl tetrazolium bromide (MTT, Sigma-Aldrich, St.
Louis, MO) for 30 minutes at 37uC in a 5% CO2humidified
incubator to determine their cytotoxic effects. Cell cytotoxicity was
expressed as percentage loss of cell viability compared with control
(DMSO), and 50% of inhibition concentration (IC50) of death cell
lines was calculated.
Cell cycle analysis
Cell cycle distribution was determined by flow cytometry. Cells
were treated with 5 mM HTPB for 24 or 48 hours, and then fixed
with 70% ethanol for at least 2 hours at 220uC. Fixed cells were
stained with a solution containing 20 mg/ml propidium iodide,
200 mg/ml RNase A, and 0.1% Triton X-100 for 20 minutes in
the dark. Cell cycle distribution was performed using FACScan
flow cytometry (BD Biosciences, Mountain View, CA) and cal-
culated with ModFIT LT 2.0 version software (BD Biosciences).
Determination of the apoptotic DNA ladder
Fixed cells were centrifuged, resuspended in 100 ml of DNA
extraction buffer (0.2 M Na2HPO4, 0.1 M citrate acid, and 0.5%
triton X-100, pH 7.8), and then incubated for 1 hour at 37uC.
After centrifugation, the supernatant was collected and incubated
with 5 ml RNase A (100 mg/ml) for 1 hour at 37uC, and followed
by digestion with 5 ml proteinase K (20 mg/ml) for 1 hour at
37uC. After electrophoresis, the gels were stained and imaged.
Caspase activity assay
Caspase activity was measured with the caspase luminescent
assay kit (Promega, Madison, WI) according to the manufacturer’s
instructions. Cells seeded in a 96-well plates were treated with
5 mM HTPB for 12 or 24 hours, followed by incubation with
various synthetic caspase substrates (Ac-DEVD-pNA, Ac-LETD-
pNA, and Ac-LEHD-pNA) to measure the activity of caspases-3,
-8, and -9, respectively. After incubation for an hour, luminescence
was detected using a SpectraMaxH M5 microplate reader
(Molecular Devices, Sunnyvale, CA).
Western blot analysis
The cells were lysed on ice. Lysates were centrifuged at 13,000
r.p.m. for 15 min at 4uC, SDS gel loading buffer was added and
samples containing equal amounts of protein (50 mg) were
separated on a 10% SDS-PAGE then electro-blotted onto a
Immobilon-P membrane (Millipore Co., Bedford, MA) in transfer
buffer. Immunoblotting was performed for various proteins, using
the antibodies with conditions described in the Table S1.
HDAC inhibition Assay
Immunoprecipitation of different HDAC isotypes from nuclear
extract were performed using specific anti-HDAC-1, -4, -6, -8, and
-11 antibodies as described in the Table S1. The HDAC activity
assay was performed using a HDAC fluorescent activity assay kit
(BIOMOL Inc, Plymouth Meeting, PA) according to the
manufacturer’s instructions. Specific HDAC isotypes were added
to the diluted HTPB (1 or 2 mM), and then the substrate was
added. Samples were incubated for 10 min at 25uC then the
reaction was stopped by adding developer. After incubation for
10 min, luminescence was recorded with a SpectraMaxH M5
microplate reader (Molecular Devices, Sunnyvale, CA).
Trans-well migration assay
Cells (,26105) suspended in serum-free DMEM medium were
pretreated with DMSO control or HTPB, and placed in the upper
chamber of culture-insert. DMEM medium containing 10% FBS
was added to the lower chamber as chemoattractants and the cells
were incubated at 37uC for 12 hours. The cells attached on the
reverse side of the membrane were stained with crystal violet and
cells in 5,10 randomly selected fields were counted under
Antitumor and Antimetastatic Activity of HTPB
PLoS ONE | www.plosone.org9January 2012 | Volume 7 | Issue 1 | e30240
inverted microscope (Nikon TS100, Nikko, Japan). Three
independent experiments were performed.
Wound healing assay
Cancer cells were treated with different concentration of HTPB
or DMSO for 48 hours. A cell-free gap of 400 mm was created
after removing the Culture-Insert (Ibidi, Martinsried, Germany).
The cells that had migrated into the wound area were calculated
as 400 mm - (5–12 hours area6400 mm)/0 hour area. Three
independent experiments were photographed and quantified un-
der a microscope.
RhoA activation assay
The RhoA activation assay was performed using active Rho
pull-down and detection Kit (Pierce, Rockford, IL). A glutathione
S-transferase (GST) fusion protein containing the Rho binding
domain (RBD) from Rhotekin was used. One mg protein lysates
were incubated with 400 mg of purified GST-Rhotekin-RBD
immobilized on agarose-glutathione beads for 1 hour at 4uC with
constant agitation. The beads were washed three times with 16
Lysis/Wash buffer and bound proteins were eluted and subjected
to Western blot analysis using RhoA antibody as described in the
Conditioned medium were collected from cells treated with
DMSO or HTPB for 48 hours, and analyzed by gelatin
zymography in 0.1% gelatin-8% acrylamide gels. After electro-
phoresis, gels were washed with 2.5% Triton X-100 to remove
SDS and renature the MMP-2 and MMP-9 in the gel. Then the
gels were incubated in the developing buffer overnight to induce
gelatin lysis by renatured MMP-2 and MMP-9. The gel was then
stained with 0.5% Coomassie blue G for 1 hour. The proteolytic
activities were identified as clear bands.
Immunofluorescence staining and confocal microscopic
To stain for DNA and F-actin, the fixed cells were stained with
DAPI and Phalloidin as described in the Table S1, respectively, for
1 hour and then the images were recorded by an OLYMPUS
FV1000 confocal microscope (Olympus America Inc., Melville,
Animal model-in vivo anti-tumor growth assay
Athymic nu/nu mice (Balb/c), 4–5 weeks of age, were obtained
from the National Laboratory Animal Center (Republic of China,
Taiwan) with the approval of Institutional Animal Care and Use
Committee (IACUC), National Cheng Kung University (IACUC
Approval No. 99131) and were maintained in pathogen free
conditions. Mice were implanted subcutaneously with 56106
A549 cells in 0.1 ml Hanks’ balanced salt solution (HBSS) in one
flank per mouse. When tumors had attained a mass of ,50 mm3,
the mice were treated intraperitoneally or orally with HTPB
(25 mg/kg, 50 mg/kg or 100 mg/kg), SAHA (50 mg/kg), DMSO
or oral solvent (0.5% methylcellulose and 0.1% Tween 80 in
ddH20) on days 1, 3, and 5 for three weeks. The tumor size was
measured according to the formula: (Length6Width2)/2. Prior to
sacrifice, the animals were anesthetized and blood samples were
collected by intracardiac puncture for the hematological biochem-
istry tests. Tumor samples and mice organ tissues were resected,
fixed and embedded in paraffin for histologic examination. To
examine the biological effects of HDAC inhibition in tumors, mice
bearing established (about 100 mm3) A549 tumors were treated
intraperitoneally with a single dose of HTPB at 50 mg/kg. After
treatment for indicated time, tumors were harvested and subjected
to Western blot or immunohistochemistry analyses.
Animal model-in vivo anti-tumor metastasis assay
The Balb/c mice were obtained and approved by Institutional
Animal Care as described above. Highly metastatic 4T1-luc
mouse breast adenocarcinoma cells were pre-treated with
1.92 mM HTPB or DMSO for 48 hours. Cells were then
trypsinized and recovered for 2 hours at 37uC in media containing
20% FBS with HTPB or DMSO. Cells (16105cells/200 ml) were
subsequently resuspended in serum-free DMEM medium and
intravenously injected via tail vein into Balb/c mice. These mice
were then given 3 mg/mice endotoxin-free luciferase substrate
(VivoGloTM, Promega) and photographed using IVIS-50 imaging
system (XENOGEN) at day-2, 4, 8 and 13.
Tissue Western Blot and immunohistochemistry (IHC)
Tumor tissues from mice were analyzed using IHC assay to
detect the expression levels of cleaved caspase-3, phospho-AKT
and phospho-FAK proteins as described in the Table S1. Tumor
tissues from mice were subjected to Western blot analysis for the
acetylated proteins and apoptotic related proteins.
Biochemistry and hematology tests
Whole blood samples of treated mice were collected by
intracardiac puncture and stored at 4uC in tube with or without
EDTA anticoagulant. Biochemistry evaluation included glutamate
oxaloacetate transaminase (GOT), glutamate pyruvate transami-
nase (GPT), albumin levels and creatinine levels. Hematology tests
included platelet count, red blood cell (RBC), and white blood cell
(WBC). All experiments and procedures were done in accordance
with the Institutional Care Use Committee guidelines.
The SPSS program (SPSS Inc. Headquarters Chicago, Illinois)
was used for all statistical analysis. Statistical analysis was
performed using Student’s t-test. Data shown were representatives
of at least three independent experiments. Data represent mean 6
SEM. P,0.05 was considered to be statistically significant.
induction of intrinsic apoptosis by HTPB. Cells were
treated with 5 mM HTPB for indicated times and then subjected to
Western blot analyses using anti-caspase-9 or anti-caspase-3
specific antibodies. The active cleaved forms of caspases are as
Caspase cleavage assay demonstrating the
growth without significant causing significant body
weight loss of tested animals. (A) Balb/c nude mice bearing
the established A549 tumors (,50 mm3) were treated with HTPB
via intraperitoneal for three weeks (3 days/week). A known HDAC
inhibitor, SAHA, was used for comparison in intraperitoneal
experiments. The tumor volumes of mice were measured twice
weekly. Six mice per group were used in the xenograft experiment.
Points, mean; bars, 6SEM. (* P,0.05, ** P,0.01) (B) HTPB
treatments did not cause significant body weight loss of tested
HTPB effectively inhibits A549 xenograft
Antitumor and Antimetastatic Activity of HTPB
PLoS ONE | www.plosone.org10January 2012 | Volume 7 | Issue 1 | e30240
and migration of 4T1-luc cells. (A) Highly metastatic 4T1-luc
breast cancer cells were treated with HTPB for 48 hours and cell
viability was assessed by MTT assay. (B) The cell cycle distribution
of treated 4T1-luc cells returned to the same distribution as
DMSO control at 5 mM treatment for 48 hours, though a
transient G1 arrest was observed for 24 hours. (C) 4T1-luc cells
treated with 1.92 mM HTPB for 48 hours decreased transwell
migration capacities to 50% compared to the un-treated control.
Data represent mean 6 SEM from three independent experi-
ments. P values are as indicated.
Effects of HTPB on cell viability, cell cycle
breast cancer cell in animal models. The treated 4T1-luc
cells were injected intravenously via tail vein into Balb/c mice and
observed for the luciferase signals and photographed using IVIS50
for 13 days after drug treatment. HTPB significantly delayed lung
HTPB delays lung metastasis of 4T1-luc
and MS275. The pan-HDAC inhibitor HTPB showed significant
inhibition of in vitro HDAC activity compared to MS275, a class I
HDAC inhibitor. A known pan-HDAC inhibitor, SAHA, was
used for comparison. Data represent mean 6 SEM from three
independent experiments. P values are as indicated.
In vitro HDAC inhibition assays for HTPB
Schematic presentation of 2-steps synthesis
used in the present study.
The antibodies and their reaction conditions
Conceived and designed the experiments: Y-CW C-TC. Performed the
experiments: J-MS T-TW Y-AT S-MH W-LW M-YC H-CC SBS PL.
Analyzed the data: J-MS T-TW Y-AT S-MH W-LW. Contributed
reagents/materials/analysis tools: H-CC SBS C-SC C-TC. Wrote the
paper: Y-CW J-MS T-TW C-TC.
1. Lazebnik Y (2010) What are the hallmarks of cancer? Nat Rev Cancer 10:
2. Gossage L, Eisen T (2010) Targeting multiple kinase pathways: a change in
paradigm. Clin Cancer Res 16: 1973–1978.
3. Hanahan D, Weinberg RA (2010) The hallmarks of cancer. Cell 100: 57–70.
4. Siddiqa A, Marciniak R (2008) Targeting the hallmarks of cancer. Cancer Biol
Ther 7: 740–741.
5. Levitzki A, Klein S (2010) Signal transduction therapy of cancer. Mol Aspects
Med 31: 287–329.
6. Witt O, Deubzer HE, Milde T, Oehme I (2009) HDAC family: What are the
cancer relevant targets? Cancer Lett 277: 8–21.
7. Ellis L, Atadja PW, Johnstone RW (2009) Epigenetics in cancer: Targeting
chromatin modifications. Mol Cancer Ther 8: 1409–1420.
8. Minucci S, Pelicci PG (2006) Histone deacetylase inhibitors and the promise of
epigenetic (and more) treatments for cancer. Nat Rev Cancer 6: 38–51.
9. Xu WS, Parmigiani RB, Marks PA (2007) Histone deacetylase inhibitors:
molecular mechanisms of action. Oncogene 26: 5541–5552.
10. Carew JS, Giles FJ, Nawrocki ST (2008) Histone deacetylase inhibitors:
mechanisms of cell death and promise in combination cancer therapy. Cancer
Lett 269: 7–17.
11. Ma X, Ezzeldin HH, Diasio RB (2009) Histone Deacetylase inhibitors, current
status and overview of recent clinical trials. Drugs 69: 1911–1934.
12. Lane AA, Chabner BA (2009) Histone deacetylase inhibitors in cancer therapy.
J Clin Oncol 27: 5459–5468.
13. Gojo I, Jiemjit A, Trepel JB, Sparreboom A, Figg WD, et al. (2007) Phase 1 and
pharmacologic study of MS-275, a histone deacetylase inhibitor, in adults with
refractory and relapsed acute leukemias. Blood 109: 2781–2790.
14. Nemunaitis JJ, Orr D, Eager R, Cunningham CC, Williams A, et al. (2003)
Phase I study of oral CI-994 in combination with gemcitabine in treatment of
patients with advanced cancer. Cancer J 9: 58–66.
15. Mann BS, Johnson JR, Cohen MH, Justice R, Pazdur R (2007) FDA approval
summary: vorinostat for treatment of advanced primary cutaneous T-cell
lymphoma. Oncologist 12: 1247–1252.
16. Marsoni S, Damia G, Camboni G (2008) A work in progress: the clinical
development of histone deacetylase inhibitors. Epigenetics 3: 164–171.
17. Lu Q, Wang DS, Chen CS, Hu YD, Chen CS (2005) Structure-based
optimization of phenylbutyrate-derived histone deacetylase inhibitors. J Med
Chem 48: 5530–5535.
18. Gui CY, Ngo L, Xu WS, Richon VM, Marks PA (2004) Histone deacetylase
(HDAC) inhibitor activation of p21WAF1 involves changes in promoter-
associated proteins, including HDAC1. Proc Natl Acad Sci U S A 101:
19. Deng C, Zhang P, Harper JW, Elledge SJ, Leder P (1995) Mice lacking
p21CIP1/WAF1 undergo normal development, but are defective in G1
checkpoint control. Cell 82: 675–684.
20. Ghobrial IM, Witzig TE, Adjei AA (2005) Targeting apoptosis pathways in
cancer therapy. CA Cancer J Clin 55: 178–194.
21. Lavrik IN, Golks A, Krammer PH (2005) Caspases: pharmacological
manipulation of cell death. J Clin Invest 115: 2665–2672.
22. Peck B, Chen CY, Ho KK, Di Fruscia P, Myatt SS, et al. (2010) SIRT inhibitors
induce cell death and p53 acetylation through targeting both SIRT1 and
SIRT2. Mol Cancer Ther 9: 844–855.
23. Mitra SK, Schlaepfer DD (2006) Integrin-regulated FAK-Src signaling in
normal and cancer cells. Curr Opin Cell Biol 18: 516–523.
24. Schaller MD, Otey CA, Hildebrand JD, Parsons JT (1995) Focal adhesion
kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic
domains. J Cell Biol 130: 1181–1187.
25. Mitra SK, Hanson DA, Schlaepfer DD (2005) Focal adhesion kinase: in
command and control of cell motility. Nat Rev Mol Cell Biol 6: 56–68.
26. Reuter JA, Ortiz-Urda S, Kretz M, Garcia J, Scholl FA, et al. (2009) Modeling
inducible human tissue neoplasia identifies an extracellular matrix interaction
network involved in cancer progression. Cancer Cell 15: 477–488.
27. Heasman SJ, Ridley AJ (2008) Mammalian Rho GTPases: new insights into
their functions from in vivo studies. Nat Rev Mol Cell Biol 9: 690–701.
28. Zhong C, Chrzanowska-Wodnicka M, Brown J, Shaub A, Belkin AM, et al.
(1998) Rho-mediated contractility exposes a cryptic site in fibronectin and
induces fibronectin matrix assembly. J Cell Biol 141: 539–551.
29. Cheng HC, Abdel-Ghany M, Pauli BU (2003) A novel consensus motif in
fibronectin mediates dipeptidyl peptidase IV adhesion and metastasis. J Biol
Chem 278: 24600–24607.
30. Tang YA, Wen WL, Chang JW, Wei TT, Tan YH, et al. (2010) A novel histone
deacetylase inhibitor exhibits antitumor activity via apoptosis induction, F-actin
disruption and gene acetylation in lung cancer. PLoS One 5(9): e12417.
31. Wong K, Ren XR, Huang YZ, Xie Y, Liu G, et al. (2001) Signal transduction in
neuronal migration: roles of GTPase activating proteins and the small GTPase
Cdc42 in the Slit-Robo pathway. Cell 107: 209–221.
32. Liu LT, Chang HC, Chiang LC, Hung WC (2003) Histone deacetylase inhibitor
up-regulates RECK to inhibit MMP-2 activation and cancer cell invasion.
Cancer Res 63: 3069–3072.
33. Takada Y, Gillenwater A, Ichikawa H, Aggarwal BB (2006) Suberoylanilide
hydroxamic acid potentiates apoptosis, inhibits invasion, and abolishes
osteoclastogenesis by suppressing nuclear factor-kB activation. J Biol Chem
Antitumor and Antimetastatic Activity of HTPB
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