Betulinic acid inhibits colon cancer cell and tumor
growth and induces proteasome-dependent and -independent downregulation of
specificity proteins (Sp) transcription factors
Abstract
Background
Betulinic
acid (BA) inhibits growth of several cancer cell lines and tumors and the
effects of BA have been attributed to its mitochondriotoxicity and inhibition
of multiple pro-oncogenic factors. Previous studies show that BA induces
proteasome-dependent degradation of specificity protein (Sp) transcription
factors Sp1, Sp3 and Sp4 in prostate cancer cells and this study focused on the
mechanism of action of BA in colon cancer cells.
Methods
The
effects of BA on colon cancer cell proliferation and apoptosis and tumor growth
in vivo were determined using standardized assays. The effects of BA on Sp
proteins and Sp-regulated gene products were analyzed by western blots, and
real time PCR was used to determine microRNA-27a (miR-27a) and ZBTB10 mRNA
expression.
Results
BA
inhibited growth and induced apoptosis in RKO and SW480 colon cancer cells and
inhibited tumor growth in athymic nude mice bearing RKO cells as xenograft. BA
also decreased expression of Sp1, Sp3 and Sp4 transcription factors which are
overexpressed in colon cancer cells and decreased levels of several
Sp-regulated genes including survivin, vascular endothelial growth factor, p65
sub-unit of NFκB, epidermal growth factor receptor, cyclin D1, and pituitary
tumor transforming gene-1. The mechanism of action of BA was dependent on cell
context, since BA induced proteasome-dependent and proteasome-independent downregulation
of Sp1, Sp3 and Sp4 in SW480 and RKO cells, respectively. In RKO cells, the
mechanism of BA-induced repression of Sp1, Sp3 and Sp4 was due to induction of
reactive oxygen species (ROS), ROS-mediated repression of microRNA-27a, and
induction of the Sp repressor gene ZBTB10.
Conclusions
These
results suggest that the anticancer activity of BA in colon cancer cells is
due, in part, to downregulation of Sp1, Sp3 and Sp4 transcription factors;
however, the mechanism of this response is cell context-dependent.
Colorectal
cancer is a leading cause of death in most developed countries including the
United States, and in 2010 it is estimated that over 102,700 new cases will be
diagnosed and 51,370 deaths will occur in the United States [1]. Genetic
susceptibility accounts for 15 - 25% of colon cancer cases, and genetic markers
provide important insights on factors important for the molecular and genetic
changes that result in development of this disease [2]. Familial
adenomatous polyposis syndromes [3,4], hereditary
non-polyposis colorectal cancer [5-8], and other
polyposis syndromes which increase the incidence of colorectal cancer including
Peutz Jegher's syndrome, familial juvenile polyposis, and hereditary mixed
polyposis syndrome, are linked to mutations in LKB1, STK11, SMAD4, PTEN,
E-cadherin, cyclin D1, and transforming growth factor β receptors [2].
The
incidence rates of sporadic colon cancer are highly variable among different
regions of the world and the changes in incidence of this disease in migrants suggests
that environmental factors related to diet contribute to development of colon
cancer [9,10].
Fruits, nuts and vegetables contain diverse anticarcinogenic phytochemicals;
however, epidemiological studies give variable results with respect to their
chemopreventive effects and similar variability among studies has been reported
for the protective effects of dietary folate [11-14].
Most colon cancer patients present with localized disease which is treated with
curative surgery; however, disease relapse is experienced by up to 40% of
patients [15-17].
Cytotoxic drugs are primarily used for colon cancer chemotherapy and there is a
increasing need to develop mechanism-based drugs for treating this disease.
Specificity
protein (Sp) transcription factors Sp1, Sp3 and Sp4 are overexpressed in colon
and other cancer cell lines [18-23],
and Sp1 is a negative prognostic factor for survival of pancreatic and gastric
cancer patients [24,25].
The potential importance of Sp transcription factors as drug targets is due not
only to their overexpression in multiple tumor types but also to their
relatively low expression in non-tumor rodent and human tissues, and this is
consistent with the reported decrease of Sp1 expression with increasing age [26-28].
RNA interference studies which knockdown Sp1, Sp3 and Sp4 (individually or
combined) have identified several Sp-regulated gene-products that are
themselves individual targets for new mechanism-based drugs. Sp-regulated genes
include several that are important for cancer cell proliferation [cyclin D1,
epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor
(c-MET)], survival (bcl-2 and survivin), angiogenesis [vascular endothelial
growth factor (VEGF) and its receptors (VEGFR1/R2) and pituitary
tumor-transforming gene 1 (PTTG-1)], and inflammation (p65 subunit of NFκB) [23,29-38].
Betulinic
acid (BA) is a naturally occurring triterpenoid which inhibits growth of
multiple tumors [39,40].
Studies in this laboratory show that BA inhibits prostate cancer cell and tumor
(xenograft) growth and this is due, in part, to proteasome-dependent
downregulation of Sp1, Sp3, Sp4 and several Sp-regulated genes [20].
In this study, we show that BA inhibits growth of colon cancer cells and tumors
and downregulates Sp transcription factors through activation of
proteasome-dependent (SW480 cells) and proteasome-independent (RKO cells)
pathways.
Cell
proliferation and cell cycle progression assays
The
RKO and SW480 colon cancer cell lines were previously characterized at the M.D.
Anderson Cancer Center (Houston, TX) and kindly provided by Dr. Stanley
Hamilton. RKO and SW480 colon cancer cells (2 × 104 per well) were plated in
12-well plates and allowed to attach for 24 h. The medium was then changed to
DMEM/Ham's F-12 medium containing 2.5% charcoal-stripped FBS, and either
vehicle [dimethyl sulfoxide (DMSO)] or different concentrations of the compound
were added. Fresh medium and compounds were added every 48 h, and cells were
then trypsinized and counted after 48 and 96 h using a Coulter Z1 cell counter.
Results are expressed as means ± SE for at least 3 replicate determinations for
each treatment group. RKO and SW480 cells were treated with either the vehicle
(DMSO) or BA for 24 h. Cells were trypsinized, centrifuged and resuspended in
staining solution containing 50 μg/ml propidium iodide, 4 mmol/L sodium
citrate, and 30 units/ml RNase. After incubation at room temperature for 1 h,
cells were analyzed on a FACS Vantage SE DiVa made by Becton Dickinson, using
FACSDiva Software V4.1.1. Propidium iodide (PI) fluorescence was collected
through a 610SP bandpass filter, and list mode data were acquired on a minimum
of 50,000 single cells defined by a dot plot of PI width vs. PI area. Data
analysis was performed in BD FACSDiva Software V4.1.1 using PI width vs. PI
area to exclude cell aggregates.
Plasmids,
transfection assay and antibodies
Sp1
and Sp3 promoter constructs were kindly provided by Drs. Carlos Cuidad and
Veronique Noe (University of Barcelona, Barcelona, Spain). The pVEGF-2068
construct contains a VEGF promoter insert (positions -2068 to +54) linked to
luciferase reporter gene. The pSurvivin-269 was kindly provided by Dr. M. Zhou
(Emory University, Atlanta, GA). The PTTG-1-luc construct containing the -1373
to +3 region of the PTTG-1 promoter was provided by Dr. Kakar (University of
Louisville, Louisville, KY). Colon cancer cells (1.5 × 105) were seeded in
12-well plates using DMEM:Ham's F-12 media containing 2.5% charcoal stripped
serum. After 24 h, cells were transfected with 0.4 μg of reporter gene
constructs and 0.04 μg of β-Gal using Lipofectamine 2000 according to
manufacturer's protocol. Reporter lysis buffer and luciferase reagent for
luciferase studies were supplied by Promega (Madison, WI). Five h after transfection,
cells were treated with control or BA for 22-24 h and luciferase activity
(normalized to β-galactosidase) was determined using Lumicount luminometer
(PerkinElmer Life and Analytical Sciences). For RNA interference assays with
iSp, a mixture of oligonucleotides containing siRNAs against Sp1, Sp3 and Sp4
(combined) was used as previously described [20,21,34].
Antibodies for Sp1, Sp3, Sp4, cyclin D1, EGFR, NFκB (p65), VEGF and VEGFR1 were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). c-PARP and survivin
antibodies were purchased from Cell Signaling Technology (Danvers, MA).
Monoclonal β-actin antibody was purchased from Sigma-Aldrich. Western blots
were determined with whole cell lysates essentially as described [20-23].
Northern
blot analysis
For
miRNA analysis, 20 μg total RNA per lane was electrophoresed on 15% TBE urea
polyacrylaminde gel (Invitrogen), electrophoretically transferred in 0.5 × TBE
at 300 mÅ for 45 min to GeneScreen Plus membrane (PerkinElmer, Boston, MA), UV
cross-linked and hybridized in ULTRAhyb-Oligo hybridization buffer (Ambion,
Austin, TX) at 42°C with 32P end-labeled DNA oligonucleotides complementary to
miR-27a. Blots were washed at 42°C in 2X SSC and 0.5% SDS for 30 min with
gentle agitation.
Semiquantitative
reverse transcription and real time PCR
RKO
and SW480 colon cancer cells were treated with BA at different concentrations
for 24 h. Total RNA was extracted using RNeasy Mini Kit (Qiagen), and 2 μg of
RNA was used to synthesize cDNA using Reverse Transcription System (Promega).
Primers were obtained from IDT and used for amplification were as follows:
ZBTB10 (sense 5'-GCT GGA TAG TAG TTA TGT TGC-3'; antisense 5'-CTG AGT GGT TTG
ATG GAC AGA G-3'). PCR products were electrophoresed on 1% agarose gels
containing ethidium bromide and visualized under UV transillumination. Real
time PCR for determining miR-27a, ZBTB10 and Myt-1 RNA levels were determined
essentially as described [20-23].
Reactive
oxygen species (ROS) and mitochondrial membrane potential assays
Cellular
ROS levels were evaluated with the cell permeant probe CM-H2DCFDA
(5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester)
from Invitrogen. Following 36 h treatment, cells plated on a 6-well cell
culture plate were loaded with 10 μM CM-H2DCFDA for 1 h, washed once with
serum-free medium, and analyzed for ROS levels using Beckman Coulter XL
four-color cytometer. Each experiment was done in triplicate and results are
expressed as mean ± S.E. for each treatment group. Mitochondrial membrane
potential (MMP) was measured with Mitochondrial Membrane Potential Detection
Kit (Stratagene) according to the manufacturer's protocol using JC-1 dye, and
mitochondrial membrane potential shift was measured using FACS Calibur flow
cytometer using CellQuest acquisition software (BD Biosciences). J-aggregates
were detected as red fluorescence and J-monomers are detected as green
fluorescence.
Xenograft
studies in athymic mice
Female
athymic nude mice were purchased from Harlan Laboratories (Indianapolis, IN)
and were cared for and used in accordance with institutional guidelines. To
produce tumors, RKO cells (5 × 106; ≥ 90% viable) were subcutaneously injected
into the flanks of individual mice. Tumors were allowed to grow for 6 days
until palpable and mice were then randomized into two groups (6 mice/group) and
dosed by oral gavage with corn oil or BA (25 mg/kg/day) every second day for 22
days. The mice were weighed, and tumor size was measured every second day with
calipers to permit calculation of tumor volumes: V = LW2/2, where L and W were
length and width, respectively. After BA treatment, the animals were
sacrificed; final body and tumor weights were determined, and major visceral
organs were collected and lysates were used for western blot analysis of Sp
proteins.
1.
BA inhibits colon cancer cell growth and induces apoptosis
BA
inhibits growth of multiple cancer cell lines [39,40],
and results in Figure 1A
demonstrate that BA inhibited RKO and SW480 cell proliferation after treatment
for 48 or 96 h. Growth inhibition was observed at concentrations of ≥ 5 μM in
both cell lines at the two time points. The effects of BA on distribution of
cells in G0/G1, S and G2/M phases of the cell cycle was cell context-dependent
(Figures 1B
and 1C).
In RKO cells, BA dramatically decreased the percentage of cells in G0/G1 and S
phase and increased the percent in G2/M, whereas in SW480 cells, there was a
slight decrease in G0/G1 and S phase and a parallel increase in the percentage
of cells in G2/M. Treatment of RKO and SW480 cells with BA also enhanced PARP
cleavage (Figure 1D)
which is consistent with induction of apoptosis in these cell lines; however,
after treatment of the cells for only 24 hr, < 10% of the cells were sub-G1
in FACS analysis.
2.
BA decreases expression of Sp1, Sp3, Sp4 and Sp-regulated gene products in
colon cancer cells
Previous
studies showed that BA decreases Sp1, Sp3 and Sp4 protein expression in
prostate and bladder cancer cells [20,32],
and results in Figure 2A
confirm that similar effects were observed in RKO and SW480 cells. These
results were similar to that observed for CDODA-Me and GT-094 in these cell
lines [38,40].
Moreover, BA also induced cleaved PARP and decreased expression of survivin, an
inhibitor of apoptosis in RKO and SW480 cells, and VEGF (Figure 2B).
These results are consistent with previous studies showing that both survivin
and VEGF are Sp-regulated genes [20,29].
RNA interference studies which knockdown Sp1, Sp3 and Sp4 (individually or
combined) have identified EGFR, p65 subunit of NFκB, PTTG1 and cyclin D1 as Sp-regulated
genes [32-35],
and results in Figure 2C
show that BA decreased expression of their corresponding gene products in RKO
and SW480 cells. Moreover combined knockdown of Sp1, Sp3 and Sp4 using an
oligonucleotide cocktail (iSp) [20,21,32-34]
also decreased expression of p65, PTTG-1, EGFR and cyclin D1 in RKO cells
(Figure 2C)
confirming their regulation by Sp transcription factors in colon cancer cells.
Figure 2. BA
decreases Sp proteins and Sp-regulated genes. BA decreases Sp proteins in RKO
and SW480 cells (A) and Sp-regulated genes (B). Cells were treated with DMSO
(0) or BA for the indicated times and whole cell lysates were analyzed by
western blots as described in the Materials and Methods. (C) BA and Sp1/Sp3/Sp4
(iSp) knockdown decrease Sp-regulated genes. Cells were either treated with BA
or transfected with iSp and whole cell lysates were analyzed by western blots
as described in the Materials and Methods. (D) Effects of proteasome
inhibitors. Cells were treated with BA ± proteasome inhibitors MG132 or lactacystin
for 24 h and whole cell lysates were analyzed by western blots as described in
the Materials and Methods.
Treatment
of RKO cells with the proteasome inhibitor MG132 alone was cytotoxic; however,
in RKO cells treated with BA or the proteasome inhibitor lactacystin alone or
in combination, BA-induced downregulation of Sp1, Sp3, and Sp4 was not
inhibited indicating that the effects were proteasome-independent (Figure 2D).
MG132 was not toxic to SW480 cells and BA-induced downregulation of Sp1, Sp3
and Sp4 was reversed in cells cotreated with BA plus MG132 (and lactacystin;
data not shown), demonstrating a proteasome-dependent pathway in this cell line
as previously observed in LNCaP cells treated with BA [20].
3.
BA decreases Sp and Sp regulated gene expression in RKO cells through
disruption of miR-27a:ZBTB10
We
further investigated BA-mediated repression of Sp and Sp-regulated genes in RKO
cells by determining the effects of BA on a series of GC-rich constructs
containing promoter inserts from the Sp1, Sp3, VEGF, survivin and PTTG-1 which
are downregulated after loss of Sp proteins [18-21,29,30].
Results in Figures 3A
and 3B
show that BA decreased luciferase activity in RKO cells transfected with
pSp1-FOR4-luc, pSp1-FOR2-luc, pSp3-FOR5-luc and pSp3-FOR2-luc constructs which
contain the GC-rich -751 to -20 and -281 to -20 region of the Sp1 gene promoter
and the GC-rich -417 to -38 and -213 to -38 regions of the Sp3 promoter,
respectively [36].
BA also decreased luciferase activity in RKO cells transfected constructs
containing VEGF (-2018 to +5), survivin (-259 to +49), and PTTG-1 (-1373 to +3)
promoter inserts (Figure 3C),
and these results are also consistent with previous studies using agents or RNA
interference that downregulate Sp protein expression [19,20,30].
Figure 3. BA
inhibits luciferase activity in RKO cells transfected with GC-rich constructs.
Transfection with constructs containing Sp1 (A), Sp3 (B) and Sp-regulated (C)
gene promoter constructs. RKO cells were transfected with the indicated
constructs, treated with DMSO or BA, and luciferase activity (normalized to
β-galactosidase) was determined as described in the Materials and Methods.
Results are expressed as means ± SE for at least 3 replicated determinations as
significant (p < 0.05) is indicated (*).
ROS
and hydrogen peroxide (H2O2) play a role in downregulation of Sp1, Sp3 and Sp4
in pancreatic and bladder cancer cells [34,37]
and treatment of RKO cells with BA for 36 h induced ROS as determined by FACS
analysis using the fluorescent ROS scavenger H2DCFDA (Figure 4A).
Moreover, in cells treated with BA plus catalase, there was a decrease in
fluorescence indicating that catalase inhibited ROS formation. Treatment of RKO
cells with BA decreased expression of Sp1, Sp3 and Sp4 proteins and this effect
was partially reversed in RKO cells cotreated with BA plus catalase (Figure 4B).
BA-decreased MMP was indicated by increased green/red fluorescence associated
with the JC-1 monomer and aggregates, respectively; moreover, BA-induced growth
inhibition was also reversed in RKO cells cotreated with BA plus catalase
(Figure 4C),
thus confirming an important role for ROS (H2O2) in mediating the growth
inhibitory effects of BA.
Figure 4. BA
decreases MMP and induces ROS in RKO cells. (A) Induction of ROS. RKO cells
were treated with 15 μM BA, catalase or BA plus catalase for 36 h and ROS
production was determined using the fluorescent probe H2DCFDA as described in
the Materials and Methods. Role of ROS in BA-induced Sp downregulation (B) and growth
inhibition and BA effects on MMP (C). Cells were treated with DMSO (control),
BA, catalase or BA plus catalase and Sp proteins (in whole cell lysates), MMP
and cell growth were determined as described in the Materials and Methods.
Results (C) are expressed as means ± SE for 3 replicate experiments and
significant (p < 0.05) growth inhibition by BA (*) and rescue by catalase
(**) are indicated. BA-induced inhibition of MMP was not affected by
cotreatment with catalase (data not shown).
Previous
studies show that ROS-dependent disruption of miR-27a:ZBTB10 is important for
Sp downregulation [33,38]
and Figure 5A
shows that BA decreased miR-27a, as determined by Northern blot analysis, and
semi-quantitative RT-PCR confirmed induction of ZBTB10. Moreover,
downregulation of miR-27a was also paralleled by decreased luciferase activity
in RKO cells transfected with a construct (pmiR-27a-luc) containing the -639 to
+36 region of the promoter for the miR-23a-miR-27a-miR-24-2 [41]
cluster (Figure 5B).
Using real time PCR, BA significantly decreased miR-27a and induced ZBTB10
(Figure 5C)
expression and these responses were all significantly attenuated in RKO cells
cotreated with BA plus catalase. These results confirm that BA-induced
suppression of Sp1, Sp3 and Sp4 is linked to induction of ROS and ROS-mediated
disruption of miR-27a:ZBTB10. The Myt-1 gene is associated with G2/M arrest and
is repressed by miR-27a in colon and breast cancer cells [22,36].
BA induced Myt-1 mRNA in RKO cells; this response was also attenuated in cells
cotreated with BA plus catalase (Figure 5D)
and this was consistent with ROS-mediated regulation of miR-27a and ZBTB10
(Figure 5C).
Figure 5. Role of
miR-27a in regulation of BA-mediated responses. (A) Repression of miR-27a and
induction of ZBTB10. RKO cells were treated with DMSO or BA and miR-27a and
ZBTB10 expression were determined by Northern blot and semi-quantitative
RT-PCR, respectively, as described in the Materials and Methods. (B) BA
decreases miR-27a promoter activity. RKO cells were transfected with
pMiR-27a(-639/+36)-luc, treated with DMSO or BA and luciferase activity
determined as described in the Materials and Methods. Role of BA-induced ROS on
expression of miR-27a and ZBTB10 (C) and Myt-1 (D). RKO cells were treated with
DMSO, BA, catalase or BA plus catalase for 36 h and miR-27a, ZBTB10 and Myt1
mRNA levels were determined by real time PCR as described in the Materials and
Methods. Results in (B) - (D) are expressed as means ± SE for at least 3
replicate determinations and significant (p < 0.05) effects by BA (*) and
reversal by catalase (**) are indicated.
4.
BA inhibits colon tumor growth
Athymic
nude mice bearing RKO cells as xenografts were treated with corn oil (control)
or BA (25 mg/kg/d). Treatment with BA significantly decreased tumor growth and
volume and this was accompanied by decreased tumor weights measured after
sacrifice (Figures 6A
and 6B).
Lysates from control and BA-treated tumors were analyzed by western blot
analysis for Sp1, Sp3 and Sp4 protein expression, and quantitated (relative to
β-actin). The results showed that BA significantly decreased expression of Sp1,
Sp3 and Sp4 (Figure 6C)
and these results were consistent with comparable effects observed in vitro
(Figure 2A).
The
anticancer activity of BA initially showed high potency against melanoma in
cell culture and animal models, and subsequent studies show the effectiveness
of this compound against multiple tumor types [39,40,42].
The low in vivo toxicity of BA coupled with supporting in vitro and in vivo
results suggest that this compound or some derivative has potential for clinical
applications in cancer chemotherapy. However, BA is a highly lipophilic
molecule with limited water solubility and this may decrease in vivo uptake of
this compound; therefore, development of specialized formulations/carriers such
as liposomes may help to enhance the in vivo efficacy of BA as an anticancer
agent [43].
Previous studies in this laboratory showed that BA inhibits prostate cancer
cell and tumor growth and this is accompanied by proteasome-dependent
degradation of Sp1, Sp3 and Sp4 and several Sp-regulated pro-oncogenic gene
products [20].
Several other anticancer agents including tolfenamic acid, curcumin, arsenic
trioxide, a nitro-NSAID (GT-094), and two synthetic triterpenoid derivatives,
CDDO-Me and CDODA-Me, also induce Sp downregulation in various cancer cell
lines via proteasome-dependent and -independent pathways [19,21,33-38].
BA
inhibits colon cancer cell growth and induces caspase-dependent PARP cleavage
in RKO and SW480 colon cancer cells (Figure 1)
and these results are consistent with other reports on the effects of BA on
colon cancer cell lines [39,40,44-46].
Moreover, BA also inhibited tumor growth in athymic nude mice bearing RKO cells
as xenografts (Figure 6).
We observed that BA decreased expression of Sp1, Sp3 and Sp4 proteins in both
RKO and SW480 colon cancer cells and tumors (Figures 2A
and 6C)
and this was accompanied by parallel decreases in survivin and VEGF (Figures 2A
and 2B),
and these results are comparable to those observed in LNCaP prostate and KU7
bladder cancer cells treated with BA [20,32].
Recent RNA interference studies show that p65 (NFκB subunit), EGFR, cyclin D1,
and pituitary tumor transforming gene-1 (PTTG-1) are also Sp-regulated genes [32-35],
and results in Figure 3C
demonstrate that BA decreased expression of these gene products in RKO and
SW480 cells. Moreover, knockdown of Sp1, Sp3 and Sp4 (in combination) in RKO
colon cancer cells also decreased expression of EGFR, cyclin D1, p65 and
PTTG-1, confirming the role of Sp transcription factors in regulating
expression of these genes. These results are consistent with the induction of
apoptosis by BA since many of these Sp-regulated genes are important for
survival pathways.
Previous
studies showed that BA-induced downregulation of Sp1, Sp3 and Sp4 was
proteasome-dependent in LNCaP cells but proteasome-independent in KU7 bladder
cancer cells [20,32].
Similar variability was observed in RKO and SW480 colon cancer cells (Figure 2D)
where BA-induced downregulation of Sp proteins was proteasome-independent and
-dependent, respectively. This demonstrates that, for BA and possibly other
drugs that downregulate Sp1, Sp3, Sp4 and Sp-regulated genes, the pathways
required for this response are variable and dependent not only on tumor type
but also cell context within the same tumor. At least two of these pathways,
namely induction of proteasome- and caspase-dependent degradation of Sp
proteins, involve activation of post-transcriptional processes [20,21,37];
however, their mechanisms have not been determined and are currently being
investigated in this laboratory.
We
have previously reported that the synthetic triterpenoid CDODA-Me and the
NO-NSAID GT-094 decrease Sp protein expression in SW480 and RKO colon cancer
cells through a transcriptional repression pathway in which miR-27a is
decreased and this results in the induction of ZBTB10, a transcriptional
repressor [36,38].
BA decreased luciferase activity in RKO cells transfected with constructs
containing several GC-rich promoter inserts (Figures 3B-D)
and also decreased expression of miR-27a and induced expression of ZBTB10 in
RKO cells (Figures 5A-C).
Since overexpression of ZBTB10 and antisense-miR-27a also decreases expression
of Sp1, Sp3, Sp4 and Sp-regulated genes in colon cancer cells [36],
the mechanism of action of BA in RKO cells is linked to disruption of miR-27a:ZBTB10
as previously reported for CDODA-Me and GT-094 in colon cancer cells [36,38].
BA
is known to be a mitochondriotoxic drug and decreases the mitochondrial
membrane potential in several different cancer cell lines leading to induction
of apoptosis [39,40,44]
and BA also decreased MMP in RKO cells (Figure 4C).
Previous studies have demonstrated that at least four agents that are
mitochondriotoxic and induce ROS also downregulate Sp proteins; this effect is
ROS-dependent and reversible with antioxidants or catalase, and compounds
activating this pathway include arsenic trioxide (bladder), curcumin and
CDDO-Me (pancreatic), and GT-094 (colon) [33,37,38].
Moreover, for GT-094 and CDDO-Me, the mechanism of ROS-dependent downregulation
of Sp1, Sp3, and Sp4 involves disruption of miR-27a:ZBTB10 [33,38].
Results of this study show that BA also induced ROS-downregulated Sp1, Sp3, Sp4
and miR-27a and induced ZBTB10 in RKO cells, and all of these responses were
significantly attenuated in cells cotreated with BA plus catalase (Figure 4).
Moreover, catalase also reversed the growth inhibitory effects of BA (Figure 4C),
further demonstrating the importance of ROS activation for the anticancer
activity of this compound in RKO cells. In contrast to previous studies showing
that CDODA-Me and GT-094 activated transcriptional repression of Sp proteins in
both RKO and SW480 cells [33,36],
BA induced transcriptional repression in RKO cells but activated the proteasome
pathway for degradation of Sp proteins in SW480 cells. The mitochondrial or
extra-mitochondrial origins of ROS in cancer cells treated with BA and other
agents that downregulate Sp transcription factors is currently being
investigated.
In
summary, we have shown that the anticancer activity of BA in colon cancer cells
is due, in part, to downregulation of Sp1, Sp3, Sp4 and Sp-regulated
prooncogenic gene products. The upstream mechanisms associated with decreased
expression of Sp1, Sp3 and Sp4 are cell context-dependent and involves
proteasome-dependent (SW480) and proteasome-independent (RKO) pathways. The
response in RKO cells involves loss of MMP and induction of ROS as previously
reported for BA in other studies [39,40]
and this is coupled with ROS-dependent disruption of miR-27a:ZBTB10. BA also
decreased luciferase activity in RKO cells transfected with a construct
containing the -639 to +39 region of the miR-27a promoter, and we are currently
examining the mechanisms associated with ROS-dependent effects on critical
transcription factors interacting with the promoter and also the functional
significance of ROS-dependent downregulation of miR-23a and miR-24-2 which form
part of the miR-23a-miR-27a-miR24-2 cluster. These results coupled with several
recent reports demonstrate potential clinical applications for BA and related
compounds alone or in combination with other anticancer agents [47-49].
The
authors declare that they have no competing interests.
SC
carried out and supervised the in vitro studies on BA-induced downregulation of
Sp proteins and Sp-regulated genes and also the RNA interference studies. SP
carried out the in vitro studies on downregulation of Sp1, Sp3 and Sp4 and
Sp-regulated genes. PL carried out some of the in vitro experiments including
the studies on miR-27a:ZBTB10. SP carried out the in vivo study and analyzed
the tumor tissue. SS carried out the experimental design and drafted the
manuscript. All authors have read and approved the final manuscript.
This
research was supported by funding from the National Institutes of Health
(R01-CA136571) and Texas AgriLife.
