siRNA-Mediated B-Catenin Knockdown in Human HepatomaCells Results in Decreased Growth and Survival1

Gang Zeng*, Udayan Apte*, Benjamin Cieply*, Sucha Singh* and Satdarshan P. S. Monga*,y

Departments of *Pathology, yMedicine (Gastroenterology), University of Pittsburgh, SOM, Pittsburgh,PA 15213, USA

Abstract

B-Catenin, the chief oncogenic component of the ca-

nonical Wnt pathway, is known to be involved in a

variety of cancers, including hepatocellular carcinoma

(HCC). Although the mechanism of B-catenin activation

in HCC is multifactorial, it is indisputably implicated

at various stages of hepatocarcinogenesis, making it

an attractive therapeutic target. Here we investigate

the effect of small interfering RNA–mediated B-catenin

knockdown on the growth and survival of human hepa-

toma cell lines with (HepG2) and without (Hep3B)

B-catenin mutations. Transfection of HepG2 and Hep3B

cells with human B-catenin (CTNNB1) small interfer-

ing RNA resulted in a significant B-catenin decrease,

as confirmed by Western blot analyses and immuno-

fluorescence, also leading to decreased expression of

known target genes such as cyclin D1 and glutamine

synthetase. The decrease in B-catenin activity was con-

firmed by TOPflash reporter luciferase assay. The func-

tional impact of diminished B-catenin was exhibited as

temporal decrease in tumor cell viability by the MTT

assay. A concomitant decrease in tumor cell prolifera-

tion was also evident with [3H]thymidine incorporation

and verified with soft agar assays. Thus, B-catenin is

essential for the survival and growth of hepatoma cells

independent of mutations in the B-catenin gene and

provide a proof of principle for the significance of the

therapeutic inhibition of B-catenin in HCC.

Neoplasia (2007) 9, 951–959

Keywords: Liver cancer, Wnt/b-catenin, development, treatment, regeneration.

Introduction

The Wnt/b-catenin pathway is important for normal growth

and development [1]. However, anomalous activation of Wnt

signaling in adults is often associated with oncogenesis [2–

4]. Aberrant signaling involving the stabilization and nuclear

translocation of b-catenin has been observed in cancers of

the colon, rectum, lung, shin, breast, liver, and pancreas [5–

12]. Interestingly, the activation of Wnt signaling can occur

due to multiple mechanisms and can result from dysregula-

tion of upstream effectors and mutations in downstream

components [13].

Hepatocellular carcinoma (HCC), which is the major primary

malignant tumor of the liver, is a disease with poor prognosis

due to lack of effective treatment, making it essential to identify

novel therapeutic targets [14]. Aberrant activation of b-catenin

has been identified in a significant subset of HCC patients with

molecular bases ranging from mutations in the �-catenin gene

(CTNNB1) (26–34%) or AXIN1/2 (5%) to upregulation of the

frizzled-7 receptor (90%) [15–17]. Although additional known

or unknown mechanisms might also contribute to b-catenin

stabilization in HCC, its role in various stages of hepatocarcino-

genesis ranging from hepatic adenoma to hepatoma is indis-

putable [18].

Based on the role of b-catenin in cellular events common to

the processes of development and oncogenesis such as prolif-

eration and survival, we initiated the current study [19–21]. We

used small interfering RNA (siRNA) directed against b-catenin

to examine the impact of successful b-catenin knockdown on

two human HCC cell lines and to demonstrate an indispensable

role of b-catenin in tumor cell survival and proliferation.

Materials and Methods

Cell Culture, Treatment, and Transient Transfection

Human HCC cell lines HepG2 and Hep3B were obtained

from the American Type Culture Collection (Manassas, VA).

Cells were plated in six-well plates and cultured in Eagle’s

minimal essential medium (EMEM) supplemented with 10% vol/

vol fetal calf serum at 37jC in a humidified 5% carbon dioxide

atmosphere. The cells were grown to 50% to 60% confluence,

followed by serum starvation for 16 hours. For siRNA inhibi-

tion studies, the cells were transfected with validated human

b-catenin (CTNNB1) siRNA or negative control siRNA 1 (Ambion,

Inc., Austin, TX) at a final concentration of 100 nM in the pres-

ence of an Oligofectamine reagent (Invitrogen, Carlsbad, CA), as

per the manufacturer’s instructions. After transfection, the cells

Address all correspondence to: Satdarshan P. S. Monga, MD, UPCI-GI Oncology/MIRM-Liver,

University of Pittsburgh School of Medicine, 200 Lothrop Street, S-421 BST, Pittsburgh, PA

15216. E-mail: smonga@pitt.edu1This report was funded, in part, by the American Cancer Society (grant RSG-03-141-01-CNE

to S.P.S.M.) and the National Institutes of Health (grants 1RO1DK62277 and 1R01CA124414

to S.P.S.M.) Rango’s Fund for Enhancement of Pathology Research.

Received 11 June 2007; Revised 1 September 2007; Accepted 7 September 2007.

Copyright D 2007 Neoplasia Press, Inc. All rights reserved 1522-8002/07/$25.00

DOI 10.1593/neo.07469

Neoplasia . Vol. 9, No. 11, November 2007, pp. 951 – 959 951

www.neoplasia.com

RESEARCH ARTICLE

were harvested at 24, 48, and 72 hours for protein extraction

and additional analysis. All experiments were performed in

triplicate, and representative results are reported.

Protein Extraction and Western Blot Analysis

Protein extraction from cell lines and Western blot anal-

ysis were performed as previously described [5,22,23].

Briefly, the HCC cell lines from siRNA treatment were used

for total cell lysate preparation. Homogenization was per-

formed in 200 ml of RIPA buffer containing fresh protease and

phosphatase inhibitors (Sigma, St. Louis, MO). The concen-

tration of the protein in lysates was determined by bicincho-

ninic acid protein assay, with bovine serum albumin as

standard. Aliquots of samples were stored at �80jC until

use. Twenty or 50 mg of proteins was resolved by SDS-

PAGE analysis using the mini-PROTEIN 3-electrophoresis

module assembly (Bio-Rad, Hercules, CA) and transferred

to Immobilon PVDF membranes (Bio-Rad). The primary

antibodies used were against b-catenin, cyclin D1, glu-

tamine synthetase (GS; Santa Cruz Biotechnology, Santa

Cruz, CA), and b-actin (Chemicon, Temecula, CA). Horse-

radish peroxidase–conjugated secondary antibodies were

purchased from Chemicon. The proteins were detected by

Super-Signal West Pico Chemiluminescent Substrate (Pierce,

Rockford, IL) and visualized by autoradiography. Densitomet-

ric analysis on blots was performed by the NIH Imager soft-

ware (NIH, Bethesda, MD), and the average integrated optical

density in the b-catenin siRNA–treated group was normalized

to control siRNA-treated group at the corresponding times.

The differences were assessed for statistical significance with

Student’s t test, and P < .05 was considered significant.

Immunofluorescence Microscopy

Cells were grown to 50% confluence on glass cover slips

in 24-well plates. After b-catenin siRNA transfection for

48 hours, the cover slips were washed once with phosphate-

buffered saline (PBS) and fixed in 100% methanol for 3 min-

utes at �20jC. Staining was performed as described else-

where [24]. The secondary antibody was Cy3, which was

conjugated and obtained from Jackson Immunoresearch

(West Grove, PA). Nuclei were counterstained with 40,6-

diamidino-2-phenylindole. The cover slips were then placed

on slides with a drop of gelvatol and viewed on a Nikon

Eclipse epifluorescence microscope (Nikon), and images

were obtained with a Sony CCD camera (Sony).

�-Catenin/Tcf Transcription Reporter Assay

b-Catenin/Tcf transcriptional reporter activity was per-

formed as previously described [23]. Briefly, after b-catenin

siRNA transfection for 24 hours, the cells were transiently

transfected with the reporter construct TOPflash or FOPflash

(Upstate, Lake Placid, NY). TOPflash has three copies of the

Tcf/Lef sites upstream of a thymidine kinase (TK) promoter

and the firefly luciferase gene. FOPflash has mutated copies

of Tcf/Lef sites and is used as a control for measuring

nonspecific activation of the reporter. All transfections were

performed using 1.8 mg of TOPflash or FOPflash plasmids

and FuGene HD reagent (Roche, Indianapolis, IN). To nor-

malize transfection efficiency in reporter assays, the cells

were cotransfected with 0.2 mg of internal control reporter

Renilla reniformis luciferase driven under the TK promoter

(pRL-TK; Promega, Madison, WI). Twenty hours after TOP-

flash or FOPflash transfection, luciferase assay was per-

formed, using the Dual Luciferase Assay System kit, in

accordance with the manufacturer’s protocols (Promega).

Relative luciferase activity (in arbitrary units) was reported as

fold induction after normalization for transfection efficiency.

The normalized luciferase activity from experiments per-

formed in triplicate was compared between the experimental

and control groups for statistical significance with unpaired

Student’s t test. P < .01 was considered significant.

Cell Viability Assay

Cell viability assay was performed as previously de-

scribed [23]. All experiments were performed in triplicate.

Briefly, at 24, 48, and 72 hours after b-catenin siRNA

transfection, 10% vol/vol of 5 mg/ml 3-(4,5-dimethylthiazol-

2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) diluted

in PBS was added to HepG2 and Hep3B cultures. After

30 minutes of incubation, the medium was aspirated and

washed with PBS. Isopropanol (600 ml) was added and

shaken gently for 5 minutes. Two hundred microliters of this

solution was transferred to a 96-well plate, and absorbance

was measured at 570 nM. The mean (±SEM) absorbance

units obtained from three experiments for each group were

compared for statistical significance with Student’s t test, and

P < .01 was considered highly significant. The data were

normalized to their respective controls and are presented as

a bar graph.

Cell Proliferation Assay

[3H]Thymidine incorporation analyses were performed as

previously described [23]. Briefly, 4 hours after b-catenin or

control siRNA transfection in Hep3B and HepG2 cells,

[3H]thymidine (2.5 mCi/ml) was added to culture media.

These cells were cultured for [3H]thymidine incorporation

for an additional 24, 48, or 72 hours. Alternatively, an

additional set of HepG2 cells was transfected with siRNA

against b-catenin a second time, at 24 hours after the initial

transfection. This was followed 4 hours later by the addition

of [3H]thymidine (2.5 mCi/ml), and the cells were cultured

for 24 or 48 hours. Next, the cells were washed with 0.9%

NaCl and lysed in 0.2% SDS, 1� SSC (150 mM NaCl and

15 mM Na–citrate, pH 7.0), and 5 mM EDTA, and aliquots

were used to determine [3H]thymidine incorporation on a

Beckman Scintillation counter (Beckman), and total DNA

using Hoechst dye H33258 and a fluorescence meter (DyNA

Quant 200; Hoefer). Counts from the experiments performed

in triplicate from each group were compared for statistical

significance with Student’s t test. P < .01 was considered

highly significant, and P < .05 was considered significant.

The analysis from sequential (2�) siRNA-treated cells was

normalized to their respective controls and is presented as a

bar graph.

952 B-Catenin Inhibition in Human Hepatoma Cells Zeng et al.

Neoplasia . Vol. 9, No. 11, 2007

Soft Agar Assay

A 1.5-ml layer of 0.5% agar (wt/vol) in EMEM with 10%

FBS was poured in 35-mm Petri dishes. HepG2 and Hep3B

cells were treated with control or b-catenin siRNA for 24 hours

at 100 nM in the presence of Oligofectamine reagent (Invi-

trogen) and resuspended in 0.35% agar (wt/vol) in EMEM with

5% FBS at a density of 5000 cells/1.5 ml. Cell suspensions

(1.5 ml) were poured on the top of the base layer, allowed to

solidify, and incubated at 37jC in the presence of 5% CO2 for

14 days (HepG2 cells) and 21 days (Hep3B cells). The

colonies were stained with 0.005% crystal violet for 1 hour.

Colonies containing > 10 cells were counted under an Olym-

pus microscope (Olympus). Absolute counts were compared

for statistical significance with Student’s t test and are pre-

sented as a bar graph.

Results

siRNA Inhibits �-Catenin Expression in HCC Cell Lines

The human hepatoma cell lines Hep3B and HepG2 were

grown to around 50% confluence, followed by transfection

with either human b-catenin (CTNNB1) or control siRNA

(referred to as control), as indicated in the Materials and

Methods section. Protein lysates obtained from the tumor

cells at different time points after CTNNB1 siRNA transfec-

tion showed a temporal decrease in total b-catenin protein

in both HepG2 and Hep3B cells compared to controls (Fig-

ure 1A). HepG2 cells harbor two species of b-catenin: wild-

type or the 96-kDa form, and a more prominent truncated

or stable 75-kDa form (tb-catenin) due to partial exon 3 de-

letion that contains phoshorylation sites critical for eventual

degradation of b-catenin. siRNA transfection led to a de-

crease in both species of b-catenin in HepG2 cells, albeit the

wild-type form showed an immediate and robust knockdown

compared to tb-catenin. Although control siRNA-transfected

HepG2 cells showed a time-dependent increase in tb-catenin

protein levels, b-catenin siRNA–treated cells failed to show

this increase, thus suggesting that de novo synthesis of

b-catenin might also contribute to the truncated species of

b-catenin. Densitometric analysis of Western immunoblots

identified a dramatic and highly significant (P < .01) decrease

in the full-length b-catenin protein in both HepG2 and Hep3B

cells within 24 hours, although a more prominent effect was

evident after 48 hours (Figure 1, B and C). In addition, a

modest but significant (P < .01) decrease in tb-catenin in

HepG2 cells was observed after 48 hours, with a more dra-

matic difference at 72 hours after a single transfection of

CTNNB1 siRNA (Figure 1B).

siRNA Inhibits �-Catenin Activation as Observed

by a Downregulation of Known Target Genes

Cyclin D1 and GS

Next, to confirm whether the decrease in total b-catenin

protein in both HepG2 and Hep3B cells following siRNA

treatment also affected the transcriptional regulatory func-

tion of b-catenin, the protein lysates were examined for

the protein expression of two known targets: cyclin D1 and

GS. Although the former is regulated by additional factors

as well, GS is a more selective Wnt/b-catenin target in the

liver [21]. After CTNNB1 siRNA transfection, a decrease in

cyclin D1 and GS was detectable in both cell lines, support-

ing a functional decrease in the b-catenin protein in both

cell lines (Figure 1A). This decrease was more dramatic in

Hep3B cells than in HepG2 cells, although it was significant

in both cells following b-catenin siRNA transfections (Figure 1,

D and E ).

Decrease in �-Catenin and Its Nuclear Localization

Are Also Detectable By Immunofluorescence in Hep3B

and HepG2 Cells after siRNA Transfection

To confirm a decrease in total b-catenin, as well as any

changes in its localization following CTNNB1 or control siRNA

transfection, Hep3B and HepG2 cells were examined for

b-catenin with immunofluorescence, as discussed in the Ma-

terials and Methods section. Although Hep3B cells transfected

with control siRNA for 48 hours showed membranous and

nuclear localization, a vivid decrease in b-catenin was evident

in CTNNB1 siRNA–transfected cultures at the same time,

which displayed occasional cells with membranous or, even

rarely, nuclear b-catenin (Figure 2, A and B). In addition,

although control HepG2 cells showed extensive membranous

and nuclear b-catenin, the CTNNB1 siRNA–transfected cells

showed a similar and pronounced b-catenin decrease at

48 hours as well (Figure 2, C and D).

�-Catenin/Tcf Reporter Assay Confirms siRNA-Mediated

Loss of �-Catenin Activity in Hep3B and HepG2 Cells

Next, we examined the effect of b-catenin knockdown on

its activity with the TOPflash reporter assay, which is a direct

and reliable measure of the b-catenin/Tcf–dependent tran-

scriptional activity. TOPflash and FOPflash activities were

measured following b-catenin siRNA transfection for 48 hours,

as described in the Materials and Methods section. A signif-

icant downregulation in TOPflash reporter activity, without any

effect on FOPflash activity, was apparent after CTNNB1

siRNA transfection in Hep3B and HepG2 tumor cells com-

pared to control siRNA–transfected cells (Figure 3). The ac-

tivity was decreased around threefold to fivefold in HepG2

cells, and by fivefold to eightfold in Hep3B cells, clearly

identifying a pronounced loss of b-catenin function in both

tumor cells.

Effect of �-Catenin Knockdown on the Survival

of Hepatoma Cells

To address any biologic relevance of the loss of b-catenin

function, we investigated the impact of b-catenin siRNA on

the survival of Hep3B and HepG2 cells. CTNNB1 siRNA–

transfected tumor cells were examined for viability by MTT

assay, as described in the Materials and Methods section.

Although a consistently small but insignificant effect on the

survival of HepG2 cells was apparent after 24 hours of

transfection, a more robust (f 35%) and significant (P <

.01) decrease was observed at 48 and 72 hours (Figure 4A).

B-Catenin Inhibition in Human Hepatoma Cells Zeng et al. 953

Neoplasia . Vol. 9, No. 11, 2007

954 B-Catenin Inhibition in Human Hepatoma Cells Zeng et al.

Neoplasia . Vol. 9, No. 11, 2007

A significant decrease in Hep3B cell viability was observed

as early as 24 hours after CTNNB1 siRNA transfection

(P < .01), which was further augmented to around a 40%

decrease in viability by 48 to 72 hours (Figure 4B). These

results demonstrate an important role in tumor cell viability.

Effect of �-Catenin siRNA on the Proliferation of HCC Cells

Next, we examined the effect of b-catenin loss on tumor

cell proliferation. Thymidine incorporation was employed as

a measure of DNA synthesis after CTNNB1 or control siRNA

transfection, as described in the Materials and Methods

section. A significant decrease in thymidine incorporation in

b-catenin siRNA–transfected HepG2 cells (P < .05), which

ranged from around 20% to 35%, was identified after 48 and

72 hours (Figure 5A). Although a 20% and significant de-

crease in b-catenin siRNA–transfected Hep3B cells was

observed at 48 hours (P < .05), a pronounced (> 50%) and

highly significant (P < .01) decrease was readily identifiable

at 72 hours (Figure 5B).

Because HepG2 cells harbor a b-catenin deletion render-

ing a more stable protein, we examined the efficacy of

employing serial CTNNB1 siRNA transfections in HepG2

cells, as detailed in the Materials and Methods section. An

additional transfection of CTNNB1 siRNA led to a dramatic

and extremely significant (P < .001) decrease in thymidine

incorporation after 24 hours (40%) and 48 hours (> 70%) in

HepG2 cells (Figure 5C). Thus, the above results demon-

strate a critical role of b-catenin in tumor cell proliferation.

Effect of �-Catenin siRNA on the Soft Agar Growth

of Hepatoma Cells

Next, we examined the effect of b-catenin loss on the

growth of hepatoma cells in soft agar. Twenty-four hours

after the control or b-catenin siRNA transfection of HepG2

and Hep3B cells, these were cultured in soft agar for 14 and

21 days, respectively. A dramatic decrease in crystal violet–

stained colonies was evident in the b-catenin siRNA–trans-

fected HepG2 and Hep3B cells, as shown in representative

cultures (Figure 5D). This difference was statistically signif-

icant, with the numbers of colonies in the b-catenin siRNA–

transfected cells being around 30% of the control siRNA–

treated group (Figure 5E ).

Discussion

HCC is the commonest primary malignant tumor of the liver

that has been observed with increasing incidence due to an

increased prevalence of risk factors, including nonalcoholic

Figure 2. Changes in �-catenin expression and localization after siRNA treatment in Hep3B and HepG2 cells by immunofluorescence. Left panel: Original mag-

nification, �600. Right panel: Original magnification, �1000. (A) Membranous and nuclear (arrowhead) �-catenin is observed in Hep3B cells at 48 hours after

control siRNA transfection. (B) Decreased expression and nuclear localization of �-catenin at 48 hours after �-CTNNB1 siRNA transfection in Hep3B cells. (C)

Membranous and nuclear (arrowhead) �-catenin is observed in HepG2 cells at 48 hours after control siRNA transfection. (D) Decreased expression and nuclear

localization of �-catenin at 48 hours after �-CTNNB1 siRNA transfection in HepG2 cells. A small subset of cells continued to show nuclear �-catenin (arrowheads).

Figure 1. Effect of CTNNB1 siRNA on �-catenin, GS, and cyclin D1 in human hepatoma cell lines (Hep3B and HepG2). All results for densitometry are presented

as the mean ± SEM of three experiments and normalized to their respective controls. (A) Western blot analysis shows a decrease in total �-catenin, GS, and cyclin

D1 in HepG2 (left panel) and Hep3B (right panel) cells. A lower-molecular-weight (75 kDa) truncated form of �-catenin (t�-catenin) in HepG2 cells showed only a

modest decrease, especially at 48 and 72 hours. �-Actin confirmed equal loading. (B) Densitometric analysis of HepG2 Western immunoblots revealed a significant

decrease in full-length �-catenin protein (left panel) at 24 hours (P < .01), and in t�-catenin (right panel) at 48 hours (P < .05), with a 40% additional loss at 72 hours

(P < .01). (C) Densitometric analysis of Hep3B immunoblots shows a significant decrease in �-catenin protein at 24 hours, with a 50% additional decrease at

48 hours and a 70% additional decrease at 72 hours. (D) A significant decrease in cyclin D1 at 48 and 72 hours, and in GS at 72 hours (P < .05), after �-catenin

siRNA transfection has been confirmed with densitometry in HepG2 cells. (E) A significant decrease in cyclin D1 and GS at 24, 48, and 72 hours (P < .01) after �-

catenin siRNA transfection has been confirmed by densitometry in Hep3B cells.

B-Catenin Inhibition in Human Hepatoma Cells Zeng et al. 955

Neoplasia . Vol. 9, No. 11, 2007

and alcoholic fatty liver disease and infections such as

hepatitis B and C [14,25]. In addition, the lack of optimal

treatment adds to the overall grim prognosis of the disease,

making it prudent to elucidate novel molecular mechanisms,

which in turn might reveal new therapeutic targets [25]. b-

Catenin, which is the chief oncogenic component of the

canonical Wnt pathway, has been identified to be active in

a significant subset of HCC, which, based on mechanisms,

ranges from 15% to 90% [12,17,26]. Furthermore, b-catenin

activation and cytoplasmic/nuclear localization have been

associated with increased proliferation and survival of

hepatocytes in normal physiology and tumor cell survival/

proliferation in HCC [2,20,21,26–28]. This implicates b-

catenin as an attractive therapeutic target in HCC. The

current study provides a proof of principle that b-catenin

inhibition is feasible, is of clinical relevance, and has func-

tional consequences in HCC cells, which show b-catenin

overexpression and/or activation.

We examined the effect of the inhibition of b-catenin by

siRNA in human HCC cell lines. In our current study, we used

two hepatoma cell lines that have appreciable b-catenin

expression but have distinct bases of b-catenin activation.

HepG2 cells have a heterozygous deletion of 348 nucleo-

tides in exon 3 of the �-catenin gene, which encode a total of

116 amino acids from trytophan-25 to asparagine-141 [29].

Critical amino acids in this region have been shown to be

necessary for GSK3b or casein kinase–driven b-catenin

phosphorylation and eventual degradation [30–32]. Hep3B

cells, however, were derived from hepatitis B–infected liver

tumor, and although they do not contain any mutations or

deletions in the �-catenin gene, these cells show high levels

of b-catenin protein [33,34]. One of the mechanisms of such

observation may be the presence of elevated levels of

inactive GSK3b (GSK3b Ser9–phosphorylated) in these

cells [34]. In our current study, we used these two cell lines

to examine the efficacy of b-catenin knockdown by siRNA

against CTNNB1 along with its functional consequences,

including effects on proliferation and survival.

A dramatic decrease in b-catenin expression, nuclear

localization, and activation was evident within 24 to 48 hours

in Hep3B cells, which exhibit elevated wild-type b-catenin. A

concomitant decrease in tumor cell proliferation, growth in

soft agar, and survival was also evident at the corresponding

times. The extent of the decrease in these biologic events

was fairly robust in these cells. A similar effect of CTNNB1

siRNA on the wild-type species of b-catenin in HepG2 cells

was evident within 24 to 48 hours. However, the effect of

siRNA on the truncated form, which is the predominant func-

tional form of b-catenin in HepG2 cells, was less dramatic

Figure 3. TOPflash reporter assay demonstrates a significant decrease in �-

catenin transcriptional activity in Hep3B and HepG2 cells following �-catenin

siRNA transfection for 48 hours (P < .01). Luciferase activity in FOPflash re-

mained unaffected, confirming a lack of nonspecific activation of the reporter

system. A vector containing Renilla luciferase was used as an internal control

for transfection efficiency, and the results are expressed as relative firefly/

Renilla luciferase activity. The results presented are the mean ± SEM for

three experiments.

Figure 4. Loss of �-catenin affects Hep3B and HepG2 cell viability by MTT assay. The results represent the mean ± SEM of three experiments and are presented

as a bar graph after normalizing to the respective controls. (A) A significant decrease in the viability of HepG2 cells was observed after 48 and 72 hours of �-catenin

siRNA transfection. (B) A significant decrease in the viability of Hep3B cells was observed at 24, 48, and 72 hours after �-catenin siRNA transfection.

956 B-Catenin Inhibition in Human Hepatoma Cells Zeng et al.

Neoplasia . Vol. 9, No. 11, 2007

and mainly evident after 48 hours. There was a modest but

significant decrease in tumor cell viability and proliferation

after 48 hours of CTNNB1 siRNA in this case, which might

be the consequence of the decrease in both wild-type and

truncated b-catenin species. In addition, the soft agar growth

of HepG2 was equally affected by siRNA against b-catenin

and was comparable to Hep3B cells, suggesting that sup-

pression of b-catenin transcription was equally efficacious in

long-term studies, irrespective of the mutational status of b-

catenin. Alternatively, tb-catenin was dramatically decreased

in HepG2 cells after two consecutive 24-hour transfections

with CTNNB1 siRNA, with a more robust decrease in tumor

cell proliferation. These findings clearly indicate the clinical

relevance of the transcriptional downregulation of b-catenin

in HCC.

Based on the above findings, there appears to be

an obvious merit of b-catenin suppression in b-catenin–

overexpressing liver tumors independent of any mutations

in CTNNB1. However, the caveat is that b-catenin sup-

pression would need to be prolonged in the event of muta-

tions in the �-catenin gene itself, as the truncated protein

is stable over prolonged time. Nonetheless, sustained

Figure 5. Loss of �-catenin compromises tumor cell proliferation by thymidine incorporation. The results represent the mean ± SEM of three experiments. (A) A

significant decrease in thymidine incorporation in HepG2 cells observed at 48 and 72 hours after �-catenin siRNA transfection (P < .05). (B) A significant decrease

in thymidine incorporation in Hep3B cells observed after 48 and 72 hours of �-catenin siRNA transfection (P < .01). (C) Bar graph shows a significant decrease in

thymidine incorporation (P < .001) by 40% and 70% in HepG2 cells at 24 and 48 hours, respectively, after two sequential siRNA transfections performed 24 hours

apart. Data presented were normalized to control siRNA for each time point and treatment. (D) A representative photograph shows a noteworthy decrease in the

numbers of soft agar colonies formed by HepG2 cells at 14 days, and by Hep3B cells at 21 days, after the initial 24-hour transfection with �-catenin siRNA

compared to the controls. (E) Histogram shows a significant decrease in the number of colonies of both HepG2 (top) and Hep3B (bottom) in soft agar in the

�-catenin siRNA– treated group compared to the respective controls at the same time (P < .01). The experiment was performed in triplicate, and average (±SD)

data were presented.

B-Catenin Inhibition in Human Hepatoma Cells Zeng et al. 957

Neoplasia . Vol. 9, No. 11, 2007

b-catenin suppression in overexpressing tumors would have

promising biologic consequences on tumor cell viability and

proliferation.

Antisense modalities are being tested preclinically and clini-

cally, and are a part of a bigger ‘‘antigene’’ strategy for cancer

prevention and treatment [35]. Antisense therapies for treat-

ment in a clinical setup are still in the stage of infancy. The

current clinical trials are mainly aimed at chemosensitization

of the tumors and similar applications [36–38]. Although no

clinical studies are available, preclinical b-catenin suppres-

sion using antisense modalities has been shown to have

relevance in colorectal cancer cells and other tumor cells such

as melanoma and sarcoma [39–41]. It remains to be seen

whether successful b-catenin suppression through antisense

techniques, including classic antisense oligodeoxynucleotide

technology, antisense RNA technology, or additional means,

will have translational significance. Successful b-catenin sup-

pression in vivo has been achieved by phosphomorpholino

oligonucleotides during liver regeneration [42].

A yet another prospective way of b-catenin suppression

in the clinical setting might be the use of agents that inhibit

�-catenin gene and protein expression, along with its activity.

PPAR-g agonists suppress b-catenin transcriptionally and

posttranslationally [43,44]. We have recently identified a role

for R-Etodolac in inhibiting b-catenin expression and activa-

tion as well [23]. Such agents would therefore have strong

clinical relevance in inhibiting b-catenin activity in liver can-

cers exhibiting b-catenin overexpression independent of mu-

tations in the �-catenin gene.

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