Telomerase antagonists GRN163 and GRN163L inhibit tumor growth and increase chemosensitivity of human hepatoma.
ABSTRACT Most cancer cells have an immortal growth capacity as a consequence of telomerase reactivation. Inhibition of this enzyme leads to increased telomere dysfunction, which limits the proliferative capacity of tumor cells; thus, telomerase inhibition represents a potentially safe and universal target for cancer treatment. We evaluated the potential of two thio-phosphoramidate oligonucleotide inhibitors of telomerase, GRN163 and GRN163L, as drug candidates for the treatment of human hepatoma. GRN163 and GRN163L were tested in preclinical studies using systemic administration to treat flank xenografts of different human hepatoma cell lines (Hep3B and Huh7) in nude mice. The studies showed that both GRN163 and GRN163L inhibited telomerase activity and tumor cell growth in a dose-dependent manner in vitro and in vivo. The potency and efficacy of the lipid-conjugated antagonist, GRN163L, was superior to the nonlipidated parent compound, GRN163. Impaired tumor growth in vivo was associated with critical telomere shortening, induction of telomere dysfunction, reduced rate of cell proliferation, and increased apoptosis in the treatment groups. In vitro, GRN163L administration led to higher prevalence of chromosomal telomere-free ends and DNA damage foci in both hepatoma cell lines. In addition, in vitro chemosensitivity assay showed that pretreatment with GRN163L increased doxorubicin sensitivity of Hep3B. In conclusion, our data support the development of GRN163L, a novel lipidated conjugate of the telomerase inhibitor GRN163, for systemic treatment of human hepatoma. In addition to limiting the proliferative capacity of hepatoma, GRN163L might also increase the sensitivity of this tumor type to conventional chemotherapy.
- SourceAvailable from: Juan Carlos Romero-Benavides[Show abstract] [Hide abstract]
ABSTRACT: Cancer remains a public health problem with a high unmet medical demand. However, in recent decades, the knowledge of several functional molecular and biological traits that distinguish tumor cells from normal cells, known as the hallmarks of cancer as described by Hannahan and Weinberg, has led to new and modern therapeutic approaches against this disease. Most cancer drugs are deliberately developed for specific molecular targets that involve these hallmarks. In this review, we address the currently available cancer drugs and development of new drugs from the perspective of their interaction with these hallmarks as well as the pathways and mechanisms involved.Tumor Biology 01/2014; · 2.52 Impact Factor
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ABSTRACT: Telomerase is required for the unlimited lifespan of cancer cells. The vast majority of pancreatic adenocarcinomas overexpress telomerase activity and blocking telomerase could limit their lifespan. GRN163L (Imetelstat) is a lipid-conjugated N3'→P5' thio-phosphoramidate oligonucleotide that blocks the template region of telomerase. The aim of this study was to define the effects of long-term GRN163L exposure on the maintenance of telomeres and lifespan of pancreatic cancer cells. Telomere size, telomerase activity, and telomerase inhibition response to GRN163L were measured in a panel of 10 pancreatic cancer cell lines. The cell lines exhibited large differences in levels of telomerase activity (46-fold variation), but most lines had very short telomeres (2-3 kb in size). GRN163L inhibited telomerase in all 10 pancreatic cancer cell lines, with IC50 ranging from 50 nM to 200 nM. Continuous GRN163L exposure of CAPAN1 (IC50 = 75 nM) and CD18 cells (IC50 = 204 nM) resulted in an initial rapid shortening of the telomeres followed by the maintenance of extremely short but stable telomeres. Continuous exposure to the drug eventually led to crisis and to a complete loss of viability after 47 (CAPAN1) and 69 (CD18) doublings. Crisis In these cells was accompanied by activation of a DNA damage response (γ-H2AX) and evidence of both senescence (SA-β-galactosidase activity) and apoptosis (sub-G1 DNA content, PARP cleavage). Removal of the drug after long-term GRN163L exposure led to a reactivation of telomerase and re-elongation of telomeres in the third week of cultivation without GRN163L. These findings show that the lifespan of pancreatic cancer cells can be limited by continuous telomerase inhibition. These results should facilitate the design of future clinical trials of GRN163L in patients with pancreatic cancer.PLoS ONE 01/2014; 9(1):e85155. · 3.73 Impact Factor
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ABSTRACT: Enhanced telomerase activity is a hallmark in the majority of cancer cells. Thus, understanding the interactions between telomerase and its inhibitors is fundamentally important for the development of novel anticancer drugs without severe side effects. In this study, the covalent binding of helenalin to CYS445 of telomerase (PDB ID: 3DU6) was simulated using combined quantum chemical and molecular mechanical (QM/MM) methods. The results showed that the reaction was a reversible Michael-type addition and a hydrogen bond was formed between helenalin and the side chain of LYS416 of telomerase during the reaction procedure. The LYS416 residue is vital to telomere DNA recognition by interacting with DNA base through hydrogen bonds. The alkylation of CYS445 of telomerase by helenalin may interfere with the telomere DNA recognition at the telomerase active site, thus resulting in inhibition of the enzyme activity.Journal of Molecular Graphics and Modelling. 01/2014;
Telomerase Antagonists GRN163 and GRN163L Inhibit
Tumor Growth and Increase Chemosensitivity of
Meta W. Djojosubroto,1Allison C. Chin,2Ning Go,2Sonja Schaetzlein,1Michael P. Manns,1Sergei Gryaznov,2
Calvin B. Harley,2and K. Lenhard Rudolph1
Most cancer cells have an immortal growth capacity as a consequence of telomerase reactivation.
Inhibition of this enzyme leads to increased telomere dysfunction, which limits the proliferative
of telomerase, GRN163 and GRN163L, as drug candidates for the treatment of human hepatoma.
xenografts of different human hepatoma cell lines (Hep3B and Huh7) in nude mice. The studies
onist, GRN163L, was superior to the nonlipidated parent compound, GRN163. Impaired tumor
growth in vivo was associated with critical telomere shortening, induction of telomere dysfunction,
ment of GRN163L, a novel lipidated conjugate of the telomerase inhibitor GRN163, for systemic
treatment of human hepatoma. In addition to limiting the proliferative capacity of hepatoma,
GRN163L might also increase the sensitivity of this tumor type to conventional chemotherapy.
increasing as a result of a growing number of carriers of
epatocellular carcinoma (HCC) is one of the
most prevalent cancers worldwide,1and the in-
cidence of HCC in developed countries is still
chronic hepatitis C virus infection.2The therapeutic op-
responds poorly to systemic treatment with chemother-
apy. Other therapeutic strategies have not revealed signif-
icant efficiency in clinical trials (reviewed by Llovet et
al.3). Therefore, there is currently no standard therapy for
advanced, multifocal HCC, indicating the need to evalu-
ate new therapeutic options for this tumor.
Similar to other malignant human tumor types,4over
80% of human HCC biopsies show activation of telom-
normal liver,6show no or very low levels of telomerase
activity. The main function of telomerase is the de novo
synthesis of telomeres, which cap the chromosome ends
of eukaryotic cells and protect chromosome ends from
fusion and DNA damage recognition.7Because of the
shorten during each cell division by 50 to 100 bp.8Telo-
mere shortening to a critical length and/or the uncapping
a finite number of cell divisions, leading to either replica-
tive senescence or crisis.9-11A critically short telomere
Abbreviations: HCC, hepatocellular carcinoma; NPS, N3?-P5? thio-phosphor-
amidate; PBS, phosphate-buffered saline; bw, body weight; BrdU, 5-bromo-2-
deoxyuridine; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP
nick end labeling; Q-FISH, quantitative fluorescence in situ hybridization; TFI,
telomere restriction fragment.
From the1Department of Gastroenterology, Hepatology, and Endocrinology,
Medical School Hannover, Hannover, Germany; and the2Geron Corporation,
Menlo Park, CA.
Received December 6, 2004; accepted June 9, 2005.
K.L.R. is supported by the Deutsche Forschungsgemeinschaft (Emmy-Noether-
Programm: Ru 745/2-3 and KFO119 and Ru 745/4-1) and by a grant from the
Deutsche Krebshilfe e.V. (10-2236-Ru2).
Address reprint requests to: K. Lenhard Rudolph, Department of Gastroenterol-
1, 30625 Hannover, Germany. E-mail: firstname.lastname@example.org; fax:
Copyright © 2005 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
Potential conflict of interest: Drs. Chin, Go, Gryaznov, and Harley own stock in
within a cell is believed to activate DNA damage signal-
ing, inducing senescence or cell death.12,13It has been
proposed that postnatal repression of telomerase func-
tions as a tumor repressor mechanism that limits the
growth of transformed cells.14This hypothesis is consis-
tent with the high frequency of telomerase reactivation
found in most human cancers, including HCC. Studies
on the inhibition of telomerase activity in cancer cell
lines15-19and in telomerase knockout mice20-22have
shown that growth and progression of malignant tumors
depend on telomerase activity and telomere stabilization.
Recently, a new class of telomerase inhibitors was de-
veloped: N3?-P5? thio-phosphoramidate (NPS) oligonu-
cleotides targeting the active site template region of the
revealed that a development candidate from this class of
oligonucleotides, GRN163, inhibited telomerase activity
in various cultured human cancer cell lines (breast, renal,
prostate, epidermoid, cervix, lung, colon, leukemia, mul-
tiple myeloma, lymphoma) leading to telomere shorten-
ing, growth arrest, and apoptosis.23-27In addition,
GRN163 inhibited tumor growth of prostate cancer,
transplant models.24,26,27To evaluate the potential use of
NPSS oligonucleotides targeting the human telomerase
RNA component for the treatment of HCC, we analyzed
GRN163 designed for increased bioavailability,28for an-
titumor effects against human hepatoma cells (Hep3B
and Huh7) in vivo and in vitro. The results suggest that
telomerase inhibition could be a valid approach for HCC
treatment and that GRN163L is a promising drug devel-
opment candidate with significant effects on tumor
growth and increased chemosensitivity to doxorubicin.
Materials and Methods
Mouse Handling. NMRI nu/nu mice were bred and
housed at the animal facility of Medical School Han-
nover, given a standard diet, and placed on a 12/12-hour
light/dark cycle in a pathogen-free barrier area. Protocols
used in this study complied with institutional guidelines.
Mice were humanely sacrificed when the total two-di-
mensional size of tumors exceeded 40 mm or after the
treatment period of 4 weeks.
Culture and Subcutaneous Inoculation of Cells.
The human hepatoma cell lines Hep3B and Huh7 were
were cultured in Dulbecco’s modified Eagle medium
(Gibco, Grand Island, NY) supplemented with 10% fetal
bovine serum (Gibco) and penicillin/streptomycin (100
5% CO2. Cells were passaged every 2 days (Huh7) and 3
to 4 days (Hep3B) 2 to 3 times before being inoculated
into nude mice. Mice were anesthetized via methyl-ether
inhalation, and 5 ? 106hepatoma cells were engrafted
subcutaneously into the right and left flanks.
Test Articles. GRN163 is a 13-mer thio-phosphor-
TAGGGTTAGACAA. GRN163L is a lipid-conjugated
derivative of GRN163 with the sequence 5?-L-
TAGGGTTAGACAA, where L ? aminoglycerol-palmi-
toyl moiety. Lyophilized powders for GRN163 and
GRN163L were formulated in phosphate-buffered saline
(PBS) at 5.0 and 3.3 mg/mL, respectively, using an ex-
tinction coefficient of 143 OD/?mol. Formulated oligo-
and stored in 5- to 10-mL aliquots at ?20°C.
Treatment Groups and Dosing Regimens. Treat-
ment via intraperitoneal injection of vehicle or drug was
started when a subcutaneous tumor became detectable
(day 1). Mice were randomized into groups (n ? 5-7
Huh7 groups). In the first round of treatment of Hep3B-
derived tumors, we injected the control group of mice
weight (bw) GRN163, 30 mg/kg bw GRN163L, and 10
istration was 5 times a week (Monday to Friday). In the
second round of study on Huh7-derived tumors, mice
were assigned to two groups—control (PBS) and
GRN163L (30 mg/kg bw)—and injections were given 5
times a week (Monday to Friday). In the third round of
treatment of Hep3B-derived tumors, mice were assigned
to five groups—control (PBS), GRN163 (50 mg/kg bw),
and GRN163L (6.1 mg/kg bw)—and the injections were
given 3 times a week (Monday, Wednesday, and Friday).
was calculated using the formula v ? (lw2)/2. Mice in
which the total two-dimensional size of tumors had not
grown beyond 40 mm were humanely sacrificed via CO2
inhalation after completion of the treatment period of 4
weeks, typically 18 to 24 hours after the last dosing. Tu-
mor samples were collected and snap-frozen in liquid ni-
Cell Proliferation Assay. Cell proliferation assay via
5-bromo-2-deoxyuridine (BrdU) incorporation was per-
formed on frozen sections of tumors as described else-
where.29The BrdU-labeling index was determined by
counting the number of BrdU-positive cells under low-
power magnification (100?) fields in a blinded manner.
Total positive cells from at least 55 fields/group were
1128 DJOJOSUBROTO ET AL.HEPATOLOGY, November 2005
counted in Hep3B tumors treated with PBS and with 30
mg/kg bw GRN163L.
In Situ Cell Death Detection. Apoptosis analysis was
performed via terminal deoxynucleotidyl transferase–me-
diated dUTP nick end labeling (TUNEL) assay. Analysis
was performed on frozen sections of tumor samples ac-
cording to a protocol provided in the In Situ Cell Death
Detection Kit (Roche, Mannheim, Germany). Slides
were mounted with Vectashield mounting solution (Vec-
tor Laboratories, Gru ¨nberg, Germany) containing DAPI.
Apoptotic areas of the tumors were classified as ?10%,
10%-20%, or ?20% according to the density of
TUNEL-positive cells under low-power magnification
in tumors treated with PBS and with 30 mg/kg bw
GRN163L. All counts were performed in a blinded man-
Anaphase Bridge Index. Hep3B and Huh7 tumors
were fixed in 4% formaldehyde, embedded in paraffin,
and subjected to hematoxylin-eosin staining. The an-
aphase bridge index was calculated by dividing the num-
ber of anaphase bridges by the total number of anaphases
in 4 to 5 tumors from four groups (Hep3B, PBS control
and GRN163L-treated [30 mg/kg bw]; Huh7, PBS con-
trol and GRN163L-treated [30 mg/kg bw]).
Telomere-Specific Quantitative Fluorescence In
Situ Hybridization. Quantitative fluorescence in situ
as described elsewhere.30Telomere fluorescence intensity
(TFI) was quantified by using TFL-TELO version 1.0a
software.31We observed TFI ranging from 100 to 2,800,
and these values were binned into groups of 1-100, 101-
200, and so forth. In total, fluorescence intensities of an
tumors treated with 30 mg/kg bw GRN163L were quan-
Telomerase Extract Preparation and Telomere Re-
peat Amplification Protocol Assay. Telomerase extrac-
tion and telomere repeat amplification protocol (TRAP)
tection System (Chemicon, Hampshire, UK) according
to the manufacturer’s instructions. Briefly, a small por-
tion of the tumor was minced in ice-cold PBS, incubated
in CHAPS lysis buffer containing 200 U/mL RNAse in-
hibitor (Sigma, Munich, Germany) on ice, and centri-
supernatant containing telomerase extract was used per
reaction for the TRAP assay. The tumor cell extract pro-
vided by the manufacturer was used as a positive control,
and the heat-inactivated extract of the same batch was
to telomerase extension at 30°C for 30 minutes and then
to polymerase chain reaction amplification in the pres-
?ATP (Amersham Biosciences, Freiburg, Germany).
TRAP products were size-fractioned on 16% polyacryl-
custom image quantitation program that extracts the
chromatogram profile from each lane and sums the area
under the peaks.
Metaphase Spreads Preparation and Staining.
Metaphase spreads were prepared from cells treated with
10 ?mol/L GRN163L for 21 days (Hep3B) and 35 days
(Huh7). Cell cultivation was performed as described
above. Demecolsine (0.2 ?g/mL [Sigma]) was adminis-
tered 10 hours before collection of cells. Cells were incu-
bated in 0.075 mol/L KCl for 40 minutes at 37°C, fixed,
and washed in methanol/acetic acid (3:1) several times
before being trickled onto slides. Telomeres were visual-
ized using telomere-specific Q-FISH as described in Te-
Hybridization and chromosomes were counterstained
with DAPI. Telomere-free ends were scored in at least 30
metaphases per sample.
DNA Damage Protein Staining.
53bp1 staining was performed on cultivated Hep3B and
70 days, respectively. Cells were grown on sterile cover
slips overnight at subconfluent density. Before antibody
staining, cells were washed with PBS, fixed in 1% form-
aldehyde, and blocked in normal goat serum. Primary
rabbit anti–?-H2ax (1:250 [Bethyl Labs, Montgomery,
TX]) or anti-53bp1 (1:250 [Abcam, Cambridge, UK])
globulin G (1:1000 [Sigma]) were used to probe and vi-
sualize the ?-H2ax and 53bp1 foci, respectively. DNA
damage foci were counted in a total of 300 nuclei per
Telomere Restriction Fragment Length Analysis.
Telomere restriction fragments (TRFs) were determined
in DNA isolated from cells cultivated for 35 days
ence of 10 ?mol/L GRN163L. TRF length analysis was
performed as previously described.30
In Vitro Combination Treatment With Doxorubi-
cin. Hep3B cells were cultured in Eagle minimal essen-
tial medium containing 10% fetal bovine serum, 0.1
mmol/L nonessential amino acids, and 0.1 mmol/L so-
times a week. At each passage, 6 to 8 ? 105cells were
seeded into 75-cm2flasks. Hep3B cells in log-growth
?mol/L for 5 to 14 days in cell culture flasks. Fresh
GRN163L-containing medium was replenished at each
HEPATOLOGY, Vol. 42, No. 5, 2005DJOJOSUBROTO ET AL. 1129
passage. One day before doxorubicin treatment, Hep3B
subconfluent density (6,000 cells/well in a 96-well dish).
Doxorubicin was added to the medium at final concen-
for an additional 24 to 48 hours. Cell viability was mea-
sured via XTT assay using the Cell Proliferation Kit II
Statistical Analysis. Statistical analysis was accom-
plished using Microsoft Excel and GraphPad Prism 3.0
(GraphPad Software, Inc.). A two-tailed Student t test
with unequal variance and ANOVA were used to calcu-
late the P values of tumor volumes. Telomeric Q-FISH
median was calculated via cumulative addition, and the P
value was calculated using a ?2test. P values for other
assays were calculated using a Student t test. In all assays,
P values of less than .05 and .001 were considered statis-
tically significant and highly significant, respectively.
GRN163 and GRN163L Inhibit Tumor Growth of
Human Hepatoma After Xenotransplantation. Hepa-
toma appeared between days 14 and 30 after subcutane-
ous injection of 5 ? 106Hep3B cells in approximately
70% of the injected nu/nu mice, and between days 7 and
10 after injection of 5? 106Huh7 cells in approximately
90% of the injected nu/nu mice (data not shown). After
subcutaneous hepatoma became visible, the mice were
grouped into cohorts, and treatment with the indicated
doses of GRN163, GRN163L, or PBS control (Fig. 1)
was started (day 1). In the first and second experiments
with Hep3B- and Huh7-derived tumors, mice were
treated 5 times a week (Monday to Friday) (Fig. 1A-B).
During the third experiment with Hep3B-derived tu-
mors, mice were treated 3 times a week (Monday,
Wednesday, and Friday) and at lower concentrations to
define the minimum effective dose. In the first experi-
icant reduction in tumor growth in the treatment groups
compared with the control group at days 18 and 27 (Fig.
1A). The late time points showed a dose-dependent re-
sponse for GRN163L, with significantly stronger inhibi-
tion of tumor growth for mice treated with 30 mg/kg bw
per injection compared with mice receiving 10 mg/kg bw
per injection (Fig. 1A). Inhibition of tumor growth was
observed earlier in the GRN163L-treated groups with
Huh7-derived tumors compared with control mice (Fig.
1B). In the third experiment, we observed a delayed anti-
tumor activity at day 27 of treatment, and only
GRN163L (18.3 mg/kg bw) induced a significant reduc-
tion of tumor growth (Fig. 1C). In contrast, GRN163 (at
50 mg/kg bw and 16.6 mg/kg bw) as well as GRN163L
(at 6.1 mg/kg bw) did not show significant inhibition of
tumor growth. Thus, in this model it appears that weekly
doses of 150 mg/kg of GRN163 or 50 mg/kg of
GRN163L are sufficient for efficacy, and that a dosing
regimen of three applications per week is less effective
than five applications per week. Further work will be re-
quired to fully define the impact of dose and schedule on
in all groups at day 27 of the third experiment compared
with that of the first experiment correlated with reduced
compared with the first experiment. No obvious toxicity,
weight loss, or other signs of morbidity were observed in
any treatment group.
GRN163L Inhibits Telomerase Activity in Hepa-
toma Cells In Vitro and in Transplanted Hepatoma
inhibition by GRN163L, we first monitored telomerase
Fig. 1. Administration of GRN163 and GRN163L restrained the growth
of tumors in a dose-dependent manner. Histograms show average tumor
volume (v ? lw2/2 ? SEM, in mm3) for control and treatment groups
from the (A) first and (C) third round in 9-day intervals (n ? 5-7
mice/group) and the (B) second round in 3-day intervals (n ? 13-18
mice/group). Treatment was administered 5 times a week (Monday to
Friday) during the first and second rounds and 3 times a week (Monday,
Wednesday, and Friday) during the third round. Note that in the second
round, only GRN163L showed significant effect of treatment, which might
relate to the different dosing regimen. PBS, phosphate-buffered saline.
1130DJOJOSUBROTO ET AL. HEPATOLOGY, November 2005
activity in the Hep3B and Huh7 cell lines that were used
for our xenotransplant experiments, as well as in another
hepatoma cell line, HepG2. This analysis showed that
GRN163L inhibited telomerase activity in Hep3B,
Huh7, and HepG2 cells with an IC50of 0.36 ?mol/L,
We next analyzed telomerase activity in xenotransplanted
Hep3B- and Huh7-derived hepatoma treated with
(P ? .001) and 34% (P ? .05) reductions of telomerase
activity in GRN163L-treated Hep3B and Huh7 tumors,
respectively, as compared with those in tumors from con-
trol mice (Fig. 3A).
previously been linked to telomere length,24we analyzed
telomere length in GRN163L-treated and PBS-treated
control Hep3B tumors. Telomere length measurements
were performed using Q-FISH, which analyzes fluores-
cent signals of telomeres after hybridization with a te-
to telomere length.31,32Our analysis revealed a significant
increase in nuclei with very low total fluorescence inten-
sity (TFI ? 400) in Hep3B tumors treated with
GRN163L compared with those from control mice
treated with PBS (Fig. 3B). The wide range of TFI in
these xenotransplanted tumors, especially the occurrence
of very strong TFIs, could be due to infiltration of host
cells (epithelial cells, fibroblasts) into the transplanted
hepatoma cells. Histological analysis via hematoxylin-eo-
composition of transplanted hepatoma between the dif-
hepatoma cells (data not shown).
TRF Length Analysis. To determine whether telo-
mere length of treated Hep3B and Huh7 cells might be
maintained by an alternative lengthening of telomeres
mechanism, TRF lengths of Hep3B and Huh7 cells were
analyzed in DNA isolated from cells cultivated with and
without addition of 10 ?mol/L GRN163L for extended
periods. After administration of 10 ?mol/L GRN163L
average TRF length of both cell lines decreased, and there
acteristic of alternative lengthening of telomeres (Fig.
3C). Moreover, growth rates were suppressed (data not
shown), and there was no indication that cells escaped
from antitelomerase treatment. Together, these data sug-
Fig. 3. (A) Telomerase activity showed an approximately 42% reduc-
tion in Hep3B tumors and and a 34% reduction in Huh7 tumors treated
with 30 mg/kg bw GRN163L compared with controls (n ? 4 tumors/
group). (B) GRN163L-treated Hep3B tumors showed more cells with
critically short telomeres (10.9% vs. 5.2% in those of the control group;
P ? .05) via Q-FISH analysis. Dashed lines show the median telomere
length. Analysis was performed using telomere-specific Q-FISH. (C) TRF
length analysis using in-gel Southern hybridization shows shorter telo-
meres in cultivated Hep3B and Huh7 cells treated with 10 ?mol/L
GRN163L for 35 and 70 days, respectively. Dashed lines show the mean
Fig. 2. Significant reduction of telomerase activity by GRN163L cor-
relating with shortened telomeres. GRN163L inhibited telomerase activity
in (A) Hep3B, (B) Huh7, and (C) HepG2 cells with an IC50of 0.36
?mol/L, 1.20 ?mol/L, and 0.63 ?mol/L, respectively. IC50values were
calculated automatically by fitting the data points to a dose–response
curve using nonlinear regression with constant values for the top (100%)
and bottom (0%) (Prism).
HEPATOLOGY, Vol. 42, No. 5, 2005DJOJOSUBROTO ET AL. 1131
gest that alternative lengthening of telomeres was not en-
gaged in the maintenance of telomere length in these cell
lines in response to telomerase inhibition. Interestingly,
the mean telomere length of Huh7 cell was longer com-
pared with Hep3B cells, yet the xenograft tumors of
Huh7 cells showed an early response to telomerase inhi-
bition (Fig. 1B)—indicating that, in addition to telomere
cells in response to telomerase inhibition.
GRN163L Inhibits Tumor Cell Proliferation, In-
creases Tumor Cell Apoptosis, and Leads to Forma-
tion of Anaphase Bridges. To evaluate the correlation
between shortened telomeres and tumor cell prolifera-
tion rates, we performed BrdU incorporation assay on
tumors treated with 30 mg/kg bw GRN163L and with
PBS. Hep3B tumor sections contained 36.5% less
BrdU-positive cells after treatment with 30 mg/kg bw
GRN163L compared with the PBS-treated controls
(145.0 ? 33.7 vs. 92.0 ? 40.0 positive cells/low power
field; P ? .001) (Fig. 4A). In addition, TUNEL assays
revealed an increased incidence of apoptosis in
GRN163L-treated tumors (Fig. 4B), which is in accor-
dance with previous in vitro studies with GRN163.24
Using hematoxylin-eosin staining, we observed a
higher percentage of anaphase bridges—a hallmark of
telomere dysfunction—in the Hep3B and Huh7 tu-
mors treated with 30 mg/kg bw GRN163L compared
with PBS-treated controls (Fig. 4C).
Increase of Telomere-Free Ends and DNA Damage
Signals in Hepatoma Cells Treated With GRN163L.
The finding on anaphase bridge formation indicated
that telomerase inhibition by GRN163L resulted in
telomere dysfunction in hepatoma cells. To directly
test this hypothesis, we analyzed metaphase prepara-
tions from Hep3B and Huh7 cells cultivated in vitro
with and without the presence of 10 ?mol/L
GRN163L. The treated cells showed a significantly
higher number of chromosomes with telomere-free
ends compared with the untreated cells (Fig. 5A). The
presence of more telomere-free ends in cells treated
with GRN163L correlated with an increased frequency
of ?-H2AX and 53bp1 foci (Fig. 5B-C), two molecular
markers previously demonstrated to localize to sites of
DNA strand breaks and dysfunctional telomeres.33-36
GRN163L Increases In Vitro Chemosensitivity of
Hep3B Cells to Doxorubicin. To explore the possibil-
Fig. 4. Lower proliferation rate,
increased tendency toward apopto-
sis, and more anaphase bridge for-
mation in tumors treated with
GRN163L. (A) The left panel shows
the average number of BrdU-positive
cells in the control and treatment
groups. The right panel shows rep-
resentative photographs of the fields
100?). (B) The left panel shows the
percentage of fields with the speci-
fied percent TUNEL-positive cells in
control and treated groups. The right
panel shows representative photo-
graphs of the fields scored (original
magnification, 100?). (C) Histo-
grams in the left panel show the
percentage of anaphase-bridges ob-
served in 50 mitotic cells/sample
(n ? 4-5 tumors/group). The right
panel shows representative pho-
bridges observed in Hep3B and
Huh7 tumors. BrdU, 5-bromo-2-de-
oxyuridine; PBS, phosphate-buffered
saline; TUNEL, terminal deoxynu-
cleotidyl transferase–mediated dUTP
nick end labeling.
1132 DJOJOSUBROTO ET AL. HEPATOLOGY, November 2005
ity of enhancing the antitumor effect of GRN163L, we
tested an in vitro combination treatment of GRN163L
and the chemotoxic agent doxorubicin. In this study,
Hep3B cells were pretreated with 0.1, 1, 3, and 10
?mol/L of GRN163L for 5 to 14 days before doxoru-
bicin was added to the medium at a final concentration
of 10 to 10,000 nmol/L and incubated for 24 to 48
hours. The XTT cell viability assay showed that pre-
treatment with 1 ?mol/L GRN163L increased the che-
mosensitivity of Hep3B cells (Fig. 6) and significantly
Fig. 5. Induction of telomere dysfunction in hepatoma cells treated with GRN163L. Treatment of in vitro cultured hepatoma cells with 10 ?mol/L
GRN163L for the indicated periods led to (A) a significantly higher prevalence of telomere-free ends in metaphase spreads of Hep3B and Huh7 cells
and an increased number of DNA damage ?-H2ax and 53bp1 foci in (B) Huh7 and (C) Hep3B cells. The photomicrographs to the right of the
histograms show representative images of (A) chromosomes and (B-C) DNA damage foci.
HEPATOLOGY, Vol. 42, No. 5, 2005DJOJOSUBROTO ET AL.1133
lowered the LD50of doxorubicin in Hep3B cells (Table
1). Cells pretreated with 0.1 ?mol/L GRN163L be-
haved similarly to untreated cells, while cells pretreated
with higher concentration of GRN163L (3 and 10
?mol/L) did not replate efficiently (data not shown).
These data strongly suggest an enhanced antitumor
effect via a combination treatment of GRN163L and
Our study provides experimental evidence that telom-
erase inhibition by NPS oligonucleotides targeting the
resent a promising approach for the treatment of HCC.
We tested two of these NPS oligonucleotides (GRN163
and GRN163L) in preclinical studies. Both compounds
significantly inhibited in vivo tumor growth after xeno-
transplantation of two different human hepatoma cell
lines. Both cell lines possessed strong telomerase activity,
characteristic of over 80% of human HCC.40Therefore,
this model appears to be appropriate to study the effect of
telomerase inhibition on human hepatoma growth.
GRN163L, the lipid-conjugated derivative, inhibited tu-
mor growth more efficiently than GRN163, the nonlipi-
dated oligonucleotide, which is consistent with its
improved potency against tumor cells in culture and its
good biodistribution and uptake by tumor cells in vivo
mor growth was correlated with telomerase inhibition,
induction of telomere dysfunction, decreased tumor cell
proliferation, and an increased incidence of tumor cell
apoptosis. These data are in agreement with studies in
mTR?/?mice showing that telomere dysfunction sup-
presses tumor progression.20-22
Tumors derived from subcutaneous engraftment of
Hep3B, Huh7, and HepG2 in nude mice have been
found to show a mild but insignificant response to doxo-
data). Interestingly, our in vivo data showed significant
antitumor effects of telomerase inhibition alone. Our in
vitro analysis showed that GRN163L led to significant
inhibition of telomerase activity in Hep3B, Huh7, and
HepG2 cells and, moreover, has the potential to augment
the chemosensitivity of Hep3B to doxorubicin. These re-
sults are consistent with studies showing increased sensi-
tivity of transformed mouse embryonal fibroblasts from
mTR?/?mice compared with mTR?/?mouse embryo-
nal fibroblasts in response to doxorubicin, and this corre-
lated with telomere shortening.37The exact mechanism
of this increased chemosensitivity has yet to be deter-
mined. Possible explanations are that functional telo-
meres are required for efficient DNA repair or that agents
such as doxorubicin can accelerate the rate of telomere
loss in the absence of telomerase activity. Recent studies
have shown that mice with shortened telomeres as well as
senescent human cells have defects in DNA repair and
sibility is that telomerase itself fulfills some function in
telomere capping and DNA repair, and that under con-
ditions in which telomerase is inhibited, genomic insta-
What mechanisms limit tumor cell growth in response
to telomerase inhibition? Studies in mTR?/?mice and
other studies in primary human cells have shown that
Table 1. Pretreatment With GRN163L Increases Hep3B
Chemosensitivity to Doxorubicin
?mol/L Treatment Period
Fig. 6. Pretreatment with GRN163L increases Hep3B chemosensitivity
to doxorubicin. GRN163L (1 ?mol/L) was given in the medium (Eagle
mimimal essential medium) for a period of (A) 5, (B) 13, and (C) 14
days. One day before doxorubicin treatment, fresh medium containing
GRN163L was added to the cultures. Doxorubicin (4-8 concentrations)
was added for a period of (A,C) 24 and (B) 48 hours.
1134 DJOJOSUBROTO ET AL.HEPATOLOGY, November 2005
our studies, we show that telomerase inhibition results in
increased rates of telomere-free ends and increased num-
bers of DNA damage foci in two different hepatoma cell
lines. Studies in mTR?/?, p53?/?double mutant mice
sponses induced by telomere dysfunction.12In liver cells,
the level of telomere dysfunction appears to determine
whether p53-dependent or p53-independent responses
are induced.39The Hep3B and Huh7 cell lines used in
strong inhibition of tumor growth in response to telom-
erase inhibition. These data, together with other reports,
suggest that p53 might not be a strong modulator in telo-
mere dysfunction–induced responses affecting the viabil-
ity of the hepatoma cells. It remains to be determined
what genetic alterations might influence the sensitivity of
hepatoma cells in cancer patients during telomerase inhi-
In conclusion, telomerase inhibition is a promising
therapy of human hepatoma. It has been suggested that
telomerase inhibition may only result in tumor growth
inhibition when telomeres have reached a critically short
length.41If this were the case, the efficacy of telomerase
inhibition in the clinical setting would depend on the
initial telomere length of each individual tumor. In this
regard, it is interesting that human HCC is characterized
by very short telomeres,30,42,43making this tumor type a
good target for telomerase inhibition therapy. In addi-
tion, our study showed that even the tumor-xenograft
from HCC cell lines with relatively long telomeres
(Huh7) responded quickly to GRN163L. Thus, our ex-
pectation is that most human HCC patients should re-
spond to GRN163L, either alone or in combination with
other therapeutic agents.
Given that the normal liver is telomerase-negative (re-
viewed in Lechel et al.44), telomerase inhibition should
have little effect on chronic liver disease and cirrhosis
progression. We did not notice any significant adverse
effects of GRN163 or GRN163L in these experiments,
suggesting that these telomerase inhibitors are well toler-
ated over this treatment interval (2-4 weeks). In humans,
most somatic tissues, including normal liver, are telomer-
ase-negative.44However, certain progenitor cells in hu-
a long-term treatment with telomerase inhibitors could
limit the proliferative potential of such progenitor cells.
Given the lack of efficient therapies and the short survival
of patients with advanced HCC, a careful evaluation of
telomerase inhibitors in clinical trials appears to be a rea-
sonable approach to hopefully improve our therapeutic
option for this devastating cancer.
GRN163L has recently received clearance by the U.S.
clinical testing in chronic lymphocytic leukemia. This
trial is designed to establish safety and tolerability of
GRN163L administered on a weekly intravenous dosing
schedule and to study human pharmacokinetic and phar-
macodynamic parameters. A safe starting dose and esca-
lation schedule of GRN163L considered sufficient to
achieve telomerase inhibition in humans was based on in
vitro potency, in vivo efficacy, and pharmacokinetic,
pharmacodynamic, biodistribution, and toxicity studies
in rodents and cynomolgus monkeys (data not shown).
hematological and solid tumors.
Hadiman, and E. Wunder for technical assistance. We
thank P. Wirapati for his help in image quantitation and
We are grateful to A. Schienke, N.
1. Fecht WJ Jr, Befeler AS. Hepatocellular carcinoma: updates in primary
prevention. Curr Gastroenterol Rep 2004;6:37-43.
2. Verucchi G, Calza L, Manfredi R, Chiodo F. Human immunodeficiency
apeutic options and clinical management. Infection 2004;32:33-46.
3. Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet 2003;
J Cancer 1997;33:787-791.
enzymatic activity in human hepatocellular carcinogenesis. J Pathol 2001;
6. Tahara H, Nakanishi T, Kitamoto M, Nakashio R, Shay JW, Tahara E, et
liver disease and hepatocellular carcinomas. Cancer Res 1995;55:2734-
7. Greider CW, Blackburn EH. Identification of a specific telomere terminal
transferase activity in Tetrahymena extracts. Cell 1985;43:405-413.
human fibroblasts. Nature 1990;345:458-460.
et al. Telomere length predicts replicative capacity of human fibroblasts.
Proc Natl Acad Sci U S A 1992;89:10114-10118.
10. Vaziri H, Schachter F, Uchida I, Wei L, Zhu X, Effros R, et al. Loss of
telomeric DNA during aging of normal and trisomy 21 human lympho-
cytes. Am J Hum Genet 1993;52:661-667.
11. Wright WE, Shay JW. The two-stage mechanism controlling cellular se-
nescence and immortalization. Exp Gerontol 1992;27:383-389.
12. Chin L, Artandi SE, Shen Q, Tam A, Lee SL, Gottlieb GJ, et al. p53
deficiency rescues the adverse effects of telomere loss and cooperates with
telomere dysfunction to accelerate carcinogenesis. Cell 1999;97:527-538.
13. Reaper PM, di Fagagna F, Jackson SP. Activation of the DNA damage
response by telomere attrition: a passage to cellular senescence. Cell Cycle
14. Wright WE, Shay JW. Cellular senescence as a tumor-protection mecha-
nism: the essential role of counting. Curr Opin Genet Dev 2001;11:98-
15. Kim JH, Kim JH, Lee GE, Kim SW, Chung IK. Identification of a qui-
noxaline derivative that is a potent telomerase inhibitor leading to cellular
senescence of human cancer cells. Biochem J 2003;373:523-529.
HEPATOLOGY, Vol. 42, No. 5, 2005DJOJOSUBROTO ET AL. 1135
16. Mo Y, Gan Y, Song S, Johnston J, Xiao X, Wientjes MG, et al. Simulta-
neous targeting of telomeres and telomerase as a cancer therapeutic ap-
proach. Cancer Res 2003;63:579-585.
17. Zaffaroni N, Lualdi S, Villa R, Bellarosa D, Cermele C, Felicetti P, et al.
Inhibition of telomerase activity by a distamycin derivative: effects on cell
proliferation and induction of apoptosis in human cancer cells. Eur J
18. Herbert BS, Pongracz K, Shay JW, Gryaznov SM, Shea-Herbert B. Oli-
gonucleotide N3?3P5? phosphoramidates as efficient telomerase inhibi-
tors. Oncogene 2002;21:638-642.
19. Damm K, Hemmann U, Garin-Chesa P, Hauel N, Kauffmann I, Priepke
H, et al. A highly selective telomerase inhibitor limiting human cancer cell
proliferation. EMBO J 2001;20:6958-6968.
20. Gonzalez-Suarez E, Samper E, Flores JM, Blasco MA. Telomerase-defi-
cient mice with short telomeres are resistant to skin tumorigenesis. Nat
21. Rudolph KL, Millard M, Bosenberg MW, DePinho RA. Telomere dys-
function and evolution of intestinal carcinoma in mice and humans. Nat
INK4a(delta2/3) cancer-prone mouse. Cell 1999;97:515-525.
23. Akiyama M, Hideshima T, Shammas MA, Hayashi T, Hamasaki M, Tai
YT, et al. Effects of oligonucleotide N3?3P5? thio-phosphoramidate
(GRN163) targeting telomerase RNA in human multiple myeloma cells.
Cancer Res 2003;63:6187-6194.
24. Asai A, Oshima Y, Yamamoto Y, Uochi TA, Kusaka H, Akinaga S, et al. A
novel telomerase template antagonist (GRN163) as a potential anticancer
agent. Cancer Res 2003;63:3931-3939.
25. Gryaznov S, Asai A, Oshima Y, Yamamoto Y, Pongracz K, Pruzan R, et al.
Oligonucleotide N3?3P5? thio-phosphoramidate telomerase template
antagonists as potential anticancer agents. Nucleosides Nucleotides Nu-
cleic Acids 2003;22:577-581.
Antitumor effects of specific telomerase inhibitor GRN163 in human gli-
oblastoma xenografts. Neuro-oncol 2004;6:218-226.
27. Wang ES, Wu K, Chin AC, Chen-Kiang S, Pongracz K, Gryaznov S, et al.
Telomerase inhibition with an oligonucleotide telomerase template antag-
onist: in vitro and in vivo studies in multiple myeloma and lymphoma.
date oligonucleotide, enhances the potency of telomerase inhibition.
Oncogene 2005 May 23; [Epub ahead of print]. DOI: 10.1038/
impairtumorigenesis in the
29. Satyanarayana A, Wiemann SU, Buer J, Lauber J, Dittmar KE, Wustefeld
cycle re-entry of a subpopulation of cells. EMBO J 2003;22:4003-4013.
30. Wiemann SU, Satyanarayana A, Tsahuridu M, Tillmann HL, Zender L,
Klempnauer J, et al. Hepatocyte telomere shortening and senescence are
general markers of human liver cirrhosis. FASEB J 2002;16:935-942.
31. Poon SS, Martens UM, Ward RK, Lansdorp PM. Telomere length measure-
ments using digital fluorescence microscopy. Cytometry 1999;36:267-278.
Measurement of telomere length in haematopoietic cells using in situ hy-
bridization techniques. Biochem Soc Trans 2000;28:245-250.
33. Rogakou EP, Boon C, Redon C, Bonner WM. Megabase chromatin do-
mains involved in DNA double-strand breaks in vivo. J Cell Biol 1999;
34. Rappold I, Iwabuchi K, Date T, Chen J. Tumor suppressor p53 binding
protein 1 (53BP1) is involved in DNA damage-signaling pathways. J Cell
35. d’Adda di Fagagna F, Reaper PM, Clay-Farrace L, Fiegler H, Carr P, Von
Zglinicki T, et al. A DNA damage checkpoint response in telomere-initi-
ated senescence. Nature 2003;426:194-198.
36. Hao LY, Strong MA, Greider CW. Phosphorylation of H2AX at short
telomeres in T cells and fibroblasts. J Biol Chem 2004;279:45148-45154.
37. Lee KH, Rudolph KL, Ju YJ, Greenberg RA, Cannizzaro L, Chin L, et al.
Telomere dysfunction alters the chemotherapeutic profile of transformed
cells. Proc Natl Acad Sci U S A 2001;98:3381-3386.
38. Wong KK, Chang S, Weiler SR, Ganesan S, Chaudhuri J, Zhu C, et al.
Telomere dysfunction impairs DNA repair and enhances sensitivity to
ionizing radiation. Nat Genet 2000;26:85-88.
39. Lechel A, Satyanarayana A, Ju Z, Plentz RR, Schaetzlein S, Rudolph C, et
al. The cellular level of telomere dysfunction determines induction of
senescence or apoptosis in vivo. EMBO Rep 2005;6:275-281.
40. Knowles BB, Howe CC, Aden DP. Human hepatocellular carcinoma cell
lines secrete the major plasma proteins and hepatitis B surface antigen.
41. Saretzki G. Telomerase inhibition as cancer therapy. Cancer Lett 2003;
42. Plentz RR, Caselitz M, Bleck JS, Gebel M, Flemming P, Kubicka S, et al.
Hepatocellular telomere shortening correlates with chromosomal instability
and the development of human hepatoma. HEPATOLOGY 2004;40:80-86.
43. Satyanarayana A, Manns MP, Rudolph KL. Telomeres and telomerase: a
dual role in hepatocarcinogenesis. HEPATOLOGY 2005;40:276-283.
1136 DJOJOSUBROTO ET AL.HEPATOLOGY, November 2005