Telomerase antagonists GRN163 and GRN163L inhibit tumor growth and increase chemosensitivity of human hepatoma


  • Potential conflict of interest: Drs. Chin, Go, Gryaznov, and Harley own stock in Geron Corp.


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. (HEPATOLOGY 2005.)

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide,1 and the incidence of HCC in developed countries is still increasing as a result of a growing number of carriers of chronic hepatitis C virus infection.2 The therapeutic options for advanced-stage HCC are limited, and this cancer responds poorly to systemic treatment with chemotherapy. Other therapeutic strategies have not revealed significant 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 evaluate new therapeutic options for this tumor.

Similar to other malignant human tumor types,4 over 80% of human HCC biopsies show activation of telomerase.5 In contrast, most somatic human tissues, including normal liver,6 show 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.7 Because of the “end replication problem” of DNA polymerase, telomeres shorten during each cell division by 50 to 100 bp.8 Telomere shortening to a critical length and/or the uncapping of the telomere limit the growth of primary human cells to a finite number of cell divisions, leading to either replicative senescence or crisis.9–11 A critically short telomere within a cell is believed to activate DNA damage signaling, inducing senescence or cell death.12, 13 It has been proposed that postnatal repression of telomerase functions as a tumor repressor mechanism that limits the growth of transformed cells.14 This hypothesis is consistent with the high frequency of telomerase reactivation found in most human cancers, including HCC. Studies on the inhibition of telomerase activity in cancer cell lines15–19 and in telomerase knockout mice20–22 have shown that growth and progression of malignant tumors depend on telomerase activity and telomere stabilization.

Recently, a new class of telomerase inhibitors was developed: N3′-P5′ thio-phosphoramidate (NPS) oligonucleotides targeting the active site template region of the human telomerase RNA component.18 Preclinical studies 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, multiple myeloma, lymphoma) leading to telomere shortening, growth arrest, and apoptosis.23–27 In addition, GRN163 inhibited tumor growth of prostate cancer, multiple myeloma, lymphoma, and glioblastoma in xenotransplant models.24, 26, 27 To evaluate the potential use of NPSS oligonucleotides targeting the human telomerase RNA component for the treatment of HCC, we analyzed GRN163 and GRN163L, a lipid-conjugated derivative of GRN163 designed for increased bioavailability,28 for antitumor 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 development candidate with significant effects on tumor growth and increased chemosensitivity to doxorubicin.


HCC, hepatocellular carcinoma; NPS, N3′-P5′ thio-phosphoramidate; 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 fluorescence intensity; TRAP, telomere repeat amplification protocol; TRF, telomere restriction fragment.

Materials and Methods

Mouse Handling.

NMRI nu/nu mice were bred and housed at the animal facility of Medical School Hannover, 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-dimensional 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 purchased from American Type Culture Collection. Cells were cultured in Dulbecco's modified Eagle medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) and penicillin/streptomycin (100 IE/100 μg/mL; Biochrom, Cambridge, UK) at 37°C and 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 × 106 hepatoma cells were engrafted subcutaneously into the right and left flanks.

Test Articles.

GRN163 is a 13-mer thio-phosphoramidate oligonucleotide with the sequence 5′-TAGGGTTAGACAA. GRN163L is a lipid-conjugated derivative of GRN163 with the sequence 5′-L-TAGGGTTAGACAA, where L = aminoglycerol-palmitoyl 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 extinction coefficient of 143 OD/μmol. Formulated oligonucleotide compounds were filter-sterilized (0.2 μmol/L) and stored in 5- to 10-mL aliquots at −20°C.

Treatment Groups and Dosing Regimens.

Treatment 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 mice/group for Hep3B groups; n = 13-18 mice/group for Huh7 groups). In the first round of treatment of Hep3B-derived tumors, we injected the control group of mice with PBS and three treatment groups with 30 mg/kg body weight (bw) GRN163, 30 mg/kg bw GRN163L, and 10 mg/kg bw GRN163L (n = 5-7 mice/group); dose administration 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), GRN163 (16.7 mg/kg bw), GRN163L (18.3 mg/kg bw), and GRN163L (6.1 mg/kg bw)—and the injections were given 3 times a week (Monday, Wednesday, and Friday). Tumor length and width were measured daily; tumor size 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. Tumor samples were collected and snap-frozen in liquid nitrogen.

Cell Proliferation Assay.

Cell proliferation assay via 5-bromo-2-deoxyuridine (BrdU) incorporation was performed on frozen sections of tumors as described elsewhere.29 The 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 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–mediated dUTP nick end labeling (TUNEL) assay. Analysis was performed on frozen sections of tumor samples according to a protocol provided in the In Situ Cell Death Detection Kit (Roche, Mannheim, Germany). Slides were mounted with Vectashield mounting solution (Vector Laboratories, Grünberg, 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 (100×) fields. In total, at least 50 fields/group were scored in tumors treated with PBS and with 30 mg/kg bw GRN163L. All counts were performed in a blinded manner.

Anaphase Bridge Index.

Hep3B and Huh7 tumors were fixed in 4% formaldehyde, embedded in paraffin, and subjected to hematoxylin-eosin staining. The anaphase bridge index was calculated by dividing the number 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 control and GRN163L-treated [30 mg/kg bw]).

Telomere-Specific Quantitative Fluorescence In Situ Hybridization.

Quantitative fluorescence in situ hybridization (Q-FISH) for telomere ends was performed as described elsewhere.30 Telomere fluorescence intensity (TFI) was quantified by using TFL-TELO version 1.0a software.31 We 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 average of 50 cells/tumor from five control tumors and six tumors treated with 30 mg/kg bw GRN163L were quantified.

Telomerase Extract Preparation and Telomere Repeat Amplification Protocol Assay.

Telomerase extraction and telomere repeat amplification protocol (TRAP) assays were performed with the TRAPeze Telomerase Detection System (Chemicon, Hampshire, UK) according to the manufacturer's instructions. Briefly, a small portion of the tumor was minced in ice-cold PBS, incubated in CHAPS lysis buffer containing 200 U/mL RNAse inhibitor (Sigma, Munich, Germany) on ice, and centrifuged at 12,000g. Two hundred nanograms of supernatant containing telomerase extract was used per reaction for the TRAP assay. The tumor cell extract provided by the manufacturer was used as a positive control, and the heat-inactivated extract of the same batch was used as a negative control. TRAP mixtures were subjected to telomerase extension at 30°C for 30 minutes and then to polymerase chain reaction amplification in the presence of telomerase substrate (TS) primer labeled with 32P-γATP (Amersham Biosciences, Freiburg, Germany). TRAP products were size-fractioned on 16% polyacrylamide gels. TRAP product ladders were quantified using a 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 μmol/L GRN163L for 21 days (Hep3B) and 35 days (Huh7). Cell cultivation was performed as described above. Demecolsine (0.2 μg/mL [Sigma]) was administered 10 hours before collection of cells. Cells were incubated 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 visualized using telomere-specific Q-FISH as described in Telomere-Specific Quantitative Fluorescence In Situ Hybridization and chromosomes were counterstained with DAPI. Telomere-free ends were scored in at least 30 metaphases per sample.

DNA Damage Protein Staining.

γ-H2ax and 53bp1 staining was performed on cultivated Hep3B and Huh7 cells treated with 10 μmol/L GRN163L for 40 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% formaldehyde, and blocked in normal goat serum. Primary rabbit anti–γ-H2ax (1:250 [Bethyl Labs, Montgomery, TX]) or anti-53bp1 (1:250 [Abcam, Cambridge, UK]) and secondary Cy3-conjugated goat anti-rabbit immunoglobulin G (1:1000 [Sigma]) were used to probe and visualize the γ-H2ax and 53bp1 foci, respectively. DNA damage foci were counted in a total of 300 nuclei per sample.

Telomere Restriction Fragment Length Analysis.

Telomere restriction fragments (TRFs) were determined in DNA isolated from cells cultivated for 35 days (Hep3B) and 70 days (Huh7), with and without the presence of 10 μmol/L GRN163L. TRF length analysis was performed as previously described.30

In Vitro Combination Treatment With Doxorubicin.

Hep3B cells were cultured in Eagle minimal essential medium containing 10% fetal bovine serum, 0.1 mmol/L nonessential amino acids, and 0.1 mmol/L sodium pyruvate (all from Gibco). The cells were passaged 3 times a week. At each passage, 6 to 8 × 105 cells were seeded into 75-cm2 flasks. Hep3B cells in log-growth phase were pretreated with GRN163L at 0.1, 1, 3, and 10 μmol/L for 5 to 14 days in cell culture flasks. Fresh GRN163L-containing medium was replenished at each passage. One day before doxorubicin treatment, Hep3B cells were reseeded in GRN163L-containing medium at a subconfluent density (6,000 cells/well in a 96-well dish). Doxorubicin was added to the medium at final concentrations of 10 to 10,000 nmol/L, and cells were incubated for an additional 24 to 48 hours. Cell viability was measured via XTT assay using the Cell Proliferation Kit II (Roche).

Statistical Analysis.

Statistical analysis was accomplished 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 calculate the P values of tumor volumes. Telomeric Q-FISH median was calculated via cumulative addition, and the P value was calculated using a χ2 test. 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 statistically significant and highly significant, respectively.


GRN163 and GRN163L Inhibit Tumor Growth of Human Hepatoma After Xenotransplantation.

Hepatoma appeared between days 14 and 30 after subcutaneous injection of 5 × 106 Hep3B cells in approximately 70% of the injected nu/nu mice, and between days 7 and 10 after injection of 5 × 106 Huh7 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 tumors, mice were treated 3 times a week (Monday, Wednesday, and Friday) and at lower concentrations to define the minimum effective dose. In the first experiment, both NPS-oligonucleotides (GRN163 and GRN163L) showed antitumor activity resulting in significant 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 response for GRN163L, with significantly stronger inhibition 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 antitumor activity at day 27 of treatment, and only GRN163L (18.3 mg/kg bw) induced a significant reduction 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 required to fully define the impact of dose and schedule on tumor growth suppression. The overall smaller tumor size in all groups at day 27 of the third experiment compared with that of the first experiment correlated with reduced starting tumor sizes in the cohorts of the third experiment compared with the first experiment. No obvious toxicity, weight loss, or other signs of morbidity were observed in any treatment group.

Figure 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.

GRN163L Inhibits Telomerase Activity in Hepatoma Cells In Vitro and in Transplanted Hepatoma In Vivo.

To understand the mechanism of tumor growth inhibition by GRN163L, we first monitored telomerase 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 IC50 of 0.36 μmol/L, 1.20 μmol/L, and 0.63 μmol/L, respectively (Fig. 2A-C). We next analyzed telomerase activity in xenotransplanted Hep3B- and Huh7-derived hepatoma treated with GRN163L or PBS as a control. TRAP assay revealed 42% (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 control mice (Fig. 3A).

Figure 2.

Significant reduction of telomerase activity by GRN163L correlating with shortened telomeres. GRN163L inhibited telomerase activity in (A) Hep3B, (B) Huh7, and (C) HepG2 cells with an IC50 of 0.36 μmol/L, 1.20 μmol/L, and 0.63 μmol/L, respectively. IC50 values 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).

Figure 3.

(A) Telomerase activity showed an approximately 42% reduction 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 telomeres in cultivated Hep3B and Huh7 cells treated with 10 μmol/L GRN163L for 35 and 70 days, respectively. Dashed lines show the mean telomere length.

Because antitumor activity of telomerase inhibition has previously been linked to telomere length,24 we analyzed telomere length in GRN163L-treated and PBS-treated control Hep3B tumors. Telomere length measurements were performed using Q-FISH, which analyzes fluorescent signals of telomeres after hybridization with a telomere-specific probe giving an intensity that corresponds to telomere length.31, 32 Our analysis revealed a significant increase in nuclei with very low total fluorescence intensity (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-eosin staining did not reveal any significant difference in the composition of transplanted hepatoma between the different groups. All tumors consisted of approximately 80% hepatoma cells (data not shown).

TRF Length Analysis.

To determine whether telomere 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 μmol/L GRN163L for extended periods. After administration of 10 μmol/L GRN163L for 35 and 70 days for Hep3B and Huh7, respectively, the average TRF length of both cell lines decreased, and there was no evidence of increased heterogeneity in length characteristic 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 suggest that alternative lengthening of telomeres was not engaged 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 compared with Hep3B cells, yet the xenograft tumors of Huh7 cells showed an early response to telomerase inhibition (Fig. 1B)—indicating that, in addition to telomere length, other factors had an impact on sensitivity of tumor cells in response to telomerase inhibition.

GRN163L Inhibits Tumor Cell Proliferation, Increases Tumor Cell Apoptosis, and Leads to Formation of Anaphase Bridges.

To evaluate the correlation between shortened telomeres and tumor cell proliferation 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 accordance 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 tumors treated with 30 mg/kg bw GRN163L compared with PBS-treated controls (Fig. 4C).

Figure 4.

Lower proliferation rate, increased tendency toward apoptosis, and more anaphase bridge formation 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 representative photographs of the fields counted (original magnification, 100×). (B) The left panel shows the percentage of fields with the specified percent TUNEL-positive cells in control and treated groups. The right panel shows representative photographs of the fields scored (original magnification, 100×). (C) Histograms in the left panel show the percentage of anaphase-bridges observed in 50 mitotic cells/sample (n = 4-5 tumors/group). The right panel shows representative photomicrographs from anaphase bridges observed in Hep3B and Huh7 tumors. BrdU, 5-bromo-2-deoxyuridine; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling.

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 preparations from Hep3B and Huh7 cells cultivated in vitro with and without the presence of 10 μmol/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 γ-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

Figure 5.

Induction of telomere dysfunction in hepatoma cells treated with GRN163L. Treatment of in vitro cultured hepatoma cells with 10 μmol/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 γ-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.

GRN163L Increases In Vitro Chemosensitivity of Hep3B Cells to Doxorubicin.

To explore the possibility 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 μmol/L of GRN163L for 5 to 14 days before doxorubicin 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 pretreatment with 1 μmol/L GRN163L increased the chemosensitivity of Hep3B cells (Fig. 6) and significantly lowered the LD50 of doxorubicin in Hep3B cells (Table 1). Cells pretreated with 0.1 μmol/L GRN163L behaved similarly to untreated cells, while cells pretreated with higher concentration of GRN163L (3 and 10 μmol/L) did not replate efficiently (data not shown). These data strongly suggest an enhanced antitumor effect via a combination treatment of GRN163L and doxorubicin.

Figure 6.

Pretreatment with GRN163L increases Hep3B chemosensitivity to doxorubicin. GRN163L (1 μmol/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.

Table 1. Pretreatment With GRN163L Increases Hep3B Chemosensitivity to Doxorubicin
Treatment Period1 μmol/LTreatment PeriodLD50 (μmol/L)
5 d24 h3.5
 +24 h1.3
13 d48 h1.6
 +48 h0.5
14 d24 h4.7
 +24 h0.9


Our study provides experimental evidence that telomerase inhibition by NPS oligonucleotides targeting the human telomerase RNA component template region represent 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 xenotransplantation of two different human hepatoma cell lines. Both cell lines possessed strong telomerase activity, characteristic of over 80% of human HCC.40 Therefore, this model appears to be appropriate to study the effect of telomerase inhibition on human hepatoma growth. GRN163L, the lipid-conjugated derivative, inhibited tumor growth more efficiently than GRN163, the nonlipidated oligonucleotide, which is consistent with its improved potency against tumor cells in culture and its good biodistribution and uptake by tumor cells in vivo following systemic administration.28 The reduction in tumor 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 suppresses 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 doxorubicin treatment (T. Wirth and S. Kubicka, unpublished 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 results are consistent with studies showing increased sensitivity of transformed mouse embryonal fibroblasts from mTR−/− mice compared with mTR+/+ mouse embryonal fibroblasts in response to doxorubicin, and this correlated with telomere shortening.37 The exact mechanism of this increased chemosensitivity has yet to be determined. Possible explanations are that functional telomeres 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 show an accumulation of DNA damage.35, 38 Another possibility is that telomerase itself fulfills some function in telomere capping and DNA repair, and that under conditions in which telomerase is inhibited, genomic instability increases.

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 critical telomere shortening induces a DNA damage–type response, including the activation of p53 signaling.12, 13 In our studies, we show that telomerase inhibition results in increased rates of telomere-free ends and increased numbers of DNA damage foci in two different hepatoma cell lines. Studies in mTR−/−, p53−/− double mutant mice have also demonstrated evidence for p53-independent responses induced by telomere dysfunction.12 In liver cells, the level of telomere dysfunction appears to determine whether p53-dependent or p53-independent responses are induced.39 The Hep3B and Huh7 cell lines used in this study are p53-mutant,40 yet show relatively rapid and strong inhibition of tumor growth in response to telomerase inhibition. These data, together with other reports, suggest that p53 might not be a strong modulator in telomere dysfunction–induced responses affecting the viability of the hepatoma cells. It remains to be determined what genetic alterations might influence the sensitivity of hepatoma cells in cancer patients during telomerase inhibition therapy.

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.41 If 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, 43 making this tumor type a good target for telomerase inhibition therapy. In addition, our study showed that even the tumor-xenograft from HCC cell lines with relatively long telomeres (Huh7) responded quickly to GRN163L. Thus, our expectation is that most human HCC patients should respond to GRN163L, either alone or in combination with other therapeutic agents.

Given that the normal liver is telomerase-negative (reviewed 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 tolerated over this treatment interval (2-4 weeks). In humans, most somatic tissues, including normal liver, are telomerase-negative.44 However, certain progenitor cells in humans are telomerase-positive; thus it remains possible that 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 reasonable approach to hopefully improve our therapeutic option for this devastating cancer.

GRN163L has recently received clearance by the U.S. Food and Drug Administration to enter human phase I/II 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 pharmacodynamic parameters. A safe starting dose and escalation 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). We look forward to future clinical testing of GRN163L in hematological and solid tumors.


We are grateful to A. Schienke, N. Hadiman, and E. Wunder for technical assistance. We thank P. Wirapati for his help in image quantitation and statistical analysis.