Molecular Cancer Therapeutics Funding
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nakase, M.
Right arrow Articles by Tagawa, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakase, M.
Right arrow Articles by Tagawa, T.
Mol Cancer Ther. 2005;4:625-631
© 2005 American Association for Cancer Research

p53 gene therapy of human osteosarcoma using a transferrin-modified cationic liposome

Minoru Nakase, Madoka Inui, Kenya Okumura, Takahiko Kamei, Shinnosuke Nakamura and Toshiro Tagawa

Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Mie University, Tsu, Japan

Requests for reprints: Minoru Nakase, Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Mie University, 2-174 Edobashi, Tsu 514-8507, Japan. Phone: 81-59-232-1111, ext. 5635; Fax: 81-59-231-5207. E-mail: nakase96{at}clin.medic.mie-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gene delivery via transferrin receptors, which are highly expressed by cancer cells, can be used to enhance the effectiveness of gene therapy for cancer. In this study, we examined the efficacy of p53 gene therapy in human osteosarcoma (HOSM-1) cells derived from the oral cavity using a cationic liposome supplemented with transferrin. HOSM-1 cells were exposed to transferrin-liposome-p53 in vitro, and the growth inhibition rate, expression of p53 and bax, and induction of apoptosis were measured 48 hours later. Treatment of HOSM-1 cells with transferrin-liposome-p53 resulted in 60.7% growth inhibition. Wild-type p53 expression and an increase in bax expression were observed following transfection with transferrin-liposome-p53, and 20.5% of the treated HOSM-1 cells were apoptotic. In vivo, the HOSM-1 tumor transplanted into nude mice grew to 5 to 6 mm in diameter. Following growth of the tumor to this size, transferrin-liposome-p53 was locally applied to the peripheral tumor (day 0) and then applied once every 5 days for a total of six times. During the administration period, tumor growth did not occur, and the mean tumor volume on the last day of administration (day 25) was 10.0% of that in the saline control group. These results suggest that p53 gene therapy via cationic liposome modification with transferrin is an effective strategy for treatment of osteosarcoma.

Key Words: Osteosarcoma • Transferrin • Liposome • p53 • Gene therapy


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Osteosarcoma is mainly treated by surgical excision or chemotherapy, but new therapies for the tumor are being developed because the prognosis of unresectable or recurrent cases is quite poor. Gene therapy is one of the most promising strategies among these therapies, but few clinical trials of gene therapy for osteosarcoma have been done to date (1). It has been assumed that mutation of tumor suppressor genes participates in the development and progression of malignant tumors, including osteosarcoma (2), and it is anticipated that introduction of normal tumor suppressor genes into osteosarcoma cells may result in tumor growth inhibition.

The p53 gene is a tumor suppressor gene that has a regulatory function associated with DNA damage in cells. Hence, the status of the gene has an effect on the growth of cancer cells, apoptosis, and sensitivity to anticancer agents (3, 4). Mutation of the p53 gene has been frequently observed in malignant tumors (5) and is found in 24% to 42% of osteosarcomas (6, 7). It has been reported that tumors with p53 mutations show resistance to chemotherapy, and the prognosis of such tumors is poor (8). Consequently, gene therapy studies aimed at introduction of a wild-type p53 gene into cancer cells have been conducted for lung cancer (9), breast cancer (10), esophageal carcinoma (11), prostate cancer (12), and colorectal cancer (13).

Both viral and nonviral vectors have been used as carriers in gene therapy approaches. Viral vectors show high efficacy of gene transfer, but it has also been pointed out that they may be biohazards and have immunogenicity concerns (14, 15). In contrast, nonviral vectors, such as cationic liposomes, can be safely administered to humans, because they have lower toxicity and immunogenicity than viral vectors (16). Nonviral vectors do, however, have the disadvantage of low efficacy of gene transfer; therefore, improvement of gene transfer using cationic liposomes is important if such nonviral vectors are to be used clinically.

The level of expression of transferrin receptors on malignant tumor cells is higher than that on normal cells (17). Cheng first reported a method to improve the efficacy of gene transfer by binding of transferrin (the ligand for the transferrin receptor) to a cationic liposome (18). Subsequently, transferrin-modified cationic liposome systemic gene therapy has been attempted in vitro and in vivo for different tumor cells and is effective (19–21). However, to date, there are no reports of its use in osteosarcoma.

In this study, we did in vitro and in vivo introduction of a p53 gene into a transferrin receptor–expressing osteosarcoma cell line using a transferrin-modified cationic liposome and studied the subsequent growth inhibition and induction of apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and Culture Conditions
The human osteosarcoma cell line HOSM-1, which is derived from the mandible, was established in our laboratory (22). HF is a normal fibroblast cell derived from human gingiva. The cells were maintained in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen) at 37°C under 5% CO2 in a humidified atmosphere. Both cell lines were tested for Mycoplasma and confirmed to be negative.

Animals and Tumor Model
Four- to 5-week-old, 19 to 22 g female BALB/cAJcl nude mice (Nihon Clea, Osaka, Japan) were used and housed at the Institute of Laboratory Animals, Faculty of Medicine, Mie University (Tsu, Japan). HOSM-1 cells (1 x 106) were inoculated s.c. in the back of nude mice. After the tumor size reached between 5 and 6 mm in diameter, treatment (see below) of the animals was initiated (day 0).

Plasmids
The plasmid pSVß contains the lacZ gene under control of the SV40 promoter (Promega, Madison, WI). The p53 expression plasmid pcGSp53 containing wild-type human p53 cDNA under control of a cytomegalovirus promoter was purchased from Invitrogen. The plasmid pcDNA3.1/Gs was used as an empty plasmid without the p53 gene (Invitrogen).

Identification of Mutations in the HOSM-1 Cells
Genomic DNA was extracted from HOSM-1 cells, and exons 4 to 9 of the p53 gene were amplified as described previously (23). Exons 8 and 9 of the p53 gene were amplified using the following primers: forward 5'-TTCTGGTAAGGACAAGGGT-3' and reverse 5'-AGGCATTGAAGTCTCATGGA-3'.

Preparation of the Transferrin-Liposome-DNA Complex
Dioleoyl trimethylammonium propane (Avanti Polar Lipid, Alabaster, AL) and dioleoyl phosphatidylethanolamine (Avanti Polar Lipid) were mixed at a 1:1 molar ratio. A lipid film was prepared with a rotary evaporator, and after addition of distilled water, the liposomes were vortexed vigorously for 2 minutes. Transferrin (ion-saturated holotransferrin; Sigma, St. Louis, MO) was dissolved in HBSS (Invitrogen). Transferrin (25 µg) in 100 µL HBSS and 100 µL liposomes (100 µmol/L) were mixed in a polypropylene tube and incubated for 15 minutes at room temperature. Then, Opti-MEM (100 µL, Invitrogen) containing 2 µg plasmid DNA was added and gently mixed. After 15-minute incubation, RPMI (700 µL) was added to the tube. The final DNA/liposome/transferrin ratio was 1:10:12.5 (µg/nmol/µg).

In vitro Transfection with the Transferrin-Liposome-pSVß Complex
Cells were plated on 24-well plates (1 x 105 per well) 24 hours before transfection. After the cells were washed with PBS, transfection medium (1 mL) containing the transferrin-liposome-pSVß complex was added to each well for a 5-hour incubation period. The transfection medium was then replaced with fresh culture medium containing 10% fetal bovine serum. After culturing for 48 hours, ß-galactosidase expression was measured by 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside staining and ß-galactosidase activity. Transfected cells were washed with PBS and fixed with 2% formaldehyde plus 0.2% glutaraldehyde for 5 minutes at room temperature. The fixed cells were washed twice with PBS and exposed to 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside solution (1 mg/mL 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside in PBS containing 5 mmol/L potassium ferricyanide, 5 mmol/L potassium ferrocyanide, and 2 mmol/L MgCl2, Cell and Molecular Technologies, Phillipsburg, NJ) at 37°C overnight. After washing, cells stained blue were counted in five random fields under a microscope to determine the percentage of blue cells per 200 cells. ß-Galactosidase activity was measured with a ß-Galactosidase Enzyme Assay System (Promega) according to the manufacturer's protocol.

Cytotoxic Assay
Cells were seeded on 96-well plates at a density of 1 x 104 cells per well. After culturing for 24 hours, the cells were treated with seven different agents (100 µL) as follows: (a) RPMI only (as the control), (b) transferrin-liposome, (c) pcGSp53 alone, (d) liposome-pcDNA3.1/Gs complex, (e) transferrin-liposome-pcDNA3.1/Gs complex, (f) liposome-pcGSp53 complex, or (g) transferrin-liposome-pcGSp53 complex. After culturing for 5 hours, the cells were washed with PBS and the medium was replaced by RPMI supplemented with 10% fetal bovine serum. After culturing for another 48 hours, the number of viable cells was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results for each agent were compared with those for the control.

Treatment of Tumor Xenografts in Nude Mice
The tumor diameter reached 5 to 6 mm in all mice by 7 to 9 days after HOSM-1 cell transfer. The animals were divided into seven groups with five mice per group, and each group was treated with one of the following: (a) saline, (b) transferrin-liposome, (c) pcGSp53 alone, (d) liposome-pcDNA3.1/Gs complex, (e) transferrin-liposome-pcDNA3.1/Gs complex, (f) liposome-pcGSp53 complex, and (g) transferrin-liposome-pcGSp53 complex. The transferrin-liposome-pcGSp53 complex contained 125 µg transferrin, 100 nmol liposome, and 10 µg pcGSp53. Each agent (100 µL) was injected along the tumor margin using a 27-gauge needle. Different sites were used for each injection such that the injection points formed a circle that surrounded the tumor. The initial day of administration was defined as day 0. Administration was then repeated five times at 5-day intervals such that day 25 was the final day of administration. The tumor volume was measured during the administration period and for 25 further days after day 25.

Western Blotting
Cells were washed twice with cold PBS and lysed at 4°C for 30 minutes in buffer (100 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 20 µg/mL aprotinin, 20 µg/mL leupeptin). Insoluble material was removed by centrifugation at 4°C for 30 minutes at 16,000 x g, and protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL). The protein contents of the cell lysates were adjusted to 4 mg/mL. The cell lysates were mixed by boiling in SDS sample buffer with reducing agents and applied to 10% to 15% SDS-polyacrylamide gradient gels. The proteins were then electrophoretically transferred to polyvinylidene difluoride membranes (Immobilon-PSQ, Millipore, Bedford, MA) using the PhastSystem (Amersham Biosciences, Piscataway, NJ). Blotted membranes were blocked with 10% dried milk in TBS plus 0.1% Tween 20 for 1 hour at room temperature and incubated for 1 hour with antibodies specific for p53, bax (mouse monoclonal anti-human antibodies, Oncogene Research Products, Boston, MA), or transferrin receptor (mouse monoclonal anti-human antibodies, Zymed Laboratories, Inc., South San Francisco, CA) diluted 1:1,000 in TBS plus 0.1% Tween 20. After incubation, the membranes were extensively washed in TBS plus 0.1% Tween 20 and incubated with anti-mouse IgG horseradish peroxidase–conjugated secondary antibody diluted 1:10,000 in TBS plus 0.1% Tween 20. Band detection was carried out using an enhanced chemiluminescence system (Amersham Biosciences).

Terminal Deoxynucleotidyl Transferase–Mediated dUTP Nick End Labeling Assay
Apoptosis-related DNA fragmentation was detected by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) using an Apop-Tag Plus Peroxidase In situ Apoptosis Detection Kit (Intergen, Oxford, United Kingdom). Cells were seeded in Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL) at a density of 1 x 104 cells per well. After culturing for 24 hours, the cells were treated with one of the above agents (100 µL) for 5 hours. Another 48 hours later, the cells were fixed by treatment with 1% paraformaldehyde for 10 minutes. The fixed cells were washed twice with PBS and preserved in precooled ethanol plus acetic acid (2:1, v/v) for 5 minutes. After washing twice with PBS, an equilibration buffer was added to the cells. Subsequently, the cells were incubated at 37°C for 1 hour with working-strength terminal deoxynucleotidyl transferase enzyme that contained digoxigenin-labeled dUTP. The reaction was stopped by addition of prewarmed stop/wash buffer. After washing twice with PBS, an anti-digoxigenin antibody fragment carrying a conjugated peroxidase was added and the mixture was incubated in a humidified chamber for 30 minutes at room temperature. The peroxidase enzyme location in the cells was detected with 3,3'-diaminobenzidine, which is a substrate for the enzyme.

Statistical Analysis
The entire experiment was done in triplicate and the data were analyzed using Student's t test. Ps < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mutation of the p53 Gene in HOSM-1 Cells
The presence of a p53 mutation in HOSM-1 cells was determined by sequencing of exons 4 to 9. This procedure confirmed that a mutation was present in one allele in codon 306, which was changed from CGA to TGA, thereby creating a stop codon in exon 8.

Expression of Transferrin Receptor
Transferrin receptor expression in HOSM-1 cells was compared with that in normal fibroblast HF cells using Western blotting. As shown in Fig. 1, bands of ~95 kDa, corresponding to the molecular weight of transferrin receptor, were identified for both HOSM-1 and HF cells, but transferrin receptor expression in HOSM-1 cells was higher than in HF cells.



View larger version (63K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Expression of transferrin receptor (Tf-R) in HF and HOSM-1 cells. Lane 1, HF; lane 2, HOSM-1. The detected band has a molecular weight of 95 kDa, which corresponds to the size of transferrin receptor. Experiments were repeated in triplicate and had similar results. ß-actin was used as a loading control.

 
Transfection Efficiency with the Transferrin-Liposome-pSVß Complex
Transfection efficiency using the transferrin-liposome or the unmodified liposome was evaluated by measurement of the ß-galactosidase activity and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside staining. The ß-galactosidase activity in HOSM-1 cells transfected with the liposome-pSVß complex was 35.6 ± 9.0 mU/mg protein, whereas that following transfection with the transferrin-liposome-pSVß complex was 151.0 ± 12.1 mU/mg protein. ß-Galactosidase activities of cells transfected with medium alone, transferrin-liposome alone, and pSVß alone were all below the detection limit. In the 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside staining assay, 17.0 ± 2.2% of cells treated with liposome-pSVß and 31.0 ± 4.0% of cells treated with transferrin-liposome-pSVß were stained blue.

Cytotoxic Effects
The numbers of viable HOSM-1 cells relative to the control in liposome-p53-treated cells and transferrin-liposome-p53-treated cells were significantly lower than those found after other treatments (P < 0.05). Growth inhibition rates of 43.6% and 60.7% were observed for cells treated with liposome-p53 and transferrin-liposome-p53, respectively, and the growth of transferrin-liposome-p53-treated cells was significantly inhibited compared with that of liposome-p53-treated cells (P < 0.05; Fig. 2). In HF cells, each treatment had little effect on cell survival.



View larger version (24K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Viability of HOSM-1 cells treated with transferrin-liposome ({04-0196fx01l}), p53 plasmid alone ({04-0196fx02l}), liposome-empty plasmid ({04-0196fx03l}), transferrin-liposome-empty plasmid ({04-0196fx04l}), liposome-p53 ({04-0196fx05l}), or transferrin-liposome-p53 ({04-0196fx06l}). The number of viable cells, relative to the control, was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. *, P < 0.05, growth of liposome-p53–treated or transferrin-liposome-p53–treated cells was significantly inhibited compared with the growth of cells that underwent other treatments. §, P < 0.05, growth inhibition of transferrin-liposome-p53–treated cells was significantly different to that of liposome-p53–treated cells. Columns, mean; bars, SD.

 
Expression of p53 and bax
As shown in Fig. 3, expression of exogenous wild-type p53 was detected in liposome-p53-treated cells (lane 4) and transferrin-liposome-p53-treated cells (lane 5) but not in cells that underwent other treatments. Furthermore, the expression of p53 following transferrin-liposome-p53 treatment was slightly higher than that following liposome-p53 treatment. On the other hand, decreased expression of endogenous mutated p53 was observed in transferrin-liposome-p53-treated cells compared with that in the control group. We also evaluated the expression of bax, which is a target gene in the p53-dependent apoptosis pathway. Bax expression increased distinctly in cells treated with transferrin-liposome-p53, but little change was observed in the liposome-p53-treated cells.



View larger version (53K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Western blots of p53 and bax in HOSM-1 cells. Expression of p53 and bax in each treatment group 48 h after completion of transfection. Lane 1, control; lane 2, p53 plasmid alone; lane 3, transferrin-liposome-empty plasmid; lane 4, liposome-p53; lane 5, transferrin-liposome-p53. Arrow a, exogenous wild-type p53; arrow b, endogenous p53. Experiments were repeated in triplicate and gave similar results. ß-actin was used as a loading control.

 
Growth Inhibitory Effect of Transferrin-Liposome-p53 on Tumors Transplanted into Nude Mice
On day 0, each group of mice had an average tumor volume in the range 60 to 70 mm3. During the administration period, the tumors in the saline group continued to grow and had increased by 5.9 times to an average of 418.7 mm3 on day 25. Tumor growth was also seen in the liposome-p53 group during the administration period, with an increase in tumor volume of 1.6 times to an average of 118.0 mm3 on day 25; this, nonetheless, reflected relative growth inhibition compared with the saline group. In contrast, during administration of transferrin-liposome-p53, the tumor gradually reduced in size, with the tumor volume significantly decreased by 36.6% from an average of 66.1 mm3 on day 0 to 41.9 mm3 on day 25. Continued observation from completion of administration (day 25) until day 50 revealed an even steeper tumor growth curve in the saline group and a growth rate in the liposome-p53 group comparable with that in the saline group, with the tumor volume on day 50 reaching 2,218.9 and 926.1 mm3 in the saline and liposome-p53 groups, respectively. In contrast, the tumors in the transferrin-liposome-p53 group only began to grow after day 35 and at a considerably slower rate compared with the other groups, with the volume on day 50 being no greater than 186.4 mm3, 8.4% of that in the saline group (Fig. 4).



View larger version (24K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Tumor volume analysis of tumor-bearing mice after intratumoral injection of various agents. Each group included five mice. Administration was started on day 0 and repeated on days 5, 10, 15, 20, and 25, for a total of six times ({downarrow}). Tumor volume was measured once every 5 d from the day of the last administration (day 25) until day 50. Tumor volume in transferrin-liposome-p53–treated animals on day 25 was significantly decreased compared with that on day 0 (P < 0.05), but the tumor volumes in animals receiving liposome-p53 treatment (P < 0.05) and in other treated groups (P < 0.01) were significantly increased compared with day 0. On day 50, the tumor volume in transferrin-liposome-p53–treated animals was significantly suppressed compared with other treated groups (P < 0.05). Points, mean; bars, SD.

 
TUNEL Assay
In vitro, TUNEL-positive cells that stained brown were found in each treatment group. The percentage of TUNEL-positive cells in a field, which is called the apoptotic index, was 3.3 ± 0.45% in the control but was markedly increased to 12.9 ± 1.6% in liposome-p53-treated cells and 20.5 ± 1.7% in transferrin-liposome-p53-treated cells (Fig. 5). In vivo, the tumor was extirpated 48 hours after completion of the final administration (day 27) and observed by the TUNEL assay. TUNEL-positive cells were sparse in the physiologic saline group but were particularly prevalent in the periphery of the tumor in the liposome-p53 or transferrin-liposome-p53 groups in proximity to the local injection site. For more central regions of the tumor, no differences in TUNEL-positive cells were noted between these groups and the saline group (Fig. 6).



View larger version (57K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Apoptotic cells were detected by the TUNEL assay (3,3'-diaminobenzidine, brown) in HOSM-1 cells: (A) control, (B) liposome-p53, and (C) transferrin-liposome-p53. TUNEL-positive cells were counted in five random fields under a microscope to determine the percentage of apoptotic cells per 200 cells. Percentages of apoptotic cells (apoptotic index) were 3.3 ± 0.45%, 12.9 ± 1.6%, and 20.5 ± 1.7% in the control, liposome-p53, and transferrin-liposome-p53 groups, respectively. Apoptotic index of transferrin-liposome-p53 group was significantly different from that of control (P < 0.01) and liposome-p53 (P < 0.05) groups. Original magnification, x200.

 


View larger version (57K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. TUNEL assay of tissue sections from tumors in the saline group (A), liposome-p53 group (B), and transferrin-liposome-p53 group (C). Tumors were extirpated 48 h after completion of administration (day 27). In saline-treated tumors, very few apoptotic cells were found. In transferrin-liposome-p53–treated tumors, many apoptotic cells were detected at the periphery of the tumor. Original magnification, x100.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The purpose of this study was to confirm whether transferrin-liposome-mediated p53 gene therapy is effective against growth inhibition of osteosarcoma with p53 mutations. Due to transferrin binding to the transferrin receptor, the efficacy of gene transfer with a transferrin-modified liposome increased by 3.7 times and the growth inhibition effect increased by 1.4 times compared with use of the unmodified liposome. The expression of exogenous wild-type p53 was higher following transfection with transferrin-liposome-p53 compared with transfection with unmodified liposome-p53. Therefore, the increase in the efficacy of p53 gene transfer and the subsequent increase in p53 expression seemed to have an effect on enhancement of HOSM-1 growth inhibition. The results of TUNEL staining suggested that this growth inhibition effect is produced by induction of apoptosis through p53. Cells are directed to cell cycle termination or apoptosis by increased p53 expression, and the transcriptional activity of the bax gene is directly associated with the p53-dependent apoptosis pathway (24). Bax activation is maintained in the presence of wild-type p53 activity, but, as shown in HOSM-1 cells carrying a mutation in codon 306 of p53, apoptosis via bax is markedly reduced when p53 function is diminished (25). The introduction of exogenous wild-type p53 enhanced bax expression in HOSM-1 cells, and induction of apoptosis by transferrin-liposome-p53 treatment occurred through a bax-dependent pathway. Slight bax expression was also seen in HOSM-1 cells in the control group, suggesting that the cells have a p53-independent pathway through which bax can be regulated (26). It has also been reported that other genes, such as PIG3 (27), Noxa (28), and p53 AIP1 (29), can act as target genes in p53-induced apoptosis. However, previous reports (30, 31) have confirmed an increase in bax expression after p53 gene therapy, and this is consistent with our results. Therefore, we conclude that bax plays an important role in the antitumor effect elicited by p53 gene therapy.

It has been noted previously that osteosarcomas with p53 mutations show resistance to chemotherapy and that their prognosis is poor (32, 33). Hence, studies of p53 gene therapy for osteosarcoma have been implemented (34). Many such studies have used viral vectors, and growth inhibition in vitro has been reported to be ~60% (34). In the current work, we obtained a growth inhibition rate for HOSM-1 cells of 60.7% following transfection with transferrin-liposome-p53. Nonspecific growth inhibition by viral infection may be a consequence of gene therapy using viral vectors, but the nonspecific growth inhibition of HOSM-1 cells caused by transfection with transferrin-liposome was only ~10% (Fig. 2). Furthermore, transferrin-liposome alone showed no cytotoxicity against normal gingiva-derived fibroblasts.

The in vivo antitumor effect of transferrin-liposome-p53 on the HOSM-1 tumor was considerably higher than expected based on the in vitro results. Treatment with liposome-p53 inhibited tumor growth during the administration period but allowed tumor growth at a rate similar to that in the saline group after completion of administration, whereas growth after completion of administration of transferrin-liposome-p53 was relatively low. In the transferrin-liposome-p53 group, the tumor volumes on days 25 and 50 were 10.0% and 8.4% of those in the saline group, respectively, showing a growth inhibitory effect higher than the ~60% growth inhibition observed in vitro. This difference is attributed to single-dose administration in vitro compared with administration once every 5 days (a total of six doses) in vivo. In contrast, in vivo TUNEL images showed apoptotic cells only in limited regions of the tumor, such as those proximal to the local injection site, suggesting that the frequency of apoptosis induction is not necessarily correlated with growth inhibition. It has been reported that p53 gene therapy inhibits tumor vascularization, thereby exerting a far-reaching effect even on nontransfected cells—the so-called bystander effect (16, 35). Hence, these findings suggest that the in vivo antitumor effect of transferrin-liposome-p53 may involve not only apoptosis induction but also bystander effects, including inhibition of tumor vascularization. Further studies will be required to investigate this possibility.

In addition to modification of liposomes through addition of transferrin, the efficacy of gene transfer with liposomes has been enhanced using ligands, such as epidermal growth factor, insulin, and lectin (21, 36). Efficacy of gene transfer is reportedly increased by 2 to 22 times by liposome modification with transferrin, 10 to 23 times with epidermal growth factor, 3 to 18 times with insulin, and 5 to 28 times with lectin compared with liposome alone. These enhancement effects vary in different target cells, and one reason may be that expression of the receptor for each ligand varies depending on the cell type (21). In the current study, enhancement of the efficacy of gene transfer occurred at 4.2 times the level of that with liposome alone, perhaps because transferrin receptor expression in HOSM-1 cells is lower than in tumor cells used in other studies, although it is higher than in normal cells.

The greatest benefit of the transferrin-liposome system is that it can be formulated by simply mixing each component with the plasmid DNA. First, the slightly negatively charged transferrin and the cationic liposome are electrostatically associated, and no chemical modification is necessary. The formulation can then be made by introduction of the negatively charged plasmid DNA, which will bind to the cationic liposome. In addition, it seems that the transferrin not only increases the uptake of DNA into cells but also plays an indirect role in enhancement of gene expression after uptake of the transferrin-liposome-DNA complex. The transferrin receptor is bound to the transferrin-liposome-DNA complex within cells, and transferrin may facilitate the escape of DNA from the endosome, because endosome escape is a normal physiologic process for transferrin and its receptor complex, thus making more DNA available for gene expression (18).

Binding of negatively charged transferrin to a cationic liposome reduces the overall charge of the assembly compared with the cationic liposome alone, and most of the complexes formed between this assembly and plasmid DNA are of neutral charge (37). Therefore, it is possible that a transferrin-modified cationic liposome might have a smaller amount of encapsulated plasmid DNA compared with a cationic liposome alone and that the transferrin-liposome-DNA complex might be less adhesive to the surface of cells. However, Xu et al. have reported that the amount of plasmid DNA bound to a transferrin-liposome is similar to that bound to an unmodified liposome. Regarding cell adhesiveness, it is thought that the transferrin-liposome-DNA complex is compressed to less than a third of an unmodified liposome-DNA complex, making it more susceptible to endocytosis and overcoming the effects of the reduced charge (37).

The sensitivity of carcinomas to anticancer agents (34) and radiosensitivity (19, 31) is increased by p53 gene therapy, so the effect of the therapy described in this study is likely to be further increased by combination with anticancer agents or radiotherapy. Regarding the transferrin-liposome-p53 administration schedule, administration once every 5 days inhibited tumor growth during the administration period and until 10 days after completion of administration, suggesting that long-term maintenance administration about once weekly may stop tumor growth; that is, it may induce "tumor dormancy." Hence, a future challenge will be to investigate the antitumor effects of long-term administration of transferrin-liposome-p53 as well as to examine survival and safety in tumor-bearing mice.


   Acknowledgments
 
We thank Dr. Junji Nishioka (Department of Laboratory Medicine) for instructions regarding the PCR technique.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/ 4/04; revised 1/ 5/05; accepted 2/ 9/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Benjamin R, Helman L, Meyers P, Reaman G. A phase I/II dose escalation and activity study of intravenous injection of OcaP1 for subjects with refractory osteosarcoma metastatic to lung. Hum Gene Ther 2001;12:1591–3.[Medline]
  2. Roth JA, Cristino RJ. Gene therapy for cancer. J Natl Cancer Inst 1997;89:21–38.[Abstract/Free Full Text]
  3. Vogelstein B, Kinzler KW. p53 function and dysfunction. Cell 1992;70:523–6.[CrossRef][Medline]
  4. Lowe SW, Bodis S, McClatchey A, et al. p53 status and efficacy of cancer therapy in vivo. Science 1994;266:807–10.[Abstract/Free Full Text]
  5. Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science 1991;253:49–53.[Abstract/Free Full Text]
  6. Toguchida J, Yamaguchi T, Ritchie B, et al. Mutation spectrum of the p53 gene in bone and soft tissue sarcomas. Cancer Res 1992;52:6194–9.[Abstract/Free Full Text]
  7. Wadayama B, Toguchida J, Yamaguchi T, Sasaki MS, Kotoura Y, Yamamura T. p53 expression and its relationship to DNA alterations in bone and soft tissue sarcomas. Br J Cancer 1993;68:1134–9.[Medline]
  8. Ruley HE. p53 and response to chemotherapy and radiotherapy. In: Devita VT, Hellman S, Rosenberg SA, editors. Important advanced in oncology. Philadelphia: Lippincott-Raven Publishers; 1996. p. 37–56.
  9. Nemunaitis J, Swisher SG, Timmons T, et al. Adenovirus-mediated p53 gene transfer in sequence with cisplatin to tumors of patients with non-small-cell lung cancer. J Clin Oncol 2000;18:609–22.[Abstract/Free Full Text]
  10. Lesoon-wood LA, Kim WH, Kleinman HK, Weintraub BD, Mixson AJ. Systemic gene therapy with p53 reduces growth and metastases of a malignant human breast cancer in nude mice. Hum Gene Ther 1995;6:395–405.[Medline]
  11. Shimada H, Matsubara H, Ochiai T. p53 gene therapy for esophageal cancer. J Gastroenterol 2002;37:87–91.[CrossRef][Medline]
  12. Eastham JA, Grafton W, Martin CM, Williams BJ. Suppression of primary tumor growth and the progression to metastasis with p53 adenovirus in human prostate cancer. J Urol 2000;164:814–9.[CrossRef][Medline]
  13. Bouvet M, Ellis LM, Nishizaki M, et al. Adenovirus-mediated wild-type p53 gene transfer down-regulates vascular endothelial growth factor expression and inhibits angiogenesis in human colon cancer. Cancer Res 1998;58:2288–92.[Abstract/Free Full Text]
  14. Check E. A tragic setback. Nature 2002;420:116–8.[CrossRef][Medline]
  15. Trapnel BC, Gorziglia M. Gene therapy using adenoviral vectors. Curr Opin Biotechnol 1994;5:617–25.[CrossRef][Medline]
  16. Xu M, Kumar D, Srinivas S, et al. Parenteral gene therapy with p53 inhibits human breast tumor in vivo through a bystander mechanism without evidence of toxicity. Hum Gene Ther 1997;8:177–85.[Medline]
  17. Elliott RL, Elliott MC, Wang F, Head JF. Breast carcinoma and the role of iron metabolism. Ann N Y Acad Sci 1993;698:159–66.[Medline]
  18. Cheng PW. Receptor ligand-facilitated gene transfer: enhancement of liposome-mediated gene transfer and expression by transferrin. Hum Gene Ther 1996;7:275–82.[Medline]
  19. Xu L, Pirollo KF, Chang EH. Transferrin-liposome-mediated p53 sensitization of squamous cell carcinoma of the head and neck to radiation in vitro. Hum Gene Ther 1997;8:467–75.[Medline]
  20. Seki M, Iwakawa J, Cheng H, Cheng PW. p53 and PTEN/MMAC/TEP 1 gene therapy of human prostate PC-3 carcinoma xenograft, using transferrin-facilitated lipofection gene delivery strategy. Hum Gene Ther 2002;13:761–73.[CrossRef][Medline]
  21. Yanagihara K, Cheng H, Cheng PW. Effects of epidermal growth factor, transferrin, and insulin on lipofection efficiency in human lung carcinoma cells. Cancer Gene Ther 2000;7:59–65.[CrossRef][Medline]
  22. Tagawa T, Nomura J, Kihira H, Murata M. A newly established osteosarcoma cell line from human mandible. Mie Med J 1989;39:279–86.
  23. Narita M, Nomura J, Nakase M, et al. Characterization of the human mandibular osteoblastic osteosarcoma cell line HOSM-2 after long-term culture. Oral Oncol 2004;40:742–50.[CrossRef][Medline]
  24. Miyashita T, Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 1995;80:293–9.[CrossRef][Medline]
  25. Kong XT, Gao H, Stanbridge. Mechanisms of differential activation of target gene promoters by p53 hinge domain mutants with impaired apoptotic function. J Biol Chem 2001;276:32990–3000.[Abstract/Free Full Text]
  26. Im E, Choi YH, Paik KJ, et al. Novel bile acid derivatives induces apoptosis via a p53-independent pathway in human breast carcinoma cells. Cancer Lett 2001;163:83–93.[CrossRef][Medline]
  27. Polyak K, Xia Y, Zweier JL. A model for p53-induced apoptosis. Nature 1997;389:300–5.[CrossRef][Medline]
  28. Oda E, Ohki R, Murasawa H. Noxa, a BH 3 only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 2000;288:1053–8.[Abstract/Free Full Text]
  29. Oda K, Arakawa H, Tanaka T. p53 AIP 1, a potential mediator of p53-induced apoptosis, and its regulation by Ser-46-phosphorylated p53. Cell 2000;102:849–62.[CrossRef][Medline]
  30. Shirakawa T, Sasaki R, Gardner TA, et al. Drug-resistant human bladder-cancer cells are more sensitive to adenovirus-mediated wild-type p53 gene therapy compared to drug-sensitive cells. Int J Cancer 2001;94:282–9.[CrossRef][Medline]
  31. Sasaki R, Shirakawa T, Zhang ZJ, et al. Additional gene therapy with AdCMV-p53 enhanced the efficacy of radiotherapy in human prostate cancer cells. Int J Radiat Oncol Biol Phys 2001;51:1336–45.[CrossRef][Medline]
  32. Pompetti F, Stuppia L, Gatta V, et al. Correlation between apoptosis and TP53 status in osteosarcoma. Cancer Genet Cytogenet 1998;105:177–81.[CrossRef][Medline]
  33. Park YB, Kim HS, Oh JH, Lee SH. The co-expression of p53 protein and P-glycoprotein is correlated to a poor prognosis in osteosarcoma. Int Orthop 2001;24:307–10.[CrossRef][Medline]
  34. Son SU, Boyce FM. Combination treatment for osteosarcoma with baculoviral vector mediated gene therapy (p53) and chemotherapy (Adriamycin). Exp Mol Med 2001;33:46–53.[Medline]
  35. Gautam A, Densmore CL, Melton S, Golunski E, Waldrep JC. Aerosol delivery of PEI-p53 complexes inhibits B16-F10 lung metastases through regulation of angiogenesis. Cancer Gene Ther 2002;9:28–36.[CrossRef][Medline]
  36. Yanagihara K, Cheng PW. Lectin enhancement of the lipofection efficiency in human lung carcinoma cells. Biochim Biophys Acta 1999;1472:25–33.[Medline]
  37. Xu L, Frederik P, Pirollo KF, et al. Self-assembly of a virus-mimicking nanostructure system for efficient tumor-targeted gene delivery. Hum Gene Ther 2002;13:469–81.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J Biomater ApplHome page
D. Li, H. Yu, H. Huang, F. Shen, X. Wu, J. Li, J. Wang, X. Cao, Q. Wang, and G. Tang
FGF Receptor-mediated Gene Delivery using Ligands Coupled to Polyethylenimine
J Biomater Appl, September 1, 2007; 22(2): 163 - 180.
[Abstract] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
T. Fukazawa, Y. Maeda, M. L. Durbin, T. Nakai, J. Matsuoka, H. Tanaka, Y. Naomoto, and N. Tanaka
Pulmonary adenocarcinoma-targeted gene therapy by a cancer- and tissue-specific promoter system
Mol. Cancer Ther., January 1, 2007; 6(1): 244 - 252.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nakase, M.
Right arrow Articles by Tagawa, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakase, M.
Right arrow Articles by Tagawa, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online