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Clinical Cancer Research Vol. 9, 1129-1135, March 2003
© 2003 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Significant Antitumoral Activity of Cationic Multilamellar Liposomes Containing Human IFN-ß Gene against Human Renal Cell Carcinoma1

Hiroyuki Nakanishi, Yoichi Mizutani2, Akihiro Kawauchi, Osamu Ukimura, Takumi Shiraishi, Manabu Hatano, Masaaki Mizuno, Jun Yoshida and Tsuneharu Miki

Department of Urology [H. N., Y. M., A. K., O. U., T. S., T. M.], Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan, and Departments of Molecular Neurosurgery [M. M.] and Neurosurgery [M. H., J. Y.], Nagoya University, Postgraduate School of Medicine, Nagoya 466-8550, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Immunotherapy is the most effective treatment against metastatic renal cell carcinoma (RCC). However, the response rate is ~15%. More effective therapy is, therefore, needed for patients with metastatic RCC. We then examined the antitumor effect of cationic multilamellar liposome containing human IFN-ß (huIFN-ß) gene (IAB-1) against RCC.

Experimental Design: Concentrations of huIFN-ß protein were measured by ELISA. The cytotoxicity of IAB-1 against human RCC (NC65, ACHN, and freshly isolated RCC cells), prostate and bladder cancer cell lines, and renal proximal tubule endothelial cells (RPTEC5899) was examined by the colorimetric method using tetrazolium salt. Apoptosis was assessed by the acridine-orange staining. For in vivo study, we used NC65 cells inoculated into severe combined immunodeficiency mouse.

Results: The RCC cells treated with IAB-1 secreted significant amounts of huIFN-ß protein continuously. Drastic in vitro cytotoxic effect of IAB-1 against RCC was observed. In contrast, treatment with 1000 IU/ml recombinant huIFN-ß protein resulted in weak cytotoxicity. The cytotoxic effect against prostate and bladder cancer cell lines was less than that against RCC. Furthermore, no significant cytotoxicity was observed in RPTEC5899 cells. Apoptosis was observed in the cells treated with IAB-1, but recombinant huIFN-ß failed to induce apoptosis. The size of NC65 tumors transfected with IAB-1 in mice was significantly smaller than that receiving injection of empty liposome or recombinant huIFN-ß protein.

Conclusion: These findings indicate that IAB-1 may have an antitumor activity against human RCC by inducing apoptosis, suggesting its potential clinical application for gene therapy against RCC.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There have been a few effective therapies against metastatic RCC.3 Immunotherapy including IFNs against metastatic RCC is relatively effective (1, 2, 3, 4) . Many investigators have tried to enhance the effectiveness of IFNs by combination treatment with other biological agents or chemotherapeutic agents (3 , 4) ; however, these response rates were ~15–20% (1, 2, 3, 4) . Furthermore, systemic side effects and high cost have limited the clinical validity of immunotherapy using recombinant cytokine protein. The development of novel strategies for patients with metastatic RCC has, therefore, been desired.

One such hope is gene therapy (5, 6, 7, 8) . Recently, Phase 1 and 2 gene therapy trials using cytokine genes such as granulocyte macrophage-colony stimulating factor (6) or interleukin 2 (7 , 8) have commenced in patients with metastatic RCC. Although these therapies appear to be safe, their efficacy is not yet clear.

Cationic liposomes are one of the most fascinating vectors for human gene therapy because they are noninfective, low in immunogenicity, low in toxicity, high in stability, and less costly to manufacture (9, 10, 11) . Although many nonviral vector systems for cancer gene therapy have been reported, only a few have been put to therapeutic use, because of poor transfection/expression efficacy and their instability. Morphologically, cationic liposomes are divided into three main types: SUVs; large unilamellar vesicles (12) ; and MLVs (13, 14, 15) . SUVs have already been used in human clinical gene therapy, especially immuno-gene therapy for cancer. SUVs bind the genes to the surfaces, producing DNA-lipid complexes; however, the transduction efficacy is relatively low (10 , 11) . In contrast, large unilamellar vesicles and MLVs generally entrap the genes within the liposomes, rather than on the surfaces (12, 13, 14, 15) . In these methods, high transfection efficiency can be achieved in the presence of serum, whereas in most SUVs, transfection was inhibited in the serum. Furthermore MLVs containing plasmids have high stability. We have already confirmed that the transfection efficacy had continued for 6 months at 4°C (data not shown). We have already reported that MLVs containing TMAG were very useful vectors for in vivo gene therapy against experimental brain tumors (14, 15, 16, 17, 18, 19, 20) . IAB-1 is composed of cationic multilamellar liposomes containing huIFN-ß gene. In 2000, we commenced clinical trials of gene therapy in Japan against malignant glioma by direct intratumoral injection of IAB-1.

Type 1 IFNs, the IFN-{alpha} family and IFN-ß, bind to the type 1 IFN receptor and are multifunctional cytokines with antiviral, antiproliferative, antiangiogenic activity and immune cell stimulation (3) . Type 1 IFNs have generally been used as antitumor agents against cancers including RCC (1, 2, 3, 4) . Although recombinant IFN-{alpha} protein is commonly used against RCC (2, 3, 4) , direct intratumoral transfection of the IFN-ß gene exhibited stronger growth-inhibitory effect than did the IFN-{alpha} gene in some in vivo experiments (21 , 22) . We postulated that the IFN-ß gene had possibilities for gene therapy against RCC. We then evaluated the in vitro cytotoxic effect of IAB-1 against RCC cells and examined the in vivo antitumor effect against human RCC tumors transplanted in SCID mice.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells.
NC65 (23) and ACHN (24) human RCC cell lines, and KC1, KC2, and KC3 primary cultures established from fresh RCC at the Department of Urology, Kyoto Prefectural University of Medicine, were used as targets. The histological classification and grading according to the TNM staging system [Unio Internationale Contra Cancrum (1997 UICC)] were as follows: KC1 and KC2, T1N0M0, Grade 2; and KC3, T3bN0M0, Grade 2. These cells were maintained in RPMI 1640 (Life Technologies Inc., Gaithersburg, MD) supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin (Life Technologies Inc.) and 10% fetal bovine serum (Life Technologies Inc., Bio-cult, Glasgow, Scotland, United Kingdom).

T24 and J82 human bladder carcinoma cell lines (25) , and LNCaP and PC-3 human prostate carcinoma cell lines (26) were maintained under the same conditions. The RPTEC5899 (Cambrex, Walkersville, MD; Ref. 27 ) normal renal proximal uriniferous tubule cell line was maintained in Renal Epithelial cell Basal Medium (Cambrex) with 100 units/ml penicillin and 100 µg/ml streptomycin.

Reagents.
IAB-1 is a plasmid DNA/lipid complex [composed of plasmids (pSV2IFNß)] that contains the SV40 early promoter and the huIFN-ß coding sequence (16) , and positively charged liposomes [TMAG, dilauroyl phosphatidylcholine (DLPC), and dioleoyl phosphatidylethanolamine (DOPE) in a molar ratio of 1:2:2] as described in our previous articles (13, 14, 15, 16, 17, 18, 19) . The type of this liposome was MLV. IAB-1 was dissolved in PBS, and the final concentration of IAB-1 was 50 nmol of lipid/µl with 1.0 µg of plasmid DNA/µl. Empty liposome is composed of liposome alone (50 nmol of lipids/µl) without plasmid DNA. The IAB-1 and empty liposome were diluted to adequate concentrations in each experiment.

The recombinant huIFN-ß protein (IFN-ß Mochida, 2.0 x 105 IU/mg) was provided by Mochida Pharmaceutical Inc., Tokyo, Japan.

In Vitro Gene Transfer.
Aliquots of 5.0 x 104 cultured cells were placed in each well of a 6-well plate with 2 ml of medium and were incubated for 24 h at 37°C in a humidified atmosphere of 5% CO2 (standard condition). Two µl of IAB-1 solution, recombinant huIFN-ß protein, or empty liposome solution were added to the medium containing 10% fetal bovine serum, and incubation was continued for an additional 4 days.

huIFN-ß ELISA.
The culture medium was collected every 24 h after the addition of IAB-1, and huIFN-ß concentration in the medium was measured by ELISA (Fijirebio Inc., Tokyo, Japan) according to the supplier’s protocol.

In Vitro Cytotoxicity Assay.
Cytotoxicity was evaluated by a colorimetric method using 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8, Nacalai Tesque, Kyoto, Japan; Ref. 28 ). Briefly, triplicate aliquots of cells were treated with IAB-1. After incubation under the standard condition until analysis, the culture supernatant was replaced by 1 ml of fresh complete medium containing 0.5 µmol of tetrazolium salt. After incubation for an additional 1 h, culture supernatant was harvested and absorbance (A) at 450 nm was measured. Cytotoxicity was calculated as follows: cytotoxicity (%) = [1 - (absorbance of experimental wells/average absorbance of control wells)] x100.

Acridine-Orange Staining.
Apoptosis was observed by the acridine-orange staining. NC65 cells were incubated with 0.1 µl/ml IAB-1 (50 nmol/µl lipid containing 1.0 µg/µl plasmid DNA), 1000 IU/ml recombinant huIFN-ß protein, and 0.1 µl/ml empty liposome (50 nmol/µl lipids) for 24 h under standard conditions. After incubation, cells were removed from the flask and washed with PBS. Acridine-orange solution (20 µg/ml; Lot No. CAE2276; Wako Pure Chemical Industry Ltd., Osaka, Japan) was added to the cells and was mixed by pipetting. The cells were then replaced on a slide glass, and the specimens were examined under a Nikon fluorescence microscope with a B2-type filter (x400). When apoptotic bodies or chromatin condensation were observed, the observation was defined as apoptosis.

Animal Care.
Female SCID mice, 8–9 weeks of age, were purchased from CLEA Japan (Osaka, Japan), and housed in a specific pathogen-free animal facility. The animals were fed irradiated mouse chow and autoclaved reverse-osmosis-treated water. The Committee for Animal Research, Kyoto Prefectural University of Medicine, permitted this experimental procedure.

In Vivo Study with the NC65 Line.
Into the right flanks of SCID mice, 6 million NC65 cells were s.c. injected with a mixture of 50 µl Matrigel (Becton-Dickinson, NJ) and 50 µl RPMI 1640 without antibiotics or serum. At 7 days after the tumor cell injection, the tumors developed to ~7 mm in length and 5 mm in width. At that point (day 0), 30 µl of IAB-1 (1.5 µmol of lipid with 30 µg of plasmid DNA), recombinant huIFN-ß protein (6000 IU), empty liposome (1.5 µmol of lipids) or PBS were injected into established tumors via a 27-gauge needle and a micro-infusion pump (Termo, Tokyo, Japan) for 3 min (10 µl/min). Injections were performed on days 0, 2, 4, 7, 9, and 11 (three times a week for two courses).

Tumor sizes were measured at the indicated days after initial treatment. Tumor diameters were scaled with a digital caliper. Tumor volume was calculated as follows: volume = a x b2/2, where a = long diameter and b = short diameter.

Statistical Evaluations.
For in vitro study, all of the determinations were made in triplicate; results were expressed as mean ± SD. For the cytotoxicity assays and IFN-ß ELISA, Fisher’s PLSD test was used to determine statistical significance. For tumor size and mouse weight changes in vivo, results were expressed as mean ± SE; and the Mann-Whitney t test was used for statistical analysis. A P of 0.05 or less was considered significant in all of the statistical evaluations.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Significant Production of IFN-ß Protein from Urological Malignant Cells by IAB-1.
huIFN-ß ELISA analysis detected huIFN-ß protein in the culture medium of NC65 cells treated with IAB-1 (Table 1)Citation . However, in the culture medium of the cells treated with PBS or empty liposome, huIFN-ß protein was not detected. Recombinant huIFN-ß protein in the culture medium decreased abruptly. This may be attributable to its short half-life even in vitro. In IAB-1-treated NC65 cells, the level of huIFN-ß protein in the culture medium increased up to day 2 after the addition of IAB-1, reaching 34.0 ± 6.8 IU/ml at maximum, and decreased on day 3. Significant huIFN-ß secretion continued until day 4.


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  		Table 1 huIFN-ß concentration (IU/ml) in the cultured medium of NC65 cellsa

 
As shown in Table 2Citation , all of the cancer cells treated with 0.1 µl/ml IAB-1 secreted significant amounts of huIFN-ß protein on day 4. When the RPTEC line was treated with IAB-1 at 0.1 µl/ml, the concentration of huIFN-ß protein in the culture medium was only 0.3 IU/ml. Compared with prostate and bladder cancer cell lines, RCC cells were more sensitive to IAB-1 and killed by IAB-1. Therefore, huIFN-ß protein concentrations per million viable cells in the culture medium of RCC cells were higher than the huIFN-ß protein concentrations per total viable cells.


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  		Table 2 huIFN-ß concentration in the cultured medium of various cells treated with 0.1 µl/ml IAB-1 on day 4 posttreatmenta

 
The concentrations of huIFN-ß in the culture medium of NC65 cells treated with 0.1 µl/ml IAB-1 were always lower than those treated with 1000 IU/ml recombinant huIFN-ß protein. We then used 1000 IU/ml recombinant huIFN-ß protein as the control for 0.1 µl/ml IAB-1 in the later experiments.

IAB-1 Was Drastically Cytotoxic against RCC Cells, but not against RPTEC 5899 Cells.
Time kinetics of cytotoxicity against NC65 cells treated with IAB-1, recombinant huIFN-ß protein, or empty liposome for up to 4 days are shown in Fig. 1ACitation . NC65 cells treated with 0.1 µl/ml IAB-1 were drastically suppressed in cell viability. Recombinant huIFN-ß protein and empty liposomes had little cytotoxic activity against NC65 cells. Cytotoxicity of IAB-1 against NC65 cells increased in a dose-dependent manner on day 4, and it was significant, compared with the cytotoxicity of recombinant huIFN-ß protein and empty liposome (Fig. 1B)Citation . No cytotoxicity was observed in RPTEC 5899 cells treated with IAB-1 (Fig. 1C)Citation . A significant in vitro cytotoxic effect of IAB-1 was also observed against ACHN RCC cells, freshly isolated RCC cells derived from three patients with RCC (Table 3)Citation . On the other hand, treatment with 1000 IU/ml recombinant huIFN-ß protein resulted in less cytotoxicity compared with IAB-1 at 0.1 µl/ml.



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  		Fig. 1. Cytotoxicity of IAB-1 against NC65 and RPTEC5899 in vitro. A, time kinetics of cytotoxicity against NC65 cells treated with IAB-1 (•) or recombinant huIFN-ß protein ({blacktriangleup}) are shown. As controls, cells treated with PBS ({diamondsuit}) or empty liposome ({blacksquare}) were also examined. Cytotoxicity was evaluated by a colorimetric method using tetrazolium salt; vertical axis is shown as absorbance at wavelength 450 nm. Results derived from three different experiments are expressed as means ± SD. *, significant differences in cell viability by addition of 0.1 µl/ml IAB-1 (5.0 nmol lipid with 0.1 µg of plasmid DNA/ml) versus control (PBS), 0.1 µl/ml empty liposome (5.0 nmol lipid/ml) and 1000 IU/ml recombinant huIFN-ß protein. *, P < 0.05 versus other groups. B and C, 50,000 of NC65 cells (Fig. 1B)Citation and RPTEC5899 (Fig. 1C)Citation were placed in each well of a 6-well plate with 2 ml of medium and incubated for 24 h. Two µl of PBS (control) or various concentrations of recombinant huIFN-ß protein, empty liposome, or IAB-1 were added to the medium, and incubation was continued for 4 days. Cytotoxic effect was evaluated by colorimetric method using tetrazolium salt as described in the "Materials and Methods" section. a, control (PBS); b, recombinant huIFN-ß (100 IU/ml); c, recombinant huIFN-ß (1000 IU/ml); d, 0.01 µl of empty liposome (0.5 nmol/ml); e, 0.1 µl of empty liposome (5.0 nmol/ml); f, 0.001 µl of IAB-1 (0.05 nmol lipid with 0.001 µg of DNA/ml); g, 0.01 µl of IAB-1 (0.5 nmol lipid with 0.01 µg of DNA/ml); h, 0.1 µl of IAB-1 (5.0 nmol lipid with 0.1 µg of DNA/ml). Results derived from three different experiments are expressed as means ± SD. *, P < 0.05 versus other groups.

 

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  		Table 3 Percentage cytotoxicity against RCC cells on day 4 posttreatmenta

 
The Antitumor Effect of IAB-1 against Human Prostate and Bladder Carcinoma Cell Lines Is Less Than That against RCC Cells.
The cytotoxicity of IAB-1 against other urological cancer lines was also examined. Although moderate cytotoxicity was observed in prostate cancer and bladder cancer cell lines, the effect was less than that against RCC. Treatment of prostate and bladder cancer cell lines with 1000 IU/ml recombinant huIFN-ß protein resulted in weak cytotoxicity (Table 4)Citation .


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  		Table 4 Percentage cytotoxicity against other urological cell lines on day 4 posttreatmenta

 
Induction of Apoptosis in NC65 Cells Transfected with huIFN-ß Gene by IAB-1.
No apoptotic cells were seen in controls; empty liposomes (Fig. 2A)Citation and recombinant huIFN-ß protein (Fig. 2B)Citation also failed to induce apoptosis. Approximately 10% of NC65 cells treated with 0.01 µl/ml IAB-1 were considered to be apoptotic (Fig. 2C)Citation . IAB-1, at a concentration of 0.1 µl/ml, was drastically cytotoxic against NC65 cells with more than 40% of the cells considered to be apoptotic (Fig. 2D)Citation .



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  		Fig. 2. Acridine-orange staining for detection of Apoptosis. Microscopic appearance of NC65 cells treated with empty liposome, recombinant huIFN-ß protein or IAB-1 is shown. Apoptosis was determined by acridine-orange staining at 24 h after transfection as described in the "Material and Methods" section. Each figure is a fluorescence microscopic view of acridine-orange staining (x400) after the treatment: A, 0.1 µl/ml empty liposome (5.0 nmol lipid/ml); B, 1000 IU/ml recombinant huIFN-ß protein; C, 0.01 µl/ml IAB-1 (0.5 nmol lipid with 0.01 µg plasmid DNA/ml); and D, 0.1 µl/ml IAB-1 (5.0 nmol lipid with 0.1 µg plasmid DNA/ml). A and B, no apoptotic cells were observed. C, red arrow, apoptotic bodies. D, many apoptotic bodies (red arrow) and condensation of chromatin (blue arrow) were observed.

 
In Vivo Treatment with IAB-1 Elicited Significant Growth Suppression of NC65 Tumors and Showed No Remarkable Toxicity for the Host.
NC65 cells were injected into the flanks of SCID mice, and treatments were performed as described in the "Materials and Methods" section. Because it was previously reported that markedly high transfection efficacy could be obtained in vivo by using a micro-infusion pump (29) , we performed our experiments by the same method. When RCC cells were treated with 0.1 µl of IAB-1 in vitro, the concentration of huIFN-ß protein in the culture medium was ~20 IU/ml. Because 30 µl of IAB-1 was injected intratumorally, we used the injection of 6000 IU (20 IU/µl x 30 µl) of recombinant huIFN-ß protein as the control. The number of mice in each group was six. No mice died until day 36. Although recombinant huIFN-ß protein treatment had stopped tumor growth during the therapies, the tumors regrew soon after the end of the treatment (Fig. 3A)Citation . In contrast, growth suppression by IAB-1 continued for at least 4 weeks. On day 30, the volumes of residual tumors into which IAB-1 had been injected were significantly smaller than those of tumors receiving PBS (P < 0.001), empty liposome (P = 0.037), or recombinant huIFN-ß protein (P = 0.010). To evaluate the possible toxic effects of IAB-1, we measured the body weights of IAB-1-treated mice as a side effect criterion. Fig. 3BCitation shows that all groups gained weight similarly to control animals. These data suggest that IAB-1 may have significant antitumor effect against RCC without irreversible toxicity.



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  		Fig. 3. In vivo therapeutic effects of IAB-1 against NC65. Seven days before the first treatment, NC65 cells (6,000,000) were s.c. injected into the right flanks of SCID mice. Thirty µl of IAB-1 (•, 1.5 µmol of lipid with 30 µg of plasmid DNA); 30 µl of recombinant huIFN-ß protein ({blacktriangleup}, 6000 IU); 30 µl of empty liposome ({blacksquare}, 1.5 µmol of lipid); or 30 µl of PBS ({diamondsuit}) had been injected into established tumors via 27-gauge needles and a micro-infusion pump for 3 min. Injections were performed on days 0, 2, 4, 7, 9, and 11 (three times a week for two courses). A, tumor volume growth curves are shown (each group, n = 6). B, changes in mouse body weight after IAB-1 injection are shown. Results are shown as the mean ± SE. *, P < 0.05.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major impediment to nonviral vectors is relatively poor transfection efficiency compared with viral vectors (9 , 10) . If we can improve the transfection efficiency, nonviral vectors may contribute greatly to gene therapy for various diseases. In our experiments, we obtained remarkable cytotoxicity against RCC using IAB-1 in vitro and in vivo. Only 0.2 µl (0.2 µg of plasmid DNA) of IAB-1 was sufficient to induce cell death against 5.0 x 105 RCC cells in vitro. This result was superior to that of all other cationic liposome systems or polymer systems previously reported (9 , 10) . In addition, significant and continuous huIFN-ß production was detected in the culture medium of RCC cells after transfection with IAB-1. These findings suggest that IAB-1 may be a potent agent for gene therapy against RCC.

In this study, remarkable IAB-1 toxicity and IFN-ß secretion were not observed in RPTEC5899 renal epithelium cells; the body weight of mice treated six times with injections of 30 µl of IAB-1 did not change as controls during the treatments. Furthermore, IAB-1 has no infectious activity and is highly stable. These data suggest that IAB-1 may be safely used clinically.

Clinical trials for RCC using recombinant huIFN-ß protein showed negative results (1) . One of the reasons may be its very short half-life in vivo. IAB-1 induced RCC cells to produce huIFN-ß protein continuously. We then postulated that IFN-ß gene therapy using IAB-1 had great possibilities against RCC. In fact, significant cytotoxic effect of IAB-1 was observed against the RCC cells, even against the relatively IFN-ß-protein-resistant NC65 cells. Because IAB-1, but not recombinant huIFN-ß protein, induced apoptosis, one of the mechanisms of enhanced cytotoxicity by IAB-1 may be caused by the induction of apoptosis. However, the reasons why only IAB-1 induces apoptosis are not clear. Therefore, we are now analyzing the differences in the type-1 IFN signal pathway between IAB-1 and recombinant huIFN-ß protein.

When we added IAB-1 to the various cell lines, the maximum secretion of the huIFN-ß protein in the culture medium was obtained on day 2 (data not shown). Therefore, in IAB-1-treated NC65 cells, the level of huIFN-ß protein in the culture medium increased up to day 2 after the addition of IAB-1. When we treated NC65 cells with IAB-1 at 0.1 µl/ml, the value of huIFN-ß protein concentration on day 3 was less than that on day 2 and was similar to that on day 4 (Table 1)Citation . RCC cells were sensitive to IAB-1, and high cytotoxicity of IAB-1 against RCC cells was observed. The concentrations of huIFN-ß protein per million viable NC65 cells in the culture medium increased until day 2 and, after that, slowly decreased until day 4 (data not shown). These results may support the finding that there was no relationship between the degree of huIFN-ß production in the culture medium and the percentage cytotoxicity of the cells.

Although the main role of cytokines including IFNs is generally an indirect effect, such as activation of host immune systems (3 , 4) , in this report, we evaluate the direct antitumor effect of IAB-1 but not its indirect effects. As previously reported, in the mouse syngeneic orthotopic model of malignant glioma, IAB-1 drastically suppressed tumor growth; its main antitumor effect presumably was dependent on the indirect effect of murine IFN-ß, especially the activation of natural killer cells or T cells (16, 17, 18) . Other groups also reported that tumor eradication by adenovirus-mediated IFN-ß gene therapy is attributable to induction of systemic immunity (30 , 31) , Furthermore, IFN-ß protein secreted from cancer cells inhibited angiogenesis, tumorigenicity, and tumor metastasis (32 , 33) . Further analysis on indirect effects of IAB-1 against RCC is necessary.

We have previously reported that IAB-1 was greatly effective against malignant glioma (14, 15, 16, 17, 18, 19) and malignant melanoma (34) in vitro and in vivo. Cytokines including IFNs have been used extensively in treating RCC (1, 2, 3, 4) . Such therapy is also effective against malignant melanoma and malignant glioma, compared with other solid tumors (3) . Despite more significant huIFN-ß production by IAB-1 in the culture medium of prostate and bladder cancer cell lines compared with the culture medium of RCC cells, there was less cytotoxic activity of IAB-1 against the prostate and bladder cancers. The reason for this may be a low sensitivity of these malignancies to IAB-1. Therefore, RCC may be one of the candidates for huIFN-ß gene therapy using IAB-1.

Interestingly, Li et al. (35) reported that some kinds of liposomal transfection reagents, themselves, induced IFN synthesis and activated IFN-stimulated genes. Freimark et al. (36) reported that plasmid/cationic lipid complexes enhanced production of IFN-{gamma} and interleukin 12 in vivo. However, in our experiments, empty liposome did not induce IFN-ß protein secretion from cancer cells in vitro.

Despite the higher cytotoxicity of IAB-1 against RCC compared with other urological cancer cell lines, RCC cells produced lower levels of huIFN-ß protein. One of the reasons responsible for this was considered to be the higher cytotoxicity of IAB-1 against RCC and the remarkably less residual RCC cells available to produce huIFN-ß protein than were available with other urological malignancies.

In this report, we did not show the cytotoxicity of naked pSV2IFNß or cationic MLV containing mock plasmid DNA as controls. However, we have already confirmed that their cytotoxicity is similar compared with empty liposome alone (14, 15, 16, 17, 18, 19) .

Improvement of the overall response rate of current recombinant cytokine protein therapy against RCC has been thought to be limited. Immuno-gene therapy may overcome this limit, because of continuous intracellular secretion of cytokine with its gene transduction. The present study shows that, compared with exogenous treatment with recombinant huIFN-ß protein, IAB-1 significantly inhibited the growth of human RCC cells by inducing apoptosis, suggesting clinical applicability of IAB-1 in gene therapy against RCC. Although additional studies are needed, our experiments imply that treatment with a novel nonviral vector system with IAB-1 may be useful for patients with RCC, as a new form of immuno-gene therapy of more selective cytotoxicity and less collateral side effects.


   ACKNOWLEDGMENTS
 
Dr. Osam Mazda, Department of Microbiology, Kyoto Prefectural University of Medicine, provided helpful support, and Yukako Morioka provided laboratory technical assistance.


   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.

1 Supported in part by Grants-in-Aid 13470339 and 14657410 from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and a 2002 Grant-in-Aid from the Japanese Urological Association, Japan. Back

2 To whom requests for reprints should be addressed, at Department of Urology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan. Phone: 81-75-251-5595; Fax: 81-75-251-5598; E-mail: ymizutan{at}koto.kpu-m.ac.jp Back

3 The abbreviations used are: RCC, renal cell carcinoma; SUV, small unilamellar vesicle; MLV, multilamellar vesicle; RPTEC, renal proximal tubule endothelial cell; SCID, severe combined immunodeficient/immunodeficiency; TMAG, N-({alpha}-trimethylammonioacetyl)-didodecyl-D-glutamine chloride; huIFN-ß, human IFN-ß; Fisher’s PLSD, Fisher’s protected least significant difference. Back

Received 8/ 6/02; revised 11/12/02; accepted 11/26/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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