Combined Liposome-Mediated Cytosine Deaminase Gene Therapy with Radiation in Killing Rectal Cancer Cells and Xenografts in Athymic Mice

Clinical management for rectal cancer includes surgical excision and adjuvant therapies, such as radiotherapy and chemotherapy. Five-year survival rates for rectal cancer patients have been ∼65% in general. Despite use of multimodality strategy, local recurrence alone or in combination with distant metastases causes severe and fatal outcome in patients who underwent excision of primary rectal carcinoma (1, 2). Only a minority of patients with local recurrent rectal cancer has limited disease amenable to surgical resection. Radiation therapy and chemotherapy have been main approaches for treatment of local recurrent rectal cancer. However, local recurrent rectal cancer is associated with poor outcome and still represents a great challenge to clinical treatment.

Suicide gene therapy, such as cytosine deaminase (CD)/5-fluorocytosine (5-FC) system, has been used in experimental treatment for colorectal carcinoma, because CD converts 5-FC to 5-fluorouracil, which is a conventional medicament for colorectal cancer (3, 4). Most studies have used a variety of viral vectors, such as retrovirus and adenovirus, which are of high transfection efficiency (5–7). However, their untoward reactions to the patients also aroused grave concern (8, 9). Liposome is often used in laboratory for mediating gene transfection. Although this method is safer, the low transfection efficiency cannot meet the clinical therapeutic requirements. Some studies have reported that radiation can promote gene transfection efficiency (10, 11). In this study, we combined liposome-mediated suicide gene therapy with radiation to improve killing effect on cancer cells. We hope it can be a new strategy for the treatment of recurrent rectal cancer and other solid tumors.

Cell cultures. HR-8348 human rectal cancer cell line was obtained from the Cell Biological Research Institute (Shanghai, China) and was inoculated in RPMI 1640 (Life Technologies, Beijing, China) supplemented with 10% fetal bovine serum. The cells were grown in 5% CO₂ at 37°C in an incubator.

Recombinant plasmid vectors. The recombinant plasmid vector plasmid PXJ41-CD constructed from plasmid PXJ41 and PCD2 bears a 1.5-kb fragment containing full length of CD cDNA sequence. Both PXJ41 and PCD2 were digested with restriction endonucleases EcoRI and BamHI. The CD cDNA sequence was cut off from PCD2. After CD and vector PXJ41 were retrieved, T4 ligase was used to ligate them subsequently. The PXJ41-CD vector was obtained and then verified by DNA sequencing analysis. Plasmid pEGFP-N1 containing green fluorescent protein cDNA was used for the observation of transfection efficiency.

Cell irradiation and transfection. The cells attached in flasks were irradiated in a ⁶⁰Co source (Beijing Radiation Medical Institute, Beijing, China) to a final dose of 2, 4, 6, and 8 Gy. One hour after radiation, PXJ41-CD and pEGFP-N1 were transfected into HR-8348 cells with a Lipofectin protocol. After 36 hours, the cells were observed under an inverse fluorescence microscope, and transfection efficiency was calculated by counting green fluorescence cells.

Detection of cytosine deaminase mRNA expression. Quantitative reverse transcription-PCR was done to confirm CD transfection and the expression of CD mRNA. Total RNA was extracted from the CD-transfected cells. For CD-specific primers, sense 5′-TGAGCAGGAAGTCGCGCC-3′ and antisense 5′-GCACTCCTATAACGGGGCG-3′, amplification fragment was 367 bp. Housekeeping β-actin mRNA expression was used as an internal control.

Cancer cell inhibition assay. HR-8348 cells were seeded into a 96-well plate in 100 μL RPMI 1640 per well and incubated for 24 hours. The cells were divided into four groups: radiation, CD/5-FC, combined radiation and CD/5-FC, and blank control. Each group of cells was treated according to the grouping arrangement. After incubation for 48 hours, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (20 μL, 5 mg/mL, Sigma) was added for further incubation of 4 hours at 37°C. The medium was removed from each well and DMSO (100 μL) was added and incubated for 15 minutes. Spectrometric absorbance at 540 nm was measured with a plate reader. The cell survival rate was calculated with the following equation: {1 − (experimental group absorbance / control group absorbance)} × 100%.

Tumor xenograft therapeutic experiments. Forty female BALB/C-nu/nu nude rats, 4 to 5 weeks old, were provided by the Centre Institute of Identifying Biological Products (Beijing, China). HR-8348 cells were inoculated (0.2 mL/rat, 5 × 10⁷ cells/mL) s.c. at the right posterior limbs of the rat. When the tumor grew up to 0.5 cm in diameter, the rats were randomly divided into four groups, each for 10 animals. In group 1, radiotherapy group, the xenograft tumor of rats received radiation 2 Gy/d for 15 days. In group 2, CD gene therapy group, Lipofectin-PXJ41-CD (200 μL) was injected into the xenograft tumor of rat through multipoint injection on the 1st, 4th, 8th, and 12th days, and on the third day, 5-FC (800 mg/kg) was injected into the abdominal cavity each day for 12 days. In group 3, radiation and CD gene therapy group, the radiation protocol was just as that in group 1 and the gene therapy protocol as that in group 2. In group 4, control group, the rats received no therapeutic intervention.

On the 30th day of treatment, the rates of all groups were sacrificed and the tumors were dissected, measured, and weighed. The equation for calculating repression rate of tumor volume is {(mean volume of the control group − mean volume of the experimental group) / mean volume of the control group} × 100%. The equation for calculating repression rate of tumor weight is {(mean weight of the control group − mean weight of the experimental group) / mean weight of the control group} × 100%. The tumor repression rate was used to evaluate the curative effect of the methods on tumors.

Tumor histopathologic examination. Xenograft tumor tissues from the rats were fixed in 10% neutral buffered formalin. Tissue samples were paraffin embedded, sectioned, and H&E stained for histopathologic evaluation.

Statistical analysis. The data from the different groups were compared statistically by one-way ANOVA and the χ² test using SPSS 10.0 statistical software. P < 0.05 was considered statistically significant.

Radiation increases liposome-mediated DNA transfection efficiency. By counting green fluorescence cells with plasmid pEGFP-N1 transfection, the transfection efficiency of HR-8348 cell line mediated with liposome was 21.3%. Transfection efficiency of the plasmid into HR-8348 cells was affected by radiation. The cells were irradiated at a dose of 2, 4, and 8 Gy. One hour later, pEGFP-N1 was transferred with liposome reagents. Eighteen hours later, green fluorescence was observed under an inverse fluorescence microscope. The number of green fluorescence cells as well as background cells was counted 36 hours after transfection, and transfection efficiency was measured 62.2%, 78.0%, and 87.8% respectively, at a dose of 2, 4, and 8 Gy. Irradiation of HR-8348 human rectal cancer cell line resulted in a 2.8- to 4.3-fold enhancement of plasmid DNA transfer compared with baseline liposome-mediated transfection (Fig. 1). This enhancement was irradiation dose dependent.

Radiation promotes cytosine deaminase transfection and expression. To assess the transcript levels of CD, we did quantitative reverse transcription-PCR and analyzed CD mRNA expression levels by single liposome-mediated transfection and the association of radiation with liposome mediating. No amplified products were detected in nontransfected HR-8348 cells, but the relative band of CD expression was found in the transfection cells. The levels of CD mRNA expression in the group of radiation followed by liposome-mediated transfection were notably higher than those in the group of simple liposome-mediated transfection (Fig. 2). Radiation improved CD transfer and expression.

Combination of radiation and cytosine deaminase gene therapy enhances killing effects on cancer cells. To prove the killing effects of combined radiation and CD/5-FC, we measured in vitro cytotoxicity with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay to evaluate the efficiency of cell inhibition. 5-FC prodrug (10 mmol) was used for each group of experimental HR-8348 cells. With this, we verified that the cells transferred with CD and selected by G418 were killed at a rate of >95%. In this experimental study, we used a radiation dose of 2 Gy, a routine dose for clinical radiotherapy. When the same dose of 5-FC was used, HR-8348 cell viabilities decreased in the different groups (Fig. 3). Combined radiation and CD/5-FC was more powerful than simple radiation or CD gene therapy in cell inhibition. Thus, a single radiation dose of 2 Gy or liposome-mediated CD gene therapy had a limited inhibitory effect on cancer cells because of a low dose of radiation and low efficiency of transfection. In contrast, combined radiation and CD gene therapy showed the effect that can meet clinical requirements for anticancer treatment.

Anticancer effect of combined liposome-mediated cytosine deaminase gene therapy with radiation on xenograft in nude rats. To evaluate the therapeutic efficacy of combined CD gene therapy and radiation, we used a tumor xenograft model. The major and minor axes of the tumor were measured on the 1st, 4th, 8th, 16th, 20th, 24th, and 28th days. The tumor volume was calculated by the equation: V = π / 6 × (major axis × minor axis)², and the tumor growth curve was drawn (Fig. 4). The growth curve of tumor xenograft was much slower on the 8th day in the combined group than in the other groups (P < 0.01). Compared with the control group, tumor volume was inhibited by 81.5%, 48.5%, and 37.4%, respectively, in the combined CD/5-FC with radiation group, CD/5-FC group, and radiation group and wet weight of tumor was decreased by 80%, 41.7%, and 37.7%, respectively. The differences of the repression rates were significant between the combined CD/5-FC with radiation group and the single CD/5-FC or radiation group (P < 0.01; Fig. 5).

CD expression was also detected in the tissues of tumor-bearing rats. Total RNA was extracted from the tissues of rectal cancer, bone marrow, cardiac muscle, and intestine. Reverse transcription-PCR showed that a 367-bp fragment could be amplified in the cancer tissues of the experimental group and the CD/5-FC group but could not be amplified in the tissues of bone marrow, cardiac muscle, and intestine (data not shown). The expression level of CD mRNA in the combined treatment group was higher than that in the CD/5-FC group, indicating that radiation might promote CD gene transfer and expression.

Histopathologic examination of the xenograft tumor tissues showed that the number of cells of xenograft tumors was reduced significantly and a great deal of adipocytes substituted for the cancer cells in the group that received combined radiation and CD gene therapy, but a slight decrease of cancer cells was observed in the group subjected to simple radiation or liposome-mediated CD gene therapy compared with the control group (Fig. 6).

Local recurrence of rectal cancer is a problem of or a challenge to clinical treatment. More effective treatment strategies and methods are needed clinically. The combination of some routine treatment methods with new biological therapy may be promising. Radiation is often used for the treatment of rectal cancer recurrence, but the effect is limited for some cases (12).

Great progress has been made in gene therapy, especially the suicide gene therapy that is considered clinically prospective (13, 14). Many gene therapy trials have been done with adenoviral or retroviral vectors (15–17). These vector systems have a relatively high efficiency of transfection, but they also have several major limitations and even cause serious side effects on human body. Liposome-mediated gene transfer has several advantages over viral vector-mediated gene transfer systems (18, 19). In particular, liposomes are not immunogenic and are safe for clinical use. The major disadvantage of liposome-mediated gene therapy is the lower efficiency of transfection. If liposome-mediated gene transfer can be enhanced sufficiently, the clinical use of noviral vectors may be possible.

Several studies have reported that radiation improves gene transfer (20, 21). In this study, we used irradiation to enhance transfection efficiency of the CD suicide gene. Thus, we combined conventional radiation with gene therapy.

The vectors and the gene transfecting methods are the main restrictions of tumor gene therapy. Clinically, liposome-mediated gene transfer is suitable, but its lower efficiency of transfection should be improved. In this study, radiation enhanced liposome-mediated gene transfer significantly. However, dose-response relationship existed between irradiation and gene transfer. In in vitro and in vivo experimental studies, an irradiation dose of 2 Gy was used according to clinical practical radiotherapy. A 2.8-fold enhancement of CD transfection efficiency resulted from irradiation of the HR-8348 human rectal cancer cell line. Cancer cell inhibition assay presented a satisfied result in killing cancer cells. Radiation can enhance CD transfer to cancer cells, and CD/5-FC can increase the sensitivity of radiation to cancer cells (22, 23). In this combination, radiotherapy and CD gene therapy facilitate each other in killing cancer cells. The lower efficiencies of liposome-mediated CD gene transfer and expression are improved in cancer cells, and the main defect of liposome is overcome. As the efficiencies of CD transfer and expression are enhanced, the radiosensitizing effect of CD/5-FC on cancer cells is increased. More powerful therapeutic effect on cancer cells can be obtained.

In this study, tumor xenograft treatment showed that combined liposome-mediated CD gene therapy with radiation has much stronger anticancer effect. Xenograft tumors can be treated with a clinical radiation dose of 2 Gy/d followed by liposome-mediated CD transfer via direct tumor injection. Although the radiation dose and the efficiency of CD transfection are limited with one-time treatment, daily radiation and repeated injection of liposome-mediated CD gene transfer may lead to the accumulation of radiation dose and increase of efficiency of CD transfection. Thus, xenograft tumor inhibition is satisfied. In this study, the growth of the tumors was inhibited significantly, and histopathologic study showed that the tumor cells and their division are reduced markedly. These evidence prove that combined liposome-mediated CD gene therapy with radiation is safe and more efficient for killing rectal carcinoma cells and that its effect meets the clinical requirement.

In conclusion, irradiation enhances liposome-mediated CD gene transfer and expression, and CD transfer facilitates radiosensitization of cancer cells. Combined radiation with CD gene therapy is adequate to improve anticancer effect and is highly potential in clinical treatment of recurrent rectal cancer and other solid tumors.