Electro-gene therapy followed by intratumoral injection of pcDNA 3 . 1p 27 Kip 1 wild type in human tongue base cancer cells spc 3 xenograft

Human tongue base cancers are characterized by a high degree of local invasion and metastasis to the regional lymph nodes and included a disease with difficult treatment. A novel method for highefficiency and region-controlled in vivo gene transfer was developed by combining electro-gene therapy and plasmid (pcDNA). The aims of the study were to examine the efficiency of transfection of p27Kip1 gene by electro-gene therapy, and to evaluate p27Kip1 gene therapy in Supri’s clone-3 (SP-C3) xenografts using pcDNA3.1-p27Kip1 wild type (wt) and pcDNA3.1 empty vector (neo) with electro-gene therapy. To investigate gene transfer method, the enhanced green fluorescence protein (EGFP) gene was transfected into xenografts by electro-gene therapy. The efficiency of p27Kip1 gene transfection at protein level was confirmed by Western blotting. To estimate the reduction of tumor size in Wistar Balb/c mice after electro-gene therapy with p27Kip1 wt gene was examined by tumorigenesis assay. To evaluate the induction of apoptosis was carried out by colorimetric assay. Result, the growth of tumors was markedly suppressed by p27Kip1 wt gene transfection. Up-regulation of p27Kip1 protein was detected in pcDNA3.1-p27Kip1 wt. Apoptosis induction through activity of caspase -3 and -9 were significantly increased in p27Kip1 wttransfected tumors. These results suggest that it is possible to transfer p27Kip1 wt into tongue base cancer cell xenografts using electro-gene therapy. p27Kip1 wt had a high-potencially to suppress the growth of tumors. Conclusion, electro-gene therapy followed by intratumoral injection of pcDNA3.1-p27Kip1 wt had a high-potencially to suppress the growth of a human tongue base cancer cell xenograft.


INTRODUCTION
Electro-gene therapy or electroporation or electric pulse has been developed for the purpose of achieving highly efficient in vitro gene or drug transfer. 1,2This system provides markedly higher efficiency transfer compared with other non-viral transfer system, including cationic liposome. 3ectro-gene therapy has been applied to in vivo drug transfer for cancer treatment and clinical trial has been started. 4Electro-gene therapy has become more and more popular as an effective technique for introduction of foreign DNA into cells of various kinds of mammalian cells. 5,6for investigation of gene regulation 7 , and has been demonstrated to be highly useful in transfecting human hematopoetic stem cells for gene therapy. 8However, the transfection efficiency in mammalian cells using in vivo electro-gene therapy has received little attention 9 and usually is still low, typically about 0.01-1%. 10Because electro-gene therapy is a physical method, it has a little biological side effect and is free of chemical toxicity. 7any types of methods and techniques for in vivo gene transfer have been developed, and some of them have already been applied in clinical trials. 1 Non-viral gene transfer, "naked" plasmid DNA is an ideal system for gene transfer.A plasmid mediated method would be economical and easy because use of this system obviates the necessity to construct viral vectors, establish clones of producer cells, access viral titers and presence of replication-competent helper virus, which has been known to activate passive oncogenes.The transfer procedure could be easily repeated because "naked" plasmid DNA has little antigenicity to the host body. 11uman tongue base cancers are characterized by a high degree of local invasion and a high rate of metastases to the cervical lymph nodes.Moreover, human tongue base cancer frequently shows local recurrence after initial treatment, probably due to micro invasion and/or metastasis of tumor cells at the primary site. 12SP-C3 cell line has an extremely growth, highly invasion and metastasis to the regional and distant lymph nodes, rapidly local recurrence after radical excision, highly mortality, and included a disease with difficult treatment. 13Despite advanced in surgery, radiotherapy and chemotherapy, the survival of patients with oral tongue cancer has not significantly improved over the past several decades.Also, treatment options for recurrent or refractory oral cancers are limited. 14Furthermore, the ratio of mortality or incidence in 1980 and 1990 was 0.48 and 0.47, respectively 15 and the prognosis has not changed during the past 10 years.However, as a new strategy for refractory cancer, gene therapy is watched with keen interest.
p27 Kip1 is a universal cyclin-dependent kinase inhibitor that directly inhibits the enzymatic activity of cyclin-CDK complexes, resulting in cell cycle arrest at G 1 . 16p27 Kip1 has an important prognostic factor in various malignancies.8][19] In addition, loss of p27 Kip1 has been associated with disease progression and an unfavorable outcome in several malignancies. 20Furthermore, mice lacking the p27 Kip1 gene show an increase in body weight, thymic hypertrophy and hyperplasia of pituitary intermediate lobe adrenocorticotropic hormon cells, adrenal glands and gonadal organ. 21oreover, malignant human oral cancer cells transfection with p27 Kip1 gene leads to inhibition of proliferation, invasion and metastasis. 22,23n the present study, the efficiency of transfection of exogenous p27 Kip1 gene by electrogene therapy, and the antitumor activity of p27 Kip1 gene therapy in human tongue base cancer cells Supri's clone-3 (SP-C3) xenografts using pcDNA3.1-p27Kip1 wild type (wt) and pcDNA3.1 empty vector (neo) with the local application of electric pulses was evaluated.

MATERIALS AND METHODS
Cell and cell culture.SP-C3 cells were isolated from a cervical lymph-node metastasis of a human tongue base cancer patient. 22The original tumor SP-C3 cells were moderately differentiated from human tongue base cancer.Cells were maintained in Dulbecco's modified eagle medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS, Moregate BioTech, Bulimba, Australia), 100 µg/ml streptomycin, and 100 units/ml penicillin (Invitrogen Corp, Carlsbad, CA, USA).
In vivo tumorigenesis and electrotransfection.The human tongue base cancer cell line, SP-C3 cells were trypsinized, washed with PBS, and suspended in saline solution at 1 x 10 6 cells in 0.1 ml.Cell suspension (0.1 ml) was injected into each male nude mouse with Balb/cA Jclnu genetic background (Clea Japan, Inc. Tokyo, Japan) subcutan in the flank area.A pair of 1 cm diameter of disc-shaped electrodes (pinsettes-type electrode 449-10 PRG, Meiwa Shoji, Tokyo, Japan) was used to nip the tumor nodule through the skin.A series of eight electrical pulses with pulse length of 1 msec was delivered with a standard square wave electroporator BTX T820 (BTX, Inc, San Diego, CA).The voltage of 80 V/1.0 cm diameters of xenografts was used.Then, it delivered an appropriate pulse length and frequency of pulses according to previous report. 24,25Immediately after electrical pulsing, 20 µg of pcDNA-neo or pcDNA3.1-p27Kip1 wt dissolved in 50 µl of Tris EDTA buffer was directly injected into the tumor nodule.This electroporation and injection were performed a total of three times at 3-day intervals.Tumor volume and body weight were measured every 3 days from the time electroporation started until the mice were sacrificed.The tumor volume was determined by measuring length (L) and width (W) diameters of the tumor and calculated as V = 0.4 x L x W 2 . 13etection of reporter gene expression in vivo.For fluorescence microscopy, pEGFP-C3 vector (BD Bioscience Clontech) -injected tumors for 48 h were sectioned and mounted in PBS for immediate microscopy.To visualize EGFP, a Xenon arch lamp and a FITC filter were used on a Zeiss Axioskop.Images were acquired with a color CCD camera and frame-grabbing equipment at identical magnification, light intensity and amplification for each sample. 13estern blotting analysis.Cell lysates were prepared from the xenograft tumor tissue.Briefly, samples containing equal amounts of protein (50 µg) were electrophoresed on a SDS-polyacrylamide gel and transferred to a nitrocellulose filter (PVDF membrane: BioRad, Hercules, CA, USA).The filters were blocked in TBS containing 5% nonfat milk powder at 37 0 C for 1 hour and then incubated with a 1: 1000 dilution of the monoclonal antibody against p27 protein (clone 1B4, monoclonal antibody, Novocastra Laboratories, New Castle, UK) and an Amersham ECL kit (Amersham Pharmacia Biotech) as the primary antibody.Mouse antibody IgM was used as the secondary antibody (1:2000).Anti-α tubulin monoclonal antibody (Zymed laboratories, San Fransisco, CA, USA) was used for normalization of Western blot analysis.
Detection of apoptosis using colorimetric assay.Induction of apoptosis was measured using the colorimetric assay kit (caspase-3 and caspase-9; BioVision Research Product, CA, USA) according to the manufacturer's directions.Briefly, equal amounts of tissue extracts prepared from SP-C3 transfected with pcDNA3.1-p27Kip1 wtor pcDNA3.1neowere incubated with the substrate (Ac-DVED-pNA or Ac-LEHD-pNA) in the assay buffer for 2 hours at 37 0 C. Absorbance was measured at 405 nm using a microplate reader (BioRad, USA).Each determination was performed in triplicate.
Statistical analysis.Statistical analysis was performed with a Stat Work program for Macintosh computers (Cricket Software, Philadelphia, PA, USA).Data were analyzed for statistical significance of 95% with two-way ANOVA and Student's t-test.

Detection of transgene expression in
Xenograft.Expression of reporter gene (EGFP) after plasmid injection and electro-gene therapy in tumor tissue was assessed in fresh tissue sections by light microscopy fluorescence imaging.Very few cells were positive when only naked DNA without consecutive electroporation was injected.The combination with electroporation resulted in consistenly efficient transduction of a higher number of cells with EGFP reporter gene (P = 0.001) (Fig. 1).
In vivo effect of p27 Kip1 wt transfection by electroporation on tumor growth.The mean relative volume for SP-C3 xenografts treated with an injection of pcDNA3.1-p27Kip1 wt or pcDNA3.1 empty vector was shown in Fig. 2A, p27 Kip1 -up-regulated tumors (pcDNA3.1-p27Kip1 wt-injected) became much smaller than pcDNA3.1 empty vector-injected tumors (P = 0.001).Interestingly, during the experimental period, no loss of body weight was observed in each treatment group, and that no skin region including a burn also was observed (Fig. 2B).
Expression of p27 Kip1 protein in xenografts.To evaluate the efficiency of transfection of p27 Kip1 gene, the expression of p27 Kip1 protein by Western blotting was evaluated.As shown in Figure 3, upregulated of p27 Kip1 protein in pcDNA3.1-p27Kip1 wt-injected tumors was detected when compared with that in pcDNA3.1 empty vector-injected tumors.The expression of α-tubulin as an internal control was approximately the same in all of the tumors.

DISCUSSION
Cell membranes electro-gene therapy has been developed for the purpose of achieving highly efficient in vitro gene and/or drug transfer. 2Interestingly, the application of electrogene therapy to cultured cells has been well established, but the use of in vivo electro-gene therapy has received little attention. 9In vivo electro-gene therapy has just recently been proposed for transdermal drug delivery 5 and for electrochemotherapy with bleomycin of superficial tumors. 6Recently, successful in vivo transfer of IL genes into muscle, and transfer of marker and therapeutic suicide genes into normal tissues and tumors have been reported. 26n the present study, electro-gene therapy with naked plasmid DNA was evaluated.The wild type p27 Kip1 gene was used as a transfection gene and was evaluated its antitumor activity in human tongue base cancer (SP-C3 cell) xenograft.The results of study demonstrated the efficiency of  α-tubulin electro-gene therapy was thought to be about 55-70% of cells, determined by EGFP expression as shown in Figure 1.Recently, the efficiency of electro-gene therapy followed by injection of pcDNA3.1-p27Kip1 wt in human salivary gland cancer was about 45-65%, and in a tongue cancer SP-C1 was about 53-75%.Interestingly, the efficiency of electro-gene therapy followed by injection of 5-fluorouracil in oral cancer was still lower than of non viral plasmid injection. 29lso, it was demonstrated that the transfection of wild type p27 Kip1 gene by electro-gene therapy could induce apoptotic cell death (Fig. 4), and inhibit the growth of SP-C3 cancer xenografts (Fig. 2).Furthermore, wild type p27 Kip1 gene by electro-gene therapy could induce the expression of p27 Kip1 protein (Fig. 3), which has the negative regulator function in the cell cycle.Therefore, electro-transfer of plasmid DNA p27 Kip1 wt into SP-C3 xenograft can be successfully achieved using disk-shaped electrodes.Suggesting that clinical application using this electro-gene therapy system for oral cancer may be possible in the future.
On the other hands, some disadvantages of this method should be considered.Although transfection by electro-gene therapy inhibited the growth of SP-C3 cancer xenografts, the target area was limited to local tumors and the growth of multiple metastatic lesions cannot be target for efficient suppression.For that reason, with a view to obtaining more effective gene therapy using electroporation for head and neck cancer and oral cancer, writter plan to attempt gene transfer with several other genes and to use various anticancer agents in combination with gene transfection by this electro-gene therapy system.
In conclusion, in vivo electro-gene therapy following by intratumoral injection of pcDNA3.1-p27Kip1 wt gene has a highly antitumor activity in human tongue base cancer xenografts.It might be possible to transfer pcDNA3.1-p27Kip1 wt gene into human tongue base cancer xenograft.In vivo gene transfer method is a simple procedure and can solve some of the critical drawbacks of the present gene transfer techniques, thus providing a new strategy for gene therapy.

Figure
Figure 2. A. Growth of tumor formed by transfectants, B. Change of body weight in mice.* P < 0.05.

Figure
Figure 4.The activity of caspase-3 and -9 in SP-C3 transfected cells.