[Antitumor activity of the recombinant rClone30-CD/5-FC system].

5-Flucytosine (5-FC) could be changed to 5-fluorouracil (5-FU) by cytosine deaminase (CD), the latter is able to kill cancer cells. However, there is no efficient method to deliver the CD gene into the tumor cells, which hampers the application of the suicide gene system. In this experiment, for the first time, the NDV has been utilized as a vector to deliver the CD gene into the cancer cells, the virus can infect the cancer cells specifically, replicate and assemble, while the cytosine deaminase is expressed. Then the CD converts the prodrug 5-FC into 5-FU to achieve the purpose of inhibiting tumor. Firstly, the whole genome of E. coli JM109 was extracted, and the CD gene was obtained by cloning method. Then the CD and IRES-EGFP were ligated into the pEE12.4 expression vector to become a recombinant pEE12.4IE-CD eukaryotic expression plasmid. The human liver cancer cells were transfected with the plasmid. The cells were treated with different concentrations of 5-FC, MTT method was used to determine the killing effect of CD/5-FC system on the human liver cancer cells. The cell deaths were 18.07%, 42.98% and 62.20% respectively when the concentrations of prodrug were at 10, 20 and 30 mmol x L(-1). In 5-FC acute toxicity experiment, Kunming mice were injected with different concentrations of 5-FC at intervals of 1:0.5. The LD50 of 5-FC through iv injection was determined by improved Karber's method, the LD50 was 507 mg x kg(-1) and the 95% confidence limit was 374-695 mg x kg(-1). According to the maximum LD0 dose of the LD50, the maximum safe dose was 200 mg x kg(-1). Body weight and clinic symptoms of the experimental animals were observed. These results laid the foundation to verify the antitumor effect and safety of CD/5-FC system in animal models. The CD gene was ligated into the NDV (rClone30) carrier, then the tumor-bearing animal was established to perform the tumor inhibiting experiment. The result showed that the recombinant rClone30-CD/5-FC system has a high antitumor activity in vivo. To summarize, CD gene has been cloned and its bioactivity has been confirmed in the mammalian cells. It is the first time in this study to utilize the recombinant NDV to deliver the CD gene into the tumor cells; our result proves the rClone30-CD/5-FC system is a potential method for cancer therapy.

[1]  Zheng Wang,et al.  [Primary safety evaluation of sulfated paeoniae radix alba]. , 2012, Yao xue xue bao = Acta pharmaceutica Sinica.

[2]  D. Singleton,et al.  Bystander or No Bystander for Gene Directed Enzyme Prodrug Therapy , 2009, Molecules.

[3]  Toshiaki Tanaka,et al.  Bystander effect from cytosine deaminase and uracil phosphoribosyl transferase genes in vitro: a partial contribution of gap junctions. , 2009, Cancer letters.

[4]  A. García-Sastre,et al.  Enhancement of oncolytic properties of recombinant newcastle disease virus through antagonism of cellular innate immune responses. , 2009, Molecular therapy : the journal of the American Society of Gene Therapy.

[5]  V. Pierrefite-Carle,et al.  Treatment of colon cancer cells using the cytosine deaminase/5-fluorocytosine suicide system induces apoptosis, modulation of the proteome, and Hsp90β phosphorylation , 2007, Molecular Cancer Therapeutics.

[6]  R. McIvor,et al.  Coexpression of the uracil phosphoribosyltransferase gene with a chimeric human nerve growth factor receptor/cytosine deaminase fusion gene, using a single retroviral vector, augments cytotoxicity of transduced human T cells exposed to 5-fluorocytosine. , 2006, Human gene therapy.

[7]  H. Kaş,et al.  5-Fluorouracil encapsulated alginate beads for the treatment of breast cancer. , 2002, International journal of pharmaceutics.

[8]  G. Milano,et al.  Cytosine deaminase/5-fluorocytosine-based vaccination against liver tumors: evidence of distant bystander effect. , 1999, Journal of the National Cancer Institute.

[9]  Xu Xiao-yu.  Study on toxicity of Yin-Ling troche , 2012 .

[10]  W. Xiaomei,et al.  Generation of Recombinant Newcastle Disease Virus LaSota Vaccine Strain Expressing VP2 Gene of Very Virulent Infectious Bursal Disease Virus Isolated from cDNA Clone , 2008 .

[11]  Zhang Kebin.  Cytosine deaminase suicide gene transfection and its expression on U251 malignant brain glioma cells , 2006 .

[12]  R. Blaese,et al.  Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: a negative selection system. , 1992, Proceedings of the National Academy of Sciences of the United States of America.