Antitumor Activity of Cationic Liposome-Mediated Bax Mrna Transfer in HOSM-1 Mandibular Osteosarcoma Cells: A Comparative Study of Local Administration and Systemic Administration

Statement of the problem: Osteosarcoma is one of the malignant tumors that has a poor prognosis. In particular, relapsed or inoperable cases may require the development of new therapeutic approaches. Gene therapy is an innovative treatment for cancer, and is currently being investigated for osteosarcoma.  Bax, one of the Bcl-2 family proteins, is a pro-apoptotic molecule that functions as a tumor suppressor, and Bax gene therapy has been investigated for various tumor cells. Gene transfer that is essential for the gene therapy is more efficient by mRNA lipofection than by plasmid DNA lipofection¹⁾. Also as an effective mRNA-based cancer vaccine by systemic injection of MART1 mRNA has been reported in B16 melanoma²⁾. We examined the antitumor effect of the Bax mRNA transfected using a cationic liposome against human mandibular osteosarcoma cells in vitro, and further studied its antitumor effects in vivo comparing the differences between local administration and systemic administration.

Material and Method: HOSM-1 is a human mandibular osteosarcoma cell line.  The liposome was made by mixing a neutral lipid, DOPE, and a cationic lipid, DOTAP, in a 1:1 molar ratio using a rotary evaporator.  Bax mRNA was produced from the Bax plasmid by the in vitro synthesis.  For in vitro study, transfection efficiency was evaluated using a GFP assay, the level of Bax protein expression was determined by Western blotting, and apoptosis was detected with a TUNEL assay.  For in vivo study, HOSM-1 cells were inoculated s.c. into the backs of the nude mice.  After the tumor size reached 5-6 mm in diameter, treatment (see below) of the animals was initiated (day 0). Liposome-Bax mRNA(50µg in total) was locally injected to the tumor periphery, or systemically administered by subcutaneous, or intravenous injection. Tumor growth was evaluated by measuring the tumor volume.

Methods of data analysis: The difference in transfection efficiency was analyzed using Student’s t-test.  Statistical analyses of other assays were performed using ANOVA with Tukey-Kramer multiple comparisons post-tests.  P values < 0.05 were considered to be statistically significant.

Results: Transfection efficiency (the percentage of GFP-positive cells) was 92.9 % for Lip-EGFP mRNA and 62.2 % for Lip-EGFP plasmid, showing an advantage of the cationic liposome-mediated transfer of mRNA. In the cells treated with Bax mRNA, expression of Bax protein was enhanced, and apoptotic cells were increased (52.9 %). TUNEL-positive cells were significantly richer after Bax mRNA transfer than Bax plasmid transfer. By local injection in mice, the mean tumor volume on day 20 was 34.8 % and 45.8 % of that in the saline control group in liposome-Bax mRNA and Bax plasmid administration groups, respectively. The tumor volume after subcutaneous injection was 79.5 % and 88.6 % with liposome-Bax mRNA and Bax plasmid, respectively. The tumor volume after intravenous administration was 23.3 % and 47.2 % with liposome-Bax mRNA and Bax plasmid, respectively.

Conclusion: Bax mRNA therapy using liposomes has more anti-tumor effects than Bax gene therapy using a plasmid, and intravenous administration was the most effective route of administration in this study. These results suggest a possibility of Bax mRNA lipofection as a treatment for a human mandibular osteosarcoma.

References: 1) Okumura K, Nakase M, Inui M, Nakamura S, Watanabe Y, Tagawa T. Bax mRNA therapy using cationic liposomes for human malignant melanoma. J Gene Med 2008; 10: 910-917.

2) Mockey M, Bourseau E, Chandrashekhar V, et al. mRNA-based cancer vaccine: prevention of B16 melanoma progression and metastasis by systemic injection of MART1 mRNA histidylated lipopolyplexes: Cancer Gene Therapy.2007;14:802-814.