Abstract: Objective: To evaluate the efficacy of cationic liposome-mediated CD40 ligand (CD40L) gene therapy for hepatocellular carcinoma. Methods: 1×10⁶ of parental H22 cells or H22 cells transfected with the expression vector containing murine CD40L cDNA encoding the entire coding region (pcDNA3.1⁺-mCD40L) were inoculated subcutaneously into the left flanks of syngenic BALB/C mice. The tumor-bearing mice (tumor nodules 10 mm in maximal diameter) received the treatment of the intratumoral injection of pcDNA3.1⁺-mCD40L/Transfectam, pcDNA3.1⁺, or phosphate-buffered saline (PBS), or no treatment. The mice were monitored for tumor growth weekly. We examined mCD40L messenger ribonucleic acid (mRNA) expression by reverse transcription polymerase chain reaction (RT-PCR) and the histologic changes in tumors at two weeks after intratumoral injection using immunohistochemical staining of tumor tissues. Results: All mice inoculated with parental H22 cells developed a tumor subcutaneously, and the tumor size increased progressively within three weeks. However, the mice receiving H22-CD40L cells exhibited complete regression of the tumor two weeks after tumor cell inoculation. The tumor-bearing animals with the treatment of pcDNA3.1⁺ or PBS, or without treatment had progressive tumor growth, while those mice treated with pcDNA3.1⁺-mCD40L exhibited a significant inhibition of tumor growth. RT-PCR analysis showed that 783-bp fragments corresponding to the mCD40L mRNA were amplified only from pcDNA3.1⁺-mCD40L treated tumors. The tumor samples from pcDNA3.1⁺-mCD40L-treated mice showed significant lymphocyte infiltration, apoptotic bodies, and confluent necrosis in the tumor tissues. Conclusion: The tumorigenicity of CD40L-expressing cells was abrogated when the cells were implanted subcutaneously. In vivo gene therapy of established liver tumor nodules in mice by the intratumoral injection of pcDNA3.1⁺-mCD40L led to significant tumor inhibition. There was mCD40L mRNA expression in the tissues from pcDNA3.1⁺-mCD40L-treated tumors. The intratumoral injection of pcDNA3.1⁺-mCD40L induced a strong inflammatory, mainly lymphocytic infiltration of the tumor, and increased the necrotic rate of the neoplastic cells.

Key words: CD40 ligand gene, Hepatocellular carcinoma (HCC), Cationic liposome, Transfection

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