Mechanisms Enhancing the Cytotoxic Effects of Bleomycin plus Suicide or Interferon-β Gene Lipofection in Metastatic Human Melanoma Cells.

Background: Three metastatic human melanoma cell lines generated from patient removed lymph nodes and spleen metastasis were established in our laboratory. Objective: To investigate the mechanisms enhancing the cytotoxic effects of Bleomycin (BLM), herpes simplex virus thymidine kinase/ganciclovir Suicide Gene (SG) and human interferon-beta gene (hIFN beta) lipofection in early passages of these melanoma cell lines. Methods: In these cell lines, we determined: cytotoxicity, bystander effect, lipofection efficiencies, apoptosis, necrosis, senescence, colony fonning capacity and mitochondrial membrane depolarization after treatments. Results: The three assayed cell lines displayed sensitivity to single and combined BIM/gene treatments. RIM improved the antitumor and anti-clonogenic effects of SG and hIFN beta genes. Considering the low lipofection efficiencies (<10%), one of the main causes of the SG and hIFN beta gene effectiveness was their bystander effect. In one of these cell lines, this effect eradicated up to 60% of the cells although <1% expressed the transgene. In the three cell lines, BLM alone or combined with SG or hIFN beta gene significantly increased the percentage of cells exhibiting membrane compromise, DNA damage, and senescence. Interestingly, the strong BLM/hIFN beta gene combination was able to generate from 73% to 98% of non-viable cells. The high proportion of senescent cells induced by BLM alone or combined with genes strongly decreased the clonogenic capacity of surviving cells. Conclusion: The presented results indicate that BLM improves the antitumor effects of SG and hIFN beta transgene expression. Altogether, these findings strongly support the clinical potential of these combined approaches.