Endogenous nitrogen and oxygen free radicals are involved in many biological processes including mediators of damage in the immune response to tumors. Nitric oxide (NO), produced from activated macrophages by immunostimulant(s), is a major effector for tumor cell killing and induces apoptosis. Reactive oxygen species (ROS), originated mostly from mitochondrial respiration, cause cell injury directly or through apoptosis. The applicant wishes to investigate the potential of NO as a chemotherapeutic agent in ovarian cancer. There is a clear need for new treatment modalities to overcome problem of drug-resistant ovarian cancer. Work by others and the applicant have shown that cisplatin can be used as a cytotoxic agent, a macrophage activator and a transduction facilitator specific to tumor cells, simultaneously. The applicant proposes to use free radical-generating genes in conjunction with cisplatin to enhance transgene expression and antitumor activity. Short-lived free radicals can be endogenously and continuously produced by direct gene transfer into the tumor microenvironment to mediate a localized or regional immune response, which ultimately inhibits tumor growth or kills tumor cells.
Three specific aims are proposed.
Aim I is to demonstrate in vitro and in vivo cytotoxicity of free radical-generating genes, inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF), in drug-resistant human epithelial ovarian carcinomas. The applicant suggests that the effectiveness of cisplatin-based gene therapy in murine ovarian carcinoma is mainly due to NO produced by activated macrophages. This observation will be confirmed by iNOS-knockout mice. Further NO-mediated tumor cell killing will be examined by using iNOS-transfectants in culture and animals. The applicant will also examine a synergistic cooperation between iNOS and the TNF genes and the effect of these genes on cisplatin sensitivity to tumor cells.
Aim II is to develop a high and sustained gene expression system using three approaches, an iNOS gene with iron-responsive element at its 3' end to stabilize iNOS mRNA, TGFb antisense plasmid to increase the stability of iNOS mRNA and protein, and cisplatin multivesicular liposome to prolong the desired cisplatin level in ascites and thus to sustain expression of the transgene.
Aim III is to critically evaluate the effectiveness of the proposed gene therapy protocol by combining Aim I and II for the treatment of peritoneal metastatic human ovarian cancer resistant to anticancer drugs including cisplatin.
| Son, K K; Tkach, D; Rosenblatt, J (2001) Delipidated serum abolishes the inhibitory effect of serum on in vitro liposome-mediated transfection. Biochim Biophys Acta 1511:201-5 |
| Son, K K; Rosenblatt, J (2001) Overexpression of protein kinase A - RIalpha reduces lipofection efficiency of cisplatin-resistant human tumor cells. Cancer Lett 165:81-6 |
| Son, K K; Tkach, D; Patel, D H (2000) Zeta potential of transfection complexes formed in serum-free medium can predict in vitro gene transfer efficiency of transfection reagent. Biochim Biophys Acta 1468:4-Nov |
| Son, K K; Tkach, D; Hall, K J (2000) Efficient in vivo gene delivery by the negatively charged complexes of cationic liposomes and plasmid DNA. Biochim Biophys Acta 1468:10-Jun |
| Son, K K; Hall, K J (2000) Nitric oxide-mediated tumor cell killing of cisplatin-based interferon-gamma gene therapy in murine ovarian carcinoma. Cancer Gene Ther 7:1324-8 |
