A. Identification of type I IFN signaling pathway associated with antitumor activity 1. TRAIL is one of the mediators for inhibiting cell viability after IFN-alpha treatment. TRAIL, but not FasL, inhibited the viability of OVCAR-3 cells sensitive to IFN-alpha 2c. Immunoaffinity capillary electrophoresis showed that there were significant increases of TRAIL in the supernatant and on the membrane, but not the cytosol, of OVACR-3 cells after IFN-alpha 2c treatment. 2. After treatment with IFN-alpha 2c, OVCAR-3 cell viability was restored by Caspase 8 inhibitor and Bid-siRNA but not by PARP inhibitor which suggests that the caspase 8/Bid pathway is predominantly responsible for both IFN-alpha 2c and TRAIL signaling pathways in the regulation of cell viability. The cleavage of Bid plays an important role in the collapse of the mitochondrial membrane potential in response to IFN-alpha 2c and TRAIL. Bid-siRNA or the Bid inhibitor BI6C9 prevented this collapse following treatment of cells with these agents. In A549 cells, Bid overexpression resulted in a significant decrease in A549 viability following treatment with IFN-alpha 2 or TRAIL. In addition, it has been shown that inhibition of Bid either by specific Bid inhibitors or Bid-siRNA attenuates the growth inhibitory and apoptotic effects of IFN-alpha 2a in OVCAR-3 cells. 3. IFN-alpha 2a induced apoptosis is accompanied by Bak oligomerization. Using a cross-linking reagent, it was shown that treatment of OVCAR-3 cells with IFN-alpha 2a resulted in Bak protein complexes but not Bax protein complexes. In addition, Bak-siRNA significantly attenuated IFN-alpha 2a-induced cytotoxicity in OVCAR-3 cells suggesting that this cytotoxicity is predominantly mediated by Bid translocation to the mitochondria which promotes Bak-controlled mitochondrial membrane potential. 4. AIF and not apoptosome formation is responsible for cell death in OVCAR3 in response to IFN-alpha 2. In the process of apoptosis, a large protein structure known as the apoptosome is sometimes formed and this is triggered by the release of cytochrome c from the mitochondria. In our studies we have shown that apoptosome formation is not required for IFN-alpha 2a-induced Bid-mediated cell death. A gene silencing approach was used to examine the potential role of AIF downstream of Bid-mediated mitochondrial depolarization through Bak oligomerization. It was shown that AIF-siRNA significantly attenuated IFN-alpha 2a-induced cell death in OVCAR-3 cells while cytochrome c-siRNA and EndoG-siRNA did not, thus suggesting that AIF but not cytochrome c and EndoG is responsible for IFN-alpha 2a-induced Bid-mediated caspase dependent execution of cell death. Bid inhibition prevents the release of AIF from the mitochondria by IFN-alpha 2a. This project has been completed. B. Antitumor Activity if type I and II human interferon-treated monocytes. 1. Examination of 7 human tumor lines and 3 diploid cell lines with physiological concentrations of IFN-alpha 2 treated monocytes showed strong cytocidal activity against 4 of the tumors, OVCAR-3, HOS osteosarcoma, A549 Lung and LOX melanoma, but not against the diploid lines. High concentrations of tumor cells required high concentrations of IFN-alpha and -gamma and monocytes to cause near eradication of tumor cells. 2. Intratumoral administration of human monocytes and IFN-alpha 2 and -gamma into established tumors using a nude mouse xenograft model reduced tumor size and prolonged survival of mice for both OVCAR-3 (ovarian) and LOX (melanoma) tumors. Treatment of 15 d established ovarian tumors resulted in significant inhibition of tumor growth and complete response in 40% of the mice. Significant tumor inhibition was also seen in treatment of 30 d established ovarian tumors. A slight delay in tumor growth was observed with either IFNs alone or monocytes alone. Repeat treatment in early or late treatment models made no additional significant difference in OVCAR-3 tumor regression. LOX melanoma tumor growth was inhibited when treatment occurred on 0 d or 3 d but this was not the case when tumors were treated later (9 d). IFN-activated macrophages accumulated in the OVCAR-3 tumors. In tissue sections of tumors harvested 2 d after combination treatment, the phenotype of the macrophages which infiltrated the OVCAR-3 tumors was CD31+/CD68+.They were shown to be classically activated macrophages (M1) by exhibiting a high IL-12/low IL-10 profile and by staining positively for the M1 biomarkers CXCL10 and NOS2. Use of the TUNEL assay allowed us to determine that the number of apoptotic cells in OVCAR-3 tumors was greater after combination therapy as opposed to single treatments with either IFNs alone or monocytes alone.
