Tongmao Zhao

IRF9 is a key factor for eliciting the antiproliferative activity of IFN-α

A number of tumors are still resistant to the antiproliferative activity of human interferon (IFN)-α. The Janus kinases/Signal Transducers and Activators of Transcription (JAK-STAT) pathway plays an important role in initial IFN signaling. To enhance the antiproliferative activity of IFN-α, it is important to elucidate which factors in the JAK-STAT pathway play a key role in eliciting this activity. In human ovarian adenocarcinoma OVCAR3 cells sensitive to both IFN-α and IFN-γ, only IFN regulatory factor 9 (IRF9)-RNA interference (RNAi) completely inhibited the antiproliferative activity of IFN-α among the intracellular JAK-STAT pathway factors. Conversely, Stat1-RNAi did not inhibit the antiproliferative activity of IFN-α, whereas it partially inhibited that of IFN-γ. As a cell death pathway, it is reported that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through TRAIL-receptor (R) 1 and TRAIL-R2. In IFN-α-treated OVCAR3 cells, IRF9-RNAi inhibited transcription of TRAIL whereas Stat1-RNAi did not, suggesting that the transcription of TRAIL induced by IFN-α predominantly required IRF9. Furthermore, IFN-stimulated response element-like motifs of TRAIL bound to IFN-stimulated gene factor 3 (ISGF3) complex after IFN-α treatment. Subsequently, TRAIL-R2-RNAi inhibited both antiproliferative activities of IFN-α and TRAIL, suggesting that TRAIL-R2 mediated both IFN-α and TRAIL signals to elicit their antiproliferative activities. Finally, IRF9 overexpression facilitated IFN-α-induced apoptosis in T98G (human glioblastoma multiforme) cells, which were resistant to IFN-α. Thus, this study suggests that IRF9 is the key factor for eliciting the antiproliferative activity of IFN-α and TRAIL may be one of the potential mediators.

Apoptosis-inducing factor (AIF) is targeted in IFN-α2a-induced Bid mediated apoptosis through Bak activation in ovarian cancer cells

Previously we have shown that interferon (IFN)-α induced apoptosis is predominantly mediated by the upregulation of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) via the caspase-8 pathway. It was also shown that recruitment of mitochondria in IFN-α induced apoptosis involves the cleavage of BH3 interacting domain death agonist (Bid) to truncated Bid (tBid). In the present study, we demonstrate that tBid induced by IFN-α2a activates mitochondrial Bak to trigger the loss of mitochondrial membrane integrity, consequently causing release of apoptosis-inducing factor (AIF) in ovarian cancer cells, OVCAR3. AIF translocates from the mitochondria to the nucleus and induces nuclear fragmentation and cell death. Both a small molecule Bid inhibitor (BI-6C9) or Bid-RNA interference (RNAi) preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated IFN-α2a-induced cell death. Cell death induced by tBid was inhibited by AIF-RNAi, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that BI-6C9 did not prevent the release of cytochrome c from mitochondria to cytosol, while the release of AIF was prevented. In conclusion, IFN-α2a-induced apoptosis is mediated via the mitochondria-associated pathway involving the cleavage of Bid followed by AIF release that involves Bak activation and translocation of AIF from the mitochondria to the nucleus in OVCAR3 cells.

BID is a critical factor controlling cell viability regulated by IFN-α

Clinical applications of human interferon (IFN)-&agr; have met with varying degrees of success. Nevertheless, key molecules in cell viability regulated by IFN-&agr; have not been clearly identified. Our previous study indicated that IFN (&agr;, &bgr;, and &ohgr;) receptor (IFNAR) 1/2- and IFN regulatory factor 9-RNA interference (RNAi) completely restored cell viability after IFN-&agr; treatment in human ovarian adenocarcinoma OVCAR3 cells sensitive to IFN-&agr;. In this study, IFNAR1/2- and IFN regulatory factor 9-RNAi inhibited the gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not of Fas ligand, after IFN-&agr; treatment. In fact, TRAIL but not Fas ligand inhibited the viability of OVCAR3 cells. IFN-&agr; notably upregulated the levels of TRAIL protein in the supernatant and on the membrane of OVCAR3 cells. After TRAIL signaling, caspase 8 inhibitor and BH3 interacting domain death agonist (BID)-RNAi significantly restored cell viability in response to IFN-&agr; and TRAIL in OVCAR3 cells. Furthermore, BID-RNAi prevented both IFN-&agr; and TRAIL from collapsing the mitochondrial membrane potential (&Dgr;&PSgr;m). Finally, we provided important evidence that BID overexpression led to significant inhibition of cell viability after IFN-&agr; or TRAIL treatments in human lung carcinoma A549 cells resistant to IFN-&agr;. Thus, this study suggests that BID is crucial for cell viability regulated by IFN-&agr; which can induce mitochondria-mediated apoptosis, indicating a notable potential to be a targeted therapy for IFN-&agr; resistant tumors.

Near Eradication of Clinically Relevant Concentrations of Human Tumor Cells by Interferon-Activated Monocytes In Vitro

We have previously reported that low concentrations of interferon (IFN)-activated monocytes exert near-eradicative cytocidal activity against low concentrations of several human tumor cells in vitro. In the present study, we examined 7 human tumor cell lines and 3 diploid lines in the presence or absence of 10 ng/mL IFNα2a and monocytes. The results confirmed strong cytocidal activity against 4 of 7 tumor lines but none against 3 diploid lines. To model larger in vivo tumors, we increased the target cell concentration and determined the concentration of IFNα2a and monocytes, required for cell death. We found that increasing the tumor cell concentration from 10- to 100-fold (10(5) cells/well) required an increase in the concentration of IFNs by over 100-fold and monocytes by 10-fold. High concentrations of monocytes could sometimes kill tumor or diploid cells in the absence of IFN. We may conclude that killing of high concentrations of tumor or diploid cells required high concentrations of monocytes that could sometimes kill in the absence of IFN. Thus, high concentrations of tumor cells required high concentrations of IFN and monocytes to cause near eradication of tumor cells. These findings may have clinical implications.