Interferon-γ (IFN-γ), as a multifunctional cytokine, exerts diverse biological functions related to host defense and immune regulation, such as inflammation, innate and acquired immunity, cell cycle and apoptosis . IFN-γ plays a critical role in promoting protective host responses to tumors, which proposed mechanisms include, (a) anti-proliferative and pro-apoptotic actions, (b) anti-angiogenesis in tumors, and (c) promoting both the innate and adoptive immune responses against tumors [20, 21].
Although IFN-γ has been investigated as a potential therapeutics for various types of tumors [6–13], the attempts to improve antitumor efficacy by increasing the dose or by repetitive continuous administration resulted in higher toxicity and low efficacy . Considerable experimental data from our group and other investigators demonstrated that intratumoral IFN-γ gene transfer to achieve long-term, continuous locoregional exposure of IFN-γ is likely an appropriate approach for improving efficacy and reducing toxicity of IFN-γ [9, 10, 15, 16, 23, 24]. In this study, we reported that adenovirus-mediated IFN-γ gene transfer (Ad-IFNγ) inhibited tumor growth of human nasopharyngeal carcinoma (NPC) cells (Figure 2 and Figure 5). Here the anti-NPC activity of IFN-γ included not only direct anti-proliferative and pro-apoptotic actions, also indirect mechanisms, such as immunomodulation and antiangiogenesis.
Ad-IFNγ efficiently expressed human IFN-γ in nasopharyngeal carcinoma cells in vitro and in vivo (Figure 1 and Figure 3), and exhibited strong antiproliferative effects (Figure 2A and Figure 6). According to previous reports, the antiproliferative mechanisms of IFN-γ seem to be cell type specific [9, 25–29], either induction of cell cycle arrest or apoptosis. Here we found that both G1 phase arrest and apoptosis contributed to Ad-IFNγ-mediated growth suppression in NPC cell lines (Figure 2B, Figure 3 and Figure 6), consistent with the report that the antiproliferative effects of minicircle-IFNγ on NPC cell lines could be attributed to G1 arrest and apoptosis . The JAK/STAT pathway may be responsible for most biological effects mediated by IFN-γ [20, 30], which regulates different cell cycle-associated proteins that control the G1-S checkpoint , or induce cell apoptosis through up-regulating the expression of various apoptosis-related proteins in different cell types [31–33].
Although antiproliferation and apoptosis induction were main effects of Ad-IFNγ-mediated anti-NPC in this study, antiangiogenesis may contribute also to growth inhibition of NPC xenografts in nude mice. Because the microvessel densities (MVDs) were found to be decreased in the xenografts treated with Ad-IFNγ compared with those treated with Ad-LacZ or PBS in our study (data not shown). Previous studies reveal that IP-10, an inhibitor of angiogenesis, could be induced by IFN-γ in endothelial cells and exert potent antiangiogenesis activity by inhibiting endothelial cell differentiation, motility and tube formation [34, 35]. However, due to highly species specificity, human unlikely exerts direct effects on murine vascular system, but likely acts on tumor cells to indirectly regulate angiogenesis. It was reported that IFN-γ acted as antiangiogenic cytokine by inhibiting the expression of angiogenic factors, such as VEGF or perlecan, in renal cell carcinoma , stromal fibroblasts , human cornea , and WiDr/HT29 colon carcinoma cells, or by inducing the expression of anti-angiogenic factor monokine induced by interferon-gamma (MIG, or CXCL9) in non-small cell lung carcinom . Here we would like to make a hypothesis that IFN-γ may exert antiangiogenic effect in nude mice carrying NPC xenografts by regulating the expression of some angiogenic or antiangiogenic factors in NPC cells. This hypothesis, of course, needs to be tested and verified.
Human and murine IFN-γ display low level of sequence homology (only 40%) at protein level, which explains why the human and murine proteins display a strict species specificity in their ability to bind to and activate human and murine cells . So human IFN-γ has less activity in the nude mouse host, though nude mice display potent macrophage and NK cell activity [42–44] and remain some basal T-cell function . Direct immunomodulation on mouse immune system by human IFN-γ unlikely contributes to its antitumor effects. However, we cannot exclude the possibility that other indirect effects are involved in Ad-IFNγ-mediated antitumor immune response. Firstly, human IFN-γ could modify the expression of MHC and costimulatory molecules or cytokines, chemokines on human NPC cells [21, 46], which may activate the residual immune system of nude mice. Secondly, adenoviral vector and heterogenous IFN-γ expression likely induce a nonspecific immune response directed by NK and/or microphage cells in nude mice .
Taken together, Ad-IFNγ displayed efficient anti-NPC activities by inhibiting tumor cell proliferation and induced cell apoptosis in this study. Additional indirect effects on antiangiogenesis and immunomodulatory may also be involved in this antitumor activity. So IFN-γ gene therapy by a replication defective adenovirus encoding the human IFN-γ (Ad-IFNγ) is likely a potential novel therapeutics on comprehensive therapy of nasopharyngeal carcinoma.
Nevertheless, recent reports have shown that the immune response activated by IFN-γ plays a dual role in cancer: It can not only suppress tumor growth by destroying cancer cells or inhibiting their outgrowth but also promote tumor progression either by selecting for tumor cells that are more fit to survive in an immunocompetent host or by establishing conditions within the tumor microenvironment that facilitate tumor outgrowth . IFN-γ treatment is a double-edged sword whose anti- and protumorigenic activities are dependent on the cellular, microenvironmental, and/or molecular context . Thereby, more investigations should be carried out to clarify the influences of cellular, microenvironmental, immunological and molecular events on the anti-NPC effects of Ad-IFNγ before its clinical application.