Coordinate regulation of IFN consensus sequence-binding protein and caspase-1 in the sensitization of human colon carcinoma cells to Fas-mediated apoptosis by IFN-gamma

Interferon-gamma is thought to be essential for the regulation of antitumor reactions. However, the degree of responsiveness of malignant cells to IFN-gamma may have a profound influence on the overall efficacy of an antitumor response. In this study, we examined the molecular basis by which IFN-gamma differentially sensitized human primary and metastatic colon carcinoma cells to Fas-mediated apoptosis. To that end, we analyzed IFN-gamma-induced gene expression at the genome scale, followed by an analysis of the expression and function of specific genes associated with IFN-gamma- and Fas-mediated signaling. We found that although both cell populations exhibited a similar gene expression profile at the genome scale in response to IFN-gamma, the expression intensities of the IFN-gamma-regulated genes were much greater in the primary tumor. Noteworthily, two genes, one involved in IFN-gamma-mediated signaling, IFN consensus sequence-binding protein (ICSBP), and one involved in Fas-mediated signaling, caspase-1, were clearly shown to be differentially induced between the two cell lines. In the primary tumor cells, the expression of ICSBP and caspase-1 was strongly induced in response to IFN-gamma, whereas they were weakly to nondetectable in the metastatic tumor cells. Functional studies demonstrated that both caspase-1 and ICSBP were involved in Fas-mediated apoptosis following IFN-gamma sensitization, but proceeded via two distinct pathways. This study also reports for the first time the expression of ICSBP in a nonhemopoietic tumor exhibiting proapoptotic properties. Overall, in a human colon carcinoma cell model, we identified important functional contributions of two IFN-gamma-regulated genes, ICSBP and caspase-1, in the mechanism of Fas-mediated death.     

Tumor suppressor IRF-1 mediates retinoid and interferon anticancer signaling to death ligand TRAIL

Retinoids and interferons are signaling molecules with pronounced anticancer activity. We show that in both acute promyelocytic leukemia and breast cancer cells the retinoic acid (RA) and interferon signaling pathways converge on the promoter of the tumoricidal death ligand TRAIL. Promoter mapping, chromatin immunoprecipitation and RNA interference reveal that retinoid-induced interferon regulatory factor-1 (IRF-1), a tumor suppressor, is critically required for TRAIL induction by both RA and IFNgamma. Exposure of breast cancer cells to both antitumor agents results in enhanced TRAIL promoter occupancy by IRF-1 and coactivator recruitment, leading to strong histone acetylation and synergistic induction of TRAIL expression. In coculture experiments, pre-exposure of breast cancer cells to RA and IFNgamma induced a dramatic TRAIL-dependent apoptosis in heterologous cancer cells in a paracrine mode of action, while normal cells were not affected. Our results identify a novel TRAIL-mediated tumor suppressor activity of IRF-1 and suggest a mechanistic basis for the synergistic antitumor activities of certain retinoids and interferons. These data argue for combination therapies that activate the TRAIL pathway to eradicate tumor cells.     

Preexisting antitumor immunity augments the antitumor effects of chemotherapy

Efficacy of cancer chemotherapy is generally believed to be the result of direct drug killing of tumor cells. However, increased tumor cell killing does not always lead to improved efficacy. Herein, we demonstrate that the status of antitumor immunity at the time of chemotherapy treatment is a critical factor affecting the therapeutic outcome in that tumor-bearing mice that possess preexisting antitumor immunity respond to chemotherapy much better than those that do not. Enhancing antitumor immunity before or at the time of chemotherapy-induced antigen release increases subsequent response to chemotherapy significantly. By in vitro and in vivo measurements of antitumor immunity, we found a close correlation between the intensity of antitumor immunity activated by chemotherapy and the efficacy of treatment. Immune intervention with interleukin-12 during the early phase of chemotherapy-induced immune activation greatly amplifies the antitumor response, often resulting in complete tumor eradication not only at the chemo-treated local site, but also systemically. These findings provide additional evidence for an immune-mediated antitumor response to chemotherapy. Further, our results show that timely immune modification of chemotherapy-activated antitumor immunity can result in enhanced antitumor-immune response and complete tumor eradication.     

Lithium sensitizes tumor cells in an NF-kappa B-independent way to caspase activation and apoptosis induced by tumor necrosis factor (TNF). Evidence for a role of the TNF receptor-associated death domain protein

We have previously shown that lithium salts can considerably increase the direct cytotoxic effect of tumor necrosis factor (TNF) on various tumor cells in vitro and in vivo. However, the underlying mechanism has remained largely unknown. Here we show that the TNF-sensitizing effect of lithium chloride (LiCl) is independent of the type of cell death, either necrosis or apoptosis. In the case of apoptosis, TNF/lithium synergism is associated with an enhanced activation of caspases and mitochondrial cytochrome c release. Sensitization to apoptosis is specific for TNF-induced apoptosis, whereas Fas-mediated or etoposide-induced apoptosis remains unaffected. LiCl also potentiates cell death induced by artificial oligomerization of a fusion protein between FKBP and the TNF receptor-associated death domain protein. TNF-induced activation of NF-kappa B-dependent gene expression is not modulated by LiCl treatment. These results indicate that LiCl enhances TNF-induced cell death in an NF-kappa B-independent way, and suggest that the TNF receptor-associated death domain protein plays a crucial role in the TNF-sensitizing effect of LiCl.     

Irradiation of tumor cells up-regulates Fas and enhances CTL lytic activity and CTL adoptive immunotherapy

CD8(+) CTL play important roles against malignancy in both active and passive immunotherapy. Nonetheless, the success of antitumor CTL responses may be improved by additional therapeutic modalities. Radiotherapy, which has a long-standing use in treating neoplastic disease, has been found to induce unique biologic alterations in cancer cells affecting Fas gene expression, which, consequently, may influence the overall lytic efficiency of CTL. Here, in a mouse adenocarcinoma cell model, we examined whether exposure of these tumor cells to sublethal doses of irradiation 1) enhances Fas expression, leading to more efficient CTL killing via Fas-dependent mechanisms in vitro; and 2) improves antitumor activity in vivo by adoptive transfer of these Ag-specific CTL. Treatment of carcinoembryonic Ag-expressing MC38 adenocarcinoma cells with irradiation (20 Gy) in vitro enhanced Fas expression at molecular, phenotypic, and functional levels. Furthermore, irradiation sensitized these targets to Ag-specific CTL killing via the Fas/Fas ligand pathway. We examined the effect of localized irradiation of s.c. growing tumors on the efficiency of CTL adoptive immunotherapy. Irradiation caused up-regulation of Fas by these tumor cells in situ, based on immunohistochemistry. Moreover, localized irradiation of the tumor significantly potentiated tumor rejection by these carcinoembryonic Ag-specific CTL. Overall, these results showed for the first time that 1) regulation of the Fas pathway in tumor cells by irradiation plays an important role in their sensitization to Ag-specific CTL; and 2) a combination regimen of tumor-targeted irradiation and CTL promotes more effective antitumor responses in vivo, which may have implications for the combination of immunotherapy and radiation therapy.     

Fas mediated apoptosis of human Jurkat T-cells: intracellular events and potentiation by redox-active alpha-lipoic acid.

Activation of caspases is required in Fas receptor mediated apoptosis. Maintenance of a reducing environment inside the cell has been suggested to be necessary for caspase activity during apoptosis. We explored the possibility to potentiate Fas mediated killing of tumor cells by alpha-lipoic acid (LA), a redox-active drug and nutrient that is intracellularly reduced to a potent reductant dihydrolipoic acid. Treatment of cells with 100 microM LA for 72 h markedly potentiated Fas-mediated apoptosis of leukemic Jurkat cells but not that of peripheral blood lymphocytes from healthy humans. In Jurkat, Fas activation was followed by rapid loss of cell thiols, decreased mitochondrial membrane potential, increased [Ca2+]i and increased PKC activity; all these responses were potentiated in LA pretreated cells. PKCdelta played an important role in mediating the effect of LA on Fas-mediated cell death. In response to Fas activation LA treatment potentiated caspase 3 activation by over 100%. The ability of LA to potentiate Fas mediated killing of leukemic cells was abrogated by a caspase 3 inhibitor suggesting that increased caspase 3 activity in LA-treated Fas-activated cells played an important role in potentiating cell death. This work provides first evidence showing that inducible caspase 3 activity may be pharmacologically up-regulated by reducing agents such as dihydrolipoic acid.     

Sensitization of tumor cells to NK cell-mediated killing by proteasome inhibition

Bortezomib is a proteasome inhibitor that has direct antitumor effects. We and others have previously demonstrated that bortezomib could also sensitize tumor cells to killing via the death ligand, TRAIL. NK cells represent a potent antitumor effector cell. Therefore, we investigated whether bortezomib could sensitize tumor cells to NK cell-mediated killing. Preincubation of tumor cells with bortezomib had no effect on short-term NK cell killing or purified granule killing assays. Using a 24-h lysis assay, increases in tumor killing was only observed using perforin-deficient NK cells, and this increased killing was found to be dependent on both TRAIL and FasL, correlating with an increase in tumor Fas and DR5 expression. Long-term tumor outgrowth assays allowed for the detection of this increased tumor killing by activated NK cells following bortezomib treatment of the tumor. In a tumor purging assay, in which tumor:bone marrow cell mixtures were placed into lethally irradiated mice, only treatment of these mixtures with a combination of NK cells with bortezomib resulted in significant tumor-free survival of the recipients. These results demonstrate that bortezomib treatment can sensitize tumor cells to cellular effector pathways. These results suggest that the combination of proteasome inhibition with immune therapy may result in increased antitumor efficacy.     

Lack of FasL-mediated killing leads to in vivo tumor promotion in mouse Lewis lung cancer

Lewis lung carcinoma (3LL) cells were constitutively resistant to Fas-mediated apoptosis, but overexpression of Fas on 3LL cells allowed Fas-mediated apoptosis after crosslinking with agonist anti-Fas antibody (Jo2) in vitro. Surprisingly, Fas-overexpressing 3LL cells showed enhanced in vivo tumor progression, whereas no promotion of in vivo tumor growth was observed for dominant negative (DN) Fas-overexpressing 3LL transfectants in which the cytoplasmic death domain was deleted. In addition, the promotion of in vivo tumor growth by Fas-overexpression was reduced in gld (FasL-mutation) mice compared to normal mice. These data indicate that intact Fas/FasL cell signaling is required for the promotion of in vivo tumor growth by Fas overexpression in 3LL cells. In contrast to the efficient Fas-mediated killing induced in vitro by crosslinking with anti-Fas antibody, Fas-overexpressing 3LL cells were resistant in vitro to Fas-mediated apoptosis by activated T cells or transient FasL transfection. These data suggest that agonist anti-Fas antibody and natural FasL can transmit qualitatively different signals, and crosslinking of Fas with natural FasL on 3LL cells does not deliver the expected death signal. Thus, our results demonstrate that in some cases overexpression of Fas can result in a survival advantage for tumor cells in vivo.     

IRF-4 suppresses BCR/ABL transformation of myeloid cells in a DNA binding-independent manner

Interferon regulatory factor 4 (IRF-4) is essential for B and T cell development and immune response regulation, and has both nuclear and cytoplasmic functions. IRF-4 was originally identified as a proto-oncogene resulting from a t(6;14) chromosomal translocation in multiple myeloma and its expression was shown to be essential for multiple myeloma cell survival. However, we have previously shown that IRF-4 functions as a tumor suppressor in the myeloid lineage and in early stages of B cell development. In this study, we found that IRF-4 suppresses BCR/ABL transformation of myeloid cells. To gain insight into the molecular pathways that mediate IRF-4 tumor suppressor function, we performed a structure-function analysis of IRF-4 as a suppressor of BCR/ABL transformation. We found that the DNA binding domain deletion mutant of IRF-4, which is localized only in the cytoplasm, is still able to inhibit BCR/ABL transformation of myeloid cells. IRF-4 also functions as a tumor suppressor in bone marrow cells deficient in MyD88, an IRF-4-interacting protein found in the cytoplasm. However, IRF-4 tumor suppressor activity is lost in IRF association domain (IAD) deletion mutants. These results demonstrate that IRF-4 suppresses BCR/ABL transformation by a novel cytoplasmic function involving its IAD domain.     

CD8+ tumor-infiltrating lymphocytes are primed for Fas-mediated activation-induced cell death but are not apoptotic in situ

Induction of Fas-mediated activation-induced cell death in antitumor T cells has been hypothesized to permit tumor escape from immune destruction. Several laboratories have proposed that expression of Fas ligand (L) by tumor is the basis for this form of T cell tolerance. In this study, we characterized murine tumor-infiltrating lymphocytes (TIL) for activation status, cell cycle status, level of apoptosis, cytokine secretion, and proliferative capacity. TILs express multiple activation markers (circa CD69, CD95L, CD122, and LFA-1) and contain IL-2 and IFN-gamma mRNAs, but are neither cycling nor apoptotic in situ. In addition, TIL are dramatically suppressed in proliferative response and do not secrete IL-2 and IFN-gamma. However, upon purification and activation in vitro, TIL secrete high levels of IL-2 and IFN-gamma, enter S phase, and then die by Fas-mediated apoptosis. Activation by injection of anti-TCR Ab or IL-2 into tumor-bearing mice induced TIL entrance into S phase preceding apoptosis, showing that TIL have functional TCR-mediated signal transduction in situ. Our data demonstrate that TIL, not tumor, express both Fas and FasL, are arrested in G(1), do not secrete cytokine in situ, and, upon activation in vitro and in vivo, rapidly die by activation-induced cell death.     

IL-17E, a proinflammatory cytokine, has antitumor efficacy against several tumor types in vivo

Interleukin-17E (IL-17E) belongs to a novel family of cytokines that possess significant homology to IL-17. IL-17E has potent inflammatory effects in vitro and in vivo. Overexpression of IL-17E in mice results in a T helper-2 (Th2)-type immune response, which includes the expansion of eosinophils through the production of IL-5, and elevated gene expression of IL-4 and IL-13 in multiple tissues. In this study, we show that IL-17E has antitumor activity in vivo, a previously unrecognized function of IL-17E. Antitumor efficacy of IL-17E was examined in a variety of human tumor xenograft models, including melanoma, breast, lung, colon, and pancreatic cancers. Injection of recombinant IL-17E every other day resulted in significant antitumor activity in these tumor models. In addition, the combination of IL-17E with chemotherapy or immunotherapy agents showed an enhanced antitumor efficacy in human tumor xenograft models in mice as compared to either agent alone. Antitumor activity was demonstrated using different routes of administration, including intraperitoneal, intravenous, and subcutaneous injection. Anticancer activity was shown for both mouse and human forms of IL-17E, which have a high degree of sequence identity. Tumor-bearing mice treated with IL-17E showed a significant increase in serum levels of IL-5 and increased numbers of eosinophils in peripheral blood compared to the control group. Spleens isolated from IL-17E-treated mice showed a significant increase in eosinophils that correlated with antitumor activity of IL-17E in a dose–response manner. Finally, we demonstrate that B cells are necessary for IL-17E-mediated antitumor activity and that IL-17E was found to activate signaling pathways in B cells in vitro. Taken together, these data demonstrate that IL-17E has antitumor activity in vivo, and support further investigation of the potential clinical use of IL-17E as an anticancer agent.     

Thymoquinone inhibits growth and augments 5-fluorouracil-induced apoptosis in gastric cancer cells both in vitro and in vivo

Thymoquinone (TQ), a component derived from the bioactive constituent of black seed (Nigella sativa), has been shown to exert biological activity on various types of human cancers. However, there are few studies addressing its effects on gastric cancer. Here, we present the first report describing the chemosensitizing effect of thymoquinone and 5-fluorouracil (5-FU) on gastric cancer cells both in vitro and in vivo. Studies have shown that pretreatment with TQ significantly increased the apoptotic effects induced by 5-FU in gastric cancer cell lines in vitro. Moreover, we found that TQ enhanced the 5-FU-induced killing of gastric cancer cells by mediating the downregulation of the anti-apoptotic protein bcl-2, the upregulation of the pro-apoptotic protein bax, and the activation of both caspase-3 and caspase-9. In addition to the in vitro results, it has been shown that the combined treatment of TQ with 5-FU represents a significantly more effective antitumor agent than either agent alone in a xenograft tumor mouse model. These data suggest that the TQ/5-FU combined treatment induces apoptosis by enhancing the activation of both caspase-3 and caspase-9 in gastric cancer cells. These results, which provide molecular evidence both in vitro and in vivo, support our conclusion that thymoquinone can activate caspase-3 and caspase-9 and thus result in the chemosensitisation of gastric cancer cells to 5-FU-induced cell death.     

p53 potentiation of tumor cell susceptibility to CTL involves Fas and mitochondrial pathways

In this study, we have investigated the mechanisms used by wild-type p53 (wtp53) to potentiate tumor cell susceptibility to CTL-mediated cell death. We report that wtp53 restoration in a human lung carcinoma cell line Institut Gustave Roussy (IGR)-Heu, displaying a mutated p53, resulted in up-regulation of Fas/CD95 receptor expression associated with an increase of tumor cell sensitivity to the autologous CTL clone, Heu127. However, when IGR-Heu cells were transfected with Fas cDNA, no potentiation to Heu127-mediated lysis was observed, indicating that induction of CD95 is not sufficient to sensitize target cells to CTL killing. Importantly, our data indicate that the effect of wtp53 on the Fas-mediated pathway involves a degradation of short cellular FLICE inhibitory protein resulting in subsequent caspase 8 activation. Furthermore, we demonstrate that wtp53 restoration also resulted in CTL-induced Bid translocation into mitochondria and a subsequent mitochondrial membrane permeabilization leading to cytochrome c release. These results indicate that tumor cell killing by autologous CTL can be enhanced by targeting degranulation-independent mechanisms via restoration of wtp53, a key determinant of apoptotic machinery regulation.     

CTL adoptive immunotherapy concurrently mediates tumor regression and tumor escape

Tumor escape and recurrence are major impediments for successful immunotherapy. It is well-documented that the emergence of Ag-loss variants, as well as regulatory mechanisms suppressing T cell function, have been linked to inadequate antitumor activity. However, little is known regarding the role of Fas-mediated cytotoxicity by tumor-specific CD8(+) CTL in causing immune evasion of Fas resistant variants during adoptive immunotherapy. In this study, we made use of an adoptive transfer model of experimental lung metastasis using tumor-specific CTL as a relevant immune-based selective pressure, and wherein the Fas ligand pathway was involved in the antitumor response. Surviving tumor cells were recovered and examined for alterations in antigenic, functional, and biologic properties. We showed that diminished susceptibility to Fas-mediated cytotoxicity in vivo was an important determinant of tumor escape following CTL-based immunotherapy. Tumor escape variants (TEV) recovered from the lungs of CTL-treated mice exhibited more aggressive behavior in vivo. However, these TEV retained relevant MHC class I and tumor Ag expression and sensitivity to CTL via the perforin pathway but reduced susceptibility to Fas-mediated lysis. Moreover, TEV were significantly less responsive to eradication by CTL adoptive immunotherapy paradigms as a consequence of increased Fas resistance. Overall, we identified that Fas(low)-TEV emerged as a direct consequence of CTL-tumor interactions in vivo, and that such an altered neoplastic Fas phenotype compromised immunotherapy efficacy. Together, these findings may have important implications for both tumor progression and the design of immunotherapeutic interventions to confront these selective pressures or escape mechanisms.     

Dynamic Rearrangement of F-Actin Is Required to Maintain the Antitumor Effect of Trichostatin A

Actin plays a role in various processes in eukaryotic cells, including cell growth and death. We investigated whether the antitumor effect of trichostatin A (TSA) is associated with the dynamic rearrangement of F-actin. TSA is an antitumor drug that induces hyper-acetylation of histones by inhibiting histone deacetylase. HeLa human cervical cancer cells were used to measure the antitumor effect of TSA. The percent cell survival was determined by an MTT assay. Hypodiploid cell formation was assessed by flow cytometry. Collapse of the mitochondrial membrane potential (MMP) was identified by a decrease in the percentage of cells with red MitoProbe J-aggregate (JC-1) fluorescence. Cell survival was reduced by treatment with TSA, as judged by an MTT assay and staining with propidium iodide, FITC-labeled annexin V, or 4′,6-diamidino-2-phenylindole (DAPI). TSA also induced an MMP collapse, as judged by the measurement of intracellular red JC-1 fluorescence. In addition, the F-actin depolymerizers cytochalasin D (CytoD) and latrunculin B (LatB) induced an MMP collapse and increased apoptotic cell death in HeLa cells. However, our data show that apoptotic cell death and the MMP collapse induced by TSA were decreased by the co-treatment of cells with CytoD and LatB. These findings demonstrate that the dynamic rearrangement of F-actin might be necessary for TSA-induced HeLa cell apoptosis involving a TSA-induced MMP collapse. They also suggest that actin cytoskeleton dynamics play an important role in maintaining the therapeutic effects of antitumor agents in tumor cells. They further suggest that maintaining the MMP could be a novel strategy for increasing drug sensitivity in TSA-treated tumors.     

CD40 ligation activates murine macrophages via an IFN-gamma-dependent mechanism resulting in tumor cell destruction in vitro

We have shown previously that agonistic anti-CD40 mAb induced T cell-independent antitumor effects in vivo. In this study, we investigated mechanisms of macrophage activation with anti-CD40 mAb treatment, assessed by the antitumor action of macrophages in vitro. Intraperitoneal injection of anti-CD40 mAb into C57BL/6 mice resulted in activation of peritoneal macrophages capable of suppressing B16 melanoma cell proliferation in vitro, an effect that was greatly enhanced by LPS and observed against several murine and human tumor cell lines. Anti-CD40 mAb also primed macrophages in vitro to mediate cytostatic effects in the presence of LPS. The tumoristatic effect of CD40 ligation-activated macrophages was associated with apoptosis and killing of tumor cells. Activation of macrophages by anti-CD40 mAb required endogenous IFN-gamma because priming of macrophages by anti-CD40 mAb was abrogated in the presence of anti-IFN-gamma mAb, as well as in IFN-gamma-knockout mice. Macrophages obtained either from C57BL/6 mice depleted of T and NK cells by Ab treatment, or from scid/beige mice, were still activated by anti-CD40 mAb to mediate cytostatic activity. These results argued against the role of NK and T cells as the sole source of exogenous IFN-gamma for macrophage activation and suggested that anti-CD40 mAb-activated macrophages could produce IFN-gamma. We confirmed this hypothesis by detecting intracytoplasmic IFN-gamma in macrophages activated with anti-CD40 mAb in vivo or in vitro. IFN-gamma production by macrophages was dependent on IL-12. Taken together, the results show that murine macrophages are activated directly by anti-CD40 mAb to secrete IFN-gamma and mediate tumor cell destruction.