Danger signalling during cancer cell death: origins, plasticity and regulation.

Accumulating data indicates that following anti-cancer treatments, cancer cell death can be perceived as immunogenic or tolerogenic by the immune system. The former is made possible due to the ability of certain anti-cancer modalities to induce immunogenic cell death (ICD) that is associated with the emission of damage-associated molecular patterns (DAMPs), which assist in unlocking a sequence of events leading to the development of anti-tumour immunity. In response to ICD inducers, activation of endoplasmic reticulum (ER) stress has been identified to be indispensable to confer the immunogenic character of cancer cell death, due to its ability to coordinate the danger signalling pathways responsible for the trafficking of vital DAMPs and subsequent anti-cancer immune responses. However, in recent times, certain processes apart from ER stress have emerged (e.g., autophagy and possibly viral response-like signature), which have the ability to influence danger signalling. In this review, we discuss the molecular nature, emerging plasticity in the danger signalling mechanisms and immunological impact of known DAMPs in the context of immunogenic cancer cell death. We also discuss key effector mechanisms modulating the interface between dying cancer cells and the immune cells, which we believe are crucial for the therapeutic relevance of ICD in the context of human cancers, and also discuss the influence of experimental conditions and animal models on these.

GRP78-targeting subtilase cytotoxin sensitizes cancer cells to photodynamic therapy.

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER)-resident chaperone and a major regulator of the unfolded protein response (UPR). Accumulating evidence indicate that GRP78 is overexpressed in many cancer cell lines, and contributes to the invasion and metastasis in many human tumors. Besides, GRP78 upregulation is detected in response to different ER stress-inducing anticancer therapies, including photodynamic therapy (PDT). This study demonstrates that GRP78 mRNA and protein levels are elevated in response to PDT in various cancer cell lines. Stable overexpression of GRP78 confers resistance to PDT substantiating its cytoprotective role. Moreover, GRP78-targeting subtilase cytotoxin catalytic subunit fused with epidermal growth factor (EGF-SubA) sensitizes various cancer cells to Photofrin-mediated PDT. The combination treatment is cytotoxic to apoptosis-competent SW-900 lung cancer cells, as well as to Bax-deficient and apoptosis-resistant DU-145 prostate cancer cells. In these cells, PDT and EGF-SubA cytotoxin induce protein kinase R-like ER kinase and inositol-requiring enzyme 1 branches of UPR and also increase the level of C/EBP (CCAAT/enhancer-binding protein) homologous protein, an ER stress-associated apoptosis-promoting transcription factor. Although some apoptotic events such as disruption of mitochondrial membrane and caspase activation are detected after PDT, there is no phosphatidylserine plasma membrane externalization or DNA fragmentation, suggesting that in DU-145 cells the late apoptotic events are missing. Moreover, in SW-900 cells, EGF-SubA cytotoxin potentiates PDT-mediated cell death but attenuates PDT-induced apoptosis. In addition, the cell death cannot be reversed by caspase inhibitor z-VAD, confirming that apoptosis is not a major cell death mode triggered by the combination therapy. Moreover, no typical features of necrotic or autophagic cell death are recognized. Instead, an extensive cellular vacuolation of ER origin is observed. Altogether, these findings indicate that PDT and GRP78-targeting cytotoxin treatment can efficiently kill cancer cells independent on their apoptotic competence and triggers an atypical, non-apoptotic cell death.

Heme oxygenase-1 protects tumor cells against photodynamic therapy-mediated cytotoxicity.

Photodynamic therapy is a promising antitumor treatment modality approved for the management of both early and advanced tumors. The mechanisms of its antitumor action include generation of singlet oxygen and reactive oxygen species that directly damage tumor cells and tumor vasculature. A number of mechanisms seem to be involved in the protective responses to PDT that include activation of transcription factors, heat shock proteins, antioxidant enzymes and antiapoptotic pathways. Elucidation of these mechanisms might result in the design of more effective combination strategies to improve the antitumor efficacy of PDT. Using DNA microarray analysis to identify stress-related genes induced by Photofrin-mediated PDT in colon adenocarcinoma C-26 cells, we observed a marked induction of heme oxygenase-1 (HO-1). Induction of HO-1 with hemin or stable transfection of C-26 with a plasmid vector encoding HO-1 increased resistance of tumor cells to PDT-mediated cytotoxicity. On the other hand, zinc (II) protoporphyrin IX, an HO-1 inhibitor, markedly augmented PDT-mediated cytotoxicity towards C-26 and human ovarian carcinoma MDAH2774 cells. Neither bilirubin, biliverdin nor carbon monoxide, direct products of HO-1 catalysed heme degradation, was responsible for cytoprotection. Importantly, desferrioxamine, a potent iron chelator significantly potentiated cytotoxic effects of PDT. Altogether our results indicate that HO-1 is involved in an important protective mechanism against PDT-mediated phototoxicity and administration of HO-1 inhibitors might be an effective way to potentiate antitumor effectiveness of PDT.

Increased local vascular endothelial growth factor expression associated with antitumor activity of proteasome inhibitor.

Inhibition of the proteasome, a multicatalytic proteinase complex, is an attractive approach to cancer therapy. Here we report that a selective inhibitor of the chymotrypsin-like activity of the proteasome, PSI (N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal) may inhibit growth of solid tumors not only through apoptosis induction, but also indirectly--through inhibition of angiogenesis. Two murine tumors: colon adenocarcinoma (C-26) and Lewis lung carcinoma (3LL) were chosen to study the antitumor effect of PSI. In an in vivo model of local tumor growth, PSI exerted significant antitumor effects against C-26 colon carcinoma, but not against 3LL lung carcinoma. Retardation of tumor growth was observed in mice treated with both 10 nmoles and 100 nmoles doses of PSI and in the latter group prolongation of the survival time of tumor-bearing mice was observed. PSI inhibited angiogenesis in the C-26 growing tumors with no such effect in 3LL tumors. Unexpectedly, that activity was associated with upregulation of vascular endothelial growth factor (VEGF) at the level of mRNA expression and protein production in C-26 tumors treated with PSI. C-26 cells treated with PSI produced increased amounts of VEGF in vitro in a dose- and time-dependent manner. We demonstrated that in C-26 colon adenocarcionoma higher VEGF production may render endothelial cells susceptible to the proapoptotic activity of PSI and is associated with inhibition of tumor growth.

Immunomodulation by anticancer chemotherapy: more is not always better (review).

Chemotherapeutics are the mainstay of the majority of antitumor treatment strategies. These agents are usually administered at or near maximum tolerated doses resulting in frequent dramatic toxicities that compromise the quality of life and the immune response towards microbial pathogens. A number of observations suggest that low-dose treatment with chemotherapeutics is sometimes equal or even superior to high-dose chemotherapy. The efficacy of low-dose chemotherapy can be at least partly explained by the regulation of the antitumor immune response. The immunomodulatory effects of some chemotherapeutics might be further potentiated by combinations with selected biological response modifiers such as recombinant cytokines (IL-2, TNF, IL-12). The effectiveness of such treatment combinations have already proved effective in preclinical animal models. However, the efficacy in humans is still to be demonstrated in rationally designed clinical trials.

Potentiated antitumor effects of interleukin 12 and interferon alpha against B16F10 melanoma in mice.

Systemic administration of cytokines has not found broad application in cancer immunotherapy due to its toxicity and lack of effectiveness in a broad spectrum of tumors. Among the most promising cytokines used often in pre-clinical and clinical trials are interferon alpha and interleukin 12. We have shown in our study that combining IL-12 with IFN-alpha in a dose which alone does not show antitumor activity results in potentiated antitumor effects without inducing toxicity.

Synergistic antitumor effects of a selective proteasome inhibitor and TNF in mice.

The ubiquitin-proteasome pathway is becoming an attractive target in cancer therapy. The inhibitors of proteasomes have recently been shown to induce apoptosis of tumor cells in vitro and to exert significant antitumor effects in murine tumor models in vivo. Proteasome inhibitors, also prevent NF-kappa B activation. Since this transcription factor is responsible for counteracting apoptosis induced by numerous agents, and proteasome inhibitors have already proved efficacious in increasing the proapoptotic activity of TNF in vitro, we decided to evaluate the antitumor effects of the combined PSI and TNF treatment against a murine C-26 carcinoma. Both agents separately exerted moderate antitumor efficacy. However, their combination proved to exert dramatic antitumor activity with retardation of tumor growth and prolongation of mice survival time. Moreover, 50% of the mice were completely cured by this drug combination. Unexpectedly, there was no potentiation of the cytostatic/cytotoxic effects of these drugs in in vitro assays which argues against the direct influence on C-26 cells. Similarly, the influence of these drugs on tumor induced angiogenesis does not seem to explain the observed antitumor effects. Further studies are necessary to explain the striking antitumor effects of the PSI and TNF combination.

Direct stimulation of macrophages by IL-12 and IL-18--a bridge too far?

A novel pathway of autocrine macrophage activation based on a positive feedback loop involving interleukin (IL)-12, IL-18 and IFN-gamma has recently been suggested. However, the macrophage isolation technique employed to describe the above phenomenon does not allow obtaining a pure population of macrophages casting some doubt to its existence. In the present study, we show that even minor contamination with lymphoid cells of a pure population of macrophage-like cells (Raw 264.7) results in a marked production of nitric oxide after stimulation with both IL-12 and IL-18. Neither macrophage-like cells nor lymphoid cells were capable of secreting high amounts of nitric oxide after stimulation with IL-12 and/or IL-18. Based on these observations we hypothesize that proposed autocrine feedback loop of macrophage activation is rather paracrine in nature and involves direct stimulation of residual lymphoid cells to secrete IFN-gamma that is then capable of activating macrophages.

Interleukin 18--interferon gamma inducing factor--a novel player in tumour immunotherapy?

The list of interleukins is growing at a steady rate. Although, it is over 8 years since the initial description of interferon gamma inducing factor (IGIF, now called IL-18), this novel cytokine is still not well characterised. However, the data were sufficient to support the testing of IL-18 in experimental tumour therapy. IL-18 is produced mainly by macrophages. Similarly to IL-1beta, IL-18 does not possess a signal sequence allowing direct secretion through the plasma membrane. Although, the exact mechanism of IL-18 secretion is not confirmed, it seems that, like IL-1beta, IGIF is processed by the cysteine proteases belonging to caspase family, especially by ICE (interleukin 1beta converting enzyme). Among the target cells responding to IL-18 are T lymphocytes and NK cells, which, under the influence of IL-18, produce substantial amounts of IFN-gamma. In this respect IL-18 seems to be even stronger than IL-12. Similarly to IL-12, IL-18 stimulates cytotoxicity of T and NK cells. Moreover, it enhances FasL-mediated cytotoxicity of CD4+ T and NK cells. A potential role of IL-18 in tumour immunotherapy is discussed in this article with special emphasis on the similarities with IL-12 and the potential mechanisms of its antitumour activity in preclinical models in mice.

Potentiation of the anti-tumour effects of Photofrin-based photodynamic therapy by localized treatment with G-CSF.

Photofrin-based photodynamic therapy (PDT) has recently been approved for palliative and curative purposes in cancer patients. It has been demonstrated that neutrophils are indispensable for its anti-tumour effectiveness. We decided to evaluate the extent of the anti-tumour effectiveness of PDT combined with administration of granulocyte colony-stimulating factor (G-CSF) as well as the influence of Photofrin and G-CSF on the myelopoiesis and functional activity of neutrophils in mice. An intensive treatment with G-CSF significantly potentiated anti-tumour effectiveness of Photofrin-based PDT resulting in a reduction of tumour growth and prolongation of the survival time of mice bearing two different tumours: colon-26 and Lewis lung carcinoma. Moreover, 33% of C-26-bearing mice were completely cured of their tumours after combined therapy and developed a specific and long-lasting immunity. The tumours treated with both agents contained more infiltrating neutrophils and apoptotic cells then tumours treated with either G-CSF or PDT only. Importantly, simultaneous administration of Photofrin and G-CSF stimulated bone marrow and spleen myelopoiesis that resulted in an increased number of neutrophils demonstrating functional characteristics of activation. Potentiated anti-tumour effects of Photofrin-based PDT combined with G-CSF observed in two murine tumour models suggest that clinical trials using this tumour therapy protocol would be worth pursuing.

Potentiated antitumor effects of interleukin 12 and matrix metalloproteinase inhibitor batimastat against B16F10 melanoma in mice.

The application of antiangiogenic agents in cancer therapy has been studied extensively. Combination of agents with antiangiogenic properties could possibly enhance antitumor effects. Interleukin 12 is a cytokine with potent antitumor activity mediated also via antiangiogenic mechanisms. These effects are attributed to IFN-gamma production stimulated by IL-12. Since IFN-gamma has been reported to augment antitumor effects when combined with one of the metalloproteinase inhibitors--batimastat (BB-94), we have examined a combined treatment with IL-12 and BB-94 in a murine melanoma model. The administration of both agents showed potentiated antitumor activity. Furthermore, we have shown in a tumor-induced angiogenesis model that the combined application of IL-12 and batimastat inhibits the formation of new blood vessels to a greater extent than either agent alone. Our observations show that antiangiogenic effects are at least partly responsible for the enhanced antitumor effects of the combined treatment with IL-12 and BB-94.

Interleukin 12 and indomethacin exert a synergistic, angiogenesis-dependent antitumor activity in mice.

Nonsteroidal anti-inflammatory drugs have been shown to reduce the incidence and mortality from colorectal cancer. It has recently been demonstrated that these drugs are capable of suppressing the production of pro-angiogenic factors from tumor cells. The mechanisms of antitumor action of interleukin 12 include the enforced secretion of anti-angiogenic factors and stimulation of antitumor immunity. Therefore, we hypothesized that the combination of a model nonsteroidal anti-inflammatory drug--indomethacin and interleukin 12--would result in enhanced angiogenesis-dependent antitumor effects against a colon-26 carcinoma cells transplanted into syngeneic mice. As expected the combined administration of both agents simultaneously resulted in a strengthened antitumor activity that was manifested as a retardation of tumor growth and prolongation of mouse survival. Importantly some mice were completely cured after the combined treatment. As administration of interleukin 12 and indomethacin resulted in enhanced inhibition of angiogenesis it seems possible that prevention of new blood vessel formation is one of the mechanisms responsible for the observed antitumor effects.

Potentiatied anti-tumor effectiveness of combined therapy with interleukin-12 and mitoxantrone of L1210 leukemia in vivo.

In previous studies we have shown that combined chemo-immunotherapy of L1210 leukemia with IL-12 and doxorubicin results in striking anti-tumor effects producing 100% of long-term survivors. In this study we investigated the efficacy of a combination of IL-12 and mitoxantrone in murine L1210 leukemia. Mice inoculated with 1x105 leukemia cells were treated with a single dose of mitoxantrone and seven daily doses of IL-12, and were daily observed for survival. Treatment with IL-12 or mitoxantrone given alone produced moderate anti-leukemic effects. However, combination of both drugs resulted in a significant prolongation of mouse survival time. Importantly, there were almost 50% of long-term (>60 days) survivors among the mice treated with both agents. This therapeutic effect was completely abrogated by sub-lethal, whole-body X-irradiation, and significantly reduced after macrophage depletion.

Potentiation of antitumor effects of IL-12 in combination with paclitaxel in murine melanoma model in vivo.

The early clinical trials with interleukin-12 (IL-12) have demonstrated substantial toxicity of this cytokine. One way to resolve this problem would be to find other antitumor agents, e.g. chemotherapeutics, synergistically interacting with IL-12. However, analysis of the recent reports on this topic leads to the conclusion that the antitumor effects achieved in such combination treatments are dependent not only on the drug applied but also on the tumor model used. We described previously lack of the potentiation of antitumor effects in combination treatment with IL-12 and paclitaxel in a murine leukemia L1210 model. We investigated whether such treatment could bring therapeutical benefit by using the murine melanoma MmB16 model. significant potentiation of antitumor effects in combination treatment with IL-12 and paclitaxel was observed. These results suggest that combination of IL-12 and paclitaxel could be beneficial in the treatment of certain types of tumors.

Subtherapeutic doses of interleukin-15 augment the antitumor effect of interleukin-12 in a B16F10 melanoma model in mice.

Interleukin-12 (IL-12) is a potent immunoregulatory cytokine that exhibits antitumor activity in many experimental tumor models. In the present study, we investigated the ability of IL-15, a cytokine sharing many functions of IL-2, to modulate antitumor effectiveness of IL-12 against B16F10 melanoma in mice. In a model of locally growing tumor, intratumoral (i.t.) administration of IL-12, in three cycles of five consecutive daily injections (0.1 mug) followed by 2 days of rest, led to considerable delay of tumor development but no curative response was achieved. When combined with IL-12, subtherapeutic doses of IL-15 (0.4 mug) pontentiated the antitumor effects of IL-12 and induced complete tumor regressions in 50% of mice. Similar results were obtained in a model in which tumor-bearing mice were intravenously co-injected with melanoma cells to induce metastases. Combined administration of IL-12 and IL-15 yielded greater antitumor activity than injections of either cytokine alone and resulted in prolonged survival of mice bearing locally growing tumor and metastases. Studies of immunological parameters in mice treated with both IL-12 and IL-15 have shown enhanced NK activity (against YAC-1 cells) in the spleen and stimulation of both NK activity and specific anti-B16F10 cytotoxic effector cells in tumor-draining lymph nodes (LN). The strong antitumor effect of the IL-12 + IL-15 combination correlated with a high serum level of IFN-gamma in the treated mice. Moreover, increased expression of IL-15Ralpha was demonstrated in LN lymphocytes isolated from mice injected with IL-12. This result together with findings of other authors showing enhanced expression of IL-12 receptor by IL-15 [1] suggests that the augmentation of the antitumor effect during the course of IL-12/IL-15-based therapy could result from reciprocal upregulation of receptors by both cytokines and synergistic effects on IFN-gamma induction.