Sensitization of head and neck squamous cell carcinoma to apoptosis by combinational SMAC mimetic and Fas ligand-Fc treatment in vitro.

This study aimed to investigate the in vitro efficacy of three different SMAC mimetics for pro-apoptotic sensitization of HNSCC cells. We evaluated BV-6 in comparison to Birinapant and LCL161, for which pro-apoptotic sensitization effects have been demonstrated. Concentration-dependent response was measured for BV-6 in each cell line with an average IC50 value 8-fold lower than of aforementioned SMAC mimetics. Combination treatment of FasL (log2) and BV-6 (IC10) showed highly significant cell count reductions even in the lowest applied concentration in five cell lines (PCI-1: p = 0.0002, PCI-13: p = 0.0002, Detroit 562: p: p < 0.0001, FaDu: p < 0.0001, SCC-25: p = 0.0047). Synergistic effects (y < 1) were evident in eight out of 10 cell lines (PCI-1, PCI-9, PCI-13, PCI-68, Detroit 562, FaDu, SCC-25 and HaCaT). Annexin V assays revealed in nine cell lines very highly significant (p < 0.001) pro-apoptotic effects of BV-6. Western blots showed a heterogeneous IAP expression following SMAC mimetic treatment. Except for two cell lines, at least the cellular inhibitor of apoptosis protein 1 (cIAP1) was degraded in response to BV-6. For prospective targeted HNSCC therapy, this study identifies SMAC mimetics, particularly BV-6 as the compound with the highest pro-apoptotic potency, as promising antitumor agents.

Fas ligand based immunotherapy: A potent and effective neoadjuvant with checkpoint inhibitor properties, or a systemically toxic promoter of tumor growth?

Fas ligand (FasL, CD95L) is a 40-kDa type II transmembrane protein that binds to Fas (CD95) receptors and promotes programmed cell death. Fas receptors are expressed at higher levels in many tumors than in normal cells; however, systemic administration of FasL or agonistic anti-Fas antibodies to mice with tumors caused lethal hepatitis. Somewhat paradoxically, elimination of Fas or FasL from tumors also leads to death induced by CD95 receptor/ligand elimination (DICE). At face value, this suggests that Fas signaling not only kills normal cells, but that it also is essential for tumor cell survival. Targeting this pathway may not only fail to kill tumors, but instead may even enhance their growth, leading some to report the demise of Fas ligand in cancer immunotherapy. But, to paraphrase Mark Twain, is this death an exaggeration? Here, we provide a careful examination of the literature exploring the merits of FasL as a novel form of cancer immunotherapy. With local administration using delivery vectors that achieve high levels of expression in the tumor environment, our results indicate that the potential for systemic toxicity is eliminated in higher mammals, and that a systemic anti-tumor response ensues, which delays or prevents progression and simultaneously attacks distant metastases.

The role of CD95 and CD95 ligand in cancer.

CD95 (Fas/APO-1) and its ligand, CD95L, have long been viewed as a death receptor/death ligand system that mediates apoptosis induction to maintain immune homeostasis. In addition, these molecules are important in the immune elimination of virus-infected cells and cancer cells. CD95L was, therefore, considered to be useful for cancer therapy. However, major side effects have precluded its systemic use. During the last 10 years, it has been recognized that CD95 and CD95L have multiple cancer-relevant nonapoptotic and tumor-promoting activities. CD95 and CD95L were discovered to be critical survival factors for cancer cells, and were found to protect and promote cancer stem cells. We now discuss five different ways in which inhibiting or eliminating CD95L, rather than augmenting, may be beneficial for cancer therapy alone or in combination with standard chemotherapy or immune therapy.

Highly efficient, in-vivo Fas-mediated apoptosis of B-cell lymphoma by hexameric CTLA4-FasL.

Non-Hodgkin lymphomas (NHLs) account for 4% of all malignancies. 5-year survival rate increased to 50% with new treatment modalities, however there is need for new effective treatment for the more aggressive, relapsing forms. Recently, CTLA4-FasL, that can bind to B7 and Fas receptor (Fas), was shown to induce robust apoptosis of cell lines originating from B cell lymphomas expressing both B7 and Fas, by activating pro-apoptotic signals in parallel to abrogating anti-apoptotic ones. The present study focuses on the unique properties of CTLA4-FasL as a potent apoptosis inducer of malignant cells in-vitro and in a xenograft model. CTLA4-FasL was found to naturally form a stable homo-hexamer. CTLA4-FasL induces robust apoptosis of a large variety of malignant cells while relatively sparing non-malignant ones, being more efficient when both receptors (B7 and Fas) are expressed on target cells. Even in non-B7 expressing cells, CTLA4-FasL exhibited better apoptotic activity than its parts, alone or in combination, however, only in B7 expressing cells apoptosis occurs at low concentrations and CTLA4-FasL induces activation of apoptotic signals and reduces anti-apoptotic ones. Importantly, CTLA4-FasL efficiently inhibited the growth of human B cell lineage tumors in a xenograft model, by provoking tumor cells' apoptosis. Thus, CTLA4-FasL, a natural homo-hexamer protein, induces robust apoptosis of malignant cells, in-vitro and in-vivo. In B-cell lymphoma, its potency stems from the combination of its synergistic effect of activating the caspases while abrogating the anti-apoptotic signaling, with its unique hexameric structure, making CTLA4-FasL a promising candidate for aggressive B cell lymphomas treatment.

Alginate encapsulated cells secreting Fas-ligand reduce lymphoma carcinogenicity.

Fas ligand (CD95L/APO-1) is considered as a potent anti-tumor agent due to its mediated cell death properties. We have designed a polymeric microencapsulation system, which encapsulates soluble FasL secreting cells. The encapsulated cells continuously release soluble FasL (sFasL) at the tumor site, while the device protects the encapsulated cells from the host immune system. The potential and efficacy of this system are demonstrated in vitro and in vivo for tumor inhibition. Polymeric microcapsules composed of Alginate Poly-l-lysine were optimized to encapsulate L5 secreting sFasL cells. The expression and anti-tumor activities of the sFasL were confirmed in vitro and tumor inhibition was studied in vivo in SCID mice bearing subcutaneous lymphoma tumors. In vitro, sFasL secreted by the encapsulated L5-sFasL cells was biologically active, inhibited proliferation and induced apoptotic cell death in Fas sensitive tumor cells. Mice injected with encapsulated L5-sFasL cells on the day of tumor injection or 10 days after tumor injection showed significant reduction in tumor volume, of 87% and 95%, respectively. Our findings show that encapsulated cells expressing sFasL can be used as a local device and efficiently suppress malignant Fas sensitive tumors with no side effects.

Targeting Fas in osteoresorptive disorders.

IMPORTANCE OF THE FIELD: Fas receptor is a mediator of the external apoptotic pathway in many cells and tissues. It is proposed that Fas receptor mediates osteoresorptive effects of estrogen deficiency and local/systemic inflammation. AREAS COVERED IN THIS REVIEW: This review covers the past two decades of research on the expression and function of the Fas-Fas ligand system on bone cells, involvement in the pathogenesis of osteoresorption and potential therapeutic modulation. WHAT THE READER WILL GAIN: We review the structure, biological function and intracellular signaling pathways of the Fas-Fas ligand system emphasizing the role of the non-apoptotic signaling pathways in bone cells, particularly osteoblast differentiation. We also present data on the in vitro expression and function of the Fas-Fas ligand system on osteoblast/osteoclast lineage cells, animal and human studies confirming its involvement in osteoresorptive disorders and potential therapeutic approaches to modulate its function. TAKE HOME MESSAGE: Tissue specific therapeutic approaches need to be established to modify the Fas-Fas ligand system in osteoresorptive disorders as systemic targeting has many side effects. The most promising approach would be to target Fas signaling molecules coupled with osteoblast/osteoclast differentiation pathways, but a precise definition of these targets is still needed.

Targeting death receptors to fight cancer: from biological rational to clinical implementation.

Considering that most currently available chemotherapeutic drugs work by inducing cell apoptosis, it is not surprising that many expectations in cancer research come from the therapeutic exploitation of the naturally occurring death pathways. Receptor mediated apoptosis depends upon the engagement of specific ligands with their respective membrane receptors and - within the frame of complex regulatory networks - modulates some key physiological and pathological processes such as lymphocyte survival, inflammation and infectious diseases. A pivotal observation was that some of these pathways may be over activated in cancer under particular circumstances, which opened the avenue for tumor-specific therapeutic interventions. Although one death-related ligand (e.g., tumor necrosis factor, TNF) is currently the basis of effective anticancer regimens in the clinical setting, the systemic toxicity is hampering its wide therapeutic exploitation. However, strategies to split the therapeutic from the toxic TNF activity are being devised. Furthermore, other death receptor pathways (e.g., Fas/FasL, TRAIL/TRAIL receptor) are being intensively investigated in order to therapeutically exploit their activity against cancer. This article summarizes the current knowledge on the molecular features of death receptor pathways that make them an attractive target for anticancer therapeutics. In addition, the results so far obtained in the clinical oncology setting as well as the issues to be faced while interfering with these pathways for therapeutic purposes will be overviewed.

Selective reduction of graft-versus-host disease-mediating human T cells by ex vivo treatment with soluble Fas ligand.

Previous work done in our laboratory, using mouse models, showed that soluble Fas ligand (sFasL) can efficiently delete donor anti-host T cells during their activation against irradiated host cells in MLCs. In the mouse models, this ex vivo sFasL treatment abrogated graft-vs-host disease (GVHD) while sparing donor T cells with antitumor reactivity. The present work was performed with human cells, to extend our work toward reduction of clinical GVHD. PBMC responders from a given individual (first party) were stimulated in vitro with irradiated PBMC stimulators from a second person (second party), in the presence of sFasL. In control MLCs without sFasL, alloreacting T cells began to up-regulate Fas (CD95) detectably and became sensitive to Fas-mediated apoptosis by as early as day 1-2. In MLCs containing sFasL, there were greatly reduced numbers of alloreacting CD3(+)CFSE(lo) cells, activation Ag-expressing CD4(hi) and CD8(hi) cells, IFN-gamma-producing CD4(+) and CD8(+) cells, and CD8(+)CD107a(+) CTLs. Furthermore, mice transplanted with the ex vivo sFasL/MLR-treated cells had prolonged time to fatal GVHD in an in vivo xenogeneic GVHD model. Responder cells harvested from primary MLCs containing sFasL had reduced proliferation in response to second party cells, but proliferated in response to CMV Ags, PHA, and third party cells. In addition, sFasL/MLR-treated cell populations contained influenza-specific T cells, CD4(+)FOXP3(+) T cells, and CD4(+)CD25(+) T cells. These data indicate that this ex vivo sFasL/MLR depletion of alloreacting human donor anti-host T cells was efficient and selective.

Enhanced allogeneic skin-graft survival using sCD95L, sCD152, interleukin-10 and transforming growth factor-beta in combination, and comparison with ciclosporin.

BACKGROUND: CD152 is a negative regulator of T-cell activation. The ligand of CD95, CD95L, induces apoptosis in CD95-expressing cells through activation of caspases. Interleukin (IL)-10 and transforming growth factor (TGF)-beta have been implicated in suppressing immune response. Independent treatment with the four factors can induce immunotolerance and prolong the survival of grafts. AIM: To investigate whether joint use of murine (m)IL-10, human (h)TGF-beta and our engineered soluble extracellular part of CD152 and CD95L (sCD152, sCD95L) has a synergistic effect on immunotolerance induction in allogeneic skin transplantation. METHODS: Recipient mice were treated with sCD152, sCD95L, mIL-10 and hTGF-beta, separately or combined. The survival time of skin allografts was observed and mixed lymphocyte reactions were performed to detect the reactivity of splenic cells from recipient mice compared with splenic cells from donors or from unrelated Institute of Cancer Research mice. The levels of cytokines such as IL-2, IL-4 and interferon (IFN)-gamma, and antibodies specific for engineered sCD152 or sCD95L in serum were determined by ELISA. RESULTS: Combined treatment with sCD152 and sCD95L synergistically prolonged the mean survival time (MST) of skin allografts, but the addition of mIL-10 or hTGF-beta to these did not improve MST further. CONCLUSION: sCD152 and/or sCD95L regulate the differentiation of T-helper (Th) cells and induce the cytokine production shift of Th1-associated and Th2-associated cells. Both IFN-gamma and IL-2 were negatively correlated and IL-4 was positively correlated with skin MST. Cytokines such as IFN-gamma, IL-2 and IL-4 might function as indicators for predicting graft survival time.

Abundant Fas expression by gastrointestinal stromal tumours may serve as a therapeutic target for MegaFasL.

Although the tyrosine kinase inhibitor imatinib has been shown to be an active agent in patients with gastrointestinal stromal tumours (GIST), complete remissions are almost never seen and most patients finally experience disease progression during their course of treatment. An alternative therapeutic option is to target death receptors such as Fas. We showed that a panel of imatinib-sensitive (GIST882) and imatinib-resistant (GIST48, GIST430 and GIST430K-) cell lines expressed Fas. MegaFasL, a recently developed hexameric form of soluble Fas ligand (FasL), appeared to be an active apoptosis-inducing agent in these cell lines. Moreover, MegaFasL potentiated the apoptotic effects of imatinib. Immunohistochemical evaluations, in 45 primary GISTs, underscored the relevance of the Fas pathway: Fas was expressed in all GISTs and was expressed strongly in 93%, whereas FasL was expressed at moderate and strong levels in 35 and 53% of GISTs, respectively. Fas and FasL expression were positively correlated in these primary GISTs, but there was no association between Fas or FasL expression and primary site, histological subtype, tumour size, mitotic index, risk classification, and KIT mutation status. The abundant immunohistochemical Fas and FasL expression were corroborated by western blot analysis. In conclusion, our data implicate Fas as a potential therapeutic target in GIST.

[CTLA4-FasL protein for the prevention of immune rejection in mouse corneal transplantation].

OBJECTIVE: To investigate the therapeutic effect of double-functioned CTLA4-FasL protein for the prevention of immune rejection in murine corneal allografts. METHODS: It was a experimental study. C57BL/6 mice (n = 45) were as donors and BALB/c mice (n = 90) as recipients. BALB/c mice accepted penetrating keratoplasty were randomly divided into 3 groups including no therapy (A), CsA DDS implanted in anterior chamber (B), 10 microg/mL CTLA4-FasL (C). Survival time of corneal allografts was observed and routine assays were performed including immunohistochemistry, reverse-transcription polymerase chain reaction (RT-PCR), TdT-mediated dUTP nick end labeling (TUNEL). RESULTS: Survival time of corneal transplants was (14.3 +/- 1.3) days, (58.0 +/- 2.8) days, (106.3 +/- 17.5) days respectively. There was significant difference between groups in statistics analysis (P = 0.000). Inflammatory cells, predominant for CD4 + T cells, showed an increasing tendency for rejected corneal allografts in A group, peaked on postoperative 7 d and then decreased in C group compared with invisible in B group. CD80 or CD86 was detected on postoperative 3 d or 7 d respectively in excised cornea and iris of both A and B group, while either showed weakening expression in C group. On postoperative 14 d, IL-10, TNF-alpha, IFN-gamma mRNA were only detected in rejected cornea allograft of A group other than clear allografts of B or C group. Compared with no apoptosis in the other groups, abundant apoptosis cells were visible in cornea and iris of C group on postoperative 7 d. CONCLUSIONS: CTLA4-FasL is able to prolong survival time of corneal allografts by dual action mechanism to exert immunosuppressive effects, both blockade of CD28-CD80/86 pathway and induction of apoptosis of T cell.

Preconditioning with death ligands FasL and TNF-alpha protects the cirrhotic mouse liver against ischaemic injury.

BACKGROUND: Ischaemic preconditioning is the preemptive proven strategy to reduce ischaemic injury in the liver, but it can be harmful in the elderly or in patients with liver diseases. Ischaemic preconditioning induces a protective effect via activation of oxidative stress. We hypothesised that Fas ligand and tumour necrosis factor alpha can induce a similar response. Therefore, we tested if death ligands could mimic ischaemic preconditioning. METHODS: Ischaemia was maintained for 60 min in cirrhotic mice. Death ligands were given 40 min before ischaemia. Ischaemic injury was assessed by histology and biochemical assays. To elucidate the mechanism, we used zinc protophorphyrin, an inhibitor of haem oxygenase-1 (HO-1), and gadolinium chloride, an inhibitor of Kupffer cells. RESULTS: Compared with the control group, death ligand preconditioning strongly reduced all markers of injury: serum transaminase levels, necrosis and apoptosis. Preconditioning caused an upregulation of HO-1, predominantly in macrophages. When zinc protophorphyrin or gadolinium chloride was applied prior to preconditioning, the beneficial effect of preconditioning was lost. CONCLUSION: These results demonstrate that ischaemic preconditioning can be replaced by death ligand preconditioning in the cirrhotic liver to prevent ischaemic injury. The protective mechanism depends on HO-1 induction in macrophages. These results open doors for novel hepato-protective strategies in liver surgery and transplantation.

In vitro apoptotic induction of human glioblastoma cells by Fas ligand plus etoposide and in vivo antitumour activity of combined drugs in xenografted nude rats.

Human glioblastomas that express Fas/CD95 receptor are highly resistant to conventional brain tumour therapies. The aim of this study is to evaluate anti-tumour properties of a combination of Fas ligand (FasL) plus etoposide with or without dexamethasone on intracerebral experimental glioblastomas. The human Fas-expressing glioblastoma cell line, U-87 MG, was firstly studied in vitro for apoptosis and proliferation assays in the presence of FasL and etoposide, separately or associated, in order to detect a supra-additive effect on FasL or etoposide-induced apoptosis. The tumourigenicity of the U-87 MG cell line and therapeutic effects of FasL-etoposide alone or combined with dexamethasone were next studied on U-87 MG cells xenografted to nude-rat brain and tumour size was hence examined by histological and immunohistochemical stainings. We demonstrated in vitro that the combination of both molecules strongly inhibited the proliferation rate and increased the sensitivity of glioblastoma cells to Fas or etoposide-mediated apoptosis. Moreover, analysis of rat brains was performed 30 days after xenograft of glioblastoma cells. These data show that the combination of FasL and etoposide could reduce significantly the tumour size and that the addition of dexamethasone enhanced the inhibiting effect of FasL and etoposide on tumour growth in vivo.

Combined therapeutic use of AdGFPFasL and small molecule inhibitors of ceramide metabolism in prostate and head and neck cancers: a status report.

As of January 2005, there were 1020 gene therapy clinical trials ongoing worldwide with 675 or 66.2% devoted to cancer gene therapy. The majority are occurring in the US and Europe (http://www.wiley.co.uk/genetherapy/clinical/). At the present time, to our knowledge there are no trials that employ gene delivery of Fas Ligand (FasL). As an important note, and in contrast to somatic cell therapy trials, there are no reported deaths due to therapeutic vector administration in any cancer gene therapy trial. That said, from our studies and from the published literature, the issue of gene delivery remains the major obstacle to successfully employing gene therapy for cancer treatment. Numerous laboratories are studying this with many different approaches. My co-workers and I have focused on the delivery issue by using various approaches that address tumor targeting and transgene expression. In addition, we are focusing on enhancing tumor cell killing via the bystander effect and through use of small molecules to enhance bystander activity.