Shilian Liu

A Novel Anti-human DR5 Monoclonal Antibody with Tumoricidal Activity Induces Caspase-dependent and Caspase-independent Cell Death *

Like anti-Fas monoclonal antibodies, some monoclonal antibodies against tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors have tumoricidal activity too. In this article we report a novel mouse anti-human DR5 monoclonal antibody, AD5–10, that induces apoptosis of various tumor cell lines in the absence of second cross-linking in vitro and showed strong tumoricidal activity in vivo. AD5–10 does not compete with TRAIL for binding to DR5 and synergizes with TRAIL to induce apoptosis of tumor cells. AD5–10 induces both caspase-dependent and caspase-independent cell death in Jurkat cells, whereas TRAIL induces only caspase-dependent cell death. We show for the first time that DR5 can mediate caspase-independent cell death, and DR5 can mediate distinct cell signals when interacting with different extracellular proteins. Studies on AD5–10 help us to understand more on the functions of DR5 and may provide new ideas for cancer immunotherapy.

Dysregulated expression of miR-146a contributes to age-related dysfunction of macrophages

Age‐associated immune dysfunction, characterized by increased systemic levels of cytokines, manifests as an increased susceptibility to infections. Thus, understanding these negative regulators of the immune response has paved the way to delineating signaling pathways that impact immune senescence. In the present study, we found that miR‐146a, which negatively regulated the expression of IL‐1β and IL‐6, was highly expressed in aged mice. However, there was a lack of response to the stimulation of lipopolysaccharide (LPS) and proinflammatory cytokines in macrophages of aged mice. As a result, the negative feedback regulation loop with miR‐146a involving down‐regulation of inflammation factors was interrupted in aged mice. Aberrant NF‐κB binding to the miR‐146a promoter was demonstrated to be associated with the abnormal expression of miR‐146a in aged mice. The DNA methyltransferase inhibitor (5‐aza‐2‐deoxycytidine) and the histone deacetylase inhibitor [trichostatin A (TSA)] both significantly up‐regulated miR‐146a transcriptional activation by altering the DNA‐binding activity of NF‐κB in macrophages isolated from aged mice, which suggests that DNA methylation and histone acetylation are involved in the suppression of age‐dependent miR‐146a expression. Additionally, high levels of histone deacetylase (HDACs) expressions contributed to the inhibition of miR‐146a expression in LPS‐stimulated macrophages from aged mice in vitro. While the suppression of HDACs activities by TSA could improve LPS‐induced inflammatory responses owing to up‐regulation of miR‐146a expression in macrophages from aged mice. These data indicate that the dysregulated expression of miR‐146a results in the age‐associated dysfunction of macrophages, and miR‐146a may be a good target for the treatment of age‐related inflammatory diseases.

HIV infection enhances TRAIL-induced cell death in macrophage by down-regulating decoy receptor expression and generation of reactive oxygen species.

Background Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) could induce apoptosis of HIV-1-infected monocyte-derived macrophage (MDM), but the molecular mechanisms are not well understood. Methodology/Principal Findings By using an HIV-1 Env-pseudotyped virus (HIV-1 PV)-infected MDM cell model we demonstrate that HIV-1 PV infection down-regulates the expression of TRAIL decoy receptor 1 (DcR1) and 2 (DcR2), and cellular FLICE-inhibitory protein (c-FLIP), but dose not affect the expression of death receptor 4 and 5 (DR4, DR5), and Bcl-2 family members in MDM cells. Furthermore, recombinant soluble TRAIL and an agonistic anti-DR5 antibody, AD5-10, treatment stimulates reactive oxygen species (ROS) generation and JNK phosphorylation. Conclusions/Significance HIV infection facilitates TRIAL-induced cell death in MDM by down-regulating the expression of TRAIL decoy receptors and intracellular c-FLIP. Meanwhile, the agonistic anti-DR5 antibody, AD5-10, induces apoptosis synergistically with TRAIL in HIV-1-infected cells. ROS generation and JNK phosphorylation are involved in this process. These findings potentiate clinical usage of the combination of TRAIL and AD5-10 in eradication of HIV-infected macrophage and AIDS.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced chemokine release in both TRAIL-resistant and TRAIL-sensitive cells via nuclear factor kappa B.

Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) induces apoptosis in a variety of tumour cells, but not in most normal cells, and has attracted considerable attention for its potential use in cancer therapy. Recently, increasing evidence has shown that TRAIL is involved in inflammation, although much of this evidence is controversial. In this article, it is shown that TRAIL induces CXCL2, CCL4 and CCL20 secretion in a nuclear factor kappa B‐dependent manner. The dominant negative constructs of tumour necrosis factor receptor‐associated death domain protein (TRADD) and tumour necrosis factor receptor‐associated factor 2 are unable to block TRAIL‐induced chemokine up‐regulation, and the dominant negative construct of TRADD may even enhance TRAIL‐triggered signals. Using small interfering RNA, receptor interacting protein has been demonstrated to be essential for TRAIL‐induced chemokine release. Furthermore, it has been demonstrated that p38 mitogen‐activated protein kinase is involved in TRAIL‐induced chemokine release without any effects on nuclear factor kappa B activation, suggesting that some unknown transcription factors may be activated by TRAIL. Using a xenograft tumour model, it has been illustrated that TRAIL can also induce chemokine release in vivo. Although these chemokines induced by TRAIL are inflammatory chemokines, their functions are not restricted to inflammation and require further examination. Our results indicate that attention should be paid to the side‐effects of TRAIL treatment, not only in TRAIL‐resistant but also in TRAIL‐sensitive tumour cells.

TRAIL receptor mediates inflammatory cytokine release in an NF-κB-dependent manner

In the present article, we report that DR4 or DR5 overexpression dramatically activates the release of the inflammatory cytokines IL-8, TNF-α, CCL20, MIP-2 and MIP-1β in an NF-κB-dependent manner in 293T, MDA-MB-231 and HCT-116 cells. We showed that death receptor-mediated signals were extracellular domain-independent, whereas the effect of overexpression of the DR4 intracellular domain was much less potent. The TRADD-TRAF2-NIK-IKKα/β signaling cascade, which plays an essential role in TNF-induced NF-κB activation, was found to be involved in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-mediated signal transduction. The FADD-caspase signaling pathway, which has been reported to be mostly related to apoptosis, was identified as being essential for DR4 or DR5 overexpression-mediated NF-κB activation and cytokine secretion and crosstalks with the TRADD-TRAF2-NIK-IKKα/β signaling cascade. Furthermore, a DR5 agonistic antibody (AD5-10) triggered the inflammatory cytokine release. These data, together with previous reports, provide strong evidence that TRAIL and TRAIL receptors play an important role in inflammation.

AAV-mediated TRAIL gene expression driven by hTERT promoter suppressed human hepatocellular carcinoma growth in mice.

A major obstacle in the development of effective recombinant adeno-associated virus (rAAV) mediated gene therapy is infection specificity and gene targeting. In the present study, we investigated whether the human telomerase reverse transcriptase (hTERT) promoter could drive tumor-specific expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), an apoptosis apoptosis-inducing protein with potential toxic effects on normal cells. Our data demonstrated that hTERT promoter-driven tumor-specific expression of TRAIL decreased the cellular viability of tumor cells, but not normal cells. TRAIL expression driven by hTERT promoter inhibited tumor growth significantly in vivo and combination of viral infection with 5-fluorouracil (5-Fu) suppressed tumor growth more efficiently. Intra-venous injection of virus showed that the recombinant virus was predominantly distributed in the liver, but not in other major tissues tested, and no transgene expression was detected in the liver. Furthermore, serum enzyme and liver histology analysis confirmed that liver function is unaffected by TRAIL expression, significant as the liver is frequently metastasized and scattered with tumors from other organs, which are unpractical to treat by intra-tumor injection. Together our results demonstrate that rAAV-mediated TRAIL expression is a promising strategy in gene therapy for treatment of cancer.

Recombinant adeno‐associated virus‐mediated TRAIL gene therapy suppresses liver metastatic tumors

To evaluate the tumoricidal activity of tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) on disseminated liver metastatic tumors, we constructed a recombinant adeno‐associated virus (rAAV) expressing the extracellular domain (95–281aa) of human TRAIL (TRAIL95–281, and the recombinant virus was designated as rAAV‐TRAIL) using the 3‐plasmid, helper‐virus‐free, packaging system. Transduction of mouse lymphoma EL‐4 cells and Jurkat T cells lead to the expression of TRAIL95–281 protein in both virus‐transduced cells and the culture media, along with apoptosis of these cells in vitro. The therapeutic potential of rAAV‐TRAIL was then evaluated in an orthotopic transplanted mouse model mimicking liver cancer metastasis, which was established by injection of EL‐4 cells into the liver of C57BL/6 mice via the hepatic portal veins. Subsequent intraportal vein injection of rAAV‐TRAIL, not the control virus, into the liver of these mice resulted in significant suppression of tumor growth and prolonged survival, while normal hepatocyte toxicity is undetectable. Histological and biochemical analysis in tumor tissue and serum confirmed that TRAIL95–281 was stably expressed in relatively high level in hepatocytes and was secreted into the serum in active trimeric form. Futhermore, the mechanism for rAAV‐TRAIL to inhibit tumor growth was by inducing apoptosis of the tumor cells metastasizing to the livers. These results strongly suggest that the rAAV‐TRAIL‐mediated gene delivery could be a promising approach for the treatment of liver metastasis cancer.

Oral adeno‐associated virus‐sTRAIL gene therapy suppresses human hepatocellular carcinoma growth in mice

The extracellular domain of the tumor necrosis factor‐related apoptosis‐inducing ligand (sTRAIL) may function as a soluble cytokine to selectively kill various cancer cells without toxicity to most normal cells. We constructed a series of recombinant adeno‐associated virus (AAV) vectors expressing the extracellular domain of human TRAIL fused with signal peptides of human insulin, interferon, human growth hormone, and serum albumin and designated them as AAV‐ISN‐T, AAV‐IFN‐T, AAV‐HGH‐T, and AAV‐Alb‐T, respectively. Transduction of human SMMC‐7721 liver cancer cells with AAV‐ISN‐T led to higher levels of TRAIL95‐281 protein expression in the cell culture media and produced more apoptosis of the cells in vitro than those with AAV‐IFN‐T, AAV‐HGH‐T, and AAV‐Alb‐T. The therapeutic potential of AAV‐ISN‐T was then evaluated in a transplanted mouse model established by injection of human liver cancer SMMC‐7721 cells subcutaneously. Subsequent oral or intraperitoneal administration of AAV‐ISN‐T resulted in a rapid, high level and long time expression of soluble TRAIL in the sera and livers of the animals, as well as effective suppression of tumor growth, with no toxicity to normal hepatocytes. These data strongly suggest that it is possible to increase soluble TRAIL expression to make full use of tumoricidal activity of TRAIL as a therapeutic strategy. In conclusion, we provide evidence that oral administration of AAV‐TRAIL might be an important alternative route with practical significance for cancer gene therapy. (HEPATOLOGY 2005;42:1355–1363.)

Actinomycin D enhances TRAIL-induced caspase-dependent and -independent apoptosis in SH-SY5Y neuroblastoma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted great attention as a promising anti-cancer reagent. Recombinant soluble TRAIL (rsTRAIL) derivatives induce apoptosis in various cancer cells, but not in most normal cells. However, a number of cancerous cell types are resistant to TRAIL cytotoxicity, limiting its application in cancer therapy. In the present study, we report that actinomycin D (Act D) pretreatment increases apoptosis in human neuroblastoma SH-SY5Y cells treated with rsTRAIL. Both caspase-9 and caspase-7, but not caspase-3, were activated during the apoptosis process. z-VAD-fmk, a pan-caspase inhibitor, only partially suppressed apoptosis of the cells, suggesting that the Act D-enhanced apoptosis of SH-SY5Y occurred via caspase-dependent and -independent manners. In cells pretreated with Act D, we found decreased mitochondrial transmembrane potential, high levels of reactive oxygen species (ROS), and up-regulated apoptotic-inducing factor (AIF). Cell death was blocked in cells stably transfected with AIF-siRNA plasmid. Taken together, these data indicate that Act D sensitizes SH-SY5Y cells to rsTRAIL-induced apoptosis via caspase activation, impairment of the mitochondrial membrane, release of ROS, and up-regulation of AIF expression. This study provides a novel strategy for the therapy of malignant neuroblastoma resistant to rsTRAIL cytotoxicity.

PKCδ protects human breast tumor MCF-7 cells against tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis

Tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) induces apoptosis in a number of tumorogenic or transformed cells, yet is relatively non‐toxic to most normal cells, therefore, it is a promising agent for cancer therapy. However, some cancer cell lines were resistant to TRAIL cytoxicity, including MCF‐7 breast cancer cells. The mechanism is not clear. Here, we report that protein kinase C delta (PKCδ) protects MCF‐7 cells from the recombinant soluble TRAIL (rsTRAIL)‐ mediated apoptosis. It was demonstrated that rottlerin, a PKCδ inhibitor, sensitized MCF‐7 cells to rsTRAIL cytoxicity. Combination of rottlerin and rsTRAIL inhibited PKCδ translocation from the cytosol to membrane, and PKCδ kinase activity on the cell membrane was kept pace with the change of PKCδ expression. Moreover, inhibition of PKCδ by interference RNA could facilitate apoptosis of MCF‐7 cells induced by rsTRAIL. Further experiments on the signal machinery showed that rottlerin increased the sensitivity of MCF‐7 cells to rsTRAIL by suppressing the transcription activity of NF‐κB, and enhancing the caspase‐processing to generate executive apoptotic signals. These findings indicate that PKCδ functions as a survival factor protecting MCF‐7 cells from the apoptosis induced by rsTRAIL.