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Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is currently being investigated as a therapeutic agent for a variety of malignancies, as it triggers apoptosis specifically in transformed cells. However, TRAIL use as a stand alone therapeutic is hampered by the fact that many primary tumor cells are resistant to TRAIL-mediated apoptosis. Here, we investigated the extent to which pretreatment of TRAIL-resistant primary B-cell chronic lymphocytic leukemia (B-CLL) cells with histone deacetylase inhibitors (HDACis) could render them susceptible to killing by TRAIL. We found that HDAC inhibition in B-CLL cells led to increased TRAIL receptor expression, increased caspase activation, decreased expression of antiapoptotic regulators such as Bcl-2, and ultimately, enhanced TRAIL-induced apoptosis. Importantly, untransformed peripheral blood mononuclear cells remained largely resistant to TRAIL, even in the presence of HDACis. These results suggest that combination therapies using HDAC inhibition and TRAIL could prove beneficial for the treatment of B-CLL.

TRAIL gene therapy: from preclinical development to clinical application.

Numerous studies have investigated the potential use of TNF-related apoptosis-inducing ligand (TRAIL) as a cancer therapeutic since its discovery in 1995--because TRAIL is a potent inducer of apoptosis in tumor cells but not in normal cells and tissues. Consequently, a great deal is known about TRAIL/TRAIL receptor expression, the molecular components of TRAIL receptor signaling, and methods of altering tumor cell sensitivity to TRAIL-induced apoptosis. Our laboratory was the first to report the possibility of TRAIL gene transfer therapy as an alternative method of using TRAIL as an antitumor therapy. As with recombinant proteins administered systemically, intratumoral TRAIL gene delivery also has limitations that can restrict its full potential. Translating the preclinical TRAIL studies into the clinic has started, with the hope that TRAIL will exhibit robust tumoricidal activity against human primary tumors in situ with minimal toxic side effects.

Activation of tumor-specific CD8+ T Cells after intratumoral Ad5-TRAIL/CpG oligodeoxynucleotide combination therapy.

CD8(+) T-cell activation via cross-presentation of antigens from apoptotic tumor cells is controversial. Dendritic cells capture naturally shed tumor antigens and cross-present them to CD8(+) T cells; unfortunately, the frequency of activated CD8(+) T cells is often too low to mount an effective response against the tumor. By increasing the amount of antigen for presentation, a larger T-cell response can be theoretically elicited. We used a recombinant adenovirus encoding full-length murine tumor necrosis factor-related apoptosis-inducing ligand (Ad5-mTRAIL) to induce tumor cell apoptosis, and when given intratumorally to mice bearing experimental renal cell carcinoma (Renca) tumors, Ad5-mTRAIL minimally prolonged survival and induced a low level of CTL activity. To enhance dendritic cell efficiency, an immunostimulatory CpG oligodeoxynucleotide (CpG ODN) was combined with Ad5-mTRAIL. This combination therapy significantly augmented in vivo antigen-specific T-cell proliferation and CTL activity, as well as prolonged survival of Renca tumor-bearing mice. Interestingly, depletion of CD4(+) or CD25(+) cells before therapy further enhanced survival and in vivo CTL activity. In addition, tumor-free mice depleted of CD4(+) cells were also able to reject a subsequent challenge of Renca cells, but not MHC-matched RM-11 prostate tumor cells, demonstrating the existence of immunologic memory. These results collectively show that local treatment with Ad5-mTRAIL and CpG ODN can augment tumor antigen cross-presentation resulting in T-cell proliferation, enhanced CTL activity, and increased animal survival.

Apoptotic cells induce tolerance by generating helpless CD8+ T cells that produce TRAIL.

The decision to generate a productive immune response or immune tolerance following pathogenic insult often depends on the context in which T cells first encounter Ag. The presence of apoptotic cells favors the induction of tolerance, whereas immune responses generated with necrotic cells promote immunity. We have examined the tolerance induced by injection of apoptotic cells, a system in which cross-presentation of Ag associated with the dead cells induces CD8+ regulatory (or suppressor) T cells. We observed that haptenated apoptotic cells induced CD8+ suppressor T cells without priming CD4+ T cells for immunity. These CD8+ T cells transferred unresponsiveness to naive recipients. In contrast, haptenated necrotic cells stimulated immunity, but induced CD8+ suppressor T cells when CD4+ T cells were absent. We further found that CD8+ T cells induced by these treatments displayed a "helpless CTL" phenotype and suppress the immune response by producing TRAIL. Animals deficient in TRAIL were resistant to tolerance induction by apoptotic cells. Thus, the outcome of an immune response taking place in the presence of cell death can be determined by the presence of CD4+-mediated Th cell function.

Histone deacetylase inhibitors enhance Ad5-TRAIL killing of TRAIL-resistant prostate tumor cells through increased caspase-2 activity.

Interest in TNF-related apoptosis-inducing ligand (TRAIL) as a cancer therapeutic has been high since its first description. Recently, the use of histone deacetylase inhibitors (HDACi) to treat cancer has progressed from the laboratory to the clinic, and the combination of HDACi and TRAIL is very powerful in killing human tumors. Using a panel of prostate tumor cell lines (ALVA-31, DU-145, and LNCaP) with varying TRAIL sensitivity, we examined their sensitization to a recombinant adenovirus encoding TRAIL (Ad5-TRAIL) by sodium butyrate and trichostatin A. HDACi treatment increased coxsackie-adenovirus receptor (CAR) expression, resulting in increased adenoviral infection, and increased TRAIL-mediated killing. In TRAIL-resistant DU-145 cells, HDAC inhibition also decreased protein kinase casein kinase (PKCK) 2 activity, leading to caspase-2 activation. The importance of PKCK2 and caspase-2 in DU-145 sensitization was demonstrated with the PKCK-2-specific inhibitor, which enhanced Ad5-TRAIL-induced death, or the caspase-2-specific inhibitor, zVDVAD, which blocked Ad5-TRAIL-induced death. Thus, our data highlight the connection between HDAC inhibition of PKCK2 activity and tumor cell sensitivity to TRAIL-induced apoptosis. Specifically, HDAC inhibition leads to decreased PCKC2 activity, which is followed by caspase-2 activation and partial cleavage of caspase-8 that sensitizes the tumor cell to TRAIL.

Histone deacetylase inhibitors modulate the sensitivity of tumor necrosis factor-related apoptosis-inducing ligand-resistant bladder tumor cells.

Urothelial carcinoma of the bladder accounts for approximately 5% of all cancer deaths in humans. The large majority of tumors are superficial at diagnosis and, after local surgical therapy, have a high rate of local recurrence and progression. Current treatments extend time to recurrence but do not alter disease survival. The objective of the present study was to investigate the tumoricidal potential of combining the apoptosis-inducing protein tumor necrosis factor-related apoptosis inducing ligand (TRAIL) with histone deacetylase inhibitors (HDACi) against TRAIL-resistant bladder tumor cells. Pretreatment with HDACi at nontoxic doses, followed by incubation with TRAIL, resulted in a marked increase in TRAIL-induced apoptosis of T24 cells but showed no significant increase in toxicity to SV40 immortalized normal human uroepithelial cell-1. HDAC inhibition, especially with sodium butyrate and trichostatin A, led to increased TRAIL-R2 gene transcription that correlated with increased TRAIL-R2 surface expression. The increased TRAIL-R2 levels also resulted in accelerated death-inducing signaling complex (DISC) formation, caspase activation, and loss of mitochondrial membrane potential, which all contributed to the increase in tumor cell death. Collectively, these results show the therapeutic potential of combining HDAC inhibition with TRAIL as an alternative treatment for bladder cancer.

Histone deacetylase inhibitors modulate renal cell carcinoma sensitivity to TRAIL/Apo-2L-induced apoptosis by enhancing TRAIL-R2 expression.

Every year, 12,000 people in the U.S. die from renal cell carcinoma. Current therapies include partial or complete nephrectomy or treatments such as administration of IFN-alpha and/or interleukins that are moderately effective, at best. Moreover, the current therapies are invasive and inefficient and new therapies are needed. Histone deacetylase (HDAC) inhibitors have recently been found to sensitize cells to apoptosis-inducing agents, although the mechanism of this action is largely unknown. The current study has investigated the potential of using five different histone deacetylase inhibitors (HDACI) (depsipeptide, MS-275, oxamflatin, sodium butyrate, and trichostatin A) to sensitize TNF-related apoptosis-inducing ligand (TRAIL)/Apo-2L-resistant renal cell carcinoma cells to TRAIL/Apo-2L-induced apoptosis. Sodium butyrate and trichostatin A each enhanced TRAIL/Apo-2L-mediated tumor cell death to a greater extent than the other HDACI. Annexin V staining and caspase activity demonstrated the mechanism of cell death was apoptosis. Both sodium butyrate and trichostatin A treatment also increased mRNA and surface expression of TRAIL receptor 2 that was dependent on the transcription factor Sp1, thus providing a possible mechanism behind the increased sensitivity to TRAIL/Apo-2L. These results indicate that combination therapy of HDACI, such as sodium butyrate and trichostatin A, and TRAIL/Apo-2L has great potential for an efficient alternative therapy for renal cell carcinoma.

Neutrophil stimulation with Mycobacterium bovis bacillus Calmette-Guerin (BCG) results in the release of functional soluble TRAIL/Apo-2L.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been used to treat bladder cancer for almost 30 years; however, the effector mechanism of the BCG-induced antitumor response remains enigmatic. Most BCG research has focused on the mononuclear-cell infiltrate, but growing evidence supports a role for neutrophils in the antitumor response. Previously, we demonstrated increased urinary tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo-2L) levels from BCG-responsive patients compared to nonresponders. Interestingly, neutrophils isolated from the urine expressed TRAIL/Apo-2L, leading us to investigate the neutrophil response to BCG. BCG-stimulated neutrophils expressed surface-bound and released functional soluble TRAIL/Apo-2L. Whereas neither interferon alpha (IFN-alpha) nor IFN-gamma directly induced TRAIL/Apo2L expression by neutrophils, IFN-alpha did stimulate TRAIL gene transcription, and IFN-primed neutrophils contained and released more TRAIL/Apo-2L after BCG stimulation than did unprimed neutrophils. In unstimulated neutrophils TRAIL/Apo-2L was present predominantly in the azurophilic granules and plasma-membrane-enriched/secretory-granule fraction. Finally, we observed that killed BCG, Toll-like receptor 2 (TLR2) and TLR4 agonists, and an M tuberculosis cell-wall fraction were each capable of inducing the release of soluble TRAIL/Apo-2L from neutrophils. These results further characterize the potential role neutrophils may play in initiating the antitumor response described with BCG treatment for superficial bladder cancer.

Depsipeptide (FR901228) enhances the cytotoxic activity of TRAIL by redistributing TRAIL receptor to membrane lipid rafts.

TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis in various tumor cell types and is under investigation as a cancer therapeutic. The development of a recombinant adenovirus encoding the full-length human TRAIL gene (Ad5-TRAIL) replaces the need for large quantities of soluble TRAIL protein in tumor suppressive therapies. However, the full potential of Ad5-TRAIL has not yet been maximized. Recent investigation of a histone deacetylase inhibitor, depsipeptide (FR901228), has demonstrated that it increases cellular susceptibility to adenovirus infection and augments adenoviral transgene expression. Thus, studies were initiated to evaluate the ability of depsipeptide to enhance the cytotoxic activity of Ad5-TRAIL against human prostate tumor cells. In vitro, depsipeptide increased expression of coxsackie-adenovirus receptor, leading to increased adenoviral infection and transgene expression. Additionally, tumor cell killing by Ad5-TRAIL was higher following depsipeptide pretreatment. More surprisingly, depsipeptide also increased prostate tumor cell sensitivity to TRAIL-induced apoptosis. Investigation into the mechanism responsible for increased TRAIL responsiveness revealed increased levels of TRAIL-R1 and -R2 in membrane lipid rafts following depsipeptide treatment. These results indicate that depsipeptide is a potent agent for enhancing the activity of Ad5-TRAIL by multiple mechanisms, allowing for a more efficient use of Ad5-TRAIL as an antitumor therapy.