Mycobacterium bovis bacillus CalmetteGué rin (BCG) has been used to treat bladder cancer for almost 30 years; however, the effector mechanism of the BCGinduced 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 ␣ (IFN-␣) nor IFN-␥ directly induced TRAIL/Apo2L expression by neutrophils, IFN-␣ 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. IntroductionUrothelial carcinoma of the bladder accounts for approximately 5% of all cancer deaths in humans. 1 A majority of the tumors (70%-80%) are superficial at diagnosis and, after local surgical therapy, have a high rate of local recurrence (70%) and progression (30%). Thus, patients require lifelong medical follow-up examinations with inspections of their bladders, and additional surgical resection and prophylactic treatments are typically needed in the presence of recurrence. Current treatments extend time to recurrence but do not alter disease survival. The resulting economic burden on the US health care system is enormous, reaching over $4 billion annually. Indeed, as measured on the basis of cumulative per patient cost from diagnosis until death, the cost to treat bladder cancer exceeds all other forms of human cancer.In 1921, bacillus Calmette-Guérin (BCG) was isolated from Mycobacterium bovis, a mycobacterium causing bovine tuberculosis. 2 Since 1976, BCG has been used with increasing success for the treatment of superficial bladder cancer and has become the treatment of choice for this tumor entity. 3 Despite its worldwide acceptance for the treatment of superficial bladder cancer, and many mechanistic studies performed, the antitumor effector mechanism remains incompletely defined. BCG therapy results in a massive local immune response characterized by the induced cytokine expression in the bladder tissue and urine, 4 and an influx of granulocytes and mononu...

CpG-containing oligodeoxynucleotides (CpG ODN) have broad-ranging immunostimulatory effects, including the generation of antitumor immune responses. Analysis of different CpG ODN have identified two classes: CpG-A ODN, which stimulate high levels of IFN-α production from plasmacytoid dendritic cells and weakly activate B cells, and CpG-B ODN, which strongly activate B cells but stimulate low production of IFN-α from plasmacytoid dendritic cells. Previously, we observed that CpG-B ODN (2006) induces TRAIL/Apo-2 ligand (Apo-2L)-mediated killing of tumor cells by CD14+ PBMC. In this study, we extend our investigation of CpG ODN-induced TRAIL/Apo-2L expression and activity in PBMC to include CpG-A ODN. Of the two classes, IFN-α production and TRAIL/Apo-2L-mediated killing of tumor cells was greatest with CpG-A ODN. Surprisingly, CD3+, CD14+, CD19+, and CD56+ PBMC expressed high levels of TRAIL/Apo-2L following CpG-A ODN stimulation. When isolated, the CD19+ PBMC (B cells) were able to kill tumor cells in a TRAIL/Apo-2L-dependent manner. As with CD14+ PBMC, CD19+ sorted B cells were capable of up-regulating TRAIL/Apo-2L expression when stimulated with IFN-α alone. Interestingly, agonist anti-CD40 mAb further enhanced the IFN-α-induced TRAIL/Apo-2L expression on CD19+ B cells. These results are the first to demonstrate human B cell-mediated killing of tumor cells in a TRAIL/Apo-2L-dependent fashion.

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.

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.

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) use in the treatment of bladder cancer was first reported in 1976, but the mechanism of the induced antitumor activity has still not been fully explained. BCG is a potent immunostimulant, normally producing a Th1 cytokine response, including IFN. Recent studies have shown CpG oligodeoxynucleotide induce tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression via IFN production. Given that Mycobacterial DNA contains high amounts of CpG motifs, we hypothesized that BCG's antitumor properties are akin to CpG oligodeoxynucleotide, where the cytokine response to BCG induces TRAIL up-regulation. Using ELISA, urine IFN-␥, and TRAIL levels were initially undetectable in BCG therapy patients but were high after later induction treatments. More importantly, patients that responded to BCG therapy had significantly higher urine TRAIL levels, which killed bladder tumor cells in vitro versus nonresponders. Flow cytometry of fresh urine revealed TRAIL-expressing neutrophils. Given these data, we propose TRAIL plays a role in BCGinduced antitumor effects.