Urothelial carcinoma of the bladder accounts for ∼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. (Cancer Res 2006; 66(1): 499-507)
- bladder tumor
Cell death can be classified into two forms based on morphologic and biochemical criteria: necrosis and apoptosis ( 1– 3). Necrosis is the nonphysiologic or passive type of cell death that is usually caused by extreme trauma or injury to the cell ( 4). It generally affects cells in groups rather than single cells and evokes inflammation when it develops in vivo. Necrotic cells cannot maintain proper plasma membrane function so they can no longer regulate osmotic pressure. The cells swell and rupture, spilling their cellular contents into the surrounding tissue, resulting in the nonspecific cellular destruction that leads to an inflammatory response necessary to remove the debris and begin tissue repair. The other morphologic pattern of cell death, apoptosis, is a more subtle process characterized by numerous cellular changes ( 3). This type of cell death usually affects single cells and is characterized histologically by the formation of small, spherical cytoplasmic fragments, some of which contain pyknotic remnants of nuclei. The fate of the apoptotic cell is to be phagocytosed by surrounding cells before it can rupture and release its potentially inflammatory contents. Thus, apoptotic cell death is well suited for a role in tissue homeostasis because it can result in the deletion of cells with little tissue disruption.
Tumor necrosis factor (TNF)–related apoptosis inducing ligand (TRAIL; Apo-2L) is a TNF family member capable of inducing apoptosis and has recently received great attention because of its therapeutic potential for cancer. Early studies identified two unique characteristics of TRAIL. First, TRAIL-induced apoptosis occurs only in tumorigenic or transformed cells and not in normal cells ( 5, 6). Soluble TRAIL has varying degrees of potency against the variety of tumor cell lines, such that 40% to 75% of the tumor cell lines tested have been declared to be TRAIL sensitive, suggesting that TRAIL could be used as a broad-spectrum, antitumor molecule in vivo ( 7, 8). Second, compared with other TNF family members whose expression is tightly regulated and often transiently expressed, TRAIL mRNA is detected in a wide range of tissues ( 5). TRAIL has proved to be a potent antitumor agent in preclinical studies but current formulations of the molecule have some limitations that restrict its potency. Consequently, there is increasing investigation into the possibility of combining TRAIL with other agents to alter tumor cell responsiveness to TRAIL. Histone deacetylase inhibitors (HDACi) are one such group of compounds that show profound tumoricidal effects when combined with TRAIL. HDAC inhibition maintains the chromatin in a state where the positive charge found on histone lysine residues is neutralized so that the chromatin can be in an open, relaxed form that favors active transcription. Moreover, deregulation of histone acetyltransferases and HDAC has been implicated in the formation and development of certain human cancers by changing the expression pattern of various genes ( 9, 10).
Annually, bladder cancer accounts for ∼13,000 deaths, and this year over 60,000 new cases will appear (accounting for ∼5% of all tumors diagnosed), making it the fourth most common cancer among men and tenth in women ( 11). At diagnosis, most bladder tumors are superficial and, after local surgical therapy, have a high rate of local recurrence (70%) and progression (30%). Thus, patients require lifelong follow-up exams 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 survival. The resulting economic burden on the U.S. 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. Over 20% of patients initially diagnosed with superficial bladder cancer will eventually die of their disease despite local endoscopic surgery ( 12). Topical application of chemotherapeutics or immune-activating substances is routinely used in over half of patients with superficial disease due to unfavorable variables that define a heightened risk of recurrence and/or progression. Thus, studies examining the effectiveness of combining TRAIL with HDAC inhibition against human bladder tumor cells were done.
Materials and Methods
Reagents and antibodies. Depsipeptide [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone; FR901228, FK228] was obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute (Bethesda, MD). Trichostatin A and sodium butyrate were purchased from Sigma (St. Louis, MO). MS-275 and oxamflatin were purchased from Calbiochem (San Diego, CA). Antibodies against TRAIL-R1 (DJR1; eBioscience, San Diego, CA) and TRAIL-R2 (DJR2-4; eBioscience) were used for flow cytometry according to the instructions of the manufacturer. Recombinant human TRAIL (rTRAIL), consisting of the 168 amino acid extracellular domain, was purchased from Peprotech (Rocky Hill, NJ). The replication-deficient adenovirus encoding human TNFSF10 (Ad5-TRAIL; ref. 13) expressed from the cytomegalovirus promoter was generated at the University of Iowa Gene Transfer Vector Core (Iowa City, IA) using the RAPAd.I system ( 14).
Cell lines. The human bladder tumor cell line T24 was obtained from American Type Culture Collection (Rockville, MD) and cultured in MEM medium supplemented with 10% FCS, 1% nonessential amino acids, 1 mmol/L sodium pyruvate, and 1% streptomycin/penicillin solution. RT-4, UMUC3, and SV40 immortalized normal human uroepithelial cells (SVHUC-1; ref. 15) were obtained from Dr. Yi Luo (University of Iowa, Iowa City, IA) and cultured in DMEM (RT-4) or RPMI (UMUC3 and SVHUC-1) medium supplemented the same as the MEM medium.
In vitro killing of human bladder tumor cells. Tumor cell sensitivity to rTRAIL or Ad5-TRAIL was assayed using the following procedure. Cells were added to 96-well plates (2 × 104 per well) in complete medium and pretreated with each HDACi for 16 hours before adding rTRAIL or Ad5-TRAIL. Cell death was determined after 24 hours by crystal violet staining ( 16). Results are presented as percentage cell death: [1 − (absorbance of cells treated / absorbance of cells not treated)] × 100.
Measurement of caspase activity or mitochondrial membrane potential. Cells (5 × 105 per well in a 24-well plate) were treated with HDACi or vehicle controls for 16 hours and then with rTRAIL (200 ng/mL) for 4 hours. The cells were harvested and caspase-8 and caspase-9 activity was measured using carboxyfluorescein-labeled leucine-glutamic acid-threonine-aspartic acid and leucine-glutamic acid-histidine-aspartic acid fluoromethyl ketone peptide caspase inhibitors, respectively, according to the protocol of the manufacturer (Immunochemistry Technologies, Bloomington, MN). To measure mitochondrial membrane potential, the MitoPT mitochondrial permeability transition detection kit was used (Immunochemistry Technologies).
Quantitative reverse transcription-PCR. Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA) from untreated T24 cells or cells treated with the HDACi or their vehicles (DMSO, ethanol, or PBS) for 24 hours. Total RNA (2 μg) was reverse-transcribed using Superscript II. The real-time quantitative reverse transcription-PCR (RT-PCR) primers and probes for TRAIL-R1 and TRAIL-R2 were designed to cross an intron using published sequences (human genome project BAC clone RP11-1149023 and RP11-875011, respectively). Sequences of the real-time quantitative RT-PCR primers and probes used were as follows: TRAIL-R1 (forward: 5′-TGTACGCCCTGGAGTGACAT-3′; reverse: 5′-CACCAACAGCAACGGAACAA-3′; probe: 5′-6FAM-TGTCCACAAAGAATC AGGCAATGGACATAAT-TAMRA-3′) and TRAIL-R2 (forward: 5′-CACTCACTGGAATGA CCTCCTTT-3′; reverse: 5′-GTGCAGGGACTTAGCTCCACTT-3′; probe: 5′-6FAM-TCACAC CTGGTGCAGCGCAAGCAG-TAMRA-3′). The Coxsackie and adenovirus receptor (CAR) and rRNA primer/probe sets were purchased from PE Applied Biosystems (Foster City, CA). cDNA (250 ng) was used as a template for TaqMan assay for both TRAIL-R1, TRAIL-R2, and CAR transcripts and the internal control of rRNA. The TaqMan PCR reaction was carried out as described previously ( 17).
Flow cytometry. Untreated or HDACi-treated cells were incubated with the phycoerythrin-conjugated anti-TRAIL-R1 or anti-TRAIL-R2 monoclonal antibody (mAb; 1:100 dilution) for 1 hour at 4°C. After three washes, cells were analyzed immediately on a FACScan (Becton Dickinson, San Jose, CA).
TRAIL receptor death-inducing signaling complex analysis. T24 cells (40 × 106 per condition) were first treated with sodium butyrate (5 mmol/L) for 16 hours and then incubated for 15, 30, or 60 minutes at 37°C with Flag-tagged rTRAIL (1 μg/mL; Axxora, San Diego, CA) and biotinylated anti-Flag mAb (M2, 2 μg/mL; Sigma). The incubation was stopped by adding 20 volumes of ice-cold PBS, and cell pellets were lysed by addition of 1 mL lysis buffer. Protein complexes were precipitated by overnight incubation with 25 μL streptavidin beads. After overnight incubation, beads were washed with lysis buffer and proteins were eluted from the beads by addition of 50 μL 2× reducing sample buffer, heated for 5 minutes at 100°C, separated by SDS-PAGE, and transferred to nitrocellulose membrane. Membranes were blocked for 4 hours with 5% nonfat dry milk in PBS containing 0.1% Tween 20, followed by overnight incubation with anti-TRAIL-R2 antiserum (Axxora) or anti-Fas-associated death domain protein (FADD) mAb (BD Transduction Laboratories, San Diego, CA). Membranes were washed and then incubated with peroxidase-conjugated donkey anti-rabbit (diluted 1:1,000, Amersham, Piscataway, NJ) or goat anti-mouse IgG (diluted 1:5,000, Jackson ImmunoResearch, West Grove, PA) for 1 hour. Following several washes, the blots were developed by chemiluminescence (SuperSignal West Pico Chemiluminescence Substrate, Pierce, Rockford, IL).
Sensitivity of human bladder cancer cell lines to TRAIL is enhanced following treatment with HDACi. We evaluated the sensitivity of various bladder cancer lines to TRAIL-induced cell death over a range of concentrations. T24 cells were the most resistant of the lines tested to TRAIL, whereas RT-4 and UMUC3 cells were quite sensitive in comparison ( Fig. 1 ). SVHUC-1 cells, an example of normal bladder epithelia, were also examined and found to be resistant to TRAIL killing. This resistance of the SVHUC-1 cells to TRAIL is consistent with previous reports demonstrating little to no TRAIL-mediated cytotoxicity on normal cells and tissues ( 18).
Observing that T24 cells were resistant to TRAIL-induced death, we tested the ability of a panel of HDACi to sensitize T24 cells to TRAIL. T24 cells were treated for 16 hours with each of the HDACi: depsipeptide (10 ng/mL), MS-275 (2.5 μmol/L), oxamflatin (2.5 μmol/L), sodium butyrate (5 mmol/L), or trichostatin A (500 ng/mL). Western blot analysis shows that each HDACi was indeed inhibiting HDAC activity because there were dramatically increased levels of acetylated histone H3 ( Fig. 2 ). Following treatment with the HDACi, the T24 cells were then exposed to rTRAIL for an additional 24 hours. Cell death was measured and showed that the T24 cells were TRAIL resistant with either medium pretreatment or vehicle controls (ethanol, DMSO, and PBS; Fig. 3A-E ). In contrast, when the cells were pretreated with any of the HDACi before adding TRAIL, TRAIL-induced death was significantly increased. To determine whether the HDACi-TRAIL combination was synergistically increasing tumor cell death, cells were treated with increasing concentrations of HDACi or TRAIL alone, or in combination. As previously observed, cells treated with rTRAIL or HDACi alone did not exhibit much cell death ( Fig. 3F-J). The combination of HDACi and rTRAIL, however, showed a significant increase in cell death that shows the therapeutics synergize to augment cell death. Together, these results show the potent tumoricidal activity of combining TRAIL with HDAC inhibition to kill bladder tumor cells.
TRAIL-mediated caspase activation is enhanced by HDAC inhibition and involves loss of mitochondrial membrane potential. It is well understood that TRAIL induces apoptosis in tumor cells via a caspase-dependent mechanism that can be amplified by the loss of mitochondrial membrane potential and release of cytochrome c ( 19). To determine whether the HDACi were increasing caspase signaling, or if there was another mechanism for cell death, caspase activation was measured. T24 cells were pretreated with each HDACi or their vehicle controls, stimulated with 200 ng/mL rTRAIL, and caspase-8 and caspase-9 activity was measured 6 hours later. Figure 4A shows histogram profiles for the results with sodium butyrate, PBS, or TRAIL alone, and the combination of TRAIL and either sodium butyrate or PBS. There was a small but measurable increase in caspase-8 and caspase-9 activity when TRAIL was alone or in combination with PBS. In contrast, sodium butyrate pretreatment significantly increased the amount of active caspase-8 and caspase-9 after TRAIL stimulation. The results for all the HDACi and vehicles alone, and in combination with TRAIL, are presented numerically in Fig. 4B. Activation of caspase-8 and caspase-9 was detected in ∼20% and 30%, respectively, of the cells that received rTRAIL stimulation alone or following the vehicle controls. In contrast, pretreatment with all the HDACi increased the percentage of caspase-8- and caspase-9-positive cells to 60% and 80%, respectively, with rTRAIL stimulation.
Caspase-9 can only be activated when procaspase-9 interacts with Apaf-1 and cytochrome c is released from mitochondria ( 20). The high degree of caspase-9 activity suggests a significant loss of mitochondrial membrane potential, which would allow cytochrome c to escape from the mitochondrial intramembrane space. Thus, mitochondrial membrane potential was analyzed in Fig. 4C. As predicted, each HDACi alone or vehicle control alone did not significantly alter the integrity of the mitochondrial membrane potential. When the combination of HDACi and rTRAIL were used, however, the loss of membrane potential was remarkably enhanced. rTRAIL stimulation alone led to ∼20% of the cells having a loss in mitochondrial membrane potential, but there was increased loss of membrane potential ranging from 64% to 95% when rTRAIL was combined with HDAC inhibition. These results clearly indicate that HDAC inhibition in TRAIL-resistant T24 cells allows the apoptotic signaling pathway to be activated more effectively after TRAIL-TRAIL death receptor ligation.
HDACi increase TRAIL-R2 expression and death-inducing signaling complex formation. One explanation for the increased caspase activation results presented in Fig. 4 is that HDAC inhibition results in the up-regulation of TRAIL-R1, TRAIL-R2, or both, so that a stronger apoptotic signal can be initiated after TRAIL ligation. To investigate this possibility, we assessed both the mRNA and protein levels of TRAIL-R1 and TRAIL-R2 after HDACi treatment. T24 cells were treated for 16 hours with each HDACi, or vehicle control, and then mRNA was isolated for quantitative RT-PCR analysis. TRAIL-R1 mRNA was unchanged by HDACi treatment, but TRAIL-R2 mRNA levels did increase especially when either sodium butyrate or trichostatin A were used ( Fig. 5A ). Surface TRAIL-R1 and TRAIL-R2 expression was also assessed by flow cytometry after the same treatment conditions. Depsipeptide, sodium butyrate, and trichostatin A increased TRAIL-R2 expression, but TRAIL-R1 remained unchanged ( Fig. 5C). To determine if this increase in TRAIL-R2 had a functional effect on the assembly of the death-inducing signaling complex (DISC), a DISC immunoprecipitation was done to assess recruitment of signaling proteins to the DISC. Thus, cells were pretreated for 16 hours with either medium alone or sodium butyrate, and then stimulated with rTRAIL for 15, 30, and 60 minutes. As expected, sodium butyrate–treated cells had more TRAIL-R2 at all time points assessed compared with that of the untreated cells ( Fig. 5D). In addition, the sodium butyrate–treated cells had more FADD recruitment, which was indicated by its association with the immunoprecipitated TRAIL-R2. FADD levels, by comparison, were undetectable in the untreated cells throughout the time course. Collectively, these results indicate that HDAC inhibition results in increased TRAIL-R2 expression on T24 that translates in accelerated and stronger DISC formation, permitting a stronger apoptotic signal to be generated.
Normal bladder cells are not sensitized by HDACi to TRAIL-induced apoptosis. Because both T24 cells and normal bladder epithelial cells (SVHUC-1) were resistant to rTRAIL-induced apoptosis, we felt it was important to determine if HDAC inhibition altered the TRAIL sensitivity of the SVHUC-1 cells as they did with the T24 cells. Thus, SVHUC-1 cells were pretreated with each of the HDACi or vehicle controls and then stimulated for 24 hours with rTRAIL. The highest concentration of TRAIL used only induced ∼20% cell death in SVHUC-1 ( Fig. 6A ). More importantly, in contrast to the T24 cells, HDACi treatment did not significantly sensitize the SVHUC-1 to rTRAIL. Cell death after pretreatment was only increased at the highest rTRAIL concentrations and did not reach >40% cell death. To understand why the SVHUC-1 cells were not sensitized to rTRAIL, TRAIL-R1 and TRAIL-R2 expression levels were assessed after HDAC inhibition. Like the T24 cells, TRAIL-R1 expression was unchanged on the SVHUC-1 cells. In contrast to the T24 cells, TRAIL-R2 expression was not changed by any of the HDACi ( Fig. 6B). These results indicate that TRAIL-HDAC inhibition combination therapy may be a viable alternative treatment for bladder cancer.
HDACi induce CAR expression and sensitize bladder tumor cells to Ad5-TRAIL-induced death. Another quality of HDACi is their ability to induce CAR expression, which allows adenovirus to infect the cell ( 21, 22). Having developed a recombinant adenovirus that encodes the human TRAIL (TNFSF10) gene ( 13), we wanted to determine if the HDACi used above also increased the tumoricidal activity of Ad5-TRAIL toward T24 bladder tumor cells. T24 normally expresses low levels of CAR ( 23– 25), so we first determined if HDAC inhibition increased CAR expression. To do this, we treated the cells with either the HDACi or vehicles and then did quantitative RT-PCR to measure CAR mRNA levels. Indeed, all five HDACi significantly increased CAR mRNA levels compared with untreated or vehicle-treated cells ( Fig. 7A ). Next, we treated T24 cells with HDACi or vehicles for 16 hours and then infected the cells with Ad5-TRAIL. After 24 hours, cell death was measured and there was significantly increased tumor cell death in the HDACi-treated cells compared with controls ( Fig. 7B). These results show the multivariable effects that HDACi can have on bladder tumor cells that not only modulate cell sensitivity to TRAIL-induced apoptosis but also permit adenoviral infection and induction of tumor cell death by expression of the TRAIL adenoviral transgene. In addition, the collective results show the potential utility of HDACi-TRAIL combination therapy for bladder cancer.
Intravesical treatment of superficial bladder cancer is done to prevent tumor recurrence after successful local surgical resection and to eradicate residual disease. Various intravesical chemotherapeutic agents, such as thiotepa, doxorubicin, and mitomycin, have been the therapeutic treatment of choice for years; however, these agents achieved short remissions with a net durable benefit for ∼7% to 14% of patients ( 26). More unconventional forms of treatment, such as immunotherapy, were introduced in the last few decades due to unsatisfactory chemotherapy results, where one of the most successful therapies for superficial bladder cancer and carcinoma in situ of the bladder is intravesical administration of Mycobacterium bovis bacillus Calmette-Guerin (BCG; ref. 27). However, this therapy has also displayed limited success because 25% to 40% of the patients never respond to BCG ( 28, 29). The development of therapeutic regimens that offer potent tumoricidal activity but no toxic side effects to normal cells and tissues is a necessary alternative. Our results show the utility of combining HDACi with TRAIL (either recombinant protein or Ad5-TRAIL) to induce bladder tumor cell death but not in normal bladder urothelium.
Tumorigenesis is a multistep process ultimately leading to genetic changes that activate oncogenes or inactivate tumor suppressor genes. Epigenetic alterations also contribute to tumor progression and histone acteylation is one example of such an alteration. HDAC catalyzes the removal of acetyl groups from the ε-amino group on lysine residues of the core nucleosomal histones H2A, H2B, H3, and H4, and, together with histone acetyltransferases, regulates histone acetylation levels. The balance of nucleosomal histone acetylation plays an important regulatory role in chromatin structure and transcription of many genes ( 30, 31). HDAC inhibition leads to the accumulation of marked amounts of acetylated histone species that result in profound effects on tumor cells, such as inhibiting cell cycle or inducing differentiation or apoptosis ( 31– 33). Consequently, there are several HDACi undergoing phases I and II testing as single-agent therapy as a treatment for cancer.
In our system, HDAC inhibition induced TRAIL-R2 expression on the bladder tumor cells, increasing their sensitivity to TRAIL-induced apoptosis. TRAIL-R2, like TRAIL-R1, contains a cytoplasmic death domain, and cross-linking by TRAIL or receptor-specific mAb activates the apoptosis signaling pathway in sensitive cells ( 34– 38). TRAIL-R1/TRAIL-R2 cross-linking leads to the formation of a multiprotein structure called the DISC ( 39) that includes the death receptor, FADD ( 40), and procaspase-8. All death receptors (Fas, TNFR-1, TRAIL-R1, and TRAIL-R2) share a homophilic protein/protein intracellular domain, the death domain ( 41, 42), which is required to attract specialized apoptosis signaling molecules that often contain a death domain themselves. In a homotypic interaction, the death domain of FADD binds to the death domain of TRAIL-R1 or TRAIL-R2. The death effector domain of FADD, in turn, interacts with the death effector domain of procaspase-8 ( 43). Procaspase-8 is proteolytically cleaved and activated at the DISC, initiating the apoptosis caspase cascade. The downstream executioner caspases, caspase-3, caspase-6, and caspase-7, are then activated to cleave the numerous structural and regulatory proteins that maintain cellular integrity. It is unknown why TRAIL-R2 was selectively increased with HDAC inhibition and not other TRAIL receptors. The genes for the four TRAIL receptors are tightly clustered on human chromosome 8p21-22 ( 37, 44, 45), suggesting that they evolved relatively recently via gene duplication. This genetic region is frequently the site of losses of heterozygosity in many types of cancer ( 46). Mutations of the TRAIL-R2 gene have been identified in multiple cancers ( 47– 53); in each case, the mutations have been located within the death domain. It remains to be determined if the other TRAIL receptor genes are also mutated in some cancers that alter tumor cell sensitivity to TRAIL-induced apoptosis.
HDAC inhibition also increased the responsiveness of the bladder tumor cells to adenoviral infection, increasing the activity of Ad5-TRAIL on these cells. This has important implications in the development and use of adenoviral vectors to treat bladder tumors. Studies on bladder cancer cell lines have revealed low expression of CAR, corresponding with the limited ability to infect these cells with adenovirus. CAR is ubiquitously expressed in most benign epithelial tissues. Yet, marked variations in CAR levels have been shown using different cancer cell lines of the same tissue origin ( 22). Despite its importance for adenovirus infection, the function of CAR is not well understood. There is evidence suggesting a role of CAR in cellular adhesion and possible association with tight junctions ( 54, 55). Furthermore, CAR expression may correlate inversely with tumor aggressiveness, and reintroduction of CAR expression may have a tumor-suppressing effect perhaps related to cell cycle phase and regulation ( 54, 56). Considering that patients enrolled in adenoviral-based cancer gene therapy trials usually have advanced disease, these associations are ominous, if confirmed. Although targeting strategies are being investigated to circumvent CAR dependence, all clinical adenoviral gene therapy approaches reported have been CAR dependent. Thus, variable CAR expression may have contributed to the disconnect between preclinical and clinical gene transfer rates.
The bladder is a unique environment when it comes to treating cancerous lesions. In 1976, Morales et al. ( 27) first described the antitumor effects of intravesical administration of BCG in bladder cancer. Today, BCG therapy remains at the forefront of treating patients with superficial bladder cancer and carcinoma in situ and continues to be one of the most successful immunotherapies for any solid human malignancy. Recent studies from our laboratory have reported increased urinary TRAIL levels in patients responsive to BCG therapy and TRAIL expression on neutrophils in voided urine following BCG instillation ( 57), suggesting the potential for soluble TRAIL and TRAIL-expressing cells as the antitumor mechanism elicited by BCG therapy. Thus, intravesical instillation of HDACi before BCG therapy may have profound effects in sensitizing the tumor cells to the TRAIL subsequently released into the bladder. Another clinical alternative could use intravesical instillation of HDACi followed by instillation of recombinant TRAIL protein or Ad5-TRAIL. This approach would circumvent any potential toxicities associated with BCG therapy and directly use the effector molecule that BCG induces. Careful consideration should be made to such possibilities.
The development of TRAIL as a potential anticancer therapeutic arose from its ability to potently induce apoptosis in tumor cells while having little or no detectable cytotoxic effects on normal cells and tissues ( 5, 6). The identification of multiple receptors that can bind TRAIL has significantly increased the potential complexity of this receptor/ligand system in terms of understanding normal physiologic functions and therapeutic applications. Not only will it be essential to optimize the form of TRAIL used in therapy, as well as the mode of delivery, it may be even more important to understand the mechanisms by which tumor cells regulate their sensitivity to TRAIL-induced apoptosis. At the same time, elucidating the molecular basis that protects normal cells and tissues from TRAIL will also be essential in formulating TRAIL-based therapies for cancer that will not be toxic to normal cells within the body. Since its identification nearly 10 years ago, there has been a great deal learned how TRAIL functions within the body in tumor and nontumor scenarios. Future studies will continue to develop TRAIL into the cancer therapeutic many believe it is destined to be.
Grant support: National Cancer Institute grant CA109446.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Drs. Bennet Elzey and Timothy Ratliff for reviewing the manuscript.
- Received August 23, 2005.
- Revision received September 28, 2005.
- Accepted October 19, 2005.
- ©2006 American Association for Cancer Research.