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CELLULAR AND MOLECULAR
James Buchanan Brady Urological Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland (C.-L.C., J.S., M.C., W.H.C., M.D.S., Y.Li., Y.La., S.E.L., R.R.); Department of Urology, Chang Gung Memorial Hospital and Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taipei, Taiwan (C.-L.C.); and Department of Pharmacology, Cancer Biochemistry Laboratory, College of Medicine, Chang Gung University, Taipei, Taiwan (B.Y.M.Y.)
Received May 15, 2006; accepted July 21, 2006.
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). Although this phenomenon was first observed over three decades ago (Allfrey, 1966), understanding of the mechanisms involved in histone modification and gene transcription has evolved only in recent years (Yoshida et al., 1990; Strahl and Allis, 2000). We now know that histone acetylation status is determined by the interplay of two classes of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDAC). Acetylation of the histones, especially H3 and H4, by HATs is associated with active transcription, whereas deacetylation by HDACs is linked to gene repression. Furthermore, treatment of cancer cells with histone deacetylase inhibitors (HDACIs) results in increased histone acetylation and ultimately, induction of growth arrest, apoptosis, and cell differentiation. These effects have been demonstrated in both hematologic (leukemias and lymphomas) and epithelial/solid (breast, prostate, lung, and neuronal) cancer cell lines (Marks et al., 2001) and are selective for transformed cancer cells. Since much of the previous research has focused on the antiproliferative and differentiation properties of HDACIs, comparatively little is known about their effects on cancer cell migration, invasion, and metastasis, which collectively result in the morbidity and mortality of cancer.
In our previous work, we have shown that treatment of bladder cancer epithelium with HDACIs can alter the expression of at least one cell-cell adhesion molecule, the coxsackie and adenovirus receptor (CAR) (Sachs et al., 2004). CAR is a member of the Ig-type superfamily of cellular adhesion molecules linked to cell motility and tumor invasiveness (Bruning and Runnebaum, 2003; Philipson and Pettersson, 2004), and it is expressed robustly in normal urothelium, but it is down-regulated in a tumor-stage and grade-dependent manner in clinical bladder cancer specimens (Sachs et al., 2002). This repressed expression level seemed to be regulated transcriptionally via epigenetic events (Li et al., 1999; Okegawa et al., 2001), because treatment of the bladder cancer cells with VPA in vitro effectively restored CAR expression (Höti, et al.). Hence, we postulated that treatment with VPA may lead to inhibition of bladder cancer cell migration and invasion. We also predicted that VPA might have the same effect on prostate cancer cells as well.
We now show that VPA treatment inhibits invasion in multiple bladder cancer cell lines, including T24, TCC-SUP, and HT1376. These effects were dose-dependent, cell line-specific, and independent of cell migration, which was not affected by treatment. When the same hypothesis was tested in multiple prostate cancer cell lines, there was no difference in migration or invasiveness between treated and untreated cells. These results demonstrate that the effects of VPA on invasion are cancer type-specific and further suggest that the pathways critical to the development of invasion differ among various cancer types. We also show that acute (72-h) treatment with VPA in vitro results in decreased in bladder cancer cell viability, especially at higher doses and that chronic administration (34 days) of lower dose (0.05–0.4 mM) VPA significantly inhibits tumor xenograft growth in vivo. Collectively, these findings suggest that VPA may represent a novel adjuvant strategy for patients at high risk of recurrence and/or progression of muscle invasive bladder cancer.
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Cell Viability Assay. Bladder cancer cell lines T24, TCC-SUP, HT1376, and RT4 were seeded at 1 x 104 cells/well in 96-well culture plates and incubated overnight with the appropriate media containing 10% FBS. The cells were then treated with media containing VPA (0, 0.6, 1.2, 2.4, and 5.0 mM) for 72 h. Doses of 0.6 to 5.0 mM were selected based on preliminary studies of cell viability in the applied cell lines as well as previously published studies in other nonbladder cell lines (Courage-Maguire et al., 1997; Kaiser et al., 2006). The fraction of cells surviving after acute VPA treatment was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (F. Hoffman-La Roche, Basel Switzerland) as per manufacturer's recommendation. Reduction of MTT to a formazan salt by viable cells was quantified by SPECTRAmax plus (Molecular Devices, Sunnyvale, CA) at 570 nm. The experiments were performed at least in quadruplicates.
Caspase Assay. Equal numbers of T24 cells were seeded and incubated overnight in T75 flasks to allow attachment. The cells were then treated with media containing 0, 0.6, 1.2, 2.4, or 5.0 mM VPA for 16 h, and then they were washed and lysed using the 1x cell lysis buffer from the ApoAlert kit (BD Biosciences, San Jose, CA). Cell lysates were then transferred to a 96-well plate and mixed with reaction buffer/dithiothreitol mix (BD Biosciences) according to the manufacturer's protocol. Caspase activity was determined by measuring fluorescence from converted substrate using the CytoFluor II Series 4000 fluorometer (Applied Biosystems, Foster City, CA) (excitation 380 nm, emission 460 nm).
Western Immunoblotting. T24 cells (70–80% confluent) in T75 flasks were treated with media containing 0, 0.6, 1.2, 2.4, or 5.0 mM VPA or 50 ng/ml TSA for 72 h. The cells were harvested with 0.05% trypsin/0.53 mM EDTA, washed in phosphate-buffered saline, and resuspended in 100 µl of mammalian protein extraction reagent) (Pierce Chemical, Rockford, IL). Tissue from T24t tumor xenografts was homogenized with the Dounce homogenizer and also resuspended in mammalian protein extraction reagent. The BCA protein assay kit (Bio-Rad, Hercules, CA) was used to determine total protein concentration and purified bovine serum albumin (Sigma-Aldrich) was used to generate the standard curve. Proteins were separated on a 12% Tris-HCl polyacrylamide gel (Bio-Rad, Hercules, CA) and transferred to polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked for 1 h in blocking buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) with 5% nonfat dry milk and then incubated overnight with a 1:1000 dilution of rabbit anti-acetyl histone H3 (Lys 9) antibody (Cell Signaling Technology Inc., Beverly, MA) followed by anti-rabbit IgG peroxidase conjugate (1:20,000) (Sigma-Aldrich) for 1.5 h at room temperature. Immunoreactive bands were detected using the ECL Plus Western blotting detection system (GE Healthcare, Piscataway, NJ), according to the manufacturer's instructions. Monoclonal anti-
-actin (1:20,000) (Sigma-Aldrich) and anti-mouse IgG-peroxidase (1:20,000) (Sigma-Aldrich) were used to detect -actin in the same blots. Anti-Cip1/WAF1/P21 mouse monoclonal IgG (Upstate Biotechnology, Charlottesville, VA) and anti-mouse IgG-peroxidase (Sigma-Aldrich) were used to test p21 expression.
Qualitative Invasion Assessment by Fluorescent Microscopy. A modified Boyden chamber invasion assay (Woodward et al., 2002) was performed using a mixed culture of human fibroblasts and primary urothelial cells. These cells were tagged with 5' (and 6')-carboxy-10-dimethylamino-3-hydroxy-spiro[7H-benzo[c]xanthene-7,1'(3H)-isobenzofuran]-3'-one (Invitrogen) and plated (1 x 104 cells/well) on top of a Matrigel-coated, fluorescence blocking, 8-µm pore, polycarbonate membrane (BD BioCoat-FluoroBlok) (BD Biosciences Discovery Labware, Bedford, MA). The BioCoat-FluoroBlok prevents fluorescent signal from traveling from the upper to lower chambers during microscopy. Next, T24 cells were treated with 0 or 5 mM VPA for 24 h and tagged with Vybrant CFDA SE (Invitrogen). The cells were then collected by brief (
1-min) trypsinization followed by blockage with an equal volume of trypsin inhibitor (Invitrogen). Using the trypan blue exclusion dye, 1 x 105 viable cells were added on top of the urothelial cells in each well of the upper chamber. The T24 cells were evaluated for invasion through the urothelial cell layer, coated Matrigel layer, and porous membrane to reach 10% FBS-enriched media in the lower wells. Fluorescent microscopy (Carl Zeiss GmbH, Jena, Germany) pictures were taken at different time intervals (1, 2, 4, 8, and 24 h) from the bottom side of fluorescence blocking membrane, and therefore they showed only the invading cells.
Endpoint (Static) Quantitative Migration and Invasion Assays. Migration and invasion studies were performed simultaneously for optimization and comparison using the ChemoTx system (Neuro Probe, Gaithersburg, MD), which consisted of a 96-well plate covered by an 8-µm porous polycarbonate membrane. Both migration and invasion assessments were performed on the same plate, with readings taken at 0.5 and 8 h. The T24, TCC-SUP, and HT1376 cell lines were treated for 72 h with 0, 1.2, or 5.0 mM VPA or 50 ng/ml TSA and then tagged with Vybrant CFDA SE (Invitrogen) as per manufacturer's recommendations. The cells were then harvested by brief (1-min) trypsinization followed by blockage with an equal volume trypsin inhibitor (Invitrogen). To evaluate migration, 5 x 104 cells/well were plated on top of the membrane in triplicates or quadruplicates in designated wells and allowed to migrate toward 20% FBS-enriched media in the lower wells. To assess invasion, 1 x 105 cells/well were plated on top of the Matrigel-coated membrane in triplicates or quadruplicates and allowed to migrate toward 20% FBS-enriched media in the lower wells. A CytoFluor, fluorescence multiwell plate reader (Perspective Biosystems, Framingham, MA) was used to quantify cell migration and invasion into the lower wells at different time points. The choice of 10 or 20% FBS as a chemoattractant and 5 x 104 or 1 x 105 cells/well for the migration and invasion assays, respectively, was made based upon results of a pilot optimization study (data not shown) on T24 cells.
Real-Time (Dynamic) Quantitative Invasion and Migration Assays. T24 cells were treated with 0, 0.6, 1.2, 2.4, or 5 mM VPA for 72 h, and then they were placed in serum-free media overnight. Next, a 10 µM staining solution was prepared by diluting 10 mM Vybrant CFDA SE (Invitrogen) with phosphate-buffered saline. Cells were prelabeled in the solution for 15 min at 37°C, and then they were trypsinized, counted, and resuspended in serum-free media. In total, 1 x 105 cells in 500 µl of serum-free media were loaded into each well of the upper chamber of a modified Boyden chamber (BD BioCoat FluoroBlok invasion system), and 750 µl of 20% FBS-enriched media was added to each bottom well. The polycarbonate membrane was either coated or uncoated with Matrigel matrix to simulate invasion and migration, respectively. Cells that crossed through the 8-µm pore size polycarbonate membrane were detected in real-time using a multiplate fluorometer (CytoFluor II plate reader) (excitation 492 nm, emission 517 nm). To account for possible effects of CFDA SE on the biological function of cells, each reading was normalized to that taken from a regular 24-well Falcon plate with the same number of CFDA SE-tagged cells. Data are shown as the mean percentage of cell invasion calculated as the mean fluorescent unit (FU) of cell invasion through the Matrigel-coated membrane divided by the mean FU of cell migration through the Matrigel-uncoated control membrane x 100, as recommended by the manufacturer (BD BioCoat FluoroBlok invasion system). Percentage of cell invasion (invasiveness) was calculated by the following equation:
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Statistical Analysis. Data are presented as means ± S.E.M. All statistical analyses were performed on an IBM compatible computer, running GraphPad Prism version 4.0 or GraphPad InStat version 3.0 (GraphPad Software Inc., San Diego, CA) on Windows XP. All experiments were done with at least four and as many as 20 replicates. All error bars represent the S.E.M. Statistical significance was calculated using the Student's t test, Wilcoxon rank sum test, or repeated measures analysis of variance with post hoc testing when appropriate. A value of p < 0.05 was considered statistically significant, and such p values are marked on graphs with an asterisk (*, p < 0.05, **, p < 0.01, and ***, p < 0.001).
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As secondary confirmation of HDACI activity, expression of p21WAF1/CIP1, a cell cycle kinase inhibitor, was also evaluated since previous studies have demonstrated that hydroxamic acid-based HDACIs, such as suberoylanilide hydroxamic acid, can increase both p21 WAF1/CIP1 mRNA and protein expression in a time- and dose-dependent manner (Richon et al., 2000). T24 cells were treated with VPA (0, 1.2, and 5.0 mM) for 4, 8, 16, and 24 h and analyzed for p21WAF1/CIP1 expression on Western blot. As expected, VPA up-regulates p21WAF1/CIP expression in a time- and dose-dependent manner with maximal effects at 16 h (Fig. 1B; Table 2). T24 cells cultured with VPA for greater than 16 h undergo cell death with up to a 40% decrease in cell viability with the 5.0 mM dose at 72 h (Fig. 2A). We also found that p21 expression increased by more than 300% in untreated cells at 24 h. This may have resulted from cell overgrowth, since p21 expression can be regulated by cell contact (Evers et al., 1996; Ritt et al., 2000), and cells were plated at 70 to 80% confluence and allowed to grow overnight. Nonetheless, it is clear that VPA causes a dose-dependent increase in p21 expression, a known HDAC inhibition effect.
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Effect of VPA on Bladder Cancer Cell Survival. To further evaluate HDACI activity of VPA in bladder cancer cells, we performed cell viability assays on a panel of bladder cancer cell lines, including T24 (anaplastic, invasive), TCC-SUP (anaplastic, invasive), HT1376 (invasive), and RT4 (noninvasive papilloma). These cell lines represent the spectrum of disease from highly invasive, anaplastic carcinoma to noninvasive papilloma. Cells were treated with 0, 0.6, 1.2, 2.4, or 5.0 mM VPA for 72 h and then evaluated by MTT. As shown in Fig. 2A, cell viability decreases in a dose-dependent manner in all four cell lines. However, the decrease in cell viability with 72-h treatment is modest except at the higher dose (5.0 mM). Of note, the IC50 value of VPA is greater than 5.0 mM in most cell lines, which is at the level of dose-limiting neurotoxicity in the clinical setting.
Of the cells that did not survive VPA treatment, we sought to determine whether activation of apoptosis was involved. In this experiment, T24 cells were VPA-treated (0, 0.6, 1.2, 2.4, or 5.0 mM) for 24 h, they were harvested, and then they were assessed for caspase activity. VPA treatment resulted in induction of both caspase-2 and -3 enzyme activity (Fig. 2B). However, maximal enhancement was less than 2-fold even at the highest dose (5.0 mM). In addition, there seemed to be little change in caspase-8 or -9 activities. This suggests that acute treatment (72 h) with VPA results in a small fraction of cells undergoing cell death via apoptosis, likely through the intrinsic (mitochondrial) pathway given the relative elevation of caspase-2. This finding is consistent with previous studies that have demonstrated HDACI induction of apoptosis often occurs via the intrinsic pathway (Peart et al., 2003).
Qualitative Analysis of VPA Effect on T24 Invasion. The effects of HDACIs on cancer cell properties, such as invasion and migration, have not been well characterized. In our previous work, we have shown that HDACIs can alter the expression of at least one cell-cell adhesion molecule, CAR, in bladder cancer cells (Sachs et al., 2004). Based on this finding, we sought to determine whether VPA by extension may affect invasion and migration properties. To investigate this hypothesis, we used both qualitative and quantitative methods and multiple bladder cancer cell lines.
A modified Boyden chamber and fluorescently labeled T24 cells were applied to evaluate the effect of VPA on cell invasion in vitro. T24 cells were treated with 0 or 5.0 mM VPA for 72 h, fluorescently tagged, and plated in equal numbers in the upper wells of a modified Boyden chamber. The chamber consisted of a urothelial layer covering a Matrigel-coated porous membrane and a lower well containing FBS-enriched media. The urothelial and Matrigel layers simulate the lamina propria and basement membrane, respectively. Invading cells were imaged in the lower chamber through fluorescent microscopy. The results are presented as paired, time-lapsed serial photomicrographs of treated versus untreated T24 cells. As shown in Fig. 3, acute VPA treatment leads to a qualitative decrease in the invasion rate of T24 cells, an effect that is evident within 2 to 4 h and up to 24 h.
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Real-Time Analysis of VPA Effect on T24 Invasion and Migration. To further investigate the effects of VPA on bladder cancer cell invasion and migration, we used the modified Boyden chamber in a dynamic invasion assay to monitor cell movement in real time. Passage of cells through the Matrigel-coated membrane simulated invasion through the basement membrane, whereas passage through the uncoated membrane simulated cellular migration only. The T24 cell line was selected for initial evaluation, because it showed promising results in the static, endpoint invasion assays. T24 cells were treated with 0, 0.6, 1.2, 2.4, or 5.0 mM VPA for 72 h, they were fluorescently tagged, and then they were harvested and plated in the upper wells of a modified Boyden chamber. Cell movement was recorded in real time for up to 72 h using a multiplate fluorometer. The data are shown in Fig. 5 as percentage of migration, percentage of invasion, and percentage of invasiveness. These experiments demonstrate that acute treatment with VPA decreases T24 cell invasion in a dose-dependent manner. The reductions in invasion were statistically significant for all tested concentrations and varied from approximately 40 to 85% between the lowest and highest doses, respectively. In contrast, VPA seemed to cause a slight increase in T24 migration at 1.2 and 5.0 mM; however, these effects were neither dose-dependent nor statistically significant (Fig. 5B). As an additional measure of VPA effect, the percentage of invasiveness was calculated and compared among treated and untreated cells. The results are shown in Fig. 5C, and they were similar to those for invasion; acute VPA treatment significantly decreases percentage of invasiveness of T24 cells in a dose-dependent manner.
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Real-Time Analysis of VPA Effect on T24, TCC-SUP, HT1376, and RT4 Invasion and Migration. After testing the effect of VPA on T24 invasion and migration, we then interrogated this effect on the other cell lines, including TCC-SUP, HT1376, and RT4, via the dynamic invasion assay. Using the same protocol described in the previous section, the cells were treated with 0, 1.2 or 5.0 mM VPA, they were fluorescently labeled, and then they were monitored in real time for migration and invasion. Data are shown in Fig. 6 as percentage of invasiveness of treated cells relative to un-treated. Our results show that acute VPA treatment decreases invasiveness in the TCC-SUP and HT1376 bladder cancer cell lines but that it has no effect on the noninvasive RT4 cell line. The effect was dose-dependent for both cell lines and statistically significant after 8 h for all doses in TCC-SUP, but it lacked sustained inhibition in HT1376. The RT4 cell line demonstrated minimal invasion even in the absence of VPA, a phenomenon that may be attributed to its derivation from noninvasive tumor. Taken together, these data confirm that VPA administered acutely for 72 h can inhibit invasion in multiple bladder cancer cell lines in vitro.
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Further confirmation of VPA activity was obtained through Western blot analysis of proteins harvested from tumor tissue. As shown in Fig. 8B, tumors treated with VPA had up-regulation of acetylated histone H3 and p21 expression compared with those in the untreated controls. Relative induction of acetylated histone H3 and p21 was 3.3- and 6.5-fold, respectively, demonstrating that the effect of tumor volume reduction correlates to HDACI activity in vivo.
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) and an estimated annual expenditure of $2.9 billion in the United States alone (Brown et al., 2002). Approximately 70% of bladder tumors present as superficial lesions, and 10 to 20% of these progress to muscle-invasive lesions (Ro et al., 1992). Once invasion outside the bladder occurs, outcomes in the absence of systemic therapy are poor with overall 5-year survival rates ranging from 4 to 35% (Herr, 1994). Given the unfavorable prognosis associated with nonorgan confined disease, therapeutic approaches targeted toward inhibiting invasion should have potentially beneficial effects in the prevention of muscle-invasive disease.
HDAC inhibitors are a new class of drugs shown to have antineoplastic activity in hematologic and solid malignancies (Sandor et al., 2002; Takai et al., 2004). A well known anti-convulsant recently discovered to have potent HDACI activity is VPA (Phiel et al., 2001). Commonly used in the treatment of seizure and bipolar disorders (Loscher, 2002), VPA is well tolerated in patients and has an established safety profile (Gottlicher et al., 2001; Blaheta and Cinatl, 2002). In our previous work, we have shown that HDACIs can alter expression of at least one cell adhesion molecule, CAR, in bladder cancer (Sachs et al., 2004). In addition, loss of CAR expression has been associated with aggressive bladder cancers (Sachs et al., 2002; Matsumoto et al., 2005). For these reasons, we chose to investigate the effects of VPA on bladder cancer cell viability, motility, and invasiveness. We further examined whether these effects were cancer type-specific by testing prostate cancer cell lines and applicable in vivo using tumor xenografts.
We first sought to confirm the HDACI activity of VPA through evaluation of acetylated H3 and p21WAF1/CIP1 expression in VPA treated T24 cells. p21 is a well known cell cycle regulatory protein and target gene of HDAC inhibition (Sowa et al., 1999; Mei et al., 2004). We found that acute (72-h) VPA treatment induces a dose-dependent increase in both acetylated H3 and p21, with maximal induction for the 5.0 mM dose at 72 and 16 h, respectively (Fig. 1). T24 cells cultured with VPA for greater than 16 h undergo cell death, contributing to the relative decrease in p21 at 24 h.
We next determined the effect of acute VPA on bladder cancer cell viability in vitro. We found that 72-h treatment with VPA results in a dose-dependent decrease in cell viability for all tested cell lines (Fig. 2A). Furthermore, caspases-2 and -3, effectors of the intrinsic (Guo et al., 2002) and final common pathways, respectively, demonstrated increased activities (Fig. 2B). However, enhancement was modest, suggesting that apoptosis comprises a small fraction of VPA-mediated cell death.
Following confirmation of the HDACI and cytotoxic activities of VPA, we studied the effect of VPA on invasion. Cancer invasion is a complex process involving activation of proteolytic activity (i.e., matrix metalloproteinases, collagenases, and others), alterations in cell-cell interactions, and coordinated motility. Recent evidence suggests that HDAC inhibitors may affect expression of some prominent effectors in these processes (Kim et al., 2000). Using time-lapse video fluorescent microscopy, we found that acute VPA treatment leads to a qualitative decrease in the invasion rate of treated versus untreated T24 cells (Fig. 3). This effect was evident within 2 to 4 h and for up to 24 h. Subsequent quantitative endpoint invasion assays confirmed these findings in multiple bladder cancer cell lines, including T24, TCC-SUP, and HT1376 (Fig. 4). These effects were significant for the 5.0 mM dose in TCC-SUP and HT1376, and they approached significance in the T24 cell line. Taken together, these data demonstrate a clear trend toward decreased invasion with the acute administration of VPA in multiple bladder cancer cell lines.
Since diminished invasion may be a nonspecific phenomenon depending on the effect of treatment on cell migration or motility (Kassis et al., 2002), we studied the effects of VPA on both migration and invasion using a real-time invasion assay. Regardless of the approach, the results were consistent. Namely, VPA does not effect migration, but it has a significant effect on invasion. Both invasion and invasiveness of T24 cells were clearly reduced in a dose-dependent manner, whereas motility was unaffected (Fig. 5, A–C). Likewise, invasiveness in the TCC-SUP and HT1376 bladder cancer cell lines was reduced, although the effects were somewhat diminished and not sustained over time in HT1376 (Fig. 6, A–C). The lack of effect in RT4 was expected, because it was derived from noninvasive papilloma. These data confirm our previous findings that VPA inhibits invasion in a dose-dependent and cell line-specific manner and further demonstrate that VPA directly affects invasion and not cell motility.
Given the varying potency of VPA to prevent invasion in different bladder cancer cell lines, we also interrogated whether this effect was cancer type-specific. We chose to evaluate prostate cancer cell lines LNCaP, PC3, and DU145, which vary in androgen dependence and aggressiveness. In contrast to our findings in bladder cancer, VPA had no effect on invasion or migration for all tested prostate cancer cells. This may be due to the fact that invasion is the net result of alterations in multiple pathways and not merely a single pathway, ultimately leading to enhanced destruction of basement membrane and extracellular matrix. Therefore, invasion may still occur if the pathways modified by VPA are not critical to or constitute a small component of the events of invasion. Together, these findings suggest that VPA exerts its effect in a cancer type-specific manner, inhibiting invasion in bladder but not prostate cancer cells.
Since the doses required for optimal activity in vitro often exceed the toxicity threshold in clinical use, the therapeutic significance of VPA in vivo remains to be seen. To assess the feasibility of VPA as an adjuvant, we performed a limited animal xenograft experiment with established T24t tumors to determine whether nontoxic levels of VPA would have measurable activity in vivo. At serum concentrations of 8 to 67 µg/ml (0.05 to 0.4 mM, respectively), well below the maximal threshold for safety in humans (135 µg/ml (0.9 mM), VPA-treated animals demonstrated a 40% reduction in tumor volume compared with untreated controls. This sensitivity to such low doses of VPA may be due to chronic (34 days) VPA treatment, in comparison with acute 3-day treatment in vitro (Fig. 2). We have previously found that prostate cancer cells are more sensitive to lower VPA doses when given chronically rather than acutely (Xia et al., 2006). Nevertheless, the lower VPA doses achieved in vivo were still capable of inducing significant HDACI activity supported by increased histone H3 acetylation and p21 induction (Fig. 8, B and C).
Prior studies of HDACIs have focused on the inhibition of cell growth and induction of apoptosis as primary mechanisms of their anticancer effects. However, newer insights have revealed that the effects of HDAC inhibition on both histone and nonhistone proteins may be even more far reaching than originally thought, modulating cancer cell differentiation, migration, invasion, metastasis, and angiogenesis (Liu et al., 2006). For example, VPA has been reported to alter the chemokine expression profile of endothelial cells, potentially effecting angiogenesis and neutrophil infiltration (Engl et al., 2004). In our previous work, we demonstrated that HDACI treatment in vitro can up-regulate expression of the cellular adhesion molecule CAR (Sachs et al., 2004). We now demonstrate that chronic low dose VPA treatment also induces CAR expression in vivo and that this treatment has profound effects on tumor growth (Fig. 8). It has been previously shown that overexpression of CAR in a glioma tumor model resulted in more than 80% decreases in tumor volume in vivo, decreased cell invasion in vitro, and that this inhibition of tumor growth required the carboxyl-terminal domain of CAR (Huang et al., 2005). Interestingly, there was no reported effect of CAR overexpression on glioma cell growth in vitro, suggesting additional microenvironment-specific effects of CAR in tumors. In contrast, overexpression of CAR in T24 bladder cancer lines has been reported to significantly inhibit cell growth in vitro, and this inhibition of cell growth also required the carboxyl-terminal domain of CAR (Okegawa et al., 2001). Collectively, it is apparent that VPA effects on tumor growth and invasion are mechanistically complex, that they may be tissue-specific, and that in bladder cancer, VPA-mediated CAR up-regulation may play an important role in controlling bladder tumor growth in vitro and in vivo.
In summary, our study provides initial evidence that VPA can inhibit the growth and invasion of bladder cancer cells. These data suggest a potential role for VPA as an adjuvant therapy for patients with recurrent, progressive, or muscle-invasive disease.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: HAT, histone acetyltransferase; HDAC, histone deacetylase; HDACI, histone deacetylase inhibitor; CAR, coxsackie and adenovirus receptor; VPA, valproic acid, 2-propylpentanoic acid; FBS, fetal bovine serum; CFDA SE, carboxyfluorescein diacetate, succinimidyl ester; TSA, trichostatin A; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FU, fluorescent unit.
Address correspondence to: Dr. Ronald Rodriguez, The Johns Hopkins Hospital, 600 North Wolfe St., Marburg 205, Baltimore, MD 21287. E-mail: rrodriguez{at}jhmi.edu
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