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BRL 49653 (rosiglitazone) is a thiazolidinedione anti-diabetic drug that activates the nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ). Pilot clinical trials have shown evidence of therapeutic activity of PPARγ agonists against prostate cancer. To more effectively use PPARγ ligands to treat this common and generally chemo-resistant type of cancer, it will be necessary to better understand the nature of PPARγ activity in prostate cancer cells. Tumor suppressor effects of activation of PPARγ may include suppression of growth and/or induction of differentiation or apoptosis. We investigated responses of primary cultures of human prostatic cancer cells to BRL 49653. PPARγ was expressed in all of the cell strains examined. BRL 49653 caused dose- and time-dependent growth inhibition that was associated with increased expression of the transcription repressor, transforming growth factor β-stimulated clone 22 (TSC-22), and markedly increased expression of the secretory differentiation-associated gene adipophilin. Adipocyte-type fatty acid binding protein (aFABP), neutrophil gelatinase-associated lipocalin (NGAL), glycerol kinase (GyK), and β-catenin, which are regulated by PPARγ ligands in certain other types of cells, were not regulated by BRL 49653 in prostate cells. Upregulation of adipophilin coincided with morphological changes and the appearance of cytoplasmic vacuoles with ultrastructural features of secondary lysosomes. These results extend previous studies with established cancer cell lines and show that PPARγ agonists can inhibit proliferation and modulate expression of secretory-associated genes in primary cultures of prostate cancer cells, further warranting consideration of these agents as pro-differentiating chemotherapeutic or chemoprevention agents for the treatment of prostate cancer. © 2003 Wiley-Liss, Inc.
Prostate cancer is the most common non-cutaneous cancer in American men and the second leading cause of death from cancer (Dennis and Resnick, 2000). Although surgical resection and radiotherapy are potentially curative for localized (organ-confined) disease, conventional chemotherapy and radiotherapy still have limited efficacy against advanced disease (Wang and Waxman, 2000). Androgen ablation often leads to symptomatic improvement in patients with advanced disease, but progression to hormone refractory disease is usually inevitable (Lytton, 2001). Therefore, new approaches to therapy of prostate cancer are greatly needed, not only as alternatives to conventional treatment for adenocarcinoma but also for chemoprevention for patients with prostate cancer precursor lesions, such as high grade prostatic intraepithelial neoplasia, or small organ-confined invasive tumors, as well as for patients with advanced and metastatic prostate cancer.
Thiazolidinediones (TZDs), a new class of anti-diabetic drugs (Murphy and Holder, 2000), have been identified as specific ligands for peroxisome proliferator-activated receptor (PPARγ), a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily (Rosen and Spiegelman, 2001). PPARγ regulates lipid and lipoprotein metabolism and influences cellular proliferation, differentiation, and apoptosis (Rosen and Spiegelman, 2001). Although PPARγ is predominately expressed in adipose tissue (one of the target tissues for insulin), it has been found in macrophages, vascular smooth muscle cells, endothelial cells, and diverse normal as well as malignant epithelial cells. Studies with cell lines suggest that activation of PPARγ by TZDs suppresses cell growth and may induce differentiation (Fujiwara and Horikoshi, 2000; Rosen and Spiegelman, 2001).
Transcriptional activity of PPARγ is increased upon binding of its ligands. Both natural and synthetic ligands have been described. Fatty acids and derivatives bind with low affinity, whereas certain eicosanoids such as 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), 13-hydroxyoctadecadienoic acid (13-HODE), and 15-hydroxyeicosatetraenoic acid (15-HETE) bind PPARγ with higher affinity (Rosen and Spiegelman, 2001). 15-HETE, derived from the activity of epithelial 15-lipoxygenase-2 (15-LOX-2), is the major arachidonic acid metabolite synthesized by benign human prostate, with reduced formation in prostate cancer, and thus represents a candidate endogenous ligand for PPARγ in prostatic epithelial cells (Shappell et al., 1999, 2001b). Rosiglitazone (BRL 49653) is a potent and selective synthetic PPARγ agonist of the TZD class of compounds (Murphy and Holder, 2000).
Previous studies using prostate cancer cell lines showed that agonists of PPARγ induced growth inhibition and cell cycle arrest in vitro, and reduced tumor size of xenografts (Kubota et al., 1998; Butler et al., 2000; Shappell et al., 2001a). Based on the previously reported preclinical data, a phase II clinical study of patients with advanced prostate cancer was conducted. Prolonged stabilization of serum prostate-specific antigen (PSA) was seen in a significant minority of patients treated with the TZD troglitazone (Mueller et al., 2000).
Given the promising preclinical and clinical data regarding the activity of TZDs on prostate cancer, it is worthwhile to further explore the mechanisms whereby PPARγ activation may be tumor suppressive. We used a model system of primary cultures of human prostatic epithelial cells derived from adenocarcinomas of Gleason patterns 3 and/or 4 (Gleason score 6 and 7 tumors), the most common grades of cancer in radical prostatectomy specimens. The expression of PPARγ and the effects of BRL 49653 on cell growth, gene expression, and differentiation were determined. We noted striking temporal and cell type-specific changes in response to BRL 49653. In particular, growth inhibition caused by BRL 49653 was reversible after up to at least 96 h of treatment, and morphologic changes induced by BRL 49653 were not fully developed until after about 7 days of treatment. The phenotype elicited by activation of PPARγ was unique in our experience with prostate cancer cells and could not be directly linked to either classic secretory or neuroendocrine differentiation of prostatic epithelial cells. The new information that we generated with primary cultures will be relevant to developing optimal strategies to target PPARγ for prevention or treatment of prostate cancer.
- Top of page
- MATERIALS AND METHODS
- LITERATURE CITED
As the mortality rate of prostate cancer continues unabated, the development of new therapeutic agents is urgently needed. The results of pilot clinical studies suggested that agonists of PPARγ may have anti-tumor activity in patients with prostate cancer (Hisatake et al., 2000; Mueller et al., 2000). PPARγ mRNA is expressed in benign and malignant prostatic tissues (Mueller et al., 2000; Shappell et al., 2001a), and the observed effects of PPARγ ligands on established prostate cancer cell lines in vitro and in xenograft models are consistent with potential therapeutic activity (Kubota et al., 1998; Hisatake et al., 2000; Shappell et al., 2001a). Arachidonic acid metabolites, including 15-HETE and 15d-PGJ2, can activate PPARγ in cell-free systems and intact cells. 15-HETE has been implicated as a possible physiological ligand of PPARγ in the prostate, as 15-LOX-2-derived 15-HETE is the major arachidonic acid metabolite synthesized by benign prostate. Both 15-LOX-2 expression and 15-HETE formation are substantially reduced or absent in most prostate cancers compared to benign prostate (Shappell et al., 2001b). 15-HETE can activate PPARγ and inhibit proliferation of PC-3 prostate carcinoma cells (Shappell et al., 2001a). 15d-PGJ2 is another suspected natural ligand of PPARγ that halts prostate cancer cell growth (Butler et al., 2000), but whether or not this metabolite is actually formed in the prostate or altered in prostate carcinoma remains to be determined.
Because of the involvement of PPARγ in so many critical physiologic and pathologic functions including regulation of insulin sensitivity, energy expenditure, the development of atherosclerosis and anti-tumorigenic effects in a variety of different cancers, great effort has been spent in trying to identify endogenous ligands for PPARγ as well as to develop and characterize the effect of high affinity synthetic ligands. In this study, we investigated the effects of a specific PPARγ agonist, BRL 49653 or rosiglitazone, on primary cultures of human prostatic cancer cells.
Our studies showed that prostate cancer cell strains expressed PPARγ mRNA and protein, similar to established prostate cancer cell lines (Kubota et al., 1998; Butler et al., 2000; Mueller et al., 2000; Shappell et al., 2001a). BRL 49653 suppressed proliferation, with half-maximal inhibition of clonal growth at about 2 μM for the majority of cell strains. This is similar to the level of BRL 49653 reported to inhibit PC-3 cells in soft agar colony-forming assays (Shappell et al., 2001a). One of the primary cancer cell strains was less sensitive than the other four to growth inhibition by BRL 49653, but the reason for this remains to be determined. As has been shown in other studies (Kubota et al., 1998; Butler et al., 2000; Mueller et al., 2000), sensitivity of the primary cultures of prostate cancer cells to growth inhibition by agonists of PPARγ did not directly correlate with levels of expression of PPARγ.
Other factors may influence response to PPARγ ligands. For instance, mitogen-activated protein kinase (MAPK) has been reported to phosphorylate and hence down-regulate PPARγ activity (Hsi et al., 2001). Whether reduced responsiveness to BRL 49653 might reflect higher levels of activated MAPK in the currently employed cell strains remains to be established. As MAPK activation has been reported in high grade human prostate cancer tissues (Gioeli et al., 1999), this pathway could possibly modulate responsiveness of prostate cancer cells in actual patients to systemically administered PPARγ agonists. This possibility needs to be considered in future clinical studies as well as in vitro. It is interesting to note that the less responsive cell strain in our study was derived from a cancer of higher Gleason grade (70% Gleason pattern 4/30% intraductal carcinoma) than the other four cell strains, which were from cancers of Gleason pattern 3 (Gleason score 3 + 3 = 6).
In the current study, growth inhibition by PPARγ stimulation was time-dependent, as shown by rather minimal inhibition after 3 days of treatment with BRL 49653 and significantly greater inhibition after 7 days of treatment. Growth inhibition was also reversible. Cells exposed to BRL 49653 for 2 days recovered almost 100% of their growth potential when inoculated into fresh medium without BRL 49653, and even after 4 days of treatment, growth potential was 75% of cells never exposed to BRL 49653. This observation is relevant to development of clinical protocols and suggests that TZD therapy of prostate cancer may have to be continuous over an extended period to be effective.
Cell cycle arrest caused by BRL 49653 was also transitory. S-phase delay was seen in treated compared to control cells at the end of 24 h with no significant difference in cell cycle distribution between the treated and untreated populations at later time points. In PC-3 cells, a slight increase of cells in G1 was noted after 3 days of treatment with BRL 49653 (Shappell et al., 2001a), somewhat similar to that observed in cancer cell lines established from other tissues (Sarraf et al., 1998). In PC-3, DU 145, and LNCaP prostate cancer cell lines, the PPARγ-ligand 15d-PGJ2 caused accumulation in the S-phase of the cell cycle (Butler et al., 2000), similar to our results with BRL 49653 on primary cultures of prostate cancer cells. However, S-phase arrest in the primary cultures was transitory and likely is not responsible for the antiproliferative effects of BRL 49653. S-phase accumulation of the established cell lines in response to 15d-PGJ2 was also followed by cell death by a non-apoptotic mechanism (Butler et al., 2000), whereas no increased death was noted in the primary cultures in our study or in prostate cell lines (Mueller et al., 2000) after treatment with BRL 49653.
Ligand activation of PPARγ induces cell-specific differentiation (Bar-Tana, 2001). Examples include the promotion of adipocytic differentiation in pre-adipocytes and other mesenchymal cells and the creation of foam cells (cholesterol-loaded macrophages) by activation of PPARγ in peripheral blood monocytes (Bar-Tana, 2001). Whether or not PPARγ agonists actually contribute to differentiation of epithelial cells, including those in the benign prostate, or can promote differentiation in prostate cancer cells, has not been thoroughly investigated. Troglitazone reportedly induced vacuolization in established prostate cancer cell lines, including ultrastructural features of surface invaginations with microvilli and of secondary lysosomes (Kubota et al., 1998). We observed similar ultrastructural features in PC-3 cells stimulated with 15-HETE and BRL 49653 (S.B.S., unpublished observations). The significance of such vacuoles remains unclear, and the effect of PPARγ agonists on other pathways paralleling prostate secretory differentiation remains unclear. Troglitazone was reported to reduce expression of PSA in LNCaP cells (Mueller et al., 2000), but as this agent can directly interfere with androgen receptor-mediated transactivation of this androgen-regulated gene (Hisatake et al., 2000), the relationship of this observation to modulation of true secretory differentiation in prostate cells remains to be defined. Hence, the relationship of the inhibition of prostate cancer cell proliferation by PPARγ agonists to possible modulation of differentiation has not been adequately addressed.
Since PPARγ was initially cloned as a master regulator of adipogenesis, this differentiation-inducing activity of PPARγ is the most thoroughly studied. The ability of PPARγ activation to convert cells to adipocytes is not confined to pre-adipocytes, but has been described as well for fibroblasts, bone marrow stromal cells, liposarcoma, and breast cancer cells (Tontonoz et al., 1997; Mueller et al., 1998). In the current study, although the prominent cytoplasmic vacuolization seen in prostate cancer cells treated with BRL 49653 was reminiscent of foam cell formation or adipogenesis, lack of staining with oil red O ruled out significant accumulation of neutral lipids in these cells, despite occasional lipid noted on ultrastructural examination. Similarly, treatment of the prostate cancer cell lines LNCaP, PC-3, and DU 145 with the PPARγ agonist 15d-PGJ2 failed to induce adipocyte differentiation as also defined by accumulation of neutral lipid (Butler et al., 2000). Vacuolization of tumor epithelial cells by PPARγ agonists has been variably associated with upregulation of a limited number of so-called adipocyte differentiation genes, such as aFABP (AP2) (Mueller et al., 1998; Butler et al., 2000; Shappell et al., 2001a), which may function in lipid processing or secretory function in other cell types.
However, other types of differentiation besides conversion to adipocytes occur in cells upon activation of PPARγ. Differentiation of physiological relevance in the prostate includes neuroendocrine and secretory differentiation. The neuroendocrine marker, chromogranin A, was not induced by treatment of primary cultures of prostate cancer cells with BRL 49653. Immunocytochemical labeling with antibodies against proteins typically expressed by differentiated secretory luminal cells of the prostatic epithelium, PSA and cytokeratin 18, also showed no significant changes in expression. We also ruled out the possibility that the ultrastructural changes induced in prostatic epithelial cells by BRL 49653 were associated with premature senescence by showing absence of an increase in SA-β-gal. At this time, we must conclude that the striking phenotype induced in prostate cancer cells by activation of PPARγ is an atypical form of differentiation. However, it must be kept in mind that development of fully differentiated prostatic secretory cells in culture has been an elusive goal, and that cancer cells might demonstrate aberrant differentiation patterns compared to normal cells. The relevance of the observed phenotype to effects that PPARγ agonists might have on prostatic epithelial cells in vivo may be discerned in future studies by examination of surgically removed prostate tissues following therapy with TZDs.
Genes up- or down-regulated by PPARγ transactivation are still being elucidated. aFABP is increased by PPARγ agonists, paralleling differentiation in adipocytes (Kletzien et al., 1992; Hauner, 2002). By RT-PCR, we have not consistently detected aFABP mRNA in snap-frozen benign or malignant human prostate tissues (Iyengar et al., 2002). In PC-3 cells, treatment with 15-HETE or BRL 49653 resulted in upregulation of aFABP (Shappell et al., 2001a). In contrast, aFABP mRNA was constitutively expressed in the cancer cells utilized in our study and was not appreciably increased by BRL 49653 treatment. GyK and β-catenin, found to be targets of PPARγ in adipocytes (Guan et al., 2002) and colon cancer cells (Girnun et al., 2002), respectively, were also not significantly regulated by BRL 49653 in primary cultures of prostate cancer cells, again demonstrating the cell-specific nature of PPARγ activity.
NGAL and adipophilin are two other genes that are upregulated by PPARγ agonists in tumor cell lines, which is prevented by pre-treatment with a specific PPARγ antagonist (Gupta et al., 2001). Adipophilin is a 48–50 kD protein, previously recognized as a marker for adipocyte differentiation, but which has been demonstrated to be present in a wide range of cell types, including epithelial cells, that have in common the normal function of lipid accumulation or steroid hormone synthesis/secretion (Heid et al., 1998). Interestingly, adipophilin was upregulated by oxidized low density lipoprotein (LDL) in macrophages (Wang et al., 1999), a process known to involve PPARγ activation (Nagy et al., 1998). TSC-22 is specifically upregulated in tumor cell lines by PPARγ agonists (Gupta et al., 2003). The TSC-22 gene product appears to be a transcriptional repressing factor (Kester et al., 1999), regulating cell growth, differentiation, and apoptosis, and which may be reduced in a variety of adenocarcinomas compared to corresponding benign epithelial tissues (Nakashiro et al., 1998; Rae et al., 2000). In contrast to our findings for aFABP, we essentially uniformly detect adipophilin and NGAL mRNA expression (Iyengar et al., 2002) in benign or malignant human prostate tissues. Expression of TSC-22 mRNA, on the other hand, is reduced in malignant compared to benign tissues (S.I., S.B.S., unpublished observations). Whether or not these genes are reduced in tumor vs. benign tissues in a manner regulated by PPARγ remains to be more fully established. Adipophilin, NGAL, and TSC-22 mRNA were constitutively expressed in the cancer cells utilized herein, but only TSC-22 and adipophilin expression was increased by BRL 49653, with adipophilin expression markedly increased. Adipophilin immunostaining is variably present in the cytoplasm of prostate secretory cells in actual human prostatic tissues (Iyengar et al., 2002), and in the current study, treatment with BRL 49653 resulted in increased cytoplasmic immunostaining of adipophilin in growth-inhibited prostate cancer cells. These results show that growth inhibition of primary prostate cancer cells by PPARγ agonists is accompanied by modulation of PPARγ-regulated genes that are normally expressed in prostate and which may contribute to normal prostate cell function and growth regulation. However, other proteins found in secretory epithelial cells of the prostate, such as PSA and cytokeratin 18, were not upregulated by BRL 49653 so that an association between activation of PPARγ and secretory differentiation remains tenuous.
In future studies, we will address the particular role of 15-HETE in regulating growth and differentiation of prostatic epithelial cells. 15-HETE is the product of 15-LOX-2 activity in the secretory cells of the normal prostatic epithelium (Shappell et al., 1999; Jack et al., 2000). This enzyme is absent or reduced in a majority of adenocarcinomas of the prostate, and levels are diminished in the premalignant lesion high grade prostatic intraepithelial neoplasia (Shappell et al., 1999, 2001b; Jack et al., 2000). 15-LOX-2 mRNA, protein, and catalytic activity are variably expressed in primary cultures of benign and malignant prostate tissues as utilized herein (S.B.S., D.M.P., manuscript in preparation). If 15-HETE is a predominant physiological ligand of PPARγ, then our studies suggest that loss of activation of PPARγ due to loss of production of 15-HETE by 15-LOX-2 in prostate cancer cells would lead to increased proliferation and diminished differentiation. Recently, Tang et al. (2002) reported that 15-LOX-2 is a negative cell regulator in normal prostatic epithelial cells, but they did not investigate the role of PPARγ in this process. We conclude that primary cell cultures provide a useful model system to investigate the role of PPARγ in prostatic biology and to generate preclinical data supporting application of PPARγ agonists for prevention or cure of prostate cancer.