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Novel pathways that contribute to the anti-proliferative and chemopreventive activities of calcitriol in prostate cancer
Journal of Steroid Biochemistry & Molecular Biology 103 (2007) 694–702
Novel pathways that contribute to the anti-proliferative and chemopreventive activities of calcitriol in prostate cancer Aruna V. Krishnan a , Jacqueline Moreno a , Larisa Nonn b , Peter Malloy a , Srilatha Swami a , Lihong Peng a , Donna M. Peehl b , David Feldman a,∗ a
Department of Medicine, Division of Endocrinology, Stanford University School of Medicine, Stanford, CA 94305, United States b Department of Urology, Stanford University School of Medicine, Stanford, CA 94305, United States Received 30 November 2006
Abstract Calcitriol, the hormonally active form of Vitamin D, inhibits the growth and development of many cancers through multiple mechanisms. Our recent research supports the contributory role of several new and diverse pathways that add to the mechanisms already established as playing a role in the actions of calcitriol to inhibit the development and progression of prostate cancer (PCa). Calcitriol increases the expression of insulin-like growth factor binding protein-3 (IGFBP-3), which plays a critical role in the inhibition of PCa cell growth by increasing the expression of the cell cycle inhibitor p21. Calcitriol inhibits the prostaglandin (PG) pathway by three actions: (i) the inhibition of the expression of cyclooxygenase-2 (COX-2), the enzyme that synthesizes PGs, (ii) the induction of the expression of 15-prostaglandin dehydrogenase (15-PGDH), the enzyme that inactivates PGs and (iii) decreasing the expression of EP and FP PG receptors that are essential for PG signaling. Since PGs have been shown to promote carcinogenesis and progression of multiple cancers, the inhibition of the PG pathway may add to the ability of calcitriol to prevent and inhibit PCa development and growth. The combination of calcitriol and non-steroidal anti-inflammatory drugs (NSAIDs) result in a synergistic inhibition of PCa cell growth and offers a potential therapeutic strategy. Mitogen activated protein kinase phosphatase 5 (MKP5) is a member of a family of phosphatases that are negative regulators of MAP kinases. Calcitriol induces MKP5 expression in prostate cells leading to the selective dephosphorylation and inactivation of the stress-activated kinase p38. Since p38 activation is pro-carcinogenic and is a mediator of inflammation, this calcitriol action, especially coupled with the inhibition of the PG pathway, contributes to the chemopreventive activity of calcitriol in PCa. Mullerian Inhibiting Substance (MIS) has been evaluated for its inhibitory effects in cancers of the reproductive tissues and is in development as an anti-cancer drug. Calcitriol induces MIS expression in prostate cells revealing yet another mechanism contributing to the anti-cancer activity of calcitriol in PCa. Thus, we conclude that calcitriol regulates myriad pathways that contribute to the potential chemopreventive and therapeutic utility of calcitriol in PCa. © 2007 Elsevier Ltd. All rights reserved. Keywords: VDR; cDNA arrays; Target genes; IGFBP-3; Prostaglandins; 15-PGDH; COX-2; NSAIDs; MKP5; p38; IL-6; MIS
1. Introduction Prostate cancer (PCa) is a common malignancy and is the second leading cause of deaths in American men [1,2]. Androgens promote PCa growth and androgen deprivation is the most useful therapy for men who fail primary therapy with surgery or radiation [1,2]. However, many patients eventually fail androgen deprivation therapy and develop ∗
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androgen-independent PCa (AIPC) and metastatic disease that is not amenable to available treatments. One of the goals of current research on PCa and AIPC is the identification of new agents that would prevent PCa development and/or slow down its progression to AIPC. In recent years, calcitriol (1,25-dihydroxyvitamin D3 ), the active metabolite of Vitamin D, has emerged as a promising therapeutic agent. Calcitriol is an important regulator of calcium homeostasis and bone metabolism through its actions in intestine, bone, kidney and the parathyroid glands [3]. However, calcitriol also exerts anti-proliferative and pro-differentiating effects in a number
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of tumors and malignant cells including PCa [4–9] raising the possibility of its use as an anti-cancer agent.
2. Calcitriol and prostate cancer 2.1. Epidemiology and genetic factors There are several risk factors for PCa including age, race and genetics [1,2]. Epidemiological studies suggest that Vitamin D deficiency increases the risk of PCa based on the observations that mortality rates due to PCa in the U.S. are inversely related to sunlight exposure and that UV light is essential for the synthesis of Vitamin D in the skin [10,11]. More recently epidemiological data suggest that Vitamin D deficiency increases PCa risk [12]. Decreased serum levels of 25-hydroxyvitamin D (25(OH)D3 ), the precursor to calcitriol, correlate with increased risk of PCa [9]. Polymorphisms in the Vitamin D receptor (VDR) gene may contribute to PCa risk as well [13–15].
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been in observed in PCa patients following calcitriol administration [21,22] indicating a beneficial effect of calcitriol in slowing the progression of the disease. Recent investigations have followed the approach of administering calcitriol intermittently in very high doses where it apparently can still elicit its anti-proliferative effects and cause only transient hypercalcemia. Thus far, these intermittent high doses do not appear to cause substantial toxicity [22–24]. Calcitriol is also being used in combination therapy with other agents that may enhance its anti-proliferative activity while reducing its hypercalcemic tendency [4]. The results of the ASCENT clinical trial in advanced PCa patients who failed other therapies is currently in press [24]. The data presented by Beer et al. [24] demonstrated that extremely high doses of calcitriol administered once weekly along with the usual regimen of the chemotherapy drug taxotere caused a statistically significant improvement in overall survival and time to progression, providing evidence that calcitriol can enhance the efficacy of active drugs in cancer patients. A large Phase III trial testing this combination is under way.
2.2. Cell culture and animal studies A number of studies have demonstrated the antiproliferative and pro-differentiating effects of calcitriol in prostatic epithelial cells and PCa cells in culture [4–9]. In the normal prostate, 25(OH)D3 -1␣ hydroxylase converts 25(OH)D3 to 1,25(OH)2 D3 [16,17] suggesting that local production of calcitriol may play an important role in normal growth and differentiation of the prostate. The antiproliferative effects of calcitriol on PCa have been observed at high concentrations of calcitriol that may cause hypercalcemia in vivo. Intense research has been undertaken by many academic investigators and pharmaceutical companies to develop calcitriol analogs/derivatives that exhibit increased anti-proliferative activity and reduced tendency to cause the hypercalcemic side-effects [18,19]. Several studies have investigated the effects of calcitriol or its analogs on the establishment and growth of human PCa xenografts in immuno-compromised mice and showed significant reduction in tumor size and volume [4–9]. Transgenic models of PCa have also been developed in animals to study the chemopreventive effects of calcitriol and its analogs [20]. These in vivo models provide a valuable tool to study the tumor inhibitory effect of calcitriol and analogs while monitoring their tendency to cause hypercalcemia and to validate their potential use in clinical trials.
3. Molecular mechanisms mediating the anti-proliferative effects of calcitriol A number of important mechanisms have been implicated in calcitriol-mediated growth inhibition. A primary mechanism of calcitriol action is to induce cell cycle arrest in the G1 /G0 phase [8,9]. The growth arrest appears to be due to an increase in the expression of cyclin-dependent kinase inhibitors p21Waf/Cip1 and p27Kip1 [25–28], a decrease in cyclin-dependent kinase 2 (Cdk2) activity, [28] and the hyperphosphorylation of the retinoblastoma protein (pRb) [29]. p53 appears to be required for calcitriol-induced G0 arrest but is not required for calcitriol induced G1 accumulation or apoptosis of LNCaP cells [6,8,9]. Loss of the expression of cell cycle regulators has been associated with a more aggressive cancer phenotype [30], decreased prognosis and poorer survival [31,32]. These data therefore suggest that calcitriol may be a suitable therapy to inhibit PCa progression. In addition, calcitriol induces apoptosis in some PCa cells and down-regulates anti-apoptotic genes like bcl-2 [33]. Other mechanisms of calcitriol actions in PCa cells include the stimulation of differentiation, modulation of growth factor actions and the inhibition of invasion and metastasis [4–9]. Some in vivo studies demonstrate an inhibition of angiogenesis that contributes to the anti-tumor effects of calcitriol [4–9].
2.3. Clinical trials Several clinical trials have been carried out in PCa patients to evaluate the safety and efficacy of treatment with calcitriol or its analogs [4–9]. Calcitriol is an FDA approved drug and has been administered at as high a dose as tolerated, limited by hypercalciuria or hypercalcemia. A decrease in the rate of rise of serum prostate specific antigen (PSA) levels has
4. Novel pathways of calcitriol actions Calcitriol actions on cell growth and differentiation are initiated by its binding to the VDR resulting in the direct activation or repression of target gene transcription. The identification of the myriad target genes that mediate calcitriol
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actions is one of the goals of our current research on Vitamin D. We have used cDNA microarrays of primary human prostatic epithelial cells as well as LNCaP human PCa cells to identify molecular targets of calcitriol involved in the regulation of prostate epithelial cell growth [34,35]. Our studies have identified several new genes as well as known, important target genes regulated by calcitriol in prostate cells. In the following sections, we will discuss some of the recently revealed novel pathways of calcitriol actions in prostate cells, focusing on new developments from our laboratory. 4.1. Regulation of insulin-like growth factor binding protein (IGFBP) expression and growth inhibition Growth factors play an important role in the regulation of prostate epithelial cell growth by autocrine and paracrine mechanisms. Expression of autocrine growth factors by the epithelium and the development of independence from epithelial–stromal interactions that modulate the growth of the normal prostate may contribute to the progression of PCa. Among the important growth factors that regulate prostate epithelial growth are insulin-like growth factors (IGFs). IGFBPs are a family of proteins that bind the IGFs with high affinity and reduce their mitogenic effects by preventing the interaction of the IGFs with the IGF receptors [36]. However, the IGFBPs can regulate cell growth through IGF-independent pathways as well [36]. In PC-3 and ALVA 31 PCa cells, calcitriol decreases the availability of IGF by increasing the expression of the IGF binding proteins IGFBP-3 and IGFBP-5 [37,38]. In LNCaP and C4-2 PCa cells, which are sensitive to calcitriol-mediated growth inhibition, calcitriol decreases the mRNA levels of IGFBP2, a growth stimulator, and increases IGFBP-3 a growth inhibitor [34,39,40]. Calcitriol does not induce IGFBP-3 expression in DU 145 cells that are resistant to the growth inhibitory effects of calcitriol [40]. Studies from our laboratory have provided evidence that the up-regulation of IGFBP-3 expression by calcitriol is a necessary component of calcitriol-mediated inhibition of LNCaP cell growth, under serum-free culture conditions, where the cells are more dependent on autocrine growth factors [39]. Under these conditions, addition of IGFBP-3 anti-sense oligonucleotides abrogate calcitriol mediated growth inhibition suggesting that in LNCaP cells the growth inhibitory action of calcitriol is dependent on IGFBP-3 up-regulation. Furthermore, anti-IGFBP-3 antibodies block calcitriol-mediated increase in p21, showing that IGFBP-3 induction is necessary for the up-regulation of p21 expression [39]. We have also shown that calcitriol induces IGFBP-3 transcription in LNCaP cells cultured in serum-containing media wherein it causes maximal growth inhibition [34]. However, the growth inhibition may not be solely dependent on IGFBP-3 induction, as siRNA oligonucleotides against IGFBP-3 did not block calcitriol-dependent inhibition of LNCaP cell growth in serum-containing media [40]. Studies from our lab have shown that calcitriol directly induces the transcription of
IGFBP-3 gene through a functional Vitamin D response element (VDRE) present in the IGFBP-3 promoter [41b]. Our recent research indicates that the transcription of the IGFBP3 gene is also directly regulated by androgens through an androgen response element (ARE) and that there is synergistic interaction between calcitriol and androgens in regulating IGFBP-3 expression in LNCaP cells [41a]. IGFBP-3 is the most abundant IGF binding protein present in the serum and IGFBP-3 levels are inversely correlated with PCa risk and metastasis [15,42]. Thus, calcitriol may elevate IGFBP-3 levels within the prostate as well as in the circulation thereby counteracting the autocrine and paracrine factors that stimulate the growth of prostate tumors. 4.2. Regulation of prostaglandin metabolism and signaling Prostaglandins (PGs) have been shown to play a role in the development and progression of many cancers including PCa [43,44]. We have recently discovered that calcitriol regulates multiple genes involved in PG the pathway. Calcitriol decreases PG synthesis, increases PG catabolism and inhibits PG signaling through their receptors thereby attenuating the growth stimulatory effects of PGs in PCa [45]. 4.2.1. Cyclooxygenase-2 Cyclooxygenase (COX)/prostaglandin endoperoxidase synthase is the rate-limiting enzyme that catalyzes the conversion of arachidonic acid to PGs and related eicosanoids. The expression of COX-2 is rapidly induced by a variety of mitogens, cytokines, tumor promoters and growth factors and therefore COX-2 is regarded as an immediate-early response gene [44]. Compelling evidence from genetic and clinical studies indicates that increased expression of COX2 is one of the key steps in carcinogenesis. Several studies have demonstrated COX-2 over-expression in prostate adenocarcinoma [46,47] and suggest a positive role for COX-2 in prostate tumorigenesis. However, not all PCa are associated with elevated COX-2 expression [48,49]. Although Zha et al. [48] did not find consistent over-expression of COX-2 in established PCa, they detected appreciable COX-2 expression in areas of proliferative inflammatory atrophy, lesions that have been implicated in prostate carcinogenesis. Both arachidonic acid, the substrate for COX, and the product prostaglandin E2 (PGE2 ) stimulate the proliferation of PCa cells causing increases in the expression of immediate-early genes including c-fos that are involved in growth regulation [50]. 4.2.2. 15-Hydroxyprostaglandin dehydrogenase The key enzyme responsible for the metabolic inactivation of PGs, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), catalyzes the conversion of PGs to their corresponding 15-keto derivatives that exhibit greatly reduced biological activity. A recent study describes 15-PGDH as an oncogene antagonist that functions as a tumor suppressor in
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colon cancer [51]. The study showed that 15-PGDH, which physiologically antagonizes COX-2, is universally expressed in normal colon specimens but is routinely absent or severely reduced in cancer specimens. Most importantly, stable transfection of a 15-PGDH expression vector into cancer cells greatly reduced the ability of the cells to form tumors and/or slowed tumor growth in nude mice [51]. In PCa cells, 15-PGDH expression is induced by high concentrations of androgens that also inhibit cell growth [52]. 4.2.3. PG receptors PGs bind to G-protein coupled membrane receptors (prostanoid receptors) which activate signal transduction pathways [53]. There are eight members in the prostanoid receptor subfamily and they are distinguished by their ligand binding profile and the signal transduction pathways that they activate upon ligand binding and account for some of the diverse and often opposing effects of PGs [53]. PCa cells express EP and FP PG receptors [45,50]. PGE and PGF are the major PGs stimulating the proliferation of PCa cells [45]. PGE acts through four different PGE receptor (EP) sub-types (EP1–EP4), while PGF activates the FP receptor. 4.2.4. Calcitriol effects on the PG pathway in prostate cells Initial analyses from our lab using cDNA microarrays to study changes in the gene expression profile following
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calcitriol treatment of prostate cells indicated that calcitriol regulated the expression of genes involved in PG metabolism [34,35]. Calcitriol treatment up-regulated the expression of 15-PGDH and down-regulated COX-2 expression [34]. We went on to demonstrate calcitriol regulation of the PG pathway genes in multiple PCa cell lines as well as primary prostatic epithelial cells established from surgically removed prostate tissue from PCa patients [45]. We found measurable amounts of COX-2 mRNA and protein in various PCa cell lines as well as primary prostatic epithelial cells derived from normal and cancerous prostate tissue, which were significantly decreased by calcitriol treatment [45]. We also found that calcitriol significantly increased the expression of 15-PGDH mRNA and protein in various PCa cells [45]. We further showed that by inhibiting COX-2 and stimulating 15-PGDH expression, calcitriol decreased the levels of biologically active PGs in PCa cells thereby reducing the growth stimulation by PGs [45]. Interestingly our data also revealed that calcitriol decreased the expression of EP and FP PG receptors. The calcitriol-induced decrease in PG receptor levels resulted in the attenuation of PG mediated functional responses even when exogenous PGs were added to the cultures [45]. Calcitriol suppressed the induction of the immediate-early gene c-fos and the growth stimulation seen following the addition of exogenous PGs or the PG precursor arachidonic acid to PCa cell cultures [45]. Thus, as illustrated in Fig. 1, calcitriol inhibits the PG pathway in
Fig. 1. Inhibition of the prostaglandin pathway by calcitriol in prostate cancer cells. Calcitriol inhibits the prostaglandin pathway by three separate mechanisms, decreasing COX-2 expression, increasing 15-PGDH expression and reducing PG receptor levels to inhibit the PG pathway in PCa cells. Non-steroidal anti-inflammatory drugs (NSAIDs) directly inhibit COX-2 enzymatic activity.
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PCa cells by three separate mechanisms, decreasing COX-2 expression, increasing 15-PGDH expression and reducing PG receptors. We believe that these actions contribute to the suppression of the proliferative stimulus provided by PGs in PCa cells. The regulation of PG metabolism and biological actions constitute an additional novel pathway of calcitriol action mediating its anti-proliferative effects in prostate cells. 4.2.5. Combination of calcitriol and non-steroidal anti-inflammatory drugs (NSAIDs) as a therapy for prostate cancer NSAIDs are a class of drugs that decrease PG synthesis by inhibiting COX-1 and COX-2 enzymatic activities. Several NSAIDs inhibit both the constitutively expressed COX-1 and the inducible COX-2 while others have been shown to be selective for COX-2. We hypothesized that the action of calcitriol to reduce COX-2 gene expression will decrease
the levels of COX-2 protein and allow the use of lower concentrations of NSAIDs to inhibit COX-2 enzyme activity [45]. In addition, an increase in the expression of 15-PGDH due to calcitriol action will lower the levels of biologically active PGs and enhance the NSAID effect. Therefore, we hypothesized that the combination of calcitriol and NSAIDs would exhibit synergistic effects to inhibit PCa cell growth. When calcitriol was combined with the COX-2-selective NSAIDs NS398 and SC-58125 or the non-selective NSAIDs, naproxen and ibuprofen, we found a synergistic enhancement of growth inhibition (Fig. 2). These results led us to further hypothesize that the combination of calcitriol and NSAIDs may have clinical utility in PCa therapy and is worthy of evaluation in a clinical trial [45]. The combination approach will allow the use of lower concentration of NSAIDs and thereby minimize their undesirable side-effects. It has very recently become clear that long-term use of COX-2selective inhibitors such as rofecoxib (Vioxx) causes an
Fig. 2. Synergistic inhibition of prostate cancer cell growth by calcitriol and NSAIDs. LNCaP or PC-3 cells were treated with 0.1% ethanol vehicle (Con) or 10 nM/L calcitriol (Cal) in the presence and absence of the indicated NSAID. Cell growth was determined by measuring DNA content. DNA values are presented as a percentage of the vehicle control value set at 100%. (A) LNCaP cells treated with a combination of calcitriol (Cal) and COX-2 specific NSAID SC-58125 (5 M). (B) PC-3 cells were treated with calcitriol (Cal) in the presence and absence of the COX-2 specific NSAID NS-398 (7.5 M). (C) LNCaP cells treated with calcitriol (Cal) in the presence and absence of the non-selective NSAID naproxen (Nap; 200 M). (D) PC-3 cells were treated with calcitriol (Cal) in the presence and absence of the non-selective NSAID ibuprofen (Ibu; 150 M). * P < 0.05; ** P < 0.01, when compared with control. ++ P < 0.01, when compared with 1 or 10 nM/L Cal alone (used with permission from [45]).
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increase in cardiovascular complications in patients [54]. In comparison, non-selective NSAIDs such as naproxen have been shown to be associated with fewer cardiovascular adverse effects [55]. Our data show the combination of calcitriol with a non-selective NSAID is equally effective in inducing synergistic growth inhibition. We proposed that the combination of calcitriol with a non-selective NSAID is a useful therapeutic approach in PCa that would allow both drugs to be used at reduced dosages leading to increased safety [45]. 4.3. Induction of mitogen-activated protein kinase phosphatase 5 (MKP5) and prostate cancer prevention PCa generally progresses very slowly, likely for decades, before symptoms become obvious and diagnosis is made. Recently, inflammation in the prostate has been proposed to be an etiological factor in the development of PCa [56]. The observed latency in PCa provides a long window of opportunity for intervention by chemopreventive agents. Our recent study using cDNA microarrays [35] revealed a newly identified calcitriol responsive gene, MAP Kinase Phosphatase 5 (MKP5) also known as dual specificity phosphatase 10 (DUSP10). As described in our recent manuscript [57], the up-regulation of MKP5 and the downstream anti-inflammatory responses elicited by calcitriol in normal prostatic epithelial cells are supportive of a role for calcitriol in PCa prevention. The effect of MKP5 regulation in the chemopreventive actions of calcitriol is discussed below. In primary cultures of normal prostatic epithelial cells from the peripheral zone (E-PZ), MKP5 transcription is increased following calcitriol addition [57]. Calcitriol treatment of a normal primary prostate epithelial cells rapidly induced the expression of MKP5 in a dose and time-dependent manner (Fig. 3A and B). In addition, we identified a putative positive VDRE in the MKP5 promoter mediating this calcitriol effect [57]. Interestingly, calcitriol up-regulation of MKP5 was observed in primary cells derived from normal prostatic epithelium and primary, localized adenocarcinoma but not in the established PCa cell lines derived from PCa metastasis such as LNCaP, PC-3 or DU145. MKP5 is a member of the dual specificity MKP family of enzymes that dephosphorylate and thereby inactivate mitogen activated protein kinases (MAPKs) as well as the stress-activated protein kinase p38. We found that calcitriol inhibited the phosphorylation and activation of p38 in normal primary prostate cells [57]. When NaCl was added to the media to induce osmotic stress it resulted in the phosphorylation of p38 in these cells. When the cells were co-treated with NaCl and calcitriol a significant decrease in phosphorylation of p38 was observed. The attenuation of p38 activation by calcitriol appeared to be dependent on MKP5 induction. MKP5 siRNA completely abolished this calcitriol effect [57]. The consequence of p38 stress-induced kinase activation is an increase in the production of pro-inflammatory cytokines
Fig. 3. Increased expression of MKP5 in primary cultures of normal prostatic epithelial cells by calcitriol. (A) RT-qPCR measurement of MKP5 mRNA in E-PZ cells after treatment with 1, 10 and 50 nM calcitriol (1,25D). (B) Time course of 1,25D regulation of MKP5 gene expression in E-PZ cells. Cells were treated with 50 nM of 1,25D. RT-qPCR results are shown relative to untreated controls and normalized to the expression of the human TATA box binding protein (TBP) gene. Each experiment was run in triplicate and results are representative of two or more separate experiments with different patient derived E-PZ cells. Used with permission from [57].
that sustain and amplify the inflammatory response [58]. As interleukin-6 (IL-6), a p38-regulated pleiotropic cytokine, is known to be associated with PCa progression [59], we investigated the effect of calcitriol on IL-6 production. Treatment of primary prostate cells with TNF␣ increased IL-6 concentrations in the conditioned media. Co-administration of calcitriol significantly attenuated IL-6 production by TNF␣. It is becoming apparent that inflammation, both chronic and acute, contributes to PCa development [2]. A model based on the results of our recent study [57] is depicted in Fig. 4. The data suggest that the ability of calcitriol to inhibit p38 signaling and reduce the subsequent production of pro-inflammatory cytokines, via MKP5 up-regulation, may contribute to the cancer preventive effects of calcitriol. In this context it is interesting (as described above) that calcitriol significantly suppresses the expression of the pro-inflammatory enzyme COX-2 and inhibits the action of PGs, well-known mediators of inflammation, lending further support to its role in the prevention of PCa. Since established metastasisderived PCa cell lines exhibit low levels of MKP5 and are
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Fig. 4. Proposed mechanism for anti-inflammatory activity and prostate cancer prevention by Vitamin D. Calcitriol stimulation of MKP5 inhibits the stressactivated kinase p38 and abrogates stimulation of pro-carcinogenic inflammatory cytokines like IL-6 (used with permission [57]).
unable to induce MKP5 in response to calcitriol, we speculate that loss of MKP5 might occur during PCa progression as a result of selective pressure to eliminate the tumor suppressor activity of MKP5 and/or calcitriol. 4.4. Up-regulation of Mullerian Inhibiting Substance (MIS) expression Mullerian Inhibiting Substance (MIS), also known as antiMullerian hormone (AMH), is a member of the transforming growth factor- (TGF) family of factors that function to regulate growth, differentiation and apoptosis in many cells [60]. In males, during fetal sexual development, MIS causes the regression of the Mullerian ducts by inducing apoptosis [60]. MIS is a secreted glycoprotein that is produced by Sertoli cells in the testes of males and by granulosa cells in the ovary of females. MIS binds to the MIS type II receptor (MISR II), initiating a signaling cascade that involves the recruitment of type I receptors ALK2 and ALK6 [61]. However, it has recently been appreciated that MIS has multiple actions in post-natal life. MIS has post-natal actions to inhibit steroidogenesis [62,63] and it may also be involved in the polycystic ovarian syndrome (PCOS) [64]. More importantly, MIS has been shown to inhibit ovarian and cervical cancers, both of which are tumors of Mullerian origin [65,66]. Recently, MIS has been shown to have anti-proliferative and pro-apoptotic effects in prostate, breast and uterine cancers [67–70]. MIS induces the expression of the NF-B target gene, X-rayinducible immediate early response factor-1 (IEX-1) [71], in breast and prostate cancer cells [67,68]. Overexpression of IEX-1 in breast cancer cells inhibits cell growth indicating a negative regulatory role for this gene [67]. Efforts are now being advanced to develop recombinant purified MIS as a therapeutic agent for ovarian and uterine cancers with the added potential to treat other cancers including breast, prostate and Leydig cell tumors [60].
Our recent observations have revealed that MIS is a novel target gene regulated by calcitriol in prostate cells. When PCa cells such as LNCaP or PC-3 cells, as well as cultures of human primary prostatic epithelial cells, were exposed to calcitriol for 24 h, we found considerable increases in the expression of MIS mRNA (Fig. 5A). When LNCaP cells were treated with calcitriol, a substantial increase in secreted MIS protein was observed (Fig. 5B). The promoter sequence for the human MIS gene is contained within a 789 bp sequence constrained by the end of the SF3A2 gene upstream and the start of the MIS open reading frame [72]. Upon in silico analysis of the MIS gene promoter sequence, we noted the presence of a putative VDRE containing two half-sites separated by a three-base spacer, the so-called DR3 motif that is highly similar to the human osteocalcin VDRE. To determine whether calcitriol is a direct regulator of the MIS gene, we cloned a 650 bp fragment containing the MIS promoter into pGL3-basic, a promoterless luciferase reporter vector. We transfected HeLa cells with the MIS promoterluciferase construct and a VDR expression vector and tested the effect of calcitriol. Calcitriol caused a significant (twoto four-fold) induction of the MIS promoter-luciferase demonstrating that the MIS promoter is responsive to calcitriol. Induction of MIS expression may play an important role in the anti-cancer actions of calcitriol. Because of its potential therapeutic benefits, MIS is under active development as a cancer therapy. We hypothesize that the ability of calcitriol to induce endogenous MIS within the prostate is an alternate approach to administering exogenous recombinant, purified MIS, which is a protein biological. A combination of calcitriol and MIS may be a more effective therapy since it is unlikely that MIS can be administered continuously in saturating concentrations. Our future research plans include studies on the contribution of MIS to the antiproliferative/pro-apoptotic actions of calcitriol.
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by calcitriol, results in the attenuation of pro-inflammatory responses in prostate cells. This novel calcitriol action pathway as well as the inhibition of the expression of the proinflammatory enzyme COX-2 suggest that calcitriol has antiinflammatory actions that may play an important role in the prevention of PCa development. Importantly, identification of novel pathways of calcitriol actions may lead to the development of new therapeutic strategies in the treatment of PCa. Acknowledgements This work was supported by Grants DK42482, DAMD1702-1-0142, and PC050074 (D.F.), DOD PC04120 (J.M.), DOD PC04616 (L.N.) and AFUD scholar (L.N.).
References
Fig. 5. Calcitriol up-regulates MIS expression in prostate cancer cells. (A) PCa cell lines (LNCaP and PC-3) and primary prostatic epithelial cells (EPZ-1) were treated with vehicle (0.1% ethanol, open bars) or 10 nM calcitriol (hatched bars) for 24 h and total RNA samples isolated. MIS mRNA levels were determined by RT-qPCR. The human TATA box binding protein (TBP) was used as a control for normalization. Changes in MIS mRNA are given as fold increase over untreated control levels. (B) LNCaP cells were treated with 10 nM calcitriol every 2 days for 6 days. MIS protein in culture medium was measured using an ELISA.
5. Conclusions Our research is aimed at gaining a better understanding the molecular mechanisms of the anti-proliferative and cancer preventive effects of calcitriol with the goal of developing strategies to improve PCa treatment. We have recently identified several new calcitriol target genes in prostate cells that have revealed novel pathways of calcitriol action. We propose that calcitriol inhibition of the PG pathway contributes significantly to its anti-proliferative action. The increase in the expression of MIS, a hormone with known anti-cancer effects in tissues of reproductive origin, also adds a new dimension to the already well-known mechanisms mediating the growth inhibitory actions of calcitriol. The induction of MKP5, and subsequent inhibition of p38 stress kinase signaling
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Novel pathways that contribute to the anti-proliferative and chemopreventive activities of calcitriol in prostate cancer Aruna V. Krishnan a , Jacqueline Moreno a , Larisa Nonn b , Peter Malloy a , Srilatha Swami a , Lihong Peng a , Donna M. Peehl b , David Feldman a,∗ a
Department of Medicine, Division of Endocrinology, Stanford University School of Medicine, Stanford, CA 94305, United States b Department of Urology, Stanford University School of Medicine, Stanford, CA 94305, United States Received 30 November 2006
Abstract Calcitriol, the hormonally active form of Vitamin D, inhibits the growth and development of many cancers through multiple mechanisms. Our recent research supports the contributory role of several new and diverse pathways that add to the mechanisms already established as playing a role in the actions of calcitriol to inhibit the development and progression of prostate cancer (PCa). Calcitriol increases the expression of insulin-like growth factor binding protein-3 (IGFBP-3), which plays a critical role in the inhibition of PCa cell growth by increasing the expression of the cell cycle inhibitor p21. Calcitriol inhibits the prostaglandin (PG) pathway by three actions: (i) the inhibition of the expression of cyclooxygenase-2 (COX-2), the enzyme that synthesizes PGs, (ii) the induction of the expression of 15-prostaglandin dehydrogenase (15-PGDH), the enzyme that inactivates PGs and (iii) decreasing the expression of EP and FP PG receptors that are essential for PG signaling. Since PGs have been shown to promote carcinogenesis and progression of multiple cancers, the inhibition of the PG pathway may add to the ability of calcitriol to prevent and inhibit PCa development and growth. The combination of calcitriol and non-steroidal anti-inflammatory drugs (NSAIDs) result in a synergistic inhibition of PCa cell growth and offers a potential therapeutic strategy. Mitogen activated protein kinase phosphatase 5 (MKP5) is a member of a family of phosphatases that are negative regulators of MAP kinases. Calcitriol induces MKP5 expression in prostate cells leading to the selective dephosphorylation and inactivation of the stress-activated kinase p38. Since p38 activation is pro-carcinogenic and is a mediator of inflammation, this calcitriol action, especially coupled with the inhibition of the PG pathway, contributes to the chemopreventive activity of calcitriol in PCa. Mullerian Inhibiting Substance (MIS) has been evaluated for its inhibitory effects in cancers of the reproductive tissues and is in development as an anti-cancer drug. Calcitriol induces MIS expression in prostate cells revealing yet another mechanism contributing to the anti-cancer activity of calcitriol in PCa. Thus, we conclude that calcitriol regulates myriad pathways that contribute to the potential chemopreventive and therapeutic utility of calcitriol in PCa. © 2007 Elsevier Ltd. All rights reserved. Keywords: VDR; cDNA arrays; Target genes; IGFBP-3; Prostaglandins; 15-PGDH; COX-2; NSAIDs; MKP5; p38; IL-6; MIS
1. Introduction Prostate cancer (PCa) is a common malignancy and is the second leading cause of deaths in American men [1,2]. Androgens promote PCa growth and androgen deprivation is the most useful therapy for men who fail primary therapy with surgery or radiation [1,2]. However, many patients eventually fail androgen deprivation therapy and develop ∗
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androgen-independent PCa (AIPC) and metastatic disease that is not amenable to available treatments. One of the goals of current research on PCa and AIPC is the identification of new agents that would prevent PCa development and/or slow down its progression to AIPC. In recent years, calcitriol (1,25-dihydroxyvitamin D3 ), the active metabolite of Vitamin D, has emerged as a promising therapeutic agent. Calcitriol is an important regulator of calcium homeostasis and bone metabolism through its actions in intestine, bone, kidney and the parathyroid glands [3]. However, calcitriol also exerts anti-proliferative and pro-differentiating effects in a number
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of tumors and malignant cells including PCa [4–9] raising the possibility of its use as an anti-cancer agent.
2. Calcitriol and prostate cancer 2.1. Epidemiology and genetic factors There are several risk factors for PCa including age, race and genetics [1,2]. Epidemiological studies suggest that Vitamin D deficiency increases the risk of PCa based on the observations that mortality rates due to PCa in the U.S. are inversely related to sunlight exposure and that UV light is essential for the synthesis of Vitamin D in the skin [10,11]. More recently epidemiological data suggest that Vitamin D deficiency increases PCa risk [12]. Decreased serum levels of 25-hydroxyvitamin D (25(OH)D3 ), the precursor to calcitriol, correlate with increased risk of PCa [9]. Polymorphisms in the Vitamin D receptor (VDR) gene may contribute to PCa risk as well [13–15].
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been in observed in PCa patients following calcitriol administration [21,22] indicating a beneficial effect of calcitriol in slowing the progression of the disease. Recent investigations have followed the approach of administering calcitriol intermittently in very high doses where it apparently can still elicit its anti-proliferative effects and cause only transient hypercalcemia. Thus far, these intermittent high doses do not appear to cause substantial toxicity [22–24]. Calcitriol is also being used in combination therapy with other agents that may enhance its anti-proliferative activity while reducing its hypercalcemic tendency [4]. The results of the ASCENT clinical trial in advanced PCa patients who failed other therapies is currently in press [24]. The data presented by Beer et al. [24] demonstrated that extremely high doses of calcitriol administered once weekly along with the usual regimen of the chemotherapy drug taxotere caused a statistically significant improvement in overall survival and time to progression, providing evidence that calcitriol can enhance the efficacy of active drugs in cancer patients. A large Phase III trial testing this combination is under way.
2.2. Cell culture and animal studies A number of studies have demonstrated the antiproliferative and pro-differentiating effects of calcitriol in prostatic epithelial cells and PCa cells in culture [4–9]. In the normal prostate, 25(OH)D3 -1␣ hydroxylase converts 25(OH)D3 to 1,25(OH)2 D3 [16,17] suggesting that local production of calcitriol may play an important role in normal growth and differentiation of the prostate. The antiproliferative effects of calcitriol on PCa have been observed at high concentrations of calcitriol that may cause hypercalcemia in vivo. Intense research has been undertaken by many academic investigators and pharmaceutical companies to develop calcitriol analogs/derivatives that exhibit increased anti-proliferative activity and reduced tendency to cause the hypercalcemic side-effects [18,19]. Several studies have investigated the effects of calcitriol or its analogs on the establishment and growth of human PCa xenografts in immuno-compromised mice and showed significant reduction in tumor size and volume [4–9]. Transgenic models of PCa have also been developed in animals to study the chemopreventive effects of calcitriol and its analogs [20]. These in vivo models provide a valuable tool to study the tumor inhibitory effect of calcitriol and analogs while monitoring their tendency to cause hypercalcemia and to validate their potential use in clinical trials.
3. Molecular mechanisms mediating the anti-proliferative effects of calcitriol A number of important mechanisms have been implicated in calcitriol-mediated growth inhibition. A primary mechanism of calcitriol action is to induce cell cycle arrest in the G1 /G0 phase [8,9]. The growth arrest appears to be due to an increase in the expression of cyclin-dependent kinase inhibitors p21Waf/Cip1 and p27Kip1 [25–28], a decrease in cyclin-dependent kinase 2 (Cdk2) activity, [28] and the hyperphosphorylation of the retinoblastoma protein (pRb) [29]. p53 appears to be required for calcitriol-induced G0 arrest but is not required for calcitriol induced G1 accumulation or apoptosis of LNCaP cells [6,8,9]. Loss of the expression of cell cycle regulators has been associated with a more aggressive cancer phenotype [30], decreased prognosis and poorer survival [31,32]. These data therefore suggest that calcitriol may be a suitable therapy to inhibit PCa progression. In addition, calcitriol induces apoptosis in some PCa cells and down-regulates anti-apoptotic genes like bcl-2 [33]. Other mechanisms of calcitriol actions in PCa cells include the stimulation of differentiation, modulation of growth factor actions and the inhibition of invasion and metastasis [4–9]. Some in vivo studies demonstrate an inhibition of angiogenesis that contributes to the anti-tumor effects of calcitriol [4–9].
2.3. Clinical trials Several clinical trials have been carried out in PCa patients to evaluate the safety and efficacy of treatment with calcitriol or its analogs [4–9]. Calcitriol is an FDA approved drug and has been administered at as high a dose as tolerated, limited by hypercalciuria or hypercalcemia. A decrease in the rate of rise of serum prostate specific antigen (PSA) levels has
4. Novel pathways of calcitriol actions Calcitriol actions on cell growth and differentiation are initiated by its binding to the VDR resulting in the direct activation or repression of target gene transcription. The identification of the myriad target genes that mediate calcitriol
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actions is one of the goals of our current research on Vitamin D. We have used cDNA microarrays of primary human prostatic epithelial cells as well as LNCaP human PCa cells to identify molecular targets of calcitriol involved in the regulation of prostate epithelial cell growth [34,35]. Our studies have identified several new genes as well as known, important target genes regulated by calcitriol in prostate cells. In the following sections, we will discuss some of the recently revealed novel pathways of calcitriol actions in prostate cells, focusing on new developments from our laboratory. 4.1. Regulation of insulin-like growth factor binding protein (IGFBP) expression and growth inhibition Growth factors play an important role in the regulation of prostate epithelial cell growth by autocrine and paracrine mechanisms. Expression of autocrine growth factors by the epithelium and the development of independence from epithelial–stromal interactions that modulate the growth of the normal prostate may contribute to the progression of PCa. Among the important growth factors that regulate prostate epithelial growth are insulin-like growth factors (IGFs). IGFBPs are a family of proteins that bind the IGFs with high affinity and reduce their mitogenic effects by preventing the interaction of the IGFs with the IGF receptors [36]. However, the IGFBPs can regulate cell growth through IGF-independent pathways as well [36]. In PC-3 and ALVA 31 PCa cells, calcitriol decreases the availability of IGF by increasing the expression of the IGF binding proteins IGFBP-3 and IGFBP-5 [37,38]. In LNCaP and C4-2 PCa cells, which are sensitive to calcitriol-mediated growth inhibition, calcitriol decreases the mRNA levels of IGFBP2, a growth stimulator, and increases IGFBP-3 a growth inhibitor [34,39,40]. Calcitriol does not induce IGFBP-3 expression in DU 145 cells that are resistant to the growth inhibitory effects of calcitriol [40]. Studies from our laboratory have provided evidence that the up-regulation of IGFBP-3 expression by calcitriol is a necessary component of calcitriol-mediated inhibition of LNCaP cell growth, under serum-free culture conditions, where the cells are more dependent on autocrine growth factors [39]. Under these conditions, addition of IGFBP-3 anti-sense oligonucleotides abrogate calcitriol mediated growth inhibition suggesting that in LNCaP cells the growth inhibitory action of calcitriol is dependent on IGFBP-3 up-regulation. Furthermore, anti-IGFBP-3 antibodies block calcitriol-mediated increase in p21, showing that IGFBP-3 induction is necessary for the up-regulation of p21 expression [39]. We have also shown that calcitriol induces IGFBP-3 transcription in LNCaP cells cultured in serum-containing media wherein it causes maximal growth inhibition [34]. However, the growth inhibition may not be solely dependent on IGFBP-3 induction, as siRNA oligonucleotides against IGFBP-3 did not block calcitriol-dependent inhibition of LNCaP cell growth in serum-containing media [40]. Studies from our lab have shown that calcitriol directly induces the transcription of
IGFBP-3 gene through a functional Vitamin D response element (VDRE) present in the IGFBP-3 promoter [41b]. Our recent research indicates that the transcription of the IGFBP3 gene is also directly regulated by androgens through an androgen response element (ARE) and that there is synergistic interaction between calcitriol and androgens in regulating IGFBP-3 expression in LNCaP cells [41a]. IGFBP-3 is the most abundant IGF binding protein present in the serum and IGFBP-3 levels are inversely correlated with PCa risk and metastasis [15,42]. Thus, calcitriol may elevate IGFBP-3 levels within the prostate as well as in the circulation thereby counteracting the autocrine and paracrine factors that stimulate the growth of prostate tumors. 4.2. Regulation of prostaglandin metabolism and signaling Prostaglandins (PGs) have been shown to play a role in the development and progression of many cancers including PCa [43,44]. We have recently discovered that calcitriol regulates multiple genes involved in PG the pathway. Calcitriol decreases PG synthesis, increases PG catabolism and inhibits PG signaling through their receptors thereby attenuating the growth stimulatory effects of PGs in PCa [45]. 4.2.1. Cyclooxygenase-2 Cyclooxygenase (COX)/prostaglandin endoperoxidase synthase is the rate-limiting enzyme that catalyzes the conversion of arachidonic acid to PGs and related eicosanoids. The expression of COX-2 is rapidly induced by a variety of mitogens, cytokines, tumor promoters and growth factors and therefore COX-2 is regarded as an immediate-early response gene [44]. Compelling evidence from genetic and clinical studies indicates that increased expression of COX2 is one of the key steps in carcinogenesis. Several studies have demonstrated COX-2 over-expression in prostate adenocarcinoma [46,47] and suggest a positive role for COX-2 in prostate tumorigenesis. However, not all PCa are associated with elevated COX-2 expression [48,49]. Although Zha et al. [48] did not find consistent over-expression of COX-2 in established PCa, they detected appreciable COX-2 expression in areas of proliferative inflammatory atrophy, lesions that have been implicated in prostate carcinogenesis. Both arachidonic acid, the substrate for COX, and the product prostaglandin E2 (PGE2 ) stimulate the proliferation of PCa cells causing increases in the expression of immediate-early genes including c-fos that are involved in growth regulation [50]. 4.2.2. 15-Hydroxyprostaglandin dehydrogenase The key enzyme responsible for the metabolic inactivation of PGs, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), catalyzes the conversion of PGs to their corresponding 15-keto derivatives that exhibit greatly reduced biological activity. A recent study describes 15-PGDH as an oncogene antagonist that functions as a tumor suppressor in
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colon cancer [51]. The study showed that 15-PGDH, which physiologically antagonizes COX-2, is universally expressed in normal colon specimens but is routinely absent or severely reduced in cancer specimens. Most importantly, stable transfection of a 15-PGDH expression vector into cancer cells greatly reduced the ability of the cells to form tumors and/or slowed tumor growth in nude mice [51]. In PCa cells, 15-PGDH expression is induced by high concentrations of androgens that also inhibit cell growth [52]. 4.2.3. PG receptors PGs bind to G-protein coupled membrane receptors (prostanoid receptors) which activate signal transduction pathways [53]. There are eight members in the prostanoid receptor subfamily and they are distinguished by their ligand binding profile and the signal transduction pathways that they activate upon ligand binding and account for some of the diverse and often opposing effects of PGs [53]. PCa cells express EP and FP PG receptors [45,50]. PGE and PGF are the major PGs stimulating the proliferation of PCa cells [45]. PGE acts through four different PGE receptor (EP) sub-types (EP1–EP4), while PGF activates the FP receptor. 4.2.4. Calcitriol effects on the PG pathway in prostate cells Initial analyses from our lab using cDNA microarrays to study changes in the gene expression profile following
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calcitriol treatment of prostate cells indicated that calcitriol regulated the expression of genes involved in PG metabolism [34,35]. Calcitriol treatment up-regulated the expression of 15-PGDH and down-regulated COX-2 expression [34]. We went on to demonstrate calcitriol regulation of the PG pathway genes in multiple PCa cell lines as well as primary prostatic epithelial cells established from surgically removed prostate tissue from PCa patients [45]. We found measurable amounts of COX-2 mRNA and protein in various PCa cell lines as well as primary prostatic epithelial cells derived from normal and cancerous prostate tissue, which were significantly decreased by calcitriol treatment [45]. We also found that calcitriol significantly increased the expression of 15-PGDH mRNA and protein in various PCa cells [45]. We further showed that by inhibiting COX-2 and stimulating 15-PGDH expression, calcitriol decreased the levels of biologically active PGs in PCa cells thereby reducing the growth stimulation by PGs [45]. Interestingly our data also revealed that calcitriol decreased the expression of EP and FP PG receptors. The calcitriol-induced decrease in PG receptor levels resulted in the attenuation of PG mediated functional responses even when exogenous PGs were added to the cultures [45]. Calcitriol suppressed the induction of the immediate-early gene c-fos and the growth stimulation seen following the addition of exogenous PGs or the PG precursor arachidonic acid to PCa cell cultures [45]. Thus, as illustrated in Fig. 1, calcitriol inhibits the PG pathway in
Fig. 1. Inhibition of the prostaglandin pathway by calcitriol in prostate cancer cells. Calcitriol inhibits the prostaglandin pathway by three separate mechanisms, decreasing COX-2 expression, increasing 15-PGDH expression and reducing PG receptor levels to inhibit the PG pathway in PCa cells. Non-steroidal anti-inflammatory drugs (NSAIDs) directly inhibit COX-2 enzymatic activity.
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PCa cells by three separate mechanisms, decreasing COX-2 expression, increasing 15-PGDH expression and reducing PG receptors. We believe that these actions contribute to the suppression of the proliferative stimulus provided by PGs in PCa cells. The regulation of PG metabolism and biological actions constitute an additional novel pathway of calcitriol action mediating its anti-proliferative effects in prostate cells. 4.2.5. Combination of calcitriol and non-steroidal anti-inflammatory drugs (NSAIDs) as a therapy for prostate cancer NSAIDs are a class of drugs that decrease PG synthesis by inhibiting COX-1 and COX-2 enzymatic activities. Several NSAIDs inhibit both the constitutively expressed COX-1 and the inducible COX-2 while others have been shown to be selective for COX-2. We hypothesized that the action of calcitriol to reduce COX-2 gene expression will decrease
the levels of COX-2 protein and allow the use of lower concentrations of NSAIDs to inhibit COX-2 enzyme activity [45]. In addition, an increase in the expression of 15-PGDH due to calcitriol action will lower the levels of biologically active PGs and enhance the NSAID effect. Therefore, we hypothesized that the combination of calcitriol and NSAIDs would exhibit synergistic effects to inhibit PCa cell growth. When calcitriol was combined with the COX-2-selective NSAIDs NS398 and SC-58125 or the non-selective NSAIDs, naproxen and ibuprofen, we found a synergistic enhancement of growth inhibition (Fig. 2). These results led us to further hypothesize that the combination of calcitriol and NSAIDs may have clinical utility in PCa therapy and is worthy of evaluation in a clinical trial [45]. The combination approach will allow the use of lower concentration of NSAIDs and thereby minimize their undesirable side-effects. It has very recently become clear that long-term use of COX-2selective inhibitors such as rofecoxib (Vioxx) causes an
Fig. 2. Synergistic inhibition of prostate cancer cell growth by calcitriol and NSAIDs. LNCaP or PC-3 cells were treated with 0.1% ethanol vehicle (Con) or 10 nM/L calcitriol (Cal) in the presence and absence of the indicated NSAID. Cell growth was determined by measuring DNA content. DNA values are presented as a percentage of the vehicle control value set at 100%. (A) LNCaP cells treated with a combination of calcitriol (Cal) and COX-2 specific NSAID SC-58125 (5 M). (B) PC-3 cells were treated with calcitriol (Cal) in the presence and absence of the COX-2 specific NSAID NS-398 (7.5 M). (C) LNCaP cells treated with calcitriol (Cal) in the presence and absence of the non-selective NSAID naproxen (Nap; 200 M). (D) PC-3 cells were treated with calcitriol (Cal) in the presence and absence of the non-selective NSAID ibuprofen (Ibu; 150 M). * P < 0.05; ** P < 0.01, when compared with control. ++ P < 0.01, when compared with 1 or 10 nM/L Cal alone (used with permission from [45]).
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increase in cardiovascular complications in patients [54]. In comparison, non-selective NSAIDs such as naproxen have been shown to be associated with fewer cardiovascular adverse effects [55]. Our data show the combination of calcitriol with a non-selective NSAID is equally effective in inducing synergistic growth inhibition. We proposed that the combination of calcitriol with a non-selective NSAID is a useful therapeutic approach in PCa that would allow both drugs to be used at reduced dosages leading to increased safety [45]. 4.3. Induction of mitogen-activated protein kinase phosphatase 5 (MKP5) and prostate cancer prevention PCa generally progresses very slowly, likely for decades, before symptoms become obvious and diagnosis is made. Recently, inflammation in the prostate has been proposed to be an etiological factor in the development of PCa [56]. The observed latency in PCa provides a long window of opportunity for intervention by chemopreventive agents. Our recent study using cDNA microarrays [35] revealed a newly identified calcitriol responsive gene, MAP Kinase Phosphatase 5 (MKP5) also known as dual specificity phosphatase 10 (DUSP10). As described in our recent manuscript [57], the up-regulation of MKP5 and the downstream anti-inflammatory responses elicited by calcitriol in normal prostatic epithelial cells are supportive of a role for calcitriol in PCa prevention. The effect of MKP5 regulation in the chemopreventive actions of calcitriol is discussed below. In primary cultures of normal prostatic epithelial cells from the peripheral zone (E-PZ), MKP5 transcription is increased following calcitriol addition [57]. Calcitriol treatment of a normal primary prostate epithelial cells rapidly induced the expression of MKP5 in a dose and time-dependent manner (Fig. 3A and B). In addition, we identified a putative positive VDRE in the MKP5 promoter mediating this calcitriol effect [57]. Interestingly, calcitriol up-regulation of MKP5 was observed in primary cells derived from normal prostatic epithelium and primary, localized adenocarcinoma but not in the established PCa cell lines derived from PCa metastasis such as LNCaP, PC-3 or DU145. MKP5 is a member of the dual specificity MKP family of enzymes that dephosphorylate and thereby inactivate mitogen activated protein kinases (MAPKs) as well as the stress-activated protein kinase p38. We found that calcitriol inhibited the phosphorylation and activation of p38 in normal primary prostate cells [57]. When NaCl was added to the media to induce osmotic stress it resulted in the phosphorylation of p38 in these cells. When the cells were co-treated with NaCl and calcitriol a significant decrease in phosphorylation of p38 was observed. The attenuation of p38 activation by calcitriol appeared to be dependent on MKP5 induction. MKP5 siRNA completely abolished this calcitriol effect [57]. The consequence of p38 stress-induced kinase activation is an increase in the production of pro-inflammatory cytokines
Fig. 3. Increased expression of MKP5 in primary cultures of normal prostatic epithelial cells by calcitriol. (A) RT-qPCR measurement of MKP5 mRNA in E-PZ cells after treatment with 1, 10 and 50 nM calcitriol (1,25D). (B) Time course of 1,25D regulation of MKP5 gene expression in E-PZ cells. Cells were treated with 50 nM of 1,25D. RT-qPCR results are shown relative to untreated controls and normalized to the expression of the human TATA box binding protein (TBP) gene. Each experiment was run in triplicate and results are representative of two or more separate experiments with different patient derived E-PZ cells. Used with permission from [57].
that sustain and amplify the inflammatory response [58]. As interleukin-6 (IL-6), a p38-regulated pleiotropic cytokine, is known to be associated with PCa progression [59], we investigated the effect of calcitriol on IL-6 production. Treatment of primary prostate cells with TNF␣ increased IL-6 concentrations in the conditioned media. Co-administration of calcitriol significantly attenuated IL-6 production by TNF␣. It is becoming apparent that inflammation, both chronic and acute, contributes to PCa development [2]. A model based on the results of our recent study [57] is depicted in Fig. 4. The data suggest that the ability of calcitriol to inhibit p38 signaling and reduce the subsequent production of pro-inflammatory cytokines, via MKP5 up-regulation, may contribute to the cancer preventive effects of calcitriol. In this context it is interesting (as described above) that calcitriol significantly suppresses the expression of the pro-inflammatory enzyme COX-2 and inhibits the action of PGs, well-known mediators of inflammation, lending further support to its role in the prevention of PCa. Since established metastasisderived PCa cell lines exhibit low levels of MKP5 and are
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Fig. 4. Proposed mechanism for anti-inflammatory activity and prostate cancer prevention by Vitamin D. Calcitriol stimulation of MKP5 inhibits the stressactivated kinase p38 and abrogates stimulation of pro-carcinogenic inflammatory cytokines like IL-6 (used with permission [57]).
unable to induce MKP5 in response to calcitriol, we speculate that loss of MKP5 might occur during PCa progression as a result of selective pressure to eliminate the tumor suppressor activity of MKP5 and/or calcitriol. 4.4. Up-regulation of Mullerian Inhibiting Substance (MIS) expression Mullerian Inhibiting Substance (MIS), also known as antiMullerian hormone (AMH), is a member of the transforming growth factor- (TGF) family of factors that function to regulate growth, differentiation and apoptosis in many cells [60]. In males, during fetal sexual development, MIS causes the regression of the Mullerian ducts by inducing apoptosis [60]. MIS is a secreted glycoprotein that is produced by Sertoli cells in the testes of males and by granulosa cells in the ovary of females. MIS binds to the MIS type II receptor (MISR II), initiating a signaling cascade that involves the recruitment of type I receptors ALK2 and ALK6 [61]. However, it has recently been appreciated that MIS has multiple actions in post-natal life. MIS has post-natal actions to inhibit steroidogenesis [62,63] and it may also be involved in the polycystic ovarian syndrome (PCOS) [64]. More importantly, MIS has been shown to inhibit ovarian and cervical cancers, both of which are tumors of Mullerian origin [65,66]. Recently, MIS has been shown to have anti-proliferative and pro-apoptotic effects in prostate, breast and uterine cancers [67–70]. MIS induces the expression of the NF-B target gene, X-rayinducible immediate early response factor-1 (IEX-1) [71], in breast and prostate cancer cells [67,68]. Overexpression of IEX-1 in breast cancer cells inhibits cell growth indicating a negative regulatory role for this gene [67]. Efforts are now being advanced to develop recombinant purified MIS as a therapeutic agent for ovarian and uterine cancers with the added potential to treat other cancers including breast, prostate and Leydig cell tumors [60].
Our recent observations have revealed that MIS is a novel target gene regulated by calcitriol in prostate cells. When PCa cells such as LNCaP or PC-3 cells, as well as cultures of human primary prostatic epithelial cells, were exposed to calcitriol for 24 h, we found considerable increases in the expression of MIS mRNA (Fig. 5A). When LNCaP cells were treated with calcitriol, a substantial increase in secreted MIS protein was observed (Fig. 5B). The promoter sequence for the human MIS gene is contained within a 789 bp sequence constrained by the end of the SF3A2 gene upstream and the start of the MIS open reading frame [72]. Upon in silico analysis of the MIS gene promoter sequence, we noted the presence of a putative VDRE containing two half-sites separated by a three-base spacer, the so-called DR3 motif that is highly similar to the human osteocalcin VDRE. To determine whether calcitriol is a direct regulator of the MIS gene, we cloned a 650 bp fragment containing the MIS promoter into pGL3-basic, a promoterless luciferase reporter vector. We transfected HeLa cells with the MIS promoterluciferase construct and a VDR expression vector and tested the effect of calcitriol. Calcitriol caused a significant (twoto four-fold) induction of the MIS promoter-luciferase demonstrating that the MIS promoter is responsive to calcitriol. Induction of MIS expression may play an important role in the anti-cancer actions of calcitriol. Because of its potential therapeutic benefits, MIS is under active development as a cancer therapy. We hypothesize that the ability of calcitriol to induce endogenous MIS within the prostate is an alternate approach to administering exogenous recombinant, purified MIS, which is a protein biological. A combination of calcitriol and MIS may be a more effective therapy since it is unlikely that MIS can be administered continuously in saturating concentrations. Our future research plans include studies on the contribution of MIS to the antiproliferative/pro-apoptotic actions of calcitriol.
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by calcitriol, results in the attenuation of pro-inflammatory responses in prostate cells. This novel calcitriol action pathway as well as the inhibition of the expression of the proinflammatory enzyme COX-2 suggest that calcitriol has antiinflammatory actions that may play an important role in the prevention of PCa development. Importantly, identification of novel pathways of calcitriol actions may lead to the development of new therapeutic strategies in the treatment of PCa. Acknowledgements This work was supported by Grants DK42482, DAMD1702-1-0142, and PC050074 (D.F.), DOD PC04120 (J.M.), DOD PC04616 (L.N.) and AFUD scholar (L.N.).
References
Fig. 5. Calcitriol up-regulates MIS expression in prostate cancer cells. (A) PCa cell lines (LNCaP and PC-3) and primary prostatic epithelial cells (EPZ-1) were treated with vehicle (0.1% ethanol, open bars) or 10 nM calcitriol (hatched bars) for 24 h and total RNA samples isolated. MIS mRNA levels were determined by RT-qPCR. The human TATA box binding protein (TBP) was used as a control for normalization. Changes in MIS mRNA are given as fold increase over untreated control levels. (B) LNCaP cells were treated with 10 nM calcitriol every 2 days for 6 days. MIS protein in culture medium was measured using an ELISA.
5. Conclusions Our research is aimed at gaining a better understanding the molecular mechanisms of the anti-proliferative and cancer preventive effects of calcitriol with the goal of developing strategies to improve PCa treatment. We have recently identified several new calcitriol target genes in prostate cells that have revealed novel pathways of calcitriol action. We propose that calcitriol inhibition of the PG pathway contributes significantly to its anti-proliferative action. The increase in the expression of MIS, a hormone with known anti-cancer effects in tissues of reproductive origin, also adds a new dimension to the already well-known mechanisms mediating the growth inhibitory actions of calcitriol. The induction of MKP5, and subsequent inhibition of p38 stress kinase signaling
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