The authors state that they have no conflicts of interest.
Calcitriol, the hormonally active form of vitamin D, inhibits the growth and development of several cancers. Inflammation has been implicated in the development and progression of many cancers, including prostate cancer (PCa). Recent research from our laboratory suggests that calcitriol exhibits anti-inflammatory actions that may contribute to its inhibitory effects in PCa. We found that calcitriol inhibits the synthesis and actions of pro-inflammatory prostaglandins (PGs) by three mechanisms: (1) inhibition of the expression of cyclooxygenase-2 (COX-2), the enzyme that synthesizes PGs, (2) induction of the expression of 15-prostaglandin dehydrogenase (15-PGDH), the enzyme that inactivates PGs, and (3) decreasing the expression of prostaglandin E and prostaglandin F PG receptors, which are the mediators of PG signaling. The combination of calcitriol and nonsteroidal anti-inflammatory drugs (NSAIDs) result in a synergistic inhibition of PCa cell growth and offers a potential therapeutic strategy. Acting on a separate anti-inflammatory pathway, calcitriol induces the expression of mitogen-activated protein kinase phosphatase 5 (MKP5), a member of a family of phosphatases that are negative regulators of MAP kinases, causing the selective dephosphorylation and inactivation of the stress-activated protein kinase p38. Because p38 activation may be both procarcinogenic and promote inflammation, this calcitriol action, especially coupled with the inhibition of the PG pathway, may contribute to the chemopreventive activity of calcitriol. We conclude that calcitriol exerts several anti-inflammatory actions in prostate cells, which contribute to its potential as a chemopreventive and therapeutic agent in PCa.
Prostate cancer (PCa) is the most commonly diagnosed malignancy and the third leading cause of cancer death among men in the United States. One of the goals of current research on early (androgen-dependent) and advanced (androgen-independent) PCa is the identification of new, less toxic agents that would prevent PCa development and/or slow its progression. Among these, calcitriol (1,25-dihydroxyvitamin D3), the active metabolite of vitamin D, has emerged as a promising therapeutic agent. Calcitriol plays an important role in calcium homeostasis and bone metabolism through its actions in intestine, bone, kidney, and the parathyroid glands. However, calcitriol also exerts anti-proliferative and pro-differentiating effects in a number of tumors and malignant cells including PCa, raising the possibility of its use as an anticancer agent.
CALCITRIOL AND PROSTATE CANCER
Epidemiology and genetic factors
There are several risk factors for PCa including age, race, and genetics. Age is the strongest risk factor for prostate cancer, and the elderly are frequently vitamin D deficient because of several factors including less exposure to UV radiation. Of particular interest is the hypothesis put forward by Schwartz and colleagues, suggesting a role for vitamin D in decreasing the risk of developing PCa based on the observation that prostate cancer mortality rates in the United States are inversely proportional to the geographically determined incident UV radiation exposure from the sun. Because UV light is essential for vitamin D synthesis, more sunlight is associated with reduced incidence of PCa. This offers a potential explanation of why black men have a higher incidence of prostate cancer than white men. Black individuals have lower serum 25-hydroxyvitamin D [25(OH)D] levels as a result of their darker skin pigmentation because the high melanin levels in darkly pigmented skin block UV radiation and inhibit the formation of vitamin D3. Epidemiological data also suggest that vitamin D deficiency increases PCa risk. Although controversial, some studies support the concept that decreased serum levels of 25(OH)D, the precursor of calcitriol, correlate with increased risk of PCa. Polymorphisms in the vitamin D receptor (VDR) gene may contribute to PCa risk, as well as the histopathological characteristics and prognosis of PCa.
Growth inhibitory effects of calcitriol in cell culture and animal models of PCa
A number of studies have shown the anti-proliferative and pro-differentiating effects of calcitriol in primary prostatic epithelial cells and human PCa cell lines. In the normal prostate, 25(OH)D3-1α hydroxylase converts 25(OH)D3 to 1,25(OH)2D3, suggesting that local production of calcitriol may play an important role in normal growth and differentiation of the prostate. The anti-proliferative effects of calcitriol in cultured cells have been observed at high concentrations. High concentrations of calcitriol in vivo may cause hypercalcemia and hypercalciuria, which may be associated with renal stone formation. Many academic investigators and pharmaceutical companies have undertaken intense research to develop calcitriol analogs/derivatives that exhibit increased anti-proliferative activity and reduced tendency to cause hypercalcemia. 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. Transgenic models of PCa have also been developed in animals to study the effects of calcitriol and its analogs to prevent or delay the development and progression of PCa. 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.
MOLECULAR MECHANISMS OF CALCITRIOL-MEDIATED GROWTH INHIBITION
A number of important mechanisms have been implicated in calcitriol-mediated growth inhibition in PCa cells. These include the induction of cell cycle arrest in the G1/G0 phase caused by an increase in the expression of cyclin-dependent kinase inhibitors, induction of apoptosis, stimulation of differentiation, and modulation of growth factor actions. Calcitriol increases the expression of insulin-like growth factor binding protein-3 (IGFBP-3) in PCa cells, and the role of IGFBP-3 induction in the growth inhibitory effect of calcitriol has been extensively studied. Calcitriol also inhibits tumor invasion and metastasis and angiogenesis.
ANTI-INFLAMMATORY ACTIONS OF CALCITRIOL IN PCa
A major goal of our current research is to gain a more complete understanding of the molecular pathways by which calcitriol mediates its anti-proliferative and pro-differentiation effects. Using cDNA microarrays, we have shown that calcitriol regulates the expression of genes involved in the metabolism and signaling of prostaglandins (PGs), pro-inflammatory molecules that promote prostate tumorigenesis and growth. We have also shown that calcitriol upregulates the expression of mitogen-activated protein kinase phosphatase-5 (MKP5) and thereby promotes downstream anti-inflammatory responses. In the following sections, we discuss the anti-inflammatory effects of calcitriol and the role of calcitriol in the chemoprevention of PCa.
Regulation of prostaglandin metabolism and signaling
PGs have been shown to play a role in the development and progression of many cancers including PCa. We have recently discovered that calcitriol regulates the expression of several genes involved in the PG pathway. These calcitriol actions result in a decrease in PG synthesis, an increase in PG catabolism, and the inhibition of PG signaling through their receptors in PCa cells.
Cyclooxygenase-2 and PCa
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. Genetic and clinical studies indicate that increased COX-2 expression is one of the key steps in carcinogenesis. Several studies have shown COX-2 overexpression in prostate adenocarcinoma and suggest a positive role for COX-2 in prostate tumorigenesis. However, not all PCa is associated with elevated COX-2 expression. Although Zha et al. did not find consistent overexpression 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. In a recent study of 91 PCa patients, Rubio et al. found COX-2 protein expression in biopsy cores from 67% of the patients and in surgically removed prostate specimens from 79% of the patients by immunohistochemistry. A recent analysis of archival radical prostatectomy specimens also found COX-2 expression in PCa cells, adjacent normal glands, and in specimens from patients who exhibited disease progression and concluded that COX-2 expression was an independent predictor of recurrence. It is clear that local production of PGs by infiltrating inflammatory cells increases the risk of carcinogenesis and/or disease progression. At the cellular level, both arachidonic acid, the substrate for COX, and the product prostaglandin E2 (PGE2) stimulate proliferation by regulating the expression of genes that are involved in growth regulation including c-fos. A recent study reported that silencing COX-2 at the mRNA level in metastatic PCa cells induced cell growth arrest and caused morphological changes associated with enhanced differentiation, highlighting the role of COX-2 in prostate carcinogenesis.
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 described 15-PGDH as an oncogene antagonist functioning as a tumor suppressor in colon cancer. The study showed that 15-PGDH, which physiologically antagonizes COX-2, is universally expressed in normal colon 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. A recent study in mice showed that 15-PGDH acts in vivo as a highly potent suppressor of colon neoplasia development.
PGE and PGF are the major PGs stimulating the proliferation of PCa cells, and they act by binding to G protein–coupled membrane receptors. PGE acts through four different receptor subtypes (EP1–EP4), whereas PGF acts through the FP receptor. PCa cells express EP and FP receptors.
Calcitriol effects on the PG pathway in prostate cells
Calcitriol regulates the expression of PG pathway genes in multiple PCa cell lines and primary prostatic epithelial cells established from surgically removed prostate tissue from PCa patients. 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. We also found that calcitriol significantly increased the expression of 15-PGDH mRNA and protein in various PCa cells. 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. Our data also showed 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. Calcitriol suppressed the induction of the immediate-early gene c-fos and the growth stimulation seen after the addition of exogenous PGs or the PG precursor arachidonic acid to PCa cell cultures. Thus, calcitriol inhibits the PG pathway in 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.
Combination of calcitriol and nonsteroidal anti-inflammatory drugs as a therapy for prostate cancer
Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of drugs that decrease PG synthesis by inhibiting COX-1 and COX-2 enzymatic activities. On the other hand, calcitriol decreases COX-2 gene expression. 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. In addition, an increase in the expression of 15-PGDH and a decrease in PG receptors caused by 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 nonselective NSAIDs, naproxen and ibuprofen, we found a synergistic enhancement of growth inhibition. These results led us to further hypothesize that the combination of calcitriol and NSAIDs may have clinical use in PCa therapy. 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-2–selective inhibitors such as rofecoxib (Vioxx) causes an increase in cardiovascular complications in patients. In comparison, nonselective NSAIDs such as naproxen have been shown to be associated with fewer cardiovascular adverse effects. Our data showed the combination of calcitriol with a nonselective NSAID is as effective in inducing synergistic growth inhibition as a combination with a COX-2–selective inhibitor. We propose that the combination of calcitriol with a nonselective NSAID is a useful therapeutic approach in PCa that would allow both drugs to be used at reduced dosages, leading to increased safety. Based on these observations, we recently initiated a phase II clinical trial of calcitriol and naproxen combination in patients with early recurrent PCa. The regimen includes very high doses of calcitriol (45 μg of DN-101; Novacea, South San Francisco, CA, USA) once weekly combined with naproxen (400 mg, twice daily), and the initial results are promising (S Srinivas and D Feldman, unpublished data, 2008) Weekly high doses of calcitriol have been shown to be well tolerated by PCa patients with little in the way of hypercalcemia or renal stone formation.
Induction of MKP5 and prostate cancer prevention
Our recent study using cDNA microarrays showed another novel calcitriol responsive gene, MKP5, also known as dual specificity phosphatase 10 (DUSP10). The upregulation of MKP5 expression by calcitriol leads to downstream anti-inflammatory responses in cells derived from normal prostatic epithelium and primary, localized adenocarcinoma, supporting a role for calcitriol in the prevention and early treatment of PCa.
In primary cultures of normal prostatic epithelial cells from the peripheral zone, calcitriol increased MKP5 transcription, and we have identified a putative positive vitamin D response element (VDRE) in the MKP5 promoter mediating this calcitriol effect. Interestingly, calcitriol upregulation of MKP5 was seen only 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). MKP5 specifically dephosphorylates p38 MAPK and stress-activated protein kinase Jun-N-terminal kinase (JNK), leading to their inactivation. Calcitriol inhibited the phosphorylation and activation of p38 in normal primary prostate cells in a MKP5-dependent manner, as MKP5 siRNA completely abolished p38 inactivation by calcitriol. A consequence of p38 stress-induced kinase activation is an increase in the production of pro-inflammatory cytokines that sustain and amplify the inflammatory response. Because interleukin-6 (IL-6), a p38-regulated pleiotropic cytokine, is known to be associated with PCa progression, we studied the effect of calcitriol on IL-6 production. Stimulation of primary prostate cells with the pro-inflammatory factor, TNFα, increased IL-6 mRNA stability and concentrations of IL-6 in the conditioned media. Pretreatment of the cells with calcitriol significantly attenuated IL-6 production by TNFα.
Our data suggest that the ability of calcitriol to inhibit p38 signaling and reduce the subsequent production of pro-inflammatory cytokines, through MKP5 upregulation, may contribute to the cancer preventive effects of calcitriol. Because established metastasis-derived PCa cell lines exhibited low levels of MKP5 and were 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.
In the various in vitro studies exploring anti-inflammatory effects, calcitriol exhibited dose-dependent activity. Significant increases in MKP5 and reductions in PGE2 were apparent at 1–5 nM calcitriol, and maximal effects were typically seen at 10–50 nM. In in vivo experimental systems, daily high concentrations of calcitriol led to hypercalcemia and hypercalciuria, which limit the dose of calcitriol that can be safely administered. Many investigators have used intermittent high dosing of calcitriol or calcitriol analogs, which exhibit improved anti-proliferative effects with reduced tendency to cause hypercalcemia. In PCa patients, intermittent administration three times a week or once weekly of high-dose calcitriol has been successfully used without causing substantial hypercalcemia (see the following section on clinical studies).
INFLAMMATION, PROSTATE CANCER, AND THE ROLE OF CALCITRIOL
Current perspectives in cancer biology suggest that inflammation plays a role in the development of cancer. Inflammation may contribute to carcinogenesis by several mechanisms including the elaboration of cytokines and growth factors that favor tumor cell growth, induction of COX-2, which leads to the synthesis of PGs that promote tumor proliferation, and generation of mutagenic reactive oxygen and nitrogen species. Epidemiological studies show that there is decreased risk of PCa associated with the intake of antioxidants and NSAIDs. De Marzo et al. have proposed that lesions in the prostate called proliferative inflammatory atrophy (PIA), which are associated with acute or chronic inflammation, are precursors of prostatic intraepithelial neoplasia (PIN) and PCa. The epithelial cells in PIA lesions have been shown to exhibit many molecular signs of stress including elevated expression of COX-2. Based on our recent research showing (1) the repression of COX-2 expression in normal and malignant prostatic epithelial cells by calcitriol and (2) the inhibition of pro-inflammatory cytokine production by calcitriol caused by MKP5-mediated p38 inactivation, we postulate that calcitriol may exert anti-inflammatory effects and thereby play a role in delaying or preventing the development and/or progression of PCa.
CALCITRIOL AND PROSTATE CANCER CHEMOPREVENTION
PCa generally progresses very slowly, likely for decades, before symptoms become obvious, and a diagnosis is made. Recently, inflammation in the prostate has been proposed to be an etiological factor in the development of PCa. The observed latency in PCa provides a long window of opportunity for intervention by chemopreventive agents. Dietary supplementation of COX-2–selective NSAIDs such as celecoxib has been shown to suppress prostate carcinogenesis in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Our studies in primary prostate cells and PCa cell lines on the inhibitory effects of calcitriol on COX-2 expression and the PG pathway and MKP induction with the resultant stress kinase inactivation and inhibition of pro-inflammatory cytokine production suggest that calcitriol exhibits anti-inflammatory effects in vitro. Therefore, we hypothesize that calcitriol has the potential to be useful as a chemopreventive agent in PCa. Recently, Alagbala et al. showed that administration of high-dose calcitriol (20 μg/kg), intermittently 3 d/wk for up to 14–30 wk, suppresses tumor development in TRAMP mice. The efficacy of calcitriol as a chemopreventive agent has also been recently examined in Nkx3.1; Pten mutant mice, which recapitulate stages of prostate carcinogenesis from PIN lesions to adenocarcinoma. The data show that calcitriol significantly reduces the progression of PIN from a lower to a higher grade. Calcitriol is more effective when administered before, rather than after, the initial occurrence of PIN. These animal studies and our in vitro observations suggest that clinical trials in PCa patients with PIN or early disease evaluating calcitriol and its analogs as agents that prevent and/or delay progression are warranted.
CLINICAL STUDIES OF CALCITRIOL USE IN PROSTATE CANCER
Several clinical trials have been carried out in PCa patients to evaluate the safety and efficacy of treatment with calcitriol or its analogs. Calcitriol is an FDA-approved drug and has been administered at the highest dose tolerated, limited by persistent hypercalcemia or hypercalciuria. A decrease in the rate of rise of serum prostate-specific antigen (PSA) levels has been in observed in PCa patients after daily modest doses of calcitriol, indicating a beneficial effect of calcitriol in slowing the progression of the disease. Recent studies have followed the approach of administering calcitriol intermittently, such as three times a week or once weekly, in very high doses when it apparently can still elicit its anti-proliferative effects but cause only transient hypercalcemia. Thus far, these intermittent high doses do not seem to cause substantial toxicity. Calcitriol is also being used in combination therapy with other agents that may enhance its anti-proliferative activity while reducing its hypercalcemic tendency. The results of the ASCENT clinical trial in advanced PCa patients who failed other therapies was recently presented at the meeting of American Society of Clinical Oncology. The data presented and published by Beer et al. showed that extremely high doses (45 μg) of calcitriol (DN-101; Novacea) administered orally once a week 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. The primary endpoint of reducing serum PSA was, however, not met. A large phase III trial testing this combination is under way. Interestingly, recent observations from the original ASCENT trial showed that high-dose calcitriol addition to docetaxel caused a statistically significant reduction in the incidence of venous and arterial thrombosis in PCa patients compared with docetaxel alone. In vitro and VDR knockout mouse studies suggest that calcitriol may act as an anti-thrombotic agent. The data from the ASCENT trial support this hypothesis and suggest that calcitriol exhibits anti-thrombotic effects in humans. ASCENT II, a large phase III trial comparing taxotere and taxotere plus large doses of DN101 once weekly, was stopped by the Data Safety Monitoring Board because of increased deaths in the DN101 arm. It is too early for an analysis of why this occurred, but the taxotere dosage schedule was different in the two arms.
Our research is aimed at gaining a better understanding of the molecular mechanisms of the anti-proliferative and cancer preventive effects of calcitriol with the goal of developing strategies to improve PCa treatment. We recently identified several new calcitriol target genes in prostate cells that have revealed novel anti-inflammatory pathways of calcitriol action (Fig. 1). We propose that calcitriol inhibition of the PG pathway contributes significantly to its anti-inflammatory actions. Also the induction of MKP5, and subsequent inhibition of p38 stress kinase signaling by calcitriol, results in the attenuation of the production of pro-inflammatory cytokines in prostate cells (Fig. 1). These novel calcitriol-regulated pathways suggest that calcitriol has anti-inflammatory actions, in addition to its other anti-cancer actions, that may play an important role in the prevention and/or treatment of PCa. Recent studies in animal models of PCa reveal that calcitriol inhibits the progression of PCa in TRAMP and Nkx3.1;Pten mice. We conclude that calcitriol and its analogs may have use as chemopreventive agents and should be evaluated in clinical trials in PCa patients with early disease.
This work was supported by Grants DK42482, DAMD17-02-1-0142, and PC050074 (DF), DOD PC04120 (JM), DOD PC04616 (LN), and AFUD scholar (LN).