Calcitriol (1,25-dihydroxyvitamin D 3 ), the hormonally active form of vitamin D, exerts growth inhibitory and prodifferentiating effects on many malignant cells and retards tumor growth in animal models. Calcitriol is being evaluated as an anticancer agent in several human cancers. The mechanisms underlying the anticancer effects of calcitriol include inhibition of cell proliferation, stimulation of apoptosis, suppression of inflammation, and inhibition of tumor angiogenesis, invasion, and metastasis. This review discusses some of the molecular pathways mediating these anticancer actions of calcitriol and the preclinical data in cell culture and animal models. The clinical trials evaluating the use of calcitriol and its analogues in the treatment of patients with cancer are described. The reasons for the lack of impressive beneficial effects in clinical trials compared with the substantial efficacy seen in preclinical models are discussed.
Calcitriol (1,25-dihydroxyvitamin D 3 ), the biologically most active form of vitamin D, maintains calcium homeostasis through its actions in intestine, bone, kidneys, and the parathyroid glands. The hormone exerts its effects through the vitamin D receptor (VDR), a member of the nuclear receptor superfamily. VDR is present not only in cells and tissues involved in calcium regulation but also a wide variety of other cells including malignant cells. In recent years it has been recognized that calcitriol exerts antiproliferative and prodifferentiating effects in many malignant cells, and retards the development and growth of tumors in animal models raising the possibility of its use as an anticancer agent.
Epidemiology
Epidemiologic studies have noted lower incidence and mortality rates from several cancers in regions with greater solar ultraviolet (UV)-B exposure. The potential benefit from sunlight is attributed to vitamin D, because UV light is essential for the cutaneous synthesis of vitamin D. The sunlight hypothesis (assuming that sunlight is a surrogate for vitamin D levels in circulation) has been proposed to determine the risk for several cancers including colorectal cancer (CRC) prostate cancer (PCa), and breast cancer (BCa). An inverse association between cancer risk and circulating levels of 25-hydroxyvitamin D (25(OH)D, the circulating precursor to calcitriol), which reflect sun exposure and dietary vitamin D intake, has also been reported. The evidence is strongest for CRC; circulating 25(OH)D levels and vitamin D intake are inversely associated with colorectal adenoma incidence and recurrence. In addition, higher prediagnosis plasma 25(OH)D levels were associated with a significant improvement in overall survival in CRC patients. A recent reanalysis of data from the Women’s Health Initiative (WHI) randomized trial concluded that concurrent estrogen therapy modified the effect of calcium and vitamin D supplementation on CRC risk and in the women assigned to placebo arms of the estrogen trials, the supplementation was beneficial. The evidence is somewhat weaker for PCa, with some studies suggesting an inverse correlation between serum 25(OH)D levels and PCa risk although others do not support such a correlation. In general, a serum 25(OH)D level exceeding 20 ng/mL was associated with a 30% to 50% reduction in the risk of developing CRC and PCa, and a level of approximately 52 ng/mL was associated with a reduction by 50% in the incidence of BCa. Higher dietary intake of vitamin D has been associated with a lower incidence of pancreatic cancer.
Mechanisms of the anticancer effects of calcitriol
In addition to the epidemiologic evidence described earlier, data from in vitro studies in cultured malignant cells reveal that calcitriol exerts antiproliferative and prodifferentiating effects; in vivo studies in animal models of cancer demonstrate that calcitriol retards tumor growth. Several important mechanisms have been implicated in the anticancer effects of calcitriol. The molecular mediators of these calcitriol actions are currently being intensively investigated and characterized.
Growth Arrest and Differentiation
Calcitriol inhibits the proliferation of many malignant cells by inducing cell cycle arrest and the accumulation of cells in the G 0 /G 1 phase of the cell cycle. In PCa cells calcitriol causes G 1 /G 0 arrest in a p53-dependent manner by increasing the expression of the cyclin-dependent kinase inhibitors p21 Waf/Cip1 and p27 Kip1 , decreasing cyclin-dependent kinase 2 (CDK2) activity, and causing the hypophosphorylation of the retinoblastoma protein (pRb). Calcitriol also enhances the expression of p73, a p53 homolog, which has been shown to be associated with apoptosis induction in several human and murine tumor systems. Suppression of p73 abrogates calcitriol-induced apoptosis and reduces the ability of calcitriol to augment the cytotoxic effects of agents such as gemcitabine and cisplatin in a squamous cell carcinoma (SCC) model. Calcitriol also increases the expression of CDK inhibitors in other cancer cells. It has been shown that calcitriol controls cell growth in part by modulating the expression and activity of key growth factors in cancer cells. For example, in PCa cells calcitriol up-regulates the expression of insulinlike growth factor binding protein-3 (IGFBP-3), which functions to inhibit cell proliferation in part by increasing the expression of p21 Waf/Cip1 .
In many neoplastic cells, calcitriol also induces differentiation resulting in the generation of cells that acquire a more mature and less malignant phenotype. The mechanisms of the prodifferentiation effects of calcitriol in various cancer cells are specific to the cell type and cell context and include, for example, the regulation of signaling pathways involving β-catenin, Jun-N-terminal kinase (JNK), phosphatidyl inositol 3-kinase, nuclear factor κB (NFκB) as well as the regulation of the activity of several transcription factors such as the activator protein-1 (AP-1) complex and CCAAT/enhancer-binding protein (C/EBP).
Apoptosis
Calcitriol induces apoptosis in several cancer cells, although this effect is not uniformly seen in all malignant cells. In PCa and BCa cells, calcitriol activates the intrinsic pathway of apoptosis causing the disruption of mitochondrial function, cytochrome release and production of reactive oxygen species. These effects are related to the repression of the expression of antiapoptotic proteins such as Bcl 2 and enhancement of the expression of proapoptotic proteins such as Bax and Bad. In some cells calcitriol also directly activates caspases to induce apoptosis. In addition, calcitriol analogues have been shown to enhance cancer cell death in response to radiation and chemotherapeutic drugs.
Inhibition of Invasion and Metastasis
Calcitriol reduces the invasive and metastatic potential of many malignant cells as demonstrated in murine models of prostate and lung cancer. The mechanisms underlying this effect include the inhibition of angiogenesis (as discussed later) and the regulation of the expression of key molecules involved in invasion and metastasis such as the components of the plasminogen activator (PA) system and matrix metalloproteinases (MMPs), decreasing the expression of tenascin-C, an extracellular matrix protein that promotes growth, invasion, and angiogenesis, down-regulation of the expression of α6 and β4 integrins, and increase in the expression of E-cadherin, a tumor suppressor gene whose expression is inversely correlated to metastatic potential. Calcitriol-mediated suppression of MMP-9 activity and increase in tissue inhibitor of metalloproteinase-1 (TIMP-1) also decrease the invasive potential of PCa cells.
Antiinflammatory Effects
Chronic inflammation, triggered by a variety of stimuli such as injury, infection, carcinogens, autoimmune disease, the development of tumors, hormonal factors, and so forth, has been recognized as a risk factor for cancer development. Cancer-related inflammation is characterized by the presence of inflammatory cells at the tumor sites and over-expression of inflammatory mediators such as cytokines, chemokines, prostaglandins (PGs) and reactive oxygen and nitrogen species in tumor tissue. Many of these proinflammatory mediators activate angiogenic switches usually under the control of vascular endothelial growth factor (VEGF) and thereby promote tumor progression, metastasis, and invasion. Epidemiologic studies show a decrease in the risk of developing several cancers associated with the intake of antioxidants and nonsteroidal antiinflammatory drugs (NSAIDs). Recent research suggests that calcitriol exhibits antiinflammatory actions that may contribute to its beneficial effects in several cancers in addition to the other antiproliferative actions discussed earlier. We used cDNA microarrays to uncover the molecular pathways that mediate the anticancer effects of calcitriol in PCa cells. The results show that calcitriol regulation of gene expression leads to the inhibition of the synthesis and biologic actions of prostaglandins (PGs), stress-activated kinase signaling, and production of proinflammatory cytokines. Calcitriol also suppresses the activation and signaling of NFκB, a transcription factor that regulates the expression of genes involved in inflammatory and immune responses and cellular proliferation and believed to play a key role in the process leading from inflammation to carcinogenesis.
Regulation of prostaglandin metabolism and signaling
In multiple PCa cell lines as well as primary prostatic epithelial cells established from prostatectomy samples, calcitriol decreases the mRNA and protein levels of cyclooxygenase-2 (COX-2), the enzyme responsible for PG synthesis, and increases the expression of 15-hyroxyprostaglandin dehydrogenase(15-PGDH), the enzyme that initiates PG catabolism. As a result calcitriol decreases the levels of biologically active PGs in these cells. In addition, calcitriol decreases the expression of EP and FP PG receptors and thereby attenuates PG-mediated functional responses including stimulation of cell growth. Thus, reduction in the levels of biologically active PGs and inhibition of PG signaling through their receptors by calcitriol results in suppression of the proliferative and angiogenic stimuli provided by PGs. Combinations of calcitriol with NSAIDs cause a synergistic enhancement of the inhibition of PCa cell proliferation, suggesting that this drug combination might have clinical usefulness in PCa therapy.
Induction of mitogen-activated protein kinase phosphatase-5 (MKP5) and inhibition of stress-activated kinase signaling
cDNA microarray analysis in normal human prostate epithelial cells revealed a novel calcitriol-responsive gene, MKP5, also known as DUSP10, 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 the stress-activated protein kinase JNK, leading to their inactivation. Calcitriol increases MKP5 transcription by the activation of VDR and its binding to a vitamin D response element (VDRE) in the MKP5 promoter. The calcitriol-mediated increase in MKP5 causes the dephosphorylation and inactivation of the p38 stress-induced kinase, resulting in a decrease in the production of proinflammatory cytokines that sustain and amplify the inflammatory response, such as interleukin-6 (IL-6). IL-6 stimulates PCa growth and progression and IL-6 synthesized in periprostatic adipose tissue has recently been shown to modulate PCa aggressiveness. Calcitriol up-regulation of MKP5 is seen in primary cells derived from normal prostate epithelium and primary localized adenocarcinoma but not in the established cell lines derived from metastatic PCa, suggesting that the loss of MKP5 might occur during PCa progression, as a result of a selective pressure to eliminate the tumor suppressor activity of MKP5 and/or calcitriol.
Inhibition of NFκB activation and signaling
NFκB comprises a family of inducible transcription factors ubiquitously present in cells that are important regulators of innate immune responses and inflammation. In the basal state, most NFκB dimers are bound to specific inhibitory proteins called IκB and proinflammatory signals activate NFκB mainly through IκB kinase (IKK)-dependent phosphorylation and degradation of the inhibitory IκB proteins. Free NFκB then translocates to the nucleus and activates the transcription of proinflammatory cytokines, chemokines, and antiapoptotic factors. In contrast to normal cells, many malignant cells have increased levels of active NFκB. Calcitriol directly modulates basal and cytokine-induced NFκB activity in many cells including human lymphocytes, fibroblasts, and peripheral blood monocytes. A reduction in the levels of the NFκB inhibitory protein IκB has been reported in mice lacking the VDR. Addition of a VDR antagonist to colon cancer cells up-regulates NFκB activity by decreasing the levels of IκB, suggesting that VDR ligands suppress NFκB activation. Calcitriol decreases the production of the angiogenic and proinflammatory cytokine IL-8 in immortalized normal human prostate epithelial cell lines and established PCa cell lines by inhibiting the nuclear translocation of the NFκB subunit p65 and subsequent transcriptional stimulation of the NFκB downstream target IL-8. Thus calcitriol could delay the progression of PCa by suppressing the expression of angiogenic and proinflammatory factors such as VEGF and IL-8. In addition, calcitriol indirectly inhibits NFκB signaling by up-regulating the expression of IGFBP-3, which has been shown to interfere with NFκB signaling in PCa cells. NFκB also provides an adaptive response to PCa cells against cytotoxicity induced by redox active therapeutic agents and is implicated in radiation resistance of cancers. Calcitriol significantly enhances the sensitivity of PCa cells to ionizing radiation by selectively suppressing radiation-mediated RelB activation. Thus calcitriol may serve as an effective agent for sensitizing PCa cells to radiation therapy via suppression of the NFκB pathway. There is also considerable evidence that calcitriol potentiates the antitumor activity of a wide variety of cytotoxic chemotherapy agents (described later). As noted earlier, the induction of p73 by calcitriol seems to contribute to the synergistic activity of calcitriol and platinum analogues and some antimetabolites.
The Role of Antiinflammatory Effects of Calcitriol in Cancer Prevention and Treatment
As discussed earlier current perspectives in cancer biology suggest that inflammation plays a role in the development of cancer. De Marzo and colleagues have proposed that precursor lesions called proliferative inflammatory atrophy (PIA) in the prostate, which are associated with acute or chronic inflammation, are the precursors of prostate intraepithelial neoplasia (PIN) and PCa. The epithelial cells in PIA lesions exhibit many molecular signs of stress including increased expression of COX-2. Similarly, inflammatory bowel disease is associated with the development of CRC. Based on the evidence demonstrating antiinflammatory effects of calcitriol, we postulate that calcitriol may play a role in delaying or preventing cancer development and/or progression. PCa generally progresses very slowly, likely over decades, before symptoms become obvious and the diagnosis is made. 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 of PCa. The inhibitory effects of calcitriol on COX-2 expression and the PG pathway, production of proinflammatory cytokines, NFκB signaling, and tumor angiogenesis suggest that calcitriol has the potential to be useful as a chemopreventive agent in malignancies such as PCa. Foster and colleagues have shown that administration of high-dose calcitriol (20 μg/kg), intermittently 3 days per week for up to 14 to 30 weeks, suppresses prostate tumor development in TRAMP mice. The efficacy of calcitriol as a chemopreventive agent has also been examined in Nkx3.1;Pten mutant mice, which recapitulate stages of prostate carcinogenesis from PIN lesions to adenocarcinoma. The data reveal 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 as well as in vitro observations suggest that clinical trials in PCa patients with PIN or early disease evaluating calcitriol and its analogues as agents that prevent and/or delay progression, are warranted.
Inhibition of Angiogenesis
Angiogenesis is the process of formation of new blood vessels from existing vasculature and is a crucial step in the continued growth, progression, and metastasis of tumors. VEGF is the most potent stimulator of angiogenesis. PGs are also important proangiogenic factors. The initiation of angiogenesis is controlled by local hypoxia, which induces the synthesis of proangiogenic factors that activate signaling pathways leading to the structural reorganization of endothelial cells favoring new capillary formation. Stimulation of angiogenesis in response to hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1), which directly increases the expression of several proangiogenic factors including VEGF. Early studies indicate that calcitriol is a potent inhibitor of tumor cell–induced angiogenesis in experimental models. Calcitriol inhibits VEGF-induced endothelial cell tube formation in vitro and decreases tumor vascularization in vivo in mice bearing xenografts of BCa cells over-expressing VEGF. Calcitriol and its analogues also directly inhibit the proliferation of endothelial cells leading to the inhibition of angiogenesis.
At the molecular level, calcitriol may exert its antiangiogenic effects through a direct antiproliferative action on endothelial cells in the tumor microenvironment and/or by regulating the expression of key factors that control angiogenesis. Calcitriol reduces VEGF expression in PCa cells through transcriptional repression of HIF-1. Calcitriol also suppresses the expression of the proangiogenic factor IL-8 in an NFκB-dependent manner. Chung and colleagues established TRAMP-2 tumors in wild-type mice and VDR knockout mice and found enlarged vessels and increased vessel volume in TRAMP tumors in the VDR knockout mice, suggesting an inhibitory role for VDR and calcitriol in tumor angiogenesis. Their study further showed increased expression of proangiogenic factors such as HIF-1α, VEGF, angiopoietin-1 and platelet-derived growth factor (PDGF) in the tumors in the VDR knockout mice. Another important mechanism by which COX-2 promotes tumor progression is through the stimulation of angiogenesis. The proangiogenic effect of COX-2–generated PGE 2 might be a result of its action to increase HIF-1α protein synthesis in cancer cells. Suppression of COX-2 expression by calcitriol therefore provides an important indirect mechanism by which calcitriol inhibits angiogenesis, in addition to its direct suppressive effects on proangiogenic factors such as HIF-1 and VEGF.
Mechanisms of the anticancer effects of calcitriol
In addition to the epidemiologic evidence described earlier, data from in vitro studies in cultured malignant cells reveal that calcitriol exerts antiproliferative and prodifferentiating effects; in vivo studies in animal models of cancer demonstrate that calcitriol retards tumor growth. Several important mechanisms have been implicated in the anticancer effects of calcitriol. The molecular mediators of these calcitriol actions are currently being intensively investigated and characterized.
Growth Arrest and Differentiation
Calcitriol inhibits the proliferation of many malignant cells by inducing cell cycle arrest and the accumulation of cells in the G 0 /G 1 phase of the cell cycle. In PCa cells calcitriol causes G 1 /G 0 arrest in a p53-dependent manner by increasing the expression of the cyclin-dependent kinase inhibitors p21 Waf/Cip1 and p27 Kip1 , decreasing cyclin-dependent kinase 2 (CDK2) activity, and causing the hypophosphorylation of the retinoblastoma protein (pRb). Calcitriol also enhances the expression of p73, a p53 homolog, which has been shown to be associated with apoptosis induction in several human and murine tumor systems. Suppression of p73 abrogates calcitriol-induced apoptosis and reduces the ability of calcitriol to augment the cytotoxic effects of agents such as gemcitabine and cisplatin in a squamous cell carcinoma (SCC) model. Calcitriol also increases the expression of CDK inhibitors in other cancer cells. It has been shown that calcitriol controls cell growth in part by modulating the expression and activity of key growth factors in cancer cells. For example, in PCa cells calcitriol up-regulates the expression of insulinlike growth factor binding protein-3 (IGFBP-3), which functions to inhibit cell proliferation in part by increasing the expression of p21 Waf/Cip1 .
In many neoplastic cells, calcitriol also induces differentiation resulting in the generation of cells that acquire a more mature and less malignant phenotype. The mechanisms of the prodifferentiation effects of calcitriol in various cancer cells are specific to the cell type and cell context and include, for example, the regulation of signaling pathways involving β-catenin, Jun-N-terminal kinase (JNK), phosphatidyl inositol 3-kinase, nuclear factor κB (NFκB) as well as the regulation of the activity of several transcription factors such as the activator protein-1 (AP-1) complex and CCAAT/enhancer-binding protein (C/EBP).
Apoptosis
Calcitriol induces apoptosis in several cancer cells, although this effect is not uniformly seen in all malignant cells. In PCa and BCa cells, calcitriol activates the intrinsic pathway of apoptosis causing the disruption of mitochondrial function, cytochrome release and production of reactive oxygen species. These effects are related to the repression of the expression of antiapoptotic proteins such as Bcl 2 and enhancement of the expression of proapoptotic proteins such as Bax and Bad. In some cells calcitriol also directly activates caspases to induce apoptosis. In addition, calcitriol analogues have been shown to enhance cancer cell death in response to radiation and chemotherapeutic drugs.
Inhibition of Invasion and Metastasis
Calcitriol reduces the invasive and metastatic potential of many malignant cells as demonstrated in murine models of prostate and lung cancer. The mechanisms underlying this effect include the inhibition of angiogenesis (as discussed later) and the regulation of the expression of key molecules involved in invasion and metastasis such as the components of the plasminogen activator (PA) system and matrix metalloproteinases (MMPs), decreasing the expression of tenascin-C, an extracellular matrix protein that promotes growth, invasion, and angiogenesis, down-regulation of the expression of α6 and β4 integrins, and increase in the expression of E-cadherin, a tumor suppressor gene whose expression is inversely correlated to metastatic potential. Calcitriol-mediated suppression of MMP-9 activity and increase in tissue inhibitor of metalloproteinase-1 (TIMP-1) also decrease the invasive potential of PCa cells.
Antiinflammatory Effects
Chronic inflammation, triggered by a variety of stimuli such as injury, infection, carcinogens, autoimmune disease, the development of tumors, hormonal factors, and so forth, has been recognized as a risk factor for cancer development. Cancer-related inflammation is characterized by the presence of inflammatory cells at the tumor sites and over-expression of inflammatory mediators such as cytokines, chemokines, prostaglandins (PGs) and reactive oxygen and nitrogen species in tumor tissue. Many of these proinflammatory mediators activate angiogenic switches usually under the control of vascular endothelial growth factor (VEGF) and thereby promote tumor progression, metastasis, and invasion. Epidemiologic studies show a decrease in the risk of developing several cancers associated with the intake of antioxidants and nonsteroidal antiinflammatory drugs (NSAIDs). Recent research suggests that calcitriol exhibits antiinflammatory actions that may contribute to its beneficial effects in several cancers in addition to the other antiproliferative actions discussed earlier. We used cDNA microarrays to uncover the molecular pathways that mediate the anticancer effects of calcitriol in PCa cells. The results show that calcitriol regulation of gene expression leads to the inhibition of the synthesis and biologic actions of prostaglandins (PGs), stress-activated kinase signaling, and production of proinflammatory cytokines. Calcitriol also suppresses the activation and signaling of NFκB, a transcription factor that regulates the expression of genes involved in inflammatory and immune responses and cellular proliferation and believed to play a key role in the process leading from inflammation to carcinogenesis.
Regulation of prostaglandin metabolism and signaling
In multiple PCa cell lines as well as primary prostatic epithelial cells established from prostatectomy samples, calcitriol decreases the mRNA and protein levels of cyclooxygenase-2 (COX-2), the enzyme responsible for PG synthesis, and increases the expression of 15-hyroxyprostaglandin dehydrogenase(15-PGDH), the enzyme that initiates PG catabolism. As a result calcitriol decreases the levels of biologically active PGs in these cells. In addition, calcitriol decreases the expression of EP and FP PG receptors and thereby attenuates PG-mediated functional responses including stimulation of cell growth. Thus, reduction in the levels of biologically active PGs and inhibition of PG signaling through their receptors by calcitriol results in suppression of the proliferative and angiogenic stimuli provided by PGs. Combinations of calcitriol with NSAIDs cause a synergistic enhancement of the inhibition of PCa cell proliferation, suggesting that this drug combination might have clinical usefulness in PCa therapy.
Induction of mitogen-activated protein kinase phosphatase-5 (MKP5) and inhibition of stress-activated kinase signaling
cDNA microarray analysis in normal human prostate epithelial cells revealed a novel calcitriol-responsive gene, MKP5, also known as DUSP10, 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 the stress-activated protein kinase JNK, leading to their inactivation. Calcitriol increases MKP5 transcription by the activation of VDR and its binding to a vitamin D response element (VDRE) in the MKP5 promoter. The calcitriol-mediated increase in MKP5 causes the dephosphorylation and inactivation of the p38 stress-induced kinase, resulting in a decrease in the production of proinflammatory cytokines that sustain and amplify the inflammatory response, such as interleukin-6 (IL-6). IL-6 stimulates PCa growth and progression and IL-6 synthesized in periprostatic adipose tissue has recently been shown to modulate PCa aggressiveness. Calcitriol up-regulation of MKP5 is seen in primary cells derived from normal prostate epithelium and primary localized adenocarcinoma but not in the established cell lines derived from metastatic PCa, suggesting that the loss of MKP5 might occur during PCa progression, as a result of a selective pressure to eliminate the tumor suppressor activity of MKP5 and/or calcitriol.
Inhibition of NFκB activation and signaling
NFκB comprises a family of inducible transcription factors ubiquitously present in cells that are important regulators of innate immune responses and inflammation. In the basal state, most NFκB dimers are bound to specific inhibitory proteins called IκB and proinflammatory signals activate NFκB mainly through IκB kinase (IKK)-dependent phosphorylation and degradation of the inhibitory IκB proteins. Free NFκB then translocates to the nucleus and activates the transcription of proinflammatory cytokines, chemokines, and antiapoptotic factors. In contrast to normal cells, many malignant cells have increased levels of active NFκB. Calcitriol directly modulates basal and cytokine-induced NFκB activity in many cells including human lymphocytes, fibroblasts, and peripheral blood monocytes. A reduction in the levels of the NFκB inhibitory protein IκB has been reported in mice lacking the VDR. Addition of a VDR antagonist to colon cancer cells up-regulates NFκB activity by decreasing the levels of IκB, suggesting that VDR ligands suppress NFκB activation. Calcitriol decreases the production of the angiogenic and proinflammatory cytokine IL-8 in immortalized normal human prostate epithelial cell lines and established PCa cell lines by inhibiting the nuclear translocation of the NFκB subunit p65 and subsequent transcriptional stimulation of the NFκB downstream target IL-8. Thus calcitriol could delay the progression of PCa by suppressing the expression of angiogenic and proinflammatory factors such as VEGF and IL-8. In addition, calcitriol indirectly inhibits NFκB signaling by up-regulating the expression of IGFBP-3, which has been shown to interfere with NFκB signaling in PCa cells. NFκB also provides an adaptive response to PCa cells against cytotoxicity induced by redox active therapeutic agents and is implicated in radiation resistance of cancers. Calcitriol significantly enhances the sensitivity of PCa cells to ionizing radiation by selectively suppressing radiation-mediated RelB activation. Thus calcitriol may serve as an effective agent for sensitizing PCa cells to radiation therapy via suppression of the NFκB pathway. There is also considerable evidence that calcitriol potentiates the antitumor activity of a wide variety of cytotoxic chemotherapy agents (described later). As noted earlier, the induction of p73 by calcitriol seems to contribute to the synergistic activity of calcitriol and platinum analogues and some antimetabolites.
The Role of Antiinflammatory Effects of Calcitriol in Cancer Prevention and Treatment
As discussed earlier current perspectives in cancer biology suggest that inflammation plays a role in the development of cancer. De Marzo and colleagues have proposed that precursor lesions called proliferative inflammatory atrophy (PIA) in the prostate, which are associated with acute or chronic inflammation, are the precursors of prostate intraepithelial neoplasia (PIN) and PCa. The epithelial cells in PIA lesions exhibit many molecular signs of stress including increased expression of COX-2. Similarly, inflammatory bowel disease is associated with the development of CRC. Based on the evidence demonstrating antiinflammatory effects of calcitriol, we postulate that calcitriol may play a role in delaying or preventing cancer development and/or progression. PCa generally progresses very slowly, likely over decades, before symptoms become obvious and the diagnosis is made. 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 of PCa. The inhibitory effects of calcitriol on COX-2 expression and the PG pathway, production of proinflammatory cytokines, NFκB signaling, and tumor angiogenesis suggest that calcitriol has the potential to be useful as a chemopreventive agent in malignancies such as PCa. Foster and colleagues have shown that administration of high-dose calcitriol (20 μg/kg), intermittently 3 days per week for up to 14 to 30 weeks, suppresses prostate tumor development in TRAMP mice. The efficacy of calcitriol as a chemopreventive agent has also been examined in Nkx3.1;Pten mutant mice, which recapitulate stages of prostate carcinogenesis from PIN lesions to adenocarcinoma. The data reveal 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 as well as in vitro observations suggest that clinical trials in PCa patients with PIN or early disease evaluating calcitriol and its analogues as agents that prevent and/or delay progression, are warranted.
Inhibition of Angiogenesis
Angiogenesis is the process of formation of new blood vessels from existing vasculature and is a crucial step in the continued growth, progression, and metastasis of tumors. VEGF is the most potent stimulator of angiogenesis. PGs are also important proangiogenic factors. The initiation of angiogenesis is controlled by local hypoxia, which induces the synthesis of proangiogenic factors that activate signaling pathways leading to the structural reorganization of endothelial cells favoring new capillary formation. Stimulation of angiogenesis in response to hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1), which directly increases the expression of several proangiogenic factors including VEGF. Early studies indicate that calcitriol is a potent inhibitor of tumor cell–induced angiogenesis in experimental models. Calcitriol inhibits VEGF-induced endothelial cell tube formation in vitro and decreases tumor vascularization in vivo in mice bearing xenografts of BCa cells over-expressing VEGF. Calcitriol and its analogues also directly inhibit the proliferation of endothelial cells leading to the inhibition of angiogenesis.
At the molecular level, calcitriol may exert its antiangiogenic effects through a direct antiproliferative action on endothelial cells in the tumor microenvironment and/or by regulating the expression of key factors that control angiogenesis. Calcitriol reduces VEGF expression in PCa cells through transcriptional repression of HIF-1. Calcitriol also suppresses the expression of the proangiogenic factor IL-8 in an NFκB-dependent manner. Chung and colleagues established TRAMP-2 tumors in wild-type mice and VDR knockout mice and found enlarged vessels and increased vessel volume in TRAMP tumors in the VDR knockout mice, suggesting an inhibitory role for VDR and calcitriol in tumor angiogenesis. Their study further showed increased expression of proangiogenic factors such as HIF-1α, VEGF, angiopoietin-1 and platelet-derived growth factor (PDGF) in the tumors in the VDR knockout mice. Another important mechanism by which COX-2 promotes tumor progression is through the stimulation of angiogenesis. The proangiogenic effect of COX-2–generated PGE 2 might be a result of its action to increase HIF-1α protein synthesis in cancer cells. Suppression of COX-2 expression by calcitriol therefore provides an important indirect mechanism by which calcitriol inhibits angiogenesis, in addition to its direct suppressive effects on proangiogenic factors such as HIF-1 and VEGF.
Anticancer effects of calcitriol in animal models
Considerable data indicate antitumor effects of vitamin D compounds in vivo models and calcitriol and calcitriol analogues also potentiate the antitumor actions of many more traditional anticancer agents. In model systems of murine SCC and human carcinomas arising in the prostate, lung, ovary, breast, bladder, pancreas, as well as neuroblastoma, calcitriol or calcitriol analogues have substantial anticancer effects. Significant inhibition of metastasis is observed in murine models of prostate and lung cancer treated with calcitriol; these effects may be based, at least in part, on antiangiogenic effects. In tumor-derived endothelial cells (TDECs), calcitriol induces apoptosis and cell cycle arrest; however, these effects are not seen in endothelial cells isolated from normal tissues or from Matrigel. Recently, Chung and colleagues reported that tumor-derived endothelial cells may be more sensitive to calcitriol as a result of novel epigenetic silencing of CYP24A1 . Because Cyp24 (24-hydroxylase) initiates the degradation of calcitriol, inhibition of this enzyme has been shown to enhance calcitriol actions and tumors expressing high levels of the enzyme are resistant to calcitriol action. Direct effects of calcitriol on endothelial cells may play a primary role in the calcitriol-mediated antitumor activity that is observed in animal tumor models. There are no in vitro data to suggest that particular histotypes of cancer are more, or less, responsive to calcitriol-mediated antitumor effects except in the case of cells that have lost their VDR or over-express CYP24.
In Vivo Animal Studies of Calcitriol in Combination Regimens
In vivo analyses in mouse tumor models indicate that calcitriol acts synergistically with a wide range of chemotherapeutic agents. Calcitriol potentiates the anticancer activity of platinum analogues, taxanes, and DNA-intercalating agents. Optimal potentiation is seen when calcitriol is administered before or simultaneously with chemotherapy treatment; administration of calcitriol after the cytotoxic agent does not provide potentiation. In SCC and PC-3 (PCa) xenografts in immunodeficient mice, pretreatment with calcitriol or calcitriol analogues followed by paclitaxel results in enhanced antitumor effects. In vivo studies also indicate that the antitumor effects of calcitriol can be potentiated by agents that inhibit calcitriol metabolism. Azole antagonists of the primary catabolic enzyme (CYP24A1) responsible for vitamin D breakdown enhance the antitumor effects of calcitriol in vitro and in vivo. Ketoconazole is the most readily available of such agents and this drug has significant utility in the treatment of men with prostate cancer in whom disease progression has occurred despite androgen deprivation (so-called androgen-independent or castration-resistant PCa). The activity of ketoconazole in tumor cells (prostate and nonprostate) that are apparently unresponsive to androgens, supports the hypothesis that there are extra-androgenic mechanisms underlying ketoconazole activity. There are more specific inhibitors of CYP24 than azoles and secosteroid vitamin D analogues. These agents have antitumor activity in in vitro and in vivo models and potentiate the antitumor activity of calcitriol.
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