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Calcitriol, the hormonally active form of vitamin D, exerts multiple anti-proliferative and pro-differentiating actions including cell cycle arrest and induction of apoptosis in many malignant cells, and the hormone is currently being evaluated in clinical trials as an anti-cancer agent. Recent research reveals that calcitriol also exhibits multiple anti-inflammatory effects. First, calcitriol inhibits the synthesis and biological actions of pro-inflammatory prostaglandins (PGs) by three mechanisms: i) suppression of the expression of cyclooxygenase-2, the enzyme that synthesizes PGs; ii) up-regulation of the expression of 15-hydroxyprostaglandin dehydrogenase, the enzyme that inactivates PGs; and iii) down-regulation of the expression of PG receptors that are essential for PG signaling. The combination of calcitriol and nonsteroidal anti-inflammatory drugs results in a synergistic inhibition of the growth of prostate cancer (PCa) cells and offers a potential therapeutic strategy for PCa. Second, calcitriol increases the expression of mitogen-activated protein kinase phosphatase 5 in prostate cells resulting in the subsequent inhibition of p38 stress kinase signaling and the attenuation of the production of pro-inflammatory cytokines. Third, calcitriol also exerts anti-inflammatory activity in PCa through the inhibition of nuclear factor-κB signaling that results in potent anti-inflammatory and anti-angiogenic effects. Other important direct effects of calcitriol as well as the consequences of its anti-inflammatory effects include the inhibition of tumor angiogenesis, invasion, and metastasis. We hypothesize that these anti-inflammatory actions, in addition to the other known anti-cancer effects of calcitriol, play an important role in its potential use as a therapeutic agent for PCa. Calcitriol or its analogs may have utility as chemopreventive agents and should be evaluated in clinical trials in PCa patients with early or precancerous disease.
Prostate cancer (PCa) is the most commonly diagnosed malignancy in men after skin cancer and is a leading cause of cancer death in men in the United States ( Jemal et al. 2008). Surgery and radiation are common initial treatment options for localized PCa. If disease recurs, it is often treated with androgen deprivation therapy (ADT). However, many patients eventually fail ADT and develop androgen-independent PCa (AIPC), more recently described as castrate-resistant PCa (CRPC). CRPC ultimately progresses to metastasis and may be lethal. This stage of PCa is not amenable to current therapy. A major goal of current research is the identification of new agents that would prevent and/or delay the development of CRPC or slow its progression.
Calcitriol (1,25-dihydroxyvitamin D3), 1,25(OH)2D3, the biologically most active metabolite of vitamin D, acts as a classical steroid hormone and regulates calcium homeostasis and bone metabolism through actions mediated by the vitamin D receptor (VDR) in intestine, bone, kidney, and the parathyroid glands ( Feldman et al. 2007). However, calcitriol also exerts anti-proliferative and pro-differentiating effects in a number of malignant cells and retards tumor growth in animal models of cancer raising the possibility of its use as an anti-cancer agent. Calcitriol has been shown to inhibit the development and progression of PCa ( Krishnan et al. 2003, Stewart & Weigel 2004, Beer & Myrthue 2006, Gombart et al. 2006, Deeb et al. 2007, Fleet 2008). U.V. light is essential for the synthesis of vitamin D in the skin, and epidemiological studies suggest that mortality rates due to PCa in the US are inversely related to sunlight exposure ( Hanchette & Schwartz 1992, Schwartz & Skinner 2007). Many studies suggest that vitamin D deficiency, characterized by low circulating 25-hydroxyvitamin D levels, is associated with increased PCa risk and progression ( Ahonen et al. 2000, Giovannucci 2005, Tretli et al. 2009, Trump et al. 2009), although all studies do not support such a correlation ( Platz et al. 2004, Ahn et al. 2008).
Inflammation and PCa
Chronic inflammation has been recognized as a risk factor for the development of many cancers ( Allavena et al. 2008, Mantovani et al. 2008). Inflammation can be triggered by a variety of stimuli such as injury, infection, exposure to toxins, autoimmune disease, the development of benign or malignant tumors, or other stresses and pathologies ( Fig. 1). Cancer-related inflammation is characterized by the presence in tumor tissue of inflammatory cells and the overexpression of inflammatory mediators such as cytokines, chemokines, prostaglandins (PGs), and reactive oxygen and nitrogen species ( Coussens & Werb 2002, Balkwill 2004, Lucia & Torkko 2004, Mantovani et al. 2008; Fig. 1). Many of these pro-inflammatory mediators activate angiogenic switches usually under the control of vascular endothelial growth factor (VEGF) and thereby promote tumor angiogenesis, metastasis, and invasion ( Angelo & Kurzrock 2007, Kundu & Surh 2008).
Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to reduce inflammation and prevent PCa development in a rat model of prostate carcinogenesis ( Narayanan et al. 2009). The use of NSAIDs such as aspirin has also been shown to lower serum prostate-specific antigen (PSA) levels in men with latent PCa ( Singer et al. 2008, Fowke et al. 2009). In PCa patients, a strong association between the levels of serum C-reactive protein (CRP), a nonspecific marker of inflammation, and serum PSA has been reported ( Lehrer et al. 2005). Recent studies also show that an elevated plasma CRP level is a strong predictor of poor prognosis in patients with metastatic AIPC ( McArdle et al. 2006, Beer et al. 2008). These observations suggest that inflammation might be a fundamental process in PCa. Several studies support a link between inflammation and the development of PCa ( De Marzo et al. 1999, 2007, Nelson et al. 2003, 2004). De Marzo et al. (2004) postulate that exposure to infectious agents, hormonal alterations, and dietary carcinogens could cause injury to the prostate epithelium leading to inflammation and the formation of lesions referred to as proliferative inflammatory atrophy (PIA), which are the precursors of prostatic intraepithelial neoplasia (PIN). PIN lesions are characterized by abnormalities that are intermediate between normal prostatic epithelium and cancer, while progression to high grade PIN is the most likely precursor of prostate carcinoma ( Montironi et al. 2004). The epithelial cells in PIA lesions have been shown to exhibit many molecular signs of stress including elevated expression of cyclooxygenase-2 (COX-2), the enzyme catalyzing the synthesis of PGs ( De Marzo et al. 1999, Zha et al. 2001, Wang et al. 2007). Thus, mediators of inflammation appear to be involved in triggering the process of prostate carcinogenesis ( Fig. 1).
Molecular mechanisms of calcitriol actions
Many molecular pathways mediate the anti-cancer effects of calcitriol in PCa cells ( Deeb et al. 2007). Calcitriol inhibits the proliferation of PCa cells through cell cycle arrest in the G1/G0 phase ( Krishnan et al. 2003, Yang & Burnstein 2003, Stewart & Weigel 2004) by increasing the expression of the cyclin-dependent kinase (CDK) inhibitors p21Waf/Cip1 and p27Kip1 ( Liu et al. 1996, Blutt et al. 1997, Campbell et al. 1997, Yang & Burnstein 2003), decreasing CDK2 activity ( Yang & Burnstein 2003), and causing the hypophosphorylation of the retinoblastoma protein ( Jensen et al. 2001). Calcitriol-induced G0 arrest appears to be p53-dependent ( Stewart & Weigel 2004). In several PCa cell lines including the androgen-independent C4-2 cells, calcitriol reduces the expression of cellular myleocytomatosis oncogene (c-MYC), a transcription factor known to promote G0/G1 to S-phase transition, by down-regulating c-MYC mRNA levels and decreasing the stability of the c-MYC protein ( Rohan & Weigel 2009). Calcitriol also induces apoptosis in some PCa cells and suppresses the expression of anti-apoptotic genes such as BCL-2 ( Blutt et al. 2000). Calcitriol stimulates differentiation and modulates growth factor actions including up-regulation of the expression of insulin-like growth factor binding protein-3 (IGFBP-3) in PCa cells, which in turn increases the expression of p21 causing cell cycle arrest ( Boyle et al. 2001, Peng et al. 2004). Other mechanisms include the inhibition of invasion and metastasis ( Sung & Feldman 2000, Krishnan et al. 2003, Stewart & Weigel 2004). Recent research, including observations from our laboratory, suggests that calcitriol also exerts anti-inflammatory actions that may contribute to its beneficial effects in several cancers in addition to its other known anti-proliferative actions ( Krishnan et al. 2007a, b). In the following sections, we will discuss the role of the anti-inflammatory actions of calcitriol and its potential chemopreventive and therapeutic utility in PCa.
Anti-inflammatory actions of calcitriol
Studies that employed cDNA microarrays to examine changes in gene expression in cancer cells treated with calcitriol or its analogs identified many novel calcitriol target genes, some of which could be important molecular mediators of its potent anti-inflammatory effects ( Krishnan et al. 2004, Peehl et al. 2004, Kriebitzsch et al. 2009). Calcitriol regulates the expression of several genes involved in PG metabolism and signaling, thereby reducing the levels and biological activity of PGs ( Moreno et al. 2005). PGs are pro-inflammatory molecules that promote tumorigenesis and cancer growth ( Badawi 2000, Hussain et al. 2003, Zha et al. 2004). Calcitriol up-regulates the expression of mitogen-activated protein kinase phosphatase-5 (MKP5; also known as dual specificity phosphatase-10 (DUSP10)) and thereby promotes downstream anti-inflammatory effects including a reduction in the level of expression of pro-inflammatory cytokines ( Nonn et al. 2006). Recent research also indicates that calcitriol interferes with the activation and signaling of nuclear factor-κ B (NFκB), a transcription factor that regulates the expression of numerous genes involved in inflammatory and immune responses and cellular proliferation ( McCarty 2004). NFκB is thought to play a key role in the process leading from inflammation to carcinogenesis ( Maeda & Omata 2008). In addition, calcitriol also exhibits important anti-angiogenic effects as detailed below. In the following sections, we will discuss the importance of these molecular pathways of inflammation in the development and progression of PCa and the therapeutic significance of the inhibition of these of pro-inflammatory signals by calcitriol.
Regulation of PG metabolism and signaling
PGs have been shown to play a role in the development and progression of many cancers, and COX-2, the enzyme that regulates PG synthesis, is an important molecular target in cancer therapy ( Badawi 2000, Hussain et al. 2003, Wang et al. 2005). PGs promote carcinogenesis by multiple mechanisms such as stimulating cellular proliferation, inhibiting apoptosis, promoting angiogenesis, and activating carcinogens ( Dubois et al. 1998, Hawk et al. 2002). Calcitriol has been shown to regulate the expression of several key genes involved in the PG pathway causing a decrease in PG synthesis, an increase in PG catabolism, and the inhibition of PG signaling through their receptors in normal and malignant prostate cells ( Moreno et al. 2005; Fig. 2).
COX/PG endoperoxidase synthase is the rate-limiting enzyme that catalyzes the conversion of arachidonic acid to PGs and related eicosanoids. COX exists as two isoforms, COX-1, which is constitutively expressed in many tissues and cell types, and COX-2, which is inducible by a variety of stimuli. COX-2 is regarded as an immediate-early response gene whose expression is rapidly induced by mitogens, cytokines, tumor promoters, and growth factors ( Hussain et al. 2003). Evidence suggests that single nucleotide polymorphisms in the COX-2 gene may affect PCa risk ( Cheng et al. 2007). Genetic and clinical studies indicate that increased COX-2 expression is one of the key steps in carcinogenesis ( Markowitz 2007). Several studies suggest a causative and/or stimulatory role for COX-2 in prostate tumorigenesis and demonstrate its overexpression in prostate adenocarcinoma ( Gupta et al. 2000, Yoshimura et al. 2000). However, not all PCa are associated with elevated COX-2 expression ( Zha et al. 2001, Wagner et al. 2005). Zha et al. (2001) did not find consistent overexpression of COX-2 in established PCa. However, they detected appreciable COX-2 expression in areas of PIA, the precursor lesions that have been implicated in prostate carcinogenesis. Silencing of COX-2 in metastatic PCa cells arrests cell growth and causes morphological changes associated with enhanced differentiation highlighting the role of COX-2 in prostate carcinogenesis ( Narayanan et al. 2006). The overexpression of COX-2 in PIA lesions also appears to be associated with elevated expression of CCAAT/enhancer-binding protein β, an important transcription factor involved in cellular proliferation and differentiation ( Wang et al. 2007). COX-2 protein expression in prostate biopsy cores and PCa surgical specimens is inversely correlated with disease-free survival ( Rubio et al. 2005). A study analyzing archival radical prostatectomy specimens concluded that COX-2 expression was an independent predictor of PCa recurrence ( Cohen et al. 2006).
Local production of PGs at the tumor sites by infiltrating inflammatory cells also increases the risk of carcinogenesis and/or cancer progression ( Zha et al. 2001, 2004, Nelson et al. 2004, Wang et al. 2005, De Marzo et al. 2007). In colon cancer, COX-2 expression is found in the carcinoma cells as well as infiltrating macrophages within the tumors ( Bamba et al. 1999, Chapple et al. 2000). In other cancers, COX-2 expression has been demonstrated in vascular endothelial cells, fibroblasts, and smooth muscle cells around the cancer ( Goluboff et al. 1999, Mifflin et al. 2002). Immunohistochemical analyses of COX-2 expression in prostate epithelium in tissue specimens from patients with PCa and benign prostatic hyperplasia (BPH) revealed high T-lymphocyte and macrophage densities in COX-2-positive areas raising the likelihood that pro-inflammatory cytokines released by these cells up-regulate COX-2 expression in the adjacent tumor cells ( Wang et al. 2004, 2005). PGs generated by COX-2 act in an autocrine and paracrine manner to stimulate cell growth. 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 ( Chen & Hughes-Fulford 2000). Studies in experimental models of cancer have shown that COX-2 enhances tumor development and progression by promoting resistance to apoptosis and stimulating angiogenesis and tumor invasion, and it is therefore regarded as an oncogene ( Zha et al. 2004, Kundu & Surh 2008).
15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is the enzyme that catalyzes the conversion of PGs to their corresponding 15-keto derivatives, which exhibit greatly reduced biological activity. Therefore, 15-PGDH can be regarded as a physiological antagonist of COX-2. 15-PGDH has been described as an oncogene antagonist and plays a tumor-suppressive role in colon cancer ( Yan et al. 2004, Myung et al. 2006). 15-PGDH is universally expressed in normal colon but is routinely absent or severely reduced in cancer specimens. Importantly, the stable transfection of a 15-PGDH expression vector into colon cancer cells greatly reduces the ability of the cells to form tumors and/or slows tumor growth in nude mice demonstrating that 15-PGDH functions as a tumor suppressor ( Yan et al. 2004). 15-PGDH also acts as a tumor suppressor in breast cancer ( Wolf et al. 2006).
Prostaglandin E (PGE) and prostaglandin F (PGF) are the major PGs stimulating the proliferation of PCa cells and they act by binding to G-protein-coupled membrane receptors (prostanoid receptors). There are eight members in the prostanoid receptor subfamily, which are distinguished by their ligand-binding profile and the signal transduction pathways that they activate, accounting for some of the diverse and often opposing effects of PGs ( Breyer et al. 2001). PGE acts through four different receptor sub-types (EP1–EP4), while PGF acts through the FP receptor. PCa cells express both EP and FP receptors ( Chen & Hughes-Fulford 2000, Moreno et al. 2005). PG receptors are also expressed in most endothelial cells, macrophages, and stromal cells found in the tumor microenvironment. PG interaction with its receptors can send a positive feedback signal to increase COX-2 mRNA levels ( Tjandrawinata & Hughes-Fulford 1997, Tjandrawinata et al. 1997, Chen & Hughes-Fulford 2000). Therefore, irrespective of the initial trigger of COX-2 expression, PGs could mediate a wave of COX-2 expression at the tumor sites not only in the cancer cells themselves, but also in the surrounding stromal cells and infiltrating macrophages as well as endothelial cells thereby promoting tumor progression.
Calcitriol effects on the PG pathway in prostate cells
As depicted in Fig. 2, calcitriol regulates the expression of several PG pathway genes in multiple PCa cell lines as well as cultured primary prostate epithelial cells established from surgically removed prostate tissue from PCa patients ( Moreno et al. 2005). The levels of COX-2 mRNA and protein are significantly decreased by calcitriol treatment of these cells ( Moreno et al. 2005). Calcitriol increases 15-PGDH mRNA and protein expression in various PCa cells ( Moreno et al. 2005). Calcitriol has also been shown to regulate COX-2 and 15-PGDH expression in other cells such as rat macrophages and human neonatal monocytes ( Pichaud et al. 1997, Aparna et al. 2008). By inhibiting COX-2 and stimulating 15-PGDH expression in PCa cells, calcitriol decreases the levels of biologically active PGs, thereby reducing the growth stimulation due to PGs. Further calcitriol also decreases the expression of EP and FP PG receptor mRNA in PCa cells ( Moreno et al. 2005). The calcitriol-induced decrease in PG receptor expression results in the attenuation of PG-mediated functional responses even when exogenous PGs are added to the cell cultures. Calcitriol suppresses 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 ( Moreno et al. 2005). The down-regulation of PG receptors by calcitriol would inhibit the positive feedback exerted by PGs on COX-2, thereby limiting the wave of COX-2 expression at the tumor sites and slowing the rate of tumor progression. Thus, calcitriol inhibits the PG pathway in PCa cells by three separate mechanisms: decreasing COX-2 expression, increasing 15-PGDH expression, and reducing PG receptor levels ( Fig. 2). We postulate that these actions contribute to the suppression of the proliferative and angiogenic stimuli provided by PGs in PCa. The regulation of PG metabolism and biological actions constitutes an important anti-inflammatory pathway of calcitriol action.
Calcitriol analogs are being developed to find a therapeutic substitute that would have increased anti-cancer efficacy and decreased side effects of hypercalcemia ( Masuda & Jones 2006). A recent study reports that the calcitriol analog elocalcitol also exhibits anti-inflammatory effects on human BPH cells ( Penna et al. 2009). The analog causes significant decreases in COX-2 mRNA levels and PGE2 synthesis resulting in the suppression of cytokine-stimulated production of the pro-inflammatory chemokine interleukin-8 (IL-8), which is involved in BPH pathogenesis ( Penna et al. 2009). Another study shows that a different calcitriol analog 1α,25-dihydroxy-16-ene-23-yne-vitamin D3 exhibits anti-inflammatory effects causing a reduction in the expression of inflammatory markers such as COX-2, inducible nitric oxide synthase, and IL-2 in experimental models of inflammation ( Aparna et al. 2008). Interestingly, this study demonstrates that calcitriol and 1α, 25-dihydroxy-16-ene-23-yne-vitamin D3 also directly inhibit COX-2 enzymatic activity ( Aparna et al. 2008).
Combination of calcitriol and NSAIDs as a therapeutic approach in PCa
NSAIDs are a class of drugs that decrease PG synthesis by inhibiting COX-1 and COX-2 enzymatic activities. Several NSAIDs nonselectively inhibit both the constitutively expressed COX-1 and the inducible COX-2, while others have been shown to be more selective in preferentially inhibiting COX-2 enzymatic activity. Based on our studies in PCa cells ( Moreno et al. 2005), we hypothesized that the action of calcitriol at the genomic level to reduce COX-2 expression, leading to decreased COX-2 protein levels, would allow the use of lower concentrations of NSAIDs to inhibit COX-2 enzyme activity ( Fig. 2). Further, an increase in the expression of 15-PGDH and a decrease in PG receptor levels due to calcitriol actions would lower the concentrations and biological activity of PGs thereby enhancing the NSAID effect ( Fig. 2). Therefore, we hypothesized that the combination of calcitriol and NSAIDs would exhibit an additive/synergistic activity to inhibit PCa cell growth. Combinations of calcitriol with the COX-2-selective NSAIDs, NS398 and SC-58125, and the nonselective NSAIDs, naproxen and ibuprofen, caused a synergistic enhancement of the inhibition of PCa cell proliferation, compared to the individual agents ( Moreno et al. 2005). These results led us to further hypothesize that the combination of calcitriol and NSAIDs may have clinical utility in PCa therapy ( Moreno et al. 2005). As discussed below (see the section on Clinical studies), we have conducted a small clinical trial examining the effects of a combination of high-dose calcitriol and the nonselective NSAID naproxen in PCa patients with far advanced disease, and the results suggest a beneficial effect of the combination to slow the course of disease progression.
Induction of MKP5 and inhibition of stress-activated kinase signaling
Data from cDNA microarray studies in normal human prostate epithelial cells ( Peehl et al. 2004) revealed another novel calcitriol-responsive gene, MKP5, also known as DUSP10. 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 the stress-activated protein kinase Jun-N-terminal kinase (JNK), leading to their inactivation ( Fig. 3). Calcitriol up-regulates MKP5 expression leading to downstream anti-inflammatory effects in cells derived from normal prostatic epithelium and primary, localized adenocarcinoma, supporting a role for calcitriol in the prevention and/or early treatment of PCa ( Nonn et al. 2006). In primary cultures of normal prostatic epithelial cells from the peripheral zone, calcitriol increases MKP5 transcription. A putative positive vitamin D response element in the MKP5 promoter mediates the stimulation of MKP5 transcription by calcitriol. Interestingly, calcitriol up-regulation of MKP5 is seen in primary cells derived from normal prostatic epithelium and primary, localized adenocarcinoma, but not in the established PCa cell lines derived from PCa metastasis. Calcitriol inhibits 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 ( Nonn et al. 2006). 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 ( Park et al. 2003). IL-6 is a p38-regulated pro-inflammatory cytokine implicated in PCa progression ( Culig et al. 2005). Stimulation of primary prostate cells with the pro-inflammatory molecule tumor necrosis factor α (TNFα) increases IL-6 mRNA stability and concentrations of IL-6 in the conditioned media. Pre-treatment of the cells with calcitriol significantly attenuates the increase in IL-6 production following TNFα treatment, adding inhibition of IL-6 production to the spectrum of calcitriol-mediated actions ( Nonn et al. 2006).
IL-6 is a major pro-inflammatory cytokine that participates in inflammation-associated carcinogenesis ( Rose-John & Schooltink 2007) and has been implicated in the pathogenesis of several cancers ( Schneider et al. 2000, Culig et al. 2005, Kai et al. 2005). Serum IL-6 levels are significantly elevated in PCa patients and in addition a positive correlation between IL-6 levels and the number of bone metastases is also seen ( Tumminello et al. 2009). IL-6 is a growth and survival factor in human PCa cells with aggressive phenotypes and has been implicated in the progression of PCa to AIPC ( Culig et al. 2005, Wegiel et al. 2008). Calcitriol reduces the production of the pro-inflammatory cytokine IL-6 by inhibiting p38 activation via MKP5 up-regulation as well as by interfering with the signaling of pleiotropic inflammatory cytokines such as TNFα ( Nonn et al. 2006; Fig. 3). These observations provide evidence for significant anti-inflammatory effects of calcitriol in prostate cells. Interestingly, established PCa cell lines derived from metastasis such as LNCaP, PC-3, and DU145 exhibit low basal levels of MKP5, and calcitriol is unable to induce MKP5 expression. We therefore speculate that a loss of MKP5 expression 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 all cells that are important regulators of innate immune responses and inflammation ( Karin & Lin 2002). In the basal state, most NFκB dimers are bound to specific inhibitory proteins called IκB. Many pro-inflammatory signals activate NFκB mainly through IκB kinase-dependent phosphorylation and degradation of the inhibitory IκB proteins ( Maeda & Omata 2008). Free NFκB then translocates to the nucleus and activates the transcription of pro-inflammatory cytokines, chemokines, and anti-apoptotic factors ( Ghosh & Karin 2002). In contrast to normal cells, many cancer cells have elevated levels of active NFκB ( Sovak et al. 1997, Palayoor et al. 1999), and constitutive activation of NFκB has been observed in AIPC ( Gasparian et al. 2002, Ismail et al. 2004, Suh & Rabson 2004). The NFκB protein RelB is uniquely expressed at high levels in PCa with high Gleason scores ( Lessard et al. 2005). NFκB plays a major role in the control of immune responses and inflammation and promotes malignant behavior by increasing the transcription of the anti-apoptotic gene BCL-2 ( Catz & Johnson 2001), cell cycle progression factors such as c-MYC and cyclin D1, proteolytic enzymes such as matrix metalloproteinase 9 (MMP-9) and urokinase-type plasminogen activator, and angiogenic factors such as VEGF and IL-8 ( Huang et al. 2001, Suh & Rabson 2004). IL-8, an angiogenic factor and a downstream target of NFκB, is also a potent chemotactic factor for neutrophils and is associated with the initiation of the inflammatory response ( Ferrer et al. 1998).
Calcitriol is known to directly modulate basal and cytokine-induced NFκB activity in many cells including human lymphocytes ( Yu et al. 1995), fibroblasts ( Harant et al. 1998), and peripheral blood monocytes ( Stio et al. 2007). A reduction in the levels of the NFκB-inhibitory protein IκBα has been reported in fibroblasts derived from knockout mice lacking the VDR ( Sun et al. 2006). Calcitriol and its analogs block NFκB activation by increasing the expression of IκB in macrophages and peripheral blood mononuclear cells ( Cohen-Lahav et al. 2006, 2007, Stio et al. 2007). VDR appears to play an inhibitory role in the regulation of basal NFκB signaling in colon cancer cells, and the VDR antagonist ZK191732 increases NFκB activity by decreasing IκBα ( Schwab et al. 2007). Calcitriol and its analogs also inhibit NFκB activation in different types of immune cells ( Dong et al. 2003, Giarratana et al. 2004, Penna et al. 2007). There is considerable evidence for the inhibition of NFκB signaling by calcitriol in PCa cells. Calcitriol decreases the levels of the angiogenic and pro-inflammatory cytokine IL-8 in immortalized normal human prostate epithelial cell lines (HPr-1 and RWPE-1) and established PCa cell lines (LNCaP, PC-3, and DU145; Bao et al. 2006a). The suppression of IL-8 by calcitriol appears to be due to the inhibition of NFκB signaling. Calcitriol reduces the nuclear translocation of the NFκB subunit p65 thereby inhibiting the NFκB complex from binding to its DNA response element and consequently suppressing the NFκB stimulation of transcription of downstream targets such as IL-8 ( Bao et al. 2006a). Thus, calcitriol could delay the progression of PCa by suppressing the expression of angiogenic and pro-inflammatory genes such as VEGF and IL-8 ( Fig. 4). The calcitriol analog elocalcitol inhibits the production of IL-8 by human BPH cells by arresting NFκB p65 nuclear translocation ( Penna et al. 2009). 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 ( Kimura et al. 1999, Criswell et al. 2003). Calcitriol significantly enhances the sensitivity of PCa cells to ionizing radiation by selectively suppressing radiation-mediated RelB activation ( Xu et al. 2007). Thus, calcitriol may serve as an effective agent for sensitizing PCa cells to radiation therapy via suppression of the NFκB pathway.
In addition to the direct inhibition, calcitriol also indirectly inhibits NFκB signaling by up-regulating the expression of other proteins that interfere with NFκB activation such as IGFBP-3 and clusterin (CLU) as discussed below. Calcitriol up-regulates the expression of IGFBP-3 ( Boyle et al. 2001, Peng et al. 2004), which has been shown to interfere with NFκB signaling in PCa cells by suppressing p65 NFκB protein levels and the phosphorylation of IκBα ( Jogie-Brahim et al. 2009; Fig. 4). Apart from its ability to inhibit NFκB, IGFBP-3 has multiple anti-PCa actions including inhibition of proliferation, stimulation of apoptosis, and a variety of anti-inflammatory effects ( Boyle et al. 2001, Ali et al. 2003, Peng et al. 2004, 2008, Jogie-Brahim et al. 2009; Fig. 4). NFκB activity is also modulated by apolipoprotein J or CLU. CLU is a protein expressed in virtually all tissues and is regulated in inflammation, apoptosis, and cancer. Its nuclear form (nCLU) is implicated in apoptosis ( Gleave & Chi 2005, Shannan et al. 2006). In human neuroblastoma cells and mouse embryonic fibroblasts, CLU has been shown to strongly inhibit NFκB activity through the stabilization of IκB proteins ( Santilli et al. 2003). Calcitriol and its analogs up-regulate CLU expression with an accompanying apoptotic response in several cells including human BPH cells ( Crescioli et al. 2004) and human breast cancer cells ( Simboli-Campbell et al. 1997).
Inhibition of angiogenesis
Angiogenesis is the process of formation of new blood vessels from existing vasculature and is a crucial step in continued tumor growth, progression, and metastasis ( Folkman 1995). VEGF is the most potent stimulator of angiogenesis. PGs, as discussed below, are also important pro-angiogenic factors. The initiation of angiogenesis is controlled by local hypoxia, which induces the synthesis of pro-angiogenic factors that activate signaling pathways leading to the structural reorganization of endothelial cells favoring new capillary formation ( Sakamoto et al. 2008). Stimulation of angiogenesis in response to hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1), which directly increases the expression of several pro-angiogenic factors including VEGF ( Giaccia et al. 2003, Rankin & Giaccia 2008). Early studies indicate that calcitriol is a potent inhibitor of tumor cell-induced angiogenesis in experimental models ( Majewski et al. 1996). Calcitriol inhibits VEGF-induced endothelial cell tube formation in vitro and decreases tumor vascularization in vivo in mice bearing xenografts of breast cancer cells overexpressing VEGF ( Mantell et al. 2000). Calcitriol and its analogs also directly inhibit the proliferation of endothelial cells ( Bernardi et al. 2002, Furigay & Swamy 2004, Ben-Shoshan et al. 2007, Chung et al. 2009) leading to the inhibition of angiogenesis.
At the molecular level, calcitriol exerts its anti-angiogenic effects by regulating the expression of key factors that control angiogenesis. Calcitriol reduces the expression of VEGF in several malignant cells including PCa cells through transcriptional repression of HIF-1 ( Ben-Shoshan et al. 2007). Furthermore, as discussed above, calcitriol inhibits PCa cell-induced angiogenesis by suppressing the expression of the pro-angiogenic factor IL-8 in an NFκB-dependent manner ( Bao et al. 2006a). Chung et al. (2009) established transgenic adenocarcinoma of the mouse prostate (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. The study further showed increased expression of pro-angiogenic factors such as HIF-1α, VEGF, angiopoietin-1, and platelet-derived growth factor in the tumors in the VDR knockout mice ( Chung et al. 2009).
Interestingly, another important mechanism by which COX-2 promotes tumor progression is through the stimulation of angiogenesis, and COX-2 inhibitors have been used to block angiogenesis and tumor proliferation ( Sahin et al. 2009). The pro-angiogenic effect of COX-2-generated PGE2 might be due to its action to increase HIF-1α protein synthesis in cancer cells ( Fukuda et al. 2003). In PCa cells, PGE2 mediates hypoxic effects on VEGF by promoting the translocation of HIF-1α protein to the nucleus ( Liu et al. 2002). The selective COX-2 inhibitor NS398 has been shown to suppresses the growth of PC-3 xenografts in vivo by a combination of the direct induction of tumor cell apoptosis and the down-regulation of tumor VEGF, leading to the inhibition of angiogenesis as demonstrated by decreased microvessel density of the tumors ( Liu et al. 2000). An immunohistochemical analysis of human PCa specimens by Wang et al. (2005) reveals that increased COX-2 immunostaining is associated with an increased infiltration of T-lymphocytes and macrophages and increased CD31-marked microvessel density, indicating a positive correlation between COX-2 expression and inflammation and angiogenesis. The authors postulate that pro-inflammatory cytokines released by tumor-adjacent inflammatory cells such as T-lymphocytes and macrophages may induce COX-2 in the epithelial cells in prostate atrophic lesions promoting tumor progression ( Wang et al. 2004). 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 pro-angiogenic factors such as HIF-1 and VEGF. It has been suggested that VEGF induction of p38 and JNK pathways is necessary for COX-2 expression in endothelial cells ( Wu et al. 2006). As discussed above, calcitriol inactivates the p38 pathway by inducing MKP5 expression. Thus, MKP5 induction and VEGF suppression by calcitriol could further contribute to its anti-angiogenic effects through p38 inactivation.
MMPs promote angiogenesis by mediating the degradation of the basement membrane of the vascular epithelium and the extracellular matrix, thereby creating a passageway in these barriers for the formation of new capillaries ( Sakamoto et al. 2008). In human PCa cells, calcitriol decreases the expression and activity of MMP-9 while increasing the activity of its counterpart tissue inhibitor of metalloproteinase-1 (TIMP-1), thereby decreasing the invasive potential of these cells ( Bao et al. 2006b).
Inhibition of invasion and metastasis
Calcitriol reduces the invasive and metastatic potential of many malignant cells including PCa cells. The mechanisms underlying this effect include the inhibition of angiogenesis (discussed above) and regulation of the expression of key molecules involved in invasion and metastasis ( Osborne & Hutchinson 2002). Calcitriol decreases the tumor size and lung metastasis of the highly metastatic Mat-ly lu and R 3327-AT-2 Dunning PCa cells in vivo ( Getzenberg et al. 1997). Calcitriol inhibits the invasiveness and migration potential of metastatic PCa cells such as DU145 and PC-3, due in part to decreasing the expression of α6 and β4 integrins ( Sung & Feldman 2000). In PCa cells, calcitriol and its analogs also increase the expression of E-cadherin, a tumor suppressor gene whose expression is inversely correlated to metastatic potential ( Campbell et al. 1997). Calcitriol-mediated suppression of MMP-9 activity and increase in TIMP-1 also decrease the invasive potential of PCa cells ( Bao et al. 2006b).
The role of anti-inflammatory effects of calcitriol in PCa prevention and treatment
PCa generally progresses very slowly, likely over decades, before symptoms become clinically detectable and a diagnosis is made ( Fang et al. 2001, Whittemore et al. 2005, Loeb et al. 2006, Steyerberg et al. 2007). Recently, inflammation in the prostate has been proposed to be an etiological factor in the development of PCa ( Nelson et al. 2004, De Marzo et al. 2007). The observed latency in PCa provides a long window of opportunity for intervention by chemopreventive agents. Treatment with COX-2-selective NSAIDs such as celecoxib has been shown to suppress prostate carcinogenesis in the TRAMP model ( Gupta et al. 2004). Our studies on the inhibitory effects of calcitriol on COX-2 expression and the PG pathway and MKP5 induction with the resultant stress kinase inactivation and inhibition of pro-inflammatory cytokine production as well as other multiple published observations of calcitriol actions to inhibit NFκB signaling and tumor angiogenesis suggest that calcitriol exhibits significant anti-inflammatory and anti-angiogenic effects in vitro and in vivo ( Fig. 4). Therefore, calcitriol has the potential to be useful as a chemopreventive agent in PCa ( Chen & Holick 2003, Banach-Petrosky et al. 2006, Krishnan et al. 2007a, b). Foster et al. have demonstrated that administration of high-dose calcitriol (20 μg/kg), intermittently 3 days/week for up to 14–30 weeks, suppresses prostate tumor development in TRAMP mice ( Alagbala et al. 2005, 2006). The efficacy of calcitriol as a chemopreventive agent has been examined in Nkx3.1; Pten mutant mice, which recapitulate stages of prostate carcinogenesis from PIN lesions to adenocarcinoma ( Banach-Petrosky et al. 2006). The data reveal that calcitriol significantly reduces the progression of PIN from low-grade to high-grade lesions. Calcitriol is more effective when administered before, rather than subsequent to, 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 analogs as agents that prevent and/or delay PCa progression are warranted.
Clinical studies of calcitriol in PCa
Several investigators have conducted clinical trials evaluating the use of calcitriol in PCa patients. In an early clinical study in PCa patients who had undergone previous radiation or surgical treatment, a statistically significant decrease in the rate of rise of serum PSA levels was seen following the administration of modest daily doses of calcitriol ( Gross et al. 1998), suggesting a beneficial effect of calcitriol in slowing the progression of the disease. Calcitriol is an FDA approved drug (for other indications). The side effects associated with calcitriol use are the development of hypercalcemia and renal stones and possibly increased soft tissue and vascular calcification. Some investigations have followed the approach of administering very high doses of calcitriol intermittently, such as three times a week ( Trump et al. 2006) or once weekly ( Beer et al. 2003), when it apparently can still elicit anti-proliferative effects but cause only transient hypercalcemia and limited toxicity ( Beer et al. 2003, Trump et al. 2006). The use of nutrient vitamin D (cholecalciferol), the biochemical precursor of calcitriol, has also been evaluated in a pilot clinical study, revealing a beneficial effect of prolongation of PSA doubling time ( Woo et al. 2005).
Many academic investigators and pharmaceutical companies have undertaken intense research to develop structural analogs of calcitriol, which exhibit increased anti-proliferative activity and reduced tendency to cause hypercalcemia ( Ma et al. 2006, Masuda & Jones 2006). A phase II study in PCa patients with advanced hormone-refractory PCa evaluated the calcitriol analog 1α-OH-D2 that had exhibited a reduced calcemic effect but an equal or greater anti-proliferative effect in comparison to calcitriol in preclinical studies ( Liu et al. 2003). PSA response was not considered a surrogate marker in this study. Although no objective responses were seen, 30% of the patients experienced stable disease for >6 months, suggesting possible cytostatic activity ( Liu et al. 2003). In a phase I/II study of the calcitriol analog 19-nor-1α-25(OH)2D2 (paricalcitol) in patients with AIPC, no PSA or objective responses were seen ( Schwartz et al. 2005). However, the data suggested that paricalcitol might be beneficial in the reduction of skeletal morbidity ( Schwartz et al. 2005). Administration of the VDR agonist elocalcitol (BXL-628) has been shown to arrest prostate growth in patients with BPH ( Maggi et al. 2006).
Calcitriol and its analogs have also been tested in PCa patients and in other cancers in combination with various therapeutic agents. Preclinical ( Torrance et al. 2000) and clinical studies ( Meyskens et al. 2008) on colon and other cancers have successfully used the strategy of combining low doses of two active drugs to achieve a more effective chemoprevention and therapeutic outcome compared to the individual agents ( Sporn & Hong 2008). The combination approach would also minimize the toxicities of the individual drugs by allowing them to be used at lower doses while achieving a significant therapeutic effect. Calcitriol is also being used in combination therapy with other agents that may enhance its anti-proliferative activity while reducing its hypercalcemic tendency ( Trump et al. 2006, Beer et al. 2007). Trump et al. (2006) carried out a phase II trial in patients with AIPC using high-dose oral calcitriol (12 μg/day 3 times/week) with dexamethasone (4 mg/day 4 times/week), and the results showed a 50% reduction in PSA in 28% of the patients and no hypercalcemia. Another phase II trial tested the combination of calcitriol, dexamethasone, and carboplatin in patients with hormone-refractory PCa and found a PSA response in 13 out of 34 patients ( Flaig et al. 2006).
Studies suggest that the addition of calcitriol to chemotherapy drugs might be beneficial in the management of advanced, hormone-refractory PCa ( Berry & Eisenberger 2005, Kantoff 2005). The ASCENT I clinical trial in advanced CRPC patients who had failed other therapies studied the administration of a very high dose (45 μg) of calcitriol (DN101, Novacea, South San Francisco, CA, USA), administered once weekly along with the chemotherapy drug docetaxel (Taxotere, sanofi-aventis, Bridgewater, NJ, USA) at the usual regimen in use at the time of that trial (once weekly) compared to docetaxel alone. The results of the trial appeared to indicate that the combination caused a very significant improvement in overall survival and time to progression, suggesting that calcitriol could enhance the efficacy of active drugs in cancer treatment ( Beer et al. 2007, 2008). The ASCENT I trial did not meet its primary endpoint, i.e. a lowering of serum PSA. However, based on the promising survival results (16.4 months in the docetaxel arm versus 24.5 months in the docetaxel plus calcitriol arm), a larger phase III trial (ASCENT II) with survival as an endpoint was initiated. A new, improved docetaxel regimen (every 3 week dosing) was used in the control arm of the ASCENT II trial, which was compared to DN101 plus the older docetaxel dose regimen (once a week), resulting in an asymmetric study design. Unfortunately, the improved survival due to the combination demonstrated in the ASCENT I trial could not be confirmed in the ASCENT II trial (Press Release, June 04, 2008, http://novacea.com/). In fact, the trial was prematurely stopped by the data safety monitoring committee after 900 patients were enrolled, when an excess number of deaths were noted in the study arm (DN101 plus old docetaxel regimen) versus the control arm (new docetaxel regimen). This negative result eventually led to the company Novacea ending its DN-101 PCa program.
Another recent study has tested the combination of high-dose calcitriol (DN-101) with mitoxantrone and prednisone in patients with metastatic AIPC without previous chemotherapy, and has concluded that calcitriol did not significantly add to the activity of mitoxantrone and prednisone as assessed by the decline in serum PSA levels ( Chan et al. 2008). In a randomized, double-blinded phase II study in a similar patient population, the addition of daily doses of doxercalciferol (1α-hydroxyvitamin D2) to weekly docetaxel did not enhance the PSA response rate or survival ( Attia et al. 2008). Interestingly, another study testing the effect of the combination of weekly high-dose calcitriol and docetaxel in PCa patients whose disease progressed after first-line chemotherapy using docetaxel alone showed that high-dose calcitriol restored the sensitivity to chemotherapy with docetaxel ( Petrioli et al. 2007).
Preclinical observations in prostate cell cultures ( Moreno et al. 2005) suggested that a combination of calcitriol with a NSAID would be a beneficial approach in PCa therapy. The combination strategy would allow the use of lower concentrations of NSAIDs thereby minimizing their undesirable side effects. It has become clear recently that the long-term use of COX-2-selective inhibitors such as rofecoxib (Vioxx) causes an increase in cardiovascular complications in patients ( Ray et al. 2002, Antman et al. 2005, Graham et al. 2005, Solomon et al. 2006). Very recently, even the use of nonselective NSAIDs has been shown to increase cardiovascular risk in patients with heart disease ( Gislason et al. 2009). However, in comparison to COX-2-selective inhibitors, nonselective NSAIDs such as naproxen may be associated with fewer cardiovascular adverse effects ( Bombardier et al. 2000, Gislason et al. 2009). Preclinical data showed that the combinations of calcitriol with nonselective or selective NSAIDs were equally effective in inducing synergistic growth inhibition ( Moreno et al. 2005). Based on these data, we have recently carried out a single-arm, open-label phase II study evaluating the combination of the nonselective NSAID naproxen and calcitriol in patients with early recurrent PCa ( Srinivas & Feldman 2009). Patients in this study had no evidence of metastases. All of the patients received 45 μg of calcitriol (DN-101) orally once a week and 375 mg naproxen twice a day for 1 year. The trial was prematurely stopped after 21 patients had been enrolled when the FDA put a temporary hold on DN-101 based on the preliminary data from the ASCENT II trial described above. The therapy was well tolerated by most patients in the trial. Serum PSA levels were monitored every 2 months. Bone scans were done every 3 months along with ultrasound of the kidney to assess the possibility of asymptomatic renal stones. One patient developed a small asymptomatic renal stone and was removed from the study. He required no intervention for his renal stone. Only four patients showed evidence of progression and they were removed from the study and offered other therapy. Changes in PSA doubling time (PSA-DT) post intervention were compared to baseline PSA-DT values. A prolongation of the PSA-DT was achieved in 75% of the patients suggesting a potential beneficial effect of the combination therapy ( Krishnan et al. 2009, Srinivas & Feldman 2009).
The use of calcitriol or various vitamin D analogs in trials in men with PCa has thus far been disappointing. Based on preclinical data from cell culture and animal models showing substantial benefits, the modest efficacy thus far achieved in patients is underwhelming. The most promising study, ASCENT I, using very high doses of calcitriol intermittently (to avoid complicating side-effects) in combination with docetaxel had very encouraging findings since the data showed improved survival and not just changes in the surrogate marker PSA ( Beer et al. 2007). However, the expanded follow-up trial, ASCENT II, not only failed to show any benefit but put a shadow over the use of calcitriol for PCa therapy when it was stopped prematurely because of increased deaths in the calcitriol arm. However, the trial was poorly designed with asymmetric placebo and treatment arms. Since the halting of the trial, further analysis (Press Release, September 11, 2008, http://novacea.com/) suggested that the increased deaths in the treatment arm compared to the control arm were not due to calcitriol toxicity, but due to better survival in the control arm that had received the new and improved docetaxel regimen compared to the older docetaxel regimen. Although both ASCENT trials seemed to indicate little problem with toxicity using very high but intermittent doses, the benefits of calcitriol were decidedly unimpressive or nonexistent in the larger ASCENT II trial. Thus, data from clinical studies using calcitriol are not as hopeful as that predicted by epidemiological and preclinical studies. One obvious and major difference in the approaches is that many of the clinical studies including the ASCENT trials were carried out in men with far advanced PCa or in men who had failed all other therapies. This suggests that calcitriol may be better used in early disease and/or in chemoprevention.
However, when used in early disease, an efficacy trial would require very large numbers of subjects and long-term studies to demonstrate improved survival or prolonged time to progression. This has led many trials examining patients with early disease to rely on changes in PSA levels as a surrogate biomarker to demonstrate benefit. There is much discussion in the urology field about whether declines in absolute PSA levels, the rate of PSA rise, PSA velocity, or PSA doubling time are valid indicators of efficacy, and criteria have been established to utilize PSA change as a biomarker of efficacy ( Petrylak et al. 2006, Scher et al. 2008). However, in clinical trials in men with early recurrent disease, whose prostates have been surgically removed or irradiated, changes in PSA levels remain a useful although not perfect tumor biomarker. Some studies do show a PSA benefit due to calcitriol by various criteria, at least in some men. Based on the strong rationale from preclinical data and epidemiological studies described in this review on control of inflammation and the many other actions of calcitriol that predict efficacy, it is our opinion that it would be premature to conclude from the limited number of clinical trials completed thus far that calcitriol has little or no efficacy in men with PCa. We need to determine the optimum regimen of calcitriol or its analogs to be used and the time during the course of PCa that is best to intervene. Since calcitriol has mild anti-proliferative and differentiating actions that are more cytostatic than cytolytic, we believe therapy would be most effectively used in chemoprevention or early disease, at the highest dose tolerated without side effects and probably in combination with other drugs.
The epidemiological studies indicating increased risk of cancer in vitamin D deficiency ( Giovannucci 2005, Garland et al. 2006) and the high frequency of vitamin D deficiency in the population ( Tangpricha et al. 2002) indicate that at a minimum, vitamin D status should be assessed and deficiency should be vigorously treated with vitamin D supplements in men at high risk for PCa or those that have already been diagnosed with cancer. The available data suggest that the likelihood of vitamin D or calcitriol having a beneficial effect in the prevention of PCa development or in treatment of early disease is better than its effects on advanced disease. Therefore, the detection and correction of vitamin D deficiency in the population at large is worthwhile for this and many other reasons. In the future, improved vitamin D analogs with increased efficacy and decreased tendency to cause hypercalcemia may be the best therapeutic option ( Ma et al. 2006, Masuda & Jones 2006). However, in the interim until those drugs are developed and become available, treatment with high doses of dietary vitamin D seems to be a safe and worthwhile approach. The prostate expresses 25-hydroxyvitamin D3-1α-hydroxylase, the enzyme that can convert circulating 25-hydroxyvitamin D to calcitriol ( Schwartz et al. 1998, Hsu et al. 2001, Chen et al. 2003). Thus, dietary vitamin D supplements can lead to local calcitriol production within the prostate where it acts as a paracrine hormone to inhibit prostate growth and perhaps to delay or prevent the progression to cancer ( Schwartz et al. 1998, Chen et al. 2003). This seems a worthwhile strategy for a safe and effective approach until new, more potent drugs become available.
Recent research has identified several new calcitriol target genes revealing multiple molecular pathways of calcitriol action in prostate cells ( Figs 1–4). The data suggest that calcitriol has several anti-inflammatory actions in addition to its other well-known therapeutic and cancer-preventive effects in PCa. We conclude that calcitriol or its analogs may have the best utility as chemopreventive agents in PCa and/or when used in early disease. Studies in animal models suggest that calcitriol is more effective when administered before, rather than subsequent to, the initial occurrence of PIN. We believe that calcitriol and its analogs should therefore be further evaluated in clinical trials in PCa patients with early or precancerous disease. The avoidance of vitamin D deficiency should be a goal for reducing the incidence of PCa. Additional trials in men with minimal or early disease, treating with high doses of dietary vitamin D, new potent analogs or, combination therapy may finally demonstrate the promise of benefit of vitamin D in the prevention and treatment of PCa.
Declaration of interest
We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Supported by NIH Grant DK042482 and AICR Grant 06A114 to D Feldman.
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domain: literature


text:
The ancient Persian and Greek Empires created some of the earliest known designed gardens, and these were all in royal residences or other hallowed grounds. With the advent of the Roman Empire, we started to see gardens in more modest residential homes.
The ancient Persian and Greek Empires created some of the earliest known designed gardens, and these were all in royal residences or other hallowed grounds. With the advent of the Roman Empire, we started to see gardens in more modest residential homes. Roman houses were built around a peristyle courtyard that contained a planted garden. This courtyard home design was popular in ancient Greece but would normally be decorated with pots, not a manicured landscape. Unlike the previous great civilizations of the western world that were mostly centered in or near the middle east, the roman empire stretched across a broad range of climates and the Roman style of garden design was not regional which led to plants being introduced to areas where they were not native. Colonists in ancient Britain created their Roman villas using popular plants from their home such as Roses and Rosemary, both of which are common sights in modern English Gardens.
Romans invented Concrete, which allowed them to really shape their landscapes in imaginative ways, however this type of material choice and the exporting of plants from Rome to far reaches of the empire is also the start of unsustainable garden design practices. As unwanted concrete patios are demolished and sent to landfills in the modern age, so too were old materials in the Roman era. Rome is said to be formed of 8 hills, one of which is completely artificial as ancient Romans used the site of Monte Testaccio as a dumping ground for used pots to create a hill over 100 feet in height.
In ancient times, villas were often self-sustaining with the use of kitchen gardens. A roman villa usually included an Herb Garden (Hortus) to supply the household kitchen with herbs and vegetables. Fruit Trees were planted for their produce, as well as a source of shade for respite from the Mediterranean sun. The Horus was often designed in a similar from to the peristyle courtyards, with bilateral symmetry the main diving force of the layout. The beneficial plants grown in the Hortus would be arranged as much by their colour, structure and fragrance as well as their medicinal/gastronomical qualities.
Gardening in the Roman age established many other practices in modern horticulture, such as topiary, planting in rows and growing plants in hothouses (greenhouses). The "Outer Peristyle" garden at the Getty Villa in California is a good representation of the grand villa gardens enjoyed by the nobles of Rome, and it does not look out of place even in the modern age. Unfortunately, after the fall of the Roman Empire in the 5th century A.D., much of the knowledge was lost as the Western World entered the Dark Ages. For a more detailed look at Roman home gardens, check out this classic history blog: https://historyandarchaeologyonline.com/ancient-roman-gardens/

domain: history


Brexit challenge: Court ruling is voter betrayal, says Farage
A High Court ruling allowing parliament a vote on the process of leaving the EU could be the start of a "deliberate wilful attempt" to "betray voters", Nigel Farage says.
The UKIP leader told Emma Barnett on BBC 5 live that the decision could be the start of an attempt to "water down what people had voted for".
The ruling means the government cannot trigger Article 50 of the Lisbon Treaty - beginning formal discussions with the EU - on their own.
Mr Farage said he believed if the vote was put to parliament, the government would lose.
The government is appealing, with a further hearing expected next month.
03 Nov 2016


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Prehistoric Times 11,600 BCE to 3,500 BCE
Many examples of well-preserved prehistoric architecture are found in southern England. Very long ago, humans built earthen mounds, stone circles, and structures that often confound today’s archaeologists. Prehistoric architecture includes monumental structures like Stonehenge, cliff dwellings in the Americas, and mud structures lost to time.
Ancient Egypt 3,050 BCE to 900 BCE
In ancient Egypt, massive pyramids, temples, and shrines were built. Ancient Egyptians were able to construct massive structures thanks to the engineering marvel of the pyramid shape. Engineering feats that could reach great heights were far from giant structures like the Giza Pyramids.
Classical – 850 BCE to CE 476
Classical architecture refers to the style and design of buildings in ancient Greece and Rome. From the rise of ancient Greece until the fall of the Roman empire, great buildings were constructed according to precise rules. The Roman architect Marcus Vitruvius believed that builders should use mathematical principles when constructing temples.
Byzantine – 527 to 565
Byzantine architecture is a style of building that grew under the rule of Roman Emperor Justinian. Its defining characteristic is interior mosaics and a heightened dome.
The characteristics of Byzantine were soaring spaces and luxurious decorations like marble columns, mosaics, and sometimes gold coffered ceilings.
Romanesque – 800 to 1200
Romanesque architecture is based on roman architecture and that is the reason it was given the name Romanesque architecture. It is determined by its massively thick walls, round arches, solid piers, groin vaults, big towers, and decorative arcades.
Gothic – 1100 to 1450
The Germanic Goths, who rebelled against Roman rule, are the inspiration for the name Gothic, and Gothic architecture’s main characteristic is the pointed arch and asymmetry.
Renaissance -1400 to 1600
Renaissance architecture is known In various parts of Europe from the early 15th and 16th centuries. The Renaissance style was first introduced in Florence and quickly spread to other Italian cities before being introduced to different parts of Europe. Symmetry, geometry, and proportion are the main elements of Renaissance design.
Baroque – 1600 to 1830
Baroque architecture appeared in Italy at the beginning of the 17th century, it is known for being a very decorative style of architecture.
Baroque architecture is characterized by its vaulted cupolas supported by rows of pillars, walls, and doorways made of unpolished stones and plaster, its interior design is one of my favorites! it is marked by luxurious furniture and materials, I really hope to visit one of them someday on my next trip to Italy.
Rococo – 1650 to 1790
Rococo architecture is similar to Baroque architecture they call it Late Baroque, it appeared in the 18th century in Paris and was known for being a more expansive aesthetic movement that includes both art and decoration. It is a kind of theatrical architecture. One of the hallmarks of baroque architecture is its richness, grandness, dynamism, and tension…
Neoclassicical Architecture – 1730 to 1925
Neoclassical architecture Emanated from Palladian architecture, and it was spread across the United States and Europe. It is known for having very simple geometrical forms and designs compared to the buildings of that time, with high columns, and grandiose scale.
Art Nouveau -1890 to 1914
Sculptural, arches, curving lines, and luscious decoration are the most defining characteristics of the Art Nouveau architectural style. The architect Charles Rennie Mackintosh is the most known for this style of architecture. This style of architecture has one of the most beautiful and stunning-looking buildings in the world, and one of my favorites is Casa Batlló which is located in Barcelona.
Beaux Arts – 1895 to 1925
This style of architecture was heavily Influenced by classical Roman and Greek structures, It then became a dominant style of architecture in the United States between the 19th and 20th centuries.
The Beaux Arts architecture uses formal symmetry, and classical Greek and Roman decorative elements like columns, pediments, and balustrades to create a grand and imposing architectural statement.
Modernist Styles – 1900 to Present
This style of architecture emphasizes mainly two things: asymmetrical compositions and minimal decor, and it particularly uses glass, steel, and reinforced concrete as the main elements in its composition.
Postmodernism – 1972 to Present
Postmodernism occurred as a reaction to the modern movement. It is a diverse and colorful style of architecture. Postmodern architecture is characterized by its asymmetry, curved forms, decorative elements, and bright color palette…
Neo-Modernism – 1997 to Present
Neo-modern architecture is one of the most dominant and relevant forms of architecture now, and it is mostly used in big projects. As for characteristics, Neomodern architecture highlights experimentation, and defects already set rules and provide freedom of expression in many different aspects such as architecture, arts…
As we’ve seen, architecture has changed and evolved over time and has had different forms, aspects, and characteristics depending on the time era. Personally, I find older architecture especially: Baroque, Rococo, and Renaissance architecture, more interesting and prefer it to the modern one, because I think it was more creative and had more interesting elements, and overall it looks better and more appealing in contrast to the one now that mostly looks bland and boring to me.
For this reason, I enjoy walking through Spain’s historic cities to watch and gather inspiration for new architectural designs. I also enjoy taking pictures of these structures, here are some that I captured in Valencia’s city center.

domain: artistic


text:
J 398.21 A Aesop Aesop's Fables (gr. 2-5)
Various classic stories and morals passed down since the time of Aesop, a slave in ancient Greece.
J 398.2 A Alderson, Brian The Arabian Nights (gr. K+)
An illustrated collection of stories from the "Arabian Nights", including those of Sinbad, Ali Baba and Aladdin.
J 398.2 A Andersen, Hans Christian Dulac's Snow Queen and Other Stories (gr. )
Contains some of the famous Hans Christian Andersen fairy tales, with beautiful illustrations by Edmund Dulac.
J 398.2 A Andersen, Hans Christian The Little Mermaid (all grades)
For years the world has delighted in the story of the mermaid who loves her prince so well that she is willing to give up life beneath the sea.
J 398.2 A Andersen, Hans Christian The Princess and the Pea (gr. K-4)
The lion that is looking for a real princess finds her when she complains about sleeping on a pea, felt through dozens of mattresses.
J 398.2 A Andersen, Hans Christian The Ugly Duckling (gr. K-3)
An ugly ducking spends an unhappy year ostracized by other animals before he grows into a beautiful swan.
J 398.0943 G Galdone, Paul The Frog Prince (gr. K-3)
The much-beloved tale of the beautiful princess who lost her plaything and the ugly frog who made her keep her promise.
J 398 G Grimm, Jakob and Wilhelm Grimm's Fairy Tales (all grades)
Two German brothers wrote many of these tales of witches, fairies, dwarfs and dragons, including the "Musicians of Bremen."
J 398.0943 G Grimm, Jakob and Wilhelm Sleeping Beauty (gr. K-4)
Enraged at not being invited to the princess's chistening, the wicked fairy casts the spell that puts the princess to sleep for one hundred years.
J 398.0943 G Grimm, Jakob and Wilhelm Snow White and the Seven Dwarfs
A princess takes refuge from her wicked step-mother in the home of the seven dwarfs.
J 292 H Hawthorne, Nathaniel A Wonder Book and Tanglewood Tales (gr. 3+)
Adaptations of the classic Greek myths of Hercules, King Midas, Pandora and other legendary figures.
J 398.22 K Malory, Thomas, Sir. King Arthur and His Knights of the Round Table
The great English hero tale of "once and future" king, his knights and their fabulous adventures in Camelot.
J 398.0943 M Mayer, Mercer Pied Piper of Hamlin (gr. K-3)
The story of the pied piper of Hamlin who rids the town of rats and then takes revenge on the townspeople.
J 398.0944 M Montresory, Beni Little Red Riding Hood (gr. K-3)
The classic story of the little girl who visits her grandmother but meets the big, bad wolf instead.
J 398.0944 P Perrault, Charles Cinderella (all ages)
The classic tale of the poor step-sister who, with the help of her fairy godmother, wins the hand of the prince.
J 398.0944 P Perrault, Charles Puss in Boots (gr. K-3)
A cunning cat wins his master a castle, a fortune and the hand of the princess.
J 398.0943 Z Zelinsky, Paul Rapunzel (gr. K-4)
Raised in solitude in a tower, lovely Rapunzel is rescued by her handsome prince and lives happily ever after.

domain: entertainment


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By James Altucher |
July 2, 2014
Entrepreneur James Altucher shares the lessons he learned from his chaotic life as a day trader.
The bears are having beef tonight
Bitreserve raises nearly $10 million; Coinbase funding global expansion
Options play: Where is the bottom in crude oil?
Fed drops "considerable time"
Lower crude produces winners & losers...
Simplifying fibonacci for traders

domain: economics


Walton County first responders extinguish fire at Taco Bell in DeFuniak Springs
DeFUNIAK SPRINGS — Firefighters extinguished a fire Monday afternoon at a fast-food restaurant in DeFuniak Springs.
A person called 911 to report a smell of smoke coming from inside a Taco Bell on U.S. Highway 331, according to a Walton County Fire Rescue news release.
More on Walton County Fire Rescue:Walton County Fire Rescue becomes first to earn EMS accreditation in Florida Panhandle
Training in Fort Walton:Fort Walton Beach, Florida firefighters use vacant building for training
No flames were visible from the outside of the building when crews from the DeFuniak Springs Fire Department, Walton County Fire Rescue, Argyle Volunteer Fire Department and Walton County Sheriff’s Office arrived.
A quick investigation showed one of the gas lines in the building had caught fire, WCFR reported. The fire was extinguished and everyone made it out of the building safely.


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An arts group run by an inspirational woman has set its sights on helping to rebuild a community hall.
Wise Up Workshops will host a family fun day at St John the Divine, Sandylands community hall this Saturday (July 29) from 10am to 4pm.
Wise Up Workshops is run by Elle Roberts, who featured in our newspaper earlier this year when we appealed for a donor who could save her life.
Elle is recovering following a stem cell bone marrow transplant after she was diagnosed with Acute Myeloid Leukaemia (AML), an aggressive form of blood cancer, for the second time.
There are plans to build a new community hall to replace the current 30-year-old building and nearby scout hut. Money from the fun day will go towards the project.
Wise Up uses art forms to promote personal development, confidence and communication skills in individuals with low confidence or additional needs.

domain: news


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People on social media are having a field day with YSL defense attorney Nicole Fegan, who was arrested on gang-related charges on Friday.
On Friday, folks on X, formerly Twitter, let their comments fly about the attorney who was representing defendant Tenquarius Mender in the YSL Rico gang trial. The woman’s Instagram page had various photos of her posing with wads of cash and multiple firearms, as well as quotes from the late Big L.
“I feel like everyone who follows her account is probably a day away from getting a RICO charge,” one person tweeted with a screenshot of Fegan pointing a gun at the camera in the picture. Another person wrote, “Young Thug getting 200 years man,” with a photo of Fegan posing with cash on the ground spelled out as “BROKE.”
A third person added, “I would prefer that my lawyer didn’t post stuff like this,” next to photos of Fegan holding firearms.
Per WSB-TV2, Fegan was taken into custody by Atlanta police on Friday after she was alleged to have contacted a suspect in the September 2022 shooting of two men.
"Investigators received information that Nicole Fegan contacted a suspect in the shooting, advising him of active warrants for his arrest," the Atlanta Police Department wrote in a statement. "Fegan provided information she had learned during a preliminary hearing related to the shooting and advised the suspect to dispose of his phone as police were going to arrest him. Investigators confirmed Fegan was not representing the suspect she called."
Fegan was taken to Fulton County jail on charges of participating in criminal street gang activity and criminal solicitation to commit the offense of tampering with evidence. One of the two men shot in the September 2022 incident shooting died.
Check out more reactions to Fegan's social media posts below.


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Microsoft still isn't divulging official Surface numbers, but it looks like it's at least making a dent in the tablet market.
If you needed further evidence that tablets are more than a mere computing fad, just look at the latest numbers from Strategy Analytics.
Confirming earlier rumors, departing Microsoft CFO Peter Klein said today on an earnings call that Microsoft is working with manufacturing partners on smaller versions of Windows 8 devices, which likely means 7- or 8-inch Windows 8 tablets.
Microsoft's next round of Surface slates may include a competitor to the iPad Mini and Kindle Fire.
Microsoft has dropped the resolution requirement for Windows 8 on tablets to 1,024 by 768, opening up the possibility for less expensive 7-inch or 8-inch tablets running the fledgling operating system.
Steve Ballmer is confirming which has been pretty obvious to everyone else: The Suface won't rule the world.
The U.S. Microsoft online store has sold out of the Surface Pro tablets, which went on sale today.
Microsoft has obviously decided that the "Pro" part of "Surface Pro" is actually meaningful, not just a marketing designation, and has provided the ability for enthusiasts to fiddle, potentially damage or even brick, but ultimately customize their devices exactly how they see fit.
For all of its failures as a pure tablet, the Surface Pro is a compelling offering as an ultraportable/tablet hybrid.
Microsoft sold a paltry number of Surface tablets in the last quarter -- somewhere between 680,000 and 750,000 units -- the research firm IHS iSuppli estimates.
$800 for 23GB of usable space? The 64GB Surface Pro is a tough sell.
Microsoft's soon-to-be-released Windows 8-running Surface Pro may cost an internal organ plus a year's servitude, but price relief -- and other Windows tablets -- sound like they're coming soon.
February is the new January for Microsoft, which is giving its Surface Pro tablet a February 9th release date.
"Mac OS is the best thing to happen to Windows RT," quip the trolls.
Advertising and analytics company Chitika released its December 2012 tablet market update, and the numbers aren't great for any tablet that doesn't start with the letter I.
Guest Post Innovation. It’s what makes new markets. It’s what creates new opportunities for companies to grow dramatically. And it’s what creates demand when consumers don’t even know they want something.
Microsoft will ring in the new year by opening six new retail locations that will help customers get better exposure to products such as its Surface tablet and Windows Phone devices.
Proving that it has at least a modicum of common sense, Microsoft says it will offer its Surface tablet in more stores starting this week.
Not surprisingly, Microsoft is pricing its Surface with Windows 8 Pro tablet — the one that will actually be able to run older Windows applications — about the same as Windows 8 Ultrabooks.
The 64 gigabyte Surface Pro (as it’s …
Unfortunately, sending a tweet about the Microsoft Surface via an Apple iPad isn't the most convincing way to make an endorsement.


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People also see good weight loss results on the keto diet because eating a low carb, high fat diet can help you feel less hungry and not have to count calories or portion sizes to lose weight. Plus, cutting out the refined carbs and sugars normally present in a traditional Western diet helps avoid crazy spikes in blood sugar that can lead to the feeling of being hungry again soon after eating.
My Husband and I started doing Keto July 2018. We got over weight after we got out of the Marine Corps. It has been hard to workout because I became disabled, but my diet was not good. After our friend Amber recommended your site and support group, we found a lot of helpful information to get us started on a successful journey. So far it’s been one month and we have lost 18 pounds each!
I should note that I fully believe the key to success on a keto diet is to be prepared. If you precook your meals, you are setting yourself up for success. You are only choosing to do keto once a week when you prepare your food. If you have no keto food at your house and its 9 pm on a Wednesday, then you are just asking to revert back to something easy like chinese food or pizza. However, if all you need to do is pull a premade meal out of the fridge and microwave it, you are much better off. Make sure to pick up some rugged, microwaveable containers to store your food. Now then, lets get started!
Wilder's colleague, paediatrician Mynie Gustav Peterman, later formulated the classic diet, with a ratio of one gram of protein per kilogram of body weight in children, 10–15 g of carbohydrate per day, and the remainder of calories from fat. Peterman's work in the 1920s established the techniques for induction and maintenance of the diet. Peterman documented positive effects (improved alertness, behaviour and sleep) and adverse effects (nausea and vomiting due to excess ketosis). The diet proved to be very successful in children: Peterman reported in 1925 that 95% of 37 young patients had improved seizure control on the diet and 60% became seizure-free. By 1930, the diet had also been studied in 100 teenagers and adults. Clifford Joseph Barborka, Sr., also from the Mayo Clinic, reported that 56% of those older patients improved on the diet and 12% became seizure-free. Although the adult results are similar to modern studies of children, they did not compare as well to contemporary studies. Barborka concluded that adults were least likely to benefit from the diet, and the use of the ketogenic diet in adults was not studied again until 1999.
That’s why you’ll find snacks of all flavors — salty, sweet, and savory — in this keto snack recipe roundup. There are grab-and-go options, as well as keto snacks requiring some preparation and cooking. We’re willing to bet that they’re in fact so tasty, you’ll forget you’re on a diet. All the while, these keto snacks will keep you going strong in ketosis — that uber fat-burning state you’re aiming for as your body shifts from burning carbs to fats for fuel.
With a ketosis diet, you tend to eliminate more calcium than usual. Additionally, foods such as cruciferous vegetables, spinach, iced tea or chocolate all have a high quantity of oxalates. Oxalates are naturally occurring substances found in a wide variety of foods; they play a supportive role in the metabolism of many plants and animals, including humans. Oxalates combined with calcium can cause kidney stones.
I have the same calories limit and macro. I easily stay within the limit but I completely stopped eating any type of bread, pasta, rice, potatoes and starchy food. I started using butter or coconut oil to cook and put mayo EVERYWHERE. It’s my new favourite topping for lettuce even XD. Then I eat plenty of eggs/meat/fatty fish and cheese. Olives are great as a snack. I cut out completely milk and use whipping cream instead. If you get into this completely counter intuitive way of thinking of food it’s super easy to get enough fat within the calorie limit.
Implementing the diet can present difficulties for caregivers and the patient due to the time commitment involved in measuring and planning meals. Since any unplanned eating can potentially break the nutritional balance required, some people find the discipline needed to maintain the diet challenging and unpleasant. Some people terminate the diet or switch to a less demanding diet, like the modified Atkins diet (MAD) or the low-glycaemic index treatment (LGIT) diet, because they find the difficulties too great.
Doing a 1:1 substitution would probably change the macros too much but that doesn’t mean that you have to eat dairy to eat a ketogenic diet. If you want to use the meal plan you’d have to adjust it with other sources of fat so that you match the macros. It will require a little work (I recommend using an online diary like MyFitnessPal for support) but you’ll end up with a plan that works for you and your needs
This is where we have to depart! Sorry to say but you’re on your own. You should have plenty of leftovers that are frozen, ready, and waiting! I know a lot of you out there have trouble with timing and are busy people – so making sure that some nights you make extras to freeze is important. All those leftovers you have in the freezer? Use them up! Create your own meal plan, at first using this as a guide, and then completely doing it yourself. Once you get the hang of it, it’ll be a sinch – I promise you 🙂
Short for “ketogenic diet,” this eating plan is all about minimizing your carbs and upping your fats to get your body to use fat as a form of energy, says Scott Keatley, R.D., of Keatley Medical Nutrition Therapy. While everyone's body and needs are slightly different, that typically translates to: 60 to 75 percent of your calories from fat, 15 to 30 percent of your calories from protein, and 5 to 10 percent of your calories from carbs.
In the 1960s, it was discovered that medium-chain triglycerides (MCTs) produce more ketone bodies per unit of energy than normal dietary fats (which are mostly long-chain triglycerides). MCTs are more efficiently absorbed and are rapidly transported to the liver via the hepatic portal system rather than the lymphatic system. The severe carbohydrate restrictions of the classic ketogenic diet made it difficult for parents to produce palatable meals that their children would tolerate. In 1971, Peter Huttenlocher devised a ketogenic diet where about 60% of the calories came from the MCT oil, and this allowed more protein and up to three times as much carbohydrate as the classic ketogenic diet. The oil was mixed with at least twice its volume of skimmed milk, chilled, and sipped during the meal or incorporated into food. He tested it on twelve children and adolescents with intractable seizures. Most children improved in both seizure control and alertness, results that were similar to the classic ketogenic diet. Gastrointestinal upset was a problem, which led one patient to abandon the diet, but meals were easier to prepare and better accepted by the children. The MCT diet replaced the classic ketogenic diet in many hospitals, though some devised diets that were a combination of the two.
For breakfast, we are going to change it up a bit. Here’s where we introduce ketoproof coffee. Now, don’t get me wrong – I know some of you won’t like it. If you’re not a fan of coffee, then try it with tea. If you’re not a fan of the taste (which is very rare), then try making a mixture of the ingredients by themselves and eating it like that. So, why ketoproof coffee?
Well, I am going to give this another try. I have great difficulty in eating greens , or drinking them, also I am not fond of fats, years and years of low fat diets have totally screwed my metabolism,and taste buds. I will read this page every day to keep my mind focused. Start tomorrow when I get up …… I work nights which can cause me problems as well. When I tried this diet before, I got terrible cramp, now I realise I wasn’t drinking enough water. Anyway.here goes.
The main ingredients in this recipe (chicken, cream cheese, cheddar cheese, and bacon) are all part of a low-carb, ketogenic diet. We skipped the store-bought Ranch seasoning mix because it usually contains sugar, quite a bit of salt, and preservatives that we try to avoid if possible. Instead we used a keto-friendly mix of dried chives, garlic powder, onion powder, crushed red pepper flakes, dried dill, salt, and black pepper to season our Crack Chicken.
Ketones are triggered by an absence of carbohydrates and sugar. There are specific steps to take to get your body into ketosis, but essentially, when carbohydrates are eliminated, the body will transfer over to using ketones. But I don’t recommend removing carbohydrates completely because I want you to be consuming lots of vegetables. One reason it is so crucial to take in lots of vegetables, and especially lots of vegetables high in potassium, such as kale, beet greens and collard greens, is because it will help you keep all this fat you’re dumping off the body off your liver. You can get a fatty liver when you start dropping lots of weight, as you will once you begin running on ketones instead of glucose. So think VEGETABLE CARBOHYDRATE ONLY.
We’re also going to keep it simple here. Most of the time, it’ll be salad and meat, slathered in high fat dressings and calling it a day. We don’t want to get too rowdy here. You can use leftover meat from previous nights or use easy accessible canned chicken/fish. If you do use canned meats, try to read the labels and get the one that uses the least (or no) additives!
Now we do have a full meal plan laid out for you below, but I would encourage you to experiment a bit with some meals that might better suit your tastes. We love all of these meals, that’s why we picked them! We know everyone is different though. If you want to sub something in, just check to make sure the nutrition is comparable. Keto on a budget can be pulled off all types of different ways. This is just one example using a lot of our favorite meals. For example, if you have a bunch of coconut oil sitting around feel free to sub it in for the butter. If you have olive oil, sub it in for the blue cheese dressing. Don’t let this meal plan feel restricting, it is best used as a reference point and not as a hard and fast rule book. We put together this full day of eating video to show you an example of how we ate on $5 a day:
Which leads us to your personality. You need to be VERY strong willed to follow this diet. It follows very strict rules and you need to be able to commit to this. It makes it hard to eat out with friends or eat with friends in general. It’s hard to find food that fits this diet in common restaurants, but and this takes us to the next point, your health goals might be more important.
One downside to a ketogenic diet for weight loss is the difficulty maintaining it. “Studies show that weight loss results from being on a low-carb diet for more than 12 months tend to be the same as being on a normal, healthy diet,” says Mattinson. While you may be eating more satiating fats (like peanut butter, regular butter, or avocado), you’re also way more limited in what’s allowed on the diet, which can make everyday situations, like eating dinner with family or going out with friends, far more difficult. Because people often find it tough to sustain, it’s easy to rely on it as a short-term diet rather than a long-term lifestyle.
Drink lots of water. This is especially crucial on a low carb or keto diet. Why? When you eat carbohydrates, your body stores the extra as glycogen in the liver, where they are bound to water molecules. Eating low carb depletes this glycogen, which allows you to burn fat – but it also means you are storing less water, making it easier to get dehydrated. Instead of the traditional recommendation of 8 cups of water per day, aim for 16 cups when following a low carb lifestyle.
With intermittent fasting, the whole goal is to burn off excess fat, right? Watch how you feel when you exercise. Do you feel best when you exercise after eating or if you exercise while fasting? Watch if your legs feel heavy or if you tire too easily. Some people do well with eating first, while some love that feeling of exercising when they have fasted and having to eat afterwards.
There are numerous benefits that come with being on keto: from weight loss and increased energy levels to therapeutic medical applications. Most anyone can safely benefit from eating a low-carb, high-fat diet. Below, you’ll find a short list of the benefits you can receive from a ketogenic diet. For a more comprehensive list, you can also read our in-depth article here >

domain: food


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Once the Raiders and Lynch have reached an agreement on a contract, Oakland will contact the Seahawks and begin the process of negotiating a trade.
- Rapoport notes that a retired player can be traded even though he has yet to be reinstated by the NFL.
- The Seahawks gave Lynch and his agents permission to speak to the Raiders about a reworked contract. If they can reach an agreement on a new deal, then trade talks can begin from there, according to Rapoport.
- Pro Football Talk reports that Lynch has yet to submit any paperwork to the NFL.
Lynch visited with the Raiders a few weeks ago and reportedly told them that he intends to play in 2017.
Lynch, 29, has two years remaining on his three-year, $31 million contract that included $12 million guaranteed. He stands to make base salaries of $9 million and $7 million over the final two years of the agreement.
During his career, Lynch rushed for 9,112 yards rushing on 2,144 carries (4.3 YPC) to go along with 252 receptions for 1,979 yards receiving and 83 total touchdowns for the Seahawks and Bills.


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Tumour-associated antigenic peptides are present in the HLA class I ligandome of cancer cell line derived extracellular vesicles
MetadataShow full item record
The recent success of monoclonal antibody checkpoint inhibitor therapies that enhance the ability of CD8+ T cells to detect cancer-related antigenic peptides has refocused the need to fully understand the repertoire of peptides being presented to the immune system. Whilst the peptide ligandome presented by cell surface human leucocyte antigen class I (HLA-I) molecules on cancer cells has been studied extensively, the ligandome of extracellular vesicles (EVs) remains poorly defined. Here we report the HLA-I ligandome of both the cell surface and EVs from eight breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-361, MDA-MB-415, MDA-MB-453, HCC 1806, HCC 1395, and HCC 1954), and additionally the melanoma cell line ESTDAB-056 and the multiple myeloma line RPMI 8226. Utilising HLA-I immunoisolation and mass spectrometry, we detected a total of 6574 peptides from the cell surface and 2461 peptides from the EVs of the cell lines studied. Within the EV HLA-I ligandome, we identified 150 peptides derived from tumour associated antigenic proteins, of which 19 peptides have been shown to elicit T cell responses in previous studies. Our data thus shows the prevalence of clinically relevant tumour-associated antigenic peptides in the HLA-I ligandome presented on EV.
Kumar , P , Boyne , C , Brown , S E , Qureshi , A , Thorpe , P J , Synowsky , S A , Shirran , S L & Powis , S J 2022 , ' Tumour-associated antigenic peptides are present in the HLA class I ligandome of cancer cell line derived extracellular vesicles ' , Immunology , vol. 166 , no. 2 , 13471 , pp. 249-264 . https://doi.org/10.1111/imm.13471
Copyright © 2022 The Authors. Immunology published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
DescriptionFunding: Breast Cancer Now (Grant Number(s): 2018JulPR1086), Wellcome Trust (GrantNumber(s): 105621/Z/14/Z), Melville Charitable Trust.
Items in the St Andrews Research Repository are protected by copyright, with all rights reserved, unless otherwise indicated.

domain: medical