EFFECTS OF 24R,25(OH)2D3 IN THE TERATMENT OF KNEE OSTEOARTHRITIS Qingfen Pan 117 Pages Directed by Dr. Barbara Boyan Osteoarthritis (OA) is a degenerative disease characterized by joint inflammation and cartilage degeneration due to matrix degradation and chondrocyte apoptosis. Previously, drug therapies have been developed that aim to ease pain and reduce local inflammation. Currently, no effective drug exists that has no significant side effects. Therefore, an unmet medical demand exists for development of tissue-engineering strategies to promote articular cartilage repair and regeneration to treat OA. 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] is an attractive option for articular cartilage repair because of its anti-inflammatory and anti-apoptotic properties. 24R,25(OH)2D3, which is a naturally occurring metabolite of vitamin D3, also has not been shown to cause toxic side effects. Results from the study demonstrate that 24R,25(OH)2D3 can inhibit chondrocyte apoptosis and suppress the production of catabolic factors that result in cartilage degeneration in the in vitro model. Furthermore, although 24R,25(OH)2D3 regulates components of TGF-β1 pathway, the effect of 24R,25(OH)2D3 is not mediated through TGF-β1 signaling. In vivo delivery of 24R,25(OH)2D3 prevented cartilage degeneration and disease progression. In addition, intraarticular injection of 24R,25(OH)2D3 had an effect on cytokines and growth factors production both locally and systemically. Human articular chondrocytes responded to 24R,25(OH)2D3 treatment in both sex and maturation dependent manner. Collectively, results from this study suggest that 24R,25(OH)2D3 ccould be used as a clinical therapy for knee OA.

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<p>Cancer or malignant tumors has a high death frequency in many countries. Nowadays many research facilities are dedicated to find new substances and techniques which would lead to better cancer therapies. Seven years ago a research team from Finland made a remarkable connection between vitamin D deficiencies and an increased chance of getting prostate cancer. The research investigating this statement has lead to findings of a new non-classical effect of the calcium controlling vitamin, 1,25(OH)2D3. This effect involves anti-proliferatory effects and more importantly apoptotic effects resulting in the hope of finding a new drug that can cure prostate cancer with the smallest amount of harm to the body.</p><p>In an attempt to find out if the signalling pathway of this apoptotic effect is fast or slow, an experiment designed to detect when the apoptotic protein caspase-3 is induced has been performed. Cells from the cell line LNCaP has been cultured and incubated with 1,25(OH)2D3 and after 0min - 48h an assay was performed to detect the relative amounts of caspase-3 present in every sample. The optimal time period (48h) was then subjected to three different concentrations of 1,25(OH)2D3 and read in the same way as the previous samples. The results showed an increase in caspase-3 expression as early as 2 min, but disappear to be seen again at 24h and are more profound in 48h samples. The caspase-3 expression was also seen to form a possible exponential curve in dose-response.</p>

Cancer or malignant tumors has a high death frequency in many countries. Nowadays many research facilities are dedicated to find new substances and techniques which would lead to better cancer therapies. Seven years ago a research team from Finland made a remarkable connection between vitamin D deficiencies and an increased chance of getting prostate cancer. The research investigating this statement has lead to findings of a new non-classical effect of the calcium controlling vitamin, 1,25(OH)2D3. This effect involves anti-proliferatory effects and more importantly apoptotic effects resulting in the hope of finding a new drug that can cure prostate cancer with the smallest amount of harm to the body. In an attempt to find out if the signalling pathway of this apoptotic effect is fast or slow, an experiment designed to detect when the apoptotic protein caspase-3 is induced has been performed. Cells from the cell line LNCaP has been cultured and incubated with 1,25(OH)2D3 and after 0min - 48h an assay was performed to detect the relative amounts of caspase-3 present in every sample. The optimal time period (48h) was then subjected to three different concentrations of 1,25(OH)2D3 and read in the same way as the previous samples. The results showed an increase in caspase-3 expression as early as 2 min, but disappear to be seen again at 24h and are more profound in 48h samples. The caspase-3 expression was also seen to form a possible exponential curve in dose-response.

The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this mainly through a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. Another major way in which 1,25(OH)2D3 inhibits cell growth in prostate cancer is via membrane-initiated steroid signalling, which triggers activation of signal cascades upon steroid binding to a receptor complex, leading to induction of genes regulating cell growth, proliferation and apoptosis. The main prostate cancer inhibiting membrane-initiated route is the TGFβ signalling pathway, elicited by the protein TGFβ. Two other important proteins downstream in this cascade are Smad2 and Smad3. In this study the early effects of 1,25(OH)2D3 on activated Smad2/3 levelsin PC-3 prostate cancer cells were examined. PC-3 cells were incubated for 3, 5, 10, 30 and 60 minutes as well as 38 hours both together with 1,25(OH)2D3 of the concentrations 10-10 and 10-7 M and without. Western Blots were then performed on supernatants from the cells treated followed by treatment of the membranes with primary antibodies against phosphorylated Smad2/3 C-terminal linker regions, alkaline phosphatase conjugated secondary antibodies and finally visualization with BCIP/ NBT tablets. As the downstream cascade protein JNK is a proposed activator of Smad2/3, this procedure was also repeated with a JNK inhibitor. This is a follow-up to an earlier study which examined the influence of 1,25(OH)2D3 on TGFβ levels using the same doses and time points and which found that 1,25(OH)2D3 initially lowered the level of active TGFβ, then increased it. The results of this study indicated a 1,25(OH)2D3 mediated induction of the same pattern in the levels of active Smad2 and 3, both with and without JNK inhibitor. The results did not indicate that 1,25(OH)2D3 activates the Smad2/3 C-terminal linker region via the JNK pathway.

The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this mainly through a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. Another major way inwhich 1,25(OH)2D3 inhibits cell growth in prostate cancer is via membrane-initiated steroid signalling, which triggers activation of signal cascades upon steroid binding to a receptor complex, leading to induction of genes regulating cell growth, proliferation and apoptosis. The main prostate cancer inhibiting membrane-initiated route is the TGFβ signalling pathway, elicited by the protein TGFβ. Another important protein downstream in this cascade is Smad2. In this study the early effects of 1,25(OH)2D3 on activated Smad2 levels in PC-3 prostate cancer cells were examined. PC-3 cells were incubated for 5, 10, 30 and 60 minutes as well as 24 and 40 hours both together with 1,25(OH)2D3 of the concentrations 10-10 and 107 M and without. An ELISA assay scanning for activated Smad2 was then performed on supernatants from both treated and untreated cells. This is a follow-up to an earlier study which examined the influence of 1,25(OH)2D3 on TGFβ levels using the same doses and similar time points and which found that 1,25(OH)2D3 initially lowered the level of active TGFβ, then increased it. The results of this study showed a statistically insignificant, time delayed 1,25(OH)2D3 mediated induction of the same pattern in the levels of active Smad2. / Project Work in Biomedicine, Advanced Level, 7.5 ECTS

<p>The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this mainly through a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. Another major way in which 1,25(OH)2D3 inhibits cell growth in prostate cancer is via membrane-initiated steroid signalling, which triggers activation of signal cascades upon steroid binding to a receptor complex, leading to induction of genes regulating cell growth, proliferation and apoptosis. The main prostate cancer inhibiting membrane-initiated route is the TGFβ signalling pathway, elicited by the protein TGFβ. Two other important proteins downstream in this cascade are Smad2 and Smad3. In this study the early effects of 1,25(OH)2D3 on activated Smad2/3 levelsin PC-3 prostate cancer cells were examined. PC-3 cells were incubated for 3, 5, 10, 30 and 60 minutes as well as 38 hours both together with 1,25(OH)2D3 of the concentrations 10-10 and 10-7 M and without. Western Blots were then performed on supernatants from the cells treated followed by treatment of the membranes with primary antibodies against phosphorylated Smad2/3 C-terminal linker regions, alkaline phosphatase conjugated secondary antibodies and finally visualization with BCIP/ NBT tablets. As the downstream cascade protein JNK is a proposed activator of Smad2/3, this procedure was also repeated with a JNK inhibitor. This is a follow-up to an earlier study which examined the influence of 1,25(OH)2D3 on TGFβ levels using the same doses and time points and which found that 1,25(OH)2D3 initially lowered the level of active TGFβ, then increased it. The results of this study indicated a 1,25(OH)2D3 mediated induction of the same pattern in the levels of active Smad2 and 3, both with and without JNK inhibitor. The results did not indicate that 1,25(OH)2D3 activates the Smad2/3 C-terminal linker region via the JNK pathway.</p>

<p>The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this mainly through a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. Another major way inwhich 1,25(OH)2D3 inhibits cell growth in prostate cancer is via membrane-initiated steroid signalling, which triggers activation of signal cascades upon steroid binding to a receptor complex, leading to induction of genes regulating cell growth, proliferation and apoptosis. The main prostate cancer inhibiting membrane-initiated route is the TGFβ signalling pathway, elicited by the protein TGFβ. Another important protein downstream in this cascade is Smad2. In this study the early effects of 1,25(OH)2D3 on activated Smad2 levels in PC-3 prostate cancer cells were examined. PC-3 cells were incubated for 5, 10, 30 and 60 minutes as well as 24 and 40 hours both together with 1,25(OH)2D3 of the concentrations 10-10 and 107 M and without. An ELISA assay scanning for activated Smad2 was then performed on supernatants from both treated and untreated cells. This is a follow-up to an earlier study which examined the influence of 1,25(OH)2D3 on TGFβ levels using the same doses and similar time points and which found that 1,25(OH)2D3 initially lowered the level of active TGFβ, then increased it. The results of this study showed a statistically insignificant, time delayed 1,25(OH)2D3 mediated induction of the same pattern in the levels of active Smad2.</p> / Project Work in Biomedicine, Advanced Level, 7.5 ECTS

Biologically active 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) binds the vitamin D receptor (VDR) to exert its effect on target cells. VDR expression is found in a number of immune cells including professional antigen-presenting cells such as dendritic cells. It has been found that the actions of 1,25-(OH)2D3 on the immune system are mainly immunosuppressive. The cross-presentation pathway allows for exogenously derived antigens to be presented by pAPCs on MHC-I molecules to CD8+ T cells. CD8+ T cell activation results in the expansion of epitope-specific T cell populations that confer host protection. These epitopes can be organized into an immunodominance hierarchy. Previous work demonstrated that introducing LCMV-NP via the cross-priming pathway significantly alters the immunodominance hierarchy of a subsequent LCMV infection. Building upon these observations, our study assessed the effects of LCMV-NP cross priming in the presence of a single dose of 1,25-(OH)2D3. Treatment with 1,25-(OH)2D3 was found to have biological effects in our model system. In vitro pAPCs were demonstrated to up-regulate IL-10 and CYP24A1 mRNA, in addition to the transactivation of cellular VDR, as demonstrated by a relocalization to the nuclear region. Mice treated with 1,25-(OH)2D3 were found to produce up-regulated IL-10 and CYP24A1 transcripts. Expression of VDR was increased at both the transcript and protein level. Our results demonstrate that a single dose of 1,25-(OH)2D3 does not affect the cross-priming pathway in this system. Treatment with 1,25-(OH)2D3 did not influence the ability of differentiated pAPCs to phagocytose or cross-present exogenous antigen to epitope-specific CD8+ T cells. Furthermore, 1,25-(OH)2D3 did not alter cross-priming or the establishment of the LCMV immunodominance hierarchy in vivo. By confirming that 1,25-(OH)2D3 does not suppress cross-priming in our model, our study helps to expand the understanding of the immunomodulatory role of exogenous 1,25-(OH)2D3 on the outcome of virus infection. Collectively, our data supports the observation that the role of 1,25-(OH)2D3 in the immune system is not always associated with suppressive effects. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-08-29 14:53:18.766

The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this mainly through a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. Another major way in which 1,25(OH)2D3 inhibits cell growth in prostate cancer is via membrane-initiated steroid signalling, which triggers activation of signal cascades upon steroid binding to a receptor complex, leading to induction of genes regulating cell growth, proliferation and apoptosis. The main prostate cancer inhibiting membrane-initiated route is the TGFβ signalling pathway, elicited by the protein TGFβ. In this experiment the activating effects of 1,25(OH)2D3 on TGFβ in prostate cancer cells, as well as two other important proteins downstream in this cascade, Smad2 and 3, were investigated. PC-3 cells were incubated for 3, 5, 10, 30 and 60 minutes as well as 38 hours both together with 1,25(OH)2D3 of the concentrations 10-10 and 10-7 M and without. As the downstream cascade protein JNK is a known activator of Smad2/3, this procedure was also repeated with a JNK inhibitor. An ELISA assay scanning for activated TGFβ was then performed on supernatants from the cells treated without JNK inhibitor. In addition, a Western Blot scanning for activated Smad2 and 3 was performed on supernatants from all groups of treatment. The analysis of the result values showed that 10-10 M 1,25(OH)2D3 significantly lowered the content of active TGFβ in PC-3 cells within 3 and 5 minutes. Unfortunately the Western Blot was unsuccessful and needs therefore be repeated.