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Cervical cancer is the fourth most common female malignancy worldwide and is substantively higher in low-income and middle-income countries. In South Africa, cervical cancer is a leading cause of mortality amongst women. The anti-cancer actions of the vitamin D and its numerous metabolites are an active field of research. The family of vitamin D metabolites regulate numerous cellular pathways which are implicated in tumorigenesis. Pre-clinical studies and clinical studies have yielded promising, although conflicting results in various cancers. Some healthy and cancerous tissue express an autocrine vitamin D metabolising system (VDMS) which is capable of tightly regulating intracellular metabolism and growth. The VDMS expresses activating and inactivating enzymes and a vitamin D receptor (VDR). At the cellular level, the VDMS can activate and inactivate vitamin D precursors and transduce signals to the nucleus to regulate various cell health genes, including cell growth, metabolism and survival. Healthy and cancerous cervical tissue express a VDMS. The anti-cancer actions of cholecalciferol, an early precursor of activated vitamin D, is poorly studied in cervical cancer. This study aimed to characterise cholecalciferol’s action on cell growth, cell death and the VDMS in a high-grade cervical cancer cell line, SiHa. SiHa cell cultures were treated with a range of cholecalciferol doses (26 nM, 104 nM, 260 nM and 2600 nM) for 72 hours. Cell count and viability were assessed by crystal violet and trypan blue assays, respectively. Cell proliferation was enumerated by Ki67 nuclear antigen and the cell cycle profile analysed by flow cytometry. Apoptotic cell death was investigated by measuring mitochondrial membrane potential (∆Ψm), phosphatidylserine (PS) externalisation, effector caspase activation and evaluation of DNA damage markers by flow cytometric analysis. The biochemical markers microtubule-associated proteins 1A/1B light chain 3B-II (LC3-II) and lactate dehydrogenase (LDH) were also measured by flow cytometry and spectrophotometric analysis to identify autophagic cell death and necrosis, respectively. In addition, brightfield microscopy and transmission electron microscopy (TEM) were respectively used to characterise morphological and ultrastructural features of apoptosis, autophagic cell death and necrosis. The VDMS in SiHa control and experimental cultures were characterised by the investigation of intracellular gene and protein expression of the cholecalciferol activating (CYP2R1 and CYP27A1) and inactivating (CYP24A1) enzymes, and the VDR. Qualitative microscopical analysis evaluated classical characteristics of cell death and semi-quantitative analysis of apoptosis was performed. Data were analysed using a one-way ANOVA and Bonferroni post-hoc test. p < 0.05 was considered statistically significant. A significant decrease in cell count and cell viability was identified in SiHa cell cultures treated with 2600 nM cholecalciferol. Furthermore, significant increase in biochemical markers of apoptosis were identified including, decreased ∆Ψm; PS exposure; terminal caspase activation; and nuclear damage at 2600 nM cholecalciferol treatment of SiHa cell cultures. Moreover, the biochemical findings were supported by brightfield microscopy and TEM, which observed classical apoptotic features viz. membrane blebbing, apoptotic bodies and nuclear fragmentation. Also, a significantly increased number of apoptotic cells were enumerated. There was no evidence of autophagic cell death and necrosis. Additionally, a significant increase in 25-hydroxylase (CYP2R1) gene and protein expression was identified in SiHa cells treated with 2600 nM cholecalciferol. Conversely, a significant decrease in 1α-hydroxylase (CYP27B1) gene and protein expression was identified in SiHa cells treated with 2600 nM cholecalciferol. Furthermore, significant increase in both 24-hydroxylase (CYP24A1) and VDR expression at gene and protein levels were observed in 2600 nM experimental SiHa cultures. In conclusion, cholecalciferol exerts growth inhibition and apoptosis in SiHa cells at 2600 nM. This is accompanied by CYP2R1 and VDR upregulation which suggests autocrine activation to calcidiol and intracellular nuclear signalling, respectively. It is therefore hypothesised that calcidiol synthesised de novo binds to VDR and induces apoptosis in SiHa cell line. / Dissertation (MSc (Chemical Pathology))--University of Pretoria, 2020. / Chemical Pathology / MSc (Chemical Pathology) / Restricted

Solar ultraviolet-B (UVB) radiation is capable of converting 7-dehydrocholesterol (7-DHC) to previtamin D3 (preD3), which undergoes thermal isomerization to produce vitamin D3. Further ultraviolet irradiation of preD3 will produce other photoproducts, including lumisterol3, tachysterol3, and 7-DHC. Continued exposure to UVB results in a photoequilibrium of these photoproducts. During the winter months, people living at latitudes greater than 32° north or south are incapable of converting cutaneous 7-DHC to preD3. Because an increased zenith angle creates a longer path-length for UVB radiation to traverse through the atmosphere, ozone can absorb a much greater proportion of this radiation. Given the absorption spectrum of tachysterol3 which absorbs UV radiation up to 340nm, it was hypothesized that winter sunlight which contains UV radiation between 315nm and 340nm would be able to convert tachysterol3 to preD3. Each hour between sunrise and sunset, ampules containing 50g/mL tachysterol3, lumisterol3, and 7-DHC in 100% ethanol were exposed to solar radiation. These samples were chromatographed on a normal phase chromatographic column. Results revealed that tachysterol3 was efficiently converted to preD3 from sunrise to sunset, whereas as 7-DHC and lumisterol3 were not. Exposure of tachysterol3 to sunlight throughout the day revealed that tachysterol3 began converting to preD3 at sunrise at 8am and the peak conversion occurred between 10:00 and 13:00. PreD3 was generated from tachysterol3 until sunset. No preD3 was observed when 7-DHC or lumisterol3 were exposed at the same time. From this data, it is feasible to use tachysterol3 to produce preD3 in a topical preparation during winter.

Der Zusammenhang zwischen Fettleibigkeit und Vitamin-D-Mangel sowie der Fettleibigkeit und der nicht-alkoholischen Fettlebererkrankung wird in der Literatur beschrieben, ist jedoch noch nicht gänzlich geklärt. In der Therapie der NAFLD wird als Ultima ratio eine bariatrische Operation erwogen. In der vorliegenden Arbeit wurde überprüft, welchen Einfluss eine bariatrische Operation auf den Outcome der Leberparameter, des NAFLD-Scores, des Ferritinwertes, des Vitamin-D-Spiegels sowie den Gewichtsverlauf bei Patienten mit NAFLD hat. Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass die meisten statistisch ausgewerteten Parameter hinsichtlich einer Fettlebererkrankung postoperativ besser ausfielen. Es kann somit von einem positiven Einfluss der bariatrischen Operation auf die Fettlebererkrankung ausgegangen werden. In dieser Arbeit kam es zu einer Besserung der Vitamin-D-Spiegel (Gruppe 2 und 4) postoperativ, jedoch bedarf es weiterer größerer Studien um den Zusammenhang zwischen Vitamin-D-Spiegel, der NAFLD und der bariatrischen Operation zu klären. / There is a correlation between obesity and vitamin D deficiency and also between NAFLD and obesity. In this study the effect of bariatric surgery was tested on the outcome of liver parameters, the NAFLD-Score, Ferritin values, vitamin D values and the weight loss after bariatric surgery. In summary, this study was able to show that most of the statistically evaluated parameters regarding fatty liver disease turned out better postoperatively. Thus, a positive influence of bariatric surgery on fatty liver disease can be assumed. In this thesis an improvement of the vitamin D levels (group 2 and 4) postoperatively occurred, but further larger studies are needed to clarify the relationship between vitamin D levels, NAFLD and bariatric surgery.

Vitamin D deficiency (25(OH)D < 20 ng/mL) is likely to be present in about 40% of veterans and is associated with much higher health care costs and service use. The prevalence of vitamin D deficiency is likely to be higher in certain subgroups such as ethnic minorities, those who are chronically ill, and nursing home residents. The lack of adequate sunlight exposure and poor dietary intake are common contributors to this deficient state. Moreover, vitamin D deficiency has also been noted in individuals taking vitamin D supplements within the recommended daily intake. To achieve a 25(OH)D value in the normal range (30-100 ng/mL), many studies indicate a much higher daily oral intake than currently recommended is needed. Inadequate vitamin D dosing may account for failure of some studies to show a benefit. Testing for vitamin D insufficiency levels remains suboptimal and serial monitoring in veterans to assess if a vitamin D-replete state has been achieved also remains less than adequate. The lack of evidence-based guidelines for testing and monitoring has hampered optimal management of this very common condition. The cardiovascular, immunologic, anti-infective, and oncologic benefits of a vitamin D-replete state are becoming recognized. Achieving a vitamin D-replete state may prolong longevity. Achieving adequate vitamin D status in US veterans is an important health measure that should be undertaken.

Vitamin D and calcium play major roles in our bone health in addition to roles in tissues and cells. These roles in tissues and cells are associated with some cancers. The purpose of this study was to determine attitudes of cancer patients towards their dietary intakes of vitamin D and calcium. A questionnaire was completed by 128 volunteers (mean age=53.5 years±16.6, 94 women, 34 men) from the Montgomery Cancer Clinic in Montgomery, Alabama. Cancer-reporting participants (n=59) were likely to agree more (p=0.048) to the statement, “I try to eat healthy every day” compared to non-cancer reporting participants (n=69) on a 5-point Likert scale; similar results occurred for the statement, “I have a healthy diet” (p=0.050). Participants without cancer consumed more fish (salmon, tuna, halibut, etc.) than cancer-reporting participants (p=.035). Women and cancer-reporting participants were more concerned with eating healthy and obtaining vitamin D and calcium than non-cancer participants and men.

Die Arbeit stellt mögliche Einflüsse durch 1,25- Dihydroxy-Vitamin D3 (1,25-VitD3) und Retinsäure (RA) in humanen mesenchymalen Stammzellen (hMSC) sowohl während der adipogenen und osteogenen Differenzierung als auch während der Kurzzeit- und Langzeitstimulation auf das Mikromilieu dar. Die Stimulation mit 1,25-VitD3 und RA verlangsamt das Wachstumsverhalten und verändert die Zellmorphologie von hMSC. Effekte auf die Genexpression werden auf mRNA-Ebene mittels RT-PCR dargestellt. Der Phänotyp als auch teilweise die Genexpression der osteogenen und adipogenen Differenzierung wird durch 1,25-VitD3 induziert und durch RA inhibiert. Zudem wird sowohl die „Mikromilieu-Zusammensetzung“ als auch das „Transkriptionssignal“ von 1,25-VitD3 und RA gegenseitig beeinflusst. / The paper reports about possible effects of 1,25-dihydroxy-vitamin D3 (1,25-VitD3) und retinoic acid (RA) in human mesenchymal stem cells (hMSC) during adipogenic and osteogenic differentiation as well as effects on the microenvironment during a short and long time stimulation. Stimulation with 1,25-VitD3 and RA slows down the growth rate and alters cell morphology of hMSC. Effects on gene expression are shown at the mRNA level by means of RT-PCR. The phenotype and partly the gene expression of adipogenic and osteogenic differentiation are stimulated by 1,25-VitD3 and are inhibited by RA. In addition, both the “microenvironment composition” and the “transcription signal” of 1,25-VitD3 and RA are mutually influenced.

In humans, the epidermis is the main site for the synthesis of Vitamin D3 (cholecalciferol) from 7-dehydrocholesterol. Cholecalciferol undergoes further hydroxylation in the liver and kidney to produce the active form of the circulating hormone 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). In target cells, 1,25(OH)2D3 interacts with the specific intracellular vitamin D receptor (VDR), a member of the nuclear receptor superfamily. However, epidermal keratinocytes, in addition to being target cells, have enzymes required for autocrine production of 1,25(OH)2D3. They can convert cholecalciferol to 1,25(OH)2D3 via 25-hydroxylase (CYP2R1) and 1α-hydroxylase (CYP27B1). Another enzyme, 24-hydroxylase (CYP24A1), regulates local levels by inactivating 1,25(OH)2D3. While recent studies have shown that absence of VDR or 1,25(OH)2D3 impairs formation of granulation tissue during wound healing in mice, little is known about the autocrine and paracrine regulation of biologically active vitamin D3 by human dermal fibroblasts during cutaneous wound healing. Primary cultures of human keratinocytes and fibroblasts expressed VDR and all the cytochrome enzymes necessary for autocrine production of vitamin D. The relative expression of VDR mRNA was higher in dermal fibroblasts than donor-matched keratinocytes. In contrast, epidermal keratinocytes had a higher mRNA expression of vitamin D3 metabolising enzymes. A scratch wound assay confirmed that 1,25(OH)2D3 stimulated keratinocyte migration, but paradoxically inhibited fibroblast migration as early as 4h, yet neither cholecalciferol nor 25-hydroxyvitamin D3 had any effect. VDR knockdown using small interfering RNA (siRNA) abolished the inhibitory effect of 1,25(OH)2D3 on fibroblast migration, demonstrating the requirement for the VDR in this response. Immunofluorescent staining revealed that 1,25(OH)2D3 increased nuclear VDR protein expression, without a corresponding increase in VDR mRNA transcription only in mechanically wounded dermal fibroblasts, indicating activation of the receptors. Incubation with either 1,25(OH)2D3, cholecalciferol or 25(OH)D3 up-regulated CYP24A1 transcription. This response was most pronounced with 1,25(OH)2D3, suggesting a tightly regulated feedback control on 1,25(OH)2D3 bioavailability within the dermis. In addition, cholecalciferol also increased CYP2R1 and CYP27B1 mRNA expression in scratched dermal fibroblasts, providing evidence for autocrine regulation of 1,25(OH)2D3 by dermal fibroblasts. Expression of α-SMA protein was up-regulated in cultured dermal fibroblasts following scratching, which was down-regulated in the presence of 1,25(OH)2D3. These observations suggest that 1,25(OH)2D3 may restrict differentiation of wounded dermal fibroblasts into pro-fibrotic myofibroblasts. 1,25(OH)2D3 also down-regulated MMP-2 secretion and collagen type I to III ratio in scratched dermal fibroblasts. Using a human ex vivo wound healing model, it was demonstrated that 1,25(OH)2D3, but not cholecalciferol, stimulated the rate of wound closure. In summary, this study has confirmed that human dermal fibroblasts express the transcriptional machinery for autocrine production of 1,25(OH)2D3, and a higher VDR expression suggests they are more responsive than keratinocytes. Changes in CYP and VDR expression in the presence of cholecalciferol, 25-hydroxyvitamin D3 or 1,25(OH)2D3 indicate fine-tuning of the bioavailability of vitamin D in the dermis after wounding. Down-regulation of α-SMA, MMP-2 secretion and the collagen type I to III ratio by 1,25(OH)2D3 highlight an important role for 1,25(OH)2D3 in modulating wound healing and the scarring process.

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