Fibroblast Growth Factor Receptor Inhibitors Decrease Proliferation of Melanoma Cell Lines and Their Activity Is Modulated by Vitamin D.

Regardless of the unprecedented progress in malignant melanoma treatment strategies and clinical outcomes of patients during the last twelve years, this skin cancer remains the most lethal one. We have previously documented that vitamin D and its low-calcaemic analogues enhance the anticancer activity of drugs including a classic chemotherapeutic-dacarbazine-and an antiangiogenic VEGFRs inhibitor-cediranib. In this study, we explored the response of A375 and RPMI7951 melanoma lines to CPL304110 (CPL110), a novel selective inhibitor of fibroblast growth factor receptors (FGFRs), and compared its efficacy with that of AZD4547, the first-generation FGFRs selective inhibitor. We also tested whether 1,25(OH)2D3, the active form of vitamin D, modulates the response of the cells to these drugs. CPL304110 efficiently decreased the viability of melanoma cells in both A375 and RPMI7951 cell lines, with the IC50 value below 1 µM. However, the metastatic RPMI7951 melanoma cells were less sensitive to the tested drug than A375 cells, isolated from primary tumour site. Both tested FGFR inhibitors triggered G0/G1 cell cycle arrest in A375 melanoma cells and increased apoptotic/necrotic SubG1 fraction in RPMI7951 melanoma cells. 1,25(OH)2D3 modulated the efficacy of CPL304110, by decreasing the IC50 value by more than 4-fold in A375 cell line, but not in RPMI7951 cells. Further analysis revealed that both inhibitors impact vitamin D signalling to some extent, and this effect is cell line-specific. On the other hand, 1,25(OH)2D3, have an impact on the expression of FGFR receptors and phosphorylation (FGFR-Tyr653/654). Interestingly, 1,25(OH)2D3 and CPL304110 co-treatment resulted in activation of the ERK1/2 pathway in A375 cells. Our results strongly suggested possible crosstalk between vitamin D-activated pathways and activity of FGFR inhibitors, which should be considered in further clinical studies.

Different impact of vitamin D on mitochondrial activity and morphology in normal and malignant keratinocytes, the role of genomic pathway.

Deregulation of mitochondria activity is one of the hallmarks of cancerogenesis and an important target for cancer therapy. Therefore, we compared the impact of an active form of vitamin D3 (1,25(OH)2D3) on mitochondrial morphology and bioenergetics in human squamous cell carcinoma (A431) and immortalized HaCaT keratinocytes. It was shown that mitochondria of cancerous A431 cells differ from that observed in HaCaT keratinocytes in terms of network, morphology, bioenergetics, glycolysis, and mitochondrial DNA copy number, while treatment of A431 with 1,25(OH)2D3 partially eliminates these differences. Furthermore, mitochondrial membrane potential, basal respiration, and mitochondrial reactive oxygen species production were decreased in A431 cells treated with 1,25(OH)2D3. Additionally, the expression and protein level of mitophagy marker PINK1 was significantly increased in A431 1,25(OH)2D3 treated cells, but not observed in treated HaCaT cells. Knockout of VDR (vitamin D receptor) or RXRA (binding partner retinoid X receptor) partially altered mitochondrial morphology and function as well as mitochondrial response to 1,25(OH)2D3. Transcriptomic analysis on A431 cells treated with 1,25(OH)2D3 revealed modulation of expression of several mitochondrial-related genes involved in mitochondrial depolarization, mitochondrial protein translation (i.e. LYRM9, MARS2), and fusion-fission (OPA1, FIS1, MFN1 and 2), however, none of the genes coded by mitochondrial DNA was affected. Interestingly, in silico analyses of nuclear-encoded mitochondrial genes revealed that they are rather activated by the secondary genomic response to 1,25(OH)2D3. Taken together, 1,25(OH)2D3 remodels mitochondrial architecture and bioenergetics through VDR-dependent and only partially RXRA-dependent activation of the genomic pathway, thus outlining a new perspective for anticancer properties of vitamin D3 in relation to mitochondria in squamous cell carcinoma.

Dissection of an impact of VDR and RXRA on the genomic activity of 1,25(OH)2D3 in A431 squamous cell carcinoma.

BACKGROUND: Human skin is the natural source, place of metabolism, and target for vitamin D3. The classical active form of vitamin D3, 1,25(OH)2D3, expresses pluripotent properties and is intensively studied in cancer prevention and therapy. To define the specific role of vitamin D3 receptor (VDR) and its co-receptor retinoid X receptor alpha (RXRA) in genomic regulation, VDR or RXRA genes were silenced in the squamous cell carcinoma cell line A431 and treated with 1,25(OH)2D3 at long incubation time points 24 h/72 h. Extending the incubation time of A431 WT (wild-type) cells with 1,25(OH)2D3 resulted in a two-fold increase in DEGs (differentially expressed genes) and a change in the amount of downregulated from 37% to 53%. VDR knockout led to a complete loss of 1,25(OH)2D3-induced genome-wide gene regulation at 24 h time point, but after 72 h, 20 DEGs were found, of which 75% were downregulated, and most of them belonged to the gene ontology group "immune response". This may indicate the existence of an alternative, secondary response to 1,25(OH)2D3. In contrast, treatment of A431 ΔRXRA cells with 1,25(OH)2D3 for 24 h only partially affected DEGs, suggesting RXRA-independent regulation. Interestingly, overexpression of classic 1,25(OH)2D3 targets, like CYP24A1 (family 24 of subfamily A of cytochrome P450 member 1) or CAMP (cathelicidin antimicrobial peptide) was found to be RXRA-independent. Also, immunofluorescence staining of A431 WT cells revealed partial VDR/RXRA colocalization after 24 h and 72 h 1,25(OH)2D3 treatment. Comparison of transcriptome changes induced by 1,25(OH)2D3 in normal keratinocytes vs. cancer cells showed high cell type specific expression pattern with only a few genes commonly regulated by 1,25(OH)2D3. Activation of the genomic pathway at least partially reversed the expression of cancer-related genes, forming a basis for anti-cancer activates of 1,25(OH)2D3. In summary, VDR or RXRA independent genomic activities of 1,25(OH)2D3 suggest the involvement of alternative factors, opening new challenges in this field.

Protein Disulfide Isomerase Family A Member 3 Knockout Abrogate Effects of Vitamin D on Cellular Respiration and Glycolysis in Squamous Cell Carcinoma.

PDIA3 is an endoplasmic reticulum disulfide isomerase, which is involved in the folding and trafficking of newly synthesized proteins. PDIA3 was also described as an alternative receptor for the active form of vitamin D (1,25(OH)2D3). Here, we investigated an impact of PDIA3 in mitochondrial morphology and bioenergetics in squamous cell carcinoma line A431 treated with 1,25(OH)2D3. It was observed that PDIA3 deletion resulted in changes in the morphology of mitochondria including a decrease in the percentage of mitochondrial section area, maximal diameter, and perimeter. The 1,25(OH)2D3 treatment of A431∆PDIA3 cells partially reversed the effect of PDIA3 deletion increasing aforementioned parameters; meanwhile, in A431WT cells, only an increase in mitochondrial section area was observed. Moreover, PDIA3 knockout affected mitochondrial bioenergetics and modulated STAT3 signaling. Oxygen consumption rate (OCR) was significantly increased, with no visible effect of 1,25(OH)2D3 treatment in A431∆PDIA3 cells. In the case of Extracellular Acidification Rate (ECAR), an increase was observed for glycolysis and glycolytic capacity parameters in the case of non-treated A431WT cells versus A431∆PDIA3 cells. The 1,25(OH)2D3 treatment had no significant effect on glycolytic parameters. Taken together, the presented results suggest that PDIA3 is strongly involved in the regulation of mitochondrial bioenergetics in cancerous cells and modulation of its response to 1,25(OH)2D3, possibly through STAT3.

PDIA3 modulates genomic response to 1,25-dihydroxyvitamin D3 in squamous cell carcinoma of the skin.

An active form of vitamin D3 (1,25-dihydroxyvitamin D3) acts through vitamin D receptor (VDR) initiating genomic response, but several studies described also non-genomic actions of 1,25-dihydroxyvitamin D3, implying the role of PDIA3 in the process. PDIA3 is a membrane-associated disulfide isomerase involved in disulfide bond formation, protein folding, and remodeling. Here, we used a transcriptome-based approach to identify changes in expression profiles in PDIA3-deficient squamous cell carcinoma line A431 after 1,25-dihydroxyvitamin D3 treatment. PDIA3 knockout led to changes in the expression of more than 2000 genes and modulated proliferation, cell cycle, and mobility of cells; suggesting an important regulatory role of PDIA3. PDIA3-deficient cells showed increased sensitivity to 1,25-dihydroxyvitamin D3, which led to decrease migration. 1,25-dihydroxyvitamin D3 treatment altered also genes expression profile of A431ΔPDIA3 in comparison to A431WT cells, indicating the existence of PDIA3-dependent genes. Interestingly, classic targets of VDR, including CAMP (Cathelicidin Antimicrobial Peptide), TRPV6 (Transient Receptor Potential Cation Channel Subfamily V Member 6), were regulated differently by 1,25-dihydroxyvitamin D3, in A431ΔPDIA3. Deletion of PDIA3 impaired 1,25-dihydroxyvitamin D3-response of genes, such as PTGS2, MMP12, and FOCAD, which were identified as PDIA3-dependent. Additionally, response to 1,25-dihydroxyvitamin D3 in cancerous A431 cells differed from immortalized HaCaT keratinocytes, used as non-cancerous control. Finally, silencing of PDIA3 and 1,25-dihydroxyvitamin D3, at least partially reverse the expression of cancer-related genes in A431 cells, thus targeting PDIA3 and use of 1,25-dihydroxyvitamin D3 could be considered in a prevention and therapy of the skin cancer. Taken together, PDIA3 has a strong impact on gene expression and physiology, including genomic response to 1,25-dihydroxyvitamin D3.

VDR and PDIA3 Are Essential for Activation of Calcium Signaling and Membrane Response to 1,25(OH)2D3 in Squamous Cell Carcinoma Cells.

The genomic activity of 1,25(OH)2D3 is mediated by vitamin D receptor (VDR), whilst non-genomic is associated with protein disulfide isomerase family A member 3 (PDIA3). Interestingly, our recent studies documented that PDIA3 is also involved, directly or indirectly, in the modulation of genomic response to 1,25(OH)2D3. Moreover, PDIA3 was also shown to regulate cellular bioenergetics, possibly through the modulation of STAT signaling. Here, the role of VDR and PDIA3 proteins in membrane response to 1,25(OH)2D3 and calcium signaling was investigated in squamous cell carcinoma A431 cell line with or without the deletion of VDR and PDIA3 genes. Calcium influx was assayed by Fura-2AM or Fluo-4AM, while calcium-regulated element (NFAT) activation was measured using a dual luciferase assay. Further, the levels of proteins involved in membrane response to 1,25(OH)2D3 in A431 cell lines were analyzed via Western blot analysis. The deletion of either PDIA3 or VDR resulted in the decreased baseline levels of Ca2+ and its responsiveness to 1,25(OH)2D3; however, the effect was more pronounced in A431∆PDIA3. Furthermore, the knockout of either of these genes disrupted 1,25(OH)2D3-elicited membrane signaling. The data presented here indicated that the VDR is essential for the activation of calcium/calmodulin-dependent protein kinase II alpha (CAMK2A), while PDIA3 is required for 1,25(OH)2D3-induced calcium mobilization in A431 cells. Taken together, those results suggest that both VDR and PDIA3 are essential for non-genomic response to this powerful secosteroid.

Current and Future Perspectives for Chimeric Antigen Receptor T Cells Development in Poland.

Chimeric antigen receptor T (CAR-T) cells are genetically modified autologous T cells that have revolutionized the treatment of relapsing and refractory haematological malignancies. In this review we present molecular pathways involved in the activation of CAR-T cells, describe in details the structures of receptors and the biological activity of CAR-T cells currently approved for clinical practice in the European Union, and explain the functional differences between them. Finally, we present the potential for the development of CAR-T cells in Poland, as well as indicate the possible directions of future research in this area, including novel modifications and applications of CAR-T cells and CAR-natural killer (NK) cells.

Diet, Sun, Physical Activity and Vitamin D Status in Children with Inflammatory Bowel Disease.

In the course of inflammatory bowel disease (IBD) malabsorption may lead to a vitamin D deficiency and calcium-phosphate misbalance. However, the reports on the vitamin D status in children with IBD are few and ambiguous. Here, we are presenting complex analyses of multiple factors influencing 25OHD levels in IBD children (N = 62; Crohn's disease n = 34, ulcerative colitis n = 28, mean age 14.4 ± 3.01 years, F/M 23/39) and controls (n = 47, mean age 13.97 ± 2.57, F/M 23/24). Additionally, calcium-phosphate balance parameters and inflammatory markers were obtained. In children with IBD disease, activity and location were defined. Information about therapy, presence of fractures and abdominal surgery were obtained from medical records. All subjects were surveyed on the frequency and extent of exposure to sunlight (forearms, partially legs for at least 30 min a day), physical activity (at least 30 min a day) and diet (3 days diary was analyzed with the program DIETA 5). The mean 25OHD level was higher in IBD patients compared to controls (18.1 ng/mL vs. 15.5 ng/mL; p = 0.03). Only 9.7% of IBD patients and 4.25% of controls had the optimal vitamin D level (30-50 ng/mL). Despite the higher level of 25OHD, young IBD patients showed lower calcium levels in comparison to healthy controls. There was no correlation between the vitamin D level and disease activity or location of gastrointestinal tract lesions. Steroid therapy didn't have much influence on the vitamin D level while vitamin D was supplemented. Regular sun exposure was significantly more common in the control group compared to the IBD group. We found the highest concentration of vitamin D (24.55 ng/mL) with daily sun exposure. There was no significant correlation between the vitamin D level and frequency of physical activity. The analysis of dietary diaries showed low daily intake of vitamin D in both the IBD and the control group (79.63 vs. 85.14 IU/day). Pediatric patients, both IBD and healthy individuals, require regular monitoring of serum vitamin D level and its adequate supplementation.

Mitochondrial potassium channels: A novel calcitriol target.

BACKGROUND: Calcitriol (an active metabolite of vitamin D) modulates the expression of hundreds of human genes by activation of the vitamin D nuclear receptor (VDR). However, VDR-mediated transcriptional modulation does not fully explain various phenotypic effects of calcitriol. Recently a fast non-genomic response to vitamin D has been described, and it seems that mitochondria are one of the targets of calcitriol. These non-classical calcitriol targets open up a new area of research with potential clinical applications. The goal of our study was to ascertain whether calcitriol can modulate mitochondrial function through regulation of the potassium channels present in the inner mitochondrial membrane. METHODS: The effects of calcitriol on the potassium ion current were measured using the patch-clamp method modified for the inner mitochondrial membrane. Molecular docking experiments were conducted in the Autodock4 program. Additionally, changes in gene expression were investigated by qPCR, and transcription factor binding sites were analyzed in the CiiiDER program. RESULTS: For the first time, our results indicate that calcitriol directly affects the activity of the mitochondrial large-conductance Ca2+-regulated potassium channel (mitoBKCa) from the human astrocytoma (U-87 MG) cell line but not the mitochondrial calcium-independent two-pore domain potassium channel (mitoTASK-3) from human keratinocytes (HaCaT). The open probability of the mitoBKCa channel in high calcium conditions decreased after calcitriol treatment and the opposite effect was observed in low calcium conditions. Moreover, using the AutoDock4 program we predicted the binding poses of calcitriol to the calcium-bound BKCa channel and identified amino acids interacting with the calcitriol molecule. Additionally, we found that calcitriol influences the expression of genes encoding potassium channels. Such a dual, genomic and non-genomic action explains the pleiotropic activity of calcitriol. CONCLUSIONS: Calcitriol can regulate the mitochondrial large-conductance calcium-regulated potassium channel. Our data open a new chapter in the study of non-genomic responses to vitamin D with potential implications for mitochondrial bioenergetics and cytoprotective mechanisms.

The Effects of Vitamin D on the Expression of IL-33 and Its Receptor ST2 in Skin Cells; Potential Implication for Psoriasis.

Interleukin 33 (IL-33) belongs to the IL-1 family and is produced constitutively by epithelial and endothelial cells of various organs, such as the skin. It takes part in the maintenance of tissue homeostasis, repair, and immune response, including activation of Th2 lymphocytes. Its involvement in pathogenesis of several inflammatory diseases including psoriasis was also suggested, but this is not fully understood. The aim of the study was to investigate expression of IL-33 and its receptor, ST2, in psoriasis, and the effects of the active form of vitamin D (1,25(OH)2D3) on their expression in skin cells. Here we examined mRNA and protein profiles of IL-33 and ST2 in 18 psoriatic patients and healthy volunteers by qPCR and immunostaining techniques. Potential effects of 1,25(OH)2D3 and its receptor (VDR) on the expression of IL-33 and ST2 were tested in cultured keratinocytes, melanocytes, fibroblasts, and basal cell carcinoma cells. It was shown that 1,25(OH)2D3 effectively stimulated expression of IL-33 and its receptor ST2's mRNAs in a time-dependent manner, in keratinocytes and to the lesser extends in melanocytes, but not in fibroblasts. Furthermore, the effect of vitamin D on expression of IL-33 and ST2 was VDR-dependent. Finally, we demonstrated that the expression of mRNA for IL-33 was mainly elevated in the psoriatic skin but not in its margin. Interestingly, ST2 mRNA was downregulated in psoriatic lesion compared to both marginal tissue as well as healthy skin. Our data indicated that vitamin D can modulate IL-33 signaling, opening up new perspectives for our understanding of the mechanism of vitamin D action in psoriasis therapy.

Modulation of dermal equivalent of hypothalamus-pituitary-adrenal axis in mastocytosis.

INTRODUCTION: Mastocytosis is a rare disease characterized by abnormal growth and accumulation of tissue mast cells (MC) in one or more organ systems and is classified as being either cutaneous mastocytosis (CM) or systemic mastocytosis (SM). In the pioneer studies of Slominski's group, a fully functional hypothalamic-pituitary-adrenal axis equivalent has been discovered in various tissues, including skin. AIM: In the present study we investigated potential involvement of hypothalamus-pituitary-adrenal (HPA) cutaneous equivalent in ongoing mastocytosis. MATERIAL AND METHODS: The expression of HPA elements: CRH, UCN1, UCN2, UCN3, CRHR1, POMC, MC1R, MC2R and NR3C1 was assessed for their mRNA level in skin biopsies of adult patients with mastocytosis and healthy donors (n = 16 and 19, respectively), while CRH, UCN1, CRHR1, ACTH and MC1R were selected for immunostaining assay (n = 13 and 7, respectively). The expression of CRH receptor 1 (CRHR1) isomers was investigated by RT-PCR. The ELISA was used for detection of cortisol, CRH, UCN and ACTH in the serum. RESULTS: The decrease in the expression of HPA element of skin equivalent was observed on both mRNA and protein levels, however quantification of immunohistochemical data was impeded due to melanin in epidermis. Furthermore, we observed infiltration of dermis with HPA elements overexpressing mononuclear cells, which is in the agreement with an in vitro study showing a high expression of HPA elements by mast cells. CONCLUSIONS: Taken together, it was confirmed that the expression elements of HPA was modulated in mastocytosis, thus the potential involvement of general and local stress responses in its pathogenesis should be postulated and further investigated.

Pathogenesis of psoriasis in the "omic" era. Part IV. Epidemiology, genetics, immunopathogenesis, clinical manifestation and treatment of psoriatic arthritis.

Psoriatic arthritis (PsA) is a chronic, progressive, inflammatory arthropathy associated with psoriasis as well as a complex pathogenesis. Genetic and environmental factors trigger the development of the immune-mediated auto-inflammatory response in different sites: skin, bone marrow, entheses and synovial tissues. Studies of the last two decades have changed the view of PsA from a mild, non-progressive arthritis to an inflammatory systemic disease with serious health consequences, not only associated with joint dysfunction, but also with an increased risk of cardiovascular disease and socioeconomic consequences with significantly reduced quality of life. The joint damage starts early in the course of the disease, thus early recognition and treatment with modern biological treatments, which may modify the natural history and slow down progression of this debilitating disease, is essential for the patient long-term outcome.

The Role of Classical and Novel Forms of Vitamin D in the Pathogenesis and Progression of Nonmelanoma Skin Cancers.

Nonmelanoma skin cancers including basal and squamous cell carcinomas (SCC and BCC) represent a significant clinical problem due to their relatively high incidence, imposing an economic burden to healthcare systems around the world. It is accepted that ultraviolet radiation (UVR: λ = 290-400 nm) plays a crucial role in the initiation and promotion of BCC and SCC with UVB (λ = 290-320 nm) having a central role in this process. On the other hand, UVB is required for vitamin D3 (D3) production in the skin, which supplies >90% of the body's requirement for this prohormone. Prolonged exposure to UVB can also generate tachysterol and lumisterol. Vitamin D3 itself and its canonical (1,25(OH)2D3) and noncanonical (CYP11A1-intitated) D3 hydroxyderivatives show photoprotective functions in the skin. These include regulation of keratinocyte proliferation and differentiation, induction of anti-oxidative responses, inhibition of DNA damage and induction of DNA repair mechanisms, and anti-inflammatory activities. Studies in animals have demonstrated that D3 hydroxyderivatives can attenuate UVB or chemically induced epidermal cancerogenesis and inhibit growth of SCC and BCC. Genomic and non-genomic mechanisms of action have been suggested. In addition, vitamin D3 itself inhibits hedgehog signaling pathways which have been implicated in many cancers. Silencing of the vitamin D receptor leads to increased propensity to develop UVB or chemically induced epidermal cancers. Other targets for vitamin D compounds include 1,25D3-MARRS, retinoic orphan receptors α and γ, aryl hydrocarbon receptor, and Wnt signaling. Most recently, photoprotective effects of lumisterol hydroxyderivatives have been identified. Clinical trials demonstrated a beneficial role of vitamin D compounds in the treatment of actinic keratosis. In summary, recent advances in vitamin D biology and pharmacology open new exciting opportunities in chemoprevention and treatment of skin cancers.

Vitamin D and its analogs as anticancer and anti-inflammatory agents.

Vitamin D3 is the precursor of the steroid hormone calcitriol (1α,25-dihydroxyvitamin D3), a potent agonist of the transcription factor vitamin D receptor (VDR). Calcitriol has received large appreciation due to the impact not only on mineral homeostasis, but also on metabolic diseases, immunological disorders, and cancer. A supraphysiological dose of calcitriol is required to reduce the proliferation of cancer cells. However, the dose will lead to calcemic side effects such as hypercalcemia and hypercalciuria. Although several analogs of 1α,25(OH)2D3 have shown potent anticancer or anti-inflammatory activity on cell cultures or in animal models, few vitamin D analogs have been successfully applied in the treatment of cancer or inflammatory diseases. This review will present and discuss vitamin D analogs that have the potential to be used as anticancer or anti-inflammatory agents.

Photoprotective Properties of Vitamin D and Lumisterol Hydroxyderivatives.

We have previously described new pathways of vitamin D3 activation by CYP11A1 to produce a variety of metabolites including 20(OH)D3 and 20,23(OH)2D3. These can be further hydroxylated by CYP27B1 to produce their C1α-hydroxyderivatives. CYP11A1 similarly initiates the metabolism of lumisterol (L3) through sequential hydroxylation of the side chain to produce 20(OH)L3, 22(OH)L3, 20,22(OH)2L3 and 24(OH)L3. CYP11A1 also acts on 7-dehydrocholesterol (7DHC) producing 22(OH)7DHC, 20,22(OH)27DHC and 7-dehydropregnenolone (7DHP) which can be converted to the D3 and L3 configurations following exposure to UVB. These CYP11A1-derived compounds are produced in vivo and are biologically active displaying anti-proliferative, anti-inflammatory, anti-cancer and pro-differentiation properties. Since the protective role of the classical form of vitamin D3 (1,25(OH)2D3) against UVB-induced damage is recognized, we recently tested whether novel CYP11A1-derived D3- and L3-hydroxyderivatives protect against UVB-induced damage in epidermal human keratinocytes and melanocytes. We found that along with 1,25(OH)2D3, CYP11A1-derived D3-hydroxyderivatives and L3 and its hydroxyderivatives exert photoprotective effects. These included induction of intracellular free radical scavenging and attenuation and repair of DNA damage. The protection of human keratinocytes against DNA damage included the activation of the NRF2-regulated antioxidant response, p53-phosphorylation and its translocation to the nucleus, and DNA repair induction. These data indicate that novel derivatives of vitamin D3 and lumisterol are promising photoprotective agents. However, detailed mechanisms of action, and the involvement of specific nuclear receptors, other vitamin D binding proteins or mitochondria, remain to be established.

Antiproliferative activity of side-chain truncated vitamin D analogs (PRI-1203 and PRI-1204) against human malignant melanoma cell lines.

Vitamin D compounds are versatile molecules widely considered as promising agents in cancer prevention and treatment, including melanoma. Previously we investigated series of double point modified vitamin D2 analogs as well as non-calcemic 20S-hydroxyvitamin D3 and 21-hydroxypregnacalciferol as to their anti-melanoma activity. Surprisingly, short side-chain vitamin D analogs were found to be biologically active compounds. Thus, here we tested novel derivatives of pregnacalciferol with an additional hydroxyl at the end of the truncated side chain, PRI-1203 and PRI-1204, as to their potency against human melanoma A375 and RPMI7951 cell lines. Tested compounds are geometric isomers, with 19-methylene positioned in PRI-1203 like in a calcitriol molecule, but reversed in the PRI-1204 analog to the (5E,7E) geometry (5,6-trans). We noticed a decrease in cells viability exerted by PRI-1203. The antiproliferative effect of PRI-1204 was very low, emphasizing the importance of the natural 19-methylene geometry in the PRI-1203. PRI-1203 was also effective in inhibition of A375 melanoma cells migration. PRI-1203, but not PRI-1204, increased the percentage of A375 and RPMI7951 melanoma cells in the G0/G1 phase of cell cycle, possibly in a p21 and p27 independent manner. Both, analogs have very low effect on the level of CYP24A1 mRNA, in comparison to active form of vitamin D - 1.25(OH)2D3. In addition, both tested compounds failed to elicit VDR translocation to the nucleus. Thus, it could be postulated that side chain shortening strongly affects binding of analogs to VDR and activation of genomic responses, however do not impair their antiproliferative activities.

The Major Heat Shock Proteins, Hsp70 and Hsp90, in 2-Methoxyestradiol-Mediated Osteosarcoma Cell Death Model.

2-Methoxyestradiol is one of the natural 17ß-estradiol derivatives and a potential novel anticancer agent currently being under evaluation in advanced phases of clinical trials. However, the mechanism of anticancer action of 2-methoxyestradiol has not been yet fully established. In our previous studies we have demonstrated that 2-methoxyestradiol selectively induces the expression and nuclear translocation of neuronal nitric oxide synthase in osteosarcoma 143B cells. Heat shock proteins (Hsps) are factors involved in the regulation of expression and activity of nitric oxide synthases. Herein, we chose osteosarcoma cell lines differed in metastatic potential, metastatic 143B and highly metastatic MG63.2 cells, in order to further investigate the anticancer mechanism of 2-methoxyestradiol. The current study aimed to determine the role of major heat shock proteins, Hsp90 and Hsp70 in 2-methoxyestradiol-induced osteosarcoma cell death. We focused on the implication of Hsp90 and Hsp70 in control under expression of neuronal nitric oxide synthase, localization of the enzyme, and further generation of nitro-oxidative stress. To give the insight into the role of Hsp90 in regulation of anticancer efficacy of 2-methoxyestradiol, we used geldanamycin as a potent Hsp90 inhibitor. Herein, we evidenced that inhibition of Hsp90 controls the protein expression of 2-methoxyestradiol-induced neuronal nitric oxide synthase and inhibits enzyme nuclear translocation. We propose that decreased level of neuronal nitric oxide synthase protein after a combined treatment with 2-methoxyestradiol and geldanamycin is directly associated with the accompanying upregulation of Hsp70 and downregulation of Hsp90. This interaction resulted in abrogation of anticancer efficacy of 2-methoxyestradiol by geldanamycin.

Vitamin D and its low calcemic analogs modulate the anticancer properties of cisplatin and dacarbazine in the human melanoma A375 cell line.

Melanoma represents a significant challenge in cancer treatment due to the high drug resistance of melanomas and the patient mortality rate. This study presents data indicating that nanomolar concentrations of the hormonally active form of vitamin D, 1α,25­dihydroxyvitamin D3 [1α,25(OH)2D3], its non­calcemic analogues 20S­hydroxyvitamin D3 and 21­hydroxypregnacalciferol, as well as the low­calcemic synthetic analog calcipotriol, modulate the efficacy of the anticancer drugs cisplatin and dacarbazine. It was observed that vitamin D analogs sensitized melanoma A375 cells to hydrogen peroxide used as an inducer of oxidative stress. On the other hand, only 1α,25(OH)2D3 resulted in a minor, but significant effect on the proliferation of melanoma cells treated simultaneously with dacarbazine, but not cisplatin. Notably, cisplatin (300 µM) exhibited a higher overall antiproliferative activity than dacarbazine. Cisplatin treatment of melanoma cells resulted in an induction of apoptosis as demonstrated by flow cytometry (accumulation of cells at the subG1 phase of the cell cycle), whereas dacarbazine caused G1/G0 cell cycle arrest, with the effects being improved by pre­treatment with vitamin D analogs. Treatment with cisplatin resulted in an initial increase in the level of reactive oxygen species (ROS). Dacarbazine caused transient stimulation of ROS levels and the mitochondrial membrane potential (Δψm) (after 1 or 3 h of treatment, respectively), but the effect was not detectable following prolonged (24 h) incubation with the drug. Vitamin D exhibited modulatory effects on the cells treated with dacarbazine, decreasing the half maximal inhibitory concentration (IC50) for the drug, stimulating G1/G0 arrest and causing a marked decrease in Δψm. Finally, cisplatin, dacarbazine and 1α,25(OH)2D3 displayed modulatory effects on the expression of ROS and vitamin D­associated genes in the melanoma A375 cells. In conclusion, nanomolar concentrations of 1,25(OH)2D3 only had minor effects on the proliferation of melanoma cells treated with dacarbazine, decreasing the relative IC50 value. However, co­treatment with vitamin D analogs resulted in the modulation of cell cycle and ROS responses, and affected gene expression, suggesting possible crosstalk between the signaling pathways of vitamin D and the anticancer drugs used in this study.

Vitamin D and Human Health.

Vitamin D is currently one of the hottest topics in research and clinics, as well as in everyday life. Over the past decades, scientists gathered overwhelming evidence indicating that the observed global vitamin D deficiency not only has a negative impact on human skeletal system, but also facilitates development and progression of multiple disease of civilization, including cardiovascular diseases, diabetes, autoimmune disease, and cancer. This Special Issue, entitled "Vitamin D and Human Health", summarizes recent advances in our understanding of pleiotropic activity of vitamin D in the form of eight comprehensive reviews. Furthermore, eight research papers provide new insight into vitamin D research and highlight new directions.

2-Methoxyestradiol and Its Combination with a Natural Compound, Ferulic Acid, Induces Melanoma Cell Death via Downregulation of Hsp60 and Hsp90.

Melanoma is an aggressive type of skin cancer with one of the highest mortality rates. Notably, its incidence in the last few decades has increased faster than any other cancer. Therefore, searching for novel anticancer therapies is of great clinical importance. In the present study, we investigated the anticancer potential of 2-methoxyestradiol, potent chemotherapeutic, in the A375 melanoma cellular model. In order to furthermore evaluate the anticancer efficacy of 2-methoxyestradiol, we have additionally combined the treatment with a naturally occurring polyphenol, ferulic acid. The results were obtained using the melanoma A375 cellular model. In the study, we used MTT assay, flow cytometry, and western blot techniques. Herein, we have evidenced that the molecular mechanism of action of 2-methoxyestradiol and ferulic acid is partly related to the reduction of Hsp60 and Hsp90 levels and the induction of nitric oxide in the A375 melanoma cell model, while no changes were observed in Hsp70 expression after 2-methoxyestradiol and ferulic acid treatment separately or in combination. This is especially important in case of chemoresistance mechanisms because the accumulation of Hsp70 reduces induction of cancer cell death, thus decreasing antitumour efficacy.