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.

Size matters: the impact of nucleus size on results from spatial transcriptomics.

BACKGROUND: Visium Spatial Gene Expression (ST) is a method combining histological spatial information with transcriptomics profiles directly from tissue sections. The use of spatial information has made it possible to discover new modes of gene expression regulations. However, in the ST experiment, the nucleus size of cells may exceed the thickness of a tissue slice. This may, in turn, negatively affect comprehensive capturing the transcriptomics profile in a single slice, especially for tissues having large differences in the size of nuclei. METHODS: Here, we defined the effect of Consecutive Slices Data Integration (CSDI) on unveiling accurate spot clustering and deconvolution of spatial transcriptomic spots in human postmortem brains. By considering the histological information as reference, we assessed the improvement of unsupervised clustering and single nuclei RNA-seq and ST data integration before and after CSDI. RESULTS: Apart from the escalated number of defined clusters representing neuronal layers, the pattern of clusters in consecutive sections was concordant only after CSDI. Besides, the assigned cell labels to spots matches the histological pattern of tissue sections after CSDI. CONCLUSION: CSDI can be applied to investigate consecutive sections studied with ST in the human cerebral cortex, avoiding misinterpretation of spot clustering and annotation, increasing accuracy of cell recognition as well as improvement in uncovering the layers of grey matter in the human brain.

Assessment of the Potential Role of Selected Single Nucleotide Polymorphisms (SNPs) of Genes Related to the Functioning of Regulatory T Cells in the Pathogenesis of Psoriasis.

Recent studies have indicated a key role of the impaired suppressive capacity of regulatory T cells (Tregs) in psoriasis (PsO) pathogenesis. However, the genetic background of Treg dysfunctions remains unknown. The aim of this study was to evaluate the association of PsO development with selected single nucleotide polymorphisms (SNPs) of genes in which protein products play a significant role in the regulation of differentiation and function of Tregs. There were three study groups in our research and each consisted of different unrelated patients and controls: 192 PsO patients and 5605 healthy volunteers in the microarray genotyping group, 150 PsO patients and 173 controls in the ARMS-PCR method group, and 6 PsO patients and 6 healthy volunteers in the expression analysis group. The DNA microarrays analysis (283 SNPs of 57 genes) and ARMS-PCR method (8 SNPs in 7 genes) were used to determine the frequency of occurrence of SNPs in selected genes. The mRNA expression of selected genes was determined in skin samples. There were statistically significant differences in the allele frequencies of four SNPs in three genes (TNF, IL12RB2, and IL12B) between early-onset PsO patients and controls. The lowest p-value was observed for rs3093662 (TNF), and the G allele carriers had a 2.73 times higher risk of developing early-onset PsO. Moreover, the study revealed significant differences in the frequency of SNPs and their influence on PsO development between early- and late-onset PsO. Based on the ARMS-PCR method, the association between some polymorphisms of four genes (IL4, IL10, TGFB1, and STAT3) and the risk of developing PsO was noticed. Psoriatic lesions were characterized with a lower mRNA expression of FOXP3, CTLA4, and IL2, and a higher expression of TNF and IL1A in comparison with unaffected skin. In conclusion, the genetic background associated with properly functioning Tregs seems to play a significant role in PsO pathogenesis and could have diagnostic value.

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.

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.

Vitamin D Enhances Anticancer Properties of Cediranib, a VEGFR Inhibitor, by Modulation of VEGFR2 Expression in Melanoma Cells.

Regardless of the recent groundbreaking introduction of personalized therapy, melanoma continues to be one of the most lethal skin malignancies. Still, a substantial proportion of patients either fail to respond to the therapy or will relapse over time, representing a challenging clinical problem. Recently, we have shown that vitamin D enhances the effectiveness of classical chemotherapeutics in the human malignant melanoma A375 cell line. In search for new combination strategies and adjuvant settings to improve melanoma patient outcomes in the current study, the effects of cediranib (AZD2171), an oral tyrosine kinase inhibitor of VEGFR1-3, PDGFR, and c-KIT, used in combination either with 1,25(OH)2D3 or with low-calcemic analog calcipotriol were tested on four human malignant melanoma cell lines (A375, MNT-1, RPMI-7951, and SK-MEL-28). Melanoma cells were pretreated with vitamin D and subsequently exposed to cediranib. We observed a marked decrease in melanoma cell proliferation (A375 and SK-MEL-28), G2/M cell cycle arrest, and a significant decrease in melanoma cell mobility in experimental conditions used (A375). Surprisingly, concurrently with a very desirable decrease in melanoma cell proliferation and mobility, we noticed the upregulation of VEGFR2 at both protein and mRNA levels. No effect of vitamin D was observed in MNT-1 and RPMI-7951 melanoma cells. It seems that vitamin D derivatives enhance cediranib efficacy by modulation of VEGFR2 expression in melanoma cells expressing VEGFR2. In conclusion, our experiments demonstrated that vitamin D derivatives hold promise as novel adjuvant candidates to conquer melanoma, especially in patients suffering from vitamin D deficiency. However, further extensive research is indispensable to reliably assess their potential benefits for melanoma patients.

Inhibition of Shh Signaling through MAPK Activation Controls Chemotherapy-Induced Alopecia.

Chemotherapy-induced hair loss (alopecia) (CIA) remains a major unsolved problem in clinical oncology. CIA is often considered to be a consequence of the antimitotic and apoptosis-promoting properties of chemotherapy drugs acting on rapidly proliferating hair matrix keratinocytes. Here, we show that in a mouse model of CIA, the downregulation of Shh signaling in the hair matrix is a critical early event. Inhibition of Shh signaling recapitulated key morphological and functional features of CIA, whereas recombinant Shh protein partially rescued hair loss. Phosphoproteomics analysis revealed that activation of the MAPK pathway is a key upstream event, which can be further manipulated to rescue CIA. Finally, in organ-cultured human scalp hair follicles as well as in patients undergoing chemotherapy, reduced expression of SHH gene correlates with chemotherapy-induced hair follicle damage or the degree of CIA, respectively. Our work revealed that Shh signaling is an evolutionarily conserved key target in CIA pathobiology. Specifically targeting the intrafollicular MAPK-Shh axis may provide a promising strategy to manage CIA.

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.

Antiproliferative Activity of Non-Calcemic Vitamin D Analogs on Human Melanoma Lines in Relation to VDR and PDIA3 Receptors.

Vitamin D is a precursor for secosteroidal hormones, which demonstrate pleiotropic biological activities, including the regulation of growth and the differentiation of normal and malignant cells. Our previous studies have indicated that the inhibition of melanoma proliferation by a short side-chain, low calcemic analog of vitamin D-21(OH)pD is not fully dependent on the expression of vitamin D receptor (VDR). We have examined the effects of classic vitamin D metabolites, 1,25(OH)2D3 and 25(OH)D3, and two low calcemic vitamin D analogs, (21(OH)pD and calcipotriol), on proliferation, mRNA expression and vitamin D receptor (VDR) translocation in three human melanoma cell lines: WM98, A375 and SK-MEL-188b (subline b of SK-MEL-188, which lost responsiveness to 1,25(OH)2D3 and became VDR-/-CYP27B1-/-). All tested compounds efficiently inhibited the proliferation of WM98 and A375 melanoma cells except SK-MEL-188b, in which only the short side-chain vitamin D analog-21(OH)pD was effective. Overall, 21(OH)pD was the most potent compound in all three melanoma cell lines in the study. The lack of responsiveness of SK-MEL-188b to 1,25(OH)2D3, 25(OH)D3 and calcipotriol is explained by a lack of characteristic transcripts for the VDR, its splicing variants as well as for vitamin D-activating enzyme CYP27B1. On the other hand, the expression of VDR and its splicing variants and other vitamin D related genes (RXR, PDIA3, CYP3A4, CYP2R1, CYP27B1, CYP24A1 and CYP11A1) was detected in WM98 and A375 melanomas with the transcript levels being modulated by vitamin D analogs. The expression of VDR isoforms in WM98 cells was stimulated strongly by calcipotriol. The antiproliferative activities of 21(OH)pD appear not to require VDR translocation to the nucleus, which explains the high efficacy of this noncalcemic pregnacalciferol analog in SK-MEL-188b melanoma, that is, VDR-/-. Therefore, we propose that 21(OH)pD is a good candidate for melanoma therapy, although the mechanism of its action remains to be defined.

Role of Oxidative and Nitro-Oxidative Damage in Silver Nanoparticles Cytotoxic Effect against Human Pancreatic Ductal Adenocarcinoma Cells.

Pancreatic ductal adenocarcinoma is one of the most aggressive human malignancies, where the 5-year survival rate is less than 4% worldwide. Successful treatment of pancreatic cancer is a challenge for today's oncology. Several studies showed that increased levels of oxidative stress may cause cancer cells damage and death. Therefore, we hypothesized that oxidative as well as nitro-oxidative stress is one of the mechanisms inducing pancreatic cancer programmed cell death. We decided to use silver nanoparticles (AgNPs) (2.6 and 18 nm) as a key factor triggering the reactive oxygen species (ROS) and reactive nitrogen species (RNS) in pancreatic ductal adenocarcinoma cells (PANC-1). Previously, we have found that AgNPs induced PANC-1 cells death. Furthermore, it is known that AgNPs may induce an accumulation of ROS and alteration of antioxidant systems in different type of tumors, and they are indicated as promising agents for cancer therapy. Then, the aim of our study was to evaluate the implication of oxidative and nitro-oxidative stress in this cytotoxic effect of AgNPs against PANC-1 cells. We determined AgNP-induced increase of ROS level in PANC-1 cells and pancreatic noncancer cell (hTERT-HPNE) for comparison purposes. We found that the increase was lower in noncancer cells. Reduction of mitochondrial membrane potential and changes in the cell cycle were also observed. Additionally, we determined the increase in RNS level: nitric oxide (NO) and nitric dioxide (NO2) in PANC-1 cells, together with increase in family of nitric oxide synthases (iNOS, eNOS, and nNOS) at protein and mRNA level. Disturbance of antioxidant enzymes: superoxide dismutase (SOD1, SOD2, and SOD3), glutathione peroxidase (GPX-4) and catalase (CAT) were proved at protein and mRNA level. Moreover, we showed cells ultrastructural changes, characteristic for oxidative damage. Summarizing, oxidative and nitro-oxidative stress and mitochondrial disruption are implicated in AgNPs-mediated death in human pancreatic ductal adenocarcinoma cells.

Differentiation of Keratinocytes Modulates Skin HPA Analog.

It is well established, that epidermal keratinocytes express functional equivalent of hypothalamus-pituitary-adrenal axis (HPA) in order to respond to changing environment and maintain internal homeostasis. We are presenting data indicating that differentiation of primary neonatal human keratinocytes (HPEKp), induced by prolonged incubation or calcium is accompanied by significant changes in the expression of the elements of skin analog of HPA (sHPA). Expression of CRF, UCN1-3, POMC, ACTH, CRFR1, CRFR2, MC1R, MC2R, and GR (coded by NR3C1 gene) were observed on gene/protein levels along differentiation of keratinocytes in culture with similar pattern seen by immunohistochemistry on full thickness skin biopsies. Expression of CRF was more pronounced in less differentiated keratinocytes, which corresponded to the detection of CRF immunoreactivity preferentially in the stratum basale. POMC expression was enhanced in more differentiated keratinocytes, which corresponded to detection of ACTH immunoreactivity, predominantly in the stratum spinosum and stratum granulosum. Expression of urocortins was also affected by induction of HPEKp differentiation. Immunohistochemical studies showed high prevalence of CRFR1 in well differentiated keratinocytes, while smaller keratinocytes showed predominantly CRFR2 immunoreactivity. MC2R mRNA levels were elevated from days 4 to 8 of in vitro incubation, while MC2R immunoreactivity was the highest in the upper layers of epidermis. Similar changes in mRNA/protein levels of sHPA elements were observed in HPEKp keratinocytes treated with calcium. Summarizing, preferential expression of CRF and POMC (ACTH) by populations of keratinocytes on different stage of differentiation resembles organization of central HPA axis suggesting their distinct role in physiology and pathology of the epidermis. J. Cell. Physiol. 232: 154-166, 2017. © 2016 Wiley Periodicals, Inc.

Assessment of Interleukin 16 Serum Levels and Skin Expression in Psoriasis Patients in Correlation with Clinical Severity of the Disease.

Interleukin 16 (IL-16) has been described as a significant cytokine involved in the recruitment of CD4+ cells during inflammation; however, its potential role in psoriasis has not been defined. Our aim was to investigate the IL-16 serum levels and IL-16 mRNA skin expression in psoriasis patients in correlation with disease severity and mRNA skin expression for CD4. Moreover, the IL-16 skin localization was assessed and the -295 T/C IL-16 polymorphism was analyzed. For this exploratory, observational, and cross-sectional study, 97 unrelated patients with chronic plaque type psoriasis and 104 healthy controls were enrolled. IL-16 serum levels were significantly increased in patients compared with controls (P = 0.000022) and positively correlated with Psoriasis Area and Severity Index (r = 0.34, P = 0.0007), Body Surface Area (r = 0.34, P = 0.01) and were significantly higher in individuals with moderate to severe psoriasis (P = 0.0029). There was no significant correlation between IL-16 serum levels and Dermatology Quality of Life Index and no differences in genotype and allele frequencies for -295 T/C IL-16 polymorphism. The expression of IL-16 (mRNA and protein) was elevated in the margin of psoriatic skin while statistically significant increase in IL-16 immunoreactivity, but not in mRNA level, was observed within plaques. Furthermore, the IL-16 mRNA levels within psoriatic lesions positively correlated with the levels of CD4 mRNA, but not with Psoriasis Area and Severity Index. In conclusion, our data revealed an association between circulating IL-16 and severity of psoriasis which indicates that this cytokine could serve as a potential marker of disease activity. However, further investigations are required.

Bioactive forms of vitamin D selectively stimulate the skin analog of the hypothalamus-pituitary-adrenal axis in human epidermal keratinocytes.

Ultraviolet radiation B stimulates both the production of vitamin D3 in the skin and the activation of the skin analog of the hypothalamic-pituitary-adrenal axis (HPA) as well as the central HPA. Since the role of vitamin D3 in the regulation of the HPA is largely unknown, we investigated the impact of 1,25(OH)2D3 and its noncalcemic analogs, 20(OH)D3 and 21(OH)pD, on the expression of the local HPA in human epidermal keratinocytes. The noncalcemic analogs showed similar efficacy to 1,25(OH)2D3 in stimulating the expression of neuropeptides, CRF, urocortins and POMC, and their receptors, CRFR1, CRFR2, MC1R, MC2R, MC3R and MC4R. Interestingly, unlike other secosteroids, the activity of 21(OH)pD did not correlate with induction of differentiation, suggesting a separate but overlapping mechanism of action. Thus, biologically active forms of vitamin D can regulate different elements of the local equivalent of the HPA with implications for the systemic HPA.

Vitamin D derivatives enhance cytotoxic effects of H2O2 or cisplatin on human keratinocytes.

Although the skin production of vitamin D is initiated by ultraviolet radiation type B (UVB), the role vitamin D plays in antioxidative or pro-oxidative responses remains to be elucidated. We have used immortalized human HaCaT keratinocytes as a model of proliferating epidermal cells to test the influence of vitamin D on cellular response to H2O2 or the anti-cancer drug, cisplatin. Incubation of keratinocytes with 1,25(OH)2D3 or its low calcemic analogues, 20(OH)D3, 21(OH)pD or calcipotriol, sensitized cells to ROS resulting in more potent inhibition of keratinocyte proliferation by H2O2 in the presence of vitamin D compounds. These results were supported by cell cycle and apoptosis analyses, and measurement of the mitochondrial transmembrane potentials (MMP), however some unique properties of individual secosteroids were observed. Furthermore, in HaCaT keratinocytes treated with H2O2, 1,25(OH)2D3, 21(OH)pD and calcipotriol stimulated the expression of SOD1 and CAT genes, but not SOD2, indicating a possible role of mitochondria in ROS-modulated cell death. 1,25(OH)2D3 also showed a short-term, protective effect on HaCaT keratinocytes, as exemplified by the inhibition of apoptosis and the maintenance of MMP. However, with prolonged incubation with H2O2 or cisplatin, 1,25(OH)2D3 caused an acceleration in the death of the keratinocytes. Therefore, we propose that lead vitamin D derivatives can protect the epidermis against neoplastic transformation secondary to oxidative or UV-induced stress through activation of vitamin D-signaling. Furthermore, our data suggest that treatment with low calcemic vitamin D analogues or the maintenance of optimal level of vitamin D by proper supplementation, can enhance the anticancer efficacy of cisplatin.

Growth Inhibition of Osteosarcoma Cell Lines in 3D Cultures: Role of Nitrosative and Oxidative Stress.

BACKGROUND: 3D cell cultures have revolutionized the understanding of cell behavior, allowing culture of cells with the possibility of resembling in vivo intercellular signaling and cell-extracellular matrix interaction. AIM: The effect of limited oxygen penetration into 3D culture of highly metastatic osteosarcoma 143B cells in terms of expression of nitro-oxidative stress markers was investigated and compared to standard 2D cell culture. MATERIALS AND METHODS: Human osteosarcoma (143B cell line) cells were cultured as monolayers, in collagen and Matrigel. Cell viability, gene expression of nitro-oxidative stress markers, and vascular endothelial growth factor were determined using Trypan blue assay, quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: Three-dimensional environments modify nitro-oxidative stress and influence gene expression and cell proliferation of OS 143B cells. CONCLUSION: Commercial cell lines might not constitute a good model of 3D cultures for bone tissue engineering, as they are highly sensitive to hypoxia, and hypoxic conditions can induce oxidation of the cellular environment.

Antiproliferative Activity of Double Point Modified Analogs of 1,25-Dihydroxyvitamin D2 Against Human Malignant Melanoma Cell Lines.

Vitamin D is a lipid soluble steroid hormone with pleiotropic biological properties, including regulation of cell proliferation, differentiation and apoptosis. As to these desirable anticancer actions, 1,25-dihydroxyvitamins D and analogs have been reported to inhibit the proliferation and to induce differentiation of a wide variety of cancer cell types, including human malignant melanoma. However, there is a need for novel and more efficacious vitamin D analogs, and how best to design such is still an open issue. A series of double point modified (DPM) analogs of 1,25-dihydroxyvitamin D2 (1,25(OH)2D2) induced differentiation of the vitamin D receptor (VDR) positive A375 and VDR negative SK-MEL 188b human malignant melanoma cell lines. Surprisingly, the dose of 1,25(OH)2D2 required to inhibit the proliferation of the A375 melanoma cell line by was several fold lower than that required in the case of 1,25(OH)2D3. To evaluate the impact of the modification in the side chain (additional 22-hydroxyl) and in the A-ring (5,6-trans modification), the regular side-chain of vitamin D2 or D3 was retained in the structure of our analogs. As expected, 5,6-trans modification was advantageous to enhancing the anti-proliferative activity of analogs, but not as a single point modification (SPM). Very unexpectedly, the additional 22-hydroxyl in the side-chain reduced significantly the anti-proliferative activity of both the natural and 5,6-trans series analogs. Finally, an induction of pigmentation in melanoma SK-MEL 188b cells was observed to sensitized cells to the effect of vitamin D analogs.

Vitamin D in the skin physiology and pathology.

Vitamin D plays important, pleiotropic role in the maintenance of global homeostasis. Its influence goes far beyond the regulation of calcium and phosphorus balance, as diverse activities of vitamin D and its natural metabolites assure proper functioning of major human organs, including skin. Recently, we reviewed the current understanding of vitamin D impact on human health from historical perspective (Wierzbicka et al. (2014) The renaissance of vitamin D. Acta Biochim Pol 61: 679-686). This article focuses on its functions in the skin. The skin and its appendages, creates a platform connecting and protecting internal organs against, usually harmful, external environment. It uppermost layer - epidermis in order to maintain a protective barrier undergoes a constant exchange of cornified keratinocytes layer. Its disturbance leads to development of serious skin disorders including psoriasis, vitiligo, atopic dermatitis and skin cancer. All of those dermatopathologies have a huge impact on modern societies, affecting not only the physical, but also mental state of patients as well as their social status. Furthermore, multiple human systemic diseases (autoimmune, blood and digestive diseases) have skin manifestation, thus "condition of the skin" often reflects the condition and pathological changes within the internal organs. In humans, the skin is the natural source of vitamin D, which is produced locally from 7-dehydrocholesterol in photoreaction induced by ultraviolet B (UVB) radiation from the sun. It is also well established, that the process of proliferation and differentiation of keratinocytes is tightly regulated by calcium and the active form of vitamin D (1,25(OH)2D3). Thus, the skin physiology is inseparably connected with vitamin D production and activity. Unfortunately, UVB, which is required for vitamin D production, is also known as the main cause of a skin cancer, including melanoma. Here, we are going to review benefits of vitamin D and its analogues in the maintenance of epidermal barrier and its potential use in the treatment of common skin diseases.