CYP24A1 affected macrophage polarization through degradation of vitamin D as a candidate biomarker for ovarian cancer prognosis.

Ovarian cancer (OC) is a fatal gynecological malignancy with a poor prognosis in which mitochondria-related genes are involved deeply. In this study, we aim to screen mitochondria-related genes that play a role in OC prognosis and investigate its effects. Through single-cell sequencing technology and bioinformatics analysis, including TCGA ovarian cancer data analysis, gene expression signature analysis (GES), immune infiltration analysis, Gene Ontology (GO) enrichment analysis, Gene Set Enrichment Analysis (GSEA), and Principal Component Analysis (PCA), our findings revealed that CYP24A1 regulated macrophage polarization through vitamin D (VD) degradation and served as a target gene for the second malignant subtype of OC through bioinformatics analyses. For further validation, the expression and function of CYP24A1 in OC cells was investigated. And the expression of CYP24A1 was much higher in carcinoma than in paracancerous tissue, whereas the VD content decreased in the OC cell lines with CYP24A1 overexpression. Moreover, macrophages were polarized towards M1 after the intervention of VD-treated OC cell lines and inhibited the malignant phenotypes of OC. However, the effect could be reversed by overexpressing CYP24A1, resulting in the polarization of M2 macrophages, thereby promoting tumor progression, as verified by constructing xenograft models in vitro. In conclusion, our findings suggested that CYP24A1 induced M2 macrophage polarization through interaction with VD, thus promoting the malignant progression of OC.

Vitamin D-metabolizing enzyme CYP24A1 affects oncogenic behaviors of oral squamous cell carcinoma and its prognostic implication.

Vitamin D is an essential molecule for cellular homeostasis, playing a critical role in cell fate decisions including cell proliferation, differentiation, and viability. Accumulating evidence has revealed that expression of the vitamin D-metabolizing enzyme CYP24A1 is dysregulated in different types of human malignancy. CYP24A1 has been shown to be involved in the oncogenic property of a variety of carcinoma cells. However, the pathological relevance of CYP24A1 expression level in human oral malignancy remains to be clarified. In the present study, suppression of CYP24A1 expression in oral squamous cell carcinoma (OSCC) cells increased cell proliferation, invasive activity, colony formation efficacy, and tumor growth in vivo. In addition, knockout of CYP24A1 expression inhibited cell death induced by two different types of anticancer drugs, i.e., fluorouracil and cisplatin. Gene clustering by RNA-sequence analysis revealed that several signaling molecules associated with MYC are involved in CYP24A1-mediated oncogenic behaviors. Furthermore, decreased expression level of CYP24A1 was observed in 124/204 cases (61%) of OSCC and was shown to be associated with short relapse-free and overall survival periods. The results showed that a low expression level of CYP24A1 promotes the oncogenic activity of OSCC and is significantly associated with poor prognosis in patients with this malignancy.

Electro-scalp acupuncture regulates the expression of CYP27a1/b1, CYP24a and related inflammatory cytokines in ischemic cortex of rats with middle cerebral artery occlusion. / ç”µå¤´é’ˆè°ƒæŽ§å¤§è„‘ä¸­åŠ¨è„‰æ “å¡žå¤§é¼ ç¼ºè¡€å¤§è„‘çš®å±‚åŒºCYP27a1/b1、CYP24aåŠç›¸å ³ç‚Žæ€§ç»†èƒžå› å­è¡¨è¾¾çš„ç ”ç©¶.

OBJECTIVES: To observe the effect of electro-scalp acupuncture (ESA) on the expression of cytochrome P450a1/b1 (CYP27a1/b1), cytochrome P45024a (CYP24a), signal transducer and activator of transcription (STAT)4, STAT6, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-4 in ischemic cerebral cortex of rats with acute ischemic stroke, so as to explore its mechanism in alleviating inflammatory reaction of ischemic stroke. METHODS: Sixty SD rats were randomly divided into sham-operation, model, vitamin D3 and ESA groups, with 15 rats in each group. The middle cerebral artery occlusion rat model was established with thread ligation according to Zea-Longa's method. Rats in the vitamin D3 group were given 1, 25-VitD3 solution (3 ng·100 g-1·d-1) by gavage, once daily for 7 days. Rats in the ESA group were treated at bilateral anterior parietotemporal slash (MS6) with ESA (2 Hz/100 Hz, 1 mA), 30 min a day for 7 days. Before and after interventions, the neurological deficit score and neurobehavioral score were evaluated. TTC staining was used to detect the volume of cerebral infarction in rats. The positive expressions of CYP24a, CYP27a1 and CYP27b1 in the cerebral cortex of ischemic area were detected by immunofluorescence. The mRNA expressions of STAT4 and STAT6 in the cerebral cortex of ischemic area were detected by quantitative real-time PCR. The protein expression levels of TNF-α, IL-1ß and IL-4 in the cerebral cortex of ischemic area were detected by Western blot. RESULTS: Compared with the sham-operation group, the neurological deficit score, neurobehavioral score, the percentage of cerebral infarction volume, the positive expression level of CYP24a and mRNA expression level of STAT4, protein expression levels of TNF-α and IL-1ß in cerebral cortex were increased (P<0.01), while the positive expression levels of CYP27a1/b1 and STAT6 mRNA, protein expression level of IL-4 were decreased (P<0.01) in the model group. After the treatment and compared with the model group, the neurological deficit score, neurobehavioral score, the percentage of cerebral infarction volume, the positive expression level of CYP24a and mRNA expression level of STAT4, protein expression levels of TNF-α and IL-1ß in cerebral cortex were decreased (P<0.01), while the positive expression levels of CYP27a1/b1 and STAT6 mRNA expression level, protein expression level of IL-4 were increased (P<0.01) in the ESA and vitamin D3 groups. CONCLUSIONS: ESA can alleviate the inflammatory response in ischemic stroke, which maybe related to its function in regulating the balance between CYP27a1/b1 and CYP24a, converting vitamin D into active vitamin D3, inhibiting vitamin D3 degradation, and regulating Th1/Th2 balance.

Persistent hypercalcaemia associated with two pathogenic variants in the CYP24A1 gene and a parathyroid adenoma-a case report and review.

Introduction: 24-Hydroxylase, encoded by the CYP24A1 gene, is a crucial enzyme involved in the catabolism of vitamin D. Loss-of-function mutations in CYP24A1 result in PTH-independent hypercalcaemia with high levels of 1,25(OH)2D3. The variety of clinical manifestations depends on age, and underlying genetic predisposition mutations can lead to fatal infantile hypercalcaemia among neonates, whereas adult symptoms are usually mild. Aim of the study: We report a rare case of an adult with primary hyperparathyroidism and loss-of-function mutations in the CYP24A1 gene and a review of similar cases. Case presentation: We report the case of a 58-year-old woman diagnosed initially with primary hyperparathyroidism. Preoperatively, the suspected mass adjoining the upper pole of the left lobe of the thyroid gland was found via ultrasonography and confirmed by 99mTc scintigraphy and biopsy as the parathyroid gland. The patient underwent parathyroidectomy (a histopathology report revealed parathyroid adenoma), which led to normocalcaemia. After 10 months, vitamin D supplementation was introduced due to deficiency, and the calcium level remained within the reference range. Two years later, biochemical tests showed recurrence of hypercalcaemia with suppressed parathyroid hormone levels and elevated 1,25(OH)2D3 concentrations. Further investigation excluded the most common causes of PTH-independent hypercalcaemia, such as granulomatous disease, malignancy, and vitamin D intoxication. Subsequently, vitamin D metabolites were measured using LC-MS/MS, which revealed high levels of 25(OH)D3, low levels of 24,25(OH)2D3 and elevated 25(OH)2D3/24,25(OH)2D3 ratios, suggesting a defect in vitamin D catabolism. Molecular analysis of the CYP24A1 gene using the NGS technique revealed two pathogenic variants: p.(Arg396Trp) and p.(Glu143del) (rs114368325 and rs777676129, respectively). Conclusions: The diagnostic process for hypercalcaemia becomes complicated when multiple causes of hypercalcaemia coexist. The measurement of vitamin D metabolites using LC-MS/MS may help to identify carriers of CYP24A1 mutations. Subsequent molecular testing may contribute to establishing the exact frequency of pathogenic variants of the CYP24A1 gene and introducing personalized treatment.

Gene expression profiling of vitamin D metabolism enzymes in leukemia and lymphoma patients: molecular aspect interplay of VDR, CYP2R1, and CYP24A1.

BACKGROUND: Vitamin D deficiency is prevalent among the Indonesian population, particularly in individuals diagnosed with leukemia-lymphoma. The regulation of vitamin D metabolism is influenced by the expression of several enzymes, such as CYP2R1, CYP24A1, and the vitamin D receptor (VDR). This study aimed to scrutinize the gene expression profiles in both mRNA and protein levels of VDR, CYP2R1, and CYP24A1 in leukemia and lymphoma patients. METHOD: The research was a cross-sectional study conducted at Cipto Mangunkusumo Hospital (RSCM) in Jakarta, Indonesia. The study included a total of 45 patients aged over 18 years old who have received a diagnosis of lymphoma or leukemia. Vitamin D status was measured by examining serum 25 (OH) D levels. The analysis of VDR, CYP2R1, and CYP24A1 mRNA expression utilized the qRT-PCR method, while protein levels were measured through the ELISA method. CONCLUSION: The study revealed a noteworthy difference in VDR protein levels between men and women. The highest mean CYP24A1 protein levels were observed in the age group > 60 years. This study found a significant, moderately positive correlation between VDR protein levels and CYP24A1 protein levels in the male and vitamin D sufficiency groups. In addition, a significant positive correlation was found between VDR mRNA levels and CYP2R1 mRNA levels, VDR mRNA levels and CYP2R1 mRNA levels, and CYP2R1 mRNA levels and CYP24A1 mRNA levels. However, the expression of these genes does not correlate with the protein levels of its mRNA translation products in blood circulation.

Effects of VDR and CYP24A1 gene polymorphisms on the outcome of supraglottic larynx cancer.

OBJECTIVE: Vitamin D has been demonstrated to play a protective role in carcinogenesis. Polymorphisms of the vitamin D receptor (VDR) genes and 24-α-hydroxylase (encoded by CYP24A1) may affect the outcome of some cancers. This study examines the effects of the VDR gene and CYP24A1 single nucleotide polymorphisms on the outcome of supraglottic larynx cancer. PATIENTS AND METHODS: Patients diagnosed with supraglottic larynx cancer between 2017 and 2022 were enrolled. Single nucleotide polymorphisms of the VDR gene (rs2228570, rs731236, rs7975232, rs11574113, rs11168267 and rs11168266) and CYP24A1 gene (rs4809960, rs6022999, rs6068816, rs2259735 and rs2296241) were investigated. All patients were followed up for any evidence of local recurrence, regional recurrence, distant metastasis, and second primary tumor development. Cox regression analysis was performed to evaluate the prognostic value of single-nucleotide polymorphisms (SNPs). Kaplan-Meier method was used for survival analysis. RESULTS: 87 patients were included. The mean follow-up time was 45.02±24.47 months. Cox regression analysis for locoregional recurrence revealed that the hazard ratio of rs731236 GG was 2.098 (95% CI, range: 1.047-4.202, p=0.037). Locoregional recurrence for rs731236 AA, AG, and GG were 38.6%, 23.1%, and 53.3%, respectively. In the presence of rs731236 GG polymorphism, disease-specific survival was significantly shorter (47.63±7.48 months, p=0.015), and disease-free survival (45.71±6.3 months) was significantly shorter (p=0.040). Rates of metastases and second primary tumors were not significantly different between SNPs. CONCLUSIONS: This study has demonstrated the possible effects of VDR rs731236 SNP on the locoregional recurrence and prognosis of supraglottic larynx cancer.

Cellular responses to silencing of PDIA3 (protein disulphide-isomerase A3): Effects on proliferation, migration, and genes in control of active vitamin D.

The active form of vitamin D, 1,25-dihydroxyvitamin D3, is known to act via VDR (vitamin D receptor), affecting several physiological processes. In addition, PDIA3 (protein disulphide-isomerase A3) has been associated with some of the functions of 1,25-dihydroxyvitamin D3. In the present study we used siRNA-mediated silencing of PDIA3 in osteosarcoma and prostate carcinoma cell lines to examine the role(s) of PDIA3 for 1,25-dihydroxyvitamin D3-dependent responses. PDIA3 silencing affected VDR target genes and significantly altered the 1,25-dihydroxyvitamin D3-dependent induction of CYP24A1, essential for elimination of excess 1,25-dihydroxyvitamin D3. Also, PDIA3 silencing significantly altered migration and proliferation in prostate PC3 cells, independently of 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 increased thermostability of PDIA3 in cellular thermal shift assay, supporting functional interaction between PDIA3 and 1,25-dihydroxyvitamin D3-dependent pathways. In summary, our data link PDIA3 to 1,25-dihydroxyvitamin D3-mediated signalling, underline and extend its role in proliferation and reveal a novel function in maintenance of 1,25-dihydroxyvitamin D3 levels.

MicroRNA-125b regulates vitamin D resistance by targeting CYP24A1 in the progression of gestational diabetes mellitus.

Vitamin D deficiency is prevalent in pregnancy and has been associated with increased occurrences of preeclampsia, cesarean delivery, neonatal bacterial vaginosis, and gestational diabetes. CYP24A1, recognized as a key factor in vitamin D metabolism homeostasis, encodes 24-hydroxylase responsible for converting 25(OH)D3 and 1,25(OH)2D3 into inactive metabolites. Recently, we have reported CYP24A1 overexpression in patients with gestational diabetes mellitus (GDM) and trophoblast cells exposed to hyperglycemia. In this study, we explored miRNA-mediated regulation of CYP24A1 in GDM progression, validating our findings through silencing experiments in a trophoblast cell line. In silico tools identified miR-125b-5p as a putative target of CYP24A1. Expression analysis revealed downregulation of miR-125b-5p in blood samples from early GDM and GDM compared to healthy pregnant women, positively correlating with vitamin D levels. Hyperglycemic exposure in human trophoblastic cell lines (BeWo) decreased miR-125b-5p expression, concomitant with an increase in CYP24A1. To confirm the regulatory role of miR-125b on CYP24A1, we transfected BeWo cells with antimiR-125b or miR-125b mimic. AntimiR-125b transfection heightened CYP24A1 levels, while miR-125b mimic overexpression resulted in decreased CYP24A1 expression. These findings establish miR-125b as a regulator of CYP24A1. To explore the influence of miR-125b on vitamin D metabolism, trophoblast cells overexpressing miR-125b were treated with 0.1 and 1 µM calcitriol. Hyperglycemic conditions exhibited a reduction in CYP24A1 levels. Collectively, our results indicate that miR-125b may regulate vitamin D metabolism by targeting CYP24A1, contributing to GDM progression. These findings may pave the way for understanding vitamin D resistance in concurrent GDM development and identifying novel miRNAs targeting CYP24A1.

Dual effect of vitamin D3 on breast cancer-associated fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) play an important role in the tumor microenvironment. Despite the well-known in vitro antitumoral effect of vitamin D3 (VD3), its impact on breast CAFs is almost unknown. In this study, we analyzed the ex vivo effects of calcitriol on CAFs isolated from breast cancer tissues. METHODS: CAFs were cultured with 1 and 10 nM calcitriol and their phenotype; gene expression, protein expression, and secretion were assessed. Calcitriol-treated CAFs-conditioned media (CM) were used to analyze the effect of CAFs on the migration and protein expression of MCF-7 and MDA-MB-231 cells. RESULTS: Tumor tissues from VD3-deficient patients exhibited lower levels of ß-catenin and TGFß1, along with higher levels of CYP24A1 compared to VD3-normal patients. In VD3-deficient patients, CAF infiltration was inversely associated with CYP24A1 levels and positively correlated with OPN levels. Calcitriol diminished CAFs' viability, but this effect was weaker in premenopausal and VD3-normal patients. Calcitriol reduced mRNA expression of CCL2, MMP9, TNC, and increased PDPN, SPP1, and TIMP1. It also decreased the secretion of CCL2, TNC, and the activity of MMP-2, while increasing cellular levels of TIMP1 in CAFs from all patient groups. In nonmetastatic and postmenopausal patients, PDPN surface expression increased, and CAFs CM from these groups decreased MCF-7 cell migration after ex vivo calcitriol treatment. In premenopausal and VD3-deficient patients, calcitriol reduced IDO1 expression in CAFs. Calcitriol-treated CAFs CM from these patients decreased OPN expression in MCF-7 and/or MDA-MB-231 cells. However, in premenopausal patients, calcitriol-treated CAFs CM also decreased E-cadherin expression in both cell lines. CONCLUSION: The effects of calcitriol on breast CAFs, both at the gene and protein levels, are complex, reflecting the immunosuppressive or procancer properties of CAFs. The anticancer polarization of CAFs following ex vivo calcitriol treatment may result from decreased CCL2, TNC (gene and protein), MMP9, and MMP-2, while the opposite effect may result from increased PDPN, TIMP1 (gene and protein), and SPP1. Despite these multifaceted effects of calcitriol on molecule expression, CAFs' CMs from nonmetastatic and postmenopausal patients treated ex vivo with calcitriol decreased the migration of MCF-7 cells.

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.

Differential gene regulation by a synthetic vitamin D receptor ligand and active vitamin D in human cells.

Vitamin D (VD) exerts a wide variety of biological functions including calcemic activity. VD nutritional status is closely associated with the onset and development of chronic diseases. To develop a VD analog with the desired VD activity but without calcemic activity, we screened synthetic VDR antagonists. We identified 1α,25-dihydroxyvitamin D3-26-23-lactams (DLAM)-2a-d (DLAM-2s) as nuclear vitamin D receptor (VDR) ligands in a competitive VDR binding assay for 1α,25(OH)2 vitamin D3 (1α,25(OH)2D3), and DLAM-2s showed an antagonistic effect on 1α,25(OH)2 D3-induced cell differentiation in HL60 cells. In a luciferase reporter assay in which human VDR was exogenously expressed in cultured COS-1 cells, DLAM-2s acted as transcriptional antagonists. Consistently, DLAM-2s had an antagonistic effect on the 1α,25(OH)2D3-induced expression of a known VD target gene [Cytochrome P450 24A1 (CYP24A1)], and VDR bound DLAM-2s was recruited to an endogenous VD response element in chromatin in human keratinocytes (HaCaT cells) endogenously expressing VDR. In an ATAC-seq assay, the effects of 1α,25(OH)2 D3 and DLAM-2b on chromatin reorganization were undetectable in HaCaT cells, while the effect of an androgen receptor (AR) antagonist (bicalutamide) was confirmed in prostate cancer cells (LNCaP) expressing endogenous AR. However, whole genome analysis using RNA-seq and ATAC (Assay for Transposase Accessible Chromatin)-seq revealed differential gene expression profiles regulated by DLAM-2b versus 1α,25(OH)2D3. The upregulated and downregulated genes only partially overlapped between cells treated with 1α,25(OH)2D3 and those treated with DLAM-2b. Thus, the present findings illustrate a novel VDR ligand with gene regulatory activity differing from that of 1α,25(OH)2D3.

Genetic polymorphisms of CYP24A1 gene and cancer susceptibility: a meta-analysis including 40640 subjects.

BACKGROUND: Whether cytochrome P450 24A1 (CYP24A1) polymorphism is associated with cancer susceptibility, the individual study results are still controversial. Therefore, we performed a comprehensive study to identify the association of CYP24A1 polymorphisms (rs4809960, rs6068816, rs2296241, rs4809957, rs2762939) with cancer susceptibility. METHODS: Electronic databases including Cochrane Library, PubMed, and Embase were systematically retrieved for relevant publications. Fixed or random-effect model was selected to calculate odds ratios (ORs) with their 95% confidence intervals (95%CI). RESULTS: Eighteen published articles were identified. The results indicated that rs4809960 polymorphism was associated with a decreased cancer risk in Caucasian (TT vs. TC+CC: P=0.035; C vs. T: P=0.016) and Asian population (CC vs. TC+TT: OR P=0.044; TT vs. TC+CC: P=0.021; CC vs. TT: P=0.020; C vs. T: P=0.008) and breast cancer risk (TT vs. TC+CC: P = 0.007; TC vs. TT: P=0.004; C vs. T: P=0.033). A significant association was found between rs2296241 polymorphism and esophageal squamous cell carcinoma risk (AA vs. GG+AG: P = 0.023) and prostate cancer susceptibility (A vs. G: P=0.022). Furthermore, rs4809957 polymorphism was associated with prostate cancer susceptibility in Caucasian (GG vs. GA+AA: P=0.029; GA vs. GG: P=0.022) and breast cancer susceptibility (AA vs. GG+GA: P=0.012; AA vs. GG, P=0.010; A vs. G: P=0.024). Additionally, rs6068816 polymorphism significantly decreased the lung cancer (CC vs. CT+TT: P = 0.016; TT vs. CC: P = 0.044; CT vs. CC: P = 0.036; T vs. C: P = 0.016) and breast cancer risk (TT vs. CC+CT: P = 0.043; TT vs. CC: P = 0.039). No association was found for rs2762939 polymorphism with overall cancer risk. However, for rs2296241, rs4809957, and rs6068816 polymorphisms, there were no significant differences after the Bonferroni correction. CONCLUSION: The meta-analysis suggested that rs4809960 was associated with cancer risk and might be a genetic marker for predicting cancer risk. More large-scale and large-sample studies are necessary to further confirm these results.

CYP24A1, AHR, CPEB4, TRIP13, and PIK3CA genes expression in colorectal cancer patients: novel diagnostic biomarkers.

OBJECTIVE: This study aimed to investigate the CYP24A1, AHR, CPEB4, TRIP13, and PIK3CA mRNA expression in the blood of colorectal cancer patients in Egypt. This was performed to elucidate if there's a link between this gene expression and other clinicopathological characteristics of the tumor. PATIENTS AND METHODS: A case-control study including 50 colorectal cancer patients and 50 healthy controls was conducted. Real-time polymerase chain reaction (rt-PCR) was utilized to assess the expression of CYP24A1, AHR, CPEB4, TRIP13, and PIK3CA mRNA in blood samples. RESULTS: Patients with colorectal cancer had significantly higher levels of mRNA for the genes CYP24A1, AHR, CPEB4, TRIP13, and PIK3CA (p<0.001, p=0.021, p<0.001, and p<0.001, respectively) compared to controls. Remarkedly, the gene expression of AHR, TRIP13, and PIK3CA genes did not exhibit a significant correlation with the tumor stages (p=0.379, p=0.095, and p=0.526, respectively). However, there was a strong correlation between CYP24A1 and CPEB4 gene expression and tumor stages (p<0.001 and p=0.002, respectively). CONCLUSIONS: Therefore, we can conclude that increased mRNA levels of CYP24A1, AHR, CPEB4, TRIP13, and PIK3CA in blood samples withdrawn from colorectal cancer patients could be a biomarker for the disease.

Association of DNA methylation of vitamin D metabolic pathway related genes with colorectal cancer risk.

BACKGROUND: Vitamin D might have anti-tumor effect, which is affected by the genes related to vitamin D metabolic pathway. Epigenetic mechanism may affect the expression level of vitamin D metabolic pathway related genes, then plays an important role in the occurrence and development of colorectal cancer. To date, no study has reported on the association between blood-based DNA methylation level of vitamin D metabolic pathway related genes and colorectal cancer risk. METHODS: A case-control study was conducted including 102 colorectal cancer cases and 102 sex- and age-frequency-matched controls in Guangzhou, China. CpG islands in the VDR, CYP24A1, CYP27B1 and CYP2R1 genes were chosen for DNA methylation analysis by MethylTarget sequencing. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of DNA methylation levels for colorectal cancer. Taking the point with the largest Youden index as the boundary value, the cumulative methylation levels of vitamin D metabolic pathway related genes were divided into hypomethylation and hypermethylation. Unconditional multivariable logistical regression model was used to calculate the adjusted odds ratio (aOR) and 95% confidence intervals (95% CIs) after adjusting for potential confounders. RESULTS: Among 153 CpG sites, 8 CpG sites were significantly different between the cases and the controls. The cumulative methylation level of all CpG sites in CYP2R1 was inversely associated with the risk of colorectal cancer (aOR, 0.49; 95% CI, 0.26-0.91). However, no significant association was found between cumulative methylation levels of all CpG sites in VDR, CYP24A1 and CYP27B1 and colorectal cancer risk. Significant inverse association was observed between cumulative methylation level of significant CpG sites in VDR (aOR, 0.28; 95% CI, 0.16-0.51) and CYP24A1 (aOR, 0.19; 95% CI, 0.09-0.40) and colorectal cancer risk. There were no significant associations between cumulative methylation levels of significant CpG sites in CYP2R1 and CYP27B1 and colorectal cancer risk. CONCLUSIONS: This study indicated that the cumulative methylation levels of significant CpG sites in VDR and CYP24A1 and all CpG sites in CYP2R1 were inversely associated with colorectal cancer risk.

Seasonal changes of vitamin D3 and ovarian steroidogenesis in the wild ground squirrels (Citellus dauricus Brandt).

There is mounting evidence that vitamin D3 regulates female reproductive function critically, while little is known about the function of seasonally variable vitamin D3 in regulating ovarian steroidogenesis. This study examined the seasonal expressions of vitamin D receptor (VDR), vitamin D metabolic molecules (CYP2R1, CYP27B1, and CYP24A1), and steroidogenic enzymes (P450scc, 3ß-HSD, P450c17, and P450arom) in the ovaries of the wild ground squirrels (Citellus dauricus Brandt) during the different breeding seasons. VDR, CYP2R1, CYP27B1, and CYP24A1 were shown to be localized in different types of ovarian cells in the wild ground squirrels during the breeding and non-breeding seasons. Meanwhile, the mRNA levels of VDR, CYP2R1, CYP27B1, CYP11A1, HSD3B1, CYP17A1, and CYP19A1 in the ovaries were remarkably higher in the breeding season. Furthermore, RNA-seq data of ovaries revealed that 6036 genes were differentially expressed genes (DEGs); further analysis revealed that several DEGs known to be involved in ovarian steroidogenesis pathway and cellular response to vitamin D pathway were identified. In addition, during the breeding season, the concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone, and 17ß-estradiol were greater in the serum of the wild female ground squirrels. This observation was positively correlated with seasonal changes in the concentration of 25(OH)D3, supporting the fact that the 25(OH)D3 content in the ovaries was significantly higher in the breeding season. These findings suggested that seasonal changes in vitamin D3 might regulate the ovarian steroidogenesis of the wild female ground squirrels.

USP11-mediated LSH deubiquitination inhibits ferroptosis in colorectal cancer through epigenetic activation of CYP24A1.

Ferroptosis is an iron-dependent form of regulated cell death characterized by lipid peroxidation. Colorectal cancer (CRC) cells evade ferroptosis despite their requirement of substantial iron and reactive oxygen species (ROS) to sustain active metabolism and extensive proliferation. However, the underlying mechanism is unclear. Herein, we report the role of lymphoid-specific helicase (LSH), a chromatin-remodeling protein, in suppressing erastin-induced ferroptosis in CRC cells. We demonstrate that erastin treatment leads to dose- and time-dependent downregulation of LSH in CRC cells, and depletion of LSH increases cell sensitivity to ferroptosis. Mechanistically, LSH interacts with and is stabilized by ubiquitin-specific protease 11 (USP11) via deubiquitination; this interaction was disrupted by erastin treatment, resulting in increased ubiquitination and LSH degradation. Moreover, we identified cytochrome P450 family 24 subfamily A member 1 (CYP24A1) as a transcriptional target of LSH. LSH binds to the CYP24A1 promoter, promoting nucleosome eviction and reducing H3K27me3 occupancy, thus leading to transcription of CYP24A1. This cascade inhibits excessive intracellular Ca2+ influx, thereby reducing lipid peroxidation and ultimately conferring resistance to ferroptosis. Importantly, aberrant expression of USP11, LSH, and CYP24A1 is observed in CRC tissues and correlates with poor patient prognosis. Taken together, our study demonstrates the crucial role of the USP11/LSH/CYP24A1 signaling axis in inhibiting ferroptosis in CRC, highlighting its potential as a therapeutic target in CRC treatment.

Vitamin D and calcium co-therapy mitigates pre-established cadmium nephropathy by regulating renal calcium homeostatic molecules and improving anti-oxidative and anti-inflammatory activities in rat.

BACKGROUND: Cadmium (Cd) is a major environmental pollutant and chronic toxicity could induce nephropathy by increasing renal oxidative stress and inflammation. Although vitamin D (VD) and calcium (Ca) prophylactic treatments attenuated Cd-induced cell injury, none of the prior studies measure their renoprotective effects against pre-established Cd-nephropathy. AIMS: To measure the alleviating effects of VD and/or Ca single and dual therapies against pre-established nephrotoxicity induced by chronic Cd toxicity prior to treatment initiation. METHODS: Forty male adult rats were allocated into: negative controls (NC), positive controls (PC), Ca, VD and VC groups. The study lasted for eight weeks and all animals, except the NC, received CdCl2 in drinking water (44 mg/L) throughout the study. Ca (100 mg/kg) and/or VD (350 IU/kg) were given (five times/week) during the last four weeks to the designated groups. Subsequently, the expression of transforming growth factor-ß (TGF-ß1), inducible nitric oxide synthase (iNOS), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), VD synthesising (Cyp27b1) and catabolizing (Cyp24a1) enzymes with VD receptor (VDR) and binding protein (VDBP) was measured in renal tissues. Similarly, renal expression of Ca voltage-dependent channels (CaV1.1/CaV3.1), store-operated channels (RyR1/ITPR1), and binding proteins (CAM/CAMKIIA/S100A1/S100B) were measured. Serum markers of renal function alongside several markers of oxidative stress (MDA/H2O2/GSH/GPx/CAT) and inflammation (IL-6/TNF-α/IL-10) together with renal cell apoptosis and expression of caspase-3 were also measured. RESULTS: The PC group exhibited hypovitaminosis D, hypocalcaemia, hypercalciuria, proteinuria, reduced creatinine clearance, and increased renal apoptosis/necrosis with higher caspase-3 expression. Markers of renal tissue damage (TGF-ß1/iNOS/NGAL/KIM-1), oxidative stress (MDA/H2O2), and inflammation (TNF-α/IL-1ß/IL-6) increased, whilst the antioxidants (GSH/GPx/CAT) and IL-10 decreased, in the PC group. The PC renal tissues also showed abnormal expression of Cyp27b1, Cyp24a1, VDR, and VDBP, alongside Ca-membranous (CaV1.1/CaV3.1) and store-operated channels (RyR1/ITPR1) and cytosolic Ca-binding proteins (CAM/CAMKIIA/S100A1/S100B). Although VD was superior to Ca monotherapy, their combination revealed the best mitigation effects by attenuating serum and renal tissue Cd concentrations, inflammation and oxidative stress, alongside modulating the expression of VD/Ca-molecules. CONCLUSIONS: This study is the first to show improved alleviations against Cd-nephropathy by co-supplementing VD and Ca, possibly by better regulation of Ca-dependent anti-oxidative and anti-inflammatory actions.

Local production of active vitamin D3 metabolites in breast cancer cells by CYP24A1 and CYP27B1.

The role of vitamin D3 and its metabolites in cancer and especially as a treatment option has been widely disputed. Clinicians noting low serum 25-hydroxyvitamin D3 [25(OH)D3] levels in their patients, recommend vitamin D3 supplementation as a method of reducing the risk of cancer; however, data supporting this are inconsistent. These studies rely on systemic 25(OH)D3 as an indicator of hormone status, but 25(OH)D3 is further metabolized in the kidney and other tissues under regulation by several factors. This study examined if breast cancer cells also possess the ability to metabolize 25(OH)D3, and if so, whether the resulting metabolites are secreted locally; if this ability reflects ERα66 status; and if they possess vitamin D receptors (VDR). To address this question, estrogen receptor alpha (ERα) positive (MCF-7) and ERα negative (HCC38 and MDA-MB-231) breast cancer cell lines were examined for expression of ERα66, ERα36, CYP24A1, CYP27B1, and VDR as well as for local production of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] after treatment with 25(OH)D3. The results showed that independent of ER status, breast cancer cells express the enzymes CYP24A1 and CYP27B1, which are responsible for converting 25(OH)D3 into its dihydroxylated forms. Moreover, these metabolites are produced at levels comparable to the levels observed in blood. They are positive for VDR, indicating that they can respond to 1α,25(OH)2D3, which can upregulate CYP24A1. These findings suggest that vitamin D metabolites may contribute to the tumorigenicity of breast cancer via autocrine and/or paracrine mechanisms.

Polymorphisms in VDR, CYP27B1, CYP2R1, GC and CYP24A1 Genes as Biomarkers of Survival in Non-Small Cell Lung Cancer: A Systematic Review.

The objective of this systematic review was to provide a compilation of all the literature available on the association between single-nucleotide polymorphisms (SNPs) in the genes involved in the metabolic pathway of vitamin D and overall survival (OS) and progression-free survival (PFS) in patients with non-small cell lung cancer (NSCLC). This systematic review was conducted in accordance with the PRISMA guidelines. It included all the literature published up to 1 November 2022 and was carried out in four databases (Medline [PubMed], Scopus, Web of Science, and Embase), using the PICO strategy, with relevant keywords related to the objective. The quality of the studies included was evaluated with an assessment tool derived from the Strengthening the Reporting of Genetic Association Studies (STREGA) statement. Six studies were included in this systematic review. Our findings showed that the BsmI (rs1544410), Cdx-2 (rs11568820), FokI (rs2228570), ApaI (rs7975232), TaqI (rs731236), rs4646536, rs6068816, rs7041, and rs10741657 SNPs in the genes that play a part in vitamin D synthesis (CYP2R1, CYP27B1), transport (GC), and metabolism (CYP24A1), as well as in the vitamin D receptor (VDR), are associated with OS and/or PFS in patients with NSCLC. The SNPs in VDR have been the most extensively analyzed. This systematic review summed up the available evidence concerning the association between 13 SNPs in the main genes involved in the vitamin D metabolic pathway and prognosis in NSCLC. It revealed that SNPs in the VDR, CYP27B1, CYP24A1, GC, and CYP2R1 genes could have an impact on survival in this disease. These findings suggest the identification of prognostic biomarkers in NSCLC patients. However, evidence remains sparse for each of the polymorphisms examined, so these findings should be treated with caution.

Suppression of the vitamin D metabolizing enzyme CYP24A1 provides increased sensitivity to chemotherapeutic drugs in breast cancer.

Vitamin D is an essential nutrient for the human body not only for the metabolism of calcium but also for homeostasis. Vitamin D contributes to cell fate decisions, including cell proliferation, differentiation and viability. Accumulated epidemiological data suggest a relationship between vitamin D deficiency and carcinogenesis in numerous organs. Furthermore, it is known that the expression of the vitamin D metabolizing enzyme, cytochrome P450 family 24 subtype A1 (CYP24A1), is increased in different types of human malignancy including breast carcinoma. However, the pathological relevance of elevated CYP24A1 expression level requires further clarification. In the present study, it was demonstrated that CYP24A1 promoted the oncogenic property of breast carcinoma cells. Consistent with previous reports, it was demonstrated that the expression of CYP24A1 was elevated in invasive breast carcinoma and significantly decreased the overall survival of patients with invasive breast carcinoma. Importantly, suppression of CYP24A1 expression significantly enhanced cell death sensitivity to two anticancer drugs with pharmacologically different modes of action, cisplatin and gefitinib. The results of the present study suggest the possibility of CYP24A1­inhibiting therapy as a novel therapy in breast cancer with overexpression of CYP24A1.