Casticin induces ferroptosis in human osteosarcoma cells through Fe2+ overload and ROS production mediated by HMOX1 and LC3-NCOA4.

Osteosarcoma is a primary solid bone malignancy, and surgery + chemotherapy is the most commonly used treatment. However, chemotherapeutic drugs can cause a range of side effects. Casticin, a polymethoxyflavonoid, has anti-tumor therapeutic effects. This study is aim to investigate the anti-osteosarcoma activity of casticin and explore the mechanism. Crystal violet staining, MTT assay, colony formation assay, wound healing assay, transwell assay, hoechst 33,258 staining, and flow cytometry analysis were used to investigate the effects of casticin on proliferation, migration, invasion, and apoptosis of osteosarcoma cells in vitro. The intracellular Fe2+, ROS, MDA, GSH/GSSG content changes were detected using the corresponding assay kits. The mRNA sequencing + bioinformatics analysis and western blot were used to detect the possible mechanism. We found that casticin caused G2/M phase cell cycle arrest in human osteosarcoma cells, inhibited the migration and invasion, and induced cell apoptosis and ferroptosis. Mechanistic studies showed the ferroptosis pathway was enriched stronger than apoptosis. Casticin up-regulated the expression of HMOX1, LC3 and NCOA4, meanwhile it activated MAPK signaling pathways. Animal experiments proved that casticin also inhibited the growth and metastasis of osteosarcoma cell xenograft tumor in vivo. In conclusion, casticin can induce ferroptosis in osteosarcoma cells through Fe2+ overload and ROS production mediated by HMOX1 and LC3-NCOA4. This provides a new strategy for osteosarcoma treatment.

Emerging contaminants: A One Health perspective.

Environmental pollution is escalating due to rapid global development that often prioritizes human needs over planetary health. Despite global efforts to mitigate legacy pollutants, the continuous introduction of new substances remains a major threat to both people and the planet. In response, global initiatives are focusing on risk assessment and regulation of emerging contaminants, as demonstrated by the ongoing efforts to establish the UN's Intergovernmental Science-Policy Panel on Chemicals, Waste, and Pollution Prevention. This review identifies the sources and impacts of emerging contaminants on planetary health, emphasizing the importance of adopting a One Health approach. Strategies for monitoring and addressing these pollutants are discussed, underscoring the need for robust and socially equitable environmental policies at both regional and international levels. Urgent actions are needed to transition toward sustainable pollution management practices to safeguard our planet for future generations.

Adipose-derived mesenchymal stem cells (MSCs) are a superior cell source for bone tissue engineering.

Effective bone regeneration through tissue engineering requires a combination of osteogenic progenitors, osteoinductive biofactors and biocompatible scaffold materials. Mesenchymal stem cells (MSCs) represent the most promising seed cells for bone tissue engineering. As multipotent stem cells that can self-renew and differentiate into multiple lineages including bone and fat, MSCs can be isolated from numerous tissues and exhibit varied differentiation potential. To identify an optimal progenitor cell source for bone tissue engineering, we analyzed the proliferative activity and osteogenic potential of four commonly-used mouse MSC sources, including immortalized mouse embryonic fibroblasts (iMEF), immortalized mouse bone marrow stromal stem cells (imBMSC), immortalized mouse calvarial mesenchymal progenitors (iCAL), and immortalized mouse adipose-derived mesenchymal stem cells (iMAD). We found that iMAD exhibited highest osteogenic and adipogenic capabilities upon BMP9 stimulation in vitro, whereas iMAD and iCAL exhibited highest osteogenic capability in BMP9-induced ectopic osteogenesis and critical-sized calvarial defect repair. Transcriptomic analysis revealed that, while each MSC line regulated a distinct set of target genes upon BMP9 stimulation, all MSC lines underwent osteogenic differentiation by regulating osteogenesis-related signaling including Wnt, TGF-ß, PI3K/AKT, MAPK, Hippo and JAK-STAT pathways. Collectively, our results demonstrate that adipose-derived MSCs represent optimal progenitor sources for cell-based bone tissue engineering.

Mitochondrial-Oriented Injectable Hydrogel Microspheres Maintain Homeostasis of Chondrocyte Metabolism to Promote Subcellular Therapy in Osteoarthritis.

Subcellular mitochondria serve as sensors for energy metabolism and redox balance, and the dynamic regulation of functional and dysfunctional mitochondria plays a crucial role in determining cells' fate. Selective removal of dysfunctional mitochondria at the subcellular level can provide chondrocytes with energy to prevent degeneration, thereby treating osteoarthritis. Herein, to achieve an ideal subcellular therapy, cartilage affinity peptide (WYRGRL)-decorated liposomes loaded with mitophagy activator (urolithin A) were integrated into hyaluronic acid methacrylate hydrogel microspheres through microfluidic technology, named HM@WY-Lip/UA, that could efficiently target chondrocytes and selectively remove subcellular dysfunctional mitochondria. As a result, this system demonstrated an advantage in mitochondria function restoration, reactive oxygen species scavenging, cell survival rescue, and chondrocyte homeostasis maintenance through increasing mitophagy. In a rat post-traumatic osteoarthritis model, the intra-articular injection of HM@WY-Lip/UA ameliorated cartilage matrix degradation, osteophyte formation, and subchondral bone sclerosis at 8 weeks. Overall, this study indicated that HM@WY-Lip/UA provided a protective effect on cartilage degeneration in an efficacious and clinically relevant manner, and a mitochondrial-oriented strategy has great potential in the subcellular therapy of osteoarthritis.

MZB1-expressing cells are essential for local immunoglobulin production in chronic rhinosinusitis with nasal polyps.

BACKGROUND: The expression of MZB1 genes is significantly elevated in patients who have chronic rhinosinusitis with nasal polyp (CRSwNP) disease compared with healthy controls. OBJECTIVE: To characterize MZB1-positive B cells in CRSwNP and to estimate the contribution of distinct subsets of B cells to the local overproduction of immunoglobulins. METHODS: Single-cell RNA-sequencing with Cellular Indexing of Transcriptomes and Epitopes by Sequencing technology, Switching Mechanism At the 5' end of RNA Template sequencing, flow cytometry, immunohistochemistry and immunofluorescence staining, Western blot, QuantiGene Plex assay, B-cell ImmunoSpot assay, Luminex assay, and enzyme-linked immunosorbent assay were performed. RESULTS: Significantly higher mRNA expression of MZB1 and HSP90B1 was found in type 2 CRSwNP compared with controls. In CRSwNP, MZB1 expression correlated with the local production of IgE. MZB1 could be colocalized with plasma and mature B cells, especially marginal zone (MZ) B cells. Single-cell transcriptome and epitope studies revealed prominent populations of B cells in type 2 CRSwNP with unexpectedly high MZB1 gene expression. The MZ B-cell population was significantly increased in CRSwNP compared with healthy controls in both peripheral blood mononuclear cells and nasal tissue single-cell suspensions. When those single cells were cultured overnight, the MZ B-cell numbers were positively correlated with local IgE production but negatively correlated with local IgM production. In vitro, MZB1 stimulation up-regulated the mRNA expression of IgE. CONCLUSION: MZB1 was primarily expressed by plasma and mature B cells in nasal mucosa. MZB1 expression level was increased in CRSwNP compared with controls. MZB1 contributed to the local IgE production in type 2 CRSwNP.

Association between serum polyunsaturated fatty acids and bone mineral density in US adults: NHANES 2011-2014.

Objective: The purpose of this study was to investigate the association between serum polyunsaturated fatty acids (PUFAs) and bone mineral density (BMD). Methods: We performed a cross-sectional study based on data from the National Health and Nutrition Examination Survey (NHANES) 2011-2014. The weighted multiple linear regression model was utilized to determine the association between serum PUFAs and BMD. Further smoothed curve fitting and threshold effect analysis were conducted. Finally, we performed a subgroup analysis. Results: In total, 1979 participants aged 20-59 years were enrolled. After adjusting for all covariates, we found that serum docosapentaenoic acid (DPA) was positively associated with head BMD (ß = 0.0015, 95% Cl: 0.0004, 0.0026, P = 0.008296) and lumbar spine BMD (ß = 0.0005, 95% Cl: 0.0000, 0.0010, P = 0.036093), and serum eicosadienoic acid (EDA) was negatively associated with thoracic spine BMD (ß = -0.0008, 95% Cl: -0.0016, -0.0000, P = 0.045355). Smoothed curve fitting revealed a nonlinear positive association between serum DPA and lumbar spine BMD. Threshold effect analysis indicated that the threshold of serum DPA was 81.4 µmol/L. Subgroup analysis revealed a positive correlation between serum DPA and head BMD in the subgroup aged 50-59 years (ß = 0.0025, 95% Cl: 0.0002, 0.0049, P = 0.035249) and females (ß = 0.0026, 95% Cl: 0.0008, 0.0044, P = 0.005005). There was a positive relationship between serum DPA and lumbar spine BMD in females (ß = 0.0008, 95% Cl: 0.0001, 0.0015, P = 0.017900) and a negative association between serum EDA and thoracic spine BMD in the subgroup aged 30-39 years (ß = -0.0016, 95% Cl: -0.0031, -0.0001, P = 0.041331), males (ß = -0.0012, 95% Cl: -0.0023, -0.0001, P = 0.039364) and other races (ß = -0.0021, 95% Cl: -0.0037, -0.0006, P = 0.008059). Conclusion: This study demonstrated a linear positive relationship between serum DPA and head BMD, a nonlinear positive association between serum DPA and lumbar spine BMD, and a linear negative correlation between serum EDA and thoracic spine BMD in US adults.

Co-exposure to organic UV filters and phthalates and their associations with oxidative stress levels in children: A prospective follow-up study in China.

Children are highly vulnerable to environmental pollutants, especially endocrine-disrupting chemicals (EDCs). Previous research has linked both organic UV filters and phthalates exposure to adiposity and pubertal development in children. Nevertheless, the individual and collective effects of these chemicals on this population remain poorly understood. In this study, twelve organic UV filters and metabolites, six phthalate metabolites and two oxidative stress biomarkers were analyzed in a prospective follow-up study in Shanghai, China after a baseline study conducted 1.5 years earlier. Results revealed a positive association between exposure to individual organic UV filters or their mixture and levels of 8-OHdG (ß ranging from 0.242 to 0.588, P < 0.05), a marker of oxidative DNA damage. BP-3 and OD-PABA made a greater contribution to oxidative DNA damage than other UV filters. Levels of 8-OHdG were also positively correlated with single phthalate metabolites and their mixture, with MnBP and MMP contributing the most. Stratified analysis found that these associations were mainly observed in girls. Our mixture analysis revealed cumulative risks of oxidative DNA damage when there was co-exposure to these two kinds of EDCs. These results underscore the importance of considering the risks associated with organic UV filters and the necessity of evaluating the effects of all these pollutants, both individually and in mixtures.

Development and validation of a novel necroptosis-related gene signature for predicting prognosis and therapeutic response in Ewing sarcoma.

Ewing sarcoma (ES) is the second most common malignant bone tumor in children and has a poor prognosis due to early metastasis and easy recurrence. Necroptosis is a newly discovered cell death method, and its critical role in tumor immunity and therapy has attracted widespread attention. Thus, the emergence of necroptosis may provide bright prospects for the treatment of ES and deserves our further study. Here, based on the random forest algorithm, we identified 6 key necroptosis-related genes (NRGs) and used them to construct an NRG signature with excellent predictive performance. Subsequent analysis showed that NRGs were closely associated with ES tumor immunity, and the signature was also good at predicting immunotherapy and chemotherapy response. Next, a comprehensive analysis of key genes showed that RIPK1, JAK1, and CHMP7 were potential therapeutic targets. The Cancer Dependency Map (DepMap) results showed that CHMP7 is associated with ES cell growth, and the Gene Set Cancer Analysis (GSCALite) results revealed that the JAK1 mutation frequency was the highest. The expression of 3 genes was all negatively correlated with methylation and positively with copy number variation (CNV). Finally, an accurate nomogram was constructed with this signature and clinical traits. In short, this study constructed an accurate prognostic signature and identified 3 novel therapeutic targets against ES.

The multi-omics single-cell landscape of sinus mucosa in uncontrolled severe chronic rhinosinusitis with nasal polyps.

Uncontrolled severe chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with elevated levels of type 2 inflammatory cytokines and raised immunoglobulin concentrations in nasal polyp tissue. By using single-cell RNA sequencing, transcriptomics, surface proteomics, and T cell and B cell receptor sequencing, we found the predominant cell types in nasal polyps were shifted from epithelial and mesenchymal cells to inflammatory cells compared to nasal mucosa from healthy controls. Broad expansions of CD4 T effector memory cells, CD4 tissue-resident memory T cells, CD8 T effector memory cells and all subtypes of B cells in nasal polyp tissues. The T and B cell receptor repertoires were skewed in NP. This study highlights the deviated immune response and remodeling mechanisms that contribute to the pathogenesis of uncontrolled severe CRSwNP. CLINICAL IMPLICATIONS: We identified differences in the cellular compositions, transcriptomes, proteomes, and deviations in the immune profiles of T cell and B cell receptors as well as alterations in the intercellular communications in uncontrolled severe CRSwNP patients versus healthy controls, which might help to define potential therapeutic targets in the future.

Metformin improves fibroblast metabolism and ameliorates arthrofibrosis in rats.

Background: Emerging studies have suggested an essential role of fibroblast metabolic reprogramming in the pathogenesis of arthrofibrosis. The metabolic modulator metformin appears to be a therapeutic candidate for fibrotic disorders. However, whether metformin could alleviate arthrofibrosis has not been defined. In this study we have determined if treatment with metformin has beneficial effect on arthrofibrosis and its underlying mechanism. Methods: Articular capsule samples were collected from patients with/without arthrofibrosis to perform gene and protein expression analysis. Arthrofibrosis animal model was established to examine the anti-fibrotic effect of metformin. Cell culture experiments were conducted to determine the mechanism by which metformin inhibits fibroblast activation. Results: We found that glycolysis was upregulated in human fibrotic articular capsules. In an arthrofibrosis animal model, intra-articular injection of metformin mitigated inflammatory reactions, downregulated expression of both fibrotic and glycolytic markers, improved range of motion (ROM) of the joint, and reduced capsular fibrosis and thickening. At the cellular level, metformin inhibited the activation of fibroblasts and mitigated the abundant influx of glucose into activated fibroblasts. Interestingly, metformin prompted a metabolic shift from oxidative phosphorylation to aerobic glycolysis in activated fibroblasts, resulting in the anti-fibrotic effect of metformin. Conclusion: Metformin decreased glycolysis, causing a metabolic shift toward aerobic glycolysis in activated fibroblasts and has beneficial effect on the treatment of arthrofibrosis.The translational potential of this article: The findings of this study demonstrated the therapeutic effect of metformin on arthrofibrosis and defined novel targets for the treatment of articular fibrotic disorders.

Corrigendum to 'Alantolactone inhibits proliferation, metastasis and promotes apoptosis of human osteosarcoma cells by suppressing Wnt/ß-catenin and MAPKs signaling pathways' [Genes & Diseases 9 (2022) 466-478].

[This corrects the article DOI: 10.1016/j.gendis.2020.07.014.].

Genipin Inhibits the Development of Osteosarcoma through PI3K/AKT Signaling Pathway.

BACKGROUND: Osteosarcoma is a highly invasive and early metastatic tumor. At present, the toxic and side effects of chemotherapy affect the quality of life of cancer patients to varying degrees. Genipin is an extract of the natural medicine gardenia with various pharmacological activities. OBJECTIVE: The purpose of this study was to investigate the effect of Genipin on osteosarcoma and its potential mechanism of action. METHODS: Crystal violet staining, MTT assay and colony formation assay were used to detect the effect of genipin on the proliferation of osteosarcoma. The effects of vitexin on migration and invasion of osteosarcoma were detected by scratch healing assay and transwell assay. Hoechst staining and flow cytometry were used to detect the effect of genipin on apoptosis of osteosarcoma cells. The expression of related proteins was detected by Western blot. An orthotopic tumorigenic animal model was used to verify the effect of genipin on osteosarcoma in vivo. RESULTS: The results of crystal violet staining, MTT method and colony formation method proved that genipin significantly inhibited the proliferation of osteosarcoma cells. The results of the scratch healing assay and transwell assay showed that gen significantly inhibited the migration and invasion of osteosarcoma cells. The results of Hoechst staining and flow cytometry showed that genipin significantly promoted the apoptosis of osteosarcoma cells. The results of animal experiments show that genipin has the same anti-tumor effect in vivo. Genipin may inhibit the growth of osteosarcoma through PI3K/AKT signaling. CONCLUSION: Genipin can inhibit the growth of human osteosarcoma cells, and its mechanism may be related to the regulation of PI3K/AKT signaling pathway.

Exploration of the Shared Hub Genes and Biological Mechanism in Osteoporosis and Type 2 Diabetes Mellitus based on Machine Learning.

A substantial amount of evidence suggests a close relationship between osteoporosis (OP) and Type 2 Diabetes Mellitus (T2DM), but the mechanisms involved remain unknown. Therefore, we conducted this study with the aim of screening for hub genes common to both diseases and conducting a preliminary exploration of common regulatory mechanisms. In the present study, we first screened genes significantly associated with OP and T2DM by the univariate logistic regression algorithm. And then, based on cross-analysis and random forest algorithm, we obtained three hub genes (ACAA2, GATAD2A, and VPS35) and validated the critical roles and predictive performance of the three genes in both diseases by differential expression analysis, receiver operating characteristic (ROC) curves, and genome wide association study (GWAS) analysis. Finally, based on gene set enrichment analysis (GSEA) and the construction of the miRNA-mRNA regulatory network, we conducted a preliminary exploration of the co-regulatory mechanisms of three hub genes in two diseases. In conclusion, this study provides promising biomarkers for predicting and treating both diseases and offers novel directions for exploring the common regulatory mechanisms of both diseases.

Echinatin inhibits the growth and metastasis of human osteosarcoma cells through Wnt/ß-catenin and p38 signaling pathways.

Osteosarcoma (OS) is a highly aggressive malignant bone tumor that mainly occurs in adolescents. At present, chemotherapy is the most commonly used method in clinical practice to treat OS. However, due to drug resistance, toxicity and long-term side effects, chemotherapy can't always provide sufficient benefits for OS patients, especially those with metastasis and recurrence. Natural products have long been an excellent source of anti-tumor drug development. In the current study, we evaluated the anti-OS activity of Echinatin (Ecn), a natural active component from the roots and rhizomes of licorice, and explored the possible mechanism. We found that Ecn inhibited the proliferation of human OS cells and blocked cell cycle at S phase. In addition, Ecn suppressed the migration and invasion, while induced the apoptosis of human OS cells. However, Ecn had less cytotoxicity against normal cells. Moreover, Ecn inhibited the xenograft tumor growth of OS cells in vivo. Mechanistically, Ecn inactivated Wnt/ß-catenin signaling pathway while activated p38 signaling pathway. ß-catenin over-expression and the p38 inhibitor SB203580 both attenuated the inhibitory effect of Ecn on OS cells. Notably, we demonstrated that Ecn exhibited synergistic inhibitory effect with cisplatin (DDP) on OS cells in vitro and in vivo. Therefore, our results suggest that Ecn may exert anti-OS effects at least partly through regulating Wnt/ß-catenin and p38 signaling pathways. Most meaningfully, the results obtained suggest a potential strategy to improve the DDP-induced tumor-killing effect on OS cells by combining with Ecn.

Innovative treatments for severe uncontrolled chronic rhinosinusitis with nasal polyps.

INTRODUCTION: In recent years, endotypes of chronic rhinosinusitis (CRS) based on the underlying immune mechanisms provided a better understanding of this heterogeneous disease and are frequently applied in diagnosis and treatment. AREAS COVERED: In this manuscript, we aim to review novel treatment approaches for this often uncontrolled disease and highlight endotype-driven medical algorithms that could be beneficial in daily clinical practice. EXPERT OPINION: With the development of endotyping and the mucosal inflammatory concept, several type 2-targeted biologics and surgical options are nowadays available for treating CRS. However, a better understanding based on clinical trials and real-life experience in daily practice is needed to optimize patient selection, biological drug selection, treatment duration, prediction, and long-term follow-up strategies. Indirect comparison analysis suggested that dupilumab might be the most effective biologic for treating CRS with nasal polyps, but the role and timing of surgery remain unclear. More real-life studies and comparative trials are needed for the optimal integration of biologics into clinical pathways in combination with established treatment approaches such as nasal and oral glucocorticosteroids and adequate surgery to provide long-term perspectives.

M2 Macrophages Upregulated by Allergen Exposure in Seasonal Allergic Rhinitis.

INTRODUCTION: Macrophages play a central role in balancing the immune response by switching phenotypes between the M1 and M2 profiles according to a delicate equilibrium. Based on a previous clinical trial (NCT03649139), this study aimed to evaluate the change in M2 macrophages during pollen exposure in seasonal allergic rhinitis (SAR). METHODS: Nasal symptom scores were recorded. Peripheral M2 macrophages were investigated according to cell surface markers, and M2-associated cytokine/chemokine release in serum and nasal secretion were assessed. In vitro pollen stimulation tests were performed, and polarized macrophage subsets were analyzed by flow cytometry. RESULTS: Compared to baseline, the percentage of peripheral CD163+ M2 macrophages in CD14+ monocytes increased during the pollen season (p < 0.001) and at the end of treatment (p = 0.004) in the SLIT group. The percentage of CD206+CD86- M2 cells in M2 macrophages during the pollen season was higher than that at baseline and at the end of SLIT. On the other hand, the percentage of CD206-CD86+ M2 cells in M2 macrophages significantly increased at the end of treatment in the SLIT group compared to baseline (p = 0.049), the peak pollen period (p = 0.017), and the placebo group (p = 0.0023). M2-associated chemokines CCL26 and YKL-40 were significantly increased during the pollen season in the SLIT group and remained higher at the end of SLIT than at baseline. Correspondingly, in vitro study demonstrated that Artemisia annua promoted M2 macrophage polarization in pollen-induced AR patients. CONCLUSION: Significant M2 macrophage polarization was promoted when patients with SAR were exposed to the allergen, either naturally exposed in pollen seasons or subjectively continuously exposed during the course of SLIT.

Inhibition of Macropinocytosis Enhances the Sensitivity of Osteosarcoma Cells to Benzethonium Chloride.

Osteosarcoma (OS) is a primary malignant tumor of bone. Chemotherapy is one of the crucial approaches to prevent its metastasis and improve prognosis. Despite continuous improvements in the clinical treatment of OS, tumor resistance and metastasis remain dominant clinical challenges. Macropinocytosis, a form of non-selective nutrient endocytosis, has received increasing attention as a novel target for cancer therapy, yet its role in OS cells remains obscure. Benzethonium chloride (BZN) is an FDA-approved antiseptic and bactericide with broad-spectrum anticancer effects. Here, we described that BZN suppressed the proliferation, migration, and invasion of OS cells in vitro and in vivo, but simultaneously promoted the massive accumulation of cytoplasmic vacuoles as well. Mechanistically, BZN repressed the ERK1/2 signaling pathway, and the ERK1/2 activator partially neutralized the inhibitory effect of BZN on OS cells. Subsequently, we demonstrated that vacuoles originated from macropinocytosis and indicated that OS cells might employ macropinocytosis as a compensatory survival mechanism in response to BZN. Remarkably, macropinocytosis inhibitors enhanced the anti-OS effect of BZN in vitro and in vivo. In conclusion, our results suggest that BZN may inhibit OS cells by repressing the ERK1/2 signaling pathway and propose a potential strategy to enhance the BZN-induced inhibitory effect by suppressing macropinocytosis.

Mixed exposure to phthalates and organic UV filters affects Children's pubertal development in a gender-specific manner.

BACKGROUND: Previous studies showed phthalates and UV filters are endocrine-disruptive and associated with puberty. However, few studies have examined effects of mixed exposure. METHODS: Six phthalate metabolites and 12 organic UV filters were detected among 223 school-age children. Puberty development was evaluated at baseline and after 18 months of follow-up. Ordered logistic regression models, least absolute shrinkage and selection operator (LASSO) regression and quantile-based g-computation (qgcomp) were used to evaluate relationships between phthalate metabolites or UV filters exposure and pubertal development. RESULTS: Six phthalate metabolites and 5 UV filters were detectable in urine samples. In boys, BP-3 and 4'-MAP were negatively associated with genital (ORBP-3 = 0.52, (0.27, 0.93), OR4'-MAP = 0.45, (0.25, 0.74)) and pubic hair development (ORBP-3:0.24, (0.05, 0.76), OR4'-MAP:0.24, (0.05, 0.77)). In girls, MEP levels were associated with advanced breast development (OR: 1.29, (1.04, 1.64)). LASSO regression identified BP-3, 4'-MAP, and OD-PABA for inverse associations with pubertal development in boys. MEP was related to an increase in girls' breast development (OR: 1.64, (1.08, 2.63)). Overall mixture was related to a 70% reduction in boys' genital development stage, with a larger effect size than a single chemical in qgcomp. Mixed exposure was associated with girls' earlier puberty onset (OR: 2.61, (1.06, 6.42)). CONCLUSIONS: Our results suggested higher levels of phthalate metabolites and UV filters were associated with delayed pubertal development in boys but with earlier puberty in girls. Higher effect size of joint exposure than single chemicals suggested phthalates and UV filters might have synergistic effects on puberty and distort adolescent endocrine function together.

Alantolactone Inhibits Melanoma Cell Culture Viability and Migration and Promotes Apoptosis by Inhibiting Wnt/ß-Catenin Signaling.

BACKGROUND: Melanoma is a highly invasive and metastatic malignant tumor originating from melanocytes and is associated with a poor prognosis. Surgical resection and chemotherapy are currently the main therapeutic options for malignant melanoma; however, their efficacy is poor, highlighting the need for the development of new, safe, and effective drugs for the treatment of this cancer. OBJECTIVE: To investigate the effects of alantolactone (ALT) on the proliferative, migratory, invasive, and apoptotic ability of malignant melanoma cells and explore its potential anticancer mechanism. METHODS: Melanoma cells (A375 and B16) were treated with different concentrations (4, 6, 8, and 10 µmol/L) of ALT, with DMSO and no treatment serving as controls. The effects of the different concentrations of the drug on cell proliferation were assessed by crystal violet staining and CCK-8 assay. The effects on cell migration and invasion were detected by wound healing and Transwell assays, respectively. Flow cytometry was used to evaluate the effects of the drug on apoptosis and the cell cycle. ALT target genes in melanoma were screened using network pharmacology. Western blotting was used to measure the expression levels of the proliferation-related protein PCNA; the apoptosisrelated proteins Bax, Bcl-2, and caspase-3; the invasion and metastasis-related proteins MMP-2, MMP-7, MMP-9, vimentin, E-cadherin, and N-cadherin; and the canonical Wnt signaling pathway-related proteins ß-catenin, c-Myc, and p-GSK3ß. In addition, an l model of melanoma was established by the subcutaneous injection of A375 melanoma cells into nude mice, following which the effects of ALT treatment on malignant melanoma were determined in vivo. RESULTS: Compared with the controls, the proliferative, migratory, and invasive capacity of ALT-treated melanoma cells was significantly inhibited, whereas apoptosis was enhanced (P<0.01), showing effects that were exerted in a dose-dependent manner. The expression levels of the pro-apoptotic proteins Bax and caspase-3, as well as those of the interstitial marker E-cadherin, were upregulated in melanoma cells irrespective of the ALT concentration (P<0.05). In contrast, the expression levels of the anti-apoptotic protein Bcl-2, the proliferation-related protein PCNA, and the invasion and metastasis-related proteins MMP-2, MMP-7, MMP-9, N-cadherin, and vimentin were downregulated (P<0.05). The network pharmacology results indicated that GSK3ß may be a key ALT target in melanoma. Meanwhile, western blotting assays showed that ALT treatment markedly suppressed the expression of ß-catenin as well as that of its downstream effector c-Myc, and could also inhibit GSK3ß phosphorylation. CONCLUSION: ALT can effectively inhibit the culture viability, migration, and invasion of A375 and B16 melanoma cells while also promoting their apoptosis. ALT may exert its anti-melanoma effects by inhibiting the Wnt/ß-catenin signaling pathway. Combined, our data indicate that ALT has the potential as an effective and safe therapeutic drug for the treatment of melanoma.