Curcumin strengthens a spontaneous self-protective mechanism-SP1/PRDX6 pathway, against di-n-butyl phthalate-induced testicular ferroptosis damage.

As one of the common plasticizers, di-n-butyl phthalate (DBP) has been using in various daily consumer products worldwide. Since it is easily released from products and exists in the environment for a long time, it has a lasting impact on human health, especially male reproductive health. However, the detailed mechanism of testicular damage from DBP and the protection strategy are still not clear enough. In this study, we found that DBP could induce dose-dependent ferroptosis in testicular tissue. Mechanism dissection indicates that DBP can upregulate SP1 expression, which could directly transcriptionally upregulate PRDX6, a negative regulator of ferroptosis. Overexpression of PRDX6 or adding SP1 agonist curcumin could suppress the DBP-induced ferroptosis on testicular cells. In vivo, rats were given 500 mg/kg/day DBP orally for 3 weeks; elevated levels of ferroptosis were detected in testicular tissue. When the above-mentioned doses of DBP and curcumin at a dose of 300 mg/kg/day were administered intragastrically simultaneously, the testicular ferroptosis induced by DBP was alleviated. Immunohistochemistry and quantitative real-time PCR of testis tissue showed that the expression of PRDX6 was upregulated under the action of DBP and curcumin. These findings suggest a spontaneous self-protection mechanism of testicular tissue from DBP damage by upregulating SP1 and PRDX6. However, it is not strong enough to resist the DBP-induced ferroptosis. Curcumin can strengthen this self-protection mechanism and weaken the level of ferroptosis induced by DBP. This study may help us to develop a novel therapeutic option with curcumin to protect the testicular tissue from ferroptosis and function impairment by DBP.

In silico, in vitro and in vivo studies: Dibutyl phthalate promotes prostate cancer cell proliferation by activating Forkhead Box M1 and remission after Natura-α pretreatment.

Dibutyl phthalate (DBP) is widely used in perfumes, cosmetics, shampoos and medical devices. It is ubiquitous in the environment and greatly endangers people's health. Several studies have reported that being exposed to it can promote the development of lung cancer, breast cancer, hepatoma, and multiple myeloma. However, there are still few studies on the specific molecular mechanism and prevention methods of DBP promoting the progression of prostate cancer. This study, in silico, in vitro and in vivo, aims to explore the promoting effect of DBP on prostate cancer cell proliferation. In silico analysis, we obtained a set of DBP interactive genes by utilizing TCGA, CTD and GEO database. These genes are mainly enriched in cell cycle regulatory pathways and they have high degree of homogeneity. We found that these genes shared one transcription factor - Forkhead Box M1 (FOXM1) by performing Chip-X Enrichment Analysis (Version 3.0). FOXM1, once called the 2010 Molecule of the Year, aberrantly expressed in up to 20 kinds of tumors. In vitro experiments, we used DBP at concentrations of 10-8 M and 5 * 10-7 M to treat C4-2 and PC3 cells for 6 days, respectively. Cell viability was promoted significantly. When Natura-α was added in the background of above-mentioned concentration of DBP, this effect was significantly inhibited. In addition, we also found that DBP can interfering with the efficacy of enzalutamide therapy. The introduction of Natura-α can also reverse this phenomenon. In vivo, subcutaneous tumor formation experiments in nude mice, 800 mg/kg/day DBP can promote the growth of prostate cancer. This phenomenon was suppressed when Natura-α (100 mg/kg/day) was added. Based on the results of the above three levels, we confirmed that DBP can target FOXM1 to promote prostate cancer cell proliferation. Natura-α can reverse its cancer-promoting effect. This study provides new insights into the impact of DBP on prostate cancer.

Comprehensive investigation into cuproptosis in the characterization of clinical features, molecular characteristics, and immune situations of clear cell renal cell carcinoma.

Background: Copper-induced cell death has been widely investigated in human diseases as a form of programmed cell death (PCD). The newly recognized mechanism underlying copper-induced cell death provided us creative insights into the copper-related toxicity in cells, and this form of PCD was termed cuproptosis. Methods: Through consensus clustering analysis, ccRCC patients from TCGA database were classified into different subgroups with distinct cuproptosis-based molecular patterns. Analyses of clinical significance, long-term survival, and immune features were performed on subgroups accordingly. The cuproptosis-based risk signature and nomogram were constructed and validated relying on the ccRCC cohort as well. The cuproptosis scoring system was generated to better characterize ccRCC patients. Finally, in vitro validation was conducted using ccRCC clinical samples and cell lines. Result: Patients from different subgroups displayed diverse clinicopathological features, survival outcomes, tumor microenvironment (TME) characteristics, immune-related score, and therapeutic responses. The prognostic model and cuproptosis score were well validated and proved to efficiently distinguish the high risk/score and low risk/score patients, which revealed the great predictive value. The cuproptosis score also tended out to be intimately associated with the prognosis and immune features of ccRCC patients. Additionally, the hub cuproptosis-associated gene (CAG) FDX1 presented a dysregulated expression pattern in human ccRCC samples, and it was confirmed to effectively promote the killing effects of copper ionophore elesclomol as a direct target. In vitro functional assays revealed the prominent anti-cancer role of FDX1 in ccRCC. Conclusion: Cuproptosis played an indispensable role in the regulation of TME features, tumor progression, and long-term prognosis of ccRCC.

KCNN4 may weaken anti-tumor immune response via raising Tregs and diminishing resting mast cells in clear cell renal cell carcinoma.

BACKGROUND: Studies over the past decade have shown that competitive endogenous RNA (ceRNA) plays an essential role in the tumorigenesis and progression of clear cell renal cell carcinoma (ccRCC). Meanwhile, immune checkpoint blocker is gradually moving towards the first-line treatment of ccRCC. Hence, it's urgent to develop a new prediction model for the efficiency of immunotherapy. At present, there is no study to reveal the effect of ceRNA network on the efficiency of immunotherapy for ccRCC. METHODS: To systematically analyze the effect of ceRNA hub genes in ccRCCon immune response, we constructed prognosis models based on ceRNAs and immune cells, respectively. We constructed ceRNA network using hypergeometric distribution test and correlation analysis with R script based on The Cancer Genome Atlas (TCGA) database. We then applied the Cibersort algorithm to simulate the infiltration overview of immune cells in kidney renal clear carcinoma (KIRC) samples. Prognosis-related immune cells were screened and a predictive model of these cells was constructed. Prognosis-related immune cells and ceRNA hub genes were performed with co-expression analysis. Finally, qRT-PCR and immunofluorescence assays were performed to validate the results. RESULTS: The construction of ceRNA related prognosis model contained 8 hub genes, including RELT, MYO9B, KCNN4, SIX1, OTOGL, MALAT1, hsa-miR-130b-3p, and hsa-miR-21-5p. The area under the receiver operating characteristic curve (AUC) was 0.77 at 5 years. For the construction of immune cells prognosis model, 3 immune cells (T cells regulatory, Macrophages, Mast cells resting) were adopted, and the AUC was 0.65 at 5 years. We then merged the two models by correlation analysis and co-expression analysis. Finally, we found that KCNN4 positively correlates with T cells regulatory (Tregs) and negatively correlates with mast cells resting significantly. Furthermore, higher expression of KCNN4 may lead to a higher potential for immune evasion and lower efficiency for immune checkpoint inhibitors (ICIs). CONCLUSIONS: Generally, this is the first study to assess the prognostic value of immune related ceRNA hub genes in ccRCC, and KCNN4 was finally demonstrated to be a key regulatory factor with strong correlation with Tregs and mast cells resting.

Clusterin suppresses invasion and metastasis of testicular seminoma by upregulating COL15a1.

Seminoma is the most common subtype of testicular germ cell tumor, with an increasing incidence worldwide. Clusterin (CLU) expression was found to be downregulated in testicular seminoma in our previous study. We now expanded the sample size, and further indicated that CLU expression correlates with tumor stage. Tcam-2 cell line was used to investigate the CLU function in testicular seminoma, and CLU was found to inhibit the proliferation and metastasis abilities. Besides, extracellular matrix protein COL15a1 was demonstrated as the downstream of CLU to affect the epithelial-mesenchymal transition (EMT) process via competitively binding to DDR1 with COL1A1 and inhibiting the phosphorylation of PYK2. MEF2A was found to interact with CLU and bind to the promoter of COL15a1 and so upregulate its expression. This is the first study using testicular xenografts in situ to simulate testicular seminoma metastatic and proliferative capacities. In conclusion, CLU acts as a tumor suppressor to inhibit the metastasis of testicular seminoma by interacting with MEF2A to upregulate COL15a1 and blocking the EMT process.

Identification of autophagy-related long non-coding RNA prognostic and immune signature for clear cell renal cell carcinoma.

BACKGROUND: Studies over the past decade have shown that long non-coding RNAs (lncRNAs) play an essential role in the tumorigenesis and progression of kidney renal clear cell carcinoma (KIRC). Meanwhile, autophagy has been demonstrated to regulate KIRC pathogenesis and targeting therapy resistance. However, the prognostic value of autophagy-related lncRNAs in KIRC patients has not been reported before. METHODS: In this study, we obtained transcriptome data of 611 KIRC cases from the TCGA database and 258 autophagy-related mRNAs from the HADb database to identify autophagy-related lncRNAs by co-expression network. A prognostic model was then established basing on these autophagy-related lncRNAs, dividing patients into high-risk and low-risk groups. Survival analysis, clinical variables dependent receiver operating characteristic (ROC) analyses, univariate/multivariate Cox analyses, and clinical correlation analysis were performed based on risk signature with R language. Gene set enrichment analysis (GSEA) was then performed to investigate the potential mechanism of the risk signature promoting KIRC progression with GSEA software. CIBERSORT algorithm was performed to assess the impact of these lncRNAs on the infiltration of immune cells. RESULTS: A total of 17 lncRNAs were screened out and all these lncRNAs were found significantly related to KIRC patients' overall survival in subsequent survival analyses. Besides, the overall survival time in the high-risk group was much poorer than in the low-risk group. The ROC analysis revealed that the prognostic value of risk signature was better than age, gender, grade, and N stage. Univariate/multivariate analyses suggested that the risk signature was an independent predictive factor for KIRC patients. Immune and autophagy related pathways were dramatically enriched in high-risk and low-risk groups, respectively, and lncRNAs related immune cells were identified by CIBERSORT. CONCLUSIONS: In summary, our identified 17 autophagy-related lncRNAs had prognostic value for KIRC patients which may function in immunomodulation.

Identification of MX2 as a Novel Prognostic Biomarker for Sunitinib Resistance in Clear Cell Renal Cell Carcinoma.

BACKGROUND: Antiangiogenic agents that specifically target vascular endothelial growth factor receptor (VEGFR), such as sunitinib, have been utilized as the standard therapy for metastatic clear cell renal cell carcinoma (ccRCC) patients. However, most patients eventually show no responses to the targeted drugs, and the mechanisms for the resistance remain unclear. This study is aimed to identify pivotal molecules and to uncover their potential functions involved in this adverse event in ccRCC treatment. METHODS: Two datasets, GSE64052 and GSE76068, were obtained from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified using the limma package in R software. The gene set enrichment analysis (GSEA) was conducted using clusterProfiler package. A protein-protein interaction (PPI) network was built using the STRING database and Cytoscape software. Kaplan-Meier survival curves were plotted using R software. qRT-PCR and Western blotting were used to detect the MX2 and pathway expression in RCC cell lines. Sunitinib-resistant cell lines were constructed, and loss-of-function experiments were conducted by knocking down MX2. All statistical analyses were performed using R version 3.6.1 and SPSS 23.0. RESULTS: A total of 760 DEGs were derived from two datasets in GEO database, and five hub genes were identified, among which high-level MX2 exhibited a pronounced correlation with poor overall survival (OS) in sunitinib-resistant ccRCC patients. Clinical correlation analysis and Gene Set Variation Analysis (GSVA) on MX2 showed that the upregulation of MX2 was significantly related to the malignant phenotype of ccRCC, and it was involved in several pathways and biological processes associated with anticancer drug resistance. qRT-PCR and Western blotting revealed that MX2 was distinctly upregulated in sunitinib-resistant RCC cell lines. Colony formation assay and Cell Counting Kit-8 (CCK8) assay showed that MX2 strongly promoted resistant capability to sunitinib of ccRCC cells. CONCLUSION: MX2 is a potent indicator for sunitinib resistance and a therapeutic target in ccRCC patients.