LncRNA JHDM1D-AS1 Is a Key Biomarker for Progression and Modulation of Gemcitabine Sensitivity in Bladder Cancer Cells.

Long non-coding RNAs are frequently found to be dysregulated and are linked to carcinogenesis, aggressiveness, and chemoresistance in a variety of tumors. As expression levels of the JHDM1D gene and lncRNA JHDM1D-AS1 are altered in bladder tumors, we sought to use their combined expression to distinguish between low-and high-grade bladder tumors by RTq-PCR. In addition, we evaluated the functional role of JHDM1D-AS1 and its association with the modulation of gemcitabine sensitivity in high-grade bladder-tumor cells. J82 and UM-UC-3 cells were treated with siRNA-JHDM1D-AS1 and/or three concentrations of gemcitabine (0.39, 0.78, and 1.56 µM), and then submitted to cytotoxicity testing (XTT), clonogenic survival, cell cycle progression, cell morphology, and cell migration assays. When JHDM1D and JHDM1D-AS1 expression levels were used in combination, our findings indicated favorable prognostic value. Furthermore, the combined treatment resulted in greater cytotoxicity, a decrease in clone formation, G0/G1 cell cycle arrest, morphological alterations, and a reduction in cell migration capacity in both lineages compared to the treatments alone. Thus, silencing of JHDM1D-AS1 reduced the growth and proliferation of high-grade bladder-tumor cells and increased their sensitivity to gemcitabine treatment. In addition, the expression of JHDM1D/JHDM1D-AS1 indicated potential prognostic value in the progression of bladder tumors.

LncRNA JHDM1D-AS1 is a key biomarker for progression and modulation of gemcitabine sensitivity in bladder cancer cells

Long non-coding RNAs are frequently found to be dysregulated and are linked to carcinogenesis, aggressiveness, and chemoresistance in a variety of tumors. As expression levels of the JHDM1D gene and lncRNA JHDM1D-AS1 are altered in bladder tumors, we sought to use their combined expression to distinguish between low-and high-grade bladder tumors by RTq-PCR. In addition, we evaluated the functional role of JHDM1D-AS1 and its association with the modulation of gemcitabine sensitivity in high-grade bladder-tumor cells. J82 and UM-UC-3 cells were treated with siRNA-JHDM1D-AS1 and/or three concentrations of gemcitabine (0.39, 0.78, and 1.56 µM), and then submitted to cytotoxicity testing (XTT), clonogenic survival, cell cycle progression, cell morphology, and cell migration assays. When JHDM1D and JHDM1D-AS1 expression levels were used in combination, our findings indicated favorable prognostic value. Furthermore, the combined treatment resulted in greater cytotoxicity, a decrease in clone formation, G0/G1 cell cycle arrest, morphological alterations, and a reduction in cell migration capacity in both lineages compared to the treatments alone. Thus, silencing of JHDM1D-AS1 reduced the growth and proliferation of high-grade bladder-tumor cells and increased their sensitivity to gemcitabine treatment. In addition, the expression of JHDM1D/JHDM1D-AS1 indicated potential prognostic value in the progression of bladder tumors.

Genetic Alterations in Patients with Two Clinical Phenotypes of Multiple Sclerosis.

The etiology of multiple sclerosis (MS) is still not known, but the interaction of genetic, immunological, and environmental factors seem to be involved. This study aimed to investigate genetic alterations and the vitamin D status in patients with relapsing-remitting MS (RRMS) and secondary progressive MS (SPMS). A total of 53 patients (29 RRMS; 24 SPMS) and 25 healthy subjects were recruited to evaluate the micronucleated cell (MNC) frequency and nuclear abnormalities in the buccal mucosa, gene expression profiling in mononuclear cells, and plasmatic vitamin D concentration in the blood. Results showed a higher frequency of cells with karyorrhexis (SPMS) and lower frequencies of nuclear pyknosis (RRMS and SPMS) and karyolysis (SPMS) in patients with MS. Significant increase in the frequency of MNC was detected in the buccal mucosa of RRMS and SPMS patients. HIF1A, IL13, IL18, MYC, and TNF were differentially expressed in MS patients, and APP was overexpressed in cells of RRMS compared to SPMS patients. No relationship was observed between vitamin D level and the differentially expressed genes. In conclusion, the cytogenetic alterations in the buccal mucosa can be important indicators of genetic instability and degenerative processes in patients with MS. Furthermore, our data introduced novel biomarkers associated with the molecular pathogenesis of MS.

BCL2 and miR-181a transcriptional alterations in umbilical-cord blood cells can be putative biomarkers for obesity.

Several findings suggest that in utero stressor stimuli can alter fetal development by promoting transcriptional changes, and predisposing the neonate to diseases later in life. This study aimed to investigate whether a hyperglycemic environment in pregnant women with gestational diabetes mellitus (GDM) is able to cause fetal genetic alterations and predispose neonates to obesity. Transcriptional alteration of SIRT1, TP53 and BCL2 genes, miR-181a (a SIRT1 or BCL2 regulator) and telomere length were evaluated in placental and umbilical-cord blood cells. Healthy (HP; n = 20) and GDM (n = 20) pregnant women and their respective neonates were included in the study. Additionally, obese (n = 20) and eutrophic (n = 20) adults also participated as reference populations. Gene expression data showed down-regulation of BCL2 in umbilical-cord and peripheral blood cells from GDM neonates and obese adults, respectively. The miR-181a was down-regulated only in umbilical-cord blood cells of GDM neonates. Telomere length presented no significant difference. In conclusion, our study demonstrated that the GDM hyperglycemic intrauterine environment promotes transcriptional alterations in BCL2 and miR-181a in neonate umbilical-cord blood cells. Furthermore, both GDM neonates and obese subjects share the same transcriptional alteration in BCL2. Considering the relationship between obesity development and the functions regulated by these two genes, BCL2 and miR-181a could be adopted as potential biomarkers for childhood obesity. However, further study designs are recommended to confirm this hypothesis.

Cytotoxic and toxicogenomic effects of silibinin in bladder cancer cells with different TP53 status.

Silibinin is a natural phenol found in the seeds of the milk thistle plant. Recent data have shown its effectiveness for preventing/treating bladder tumours. Therefore, in this study we investigated the cytotoxic and toxicogenetic activity of silibinin in bladder cancer cells with different TP53 statuses. Two bladder urothelial carcinoma cell lines were used: RT4 (wild-type TP53 gene) and T24 (mutated TP53 gene). Cell proliferation, clonogenic survival, apoptosis rates, genotoxicity and relative expression profile of FRAP/mTOR, FGFR3, AKT2 and DNMT1 genes and of miR100 and miR203 were evaluated. Silibinin promoted decreased proliferation and increased late apoptosis in TP53 mutated cells. Increased early apoptosis rates, primary DNA damage, and decrease of cell colonies in the clonogenic survival assay were detected in both RT4 and T24 cell lines. Down-regulation of FRAP/mTOR, AKT2, FGFR3, DNMT1 and miR100 expression occurred in RT4 cells. Modulation of miR203 was observed in both cell lines. In conclusion, despite the reduction of clone formation in both cell lines, the toxicogenomic effect of silibinin on FRAP/mTOR, AKT2, FGFR3, DNMT1 and miR100 was dependent on the TP53 status. Taken together, the data confirmed the role of silibinin as an antiproliferative compound, whose mechanism of action was related to the TP53 status.

Inhibition of bladder cancer cell proliferation by allyl isothiocyanate (mustard essential oil).

Natural compounds hold great promise for combating antibiotic resistance, the failure to control some diseases, the emergence of new diseases and the toxicity of some contemporary medical products. Allyl isothiocyanate (AITC), which is abundant in cruciferous vegetables and mustard seeds and is commonly referred to as mustard essential oil, exhibits promising antineoplastic activity against bladder cancer, although its mechanism of action is not fully understood. Therefore, the aim of this study was to investigate the effects of AITC activity on bladder cancer cell lines carrying a wild type (wt; RT4) or mutated (T24) TP53 gene. Morphological changes, cell cycle kinetics and CDK1, SMAD4, BAX, BCL2, ANLN and S100P gene expression were evaluated. In both cell lines, treatment with AITC inhibited cell proliferation (at 62.5, 72.5, 82.5 and 92.5µM AITC) and induced morphological changes, including scattered and elongated cells and cellular debris. Gene expression profiles revealed increased S100P and BAX and decreased BCL2 expression in RT4 cells following AITC treatment. T24 cells displayed increased BCL2, BAX and ANLN and decreased S100P expression. No changes in SMAD4 and CDK1 expression were observed in either cell line. In conclusion, AITC inhibits cell proliferation independent of TP53 status. However, the mechanism of action of AITC differed in the two cell lines; in RT4 cells, it mainly acted via the classical BAX/BCL2 pathway, while in T24 cells, AITC modulated the activities of ANLN (related to cytokinesis) and S100P. These data confirm the role of AITC as a potential antiproliferative compound that modulates gene expression according to the tumor cell TP53 genotype.

Cell cycle kinetics, apoptosis rates, DNA damage and TP53 gene expression in bladder cancer cells treated with allyl isothiocyanate (mustard essential oil).

Allyl isothiocyanate (AITC) is present in plants of the cruciferous family and is abundant in mustard seed. Due to its high bioavailability in urine after ingestion, AITC has been considered a promising antineoplastic agent against bladder cancer. Because TP53 mutations are the most common alterations in bladder cancer cells and are frequently detected in in situ carcinomas, in this study, we investigated whether the AITC effects in bladder cancer cells are dependent on the TP53 status. Two bladder transitional carcinoma cell lines were used: RT4, with wild-type TP53; and T24, mutated TP53 gene. AITC was tested at concentrations of 0.005, 0.0625, 0.0725, 0.0825, 0.0925, 0.125 and 0.25 µM in cytotoxicity, cell and clonogenic survival assays, comet and micronucleus assays and for its effects on cell cycle and apoptosis by flow cytometry and on TP53 gene expression. The data showed increased primary DNA damage in both cell lines; however, lower concentrations of AITC were able to induce genotoxicity in the mutant cells for the TP53 gene. Furthermore, the results demonstrated increased apoptosis and necrosis rates in the wild-type cells, but not in mutated TP53 cells, and cell cycle arrest in the G2 phase for mutated cells after AITC treatment. No significant differences were detected in TP53 gene expression in the two cell lines. In conclusion, AITC caused cell cycle arrest, increased apoptosis rates and varying genotoxicity dependent on the TP53 status. However, we cannot rule out the possibility that those differences could reflect other intrinsic genetic alterations in the examined cell lines, which may also carry mutations in genes other than TP53. Therefore, further studies using other molecular targets need to be performed to better understand the mechanisms by which AITC may exert its antineoplastic properties against tumor cells.

MRE11A and SKP2 genes are associated with the increased cytotoxicity induced by the synergistic effects of cisplatin and gemcitabine in bladder cancer cells.

The combination of gemcitabine and cisplatin has been shown previously to elicit a synergistic therapeutic effect on bladder cancer cell lines and result in reduced cell survival. However, the precise mechanism by which cells die has not been elucidated. Cell cycle-related genes are the predominant targets of chemotherapeutic protocols. Therefore, molecular biomarkers that are predictive of therapeutic outcomes associated with tumor sensitivity might be important for optimal treatment protocol selection. The aim of this study was to investigate the changes in gene expression in cell cycle-related genes that were induced by cisplatin, gemcitabine or a combined treatment using both agents in a low-grade urinary bladder transitional carcinoma cell line (RT4). The following three treatment protocols were used: 1.0 µM cisplatin, 1.56 µM gemcitabine and a combination of 1.0 µM cisplatin and 1.56 µM gemcitabine. Cytometry and morphology analysis (by phase-contrast photomicrography) were performed in addition to pathway-specific gene expression analysis using quantitative RT-PCR gene arrays. The following results were observed after 1.0 µM cisplatin treatment: (1) a decrease in cell number, (2) an increased percentage of scattered cells and (3) downregulated expression of genes related to cell cycle arrest, G1/S-to-mitotic cell cycle transition, DNA repair, apoptosis, transcription and mitosis. Treatment with 1.56 µM gemcitabine, or with both drugs simultaneously, induced the following effects: (1) a decrease in cell number, (2) an increased percentage of scattered and elongated cells, (3) the modulation of genes that are predominantly involved in DNA repair and (4) a significant upregulation of genes related to cell cycle arrest. Reduced cell density was observed after the combined treatment compared to the two other single-agent protocols. The downregulation of MRE11A and SKP2 was observed only in cells subjected to the combined treatment. In conclusion, cisplatin, gemcitabine and the combination of both drugs elicited distinct toxicogenomic effects in the RT4 bladder transitional carcinoma cell line, although disruptions in the expression of cell cycle control-related genes and other pathways responsible for cell survival were observed for all of the protocols. MRE11A and SKP2 downregulation appeared to be responsible for the synergistic therapeutic effects elicited by cisplatin and gemcitabine.