Objective To study the impacts of curcumin on the proliferation, migration and cisplatin (DDP) resistance of bladder cancer cells by regulating the liver kinase B1-AMP activated protein kinase-microtubule-associated protein 1 light chain 3 (LKB1-AMPK-LC3) signaling pathway. Methods Human bladder cancer cell line T24 was cultured in vitro, and its DDP resistant T24/DDP cells were induced by cisplatin (DDP). After treating T24 and T24/DDP cells with different concentrations of curcumin, the optimal concentration of curcumin was screened by MTT assay. T24 cells were randomly grouped into control group, curcumin group, metformin group, and combination group of curcumin and metformin. After treatment with curcumin and LKB1-AMPK activator metformin, the proliferation, autophagy, migration, and apoptosis of T24 cells in each group were detected by MTT assay, monodansylcadavrine (MDC) fluorescence staining, cell scratch assay, and flow cytometry, respectively. Western blot was used to detect the expression of proteins related to LKB1-AMPK-LC3 signaling pathway in T24 cells of each group. T24/DDP cells were randomly assigned into control group, curcumin group, metformin group, and combination group of curcumin and metformin. Cells were treated with curcumin and metformin according to grouping and treated with different concentrations of DDP simultaneously. Then, the effect of curcumin on the DDP resistance coefficient of T24/DDP cells was detected by MTT assay. T24/DDP cells were randomly grouped into control group, DDP group, combination groups of DDP and curcumin, DDP and metformin, DDP, curcumin and metformi. After treatment with DDP, curcumin, and metformin, the proliferation, autophagy, migration, apoptosis, drug resistance, and the expression of proteins related to LKB1-AMPK-LC3 signaling pathway in T24/DDP cells of each group were detected with the same methods. Results Compared with the control group, the activity of T24 cells, relative number of autophagosomes, migration rate, Phosphorylated-LKB1 (p-LKB1)/LKB1, Phosphorylated-AMPK (p-AMPK)/AMPK, LC3II/LC3I, and the DDP resistance coefficient of T24/DDP cells in the curcumin group were lower, and the apoptosis rate of T24 cells was higher; the changes in various indicators in the metformin group were opposite to those in the curcumin group. Compared with the curcumin group, the activity of T24 cells, relative number of autophagosomes, migration rate, p-LKB1/LKB1, p-AMPK/AMPK, LC3II/LC3I, and the DDP resistance coefficient of T24/DDP cells in the combination group of curcumin and metformin were higher, and the apoptosis rate of T24 cells was lower. Compared with the control group, there were no obvious changes in various indicators of T24/DDP cells in the DDP group. Compared with the control group and DDP group, the viability of T24/DDP cells, relative number of autophagosomes, migration rate, P-glycoprotein (P-gp) protein expression, p-LKB1/LKB1, p-AMPK/AMPK, and LC3II/LC3I in the combination group of DDP and curcumin were lower, and the apoptosis rate of T24/DDP cells was higher; the changes in the above indicators in the combination group of DDP and metformin were opposite to those in the combination group of DDP and curcumin. Compared with the combination group of DDP and curcumin, the viability of T24/DDP cells, relative number of autophagosomes, migration rate, P-gp protein expression, p-LKB1/LKB1, p-AMPK/AMPK, and LC3II/LC3I in the combination group of DDP, curcumin and metformin were higher, and the apoptosis rate of T24/DDP cells was lower. Conclusion Curcumin can reduce the activity of LKB1-AMPK-LC3 signaling pathway, thereby inhibiting autophagy, proliferation and migration of bladder cancer cells, promoting their apoptosis, and weakening their resistance to DDP.
Humans ; Cisplatin/pharmacology* ; Curcumin/pharmacology* ; Cell Proliferation/drug effects* ; Signal Transduction/drug effects* ; Protein Serine-Threonine Kinases/genetics* ; AMP-Activated Protein Kinases/metabolism* ; Drug Resistance, Neoplasm/drug effects* ; Urinary Bladder Neoplasms/pathology* ; Cell Line, Tumor ; Cell Movement/drug effects* ; AMP-Activated Protein Kinase Kinases ; Microtubule-Associated Proteins/metabolism* ; Apoptosis/drug effects* ; Antineoplastic Agents/pharmacology* ; Metformin/pharmacology* ; Autophagy/drug effects*

OBJECTIVE: To investigate the safety and prognostic factors influencing the treatment of upper urinary tract urothelial carcinoma (UTUC) combined with bladder cancer (BCa) by laparoscopic simultaneous radical cystectomy and nephroureterectomy (RCNU). METHODS: The clinical data of patients admitted to Peking University Third Hospital for laparoscopic RCNU surgery from January 2009 to September 2023 were analyzed retrospectively. Based on the same gender, age (±5 years), history of uroepithelial tumors, underlying diseases, T-stage, N-stage, M-stage, American Society of Anesthesiologists (ASA) score, Charlson comorbidity index, and body mass index (BMI) (±5), 34 patients with RCNU were matched 1 ∶1 with patients with bladder cancer who underwent laparoscopic radical cystectomy (RC) alone. Kaplan-Meier survival analysis was used to calculate patient survival, and Cox proportional regression risk model was used to analyze clinical factors affecting prognosis. RESULTS: Of the 68 patients enrolled, the follow-up rate was 100% with a median follow-up time of 27.0 (11.7, 60.2) months. Comparison of intraoperative conditions (including operation time, estimated intraoperative bleeding, intra-operative blood transfusion, etc.) between the two groups of patients showed no significant difference (P>0.05). Comparison of preoperative creatinine and postoperative creatinine between the two groups of patients showed significant differences (P < 0.05). The perioperative Clavien grade Ⅲ-Ⅳ complication rates were 2.9% (1/34) in the RC group and 5.9% (2/34) in the RCNU group. There was no significant difference in terms of perioperative complications between the two groups. Overall survival was significantly lower in the patients receiving RCNU compared with the matched group receiving RC alone (P < 0.05). Cox regression analysis suggested that two factors, high N stage and high postoperative creatinine, were independent risk factors affecting the prognosis of patients in the 2 groups (P < 0.05). CONCLUSION The overall survival prognosis of patients undergoing RCNU surgery was worse compared with laparoscopic RC surgery alone during the same period. There was no clinically significant difference between the two groups in terms of operation time, intraoperative bleeding, and perioperative complications, and there were clinically significant differences in preoperative renal function and post-operative renal function.
Humans ; Laparoscopy/methods* ; Nephroureterectomy/methods* ; Cystectomy/methods* ; Prognosis ; Male ; Retrospective Studies ; Female ; Urinary Bladder Neoplasms/mortality* ; Middle Aged ; Aged

OBJECTIVES: Bladder cancer is a common malignancy with high incidence and poor prognosis. N⁶-methyladenosine (m⁶A) modification is widely involved in diverse physiological processes, among which the m⁶A recognition protein YTH N⁶-methyladenosine RNA binding protein F2 (YTHDF2) plays a crucial role in bladder cancer progression. This study aims to elucidate the molecular mechanism by which O-linked N-acetylglucosamine (O-GlcNAc) modification of YTHDF2 regulates its downstream target, period circadian regulator 1 (PER1), thereby promoting bladder cancer cell proliferation. METHODS: Expression of YTHDF2 in bladder cancer was predicted using The Cancer Genome Atlas (TCGA). Twenty paired bladder cancer and adjacent normal tissues were collected at the clinical level. Normal bladder epithelial cells (SV-HUC-1) and bladder cancer cell lines (T24, 5637, EJ-1, SW780, BIU-87) were examined by quantitative real-time PCR (RT-qPCR), Western blotting, and immunohistochemistry for expression of YTHDF2, PER1, and proliferation-related proteins [proliferating cell nuclear antigen (PCNA), minichromosome maintenance complex component 2 (MCM2), Cyclin D1]. YTHDF2 was silenced in 5637 and SW780 cells, and cell proliferation was assessed by Cell Counting Kit-8 (CCK-8), colony formation, and EdU assays. Bioinformatics was used to predict glycosylation sites of YTHDF2, and immunoprecipitation (IP) was performed to detect O-GlcNAc modification levels of YTHDF2 in tissues and cells. Bladder cancer cells were treated with DMSO, OSMI-1 (O-GlcNAc inhibitor), or Thiamet G (O-GlcNAc activator), followed by cycloheximide (CHX), to assess YTHDF2 ubiquitination by IP. YTHDF2 knockdown and Thiamet G treatment were further used to evaluate PER1 mRNA stability, PER1 m⁶A modification, and cell proliferation. TCGA was used to predict PER1 expression in tissues; SRAMP predicted potential PER1 m⁶A sites. Methylated RNA immunoprecipitation (MeRIP) assays measured PER1 m⁶A modification. Finally, the effects of knocking down YTHDF2 and PER1 on 5637 and SW780 cell proliferation were assessed. RESULTS: YTHDF2 expression was significantly upregulated in bladder cancer tissues compared with adjacent tissues (mRNA: 2.5-fold; protein: 2-fold), which O-GlcNAc modification levels increased 3.5-fold (P<0.001). YTHDF2 was upregulated in bladder cancer cell lines, and its knockdown suppressed cell viability (P<0.001), downregulated PCNA, MCM2, and CyclinD1 (all P<0.05), reduced colony numbers 3-fold (P<0.01), and inhibited proliferation. YTHDF2 exhibited elevated O-GlcNAc modification in cancer cells. OSMI-1 reduced YTHDF2 protein stability (P<0.01) and enhanced ubiquitination, while Thiamet G exerted opposite effects (P<0.001). Thiamet G reversed the proliferation-suppressive effects of YTHDF2 knockdown, promoting cell proliferation (P<0.01) and upregulating PCNA, MCM2, and CyclinD1 (all P<0.05). Mechanistically, YTHDF2 targeted PER1 via m⁶A recognition, promoting PER1 mRNA degradation. Rescue experiments showed that PER1 knockdown reversed the inhibitory effect of YTHDF2 knockdown on cell proliferation, upregulated PCNA, MCM2, and Cyclin D1 (all P<0.05), and promoted bladder cancer cell proliferation (P<0.001). CONCLUSIONS O-GlcNAc modification YTHDF2 promotes bladder cancer development by downregulating the tumor suppressor gene PER1 through m⁶A-mediated post-transcriptional regulation.
Humans ; Urinary Bladder Neoplasms/metabolism* ; RNA-Binding Proteins/genetics* ; Cell Proliferation ; Cell Line, Tumor ; Disease Progression ; Acetylglucosamine/metabolism* ; Adenosine/metabolism* ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor

OBJECTIVES: To explore the potential of pyrroline-5-carboxylate reductase 1 (PYCR1) as a pan-cancer biomarker and investigate its expression, function, and clinical significance in bladder cancer (BLCA). METHODS: Bioinformatics analysis was conducted to evaluate the associations of PYCR1 with prognosis, immune microenvironment remodeling, tumor mutation burden (TMB), and microsatellite instability (MSI) in cancer patients. Using the TCGA-BLCA dataset, univariate and multivariate regression analyses were performed to assess the potential of PYCR1 as an independent prognostic risk factor for BLCA, and a clinical decision model was constructed. The IMvigor210 cohort was utilized to evaluate the potential of PYCR1 for independently predicting the efficacy of immunotherapy. The pRRophetic was employed to screen candidate chemotherapeutic agents for treating BLCA with high PYCR1 expression. The CMap-XSum algorithm and molecular docking techniques were used to explore and validate small molecule inhibitors of PYCR1. RESULTS: A high expression of PYCR1 was significantly associated with poor prognosis, immune cell infiltration, TMB and MSI in various tumors (r>0.3). PYCR1 was overexpressed in BLCA, and high PYCR1 expression was closely related to poor prognosis in BLCA patients (HR: 1.14, 95% CI: 1.02-1.68, P=0.006). The IC₅₀ of the anti-cancer drugs cetuximab, 5-fluorouracil, and doxorubicin increased significantly in BLCA cell lines with high PYCR1 expressions (P<0.0001). CONCLUSIONS High PYCR1 expression is an independent risk factor for poor prognosis in BLCA patients and can serve as a significant indicator for clinical decision-making as well as a marker for predicting sensitivity to chemotherapeutic agents and the efficacy of immunotherapy.
Humans ; Urinary Bladder Neoplasms/genetics* ; Immunotherapy ; Prognosis ; Pyrroline Carboxylate Reductases/metabolism* ; Biomarkers, Tumor/genetics* ; delta-1-Pyrroline-5-Carboxylate Reductase ; Microsatellite Instability ; Tumor Microenvironment ; Mutation ; Computational Biology ; Molecular Docking Simulation

OBJECTIVES: To investigate the role of apelin in regulating proliferation, migration and angiogenesis of bladder cancer cells and the possible regulatory mechanism. METHODS: GEO database was used to screen the differentially expressed genes in bladder cancer tissues and cells. Bladder cancer and paired adjacent tissues were collected from 60 patients for analysis of apelin expressions in relation to clinicopathological parameters. In cultured bladder cancer J82 cells and human umbilical vein endothelial cells (HUVECs), the effects of transfection with an apelin-overexpressing plasmid or specific siRNAs targeting apelin, fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 1 (FGFR1) on proliferation and migration of J82 cells and tube formation in HUVECs were examined using plate cloning assay, Transwell assay, and angiogenesis assay; the changes in FGF2 expression and FGFR1 phosphorylation were detected using Western blotting. RESULTS: The expression level of apelin was significantly higher in bladder cancer tissues than adjacent tissues, and bladder cancer cell lines (T24 and J82) also expressed higher mRNA and protein levels of apelin than SV-HUC-1 cells. Apelin expression level in bladder cancer tissues was correlated with tumor invasion, distant metastasis and advanced TNM stages. Apelin knockdown significantly suppressed proliferation and migration of J82 cells and decreased the total angiogenic length of HUVECs. In contrast, apelin overexpression significantly promoted proliferation and migration and enhanced FGFR1 phosphorylation in J82 cells, and increased the total angiogenesis length in HUVECs, but this effects were effectively mitigated by transfection of the cells with FGF2 siRNA or FGFR1 siRNA. CONCLUSIONS High expression of apelin promotes J82 cell proliferation and migration and HUVEC angiogenesis by promoting activation of the FGF2/FGFR1 pathway.
Humans ; Urinary Bladder Neoplasms/blood supply* ; Receptor, Fibroblast Growth Factor, Type 1/metabolism* ; Cell Proliferation ; Cell Movement ; Fibroblast Growth Factor 2/metabolism* ; Neovascularization, Pathologic ; Human Umbilical Vein Endothelial Cells ; Cell Line, Tumor ; Signal Transduction ; Apelin ; Intercellular Signaling Peptides and Proteins/genetics* ; Female ; Male ; Angiogenesis

OBJECTIVES: To investigate the inhibitory effect of pirfenidone (PFD) on growth of bladder cancer xenograft and its regulatory effect on Treg cells in tumor-bearing mice. METHODS: Thirty-two C57BL/6 mice bearing ectopic bladder tumors were randomized into control and PFD groups (n=16). In PFD group, PFD was administered orally at the daily dose of 500 mg/kg, and tumor growth and survival of the mice were monitored. After treatment for 21 days, the tumors and vital organs were harvested for analysis. Immunohistochemistry was used to assess CD3, CD4, CD8, and FOXP3 expressions in the tumors. Flow cytometry and RT-qPCR were used to analyze the percentage of CD4⁺CD25⁺FOXP3⁺ Treg cells and IL-2, IL-10, and IL-35 expressions in the tumors and spleens; organ damage of the mice was examined with HE staining. RESULTS: Compared with the control group, the PFD-treated mice exhibited significantly lower tumor growth rate with smaller tumor volumes at day 21, along with improved survival at day 28. Immunohistochemistry revealed no significant differences in the infiltration of CD3⁺ and CD8⁺ cells between the two groups, but the percentages of CD4⁺ and FOXP3⁺ cells were significantly lower in the tumors of PFD-treated mice. Flow cytometric analysis confirmed a decrease in CD4⁺CD25⁺FOXP3⁺ Treg cells in the tumors from PFD-treated mice, which also had reduced expression levels of IL-2, IL-10 and IL-35 mRNAs in the tumors. No significant differences were found in Treg cell populations or cytokine expressions in the spleen tissues between the two groups. HE staining showed obvious organ damage in neither of the groups. CONCLUSIONS PFD inhibits bladder cancer growth and enhances survival of tumor-bearing mice possibly by suppressing Treg cells in the tumor microenvironment.
Animals ; Urinary Bladder Neoplasms/drug therapy* ; Mice ; T-Lymphocytes, Regulatory/metabolism* ; Mice, Inbred C57BL ; Interleukins/metabolism* ; Interleukin-10/metabolism* ; Cell Line, Tumor ; Interleukin-2/metabolism* ; Xenograft Model Antitumor Assays ; Female

Humans ; Urinary Bladder Neoplasms/pathology* ; Carcinoma, Transitional Cell/pathology* ; Genetic Markers ; Biomarkers, Tumor/genetics* ; Urothelium/pathology*

OBJECTIVE: To investigate the clinicopathological features and prognosis of urinary bladder urothelial carcinoma (UBUC) with incidental prostate cancer (IPCa). METHODS: We retrospectively analyzed the clinicopathological features of 65 cases of UBUC and 38 cases of UBUC + IPCa after radical cystoprostatectomy (RCP) from January 2017 to February 2020. We compared their expressions of the immunohistochemical markers androgen receptor (AR) and (human epidermal growth factor receptor 2,HER2) between the two groups of patients, and analyzed their clinicopathological characteristics by chi-square test and their survival rates using the Kaplan-Meier method and log-rank test. RESULTS: The detection rate of UBUC + IPCa was 16.5%, and that of clinically significant IPCa was 39.5%, with preoperative PSA≥4 μg/L in 23.7% of the patients. Compared with the patients with UBUC, most of the UBUC + IPCa cases had no smoking history (73.8% vs 92.1%, P = 0.024), and fewer had histological variants (43.1% vs 10.5%, P = 0.003). The incidence rate of vascular invasion was significantly higher in the UBUC than in the UBUC + IPCa group (49.2% vs 21.1%, P = 0.005), and so was the rate of advanced cases (67.7% vs 31.6%, P<0.001). In comparison with the patients of the UBUC group, those of the UBUC + IPCa group showed remarkably higher expressions of AR (9.2% vs 31.6%, P = 0.004) and HER2 (43.1% vs 71.1%, P = 0.006). The mean overall survival time was longer in the UBUC + IPCa than in the UBUC group (48.8 mo ［95% CI: 2.5－42.6 mo］ vs 39.9 mo ［95% CI: 2.8－34.5 mo］), but with no statistically significant difference between the two groups (P = 0.608). CONCLUSION Standardized sampling of prostate samples after RCP helps to improve the detection rate of IPCa. Preoperative level of PSA is not a good predictor of IPCa. Few patients with UBUC + IPCa have a history of cigarette smoking, and the predominant histological type of the malignancy is high-grade invasive urothelial carcinoma, which is not significantly different from UBUC in prognosis. The expressions of HER2 and AR are significantly higher in UBUC + IPCa than in UBUC, suggesting that UBUC + IPCa may benefit from HER2- and AR-targeted therapy.
Humans ; Male ; Urinary Bladder Neoplasms/metabolism* ; Receptors, Androgen/metabolism* ; Prostatic Neoplasms/metabolism* ; Retrospective Studies ; Receptor, ErbB-2/metabolism* ; Prognosis ; Middle Aged ; Aged ; Survival Rate ; Prostatectomy

OBJECTIVES: To construct a prediction model for the prognosis of bladder cancer patients based on the expression of ion channel-related genes (ICRGs). METHODS: ICRGs were obtained from the existing researches. The clinical information and the expression of ICRGs mRNA in breast cancer patients were obtained from the Cancer Genome Atlas database. Cox regression analysis, minimum absolute shrinkage and selection operator regression analysis were used to screen breast cancer prognosis related genes, which were verified by immunohistochemistry and qRT-PCR. The risk scoring equation for predicting the prognosis of patients with bladder cancer was constructed, and the patients were divided into high-risk group and low-risk group according to the median risk score. Immune cell infiltration was compared between the two groups. Kaplan-Meier survival curve and receiver operating characteristic (ROC) curve were used to evaluate the accuracy and clinical application value of the risk scoring equation. The factors related to the prognosis of bladder cancer patients were analyzed by univariate and multivariate Cox regression, and a nomogram for predicting the prognosis of bladder cancer patients was constructed. RESULTS: By comparing the expression levels of ICRGs in bladder cancer tissues and normal bladder tissues, 73 differentially expressed ICRGs were dentified, of which 11 were related to the prognosis of bladder cancer patients. Kaplan-Meier survival curve suggested that the risk score based on these 11 genes was negatively correlated with the prognosis of patients. The area under the ROC curve of the risk score for predicting the prognosis of patients at 1, 3 and 5 year was 0.634, 0.665 and 0.712, respectively. Stratified analysis showed that the ICRGs-based risk score performed well in predicting the prognosis of patients with American Joint Committee on Cancer (AJCC) stage Ⅲ-Ⅳ bladder cancer (P<0.05), while it had a poor value in predicting the prognosis of patients with AJCC stage Ⅰ-Ⅱ (P>0.05). There were significant differences in the infiltration of plasma cells, activated natural killer cells, resting mast cells and M2 macrophages between the high-risk group and the low-risk group. Cox regression analysis showed that risk score, smoking, age and AJCC stage were independently associated with the prognosis of patients with bladder cancer (P<0.05). The nomogram constructed by combining risk score and clinical parameters has high accuracy in predicting the 1, 3 and 5 year overall survival rate of bladder cancer patients. CONCLUSIONS The study shows the potential value of ICRGs in the prognostic risk assessment of bladder cancer patients. The constructed prognostic nomogram based on ICRGs risk score has high accuracy in predicting the prognosis of bladder cancer patients.
Humans ; Female ; Prognosis ; Urinary Bladder Neoplasms/genetics* ; Urinary Bladder ; Ion Channels ; Breast Neoplasms

Objective To investigate the clinicopathological features,immunohistochemical features,diagnosis,and relationship with sporadic prostate cancer in primary small cell neuroendocrine carcinoma of the bladder. Methods We retrospectively analyzed the clinical characteristics of 12 patients with primary small cell neuroendocrine carcinoma of the bladder diagnosed at Beijing Chao-Yang Hospital affiliated to Capital Medical University from January 2013 to September 2022.The histological features of primary small cell neuroendocrine carcinoma of the bladder were re-evaluated by two pathologists according to the 2022 revision of the World Health Organization Classification of Tumors of the Urinary System and Male Genital Organs.Electronic medical records were retrieved,and telephone follow-up was conducted from the time of histopathological diagnosis to the death or the end of the last follow-up until January 31,2023. Results The 12 patients include 7 patients in pT3 stage and 1 patient in pT4 stage.Eight patients were complicated with other types of tumors,such as high-grade urothelial carcinoma of the bladder and squamous cell carcinoma.Five patients had sporadic prostate cancer.Immunohistochemical staining showed that 12 (100.0%),10 (83.3%),and 8 (66.7%) patients were tested positive for CD56,Syn,and CgA,respectively.The Ki67 proliferation index ranged from 80% to 90%.Five patients with urothelial carcinoma were tested positive for CK20,GATA3,and CK7.P504S was positive in all the 5 patients with prostate cancer,while P63 and 34βE12 were negative.The follow-up of the 12 patients lasted for 3-60 months.Eight of these patients died during follow-up,with the median survival of 15.5 months.Four patients survived. Conclusions Primary small cell neuroendocrine carcinoma of the bladder is a rare urological tumor with high aggressiveness and poor prognosis.In male patients with bladder prostatectomy,all prostate tissue should be sampled.If prostate cancer is detected,the prostate-specific antigen level should be monitored.
Humans ; Male ; Carcinoma, Transitional Cell/pathology* ; Carcinoma, Neuroendocrine/pathology* ; Urinary Bladder Neoplasms/pathology* ; Urinary Bladder/pathology* ; Retrospective Studies ; Prostatic Neoplasms ; Biomarkers, Tumor