Bladder Cancer Cell Growth Research Articles

Gefitinib Reverses PD-L1-Mediated Immunosuppression Induced by long-term Glutamine blockade in Bladder Cancer.

Glutamine is a major energy source for tumor cells and blocking glutamine metabolism is being investigated as a promising strategy for cancer therapy. However, the antitumor effect of glutamine blockade in bladder cancer remains unclear, necessitating further investigation. Here, we demonstrated that glutamine metabolism was involved in the malignant progression of bladder cancer. Treatment with the glutamine antagonist 6-Diazo-5-oxo-L-norleucine (DON) inhibited the growth of bladder cancer cells in vitro in several ways. In addition, we observed inhibition of tumor growth in bladder cancer-bearing mice using JHU083, a prodrug that was designed to prevent DON-induced toxicity. However, the antitumor immune effect of T cells changed from activation to inhibition as the administrated time extended. We found that both in vitro treatment with DON and in vivo prolonged administration of JHU083 led to the upregulation of PD-L1 in bladder cancer cells. Mechanistically, glutamine blockade up-regulated PD-L1 expression in bladder cancer cells by accumulating ROS, subsequently activating the EGFR/ERK/C-Jun signaling pathway. Combination treatment of JHU083 and gefitinib reversed the up-regulation of PD-L1 in bladder cancer cells induced by prolonged glutamine blockade, resulting in the alleviation of T-cell immunosuppression and a significant improvement in therapeutic outcome. These preclinical findings show promise for glutamine metabolism targeting as a viable therapeutic strategy for bladder cancer, with the potential for further enhancement through combined treatment with gefitinib.

Clitoria ternatea L. flower-derived anthocyanins and flavonoids inhibit bladder cancer growth by suppressing SREBP1 pathway-mediated fatty acid synthesis.

Clitoria ternatea L. flowers are used as traditional herbal medicines and are known for their advanced pharmacological activities. Flavonoids and anthocyanins reportedly contribute to the therapeutic properties of C. ternatea flowers; however, their potential anti-bladder cancer effects and molecular mechanisms remain unknown. In this study, flavonoid- and anthocyanin-rich samples from C. ternatea flowers (DDH) are prepared via macroporous resin-based extraction coupled with an efficient and reliable two-dimensional UPLC-DAD-MS/MS method. In vitro and in vivo studies reveal that DDH can inhibit bladder cancer cell growth and enhance the anti-bladder cancer activity of cisplatin. RNA-seq combined with KEGG analysis reveals that fatty acid synthesis is closely related to the anti-bladder cancer effect of DDH. Furthermore, DDH dose-dependently reduces cellular fatty acid levels in bladder cancer cells, and the addition of fatty acids significantly mitigates DDH-induced cell growth inhibition. Subsequent findings reveal that DDH downregulates sterol regulatory element-binding protein 1 (SREBP1), a key transcriptional regulator of de novo fatty acid synthesis in cancer cells, and its downstream targets (FASN, SCD1, and ACC). Additionally, this study demonstrates that gallic acid not only enhances the stability of DDH but also synergistically potentiates its anti-bladder cancer activity. Our study suggests that targeting the SREBP1 pathway is an effective strategy in bladder cancer therapy, and the ability of DDH to induce cell death by inhibiting the SREBP1 pathway and its good tolerance in mice make it a promising strategy for preventing and treating bladder cancer.

ARNTL2 facilitates bladder cancer progression through potentiating ENO1-mediated glycolysis in a SLC31A1-independent and -dependent manner

BackgroundBasic helix-loop-helix ARNT like 2 (ARNTL2) is a transcription factor that controls the circadian rhythm. Amounts of studies have demonstrated the carcinogenic function of ARNTL2 in human malignant tumors albeit the underlying mechanisms remain poorly understood. We aimed to study the significance of ARNTL2 in bladder cancer (BLCA). MethodsImmunohistochemical staining, immunoblotting and the database from TCGA were used to analyze the clinical relevance of ARNTL2, enolase 1 (ENO1) and solute carrier family 31 member 1 (SLC31A1) in BLCA. The function of ARNTL2 was explored by cell proliferation assay, apoptosis, colony formation and xenografted tumorigenesis. The molecular mechanisms of ARNTL2-driving BLCA development were investigated by RT-qPCR, immunoblotting and luciferase assays. Glycolysis was checked by measuring glucose consumption and lactate production. ENO1 activity was assessed by using indicated assay kit. ResultsOverexpression of ARNTL2 facilitates the proliferation and tumorigenesis of BLCA cells through suppression of apoptosis and enhancement of glycolysis. Up-regulation of SLC31A1, ENO1, and enhancement of SLC31A1-mediated ENO1 activity were critical for ARNTL2-triggered glycolysis and malignant growth in BLCA cells. ARNTL2 was positively correlated with SLC31A1 and ENO1 in BLCA patients. High expression of ARNTL2, SLC31A1 or ENO1 predicted the poor prognosis of BLCA patients. Depletion of SLC31A1 and inhibition of glycolysis completely blunted the growth ability of BLCA cells. ConclusionIn summary, ARNTL2 facilitates the progression of BLCA via activating ENO1-mediated glycolysis in a SLC31A1-independent and -dependent manner. Inhibiting SLC31A1 and glycolysis may be an aspirational approach for the treatment of BLCA patients with overexpression of ARNTL2.

Knockdown of CPSF4 Inhibits Bladder Cancer Cell Growth by Upregulating NRF1.

Increasing studies have shown that nuclear respiratory factor 1 (NRF1) deficiency frequently occurs in many human diseases, and its activation can protect neurons and other cells from degenerative diseases and malignant tumors. However, how NRF1 is regulated in bladder cancer remains unknown. Our research aims to reveal the role of leavage and polyadenylation-specific factor 4 (CPSF4) on the growth inhibition effect of bladder cancer and clarify its relationship with NRF1. Here, cell proliferation assay, transwell migration assay and multicellular tumor spheroids (MCTS) formation assay in the bladder cancer cell lines were carried out to measure tumor cell growth. Western bolt assay was carried out to identify the relationship between NRF1 and CPSF4. Also, subcutaneous xenograft tumors in nude mice were established to further validate the inhibition effect of CPSF4 on bladder tumor and the regulation on NRF1. The results in vitro showed that knockdown of CPSF4 strongly reduced the proliferation and migration, and inhibited MCTS formation in 5637 and HT1376 cell lines, while an additional knockdown of increased NRF1 induced by CPSF4 knockdown partially abolished these effects. The results in vivo showed that knockdown of CPSF4 strongly reduced the volume and weight of subcutaneous tumor, and decreased the expression of Ki-67 in tumor tissue, while NRF1 knockdown partially reversed these effects induced by CPSF4 knockdown. Western bolt assay demonstrated that CPSF4 could negatively regulate NRF1. Our results indicated that knock-down of CPSF4 inhibited bladder cancer cell growth by upregulating NRF1, which might provide evidence of CPSF4 as a therapeutic target for bladder cancer.

M6A reader IGF2BP2 promotes M2 macrophage polarization and malignant biological behavior of bladder cancer by stabilizing NRP1 mRNA expression

BackgroundInsulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) has been confirmed to play oncogenic role in many cancers. However, the role and mechanism of IGF2BP2 in bladder cancer (BCa) still deserves to be further revealed.MethodsThe mRNA and protein levels of IGF2BP2 and neuronilin-1 (NRP1) were detected by real-time quantitative PCR (RT-qPCR) and western blot. Cell proliferation, apoptosis, migration and invasion were determined using colony formation assay, EdU assay, CCK8 assay, flow cytometry and transwell assay. Xenograft tumor model was conducted to evaluate the role of IGF2BP2 in vivo. THP-1-M0 macrophages were co-cultured with the condition medium (CM) of BCa cells to induce polarization. M2 macrophage polarization was assessed by detecting the mRNA levels of M2 macrophage markers using RT-qPCR and measuring the proportion of M2 macrophage markers using flow cytometry. Moreover, MeRIP and RIP assay were performed to assess m6A level and the interaction between IGF2BP2 and NRP1.ResultsIGF2BP2 and NRP1 were upregulated in BCa tissues and cells. IGF2BP2 knockdown suppressed BCa cell growth and metastasis, as well as inhibited BCa tumor growth. After THP-1-M0 macrophages were co-cultured with the CM of BCa cells, the levels of M2 macrophage markers were markedly enhanced, while this effect was abolished by IGF2BP2 knockdown. IGF2BP2 level was positively correlated with NRP1 level, and it could increase NRP1 mRNA stability. NRP1 overexpression reversed the suppressive effect of IGF2BP2 knockdown on M2 macrophage polarization and BCa cell progression.Conclusionm6A-reader IGF2BP2 enhanced M2 macrophage polarization and BCa cell progression by promoting NRP1 mRNA stability.

GINS1 promotes the initiation and progression of bladder cancer by activating the AKT/mTOR/c-Myc signaling pathway

Bladder cancer(BC) is one of the most prevalent cancers in the urinary tract, with high recurrence and fatality rates. Research indicates that go-ichi-ni-san complex subunit 1 (GINS1) crucially influences cancer progression by regulating DNA replication through cell cycle modulation. Thus, suppressing the active proliferation of cells in tumor tissues may require silencing GINS1. However, the consequences of GINS1 in bladder cancer aren't to be determined. In this paper, we examine the role and mechanism of GINS1 in the development of bladder cancer. GINS1 expression levels and prognostic relevance in bladder cancer were validated using Western blotting, immunohistochemistry, and Kaplan-Meier survival analysis. The influence of GINS1 on bladder cancer was investigated using a variety of approaches, including cell transfection, cell counts, transwell migrations, colony formation, and flow cytometry. Immunohistochemistry studies demonstrate that GINS1 expression is increased in bladder cancer tissues. GINS1 silencing resulted in an arrest of the cell cycle at the phase of G0/G1, which inhibited BC cell growth both in vitro and in vivo. GINS1 knockdown also hindered the AKT/mTOR pathway. Furthermore, increased GINS1 expression affects the cell cycle and stimulates the AKT/mTOR pathway, allowing BC to develop more quickly. Consequently, GINS1 occurs as a latent therapeutic target, particularly for individuals with BC.

Circ_0000758 Facilitates Bladder Cancer Cell Growth, Migration and Angiogenesis Via Severing as miR-1236-3p Sponge.

Circular RNA (circRNA) has been reported to regulate the development of bladder cancer (BCa). However, the role of circ_0000758 in BCa progression is unknown. Circ_0000758 and miR-1236-3p expression, as well as ZEB2 mRNA expression were determined by qRT-PCR. BCa cell biological functions were determined by MTT assay, EdU assay, flow cytometry, wound healing assay and tube formation assay. Protein expression was detected by western blot analysis. RNA pull-down assay and dual-luciferase reporter assay were used to confirm RNA interaction. Xenograft mice models were constructed to assess the effect of circ_0000758 on BCa tumor growth. Circ_0000758 had increased expression in BCa tissues and cells. Circ_0000758 silencing could inhibit BCa cell growth, migration, angiogenesis in vitro, and tumor growth in vivo. Circ_0000758 served as a molecular sponge for miR-1236-3p, and miR-1236-3p inhibitor reversed circ_0000758 knockdown-mediated the inhibition effect on BCa cell progression. ZEB2 was targeted by miR-1236-3p, and its overexpression also revoked the suppressive effect of miR-1236-3p on BCa cell growth, migration, and angiogenesis. Besides, circ_0000758 knockdown also restrained BCa tumor growth. Circ_0000758 might promote BCa cell growth, migration, and angiogenesis by regulating the miR-1236-3p/ZEB2 axis.

Transcription Factor yin-Yang 1 augments nucleoporin 93 oncogene activity and modulates bladder Cancer malignancy

ObjectiveThis study aims to investigate the functional interplay between transcription factor YY1 and nucleoporin 93 (NUP93) in regulating the malignancy of bladder cancer cells. MethodsNUP93 expressions in bladder cancer tissues and normal counterparts were analyzed using a public dataset and clinical samples. NUP93 and Yin Yang 1 (YY1) mRNA expression and protein levels in T24 and RT4 cells were determined by Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The effect of NUP93 knockdown on the proliferation, migration, and invasion capabilities of cells was evaluated. Concurrently, transcriptional regulation of NUP93 by YY1 was confirmed using a dual luciferase assay. The effect of NUP93 knockdown on tumorigenesis was evaluate in a subcutaneous xenograft mouse model. ResultsElevated levels of NUP93 in bladder cancer tissues and cell lines were observed. Silencing NUP93 significantly suppressed glycolysis, impeded the growth, migration, invasion and tumor formation of bladder cancer cells. The transcription factor YY1 acted as a positive regulator to upregulate NUP93 expression. YY1 overexpression partially rescued the effects of NUP93 silencing on bladder cancer cells. ConclusionOur results uncovered transcription factor YY1 as a positive regulator of NUP93 expression, and NUP93 serves as an oncogenic factor to sustain the malignancy of bladder cancer cells. These findings suggest that targeting the YY1-NUP93 axis could offer novel therapeutic strategies for bladder cancer treatment.

Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

BackgroundThe functions of activating transcription factor 3 (ATF3) within the human bladder remain unexplored. This study delves into the expressions, functions, and regulatory mechanisms of ATF3 in human bladder cancer. Material and methodsGene expressions were determined by immunoblot, RT-qPCR, and reporter assays. Assays of Ki67, colony formation, Matrigel invasion, and the xenograft animal study were used to assess the cell proliferation, invasion, and tumorigenesis in vitro and in vivo. Silico analysis from TCGA database examined the correlations between GDF15 and ATF3 expressions, clinicopathologic features, and progression-free survival rates. ResultsSilico analysis confirmed that ATF3 is an antitumor gene, and the expression positively correlates with GDF15 in bladder cancer tissues. Multivariate analysis revealed that low ATF3/GDF15 but not a single low expression of ATF3 is an independent prognostic factor for progression-free survival of bladder cancer patients. Ectopic overexpression of ATF3 downregulated cell proliferation and invasion in bladder cancer cells in vitro, while ATF3-knockdown reversed these results. Knockdown of ATF3 upregulated EMT markers to enhance cell invasion in vitro and downregulated GDF15, NDRG1, and KAI-1 to elevate tumor growth in vivo. The activation of metformin on ATF3 and GDF15 in bladder cancer cells was blocked by SB431542, a TGFβ receptor inhibitor. ATF3 positively regulated GDF15 expression in bladder cancer cells through a feedback loop. ConclusionsOur results identify that ATF3 is a metformin-upregulated antitumor gene. Results of Silico analysis align with cell-based studies suggesting that low ATF3/GDF15 could be a negative prognostic marker for bladder cancer.

Nucleoporin 93 Regulates Cancer Cell Growth and Stemness in Bladder Cancer via Wnt/β-Catenin Signaling.

Bladder cancer (BLCA) is a prevalent cancer type with an unmet need for new therapeutic strategies. Nucleoporin 93 (Nup93) is implicated in the pathophysiology of several cancers, but its relationship with bladder cancer remains unclear. Nup93 expression was analyzed in TCGA datasets and 88 BLCA patient samples. Survival analysis and Cox regression models evaluated the association between Nup93 levels and patient prognosis. BLCA cells were used to investigate the effects of Nup93 overexpression or knockdown on cell growth, invasion, stemness (sphere formation and ALDH2 + cancer stem cell marker), and Wnt/β-catenin signaling in vitro. The Wnt activator BML-284 was used to confirm the involvement of Wnt/β-catenin signaling pathway. A xenograft mouse model validated the in vitro findings. Nup93 was highly expressed in BLCA tissues and cell lines, and high Nup93 expression correlated with poor prognosis in BLCA patients. Nup93 silencing inhibited BLCA cell proliferation, Wnt/β-catenin activation, and cancer cell stemness. Conversely, Nup93 overexpression promoted these effects. BML-284 partially rescued the reduction in cell growth and stemness markers caused by Nup93 knockdown. Nup93 knockdown also suppressed the tumor formation of BLCA cells in vivo. Nup93 regulates BLCA cell growth and stemness via the Wnt/β-catenin pathway, suggesting its potential as a prognostic marker and therapeutic target in BLCA.

Novel artesunate-metformin conjugate inhibits bladder cancer cell growth associated with Clusterin/SREBP1/FASN signaling pathway.

Bladder cancer remains the 10th most common cancer worldwide. In recent years, metformin has been found to have potential anti-bladder cancer activity while high concentration of IC50 at millimolar level is needed, which could not be reached by regular oral administration route. Thus, higher efficient agent is urgently demanded for clinically treating bladder cancer. Here, by conjugating artesunate to metformin, a novel artesunate-metformin dimer triazine derivative AM2 was designed and synthesized. The inhibitory effect of AM2 on bladder cancer cell line T24 and the mechanism underlying was determined. Anti-tumor activity of AM2 was assessed by MTT, cloning formation and wound healing assays. Decreasing effect of AM2 on lipogenesis was determined by oil red O staining. The protein expressions of Clusterin, SREBP1 and FASN in T24 cells were evaluated by Western blotting. The results show that AM2 significantly inhibited cell proliferation and migration at micromolar level, much higher than parental metformin. AM2 reduced lipogenesis and down-regulated the expressions of Clusterin, SREBP1 and FASN. These results suggest that AM2 inhibits the growth of bladder cancer cells T24 by inhibiting cellular lipogenesis associated with the Clusterin/SREBP1/FASN signaling pathway.

A study on the significance of serine hydroxymethyl transferase expression and its role in bladder cancer

Bladder cancer (BLCA) is a common malignant tumor in urinary system all over the world. However, due to its high recurrence rate and complex causes, clinicians often have limited options for surgical and drug treatments. Recent researchs on the molecular mechanism of BLCA have reveals its biological progress and potential for early diagnosis. Serine hydroxymethyltransferase 1/2 (SHMT1/2) is a crucial enzyme in the one-carbon metabolism of tumor cells, and the expression levels of these isozymes have been found to be associated with the biological progression of various malignant tumors. However, the impact of SHMT1/2 on the biological progression of bladder cancer and its molecular regulation mechanism remain unclear. In this research utilizes BLCA clinical sample data, the TCGA database, and in vitro cell experiments to predict the expression levels of SHMT1/2 in BLCA. The findings indicate that SHMT1 remained unchanged, while SHMT2 expression is increased in BLCA, which was related to poor prognosis. Additionally, SHMT2 affects the growth, migration, and apoptosis of bladder cancer cells in vitro. It also influences the expression levels of E-cadherin and N-cadherin, ultimately impacting the malignant biological progression of bladder tumors. These results establish a correlation between SHMT2 and the malignant biological progression of BLCA, providing a theoretical basis for the early diagnosis and treatment of bladder cancer.

Targeting the KAT8/YEATS4 Axis Represses Tumor Growth and Increases Cisplatin Sensitivity in Bladder Cancer.

Bladder cancer (BC) is one of the most common tumors characterized by a high rate of relapse and a lack of targeted therapy. Here, YEATS domain-containing protein 4 (YEATS4) is an essential gene for BC cell viability using CRISPR-Cas9 library screening is reported, and that HUWE1 is an E3 ligase responsible for YEATS4 ubiquitination and proteasomal degradation by the Protein Stability Regulators Screening Assay. KAT8-mediated acetylation of YEATS4 impaired its interaction with HUWE1 and consequently prevented its ubiquitination and degradation. The protein levels of YEATS4 and KAT8 are positively correlated and high levels of these two proteins are associated with poor overall survival in BC patients. Importantly, suppression of YEATS4 acetylation with the KAT8 inhibitor MG149 decreased YEATS4 acetylation, reduced cell viability, and sensitized BC cells to cisplatin treatment. The findings reveal a critical role of the KAT8/YEATS4 axis in both tumor growth and cisplatin sensitivity in BC cells, potentially generating a novel therapeutic strategy for BC patients.

Abstract 1783: Understanding lipogenesis in bladder cancer

Abstract Cancer cells are known to have different metabolic needs for growth and survival and by understanding these demands of individual cancers, targeted novel therapies may be developed. Lipogenesis and ß-oxidation serve to balance lipid levels in the cell. Lipogenesis is rate limited by a series of reactions that include enzymes fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC1) that facilitate lipid formation; whereas ß-oxidation is rate limited by the transporter carnitine palmitoyltransferase 1 (CPT1) and the enzyme thiolase that catalyzes the final step of the ß-oxidation cycle. Many cancers are dependent on lipogenesis, but the role of these factors in BCa is poorly defined. The Cancer Genome Atlas was used to assess levels of relevant metabolic proteins in normal and cancerous BCa tissue, and showed FASN levels were elevated in BCa tissue and were associated with worse survival. The purpose of this project is to better understand the metabolic demands of bladder cancer (BCa) cell lines, and we hypothesized lipogenesis is a major metabolic pathway for BCa cell growth. T24, HTB-5, and HTB-9 BCa cell lines were used to evaluate the role of metabolism. CPT1 was inhibited with etomoxir, thiolase with trimetazidine, FASN with cerulenin, and ACC1 with 5-(Tetradecyloxy)-2-furoic acid (TOFA). Cell viability was assessed at 48 and 72 hours with an enzymatic activity assay measuring ß-hexosaminidase activity as a surrogate for cell proliferation. The IC50 (concentration of a drug to reduce cell proliferation by 50%) was calculated from this data. At 48 hours, cerulenin-treated cells had an IC50 of 11.80 µM, 13.28 µM, 18.31 µM for HTB-9, T24, and HTB-5 respectively. At 48 hours, TOFA-treated cells had an IC50 of 64.25 µM, 59.29 µM, 32.03 µM for HTB-9, T24, and HTB-5 respectively. Cellular proliferation was not inhibited by CPT1 or trimetazidine at enzymatic IC50 concentrations consistent with their known levels of efficacy. When adding the lipid palmitate (20µM) to TOFA-treated cells, it appeared to mediate a rescue of cell proliferation almost to control, although statistical significance was not achieved. Crystal violet assays were performed to assess long-term cell viability. There was visible reduction in colony formation in a dose-dependent manner at 5 µM and 10 µM cerulenin in T24 cells at 14 days of culture, and there were similar results in HTB-9 cells treated with 20 µM and 40 µM TOFA at 17 days. In both cerulenin and TOFA-treated cells, there was elevated cleaved caspase-3 protein expression, indicating initiation of apoptosis. In conclusion, inhibition of lipogenesis in BCa cell lines reduces cell viability by reducing proliferation and inducing apoptosis whereas inhibitors of ß-oxidation did not produce the same effect at relevant concentrations. These data indicate lipogenesis is a key metabolic pathway for BCa cell growth. Citation Format: Juliann Leak, David Matye, Erika Abbott, Benjamin Woolbright, John A. Taylor. Understanding lipogenesis in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1783.

The RNA-binding protein sorbin and SH3 domain-containing 2 are transcriptionally regulated by specificity protein 1 and function as tumor suppressors in bladder cancer by stabilizing tissue factor pathway inhibitor.

Sorbin and SH3 domain-containing 2 (SORBS2) is anRNA-binding protein and has been implicated in the development of some cancers. However, its role in bladder cancer (BC) is yet to be established. The expression of SORBS2 in BC tissues was determined from the Gene Expression Omnibusand Gene Expression Profiling Interactive Analysisdatabases and collected paired tumor/normal samples. The effects of SORBS2 on BC cells were detected by CCK-8, colony formation, Transwell, dual-luciferase, RNA immunoprecipitation, chromatin immunoprecipitation, and DNA pull-down assays. In vivo, BC cell growth and metastasis were studied by a xenograft subcutaneous model and a tail-vein metastasis model. The results showed that SORBS2 expression was significantly decreased in BC tissues and cells. SORBS2 overexpression inhibited cell proliferation, migration, invasion, and epithelial-mesenchymal transitionin vitro and tumor growth and metastasis in vivo, while silencing SORBS2 produced the opposite effect. Mechanistically, we found that SORBS2 enhanced the stability of tissue factor pathway inhibitor (TFPI) mRNA via direct binding to its 3' UTR. Restoration of TFPI expression reversed SORBS2 knockdown-induced malignant phenotypes of BC cells. In addition, SORBS2 expression was negatively regulated by the transcription factor specificity protein 1 (SP1). Conversely, SORBS2 can be transcriptionally regulated by SP1 and inhibit BC cell growth and metastasis via stabilization of TFPI mRNA, indicating SORBS2 may be a promising therapeutic target for BC.

CircMCTP2 enhances the progression of bladder cancer by regulating the miR-99a-5p/FZD8 axis

BackgroundCircRNAs and miRNAs are involved in the progression of tumor. CircMCTP2 is considered as a novel tumor promoter. However, the exact functions of circMCTP2 in bladder cancer are still unclear. This study was designed to explore the underlying mechanisms of circMCTP2-modulated tumor development in bladder cancer.MethodsThe present study is an original research. The levels of circMCTP2 in a total of 39 bladder cancer specimens and cell lines were determined by RT-qPCR. The expression of FZD8 in T24 and RT-4 cells treated with miR-99a-5p mimics were examined using western blotting. In addition, the proliferative, migrative and invasive abilities of transfected cells were determined by CCK8 and Transwell assays. Furthermore, the apoptosis of transfected cells was evaluated using flow cytometry. Dual luciferase reporter assay was performed to elucidate the relationship between miR-99a-5p and circMCTP2/FZD8.ResultsThe levels of circMCTP2 were elevated in bladder cancer samples and cells, and this was related to worse survival rate. Downregulation of circMCTP2 suppressed growth and metastasis of cells, whereas the apoptotic rate of cells was enhanced. The levels of miR-99a-5rp was elevated after the downregulation of circMCTP2. Moreover, reverse correlation between the expression of miR-99a-5p and circMCTP2 was revealed in bladder cancer specimens. Additionally, FZD8 was the putative target of miR-99a-5p and the mimics of miR-99a-5p inhibited the proliferation, migration and invasion of bladder cancer cells via the FZD8/Wnt-b-catenin axis. Moreover, circMCTP2 regulated the growth and metastasis of bladder cancer cells potentially through regulating the miR-99a-5p/FZD8/Wnt-b-catenin axis. In summary, circMCTP2 was considered as an oncogenic factor through regulating the miR-99a-5p/FZD8/Wnt-b-catenin axis.ConclusionsThis novel signaling could regulate the biological behaviours of bladder cancer cells, and these findings highlighted circMCTP2 as a critical target for treating bladder cancer.

Securinine inhibits the tumor growth of human bladder cancer cells by suppressing Wnt/β-catenin signaling pathway and activating p38 and JNK signaling pathways

Bladder cancer (BC) is the most common malignant tumor in urinary system. Although chemotherapy is one of the most important adjuvant treatments for BC, drug resistance, non-specific toxicity and severe side effects are the major obstacles to BC chemotherapy. Natural products have always been a leading resource of antitumor drug discovery, with the advantages of excellent effectiveness, low toxicity, multi-targeting potency and easy availability.In this study, we evaluated the potential anti-tumor effect of securinine (SEC), a natural alkaloid from Securinega suffruticosa, on BC cells in vitro and in vivo, and delineated the underlying mechanism. We found that SEC inhibited the proliferation, migration and invasion, induced the apoptosis of BC cells in vitro, and retarded the xenograft tumor growth of BC cell in vivo. Notably, SEC had a promising safety profile because it presented no or low toxicity on normal cells and mice. Mechanistically, SEC inactivated Wnt/β-catenin signaling pathway while activated p38 and JNK signaling pathway. Moreover, β-catenin overexpression, the p38 inhibitor SB203580 and the JNK inhibitor SP600125 both mitigated the inhibitory effect of SEC on BC cells. Furthermore, we demonstrated a synergistic inhibitory effect of SEC and gemcitabine (GEM) on BC cells in vitro and in vivo.Taken together, our findings suggest that SEC may exert anti-BC cell effect at least through the activation of p38 and JNK signaling pathways, and the inhibition of Wnt/β-catenin signaling pathway. More meaningfully, the findings indicate that GEM-induced BC cell killing can be enhanced by combining with SEC.

A first-in-class TIMM44 blocker inhibits bladder cancer cell growth

Mitochondria play a multifaceted role in supporting bladder cancer progression. Translocase of inner mitochondrial membrane 44 (TIMM44) is essential for maintaining function and integrity of mitochondria. We here tested the potential effect of MB-10 (MitoBloCK-10), a first-in-class TIMM44 blocker, against bladder cancer cells. TIMM44 mRNA and protein expression is significantly elevated in both human bladder cancer tissues and cells. In both patient-derived primary bladder cancer cells and immortalized (T24) cell line, MB-10 exerted potent anti-cancer activity and inhibited cell viability, proliferation and motility. The TIMM44 blocker induced apoptosis and cell cycle arrest in bladder cancer cells, but failed to provoke cytotoxicity in primary bladder epithelial cells. MB-10 disrupted mitochondrial functions in bladder cancer cells, causing mitochondrial depolarization, oxidative stress and ATP reduction. Whereas exogenously-added ATP and the antioxidant N-Acetyl Cysteine mitigated MB-10-induced cytotoxicity of bladder cancer cells. Genetic depletion of TIMM44 through CRISPR-Cas9 method also induced robust anti-bladder cancer cell activity and MB-10 had no effect in TIMM44-depleted cancer cells. Contrarily, ectopic overexpression of TIMM44 using a lentiviral construct augmented proliferation and motility of primary bladder cancer cells. TIMM44 is important for Akt-mammalian target of rapamycin (mTOR) activation. In primary bladder cancer cells, Akt-S6K1 phosphorylation was decreased by MB-10 treatment or TIMM44 depletion, but enhanced after ectopic TIMM44 overexpression. In vivo, intraperitoneal injection of MB-10 impeded bladder cancer xenograft growth in nude mice. Oxidative stress, ATP reduction, Akt-S6K1 inhibition and apoptosis were detected in MB-10-treated xenograft tissues. Moreover, genetic depletion of TIMM44 also arrested bladder cancer xenograft growth in nude mice, leading to oxidative stress, ATP reduction and Akt-S6K1 inhibition in xenograft tissues. Together, targeting overexpressed TIMM44 by MB-10 significantly inhibits bladder cancer cell growth in vitro and in vivo.

SRT1720 inhibits bladder cancer cell progression by impairing autophagic flux

Bladder cancer (BC) is the most common cancer of the urinary tract, with poor survival, high recurrence rates, and lacking of targeted drugs. In this study, we constructed a library to screen compounds inhibiting bladder cancer cells growth. Among them, SRT1720 was identified to inhibit bladder cancer cell proliferation in vitro and in vivo. SRT1720 treatment also suppressed bladder cancer cells migration, invasion and induced apoptosis. Mechanism studies shown that SRT1720 promoted autophagosomes accumulation by inducing early-stage autophagy but disturbed the late-stage of autophagy by blocking fusion of autophagosomes and lysosomes. SRT1720 appears to induce autophagy related proteins expression and alter autophagy-related proteins acetylation to impede the autophagy flux. LAMP2, an important lysosomal associated membrane protein, may mediate SRT1720-inhibited autophagy flux as SRT1720 treatment significantly deacetylated LAMP2 which may influence its activity. Taken together, our results demonstrated that SRT1720 mediated apoptosis and autophagy flux inhibition may be a novel therapeutic strategy for bladder cancer treatment.

Bioinformatics analysis and experimental validation reveal that CDC20 overexpression promotes bladder cancer progression and potential underlying mechanisms.

Bladder cancer is a prevalent malignancy. CDC20, a pivotal cell cycle regulator gene, plays a significant role in tumour cell proliferation, but its role in bladder cancer remains unclear. This study aimed to analyse CDC20 expression in bladder cancer and explore its roles in tumour progression, treatment response, patient prognosis, and cellular proliferation mechanisms. We systematically analysed CDC20 expression in bladder cancer using bioinformatics. Our study investigated the impact of CDC20 on chemotherapy and radiotherapy sensitivity, patient prognosis, and changes in CDC20 methylation levels. We also explored the role and potential underlying mechanisms of CDC20 in bladder cancer cell growth. We used lentiviral transfection to downregulate CDC20 expression in 5637 and T24 cells, followed by CCK-8, colony formation, scratch, invasion, apoptosis, and cell cycle analyses. CDC20 is highly expressed in bladder cancer and is significantly correlated with poor prognosis. Moreover, CDC20 demonstrated high diagnostic potential for bladder cancer (AUC > 0.9). The tumour methylation levels of CDC20 in tumour tissues markedly decreased compared with those in normal tissues, and lower methylation levels were associated with a worse prognosis. Elevated CDC20 expression is linked to increased mutation burden. Our findings suggested a potential association between high CDC20 expression and resistance to chemotherapy and radiotherapy, as CDC20 expression may impact immune cell infiltration levels. Mechanistic analysis revealed the influence of CDC20 on bladder cancer cell proliferation through cell cycle-related pathways. According to the cell experiments, CDC20 downregulation significantly impedes bladder cancer cell proliferation and invasion, leading to G1 phase arrest. Aberrantly high CDC20 expression promotes tumour progression in bladder cancer, resulting in a poor prognosis, and may also constitute a promising therapeutic target.