Molecular Dynamic Stability Study of VEGF Inhibitor in Patients with Bladder Cancer

Vascular endothelial growth factor (VEGF) plays a crucial role in bladder cancer progression. Brolucizumab, an anti-VEGF agent, has been studied in various diseases; however, its potential in bladder cancer remains largely unexplored. This study aimed to analyze the molecular docking and dynamic stability of Brolucizumab as a VEGF inhibitor in bladder cancer. Target protein and ligand data mining were conducted. Proteins were prepared by removing water molecules using Discovery Studio 2019. Ligand energy minimization was performed using Pyrx v.0.9.8. Protein-ligand docking was conducted, and protein-protein docking was performed using the HADDOCK server. The interactions between compounds and proteins were visualized with BioVia Discovery Studio 2019. Molecular dynamics simulations were carried out using the YASARA Dynamic program. Brolucizumab binding induced smaller conformational changes compared to VEGFR2 binding. When VEGFR2 interacted with the VEGFA-Brolucizumab complex, significant conformational changes occurred, suggesting an inhibitory and blocking effect of Brolucizumab. Bond relaxation was observed when Brolucizumab bound to VEGFA and VEGFR, initiating conformational changes as part of its inhibitory activity. Brolucizumab demonstrated strong and competitive binding to VEGFA, with greater affinity than VEGFR2. Brolucizumab exhibits inhibitory and blocking activity against VEGFR2, suggesting its potential as a therapeutic agent in bladder cancer.

Hypertension Editors' Picks Cardio-oncology and Hypertension.

Unlike prostate and breast cancers, urothelial carcinoma of the urinary bladder is not yet considered as an endocrine-related neoplasm, and hormonal therapy for bladder cancer remains experimental. Nonetheless, there is increasing evidence indicating that nuclear hormone receptor signals are implicated in the development and progression of bladder cancer. Androgen-mediated androgen receptor (AR) signals have been convincingly shown to induce bladder tumorigenesis. Androgens also promote the growth of AR-positive bladder cancer cells, although it is controversial whether AR plays a dominant role in bladder cancer progression. Both stimulatory and inhibitory functions of estrogen receptor signals in bladder cancer have been reported. Various studies have also demonstrated the involvement of other nuclear receptors, including progesterone receptor, glucocorticoid receptor, vitamin D receptor, and retinoid receptors, as well as some orphan receptors, in bladder cancer. This review summarizes and discusses available data suggesting the modulation of bladder carcinogenesis and cancer progression via nuclear hormone receptor signaling pathways. These pathways have the potential to be an extremely important area of bladder cancer research, leading to the development of effective chemopreventive/therapeutic approaches, using hormonal manipulation. Considerable uncertainty remains regarding the selection of patients who are likely to benefit from hormonal therapy and optimal options for the treatment.

Introduction: Bladder cancer represents a significant public health concern with diverse genetic alterations influencing disease onset, progression, and therapy response. In this study, we explore the multifaceted role of Solute Carrier Family 31 Member 1 (SLC31A1) in bladder cancer, a pivotal gene involved in copper homeostasis. Methods: Our research involved analyzing the SLC31A1 gene expression via RT-qPCR, promoter methylation via targeted bisulfite sequencing, and mutational status via Next Generation Sequencing (NGS) using the clinical samples sourced by the local bladder cancer patients. Later on, The Cancer Genome Atlas (TCGA) datasets were utilized for validation purposes. Moreover, prognostic significance, gene enrichment terms, and therapeutic drugs of SLC31A1 were also explored using KM Plotter, DAVID, and DrugBank databases. Results: We observed that SLC31A1 was significantly up-regulated at both the mRNA and protein levels in bladder cancer tissue samples, suggesting its potential involvement in bladder cancer development and progression. Furthermore, our investigation into the methylation status revealed that SLC31A1 was significantly hypomethylated in bladder cancer tissues, which may contribute to its overexpression. The ROC analysis of the SLC31A1 gene indicated promising diagnostic potential, emphasizing its relevance in distinguishing bladder cancer patients from normal individuals. However, it is crucial to consider other factors such as cancer stage, metastasis, and recurrence for a more accurate evaluation in the clinical context. Interestingly, mutational analysis of SLC31A1 demonstrated only benign mutations, indicating their unknown role in the SLC31A1 disruption. In addition to its diagnostic value, high SLC31A1 expression was associated with poorer overall survival (OS) in bladder cancer patients, shedding light on its prognostic relevance. Gene enrichment analysis indicated that SLC31A1 could influence metabolic and copper-related processes, further underscoring its role in bladder cancer. Lastly, we explored the DrugBank database to identify potential therapeutic agents capable of reducing SLC31A1 expression. Our findings unveiled six important drugs with the potential to target SLC31A1 as a treatment strategy. Conclusion: Our comprehensive investigation highlights SLC31A1 as a promising biomarker for bladder cancer development, progression, and therapy.

KiSS-1 is a metastasis suppressor gene reported to be involved in the progression of several solid neoplasias. The loss of KiSS-1 gene expression has been shown to be inversely correlated with increasing tumor stage, distant metastases, and poor overall survival in bladder tumors. To identify the molecular pathways associated with the metastasis suppressor role of KiSS-1 in bladder cancer, we carried out a proteomics analysis of bladder cancer cells (EJ138) transiently transfected with a vector encompassing the full-length KiSS-1 gene using an iTRAQ (isobaric tags for relative and absolute quantitation) approach. Protein extracts collected after 24- and 48-h transfection were fractionated and cleaved with trypsin, and the resulting peptides were labeled with iTRAQ reagents. The labeled peptides were separated by strong cation exchange and reversed phase LC and analyzed by MALDI-TOF/TOF MS. Three software packages were utilized for data analysis: ProteinPilot for identification and quantification of differentially expressed proteins, Protein Center for gene ontology analysis, and Ingenuity Pathways Analysis to provide insight into biological networks. Comparative analysis among transfected, mock, and empty vector-exposed cells identified 1529 proteins with high confidence (>99%) showing high correlation rates among replicates (70%). The involvement of the identified proteins in biological networks served to characterize molecular pathways associated with KiSS-1 expression and to select critical candidates for verification analyses by Western blot using independent transfected replicates. As part of complementary clinical validation strategies, immunohistochemical analyses of proteins regulated by KiSS-1, such as Filamin A, were performed on bladder tumors spotted onto tissue microarrays (n = 280). In summary, our study not only served to uncover molecular mechanisms associated with the metastasis suppressor role of KiSS-1 in bladder cancer but also to reveal the biomarker role of Filamin A in bladder cancer progression and clinical outcome.

Bladder cancer remains a significant health challenge due to its high recurrence and progression rates. This study aims to evaluate the role of POLR3G in the development and progression of bladder cancer and the potential of POLR3G to serve as a novel therapeutic target. We constructed a bladder cancer model in Wistar rats by administering N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), which successfully induced a transition from normal mucosa to hyperplasia and ultimately to urothelial carcinoma. We observed a progressive upregulation of POLR3G expression during the bladder cancer development and progression. To investigate the functional role of POLR3G, we performed functional experiments in bladder cancer cell lines. The results demonstrated that knocking down POLR3G significantly inhibited cell proliferation, migration, and invasion. We further conducted RNA sequencing on POLR3G-knockdown bladder cancer cells, and Metascape was employed to perform the functional enrichment analysis of the differentially expressed genes (DEGs). Enrichment analysis revealed the enrichment of DEGs in the RNA polymerase and apoptotic cleavage of cellular proteins pathways, as well as their involvement in the Wnt and MAPK signaling pathways. The downregulation of Wnt pathway-related proteins such as Wnt5a/b, DVL2, LRP-6, and phosphorylated LRP-6 upon POLR3G knockdown was further confirmed by Western blotting, indicating that POLR3G might influence bladder cancer behavior through the Wnt signaling pathway. Our findings suggest that POLR3G plays a crucial role in bladder cancer progression and could serve as a potential therapeutic target. Future studies should focus on the detailed mechanisms by which POLR3G regulates these signaling pathways and its potential as a biomarker for early detection and prognosis of bladder cancer.

ANALYSIS OF THE GENE EXPRESSION OF SPARC AND ITS PROGNOSTIC VALUE FOR BLADDER CANCER

Emerging evidence highlights dioscin, a bioactive compound derived from Dioscoreaceae plants, as a promising antitumor agent, yet its regulatory mechanisms in bladder cancer and interaction with microRNAs remain unclear. This study systematically investigated dioscin's dual roles in bladder cancer progression through in vitro and in vivo models. Functional assays demonstrated that dioscin significantly upregulated miR-195-5p expression in bladder cancer cells, while both dioscin and miR-195-5p suppressed T24/EJ cell proliferation, migration, and invasion. Mechanistically, RNA-seq and molecular docking revealed dioscin directly bound to fatty acid synthase (FASN), which then regulated the SLC3A2 expression. Strikingly, miR-195-5p mimic transfection downregulated FASN, whereas its inhibitor reversed this effect, confirming miR-195-5p's pivotal role in dioscin-mediated FASN/SLC3A2 inhibition. Notably, dioscin potentiated cisplatin's antitumor efficacy against both BIU87 and cisplatin-resistant BIU87 bladder cancer cells at low micromolar concentrations. Intriguingly, the bladder cancer cell induced by dioscin could be counteracted by inhibitors of apoptosis, necroptosis, and ferroptosis, particularly in the presence of gap junction inhibitor carbenoxolone. However, in vivo studies uncovered a paradoxical duality: dioscin enhanced N-methyl-N-nitrosourea (MNU)-induced bladder tumorigenesis. Its combination with MNU exacerbated renal toxicity and bladder stone formation in rats, accompanied by elevated creatinine and uric acid levels. Crucially, dioscin exhibited cytotoxicity against normal urothelial (SV-HUC-1) and renal (MDCK) cells, warranting cautious therapeutic application. These findings unveil a novel miR-195-5p/FASN/SLC3A2 axis through which dioscin initiates bladder cancer cell death, while highlighting dual roles of dioscin in bladder cancer and the necessity for dosage optimization to balance its antitumor potency and off-target toxicity.

It was the aim of this study to explore the role and mechanism of long non-coding RNA (lncRNA) AFAP1-AS1 in the progression of bladder cancer (BCa) by in vitro experiments. AFAP1-AS1 levels in 40 pairs of clinical BCa tissue samples and normal ones collected from BCa patients were determined, and paired sample t-test was applied to compare the differences between groups. The prognosis data of patients with BCa were collected, and survival analysis and t-test were performed to specify the interplay between AFAP1-AS1 and the prognosis of BCa patients. Subsequently, AFAP1-AS1 expression level in BCa and normal cells were further confirmed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), and transwell assays were performed to figure out the influence of this lncRNA on the proliferation ability and invasiveness of BCa cells. Meanwhile, the interaction between AFAP1-AS1 and its sense mRNA was analyzed. We used co-transfection technology to simultaneously transfect si-AFAP1-AS1 and pcDNA3.1-AFAP1 or their corresponding negative controls into BCa cells, and cell proliferation and invasion ability in different subgroups were determined to explore the underlying mechanism through which AFAP1-AS1 plays a role in BCa progression. No matter in BCa tissues or in cell samples, compared to the corresponding normal controls, AFAP1-AS1 was found highly expressed; at the same time, in invasive bladder cancer tissues, the expression level of AFAP1-AS1 was also higher than that in non-invasive tissues. Meanwhile, survival analysis revealed that patients with BCa with high expression of AFAP1-AS1 owned a shorter overall survival rate than those with low expression, indicating a negative interplay between AFAP1-AS1 expression and patients' prognosis. In addition, in BCa cell lines, according to the results of CCK-8, EDU, and transwell assays, the proliferative capacity, as well as the invasive ability of BCa cells, were found weakened after downregulation of AFAP1-AS1. Meanwhile, a negative interplay was discovered between AFAP1-AS1 and its sense mRNA. Finally, the results of cell reversal experiment using co-transfection technique revealed that overexpression of AFAP1 can reverse the inhibitory impact of lncRNAAFAP1-AS1 on the malignant ability of BCa cells. AFAP1-AS1 may enhance the proliferation ability as well as the invasiveness of BCa cells so as to aggravate the degree of BCa malignancy.

BackgroundThe regulator of cullins-1 (ROC1) is an essential subunit in the cullin-RING ligase (CRL) protein complex and has been shown to be critical in bladder cancer cell survival and progression. This study aimed to explore the molecular mechanism of ROC1 action in the malignant progression of bladder cancer.MethodsThis study utilized ex vivo, in vitro, and in vivo nude mouse experiments to assess the underlying mechanisms of ROC1 in bladder cancer cells. The expression of the components of the sonic hedgehog (SHH) pathway was determined by western blot analysis. ROC1 expression in human tumors was evaluated by immunohistochemistry.ResultsROC1 overexpression promoted the growth of bladder cancer cells, whereas knockdown of ROC1 expression had the opposite effect in bladder cancer cells. Mechanistically, ROC1 was able to target suppressor of fused homolog (SUFU) for ubiquitin-dependent degradation, allowing Gli2 release from the SUFU complex to activate the SHH pathway. Furthermore, knockdown of SUFU expression partially rescued the ROC1 knockdown-suppressed SHH activity as well as cancer cell growth inhibition. In ex vivo experiments, tissue microarray analysis of human bladder cancer specimens revealed a positive association of ROC1 expression with the SHH pathway activity.ConclusionThis study demonstrated that dysregulation of the ROC1–SUFU–GLI2 axis plays an important role in bladder cancer progression and that targeting ROC1 expression is warranted in further investigations as a novel strategy for the future control of bladder cancer.

Loss of 15-Hydroxyprostaglandin Dehydrogenase Expression Contributes to Bladder Cancer Progression

Vascular Endothelial Growth Factor in Patients with Exudative Age-related Macular Degeneration Treated with Ranibizumab

BackgroundCircular RNAs (circRNAs) have important functions in many fields of cancer biology. In particular, we previously reported that the oncogenic circRNA, circPRMT5, has a major role in bladder cancer progression. Therapy based on circRNAs have good prospects as anticancer strategies. While anti-circRNAs are emerging as therapeutics, the specific in vivo delivery of anti-circRNAs into cancer cells has not been reported and remains challenging.MethodsSynthesized chrysotile nanotubes (SCNTs) with a relatively uniform length (~ 200 nm) have been designed to deliver an siRNA against the oncogenic circPRMT5 (si-circPRMT5) inhibit circPRMT5. In addition, the antitumor effects and safety evaluation of SCNTs/si-circPRMT5 was assessed with a series of in vitro and in vivo assays.ResultsThe results showed that SCNTs/si-circPRMT5 nanomaterials prolong si-circPRMT5’s half-life in circulation, enhance its specific uptake by tumor cells, and maximize the silencing efficiency of circPRMT5. In vitro, SCNTs encapsulating si-circPRMT5 could inhibit bladder cancer cell growth and progression. In vivo, SCNTs/si-circPRMT5 inhibited growth and metastasis in three bladder tumor models (a subcutaneous model, a tail vein injection lung metastatic model, and an in situ model) without obvious toxicities. Mechanistic study showed that SCNTs/si-circPRMT5 regulated the miR-30c/SNAIL1/E-cadherin axis, inhibiting bladder cancer growth and progression.ConclusionThe results highlight the potential therapeutic utility of SCNTs/si-circPRMT5 to deliver si-circPRMT5 to treat bladder cancer.

With the advent of Bacille Calmette Guerin (BCG), bladder cancer was one of the earliest cancers where the concept of immunotherapy was utilized. While this is true, recent advances in the use of immunotherapy are enabling oncologists to expand the armamentarium for the treatment of bladder cancer. Unacceptable side effects and failure to produce a durable response with the use of chemotherapeutic agents in bladder cancer has led to the evaluation of more targeted and personalized approaches. Increased understanding of the underlying carcinogenesis of bladder cancer, coupled with the ability to engineer targeted agents implicated in bladder cancer associated pathways has provided new avenues for the management of this disease. Newer immunotherapeutic approaches have generated a great deal of interest in bladder cancer along with other diseases. In this article we will focus on various forms of immunotherapies that may have a therapeutic potential in bladder cancer. We will briefly review the current status of "non-targeted" immunotherapeutic agents like BCG, interferons and interleukins in bladder cancer. But the main focus of this article is to discuss the emerging role of "targeted" immunotherapeutic agents like cytotoxic T cell lymphocyte associated protein-4 blocking antibody and programmed death pathway blocking antibodies in localized or metastatic bladder cancer.

In the United States, it is predicted that there will be over 70,000 new cases of bladder cancer this year. Bladder cancer is the fourth most common cancer in men and eighth in women. Our lab has previously identified Thromboxane receptor-β (TP-β) as potentially playing a role in bladder cancer progression. TP-β is a G-protein coupled receptor that is over-expressed in bladder cancer tumors and high levels of TP-β in bladder cancer patients correlate with decreased survival. G-proteins are involved in second messenger cascades that influence a variety of cellular functions, including transcription and motility. In multiple cellular systems TP-β has been found to interact with multiple G proteins such as Gαi, Gαs, Gα12, and Gα13. We found high levels of mRNA and protein expression of the G-protein, Gα12, in bladder cancer cell lines. We also observe high levels of Gα12 expression in human bladder cancer tissues. Studies using a constitutively active Gα12 mutant, suggest that signaling through Gα12 can contribute to oncogenic transformation. In this study we aim to elucidate the TP-β associated Gα12 dependent pathways that contribute to bladder cancer progression. We found that TP agonist U46619 increases cell migration and that treatment with TP antagonist blocks this increase in cell migration induced by U46619. TP agonist stimulation also increases the phosphorylation of extracellular regulated kinase (ERK) and U46619-induced phosphorylation of ERK is blocked with TP agonist treatment. Using a shRNA to knockdown Gα12 we observe a reduction in U46619-induced cell migration and ERK phosphorylation. Together our results suggest that TP-β mediated cell migration and the MAPK signaling pathway is in part regulated through Gα12. This work could have clinical implications by targeting the TP-β and Gα12 interaction as a potential therapeutic approach for bladder cancer treatment. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B26