Natural products and derivatives in renal, urothelial and testicular cancers: Targeting signaling pathways and therapeutic potential.

BACKGROUND: Natural products have demonstrated significant potential in cancer drug discovery, particularly in renal cancer (RCa), urothelial carcinoma (UC), and testicular cancer (TC). PURPOSE: This review aims to examine the effects of natural products on RCa, UC and TC. STUDY DESIGN: systematic review METHODS: PubMed and Web of Science databases were retrieved to search studies about the effects of natural products and derivatives on these cancers. Relevant publications in the reference list of enrolled studies were also checked. RESULTS: This review highlighted their diverse impacts on key aspects such as cell growth, apoptosis, metastasis, therapy response, and the immune microenvironment. Natural products not only hold promise for novel drug development but also enhance the efficacy of existing chemotherapy and immunotherapy. Importantly, we exert their effects through modulation of critical pathways and target genes, including the PI3K/AKT pathway, NF-κB pathway, STAT pathway and MAPK pathway, among others in RCa, UC, and TC. CONCLUSION: These mechanistic insights provide valuable guidance for researchers, facilitating the selection of promising natural products for cancer management and offering potential avenues for further gene regulation studies in the context of cancer treatment.

Unveiling nature's potential weapon: Magnolol's role in combating bladder cancer by upregulating the miR-124 and inactivating PKC-δ/ERK axis.

BACKGROUND: Bladder cancer (BC) is a challenging disease to manage. Researchers have been investigating the potential of magnolol, a compound derived from Magnolia officinalis, as an anti-cancer agent. However, the exact regulatory mechanism of magnolol and its impact on the NF-κB signaling pathway in BC remain unclear. MATERIALS: To comprehensively evaluate its therapeutic potential, the researchers conducted a series of experiments using BC cell lines (TSGH8301, T24, and MB49) and in vivo animal models. RESULTS: The results of the study demonstrated that magnolol exhibits cytotoxic effects on BC cells by activating both the extrinsic and intrinsic apoptosis signaling pathways. Additionally, the expression of anti-apoptotic genes was downregulated by magnolol treatment. The researchers also uncovered the regulatory role of PKCδ/ERK and miR-124-3p in the NF-κB pathway, which may be influenced by magnolol. Treatment with magnolol led to the inactivation of PKCδ/ERK and an increase in miR-124-3p expression, effectively inhibiting NF-κB-mediated progression of BC. Importantly, the administration of magnolol did not result in significant toxicity in normal tissues, highlighting its potential as a safe adjunctive therapy with minimal adverse effects. CONCLUSION: These findings position magnolol as a promising therapeutic agent for the treatment of BC. By activating apoptosis signaling pathways and inhibiting NF-κB pathway through the upregulation of miR-124-3p and downregulation of PKCδ/ERK activation, magnolol holds promise for suppressing tumor progression and improving patient outcomes in BC. Further research and clinical trials are warranted to explore the full potential of magnolol in the future.

Hyperbaric oxygen enhanced the chemotherapy of mitochondrial targeting molecule IR-780 in bladder cancer.

BACKGROUND: Bladder cancer has a high rate of recurrence and drug resistance due to the lack of effective therapies. IR-780 iodide, a near-infrared (NIR) mitochondria-targeting fluorescent agent, has been demonstrated to achieve higher selectivity than other drugs in different tumor types and exhibited tumor-killing effects in some cancers. However, this therapeutic strategy is rarely studied in bladder cancer. MATERIAL AND METHODS: The accumulation of IR-780 in bladder cancer was measured by NIR imaging. Human bladder cell lines (T24, 5637, and TCCSUP) were treated with IR-780 or combined IR-780 and hyperbaric oxygen (HBO). Cell viability, cell apoptosis, cellular ATP production, mitochondrial reactive oxygen species (ROS), and plasma membrane potential were detected. Mitochondrial complex I protein NDUFS1 was measured by western blot. To confirm the anti-tumor efficacy of IR-780 + HBO, mouse bladder cell line (MB49) tumor-bearing mice were established and tumor size and weight were recorded. Besides, cell apoptosis and tumor size were assessed in drug-resistant bladder cancer cells (T24/DDP) and xenografts to evaluate the effect of IR-780 + HBO on drug-resistant bladder cancer. RESULTS: IR-780 selectively accumulated in bladder cancer (bladder cancer cells, transplanted tumors, and bladder cancer tissue from patients) and could induce cancer cell apoptosis by targeting the mitochondrial complex I protein NDUFS1. The combination with HBO could significantly enhance the anti-tumor effect of IR-780 in vitro by promoting cancer cell uptake and inducing excessive mitochondrial ROS production, while suppressing tumor growth and recurrence in animal models without causing apparent toxicity. Moreover, this combination antitumor strategy was also demonstrated in drug-resistant bladder cancer cells (T24/DDP) and xenografts. CONCLUSION: We identified for the first time a combination of IR-780 and HBO (IR-780 + HBO), which exhibits mitochondria-targeting and therapeutic capabilities, as a novel treatment paradigm for bladder cancer.

Aesculetin Inhibits Proliferation and Induces Mitochondrial Apoptosis in Bladder Cancer Cells by Suppressing the MEK/ERK Signaling Pathway.

BACKGROUND: Aesculetin (AE), a natural coumarin derivative found in traditional medicinal herbs, has a variety of pharmacological effects. However, the role of AE and its molecular mechanisms of action on bladder cancer remains undefined. OBJECTIVE: The study aims to explore the anti-tumor effects of AE on bladder cancer cells and the associated molecular mechanisms. METHODS: We performed a Cell Counting Kit-8 assay to examine the inhibitory effects of AE on 5637 and T24 cells. The anti-tumor effects of AE on 5637 cells were evaluated by performing colony formation, living/dead cell staining, apoptosis, cell cycle, migration and invasion assays. The expression levels of related proteins were determined using western blotting. RESULTS: The viability of 5637 and T24 cells was decreased by AE. AE significantly inhibited colony formation, arrested the cell cycle at the G0/G1 phase, decreased migration and invasion, decreased the mitochondrial membrane potential and increased apoptosis in 5637 cells. Western blotting results showed the release of cytochrome C from mitochondria; the activation of caspase-9 and caspase-3; decrease in CDK4, CCND1, MMP2 and MMP9 levels and an increase in the BAX/BCL-2 protein ratio after treatment with AE. AE also downregulated the levels of p-ERK and p- MEK proteins. Pre-treatment with U0126 significantly enhanced the anti-tumor effects of AE. CONCLUSIONS: AE inhibited the proliferation and induced the apoptosis of bladder cancer cells through the MEK/ERK pathway. These findings provide possible therapeutic strategies for bladder cancer.

Effects of Supplemental Drugs on Hexaminolevulinate (HAL)-Induced PpIX Fluorescence in Bladder Cancer Cell Suspensions.

Seven different inhibitors of the heme metabolic pathway were applied in combination with HAL to study the formation of PpIX in bladder cancer HT1197 and normal fibroblast HFFF2 cells ex vivo, specifically with the aim to increase the fluorescence contrast between cancer and non-cancer cells. The mRNA expression of enzymes involved in the heme biosynthesis pathway were measured via PCR following incubation with the drugs in order to link the fluorescence levels and metabolic activity. The exogenous administration of HAL does lead to cancer-specific PpIX accumulation. However, the contrast between cancer and normal cells in suspension was not enhanced by the enzyme inhibitors and iron-chelating agents tested, nor did the mRNA expression necessarily correlate with the fluorescence intensity. The results indicate that a difference in the metabolic activity of cells in suspension may limit the applicability of exogenous enzyme inhibitor administration as a mean to improve the fluorescence-based detection of cancer cells shed in body fluids.

Thymoquinone Suppresses the Proliferation, Migration and Invasiveness through Regulating ROS, Autophagic Flux and miR-877-5p in Human Bladder Carcinoma Cells.

Bladder carcinoma is among the top 10 most frequently diagnosed cancer types in the world. As a phytochemical active metabolic, thymoquinone (TQ) is extracted from seeds of Nigella sativa, possessing various biological properties in a wide range of diseases. Moreover, the outstanding anti-cancer effect of TQ is attracting increasing attentions. In certain circumstances, moderate autophagy is regarded to facilitate the adaptation of malignant cells to different stressors. Conversely, closely linked with the mitochondrial membrane potential (MMP) loss, the upregulation of intracellular reactive oxygen species (ROS) is reported to activate the cell apoptosis in many cancer types. Furthermore, the vital effects of microRNAs in the pathological processes of cancer cells have also been confirmed by previous studies. The present research confirms that TQ restrains the viability, proliferation, migration and invasion through activating caspase-dependent apoptosis in bladder carcinoma cells, which is mediated by TQ induced ROS increase in bladder carcinoma cells. Furthermore, TQ is proved to block the fusion of autophagosomes and lysosomes, causing the accumulation of autophagosomes and subsequent cell apoptosis. In addition, TQ is also found to initiate the miR-877-5p/PD-L1 axis, which suppresses the epithelial mesenchymal transition (EMT) and invasion of bladder carcinoma cells. Taken together, TQ induces the apoptosis through upregulating ROS level and impairing autophagic flux, and inhibiting the EMT and cell invasion via activating the miR-877-5p/PD-L1 axis in bladder carcinoma cells.

Grape Seed Proanthocyanidins Inhibit Migration and Invasion of Bladder Cancer Cells by Reversing EMT through Suppression of TGF-ß Signaling Pathway.

Bladder cancer (BC) is the most common cancer of the urinary system. Despite advances in diagnosis and therapy, the prognosis is still poor because of recurrence and metastasis. Epithelial-mesenchymal transition (EMT) is considered to play an important role in the invasion and metastasis of BC. Grape seed proanthocyanidins (GSPs) exhibit chemopreventive and chemotherapeutic activities against several types of cancer. However, their effects and underlying mechanisms on the invasive potential of BC remain unclear. In this study, we found that GSPs inhibited migration, invasion, and MMP-2/-9 secretion of both T24 and 5637 bladder cancer cells at noncytotoxic concentrations. We also discovered that 5637 cells were more suitable than T24 cells for the EMT study. Further study showed that GSPs inhibited EMT by reversing the TGF-ß-induced morphological change and upregulation of mesenchymal markers N-cadherin, vimentin, and Slug as well as downregulation of epithelial markers E-cadherin and ZO-1 in 5637 cells. GSPs also inhibited TGF-ß-induced phosphorylation of Smad2/3, Akt, Erk, and p38 in 5637 cells without affecting the expression of total Smad2/3, Akt, Erk, and p38. Taken together, the results of the present study demonstrate that GSPs effectively inhibit the migration and invasion of BC cells by reversing EMT through suppression of the TGF-ß signaling pathway, which indicates that GSPs could be developed as a potential chemopreventive and therapeutic agent against bladder cancer.

Guggulsterone induces apoptosis and inhibits lysosomal-dependent migration in human bladder cancer cells.

BACKGROUND: The survival rate and therapeutic options for patients with bladder cancer have improved little in recent decades. Guggulsterone (GS), a phytoestrogen, has been investigated as an anticancer drug in various malignancies. PURPOSE: The present study aimed to evaluate the anticancer effects of E-isomer and Z-isomer GS in the human bladder cancer cell lines TSGH8301 (low-grade) and T24 (high-grade) and their underlying mechanisms. METHODS: The cell survival effect of GS was investigated by the MTT and colony formation assays in bladder cancer cell lines. Flow cytometry was used to analyze the cell cycle and cell death. Migration ability was measured by wound healing and transwell assays. Protein expression was determined by Western blot after GS treatment. The potency of GS on subcutaneous TSGH8301 bladder tumors was evaluated using an in vivo imaging system. RESULTS: E-isomer GS reduced the survival rate of both low- and high-grade human bladder cancer cells. GS caused cell cycle arrest, accompanied by the decrease and increase in cyclin A and p21 levels, respectively. Additionally, caspase-dependent apoptosis was observed following GS treatment. Furthermore, GS treatment downregulated mTOR-Akt signaling and induced autophagy with p62 and LC3ß-II expression. Moreover, the farnesoid X receptor was involved in GS-inhibited cell growth. In addition, GS reduced the migration ability with a decrease in integrin-focal adhesion kinase and myosin light chain. Interestingly, the suppression of GS-mediated migration was prevented by the lysosomal inhibitor ammonium chloride (NH4Cl). GS also reduced TSGH8301 bladder cancer cell progression by increasing the level of p21, cleaved caspase 3, cleaved poly (ADP-ribose) polymerase (PARP), and LC3ß-II in vivo. CONCLUSIONS: The current findings suggest that GS treatment may serve as a potential anticancer therapy for different grades of urothelial carcinoma.

A network pharmacology perspective for deciphering potential mechanisms of action of Solanum nigrum L. in bladder cancer.

BACKGROUND: Solanum nigrum L. decoction has been used as a folklore medicine in China to prevent the postoperative recurrence of bladder cancer (BC). However, there are no previous pharmacological studies on the protective mechanisms of this activity of the plant. Thus, this study aimed to perform a systematic analysis and to predict the potential action mechanisms underlying S. nigrum activity in BC based on network pharmacology. METHODS: Based on network pharmacology, the active ingredients of S. nigrum and the corresponding targets were identified using the Traditional Chinese Medicines for Systems Pharmacology Database and Analysis Platform database, and BC-related genes were screened using GeneCards and the Online Mendelian Inheritance in Man database. In addition, ingredient-target (I-T) and protein-protein interaction (PPI) networks were constructed using STRING and Cytoscape, Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted, and then the pathways directly related to BC were integrated manually to reveal the pharmacological mechanism underlying S. nigrum-medicated therapeutic effects in BC. RESULTS: Seven active herbal ingredients from 39 components of S. nigrum were identified, which shared 77 common target genes related to BC. I-T network analysis revealed that quercetin was associated with all targets and that NCOA2 was targeted by four ingredients. Besides, interleukin 6 had the highest degree value in the PPI network, indicating a hub role. A subsequent gene enrichment analysis yielded 86 significant GO terms and 89 significant pathways, implying that S. nigrum had therapeutic benefits in BC through multi-pathway effects, including the HIF-1, TNF, P53, MAPK, PI3K/Akt, apoptosis and bladder cancer pathway. CONCLUSIONS: S. nigrum may mediate pharmacological effects in BC through multi-target and various signaling pathways. Further validation is required experimentally. Network pharmacology approach provides a predicative novel strategy to reveal the holistic mechanism of action of herbs.

Therapeutic Applications of Curcumin and its Novel Formulations in the Treatment of Bladder Cancer: A Review of Current Evidence.

Bladder cancer, a life-threatening serious disease, is responsible for thousands of cancer-associated deaths worldwide. Similar to other malignancies, standard treatments of bladder cancer, such as Chemoradiotherapy, are not efficient enough in the affected patients. It means that, according to recent reports in the case of life quality as well as the survival time of bladder cancer patients, there is a critical requirement for exploring effective treatments. Recently, numerous investigations have been carried out to search for appropriate complementary treatments or adjuvants for bladder cancer therapy. Curcumin, a phenolic component with a wide spectrum of biological activities, has recently been introduced as a potential anti-cancer agent. It has been shown that this agent exerts its therapeutic effects via targeting a wide range of cellular and molecular pathways involved in bladder cancer. Herein, the current data on curcumin therapy for bladder cancer are summarized.

Artesunate Impairs Growth in Cisplatin-Resistant Bladder Cancer Cells by Cell Cycle Arrest, Apoptosis and Autophagy Induction.

Cisplatin, which induces DNA damage, is standard chemotherapy for advanced bladder cancer (BCa). However, efficacy is limited due to resistance development. Since artesunate (ART), a derivative of artemisinin originating from Traditional Chinese Medicine, has been shown to exhibit anti-tumor activity, and to inhibit DNA damage repair, the impact of artesunate on cisplatin-resistant BCa was evaluated. Cisplatin-sensitive (parental) and cisplatin-resistant BCa cells, RT4, RT112, T24, and TCCSup, were treated with ART (1-100 µM). Cell growth, proliferation, and cell cycle phases were investigated, as were apoptosis, necrosis, ferroptosis, autophagy, metabolic activity, and protein expression. Exposure to ART induced a time- and dose-dependent significant inhibition of tumor cell growth and proliferation of parental and cisplatin-resistant BCa cells. This inhibition was accompanied by a G0/G1 phase arrest and modulation of cell cycle regulating proteins. ART induced apoptos is by enhancing DNA damage, especially in the resistant cells. ART did not induce ferroptosis, but led to a disturbance of mitochondrial respiration and ATP generation. This impairment correlated with autophagy accompanied by a decrease in LC3B-I and an increase in LC3B-II. Since ART significantly inhibits proliferative and metabolic aspects of cisplatin-sensitive and cisplatin-resistant BCa cells, it may hold potential in treating advanced and therapy-resistant BCa.

Transcription factor YY1 mediates epithelial-mesenchymal transition through the TGFß signaling pathway in bladder cancer.

Bladder cancer is one of the most aggressive urothelial tumors. Previous studies have suggested that epithelial-mesenchymal transition (EMT) contributes to bladder cancer progression. However, the regulatory network of EMT in bladder cancer remains elusive. In this study, we found Yin Yang 1 (YY1) is a critical regulator of EMT in bladder cancer. First, we showed that YY1 was upregulated in bladder cancer tissues than that in adjacent normal tissues. Then, we proved that YY1 promoted EMT of bladder cancer cells. Further experiments indicated that YY1 affected the EMT of bladder cancer through transforming growth factor-ß (TGFß) signaling pathway. Taken together, our study identifies YY1 as a key EMT driver in bladder cancer, suggesting it as a potential therapeutic target.

An ATF24 peptide-functionalized ß-elemene-nanostructured lipid carrier combined with cisplatin for bladder cancer treatment.

Objective: In this study, we aimed to develop an amino-terminal fragment (ATF) peptide-targeted liposome carrying ß-elemene (ATF24-PEG-Lipo-ß-E) for targeted delivery into urokinase plasminogen activator receptor-overexpressing bladder cancer cells combined with cisplatin (DDP) for bladder cancer treatment. Methods: The liposomes were prepared by ethanol injection and high-pressure microjet homogenization. The liposomes were characterized, and the drug content, entrapment efficiency, and in vitro release were studied. The targeting efficiency was investigated using confocal microscopy, ultra-fast liquid chromatography, and an orthotopic bladder cancer model. The effects of ATF24-PEG-Lipo-ß-E combined with DDP on cell viability and proliferation were evaluated by a Cell Counting Kit-8 (CCK-8) assay, a colony formation assay, and cell apoptosis and cell cycle analyses. The anticancer effects were evaluated in a KU-19-19 bladder cancer xenograft model. Results: ATF24-PEG-Lipo-ß-E had small and uniform sizes (˜79 nm), high drug loading capacity (˜5.24 mg/mL), high entrapment efficiency (98.37 ± 0.95%), and exhibited sustained drug release behavior. ATF24-PEG-Lipo-ß-E had better targeting efficiency and higher cytotoxicity than polyethylene glycol (PEG)ylated ß-elemene liposomes (PEG-Lipo-ß-E). DDP, combined with ATF24-PEG-Lipo-ß-E, exerted a synergistic effect on cellular apoptosis and cell arrest at the G2/M phase, and these effects were dependent on the caspase-dependent pathway and Cdc25C/Cdc2/cyclin B1 pathways. Furthermore, the in vivo antitumor activity showed that the targeted liposomes effectively inhibited the growth of tumors, using the combined strategy. Conclusions: The present study provided an effective strategy for the targeted delivery of ß-elemene (ß-E) to bladder cancer, and a combined strategy for bladder cancer treatment.

Radix Sophorae Flavescentis induces apoptosis through by Caspase, MAPK Activation and ROS Signaling Pathways in 5637 Human Bladder Cancer Cells.

The anti-cancer mechanisms of Radix Sophorae Flavescentis were investigated in 5637 bladder cancer cells. Radix Sophorae Flavescentis extract (RSF) (50­400 µg/ml) inhibited the proliferation of 5637 cells and increased sub­G1 phase ratios. RSF­induced cell death was associated with the down-regulation of B­cell lymphoma 2 (Bcl­2) and the up-regulation of Bcl­2 X­associated protein (Bax). RSF also activated caspase­3 and -9 and regulated the activations of mitogen-activated protein kinases (MAPKs). In addition, RSF increased intracellular reactive oxygen species (ROS) levels and depolarized the mitochondrial membrane potential. These findings suggest RSF induces apoptosis in 5637 bladder cancer cells and that it has potential use as a novel anti-cancer drug for bladder cancer.

Ovatodiolide, isolated from Anisomeles indica, suppresses bladder carcinogenesis through suppression of mTOR/ß-catenin/CDK6 and exosomal miR-21 derived from M2 tumor-associated macrophages.

Bladder cancer (BCa) is the fourth leading cause of cancer deaths worldwide due to its aggressiveness and resistance against therapies. Intricate interactions between cancer cells and the tumor microenvironment (TME) are essential for both disease progression and regression. Thus, interrupting molecular communications within the TME could potentially provide improved therapeutic efficacies. M2-polarized tumor-associated macrophages (M2 TAMs) were shown to contribute to BCa progression and drug resistance. We attempted to provide evidence for ovatodiolide (OV) as a potential therapeutic agent that targets both TME and BCa cells. First, tumor-suppressing functions of OV were determined by cell viability, colony, and tumor-sphere formation assays using a coculture system composed of M2 TAMs/BCa cells. Subsequently, we demonstrated that extracellular vesicles (EVs) isolated from M2 TAMs containing oncomiR-21 and mRNAs, including Akt, STAT3, mTOR, and ß-catenin, promoted cisplatin (CDDP) resistance, migration, and tumor-sphere generation in BCa cells, through increasing CDK6, mTOR, STAT3, and ß-catenin expression. OV treatment also prevented M2 polarization and reduced EV cargos from M2 TAMs. Finally, in vivo data demonstrated that OV treatment overcame CDDP resistance. OV only and the OV + CDDP combination both resulted in significant reductions in mTOR, ß-catenin, CDK6, and miR-21 expression in tumor samples and EVs isolated from serum. Collectively, we demonstrated that M2 TAMs induced malignant properties in BCa cells, in part via oncogenic EVs. OV treatment prevented M2 TAM polarization, reduced EV cargos derived from M2 TAMs, and suppressed ß-catenin/mTOR/CDK6 signaling. These findings provide preclinical evidence for OV as a single or adjuvant agent for treating drug-resistant BCa.

A Peptidomimetic Fluorescent Probe to Detect the Trypsin ß2 Subunit of the Human 20S Proteasome.

This work describes the chemical synthesis, combinatorial selection, and enzymatic evaluation of peptidomimetic fluorescent substrates specific for the trypsin-like (ß2) subunit of the 20S human proteasome. After deconvolution of a library comprising nearly 6000 compounds composed of peg substituted diaminopropionic acid DAPEG building blocks, the sequence ABZ-Dap(O2(Cbz))-Dap(GO1)-Dap(O2(Cbz))-Arg-ANB-NH2, where ABZ is 2-aminobenzoic acid, and ANB- 5 amino 2- nitro benzoic acid was selected. Its cleavage followed sigmoidal kinetics, characteristic for allosteric enzymes, with Km = 3.22 ± 0.02 µM, kcat = 245 s-1, and kcat/Km = 7.61 × 107 M-1 s-1. This process was practically halted when a selective inhibitor of the ß2 subunit of the 20S human proteasome was supplemented to the reaction system. Titration of the substrate resulting in decreased amounts of proteasome 20S produced a linear signal up to 10-11 M. Using this substrate, we detected human proteasome 20S in human urine samples taken from the bladders of cancer patients. This observation could be useful for the noninvasive diagnosis of this severe disease.

Effect of nitric oxide inhibition in Bacillus Calmette-Guerin bladder cancer treatment.

BACKGROUND: Bacillus Calmette-Guerin (BCG) is the standard treatment for patients with high-risk non-muscle invasive bladder cancer (BC). Despite its success, about 30-50% of patients are refractory. It was reported that inducible nitric oxide synthase (iNOS) tumor expression is presented in 50% of human BC, associated with bad prognosis and BCG failure. OBJECTIVE: to evaluate in human bladder tumors the association between iNOS expression and the tumor microenvironment focusing on the immunosuppressive protein S100A9. Also, investigate in a preclinical murine MB49-BC model the tumor immunoresponse induced by BCG in combination with the nitric oxide production inhibitor l-NAME. RESULTS: In human bladder tumors, we detected a positive association between iNOS and S100A9 tumor expression, suggesting a relationship between both immunomodulatory proteins. We also found a positive correlation between iNOS tumor expression and the presence of S100A9+ tumor-infiltrating cells, suggesting an immunosuppressive tumor microenvironment induced by the nitric oxide production. Using the subcutaneous murine BC model, we show that similarly to the human pathology, MB49 tumors constitutively expressed iNOS and S100A9 protein. MB49 tumor-bearing mice presented an immunosuppressive systemic profile characterized by fewer cytotoxic cells (CD8+ and NK) and higher suppressor cells (Treg and myeloid-derived suppressor cells -MDSC-) compared to normal mice. BCG treatment reduced tumor growth, increasing local CD8+-infiltrating cells and induced a systemic increase in CD8+ and a reduction in Treg. BCG combined with l-NAME, significantly reduced tumor growth compared to BCG alone, diminishing iNOS and S100A9 tumor expression and increasing CD8+-infiltrating cells in tumor microenvironment. This local response was accompanied by the systemic increase in CD8+ and NK cells, and the reduction in Treg and MDSC, even more than BCG alone. Similar results were obtained using the orthotopic BC model, where an increase in specific cytotoxicity against MB49 tumor cells was detected. CONCLUSION: The present study provides preclinical information where NO inhibition in iNOS-expressing bladder tumors could contribute to improve BCG antitumor immune response. The association between iNOS and S100A9 in human BC supports the hypothesis that iNOS expression is a negative prognostic factor and a promising therapeutic target.

Therapeutic Effects of Curcumin against Bladder Cancer: A Review of Possible Molecular Pathways.

There are concerns about the increased incidence of cancer both in developing and developed countries. In spite of recent progress in cancer therapy, this disease is still one of the leading causes of death worldwide. Consequently, there have been rigorous attempts to improve cancer therapy by looking at nature as a rich source of naturally occurring anti-tumor drugs. Curcumin is a well-known plant-derived polyphenol found in turmeric. This compound has numerous pharmacological effects such as antioxidant, anti-inflammatory, antidiabetic and anti-tumor properties. Curcumin is capable of suppressing the growth of a variety of cancer cells including those of bladder cancer. Given the involvement of various signaling pathways such as PI3K, Akt, mTOR and VEGF in the progression and malignancy of bladder cancer, and considering the potential of curcumin in targeting signaling pathways, it seems that curcumin can be considered as a promising candidate in bladder cancer therapy. In the present review, we describe the molecular signaling pathways through which curcumin inhibits invasion and metastasis of bladder cancer cells.

RASSF1A-Hippo pathway link in patients with urothelial carcinoma of bladder: plausible therapeutic target.

RASSF1A is a tumor suppressor gene, and its hypermethylation has been observed in cancers. RASSF1A acts as an upstream regulator of Hippo pathway and modulates its function. The aim of this study was to analyze expression of RASSF1A, Hippo pathway molecules (YAP, MST) and downstream targets (CTGF, Cyr61 and AREG) in bladder cancer patients. Later, the link between RASSF1A and Hippo pathway and a potential therapeutic scope of this link in UBC were also studied. MSPCR was performed to study methylation of RASSF1A promoter. Expression of molecules was studied using qPCR, Western blot and IHC. The link between RASSF1A and Hippo pathway was studied using Spearman's correlation in patients and validated by overexpressing RASSF1A in HT1376 cells and its effect on Hippo pathway was observed using qPCR and Western blot. Further therapeutic potential of this link was studied using MTT and PI assays. The expression of RASSF1A was lower, whereas the expression of YAP, CTGF and CYR61 was higher. The expression of RASSF1A protein gradually decreased, while the expression of YAP, CTGF and CYR61 increased with severity of disease. Based on Spearman's correlation, RASSF1A showed a negative correlation with YAP, CTGF and CYR61. YAP showed a positive correlation with CTGF and CYR61. To validate this link, RASSF1A was overexpressed in HT1376 cells. Overexpressed RASSF1A activated Hippo pathway, followed by a decrease in CTGF and CYR61 at mRNA, and enhanced cytotoxicity to chemotherapeutic drugs. This study finds a previously unrecognized role of RASSF1A in the regulation of CTGF and CYR61 through mediation of Hippo pathway in UBC and supports the significance of this link as a potential therapeutic target for UBC.

Development of a high-precision bladder hyperthermic intracavitary chemotherapy device for bladder cancer and pharmacokinetic study.

BACKGROUND: Bladder hyperthermic intracavitary chemotherapy (HIVEC) has good effectiveness for bladder cancer, but conventional HIVEC systems lack precision and convenient application. To test the safety of a new HIVEC device (BR-TRG-II-type) in pigs and to perform a preliminary clinical trial in patients with bladder cancer. METHODS: This device was tested on six pigs to optimize the temperature and time parameters. Then, 165 patients (HIVEC after transurethral resection (TUR), n = 128; or HIVEC, n = 37) treated between December 2006 and December 2016 were recruited. Mitomycin C (MMC) was the chemotherapeutic agent. A serum pharmacokinetic study was performed. The primary endpoints were tumor recurrence, disease-free survival (DFS), and cumulative incidence rate (CIR) during follow-up. The adverse effects were graded. RESULTS: The animal experiment showed that 45 °C for 1 h was optimal. HIVEC was successful, with the infusion tube temperature stably controlled at about 45 °C, and outlet tube temperature of about 43 °C in all patients, for three sessions. Serum MMC levels gradually increased during HIVEC and decreased thereafter. The mean DFS was 39 ± 3.21 months (ranging from 8 to 78 months), and the DFS rate was 89.1% during follow-up. No adverse events occurred. CONCLUSION: The use of the BR-TRG-II-type HIVEC device is feasible for the treatment of bladder cancer. Future clinical trials in patients with different stages of bladder cancer will further confirm the clinical usefulness of this device. TRIAL REGISTRATION: chictr.org.cn: ChiCTR1900022099 (registered on Mar. 252,019). Retrospectively registered.