ABSTRACT
Bladder cancer (BC) is a malignant tumor of the urinary system with high mortality and recurrence rates. Proteasome subunit type 4 (PSMB4) is highly expressed and has been identified as having oncogenic properties in a variety of cancer types. This study aimed to explore the effect of PSMB4 knockdown on the survival, migration, and angiogenesis of human bladder cancer cells with different degrees of malignancy. We analyzed the effects of PSMB4 knockdown in bladder cancer cells and endothelial cells in the tumor microenvironment. PSMB4 was highly expressed in patients with low- and high-grade urothelial carcinoma. Inhibition of PSMB4 reduced protein expression of focal adhesion kinase (FAK) and myosin light chain (MLC), leading to reduced migration. Furthermore, the suppression of PSMB4 decreased the levels of vascular endothelial factor B (VEGF-B), resulting in lower angiogenic abilities in human bladder cancer cells. PSMB4 inhibition affected the migratory ability of HUVECs and reduced VEGFR2 expression, consequently downregulating angiogenesis. In the metastatic animal model, PSMB4 knockdown reduced the relative volumes of lung tumors. Our findings suggest the role of PSMB4 as a potential target for therapeutic strategies against human bladder cancer.
Subject(s)
Cell Movement , Neovascularization, Pathologic , Proteasome Endopeptidase Complex , Urinary Bladder Neoplasms , Humans , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/metabolism , Cell Movement/genetics , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/genetics , Cell Line, Tumor , Animals , Mice , Human Umbilical Vein Endothelial Cells/metabolism , Endothelial Cells/metabolism , Endothelial Cells/pathology , Gene Knockdown Techniques , Gene Expression Regulation, Neoplastic , Tumor Microenvironment/genetics , Male , Vascular Endothelial Growth Factor Receptor-2/metabolism , Vascular Endothelial Growth Factor Receptor-2/genetics , Female , Angiogenesis , Cysteine EndopeptidasesABSTRACT
The ability of bladder cancer to invade and metastasize often leads to poor prognosis in bladder cancer patients. The aim of this study was to evaluate the effect of the farnesoid X receptor (FXR) agonist GW4064 on the migration and invasion of human bladder cancer cells. Long-term exposure to GW4064 decreased the colony formation of RT4 and T24 cells. The wound healing migration assay revealed an inhibitory effect of GW4064 on both of these bladder cancer cell lines. In addition, integrin ß3 expression and myosin light chain phosphorylation were decreased after GW4064 treatment. Immunocytochemistry showed an increase in E-cadherin and a decrease in ß-catenin in the cell membrane of bladder cancer cells. Total protein expression and membrane fractionation assays also indicated upregulation of E-cadherin and downregulation of ß-catenin. Moreover, GW4064 reduced the invasion of muscle-invasive T24 cells. The GW4064-decreased migration and invasion were reversed by the proteasome inhibitor MG132 and the lysosome inhibitor NH4Cl. Furthermore, the GW4064-induced inhibition of matrix metalloproteinase-2 (MMP2) and cathepsin B expression was reversed by NH4Cl. Xenograft animal studies revealed that GW4064 declined MMP2, cathepsin B and lung metastasis of bladder cancer. In conclusion, GW4064 decreases the migration and invasion of human bladder cancer cells, which may provide a new therapeutic strategy for the treatment of human bladder cancer.
Subject(s)
Isoxazoles , Urinary Bladder Neoplasms , beta Catenin , Animals , Humans , beta Catenin/metabolism , Matrix Metalloproteinase 2/metabolism , Down-Regulation , Cathepsin B , Cell Line, Tumor , Urinary Bladder Neoplasms/metabolism , Cadherins/metabolism , Cell Movement , Neoplasm InvasivenessABSTRACT
Bladder cancer is one of the most prevailing cancers worldwide. Although treatments for urothelial carcinoma have improved, the rate of recurrence observed in the clinic is still high. The aim of this study was to evaluate whether cholesterol biosynthesis is involved in the effect of Farnesoid X Receptor (FXR) on bladder cancers. FXR overexpression contributed to activation of 5' AMP-activated protein kinase (AMPK) and decreased cholesterol levels. FXR overexpression reduced cholesterol biosynthesis and secretion by downregulating Sterol Regulatory Element Binding Protein 2 (SREBP2) and 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR) expression. In addition, an AMPK inhibitor, dorsomorphin, reversed the inhibition of migration, invasion and angiogenesis by FXR overexpression. In a metastatic xenograft animal study, FXR overexpression suppressed bladder cancer lung metastasis by decreasing matrix metalloproteinase-2 (MMP2), SREBP2 and HMGCR expression. Moreover, FXR overexpression combined with atorvastatin treatment further enhanced the downregulation of the migratory, adhesive, invasive and angiogenic properties in human urothelial carcinoma. In clinical observations, statin administration was associated with better survival rates of early-stage bladder cancer patients. Our results may provide guidance for improving therapeutic strategies for the treatment of urothelial carcinoma.
ABSTRACT
(1) Background: Bladder cancer is a malignant tumor mainly caused by exposure to environmental chemicals, with a high recurrence rate. NR1H4, also known as Farnesoid X Receptor (FXR), acts as a nuclear receptor that can be activated by binding with bile acids, and FXR is highly correlated with the progression of cancers. The aim of this study was to verify the role of FXR in bladder cancer cells. (2) Methods: A FXR overexpressed system was established to investigate the effect of cell viability, migration, adhesion, and angiogenesis in low-grade TSGH8301 and high-grade T24 cells. (3) Results: After FXR overexpression, the ability of migration, adhesion, invasion and angiogenesis of bladder cancer cells declined significantly. Focal adhesive complex, MMP2, MMP9, and angiogenic-related proteins were decreased, while FXR was overexpressed in bladder cancer cells. Moreover, FXR overexpression reduced vascular endothelial growth factor mRNA and protein expression and secretion in bladder cancer cells. After treatment with the proteosome inhibitor MG132, the migration, adhesion and angiogenesis caused by FXR overexpression were all reversed in bladder cancer cells. (4) Conclusions: These results may provide evidence on the role of FXR in bladder cancer, and thus may improve the therapeutic efficacy of urothelial carcinoma in the future.
Subject(s)
Carcinoma, Transitional Cell , Receptors, Cytoplasmic and Nuclear/metabolism , Urinary Bladder Neoplasms , Cell Line, Tumor , Female , Humans , Male , Neovascularization, Pathologic/genetics , Proteasome Endopeptidase Complex , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathology , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth FactorsABSTRACT
Bladder cancer (BC) has a high recurrence rate worldwide. The aim of this study was to evaluate the role of fatty acid binding protein 6 (FABP6) in proliferation and migration in human bladder cancer cells. Cell growth was confirmed by MTT and colony formation assay. Western blotting was used to explore protein expressions. Wound healing and Transwell assays were performed to evaluate the migration ability. A xenograft animal model with subcutaneous implantation of BC cells was generated to confirm the tumor progression. Knockdown of FABP6 reduced cell growth in low-grade TSGH-8301 and high-grade T24 cells. Cell cycle blockade was observed with the decrease of CDK2, CDK4, and Ki67 levels in FABP6-knockdown BC cells. Interestingly, knockdown of FBAP6 led to downregulation of autophagic markers and activation of AKT-mTOR signaling. The application of PI3K/AKT inhibitor decreased cell viability mediated by FABP6-knockdown additionally. Moreover, FABP6-knockdown reduced peroxisome proliferator-activated receptor γ and retinoid X receptor α levels but increased p-p65 expression. Knockdown of FABP6 also inhibited BC cell motility with focal adhesive complex reduction. Finally, shFABP6 combined with cisplatin suppressed tumor growth in vivo. These results provide evidence that FABP6 may be a potential target in BC cells progression.
Subject(s)
Autophagy/physiology , Cell Cycle/physiology , Cell Movement/physiology , Fatty Acid-Binding Proteins/metabolism , Gastrointestinal Hormones/metabolism , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathology , Animals , Cell Line, Tumor , Cell Proliferation/physiology , Cell Survival/physiology , Down-Regulation/physiology , Gene Expression Regulation, Neoplastic/physiology , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/physiologyABSTRACT
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.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Lysosomes/drug effects , Pregnenediones/pharmacology , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/pathology , Animals , Apoptosis/drug effects , Autophagy/drug effects , Caspase 3/metabolism , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Humans , Lysosomes/metabolism , Lysosomes/pathology , Mice, Inbred BALB C , TOR Serine-Threonine Kinases/metabolism , Urinary Bladder Neoplasms/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Among herbal medicines, magnolia bark extract, particularly its components honokiol (Hono) and magnolol (Mag), has been widely documented to have antineoplastic properties. The present study aimed to evaluate the synergism of Hono and Mag in bladder cancer therapy both in vitro and in vivo. Treatment with Mag alone at concentrations up to 80 µM failed to have an antiproliferative effect. In contrast, the combination of Hono and Mag at 40 µM decreased viability, caused cell cycle arrest and enhanced the proportion of Annexin V/7AAD-positive cells. Moreover, Mag with Hono at 40 µM induced caspase 3-dependent apoptosis and autophagy. Neither Hono nor Mag alone had an anti-migratory effect on bladder cancer cells. In contrast, Hono and Mag at 20 µM inhibited the motility of TSGH8301 and T24 cells in wound-healing and Transwell assays. The above phenomena were further confirmed by decreased phosphorylated focal adhesion kinase (p-FAK), p-paxillin, integrin ß1, and integrin ß3 protein levels. In a nude mouse xenograft model, Mag/Hono administration preferentially retarded T24 tumor progression, which was consistent with the results of cellular experiments. Current findings suggest Hono and Mag treatment as a potential anticancer therapy for both low- and high-grade urothelial carcinoma.
