Guggulsterone inhibits migration and invasion through proteasomal and lysosomal degradation in human glioblastoma cells.

Glioblastoma multiforme (GBM) is a deadly brain malignancy, and current therapies offer limited survival benefit. The phytosterol guggulsterone (GS) has been shown to exhibit antitumor efficacy. This study aimed to investigate the effects of GS on migration and invasion and its underlying mechanisms in human GBM cell lines. After GS treatment, the survival rate of GBM cells was reduced, and the migration and invasion abilities of GBM cells were significantly decreased. There was also concomitant decreased expression of focal adhesion complex, matrix metalloproteinase-2 (MMP2), MMP9 and cathepsin B. Furthermore, GS induced ERK phosphorylation and autophagy, with increased p62 and LC3B-II expression. Notably, treatment of in GBM cells with the proteasome inhibitor MG132 or the lysosome inhibitor NH4Cl reversed the GS-mediated inhibition of migration and invasion. In an orthotopic xenograft mouse model, immunohistochemical staining of brain tumor tissues demonstrated that MMP2 and cathepsin B expression was reduced in GS-treated mice. GS treatment inhibited GBM cell migration and invasion via proteasomal and lysosomal degradation, suggesting its therapeutic potential in clinical use in the future.

ONX-0914 Induces Apoptosis and Autophagy with p53 Regulation in Human Glioblastoma Cells.

Glioblastoma is believed to be one of the most aggressive brain tumors in the world. ONX-0914 (PR957) is a selective inhibitor of proteasome subunit beta type-8 (PSMB8). Previous studies have shown that inhibiting PSMB8 expression in glioblastoma reduces tumor progression. Therefore, this study aimed to determine whether ONX-0914 has antitumor effects on human glioblastoma. The results indicated that ONX-0914 treatment inhibited survival in LN229, GBM8401, and U87MG glioblastoma cells. Cell cycle analysis showed that ONX-0914 treatment caused cell cycle arrest at the G1 phase and apoptosis in glioblastoma cells. The protein expression of BCL-2 was reduced and PARP was cleaved after ONX-0914 treatment. Furthermore, the levels of p53 and phosphorylated p53 were increased by ONX-0914 treatment in glioblastoma cells. ONX-0914 also induced autophagy in glioblastoma cells. Furthermore, the p53 inhibitor pifithrin attenuated apoptosis but enhanced autophagy caused by ONX-0914. In an orthotopic mouse model, TMZ plus ONX-0914 reduced tumor progression better than the control or TMZ alone. These data suggest that ONX-0914 is a novel therapeutic drug for glioblastoma.

Farnesoid X Receptor Overexpression Decreases the Migration, Invasion and Angiogenesis of Human Bladder Cancers via AMPK Activation and Cholesterol Biosynthesis Inhibition.

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.

Survival Benefit of Statin with Anti-Angiogenesis Efficacy in Lung Cancer-Associated Pleural Fluid through FXR Modulation.

Lung cancer-related pleural fluid (LCPF) presents as a common complication with limited treatment. Beyond its function in lipid digestion, bile acid was identified as a potent carcinogen to stimulate tumor proliferation. Previous research indicated a correlation between serum bile acid levels and the risk of developing several gastrointestinal cancers. Our study identified elevated bile acid levels in LCPF and increased farnesoid X receptor (FXR) expression as bile acid nuclear receptors in pleural microvessels of lung adenocarcinoma. Additionally, LCPF stimulated the expression of proteins involved in bile acid synthesis and cholesterol metabolism in HUVECs including CYP7A1, StAR, HMGCR, and SREBP2. LCPF-induced endothelial motility and angiogenesis were counteracted by using ß-muricholic acid as an FXR antagonist. Moreover, we investigated the efficacy of cholesterol-lowering medications, such as cholestyramine, fenofibrate, and atorvastatin, in regulating LCPF-regulated angiogenesis. Along with suppressing endothelial proliferation and angiogenesis, atorvastatin treatment reversed cholesterol accumulation and endothelial junction disruption caused by LCPF. Statin treatment inhibited LCPF-induced endothelial FXR expression as well as the downstream proteins RXR and SHP. Based on the positive findings of suppressing endothelial angiogenesis, our group further incorporated the effect of statin on clinical patients complicated with LCPF. A Kaplan-Meier analysis revealed the clinical benefit of statin exposure in patients with lung adenocarcinoma with LCPF. Conclusively, our study demonstrated the ability of statin to alleviate LCPF-induced angiogenesis in patients with LCPF via FXR modulation.

Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers.

(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.

Accelerated Wound Healing and Keratinocyte Proliferation through PI3K/Akt/pS6 and VEGFR2 Signaling by Topical Use of Pleural Fluid.

Impaired wound healing is an ongoing issue that cancer patients undergoing chemotherapy or radiotherapy face. Our previous study regarding lung-cancer-associated pleural fluid (LCPF) demonstrated its propensity to promote endothelial proliferation, migration, and angiogenesis, which are crucial features during cutaneous wound healing. Therefore, the current study aimed to investigate the effect of pleural fluid on cutaneous wound closure in vitro and in vivo using HaCaT keratinocytes and a full-thickness skin wound model, respectively. Both heart-failure-associated pleural fluid (HFPF) and LCPF were sequentially centrifuged and filtered to obtain a cell-free status. Treatment with HFPF and LCPF homogeneously induced HaCaT proliferation with cell cycle progression, migration, and MMP2 upregulation. Western blotting revealed increased PI3K/Akt phosphorylation and VEGFR2/VEGFA expression in HaCaT cells. When treated with the PI3K inhibitor, LCPF-induced keratinocyte proliferation was attenuated with decreased pS6 levels. By applying the VEGFR2 inhibitor, LCPF-induced keratinocyte proliferation was ameliorated by pS6 and MMP2 downregulation. The effect of LCPF-induced cell junction rearrangement was disrupted by co-treatment with a VEGFR2 inhibitor. Compared with a 0.9% saline dressing, LCPF significantly accelerated wound closure and re-epithelization when used as a dressing material in a full-thickness wound model. Histological analysis revealed increased neo-epidermis thickness and dermis collagen synthesis in the LCPF-treated group. Furthermore, LCPF treatment activated basal keratinocytes at the wound edge with the upregulation of Ki-67, VEGFA, and MMP2. Our preliminaries provided the benefit of wet dressing with pleural fluid to improve cutaneous wound closure through enhanced re-epithelization and disclosed future autologous application in cancer wound treatment.

Fatty Acid Binding Protein 6 Inhibition Decreases Cell Cycle Progression, Migration and Autophagy in Bladder Cancers.

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.

Concurrent Blockade of Endothelial EGFR and VEGF Signaling on Malignant Associated Pleural Fluid Induced Angiogenesis: From Clinic to Bench.

Malignant-associated pleural fluid (MAPF) represented an unsolved problem in advanced lung cancer. Our previous work characterized increased pleural angiogenesis in lung adenocarcinoma and the propensity of MAPF on endothelial angiogenesis. This study investigated the combined efficacy of the tyrosine kinase inhibitor (gefitinib) and bevacizumab in opposing MAPF-induced angiogenesis. In lung adenocarcinoma patients with malignant pleural effusion (MPE), Kaplan-Meier analysis revealed the benefit of cotreatment with target therapy and bevacizumab. Increased EGFR expression was observed in the pleural microvessels of patients with lung adenocarcinoma both with and without mutations in EGFR. MAPF was obtained from lung adenocarcinoma patients both wild-type and mutant EGFRs. Total and phosphorylated EGFR were upregulated in HUVEC cultured with MAPF. Treatment with gefitinib as an EGFR inhibitor suppressed MAPF-induced endothelial migration and partially attenuated endothelial proliferation in both wild-type and mutant EGFR lung adenocarcinoma. Cotreatment with gefitinib and bevacizumab produced better inhibition of MAPF-induced endothelial angiogenesis than gefitinib alone in the mutant EGFR subgroup. Protein analysis of MAPF-derived exosomes revealed abundant EGFR and p-EGFR components that implied possible transfer to endothelial cells. Concluding Kaplan-Meier analysis and in vitro studies, the results indicated that the addition of bevacizumab on gefitinib treatment could suppress MAPF-induced angiogenesis in lung adenocarcinoma patients.

Inhibition of FABP6 Reduces Tumor Cell Invasion and Angiogenesis through the Decrease in MMP-2 and VEGF in Human Glioblastoma Cells.

Malignant glioma is one of the most lethal cancers with rapid progression, high recurrence, and poor prognosis in the central nervous system. Fatty acid-binding protein 6 (FABP6) is a bile acid carrier protein that is overexpressed in colorectal cancer. This study aimed to assess the involvement of FABP6 expression in the progression of malignant glioma. Immunohistochemical analysis revealed that FABP6 expression was higher in glioma than in normal brain tissue. After the knockdown of FABP6, a decrease in the migration and invasion abilities of glioma cells was observed. The phosphorylation of the myosin light chain was inhibited, which may be associated with migration ability. Moreover, expression levels of invasion-related proteins, matrix metalloproteinase-2 (MMP-2) and cathepsin B, were reduced. Furthermore, tube formation was inhibited in the human umbilical vein endothelial cells with a decreased concentration of vascular endothelial growth factor (VEGF) in the conditioned medium after the knockdown of FABP6. The phosphorylation of the extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p65 were also decreased after FABP6 reduction. Finally, the bioluminescent images and immunostaining of MMP-2, cluster of differentiation 31 (CD31), and the VEGF receptor 1 (VEGFR1) revealed attenuated tumor progression in the combination of the FABP6-knocked-down and temozolomide (TMZ)-treated group in an orthotopic xenograft mouse tumor model. This is the first study that revealed the impact of FABP6 on the invasion, angiogenesis, and progression of glioma. The results of this study show that FABP6 may be a potential therapeutic target combined with TMZ for malignant gliomas.

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.

Distinct JNK/VEGFR signaling on angiogenesis of breast cancer-associated pleural fluid based on hormone receptor status.

Malignant pleural effusion is a common complication in metastatic breast cancer (MBC); however, changes in the pleural microenvironment are poorly characterized, especially with respect to estrogen receptor status. Histologically, MBC presents with increased microvessels beneath the parietal and visceral pleura, indicating generalized angiogenic activity. Breast cancer-associated pleural fluid (BAPF) was collected and cultured with HUVECs to recapitulate the molecular changes in subpleural endothelial cells. The clinical progression of triple-negative breast cancer (TNBC) is much more aggressive than that of hormone receptor-positive breast cancer (HPBC). However, BAPF from HPBC (BAPF-HP) and TNBC (BAPF-TN) homogeneously induced endothelial proliferation, migration, and angiogenesis. In addition, BAPF elicited negligible changes in the protein marker of endothelial-mesenchymal transition. Both BAPF-HP and BAPF-TN exclusively upregulated JNK signaling among all MAPKs in HUVECs. By contrast, the response to the JNK inhibitor was insignificant in Transwell and tube formation assays of the HUVECs cultured with BAPF-TN. The distinct contribution of p-JNK to endothelial angiogenesis was consequently thought to be induced by BAPF-HP and BAPF-TN. Due to increased angiogenic factors in HUVECs cultured with BAPF, vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor was applied accordingly. Responses to VEGFR2 blockade were observed in both BAPF-HP and BAPF-TN concerning endothelial migration and angiogenesis. In conclusion, the above results revealed microvessel formation in the pleura of MBC and the underlying activation of p-JNK/VEGFR2 signaling. Distinct responses to blocking p-JNK and VEGFR2 in HUVECs cultured with BAPF-HP or BAPF-TN could lay the groundwork for future investigations in treating MBC based on hormone receptor status.

Pharmaceutical Evaluation of Honokiol and Magnolol on Apoptosis and Migration Inhibition in Human Bladder Cancer Cells.

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.

Molecular Mechanisms of KDELC2 on Glioblastoma Tumorigenesis and Temozolomide Resistance.

The activation of the Notch pathway induces glioblastoma (GBM) development. Since KDEL (Lys-Asp-Glu-Leu) containing 2 (KDELC2) is involved in the Notch pathway, the detailed mechanism is still undetermined. The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases revealed that KDELC2 mRNA was associated with oncologic factors of GBM. U87, LN229, LNZ308, U118MG, and GBM8401 cells showed higher KDELC2 expression than normal brain tissues. The results of MTT, wound healing, and invasion assays proved that KDELC2 knockdown suppressed GBM-aggressive behaviors. The inhibitory properties of GBM stemness and angiogenesis under KDELC2 knockdown were evaluated by tumor spheroid and tube formation assays. Suppression of KDELC2 downregulated Notch factors' expressions, including KDELC1, pofut1, Notch receptors 1-3, and HES-1. Immunoblot assay showed that KDELC2 knockdown promoted tumor apoptosis by downregulating PI3k/mTOR/Akt, MAPK/ERK, and NF-kB pathways. The combination of KDELC2 knockdown and temozolomide (TMZ) treatment had an optimal therapeutic effect by suppressing MGMT expression. Results of an orthotopic xenograft animal model and human tissue confirmed that KDELC2 correlated with glioma proliferation, advanced grades, and poor prognosis. Therefore, KDELC2 might be a potential pharmacological target to inhibit tumorigenesis, epithelial-mesenchymal transition, angiogenesis, and chemo-resistance of GBM.

PSMB8 inhibition decreases tumor angiogenesis in glioblastoma through vascular endothelial growth factor A reduction.

Glioblastoma, also known as glioblastoma multiforme (GBM), is a fast-growing tumor and the most aggressive brain malignancy. Proteasome subunit beta type-8 (PSMB8) is one of the 17 essential subunits for the complete assembly of the 20S proteasome complex. The aim of the present study was to evaluate the role of PSMB8 expression in GBM progression and angiogenesis. PSMB8 expression in glioblastoma LN229 and U87MG was knocked down by siRNA or inducible shRNA both in vitro and in vivo. After PSMB8 reduction, cell survival, migration, invasion, angiogenesis, and the related signaling cascades were evaluated. An orthotopic mouse tumor model was also provided to examine the angiogenesis within tumors. A GEO profile analysis indicated that high expression of PSMB8 mRNA in GBM patients was correlated with a low survival rate. In immunohistochemistry analysis, PSMB8 expression was higher in high-grade than in low-grade brain tumors. The proliferation, migration, and angiogenesis of human GBM cells were decreased by PSMB8 knockdown in vitro. Furthermore, phosphorylated focal adhesion kinase (p-FAK), p-paxillin, MMP2, MMP9, and cathepsin B were significantly reduced in LN229 cells. Integrin ß1 and ß3 were reduced in HUVEC after incubation with LN229-conditioned medium. In an orthotopic mouse tumor model, inducible knockdown of PSMB8 reduced the expression of vascular endothelial growth factor (VEGF), VEGF receptor, and CD31 as well as the progression of human glioblastoma. In this article, we demonstrated the role of PSMB8 in glioblastoma progression, especially neovascularization in vitro and in vivo. These results may provide a target for the anti-angiogenic effect of PSMB8 in glioblastoma therapy in the future.

Effect of malignant-associated pleural effusion on endothelial viability, motility and angiogenesis in lung cancer.

Malignant pleural effusion (MPE) and paramalignant pleural effusion (PPE) remain debilitating complications in lung cancer patients with poor prognosis and limited treatment options. The role of vascular endothelial cells has not been explored in the pleural environment of lung cancer. By integrating MPE and PPE as malignant-associated pleural fluid (MAPF), the current study aimed to evaluate the effect of MAPF on cell proliferation, migration and angiogenesis of HUVEC. First, increased capillaries were identified in the subpleural layer of lung adenocarcinoma. Compatible with pathological observations, the ubiquitous elevation of HUVEC survival was identified in MAPF culture regardless of the underlying cancer type, the driver gene mutation, prior treatments and evidence of malignant cells in pleural fluid. Moreover, MAPF enhanced HUVEC motility with the formation of lamellipodia and filopodia and focal adhesion complex. Tube formation assay revealed angiogenic behavior with the observation of sheet-like structures. HUVEC cultured with MAPF resulted in a significant increase in MAPK phosphorylation. Accompanied with VEGFR2 upregulation in MAPF culture, there was increased expressions of p-STAT3, HIF-1α and Nf-kB. VEGF/VEGFR2 blockade regressed endothelial migration and angiogenesis but not cell proliferation. Our data indicate the angiogenic activities of MAPF on vascular endothelial cells that revealed increased pleural capillaries in lung cancer. Targeting the VEGF/VEGFR2 pathway might modulate the angiogenic propensity of MAPF in future clinical investigations.

Uremic toxin indoxyl sulfate suppresses myocardial Cx43 assembly and expression via JNK activation.

Heart rhythm disturbances have been widely recognized as major triggers of cardiovascular (CV) mortality in chronic kidney disease (CKD) patients. Connexin 43 (Cx43)-composed gap junctions are essential in cardiomyocyte synchronization and may be involved in the pathological response to uremic toxins. Indoxyl sulfate (IS) is one of the most dominant uremic toxins that contribute to CKD-related cardiovascular diseases. In primary cultures of rat neonatal cardiomyocytes, we demonstrated that IS treatment decreased spontaneous contraction without impairing viability. In addition, there was disruption of gap junction intercellular communication (GJIC) between cardiomyocytes after 30 min of IS stimulation. IS caused time- and dose-dependent Cx43 redistribution, and the patterns of Cx43 immunostaining returned to baseline while IS stimulation was removed. Furthermore, IS exposure downregulated Cx43 protein and mRNA levels. Elevated JNK1 and JNK2 phosphorylation was further identified after IS exposure in both rat cardiomyocytes and H9c2 cells. The above changes as well as GJIC and Cx43 suppression were reversed by pretreatment with a JNK inhibitor (SP600125). Inhibition of p-JNK attenuated IS-mediated downward trends in Cx43 transcription and translation. In cardiac muscle from nephrectomy-induced CKD mice, an alteration in Cx43 level was identified at intercalated discs. Our findings disclosed that JNK activation might participate in the remodeling of gap junction and Cx43 expression by uremic toxin-IS both in vitro and in vivo.

Indoxyl sulfate induces myotube atrophy by ROS-ERK and JNK-MAFbx cascades.

Accumulations of uremic toxins has been widely recognized as the major trigger of skeletal muscle loss in chronic kidney disease (CKD), which is defined as uremic sarcopenia. Current study was aimed to examine the effects of representative uremic toxin, indoxyl sulfate (IS), on C2C12 myotubes. The incubation of IS (from 0.1 mM to 1.2 mM) exerted the reduction in myotube diameter without cell survival impairment. Elevated oxidative stress and mitogen-activated protein kinase (MAPKs) phosphorylation were observed after IS stimulation for 1 and 24 h. After N-acetylcysteine (NAC) treatment as antioxidants, the recovery in IS-induced decrease myotube diameter and ERK phosphorylation was observed. This findings were implicit the transduction of p-ERK in IS-induced ROS toxicity. Moreover, the increase of LC3ß was found closely with IS treatment in C2C12 myotubes. The reverse effect of NAC on LC3ß expression revealed the ROS-responsibility in autophagy regulation of CKD myopathy. The evaluation of IS-treated proteasome system showed increased phospho-myosin light chain, along with the upregulation of muscle atrophy F-box (MAFbx) mRNA and protein. This alteration in MAFbx was also identified in nephrectomy-induced CKD model. Besides, the inhibition of p-JNK was capable to attenuate IS-induced upward change in MAFbx protein expression. These findings indicated that IS-mediated myotube atrophy may manipulate through ROS-ERK axis and JNK-MAFbx regulation in C2C12 cells.

Pyr3 Induces Apoptosis and Inhibits Migration in Human Glioblastoma Cells.

BACKGROUND/AIMS: Glioblastoma, also known as glioblastoma multiforme (GBM), is a fast-growing type of tumor that is the most aggressive brain malignancy in adults. According to GEO profile analysis, patients with high transient receptor potential canonical 3 (TRPC3) expression have poor survival rates. The aim of this study is to evaluate the effects of Ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (Pyr3), a selective TRPC3 channel blocker, on the proliferation and migration of human glioblastoma cells. METHODS: We first analyzed the TRPC3 mRNA expression in Gene Expression Omnibus (GEO) database. Then, TRPC3 protein expression was analyzed by Western blotting in three human GBM cell lines. The survival rate was measured by sulforhodamine B. JC1 staining was used to analyze the mitochondria membrane potential by flow cytometric analysis. Besides, the migration and invasion were evaluated by wound healing and Transwell assays. Annexin V and 7-aminoactinomycin D staining was used to monitor the apoptosis by flow cytometric analysis. The expression of apoptotic-related and migration-related proteins after Pyr3 treatment was detected by Western blotting. In addition, an orthotropic xenograft mouse model was used to assay the effect of Pyr3 in the in vivo study. RESULTS: Basis on the results of bioinformatics study, glioma patients with higher TRPC3 expression had a shorter survival time than those with lower TRPC3 expression. GBM cell proliferation was decreased by Pyr3 treatment. The migration and invasion abilities of glioma cells were also inhibited via focal adhesion kinase and myosin light chain dephosphorization after Pyr3 treatment. Moreover, Pyr3 induced caspase-dependent apoptosis and mitochondria membrane potential imbalance in the GBM cells. In a xenograft animal model, Pyr3 in combination with temozolomide (TMZ) inhibited GBM tumor growth. CONCLUSION: Pyr3 inhibited GBM tumor growth in vitro and in vivo. Pyr3-TMZ combination therapy could be used to treat glioblastoma in the future.

Interference with PSMB4 Expression Exerts an Anti-Tumor Effect by Decreasing the Invasion and Proliferation of Human Glioblastoma Cells.

BACKGROUND/AIMS: Glioblastoma (GBM) is a malignant brain tumor with a poor prognosis. Proteasome subunit beta type-4 (PSMB4) is an essential subunit that contributes to the assembly of the 20S proteasome complex. However, the role of PSMB4 in glioblastomas remains to be clarified. The aim of this study was to investigate the role of PSMB4 in GBM tumor progression. METHODS: We first analyzed the PSMB4 protein and mRNA expression in 80 clinical brain specimens and 77 datasets from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. Next, we inhibited the PSMB4 expression by siRNA in cellular and animal models to explore PSMB4's underlying mechanisms. The cell survival after siPSMB4 transfection was assayed by MTT assay. Annexin V and propidium iodide staining was used to monitor the apoptosis by flow cytometric analysis. Moreover, the migration and invasion were evaluated by wound healing and Transwell assays. The expression of migration-related and invasion-related proteins after PSMB4 inhibition was detected by Western blotting. In addition, an orthotropic xenograft mouse model was used to assay the effect of PSMB4 knockdown in the in vivo study. RESULTS: Basis on the results of bioinformatics study, glioma patients with higher PSMB4 expression had a shorter survival time than those with lower PSMB4 expression. The staining of clinical brain tissues showed elevated PSMB4 expression in GBM tissues compared with normal brain tissues. The PSMB4 inhibition decreased proliferation, migration and invasion abilities in human GBM cells. Downregulated PSMB4 resulted in cell cycle arrest and apoptosis in vitro. In an orthotropic xenograft mouse model, the glioma tumors progression was reduced when PSMB4 was down-regulated. The decreased PSMB4 enhanced the anti-tumor effect of temozolomide (TMZ) on tumor growth. In addition, the absence of PSMB4 decreased the expression of phosphorylated focal adhesion kinase and matrix metallopeptidase 9 in vivo. CONCLUSION: PSMB4 inhibition in combination with TMZ may exert an anti-tumor effect by decreasing cell proliferation and invasion as well as by promoting apoptosis in human glioblastoma cells. This research may improve the therapeutic efficacy of glioblastoma treatment.