Human Bladder Cancer Cell Research Articles

DNA Methylation Aberrations in Dimethylarsinic Acid-Induced Bladder Carcinogenesis.

Arsenic is a known human urinary bladder carcinogen. While arsenic is known to cause aberrant DNA methylation, the mechanism of arsenic-triggered bladder carcinogenesis is not fully understood. The goal of this study was to identify aberrant DNA methylation in rat bladder urothelial carcinoma (UC) induced by dimethylarsinic acid (DMAV), a major organic metabolite of arsenic. We performed genome-wide DNA methylation and microarray gene expression analyses of DMAV-induced rat UCs and the urothelium of rats treated for 4 weeks with DMAV. We identified 40 genes that were both hypermethylated and downregulated in DMAV-induced rat UCs. Notably, four genes (CPXM1, OPCML, TBX20, and KCND3) also showed reduced expression in the bladder urothelium after 4 weeks of exposure to DMAV. We also found that CPXM1 is aberrantly methylated and downregulated in human bladder cancers and human bladder cancer cells. Genes with aberrant DNA methylation and downregulated expression in DMAV-exposed bladder urothelium and in DMAV-induced UCs in rats, suggest that these alterations occurred in the early stages of arsenic-induced bladder carcinogenesis. Further study to evaluate the functions of these genes will advance our understanding of the role of aberrant DNA methylation in arsenic bladder carcinogenesis, and will also facilitate the identification of new therapeutic targets for arsenic-related bladder cancers.

P190 - Studied on the effect of metformin combined with epirubicin on apoptosis of human bladder cancer cell line through PI3K/AKT/mTOR signaling pathway

P190 - Studied on the effect of metformin combined with epirubicin on apoptosis of human bladder cancer cell line through PI3K/AKT/mTOR signaling pathway

Cell Line-Based Human Bladder Organoids with Bladder-like Self-Organization-A New Standardized Approach in Bladder Cancer Research.

Three-dimensional tumor models have gained significant importance in bladder cancer (BCa) research. Organoids consisting of different cell types better mimic solid tumors in terms of 3D architecture, proliferation, cell-cell interaction and drug responses. We developed four organoids from human BCa cell lines with fibroblasts and smooth muscle cells of the bladder, aiming to find models for BCa research. The organoids were characterized in terms of cytokeratins, vimentin, α-actin and KI67 by immunoreactivity. Further, we studied ligand-dependent activation of the Wnt/β-catenin pathway and investigated the responses to anti-tumor therapies. The organoids mimicked the structure of an inverse bladder wall, with outside urothelial cells and a core of supportive cells. The cytokeratin staining patterns and proliferation rate were in conjunction with the origins of the BCa cells. RT-112 even showed stratification of the epithelium. Treatment with Wnt10B led to increased β-catenin (active) levels in high-grade organoids, but not in low-grade BCa cells. Doxorubicin treatment resulted in clearly reduced viability (10-30% vs. untreated). In contrast, the effectivity of radiotherapy depended on the proliferation status of BCa cells. In conclusion, cell-line-based organoids can form bladder-like structures and reproduce in vivo features such as urothelial differentiation and stratification. Thus, they can be useful tools for functional studies in BCa and anti-cancer drug development.

Sakuranin represses the malignant biological behaviors of human bladder cancer cells by triggering autophagy via activating the p53/mTOR pathway

ObjectiveSakura extract is a natural flavonoid compound that may have potential anti-tumor effects. The paper focuses on investigating Sakuranin mechanism on bladder cancer (BC) cells.MethodsBC cells (T24) were treated with different concentrations of Sakuranin, with 48-h IC50 determined. T24 cells were treated with Sakuranin at IC50, followed by assessment of cell proliferative/apoptotic/migrative/invasive activities by CCK-8, EdU and plate clone formation assays/flow cytometry/Transwell/scratch test. MMP-2 (migration and invasion-related protein) protein level was assessed by Western blot. Cell autophagy was evaluated by measuring the protein levels of autophagy markers (LC3-I/LC3-II/p62) through Western blot. The autophagy inhibitor 3-MA was used to validate the role of autophagy in the regulatory mechanism of Sakuranin in T24 cell behaviors. Furthermore, the activation of the p53/mTOR pathway in cells was detected and a combination of Sakuranin and p53 inhibitor Pifithrin-µ was adopted to explore the involvement of this pathway.ResultsSakuranin decreased T24 cell proliferation/EdU positive cell percentage/colony formation number and area/migration/invasion/scratch healing/MMP-2 protein level, and accelerated apoptosis. Sakuranin elevated the LC3-II/I ratio and lowered p62 level in T24 cells. 3-MA partially averted Sakuranin-mediated repression on cell malignant behaviors. Sakuranin upregulated p-p53 and p53 levels, and decreased the p-mTOR/mTOR ratio in T24 cells. The effects of Sakuranin on cell biological behaviors were partly annulled by Pifithrin-µ treatment.ConclusionSakuranin suppressed T24 cell proliferation/migration/invasion, and enhanced apoptosis by potentiating autophagy through activating the p53/mTOR pathway. This study provided a theoretical basis for Sakuranin as a potential drug for clinical treatment of BC.

The p38-MITOGEN-ACTIVATED PROTEIN KINASE Signaling Pathway Is Involved in Leonurus artemisia Extract-Induced Inhibition of the Proliferation of Human Bladder Cancer BFTC-905 Cells via G1/G0 Arrest and Causes Apoptosis In Vitro.

Bladder cancer is a urothelial malignancy. Bladder cancer starts in the urothelial cells lining the inside of the bladder. The 5-year recurrence rate for bladder cancer ranges from 31% to 78%, and the progression rate is approximately 45%. To treat bladder cancer, intravesical drug therapy is often used. Leonurus artemisia extract (LaE) was obtained from medicinal samples of Chinese motherwort Scientific Chinese Medicine; L. artemisia has various biological effects. This study investigated the impact of LaE on human bladder cancer cells (the BFTC-905 cell line) and the molecular mechanism underlying apoptosis resulting from the activation of cell signal transduction pathways in bladder cancer cells. A cell counting kit-8 (CCK-8) assay was used to determine the effect of LaE on cell growth. The effect of LaE on migration ability was observed using a wound healing assay. The effects of LaE on the cell cycle, reactive oxygen species production, and apoptosis were investigated. Western blot analysis detected apoptosis-related and mitogen-activated protein kinase signaling pathway-related protein concentrations. At non-toxic concentrations, LaE inhibited the proliferation of BFTC-905 cells in a concentration-dependent manner, and the half-maximal inhibitory concentration (IC50) was 24.08172 µg/µL. LaE impaired the migration ability of BFTC-905 cells. LaE arrested the cell cycle in the G1 and G0 phases, increased reactive oxygen species production, and induced apoptosis. LaE increased Bax and p-ERK concentrations and decreased Bcl-2, cleaved caspase-3, and p-p38 concentrations. No differences in PARP, C-PARP, vimentin, e-cadherin, p-JNK, or TNF-alpha concentrations were observed. These results suggest that LaE inhibits the proliferation of human bladder cancer cells. Moreover, the mitogen-activated protein kinase signaling pathway is involved in the inhibition of the proliferation of BFTC-905 cells.

Cardamonin suppresses glycolysis and induces oxidative stress by inhibiting PI3K/AKT/mTOR pathway in bladder cancer cells

Purpose: To evaluate the effect and underlying mechanisms of action of cardamine on the progression of bladder cancer (BC).Methods: Human bladder epithelium immortalized cell line (SV-HUC-1) and human bladder cancer (BC) cell lines (T24 and UM-UC-3) were used in this investigation. They were treated with cardamine at concentrations of 0, 15, 30, 60 or 120 μmol/L. Cell viability was determined using cell counting kit 8(CCK-8) assay while 5-ethynyl-2'-deoxyuridine (Edu) assay was used to assess cell proliferation. Cell apoptosis as well as reactive oxygen species (ROS) accumulation were determined by flow cytometry whereas glucose uptake, adenosine triphosphate (ATP) level and lactate production were determined using their respective assay kits. Furthermore, the expression levels of nuclear factor level (erythroidderived 2)-like 2 (Nrf2), NAD(P)H, quinone oxidoreductase 1 (NQO1), protein kinase B (AKT), phosphorylated-AKT (p-AKT), phosphatidylinositol 3-kinase (PI3K), p-PI3K, mechanistic target of rapamycin kinase (mTOR) and p-mTOR were evaluated by western blot analysis.Results: Cardamine significantly reduced cell viability and inhibited cell proliferation in BC cells in a dose-dependent manner, but did not affect human normal cells. In addition, treatment with the compound induced apoptosis in BC cells; the higher the concentration, the higher the apoptosis level. Besides, cardamine administration suppressed aerobic glycolysis, and decreased the nuclear factor level (Nrf2) level, thereby increasing ROS production in a concentration-dependent manner.Furthermore, it blocked the activation of PI3K/AKT/mTOR signal cascade.Conclusion: Cardamine inhibits glycolysis and PI3K/AKT/mTOR pathway, and also promotes apoptosis as well as oxidative stress in BC cells. Thus, the compound is a potential therapeutic reagent for BC.

Isolation of adipose derived mesenchymal stem cells by using anti-CD105 VHH-magnetic beads

BackgroundHuman adipose tissue-derived stem cells (ASCs) are widely used in regenerative medicine and tissue engineering. Magnetic-activated cell sorting (MACS) with monoclonal antibodies (mAbs) that bind surface markers of stem cells such as CD105, CD73, and CD90 is currently applied for the enrichment and isolation of ASCs. Alternatives to mAbs are the variable domains of heavy chain antibodies (VHHs), which are naturally produced in camelids. We report the application of an anti-CD105 VHH conjugated to magnetic beads in the isolation of ASCs in vitro. ResultsTwo identical anti-CD105 VHHs (EngVHH17) were screened by phage display from a VHH cDNA library constructed from the PBMCs of an alpaca immunized with a lysate of human bladder cancer cell line (T24 cells). EngVHH17 was cloned in pET22b(+), expressed in Escherichia coli BL21 and purified in Ni-NTA chromatography resulting in a ∼15 kDa VHH antibody. EngVHH17 binds CD105 with high affinity (Kd = 3.9 × 10−10 M). The specific binding of EngVHH17 to CD105 was visualized by fluorescence inmunolabeling of phorbol-12-myristate-13-acetate (PMA)-differentiated THP1 cells using FITC labeled anti-6 × His Tag IgG1 mouse mAb. EngVHH17-magnetic beads selectively sorted human ASCs from osteoblasts in a mixed culture in vitro. The selective recovery of ASC cells using different ASC/Osteoblast ratios was higher than 85%. ConclusionsEngVHH17 coupled to magnetic beads binds CD105 expressed on the cell surface of ASCs and isolates them from osteoblasts in mixed cultures in vitro by application of an external magnetic field. EngVHH17 can be further evaluated for the isolation of MSCs by MACS.How to cite: Gushiken E, Quintana M, Perez L, et al. Isolation of adipose derived mesenchymal stem cells by using anti-CD105 VHH-magnetic beads. Electron J Biotechnol 2023;66. https://doi.org/10.1016/j.ejbt.2023.06.004.

Anticancer effects of cell-free culture supernatant of Escherichia coli in bladder cancer cell line: New insight into the regulation of inflammation

Bladder cancer (BC) is the 10th most common malignancy in worldwide, with substantial mortality and morbidity if not treated effectively. According to various research, inflammatory circumstances majorly impact the microenvironment of bladder cancer, and the chronic presence of cytokines and chemokines promotes tumor progression. In this investigation, we explored the impact of cell-free culture supernatant ofEscherichia colistrain 536 on inflammatory cytokines and chemokines in bladder cancer model microarray data (GSE162251). Then we examined in silico outcomes on human bladder cancer cell line 5637 to verify and extrapolate findings. This investigation revealed for the first time that this compound has potent suppressor effects on interleukin 1 beta (IL-1β), C–C motif chemokine ligand 2 (CCL2), and C-X3-C motif chemokine ligand 1 (CX3CL1) gene expression as well as increased NAD(P)H quinone dehydrogenase 1 (NQO1), as an anti-oxidant agent, gene expression in 4, 8, and 24 h. Moreover, we confirmed that c-MYC, a member of the MYC proto-oncogene family, gene expression reduced in 5637 cells in 4 h and then followed up its expression in 8 and 24 h. In addition, our investigation demonstrated that the supernatant raised the BCL2-Associated X Protein/B-cell lymphoma 2 (BAX/BCL2) ratio, and subsequent flow cytometry analysis demonstrated that the supernatant induction apoptosis and necrosis. In conclusion, our findings demonstrate that this compound is a potential candidate for the suppression of bladder cancer progression.

The Role of Specificity Protein 1 (SP1) in Bladder Cancer Progression through PTEN-Mediated AKT/mTOR Pathway

Introduction: The aim of the study was to investigate the potential mechanism of specificity protein 1 (SP1) in bladder cancer progression through the PTEN-mediated AKT/mTOR pathway. Methods: Human bladder cancer cell lines (HT-1197, HT-1376, and T24) and normal ureteral epithelial cell line SV-HUC-1 were used. SP1 expression was detected via quantitative real-time PCR and Western blotting. Cell viability, migration, invasion, and apoptosis were assessed using CCK-8, transwell, and flow cytometry assays, respectively. The involvement of the PTEN-mediated AKT/mTOR pathway was evaluated by Western blot. A mouse xenograft model was built, and immunohistochemical staining was applied to visualize SP1 and Ki67 expression in tumor tissues. Results: SP1 was overexpressed in bladder cancer cells. SP1 knockdown inhibited viability, migration, and invasion and promoted apoptosis in bladder cancer cells. PTEN intervention increased cell viability, migration, and invasion and decreased apoptosis, which was reversed by SP1 knockdown. The activation of the AKT/mTOR pathway resulting from PTEN knockdown was attenuated by SP1 knockdown. In vivo results showed that SP1 knockdown suppressed tumor growth, increased PTEN expression, and decreased AKT/mTOR pathway-related protein levels. Conclusion: SP1 promotes bladder cancer progression by inhibiting the PTEN-mediated AKT/mTOR pathway. Targeting SP1 may be a potential therapeutic strategy for treating bladder cancer.

Synthetic lethality in human bladder cancer cells by curcumin via concurrent Aurora A inhibition and autophagy induction

Combination therapies to induce mixed-type cell death and synthetic lethality have the potential to overcome drug resistance in cancer. In this study, we demonstrated that the curcumin-enhanced cytotoxicity of cisplatin/carboplatin in combination with gemcitabine was associated with Aurora A suppression-mediated G2/M arrest, and thus apoptosis, as well as MEK/ERK-mediated autophagy in human bladder cancer cells. Animal study data confirmed that curcumin combined with cisplatin/gemcitabine reduced tumorigenesis of xenograft in mice and this phenomenon was associated with elevated expressions of p-ERK and reduced p-Aurora A in tumors. Gene analyses using data repositories further revealed that reduced Aurora A expression alone did not significantly elevate the sensitivity of human bladder carcinoma cells to these anticancer drugs. Unlike other major cancer types, human bladder urothelial carcinoma tissue coexpressed higher AURKA and lower MAP1LC3B than normal tissue, and reduced Aurora A and induction of autophagy have been clinically associated with a better prognosis in patients with early but not advanced stage bladder cancer. Therefore, our results suggest that treatment strategies can utilize the synthetic lethal pair to concurrently suppress oncogenic Aurora A and induce autophagy by coadministrating curcumin with anticancer drugs for early-stage bladder cancer with high expression of Aurora A.

Sertindole, an Antipsychotic Drug, Curbs the STAT3/BCL-xL Axis to Elicit Human Bladder Cancer Cell Apoptosis In Vitro.

Bladder cancer is the leading urinary tract malignancy. Epidemiological evidence has linked lower cancer incidence in schizophrenia patients to long-term medication, highlighting the anticancer potential of antipsychotics. Sertindole is an atypical antipsychotic agent with reported anticancer action on breast and gastric cancers. Yet, sertindole's effect on bladder cancer remains unaddressed. We herein present the first evidence of sertindole's antiproliferative effect and mechanisms of action on human bladder cancer cells. Sertindole was cytotoxic against bladder cancer cells while less cytotoxic to normal urothelial cells. Apoptosis was a primary cause of sertindole's cytotoxicity, as the pan-caspase inhibitor z-VAD-fmk rescued cells from sertindole-induced killing. Mechanistically, sertindole inhibited the activation of signal transducer and activator of transcription 3 (STAT3), an oncogenic driver of bladder cancer, as sertindole lowered the levels of tyrosine 705-phosphorylated STAT3 along with that of STAT3's target gene BCL-xL. Notably, ectopic expression of the dominant-active STAT3 mutant impaired sertindole-induced apoptosis in addition to restoring BCL-xL expression. Moreover, bladder cancer cells overexpressing BCL-xL were refractory to sertindole's proapoptotic action, arguing that sertindole represses STAT3 to downregulate BCL-xL, culminating in the induction of apoptosis. Overall, the current study indicated sertindole exerts bladder cancer cytotoxicity by provoking apoptosis through targeted inhibition of the antiapoptotic STAT3/BCL-xL signaling axis. These findings implicate the potential to repurpose sertindole as a therapeutic strategy for bladder cancer.

Microtubule Dynamics Deregulation Induces Apoptosis in Human Urothelial Bladder Cancer Cells via a p53-Independent Pathway.

Bladder cancer (BLCA) is the sixth most common type of cancer and has a dismal prognosis if diagnosed late. To identify treatment options for BLCA, we systematically evaluated data from the Broad Institute DepMap project. We found that urothelial BLCA cell lines are among the most sensitive to microtubule assembly inhibition by paclitaxel treatment. Strikingly, we revealed that the top dependencies in BLCA cell lines include genes encoding proteins involved in microtubule assembly. This highlights the importance of microtubule network dynamics as a major vulnerability in human BLCA. In cancers such as ovarian and breast, where paclitaxel is the gold standard of care, resistance to paclitaxel treatment has been linked to p53-inactivating mutations. To study the response of BLCA to microtubule assembly inhibition and its mechanistic link with the mutational status of the p53 protein, we treated a collection of BLCA cell lines with a dose range of paclitaxel and performed a detailed characterization of the response. We discovered that BLCA cell lines are significantly sensitive to low concentrations of paclitaxel, independently of their p53 status. Paclitaxel induced a G2/M cell cycle arrest and growth inhibition, followed by robust activation of apoptosis. Most importantly, we revealed that paclitaxel triggered a robust DNA-damage response and apoptosis program without activating the p53 pathway. Integration of transcriptomics, epigenetic, and dependency data demonstrated that the response of BLCA to paclitaxel is independent of p53 mutational signatures but strongly depends on the expression of DNA repair genes. Our work highlights urothelial BLCA as an exceptional candidate for paclitaxel treatment. It paves the way for the rational use of a combination of paclitaxel and DNA repair inhibitors as an effective, novel therapeutic strategy.

FLRT2 suppresses bladder cancer progression through inducing ferroptosis.

Bladder cancer is a common tumour worldwide and exhibits a poor prognosis. Fibronectin leucine rich transmembrane protein 2 (FLRT2) is associated with the regulation of multiple tumours; however, its function in human bladder cancer remain unclear. Herein, we found that FLRT2 level was reduced in human bladder cancer and that higher FLRT2 level predicted lower survival rate. FLRT2 overexpression inhibited, while FLRT2 silence facilitated tumour cell growth, migration and invasion. Mechanistic studies revealed that FLRT2 elevated acyl-CoA synthetase long-chain family member 4 (ACSL4) expression, increased lipid peroxidation and subsequently facilitated ferroptosis of human bladder cancer cells. In summary, we demonstrate that FLRT2 elevates ACSL4 expression to facilitate lipid peroxidation and subsequently triggers ferroptosis, thereby inhibiting the malignant phenotype of human bladder cancer cells. Overall, we identify FLRT2 as a tumour suppressor gene.

Network pharmacology and in vitro experiments reveal that Noscapine induces ROS-mediated apoptosis and cell cycle arrest via PI3K/Akt/FoxO3a signaling pathway in human bladder cancer cells.

Noscapine (NA) has been demonstrated to have antitussive and antitumoral activities. Nonetheless, the potential mechanism of action on Bladder Cancer (BLCA) is yet to be completely grasped. The targets of NA action and bladder cancer disease targets were found by the database. Construct the PPI network. Subsequently, conduct pathway enrichment of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) on core targets. A "drug-disease-target-pathway" network map was made. Cytotoxicity was examined via CCK-8 and colony formation assays. Both a scratch test and a transwell assay confirmed that NA was capable of suppressing the invasiveness and migratory potential of bladder cancer cells. Hoechst 33342 staining was used to visualize NA-induced apoptosis in bladder cancer cells. Flow cytometry was employed to investigate the induction of apoptosis, the distribution of the cell cycle, the production of Reactive Oxygen Species (ROS), and the Mitochondrial Membrane Potential (MMP). The Western blot was applied to show the expression of proteins that are implicated in the pathway, cell cycle, apoptotic process, and proliferation. 198 Noscapine-BLCA-related targets were obtained. GO functional enrichment analysis yielded 428 entries (P < 0.05 and FDR < 0.05). KEGG pathway enrichment analysis identified 138 representative signaling pathways (P < 0.01 and FDR < 0.01). NA concentration-dependently suppressed cell growth and colony formation, along with the invasiveness and migratory potential of bladder cancer cells, by promoting apoptosis, a cell cycle arrest in the G2/M phase, generation of ROS, and depolarization of MMPs. In addition, Western blotting illustrated that NA down-regulated the protein levels associated with pathway, anti-apoptotic proteins, proliferation-related proteins, and cell cycle promoters but up-regulated pro-apoptotic proteins, cell cycle modulators, and Endoplasmic Reticulum (ER) stress expression. Pretreatment with Acetylcysteine N-acetyl-L-cysteine (NAC) and YS-49 counteracted the influence of NA on ROS induction and apoptosis. Noscapine induces ROS-mediated apoptosis and cell cycle arrest via PI3K/Akt/FoxO3a signaling pathway in human BLCA cells.

Exploring the anticancer potential of thiadiazole derivatives of substituted thiosemicarbazones formed via copper‐mediated cyclization

A couple of N‐(4)‐morpholine/pyrrolidine‐substituted thiosemicarbazones (TSCs) of fluorene‐2‐carboxaldehyde (FM and FP), and their corresponding thiadiazoles (TDZs) (CFM and CFP), were synthesized and characterized (elemental analysis, ultraviolet–visible [UV–Visible], Fourier transform infrared [FT‐IR], nuclear magnetic resonance [NMR; 1H &amp; 13C], high‐resolution mass spectrometry [HRMS], and single‐crystal X‐ray diffraction [SCXRD]) for the evaluation of their anticancer potential. The TDZs were obtained unexpectedly and are possibly formed via single‐step metal (copper)‐mediated oxidative cyclizations of the TSCs. The synthesized compounds are fairly stable in phosphate buffer at the biological pH of 7.4. The density functional theory [DFT] studies were performed to predict the optimized structures and physicochemical properties of these compounds. The compounds were further subjected to computational and experimental biomolecular investigations in order to evaluate their anticancer activity in detail. CFM had the most potent activity against human breast adenocarcinoma (MCF‐7) and human urinary bladder (T24) cancer cells, with IC50 values of 12.00 and 24.80 μM, respectively. In contrast, CFM had negligible cytotoxicity (IC50 = 98.70 μM) against kidney epithelial cells extracted from an African green monkey (Vero) normal cells. This outcome was preferable to that of the widely used medicine Cisplatin. Molecular docking studies were performed with the breast cancer protein “cytochrome P450 1A1” (CYP1A1) and bovine serum albumin (BSA) to predict how effectively the compounds bind to the receptor. The ADMET findings suggest that these compounds have considerable drug‐likeness and oral bioavailability. These insights may open the door for additional medical research into the bioactivities of TSCs and TDZs produced from bioactive carbonyl compounds.

Brazilin inhibits bladder cancer by promoting cell necroptosis.

Brazilin possesses anticancer effects, but the mechanisms are poorly understood. This study investigated the mechanisms of brazilin-induced cell death in the T24 human bladder cancer cell line. Low serum cell culture and the lactate dehydrogenase assay were used to confirm the antitumor effect of brazilin. Annexin V and propidium iodide double staining, transmission electron microscopy, fluo-3-AM assay for Ca2+ mobilization and caspase activity assay were performed to identify the type of cell death after brazilin treatment. Mitochondria membrane potentials were measured using JC-1. Quantitative real-time polymerase chain reaction and western blot analyses were performed to verify the expression of the necroptosis-related genes and proteins receptor interacting protein 1 (RIP1), RIP3 and mixed lineage kinase domain-like (MLKL). The results showed that brazilin induced necrosis in T24 cells and upregulated the mRNA and protein levels of RIP1, RIP3 and MLKL and Ca2+ influx. The necroptosis-mediated cell death was rescued by the necroptosis inhibitor necrostatin-1 (Nec-1), but not by the apoptosis inhibitor z-VAD-fmk. Brazilin repressed caspase 8 expression and decreased the mitochondrial membrane potentials; both effects were partially reversed by Nec-1. Brazilin induced physiological and morphological changes in T24 cells and RIP1/RIP3/MLKL-mediated necroptosis might be involved. In conclusion, the results confirm the involvement of necroptosis in brazilin-induced cell death and suggest that brazilin could be explored as an anticancer agent against bladder cancer.

Elemene inhibits the growth and promotes apoptosis of bladder cancer cells through PTEN-Akt signaling pathway

Purpose: To investigate the influence of elemene on growth and apoptotic changes in bladder cancer cells, and the involvement of the phosphatase and tensin homologous protein (PTEN)-serine/threonine kinase (Akt) signal pathway in the process.&#x0D; Methods: Human bladder cell line SV-HUC-1 and human bladder cancer cell line T24 were randomly assigned to blank control group and elemene groups. Protein expression levels of apoptosis-related factors (Bcl-2, Bax, and caspase-3] and PTEN, Akt, and p-Akt in both cells were determined using western blot assay.&#x0D; Results: In T24 cells, the protein level of Bcl-2 was significantly higher in elemene group than in blank control, while the protein expression levels of Bax and caspase-3 in elemene group were significantly lower than the corresponding levels in blank control group (p &lt; 0.05). The protein expression of PTEN level in T24 cells was significantly higher in elemene group than in blank control group, while protein expression level of p-Akt was significantly lower in elemene group than in blank control (p &lt; 0.05). The Akt protein expression was comparable in elemene and blank control groups.&#x0D; Conclusion: Elemene inhibits the growth and enhances apoptosis of bladder cancer cells through a mechanism involving up-regulation of the expression of PTEN and suppression of the expression of pAkt.

Methylseleninic acid induces apoptosis of human bladder cancer cells through the ROS-mediated mitochondrial pathway.

As the most common selenium derivative, methylseleninic acid (MSA) has attracted wide attention. Its apoptotic induction ability and the possible molecular mechanism in human bladder cancer (BC) J82 and T24 cells were investigated in the present study. We found that the survival of J82 and T24 cells were inhibited in a dose-dependent manner after MSA treatment. Propidium iodide (PI) staining and Annexin V-fluorescein isothiocyanate/PI double staining clarified that MSA stocked cells at G2 /M phase and caused apoptosis in J82 and T24 cells. Further, typical morphological features of apoptotic cells were also observed. Accumulation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential were also detected by dichlorodihydrofluorescein diacetate and Rhodamin123 staining. Meanwhile, pretreatment with N-acetylcysteine, an ROS scavenging agent, found that the apoptosis of BC cells induced by MSA was related to the production of ROS. Western blot analysis results showed that MSA interrupted Bax/Bcl-2 balance, stimulated cytochrome c release into the cytoplasm, activated caspase-9 and caspase-3, and finally induced the apoptosis of the BC cells. These findings demonstrated that MSA was able to induce apoptosis in J82 and T24 cells through ROS-mediated mitochondrial apoptosis.

Echinatin inhibits tumor growth and synergizes with chemotherapeutic agents against human bladder cancer cells by activating p38 and suppressing Wnt/β-catenin pathways

Bladder cancer (BC) is one of the most common malignant tumors in the urinary system. Due to the poor prognosis and high mortality rate of the disease, it is urgent to develop new drugs with high efficacy and low toxicity to treat BC. Echinatin (Ecn) is a bioactive natural flavonoid oflicorice that has attracted special attention for its promising anti-tumor potential. Herein, we explored the inhibitory effects of Echinatin on BC cells and probed the possible molecular mechanism. We found that Ecnin vitro inhibited the proliferation, migration, and invasion, arrested the cell cycle at the G2/M phase, and promoted apoptosis in BC cells. Besides, Ecn had no notable cytotoxicity towards human normal cells. We subsequently confirmed that Ecn restrained xenograft tumor growth and metastasis of BC cells in vivo. Mechanistically, Ecn activated the p38 signaling pathway but inactivated the Wnt/β-catenin signaling pathway, while over-expression of β-catenin and the p38 inhibitor both attenuated the inhibitory effects of Ecn on BC cells. Remarkably, Ecn combined with cisplatin (DDP) or gemcitabine (Gem) had synergistic inhibitory effects on BC cells. In summary, our results validate that Ecn inhibits the tumor growth of human BC cells via p38 and Wnt/β-catenin signaling pathways. More meaningfully, our results suggest a potential strategy to enhance DDP- or Gem-induced inhibitory effects on BC cells by combining with Ecn.

New cyclic C-geranylflavanones isolated from Paulownia fortunei fruits with their anti-proliferative effects on three cancer cell lines

Seven new C-geranylated flavanones, fortunones F - L (1–7), were isolated from the fresh mature fruits of Paulownia fortunei (Seem.) Hemsl. Their structures were determined by extensive spectroscopic data interpretation (UV, IR, HRMS, NMR, and CD). These new isolated compounds were all with a cyclic side chain modified from the geranyl group. Among them, compounds 1–3 all possessed a dicyclic geranyl modification, which was described firstly for Paulownia C-geranylated flavonoids. All the isolated compounds were subjected to the cytotoxic assay on human lung cancer cell A549, mouse prostate cancer cell RM1 and human bladder cancer cell T24, respectively. Results indicated A549 cell line was more sensitive to C-geranylated flavanones than the other two cancer cell lines and compounds 1, 7 and 8 exhibited potential anti-tumor effects (IC50 ˂ 10 μM). Further research revealed the effective C-geranylated flavanones could exert their anti-proliferative activity on A549 cells by inducing apoptosis and blocking cells in G1 phase.