RT4 Cells Research Articles

Osmotic regulation of UT-B urea transporters in the RT4 human urothelial cell line.

Facilitative UT‐B urea transporters play important physiological roles in numerous tissues, including the urino‐genital tract. Previous studies have shown that urothelial UT‐B transporters are crucial to bladder function in a variety of mammalian species. Using the RT4 bladder urothelial cell line, this study investigated the potential osmotic regulation of human UT‐B transporters. Initial end‐point PCR experiments confirmed expression of both UT‐B1 and UT‐B2 transcripts in RT4 cells. Western blotting analysis revealed glycosylated UT‐B protein to be highly abundant and immunolocalization experiments showed it was predominantly located on the plasma membrane. Further PCR experiments suggested that a 48 hr, NaCl‐induced raise in external osmolality increased expression of UT‐B transcripts. Importantly, these NaCl‐induced changes also significantly increased UT‐B protein abundance (p < .01, n = 7, ANOVA), whereas mannitol‐induced changes in external osmolality had no effect (NS, n = 4, ANOVA). Finally, similar increases in both UT‐B RNA expression and protein abundance were observed with urea‐induced changes to external osmolality (p < .05, n = 4, ANOVA). In conclusion, these findings strongly suggest that increases in external osmolality, via either NaCl or urea, can regulate human urothelial UT‐B transporters.

HOXB2 is a Putative Tumour Promotor in Human Bladder Cancer.

The present study aimed to investigate the role of Homebox B2 (HOXB2) in bladder cancer (BC). The Cancer Genome Atlas (TCGA) dataset was used to analyse HOXB2 expression in BC. The influence of HOXB2 on the cellular functions of BC cells was determined in both HOXB2 knockdown and HOXB2 overexpressed BC cell lines using in vitro assays. HOXB2 mRNA was significantly upregulated in luminal infiltrated and luminal papillary subtypes of BC. Drug Metabolism Cytochrome P450 was significantly enriched in BCs expressing high levels of HOXB2. Knockdown of HOXB2 from EJ138 cells reduced growth, adhesion and invasion. In contrast, overexpression of HOXB2 in RT112 cells induced growth and adhesion of bladder cancer cells. Increased HOXB2 expression in papillary BC can promote cell growth and adhesion of BC cells. Drug Metabolism Cytochrome P450 pathway was enriched in BCs overexpressing HOXB2.

Cotinine, a major nicotine metabolite, induces cell proliferation on urothelium in vitro and in vivo

Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. In a previous study, nicotine enhanced rat urinary bladder carcinogenesis using a rat urinary bladder two-stage carcinogenesis model. In the present study, nicotine metabolites (cotinine, trans-3’-hydroxy cotinine and N’-nitrosonornicotine) were evaluated in a cell proliferation assay using urinary bladder urothelial cell lines. Cotinine (0.1 to 1 mM) induced the highest cell proliferation compared to the others, including nicotine, in three bladder cancer cell lines (RT4, T24 and UMUC3 cells). By Western blot, cotinine induced phosphorylation of Stat3 and expression of cyclin D1 in UMUC3 cells. The cell proliferation induced by cotinine was blocked by inhibitors of nicotinic receptors (10 nM SR16584 or 10 μM methyllycaconitine citrate) and Stat3 (100 nM stattic). In an in vivo study, cotinine (13, 40 and 120 ppm) in drinking water also induced cell proliferation and simple hyperplasia in urinary bladder and renal pelvis urothelium of rats, but to a lesser degree compared to nicotine (40 ppm). Cytotoxicity detected by scanning electron microscopy and apoptosis in the bladder urothelium were induced by nicotine but not cotinine. These data suggest that cotinine is able to induce urothelial cell proliferation both in vitro and in vivo, and high urinary concentrations may enhance urothelial carcinogenesis.

Novel insights into the mechanism of cyclophosphamide-induced bladder toxicity: chloroacetaldehyde's contribution to urothelial dysfunction in vitro.

The clinical use of cyclophosphamide and ifosfamide is limited by a resultant bladder toxicity which has been attributed to the metabolite acrolein. Another metabolite chloroacetaldehyde (CAA) associated with nephrotoxicity, has not been investigated for toxicity in the bladder and this study investigates the effects of acrolein and CAA on human urothelial cells in vitro. Human urothelial cells (RT4 and T24) were treated with acrolein or CAA and changes in cell viability, reactive oxygen species, caspase-3 activity and release of urothelial mediators ATP, acetylcholine, PGE2 were measured. The protective effects of N-acetyl cysteine (NAC) were also assessed. Both metabolites were toxic to human urothelial cells, however, CAA significantly decreased cell viability at a ten-fold lower concentration (10µM) than acrolein (100µM). This was associated with increased ROS production and caspase-3 activity. NAC protected cells from these changes. In RT4 cells 100µM acrolein caused a significant increase in basal and stretch-induced ATP, Ach and PGE2 release. In T24 cells chloroacetaldehyde (10µM) increased basal and stimulated ATP and PGE2 levels. Again, NAC protected against changes in urothelial mediator release following acrolein or CAA. This study is the first to report that CAA in addition to acrolein contributes to the urotoxicity of cyclophosphamide and ifosfamide. Both metabolites altered urothelial mediator levels which could contribute to the sensory and functional bladder changes experienced by patients after treatment with cyclophosphamide or ifosfamide. Alterations in urothelial cell viability and mediator release may be causally linked to oxidative stress, with NAC providing protection against these changes.

Apoptotic Fragmentation of Tricellulin.

Apoptotic extrusion of cells from epithelial cell layers is of central importance for epithelial homeostasis. As a prerequisite cell–cell contacts between apoptotic cells and their neighbors have to be dissociated. Tricellular tight junctions (tTJs) represent specialized structures that seal polarized epithelial cells at sites where three cells meet and are characterized by the specific expression of tricellulin and angulins. Here, we specifically addressed the fate of tricellulin in apoptotic cells. Methods: Apoptosis was induced by staurosporine or camptothecin in MDCKII and RT-112 cells. The fate of tricellulin was analyzed by Western blotting and immunofluorescence microscopy. Caspase activity was inhibited by Z-VAD-FMK or Z-DEVD-FMK. Results: Induction of apoptosis induces the degradation of tricellulin with time. Aspartate residues 487 and 441 were identified as caspase cleavage-sites in the C-terminal coiled-coil domain of human tricellulin. Fragmentation of tricellulin was inhibited in the presence of caspase inhibitors or when Asp487 or Asp441 were mutated to asparagine. Deletion of the tricellulin C-terminal amino acids prevented binding to lipolysis-stimulated lipoprotein receptor (LSR)/angulin-1 and thus should impair specific localization of tricellulin to tTJs. Conclusions: Tricellulin is a substrate of caspases and its cleavage in consequence contributes to the dissolution of tTJs during apoptosis.

Sulfated polysaccharide of Sepiella maindroni ink targets Akt and overcomes resistance to the FGFR inhibitor AZD4547 in bladder cancer.

Rapid appearance of resistance to fibroblast growth factor receptor (FGFR) inhibitors hampers targeted regimens in bladder cancer. In the present study, we evaluated whether SIP-SII, a sulphated derivative of the polysaccharide in Sepiella maindroni (spineless cuttlefish) ink used in traditional Chinese medicine, could attenuate resistance to FGFR inhibition in bladder cancer cells. In vitro assays indicated that SIP-SII reduced cell viability and migration, restricted cell cycle progression, and increased apoptosis in parallel with decreased AKT phosphorylation and downregulation of CDK4, MMP2, and Bcl-2 in RT112 and JMSU1 cells. Synergistic effects on cell viability were observed when SIP-SII was combined with the small-molecule FGFR inhibitor AZD4547. Specific Akt targeting by SIP-SII was suggested by the fact that neither Akt knockdown nor the selective PI3K inhibitor BKM120 enhanced the inhibitory effects of SIP-II, while expression of a constitutively active Akt mutant rescued SIP-SII effects. Furthermore, subcutaneous transplantation of RT112 xenografts confirmed the superiority and tolerability of combined SIP-SII and AZD4547 administration over monotherapy regimens. The present study thus provides pre-clinical evidence of the ability of SIP-SII to improve FGFR-targeted therapies for bladder cancer by inhibiting Akt.

Assessing the cytotoxic potential of glycoalkaloidic extract in nanoparticles against bladder cancer cells.

This study proposed to use the nanotechnology to deliver glycoalkaloidic extract (AE) to bladder cancer cells, evaluating their activity in 2D and 3D models and the biological mechanism of cell death. NPs were prepared by nanoprecipitation method using polylactic acid (PLA) and characterized considering their size, charge, particle concentration and stability. The cytotoxicity was evaluated in 2D and 3D model, and the apoptosis and cell cycle were investigated using flow cytometry. NPs loading AE (NP-AE) had diameter around 125±6nm (PdI <0.1) and negative charge. The encapsulation efficiency of SM and SS was higher than 85% for both compounds. The obtained formulation showed a significant in-vitro cytotoxic effect against RT4 cells in a dose-dependent manner with IC50 twofold lower than the free AE. The cytotoxic effect of NP-AE was mediated by apoptosis and cell cycle arrested in the S phase. RT4 cells cultured under 3D conditions exhibited a higher resistance to the treatments (IC50 ~ threefold higher than in 2D cell culture). The NP-AE might be a promising nanocarrier to load and deliver glycoalkaloids against bladder cancer.

Abstract 3818: BGJ398, a pan-FGFR inhibitor, overcomes paclitaxel resistance in urothelial carcinoma with FGFR1 overexpression

Abstract Introduction: Paclitaxel (PTX) is commonly used to treat urothelial carcinoma (UC) after platinum-based chemotherapy has failed. However, single-agent taxane therapy is not sufficient to inhibit tumor progression and drug resistance in advanced UC. Epithelial-to-mesenchymal transition (EMT) induced by fibroblast growth factor receptor (FGFR)1 signaling has been proposed as a mechanism of PTX resistance, but it is unclear whether this can be overcome by FGFR1 inhibition. The present study investigated whether FGFR1 overexpression contributes to PTX resistance and whether FGFR inhibition can enhance PTX efficacy in UC. Materials and Methods: UC cell lines: UMUC-14, RT4, T24, J82, HTB5 and HTB9 cells were used. Cells were treated with various concentration of PTX (0 to 20 nM) and BGJ398 (0 to 6 μM). The half-maximal inhibitory concentrations (IC50) were measured by the CellTiter-Glo assay. Cell cycle was analyzed after propidium iodide (PI) staining via flow cytometry. Changes in cell cycle, apoptosis and EMT-associated protein levels were assessed by Western Blot analysis. The migratory and colony formative property were analyzed by wound healing assay and colony forming assay, respectively. RNA interference was performed to assess the dependence of PTX sensitivity on pro-apoptotic proteins. Results: We showed that mesenchymal-type T24 and J82 cell lines were highly resistant to PTX (0 to 20 nM) or BGJ398 (0 to 6 μM) monotherapy compared to FGFR3-positive epithelial-type (i.e., RT4 and UMUC-14) UC cell lines. We further found that PTX (10 nM) combined with BGJ398 (6 μM) caused synergistic effects on sub G1 accumulation and apoptosis compared to PTX or BGJ398 monotherapy in T24 and J82 cells, which were accompanied by downregulation of cyclin D1 protein and upregulation of gamma-histone 2A family member X, cleaved caspase-9 and cleaved poly (ADP-ribose) polymerase (PARP). Additionally, 10 nM of PTX combined with 6 μM of BGJ398 synergistically suppressed mesenchymal-type UC cell migration and colony formation via downregulation of EMT-associated factors including snail, slug and ZEB1. FGFR1 knockdown enhanced the antitumor effect of PTX (10 nM) by upregulation of apoptosis-associated markers, cleaved caspase-9 and PARP. Conclusions: We investigated the mechanism of resistance to PTX in UC mediated by FGFR1 and found that combined treatment with BGJ398 can enhance the efficacy of PTX in UC cell lines with mesenchymal features. Our results demonstrate that combination with FGFR1-targeted therapy improves the antitumor efficacy of standard cytotoxic chemotherapy, which is a strategy that warrants further investigation in clinical trials in selected UC patients with FGFR1 overexpression. Citation Format: Haram Ryu, Se Hyun Kim, Chan-Young Ock, Koung Jin Suh, Ji Yun Lee, Ji-Won Kim, Jin Won Kim, Jeong-Ok Lee, Yu Jung Kim, Keun-Wook Lee, Soo-Mee Bang, Jee Hyun Kim, Jong Seok Lee, Joong Bae Ahn, Kui-Jin Kim, Sun Young Rha. BGJ398, a pan-FGFR inhibitor, overcomes paclitaxel resistance in urothelial carcinoma with FGFR1 overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3818.

Abstract 1026: Oncogenic alterations in FGFR3 and ERBB2 lead to ligand-independent activation of PPARG in bladder cancer

Abstract PPARG is genomically activated in muscle-invasive bladder cancer through focal PPARG gene amplification and hotspot mutations in its heterodimer partner, RXRA. However, more than half of the PPARG-activated bladder tumors and cell lines do not have identifiable somatic alterations in either PPARG or RXRA. Using a PPARG-driven reporter assay in RT112 bladder cancer cell line, we screened probe compounds to identify candidate drivers of ligand-independent PPARG activation. We found that pan-FGFR inhibitors and MEK1/2 inhibitors antagonized the PPARG-driven reporter assay with potency similar to reported values for their cognate targets (1-20 nM) and interestingly, RT112 cells carry an FGFR3-TACC3 oncogenic fusion. In addition to the expected effects of these inhibitors on phospho-MEK1/2 and phospho-ERK1/2, they also inhibited production of canonical PPARG targets, including FABP4. In a second subset of cell lines, a parallel story was also observed for the effects of ERBB2 inhibitors in ERBB2 hotspot mutant bladder cancer. Taken together, these data uncover additional mechanisms for functional activation of PPARG in bladder cancer, suggest potential resistance mechanisms for FGFR and ERBB2 inhibitors, and provide a rationale for therapeutic combinations of PPARG modulators. Citation Format: Jonathan T. Goldstein, Ashton C. Berger, Craig A. Strathdee, Matthew Meyerson. Oncogenic alterations in FGFR3 and ERBB2 lead to ligand-independent activation of PPARG in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1026.

Abstract 3080: Activity of pan-FGFR inhibitor rogaratinib and PI3K inhibitor copanlisib in preclinical urothelial bladder cancer models

Abstract Rogaratinib is a potent small molecule pan-FGFR inhibitor that leads to downregulation of MAPK and PI3K signaling (1). In a recent Phase I study rogaratinib demonstrated higher ORR in locally advanced or metastatic urothelial bladder cancer (UBC) with FGFR mRNA overexpression in patients with PIK3CA and RASwildtype than in those harboring PIK3CA or RAS mutations (2). In UBC, about 25% of tumors contain mutated PIK3CA of which 75% represent hot spot mutations E545K or E542K (3). Copanlisib is a potent pan class I PI3K inhibitor with predominant activity against the α and δ isoforms being approved for treatment of relapsed follicular lymphoma (4). We evaluated the activity of rogaratinib in cellular and in vivo efficacy studies in bladder cancer models in monotherapy and in combination with copanlisib. Cell proliferation and viability in response to rogaratinib, copanlisib or the combination of both was studied in rogaratinib-sensitive UBC cell lines like RT112 as well as in a recombinant RT112-PIK3CAE545K cell line. Rogaratinib potently inhibited cell proliferation with IC50 values in the double nM range. Copanlisib’s potency varied between 20 and 900 nM. In combination, rogaratinib and copanlisib increased cell death and were synergistic as shown by their proliferation combination indices for the absolute IC50 and IC80. Expression of the PIK3CAE545K hot spot mutant in RT112 cells decreased the anti-proliferative efficacy of rogaratinib but did not change the sensitivity to copanlisib. In vivo, rogaratinib led to tumor regression in the FGFR1-expressing JMSU1 xenograft and strongly inhibited tumor growth in the FGFR3-driven RT112 model. Copanlisib did not show significant inhibition of tumor growth in monotherapy in either model. In the JMSU1 model the combination was not superior to rogaratinib alone at maximal tolerated doses. In the RT112 as well as in the RT112-PIK3CAE545K model, the combination significantly improved anti-tumor activity. In a PDX model with FGFR3 overexpression and a PIK3CAH1047R hot spot mutation rogaratinib did not inhibit tumor growth significantly while copanlisib displayed significant anti-tumor effects. The combination of both drugs reduced tumor growth compared to either monotherapy group. In conclusion, in UBC tumor models overexpressing FGFR with differing sensitivities to rogaratinib, its anti-tumor activity could be further enhanced when combined with copanlisib and suggests that PIK3CA mutations may play a role in reduced sensitivity of UBC to FGFR inhibition. These promising results warrant further development of rogaratinib in monotherapy and in combination. Clinical trials with rogaratinib are currently recruiting (NCT03517956, NCT03410693, NCT03473756).

Sulforaphane Inhibits Nonmuscle Invasive Bladder Cancer Cells Proliferation through Suppression of HIF-1α-Mediated Glycolysis in Hypoxia.

Bladder cancer is the fourth common cancer among men and more than 70% of the bladder cancer is nonmuscle invasive bladder cancer (NMIBC). Because of its high recurrence rate, NMIBC brings to patients physical agony and high therapy costs to the patients' family and society. It is imperative to seek a natural compound to inhibit bladder cancer cell growth and prevent bladder cancer recurrence. Cell proliferation is one of the main features of solid tumor development, and the rapid tumor cell growth usually leads to hypoxia due to the low oxygen environment. In this study we found that sulforaphane, a natural chemical which was abundant in cruciferous vegetables, could suppress bladder cancer cells proliferation in hypoxia significantly stronger than in normoxia (p < 0.05): 20 μM sulforaphane inhibited bladder cancer cell proliferation by 26.1 ± 4.1% in normoxia, while it inhibited cell proliferation by 39.7 ± 5.2% in hypoxia in RT112 cells. Consistently, sulforaphane inhibited cell proliferation by 29.7 ± 4.6% in normoxia, while it inhibited cell proliferation by 48.3 ± 5.2% in hypoxia in RT4 cells. Moreover, we revealed that sulforaphane decreased glycolytic metabolism in a hypoxia microenvironment by downregulating hypoxia-induced HIF-1α and blocking HIF-1α trans-localization to the nucleus in NMIBC cell lines. This study discovered a food sourced compound inhibiting bladder cancer cells proliferation and provided experimental evidence for developing a new bladder cancer preventive and therapeutic strategy.

Bioactivation of the tobacco carcinogens 4-aminobiphenyl (4-ABP) and 2-amino-9H-pyrido[2,3-b]indole (AαC) in human bladder RT4 cells.

Occupational and tobacco exposure to aromatic amines (AAs) including 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA) are associated with bladder cancer (BC) risk. Several epidemiological studies have also reported a possible role for structurally related heterocyclic aromatic amines (HAAs) formed in tobacco smoke or cooked meats with BC risk. We had screened for DNA adducts of 4-ABP, 2-NA, and several prominent HAAs formed in tobacco smoke or grilled meats including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-9H-pyrido[2,3-b]indole (AαC) in the bladder DNA of BC patients, using liquid chromatography/mass spectrometry. We detected DNA adducts of 4-ABP, but not adducts of the other carcinogens. In this study, we have examined the capacity of RT4 cells, an epithelial human bladder cell line, to bioactivate AAs and HAAs to DNA damaging agents, which may contribute to BC. 4-ABP and AαC formed DNA adducts, but DNA adducts of 2-NA, PhIP, and MeIQx were not detected. 4-ABP DNA adducts were formed at tenfold higher levels than AαC adducts. Pretreatment of RT4 cells with α-naphthoflavone (1-10µM), a specific cytochrome P450 1 (CYP1) inhibitor, decreased AαC adduct formation by 50% but did not affect the level of 4-ABP adducts. However, cell pretreatment with 8-methoxypsoralen (0.1-1µM), a potent inhibitor of CYP2A, resulted in a 90% decrease of 4-ABP DNA adducts levels. These data signify that CYP2A and CYP1A isoforms expressed in the target urothelium bioactivate 4-ABP and AαC, respectively, and may be a critical feature of aromatic amine-induced urinary bladder carcinogenesis. The bioactivation of other tobacco and environmental AAs by bladder CYPs and their ensuing bladder DNA damage warrants further study.

Mechanisms behind Temsirolimus Resistance Causing Reactivated Growth and Invasive Behavior of Bladder Cancer Cells In Vitro.

Background: Although mechanistic target of rapamycin (mTOR) inhibitors, such as temsirolimus, show promise in treating bladder cancer, acquired resistance often hampers efficacy. This study evaluates mechanisms leading to resistance. Methods: Cell growth, proliferation, cell cycle phases, and cell cycle regulating proteins were compared in temsirolimus resistant (res) and sensitive (parental—par) RT112 and UMUC3 bladder cancer cells. To evaluate invasive behavior, adhesion to vascular endothelium or to immobilized extracellular matrix proteins and chemotactic activity were examined. Integrin α and β subtypes were analyzed and blocking was done to evaluate physiologic integrin relevance. Results: Growth of RT112res could no longer be restrained by temsirolimus and was even enhanced in UMUC3res, accompanied by accumulation in the S- and G2/M-phase. Proteins of the cdk-cyclin and Akt-mTOR axis increased, whereas p19, p27, p53, and p73 decreased in resistant cells treated with low-dosed temsirolimus. Chemotactic activity of RT112res/UMUC3res was elevated following temsirolimus re-exposure, along with significant integrin α2, α3, and β1 alterations. Blocking revealed a functional switch of the integrins, driving the resistant cells from being adhesive to being highly motile. Conclusion: Temsirolimus resistance is associated with reactivation of bladder cancer growth and invasive behavior. The α2, α3, and β1 integrins could be attractive treatment targets to hinder temsirolimus resistance.

NRF2 activates a partial epithelial-mesenchymal transition and is maximally present in a hybrid epithelial/mesenchymal phenotype.

The epithelial-mesenchymal transition (EMT) is a key process implicated in cancer metastasis and therapy resistance. Recent studies have emphasized that cells can undergo partial EMT to attain a hybrid epithelial/mesenchymal (E/M) phenotype – a cornerstone of tumour aggressiveness and poor prognosis. These cells can have enhanced tumour-initiation potential as compared to purely epithelial or mesenchymal ones and can integrate the properties of cell-cell adhesion and motility that facilitates collective cell migration leading to clusters of circulating tumour cells (CTCs) – the prevalent mode of metastasis. Thus, identifying the molecular players that can enable cells to maintain a hybrid E/M phenotype is crucial to curb the metastatic load. Using an integrated computational-experimental approach, we show that the transcription factor NRF2 can prevent a complete EMT and instead stabilize a hybrid E/M phenotype. Knockdown of NRF2 in hybrid E/M non-small cell lung cancer cells H1975 and bladder cancer cells RT4 destabilized a hybrid E/M phenotype and compromised the ability to collectively migrate to close a wound in vitro. Notably, while NRF2 knockout simultaneously downregulated E-cadherin and ZEB-1, overexpression of NRF2 enriched for a hybrid E/M phenotype by simultaneously upregulating both E-cadherin and ZEB-1 in individual RT4 cells. Further, we predict that NRF2 is maximally expressed in hybrid E/M phenotype(s) and demonstrate that this biphasic dynamic arises from the interconnections among NRF2 and the EMT regulatory circuit. Finally, clinical records from multiple datasets suggest a correlation between a hybrid E/M phenotype, high levels of NRF2 and its targets and poor survival, further strengthening the emerging notion that hybrid E/M phenotype(s) may occupy the ‘metastatic sweet spot’.

Flaccidoxide-13-Acetate-Induced Apoptosis in Human Bladder Cancer Cells is through Activation of p38/JNK, Mitochondrial Dysfunction, and Endoplasmic Reticulum Stress Regulated Pathway.

Flaccidoxide-13-acetate, an active compound isolated from cultured-type soft coral Sinularia gibberosa, has been shown to have inhibitory effects against invasion and cell migration of RT4 and T24 human bladder cancer cells. In our study, we used an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), colony formation assay, and flow cytometry to determine the mechanisms of the anti-tumor effect of flaccidoxide-13-acetate. The MTT and colony formation assays showed that the cytotoxic effect of flaccidoxide-13-acetate on T24 and RT4 cells was dose-dependent, and the number of colonies formed in the culture was reduced with increasing flaccidoxide-13-acetate concentration. Flow cytometry analysis revealed that flaccidoxide-13-acetate induced late apoptotic events in both cell lines. Additionally, we found that flaccidoxide-13-acetate treatment upregulated the expressions of cleaved caspase 3, cleaved caspase 9, Bax, and Bad, and down-regulated the expressions of Bcl-2, p-Bad, Bcl-x1, and Mcl-1. The results indicated that apoptotic events were mediated by mitochondrial dysfunction via the caspase-dependent pathway. Flaccidoxide-13-acetate also provoked endoplasmic reticulum (ER) stress and led to activation of the PERK-eIF2α-ATF6-CHOP pathway. Moreover, we examined the PI3K/AKT signal pathway, and found that the expressions of phosphorylated PI3K (p-PI3K) and AKT (p-AKT) were decreased with flaccidoxide-13-acetate concentrations. On the other hand, our results showed that the phosphorylated JNK and p38 were obviously activated. The results support the idea that flaccidoxide-13-acetate-induced apoptosis is mediated by mitochondrial dysfunction, ER stress, and activation of both the p38 and JNK pathways, and also relies on inhibition of PI3K/AKT signaling. These findings imply that flaccidoxide-13-acetate has potential in the development of chemotherapeutic agents for human bladder cancer.

ALDOLASE A regulates invasion of bladder cancer cells via E-cadherin-EGFR signaling.

Glycolysis and glycogenesis are known to be tightly associated with cancer cell migration. However, their roles in bladder cancer have not been reported. In this study, ALDOLASE A (ALDOA) was identified in a coexpression network generated using glycolysis- and glycogenesis-related genes in Kyoto Encyclopedia of Genes and Genomes. ALDOA was located in the central region in the network, and the cancer genome atlas (TCGA) data suggest that ALDOA expression levels are associated with viability in patients with cancer at the middle and late stages. Bladder cancer cell lines, T24 and RT4, were used to knockdown (sh) or overexpress (OE) ALODA to analyze its role. The sh-ALDOA reduced cell viability, colony formation rate, and invasion cell number; while OE had an opposite effect compared with sh-ALDOA. Further, the sh-ALDOA expression induced E-cadherin level while reduced N-cadherin and vimentin levels. The OE cells reduced E-cadherin and induced N-cadherin and vimentin levels. In addition, epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK), and AKT serine/threonine kinase (AKT) phosphorylation levels are all reduced in sh-ALODA while activated in OE cells compared with the control group. But either sh-ALODA or OE did not change total protein levels of EGFR, MAPK, and AKT. To further analyze E-cadherin function in ALDOA regulation on bladder cancer cells, sh-ALDOA and sh-E-cadherin were cotransfected in T24 and RT4 cells. The results indicated that sh-ALDOA and sh-E-cadherin expressions eliminated sh-ALDOA function, resulting similar cell viability, colony formation rate, and invasion cell number with control group. Also, sh-ALDOA and shE-cadherin expressions increased EGFR, MAPK, and AKT phosphorylation levels; and the levels were similar to the control group. But, sh-ALDOA and sh-E-cadherin expressions did not change N-cadherin and vimentin levels, which maintain similar levels with sh-ALDOA-expressing cells. Taken together, these results suggest that ALDOA might play an important function in bladder cancer and its action may be though E-cadherin-EGFR signaling.

Aminosalicylic acid reduces ER stress and Schwann cell death induced by MPZ mutations.

Mutations in myelin protein zero (MPZ) cause inherited peripheral neuropathies, including Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas neuropathy. Mutant MPZ proteins have previously been reported to cause CMT via enhanced endoplasmic reticulum (ER) stress and Schwann cell (SC) death, although the pathological mechanisms have not yet been elucidated. In this study, we generated an in vitro model of rat SCs expressing mutant MPZ (MPZ V169fs or R98C) proteins and validated the increase in cell death and ER stress induced by the overexpression of the MPZ mutants. Using this model, we examined the efficacy of 3 different aminosalicylic acids (ASAs; 4-ASA, sodium 4-ASA and 5-ASA) in alleviating pathological phenotypes. FACS analysis indicated that the number of apoptotic rat SCs, RT4 cells, induced by mutant MPZ overexpression was significantly reduced following treatment with each ASA. In particular, treatment with 4-ASA reduced the levels of ER stress markers in RT4 cells induced by V169fs MPZ mutant overexpression and relieved the retention of V169fs mutant proteins in the ER. Additionally, the level of an apoptotic signal mediator (p-JNK) was only decreased in the RT4 cells expressing R98C MPZ mutant protein following treatment with 4-ASA. Although 4-ASA is known as a free radical scavenger, treatment with 4-ASA in the in vitro model did not moderate the level of reactive oxygen species, which was elevated by the expression of mutant MPZ proteins. On the whole, the findings of this study indicate that treatment with 4-ASA reduced the ER stress and SC death caused by 2 different MPZ mutants and suggest that ASA may be a potential therapeutic agent for CMT.

Understandings of Immunomodulatory Pathways of Indoleamine 2,3‐dioxygenase (IDO) in Bladder Cancer Cells

Bladder cancer (BC) is the fourth most common cancer between men and the ninth in women, with a high recurrence rate and progression after conventional treatments. A growing body of evidence suggests that immunomodulatory molecules protect bladder tumors against immune surveillance by triggering signaling pathways that modulate locally the immune system. One important molecule is indoleamine 2,3‐dioxygenase (IDO), which promotes tryptophan breakdown, activating sensitive pathways such as GCN2 in immune cells. In addition, tryptophan breakdown leads to the production of kynurenine compounds, which activate aryl hydrocarbon receptor (AHR), promoting anergy and apoptosis on T cells and Natural killer cells. Although these IDO‐mediated pathways are well recognized in immune cells, they were not yet analyzed in bladder cancer cells. The use of IDO inhibitors arises as a promise anti‐tumor strategy, however, they have divergent effects depending on the activated pathway. Here, we analyzed the expression of IDO and the activation of its pathways in non‐invasive muscle and in invasive muscle bladder cancer cells, and the effect of two IDO inhibitors on these pathways.RT4 and T24 cells were maintained in RPMI medium and incubated for 48h with the following stimuli: IFN‐γ (1000U/mL), Methyl‐D‐tryptophan (MT) (500μM), Incb024360 analog (INCB) (1μM). Control cells were maintained in RPMI medium. After the stimuli, RNA was extracted and qRT‐PCR for IDO, CHOP (a marker of GCN2 activation), CYP1A1 (an AHR activation marker), and AHR was performed.T24 cells constitutively express higher levels of IDO when compared to RT4 cells (relative expression (RE) of 1.0±0.1 in RT4 vs. 73.8±17.0 in T24), accompanied by high expression of CHOP (RE of 1.0±1.2 in RT4 vs. 10.5±4.5 in T24) and CYP1A1 (RE 1.0±0.0 in RT4 vs. 28.2±1.5 in T24), with no differences in AHr expression. When stimulated with IFN‐γ, both RT4 and T24 cells showed increased IDO expression, CHOP and CYP1A1 (p&lt;0.001). While INCB significantly reduced the IDO‐mediated pathways (CHOP and CYP1A1 expression), MT treatment increased CYP1A1 expression (p&lt;0.05).IDO‐regulated pathways are differentiated in invasive and noninvasive bladder cancer cells. As such, adjuvant treatments to modulate IDO are indeed promising. The use of certain inhibitors, however, such as MT, while may inhibit tryptophan depletion, also lead to activation of AHR, promoting kynurenine‐like effects. The use of non AHR binding compounds, such as Incb024360 analogs are promising then proposed as an alternative and safer approach for regulating IDO‐mediated events without nonspecific bindings.Support or Funding InformationFundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Assessing Radiosensitivity of Bladder Cancer in vitro: A 2D vs. 3D Approach.

Background: Bladder cancer is the fourth most commonly diagnosed cancer among males worldwide. Current treatment strategies established for bladder cancer mainly consist of cystectomy yet advances in radiation therapy have pointed to the value of organ-preserving strategies in preserving patients' quality of life.Aim: To study and compare the radiosensitivity in two-dimension (2D) and physiologically-relevant three-dimension (3D) in vitro culture of three human bladder cancer cell lines, RT4, T24, and UM-UC-3.Materials and Methods: Clonogenic assay was performed to assess cells' radiosensitivity in 2D. Employing the 3D Matrigel™-based cultures to enrich for cancer stem cells (CSCs) allowed us to assess the survival of this subpopulation of cells via evaluating the number, i.e., sphere forming unit (SFU), and the sizes of cultured spheres, formed from cells exposed to different radiation doses compared to non-irradiated cells.Results: Irradiating cells with increasing radiation doses revealed highest survival rates with RT4 cells in 2D, followed by T24 and UM-UC-3. In 3D, however, UM-UC-3 cells were shown to be the most radio-resistant as evidenced by the number of spheres formed, yet they displayed the least efficient volume reduction/regression (VR), whilst the volume decreased significantly for both RT4 and T24 cells. Sphere VR and sphere ratio (SR) values were then plotted against each other demonstrating a linear correlation between volume and number with RT4 and UM-UC-3 cell lines, but not T24. Lastly, multiple regression model was employed to evaluate the possibility of obtaining a function combining both 3D parameters, SR and VR, with the surviving fraction (SF) in 2D, and showed a linear regression for T24 cells only, with a correlation coefficient of 0.97 for the combined parameters.Conclusion: We were able to radiobiologically characterize 3 human bladder cancer cell lines showing differential effects of radiation between 2D and 3D culture systems, paving the way for achieving better assessment of radiosensitivity of bladder cancer in vitro.

The Antitumor Effect of Curcumin in Urothelial Cancer Cells Is Enhanced by Light Exposure In Vitro.

The natural compound curcumin exerts antitumor properties in vitro, but its clinical application is limited due to low bioavailability. Light exposure in skin and skin cancer cells has been shown to improve curcumin bioavailability; thus, the object of this investigation was to determine whether light exposure might also enhance curcumin efficacy in bladder cancer cell lines. RT112, UMUC3, and TCCSUP cells were preincubated with low curcumin concentrations (0.1-0.4 μg/ml) and then exposed to 1.65 J/cm2 visible light for 5 min. Cell growth, cell proliferation, apoptosis, cell cycle progression, and cell cycle regulating proteins along with acetylation of histone H3 and H4 were investigated. Though curcumin alone did not alter cell proliferation or apoptosis, tumor cell growth and proliferation were strongly blocked when curcumin was combined with visible light. Curcumin-light caused the bladder cancer cells to become arrested in different cell phases: G0/G1 for RT112, G2/M for TCCSUP, and G2/M- and S-phase for UMUC3. Proteins of the Cdk-cyclin axis were diminished in RT112 after application of 0.1 and 0.4 μg/ml curcumin. Cell cycling proteins were upregulated in TCCSUP and UMUC3 in the presence of 0.1 μg/ml curcumin-light but were partially downregulated with 0.4 μg/ml curcumin. 0.4 μg/ml (but not 0.1 μg/ml) curcumin-light also evoked late apoptosis in TCCSUP and UMUC3 cells. H3 and H4 acetylation was found in UMUC3 cells treated with 0.4 μg/ml curcumin alone or with 0.1 μg/ml curcumin-light, pointing to an epigenetic mechanism. Light exposure enhanced the antitumor potential of curcumin on bladder cancer cells but by different molecular action modes in the different cell lines. Further studies are necessary to evaluate whether intravesical curcumin application, combined with visible light, might become an innovative tool in combating bladder cancer.