Activity In Bladder Cancer Cells Research Articles

Insights into the Mode of Action of Novel Morpholinated Curcumin Derivatives Exhibiting Potent Antitumor Activity in Bladder Cancer Cells In Vitro.

Although curcumin is a well-known natural polyphenol with many biological activities, its clinical application has been limited by low aqueous solubility and stability. Therefore, curcumin derivatives have been proposed to overcome these limitations and increase anticancer activity. This study tested curcumin derivatives with modified feruloyl moieties (2a and 2a-B) and the β-diketo moiety (2a-B) to better understand their anticancer mechanism against human bladder cancer cells. The anticancer activity of 2a and 2a-B was determined using MTT (hypoxic conditions) and LDH (normoxic conditions) assays. An ELISA-based protein panel was used to find the potential molecular targets, while flow cytometric, colorimetric, fluorescent, and luminescent assays were used to investigate the cell death mechanism. It was shown that compound 2a exerted a more potent cytotoxic effect under hypoxic conditions, while compound 2a-B demonstrated a comparable effect in normoxic and hypoxic conditions. The potential molecular targets modified by 2a and 2a-B depending on oxygen concentration were also proposed. Both compounds alter cell cycle progression by blocking the cell cycle in the G2/M phase and decreasing the percentage of cells in the G0/G1 phase. Compound 2a-B led to phosphatidylserine translocation, increased caspase 3/7 activity, and decreased mitochondrial membrane potential, suggesting a mitochondrial apoptosis pathway. We found that the Akt signaling pathway may modulate the activity of compound 2a-B, as evidenced by enhanced cytotoxic activity in combination with MK-2206, an Akt 1/2/3 inhibitor. Thus, our results provide new insights into the anticancer activity of compounds 2a and 2a-B; however, further studies are needed to better understand their therapeutic potential.

NEDD4L inhibits migration, invasion, cisplatin resistance and promotes apoptosis of bladder cancer cells by inactivating the p62/Keap1/Nrf2 pathway.

This study identified the function of neural precursor cell expressed developmentally down-regulated 4-like (NEDD4L) on bladder cancer (BLCA). NEDD4L expression in BLCA patients was scrutinized. The function of NEDD4L on the viability, apoptosis, migration and invasion of BLCA cells was evaluated by cell counting kit-8, flow cytometry and Transwell assays. The effect of NEDD4L on the cisplatin (DDP) resistance of the DDP-resistant BLCA cells was explored. The influence of NEDD4L on the p62/Keap1/Nrf2 pathway activity in BLCA cells was tested by Western blot. Rescue experiments were implemented to verify whether NEDD4L regulated BLCA cell malignant behavior by mediating the Keap1/Nrf2 pathway activity via p62. The effect of NEDD4L on the growth and the p62/Keap1/Nrf2 pathway activity in vivo was researched in xenograft tumor nude mice models. The down-regulated NEDD4L in BLCA patients was associated with unfavorable survival. NEDD4L suppressed the viability (inhibition rate 57.1%/49.0%), migration (inhibition rate 49.7%/77.1%), invasion (inhibition rate 50.6%/75.7%), promoted the apoptosis of T24/5637 cells (promotion rate 243.8%/201.9%), reduced IC 50 of DDP-resistant T24/5637 cells from 132.2/101.8 to 57.81/59.71 μM, respectively, and inactivated the p62/Keap1/Nrf2 pathway in T24/5637 cells. p62 up-regulation partially abrogated the inhibition of NEDD4L on the Keap1/Nrf2 pathway activity, the malignant behavior of BLCA cells, and the DDP resistance of DDP-resistant BLCA cells. NEDD4L overexpression inhibited the tumor growth and the p62/Keap1/Nrf2 pathway activity in vivo in BLCA. NEDD4L inhibits the progression of BLCA by inactivating the p62/Keap1/Nrf2 pathway. It may be an effective target for BLCA treatment.

LncRNA UCA1 Participates in De Novo Synthesis of Guanine Nucleotides in Bladder Cancer by Recruiting TWIST1 to Increase IMPDH1/2.

Metabolic dysregulation has been identified as one of the hallmarks of cancer biology. Based on metabolic heterogeneity between bladder cancer tissues and adjacent tissues, we discovered several potential driving factors for the bladder cancer occurrence and development. Metabolic genomics showed purine metabolism pathway was mainly accumulated in bladder cancer. Long noncoding RNA urothelial carcinoma-associated 1 (LncRNA UCA1) is a potential tumor biomarker for bladder cancer diagnosis and prognosis, and it increases bladder cancer cell proliferation, migration, and invasion via the glycolysis pathway. However, whether UCA1 plays a role in purine metabolism in bladder cancer is unknown. Our findings showed that UCA1 could increase the transcription activity of guanine nucleotide de novo synthesis rate limiting enzyme inosine monophosphate dehydrogenase 1 (IMPDH1) and inosine monophosphate dehydrogenase 2 (IMPDH2), triggering in guanine nucleotide metabolic reprogramming. This process was achieved by UCA1 recruiting the transcription factor TWIST1 which binds to the IMPDH1and IMPDH2 promoter region. Increased guanine nucleotide synthesis pathway products stimulate RNA polymerase-dependent production of pre-ribosomal RNA and GTPase activity in bladder cancer cells, hence increasing bladder cancer cell proliferation, migration, and invasion. We have demonstrated that UCA1 regulates IMPDH1/2-mediated guanine nucleotide production via TWIST1, providing additional evidence of metabolic reprogramming.

Identification and Validation of N6-Methyladenosine-Related Biomarkers for Bladder Cancer: Implications for Immunotherapy.

N6-methyladenosine (m6A) has emerged as one of the most important modifications of RNA. Based on the expression of 23 different modes of m6A regulatory factors, we identified three different m6A modification patterns in bladder cancer. The effects of the three different modes of m6A modification on clinicopathological characteristics, immune cell infiltration levels and expression levels of immune checkpoint genes were comprehensively analyzed. In addition, the effects of different modes of m6A modification on the therapeutic efficacy of anti-PD-L1 immunotherapy (atezolizumab) are also discussed. Our results confirm that m6A methylation plays an important role in immune cell recruitment in the tumor microenvironment of bladder cancer, which influences the efficacy of anti-PD-L1 therapy for bladder cancer. We further confirmed the important role of FTO protein in the biological function of bladder cancer cells by performing in vitro experiments. FTO functions as an oncogene in bladder cancer cells, and upon FTO knockdown, the level of m6A enzyme activity in bladder cancer cells was significantly increased, apoptosis was increased, and cell proliferation and cell invasion were reduced. In addition, our study also confirmed that K216H and K216E are probably important targets for regulating FTO. We provide new insights into the regulatory pathways of the immune microenvironment and the methylation function of m6A in bladder cancer, which will help in designing novel diagnostic methods, prognostic tools, and therapeutic targets.

Hybrid Nanoparticles as an Efficient Porphyrin Delivery System for Cancer Cells to Enhance Photodynamic Therapy.

Photodynamic therapy (PDT) is a potential non-invasive approach for application in oncological diseases, based on the activation of a photosensitizer (PS) by light at a specific wavelength in the presence of molecular oxygen to produce reactive oxygen species (ROS) that trigger the death tumor cells. In this context, porphyrins are interesting PS because they are robust, have high chemical, photo, thermal, and oxidative stability, and can generate singlet oxygen (1O2). However, porphyrins exhibit low solubility and a strong tendency to aggregate in a biological environment which limits their clinical application. To overcome these challenges, we developed hybrid nanostructures to immobilize 5,10,15,20-tetrakis[(4-carboxyphenyl) thio-2,3,5,6-tetrafluorophenyl] (P), a new third-generation PS. The biological effect of this system was evaluated against bladder cancer (BC) cells with or without light exposition. The nanostructure composed of lipid carriers coated by porphyrin-chitosan (P-HNP), presented a size of ca. 130 nm and low polydispersity (ca. 0.25). The presence of the porphyrin-chitosan (P-chitosan) on lipid nanoparticle surfaces increased the nanoparticle size, changed the zeta potential to positive, decreased the recrystallization index, and increased the thermal stability of nanoparticles. Furthermore, P-chitosan incorporation on nanoparticles increased the stability and enhanced the self-organization of the system and the formation of spherical structures, as observed by small-angle X-ray scattering (SAXS) analysis. Furthermore, the immobilization process maintained the P photoactivity and improved the photophysical properties of PS, minimizing its aggregation in the cell culture medium. In the photoinduction assays, the P-HNP displayed high phototoxicity with IC50 3.2-folds lower than free porphyrin. This higher cytotoxic effect can be correlated to the high cellular uptake of porphyrin immobilized, as observed by confocal images. Moreover, the coated nanoparticles showed mucoadhesive properties interesting to its application in vivo. Therefore, the physical and chemical properties of nanoparticles may be relevant to improve the porphyrin photodynamic activity in BC cells.

Microbial Metabolites of Flavanols in Urine are Associated with Enhanced Anti-Proliferative Activity in Bladder Cancer Cells In Vitro

Flavanols are metabolized by the gut microbiota to bioavailable metabolites, and the absorbed fraction is excreted primarily via urine. Uroepithelial cells are thus a potential site of activity due to exposure to high concentrations of these compounds. Chemoprevention by flavanols may be partly due to these metabolites. In Vitro work in this area relies on a limited pool of commercially available microbial metabolites, and little has been done in bladder cancer. The impact of physiologically relevant mixtures of flavanols and their metabolites remains unknown. Rats were fed various flavanols and urine samples, approximating the bioavailable metabolome, were collected. Urines were profiled by UPLC-MS/MS, and their anti-proliferative activities were assayed In Vitro in four bladder cancer models. Significant interindividual variability was observed for composition and proliferation. Microbial metabolite concentrations (valerolactones, phenylalkyl acids and hippuric acids) were positively associated with reduced bladder cancer proliferation In Vitro, while native flavanols were poorly correlated with activity. These results suggest that microbial metabolites may be responsible for chemoprevention in uroepithelial cells following flavanol consumption. This highlights the potential to use individual genetics and microbial metabotyping to design personalized dietary interventions for cancer prevention and/or adjuvant therapy to reduce bladder cancer incidence and improve outcomes.

MicroRNA-27b inhibits cell proliferation and invasion in bladder cancer by targeting engrailed-2.

Background: Bladder cancer is considered a malignant tumour characterised by great heterogeneity. Engrailed-2 may be a gene implicated in bladder cancer. Bioinformatics analysis found base pair complementation between microRNA-27b and engrailed-2. The present study aimed to investigate the reciprocal association between microRNA-27b and engrailed-2 in bladder cancer.Methods: The microRNA-27b and the protein of engrailed-2 in the tissues and cells of the bladder were detected. The processes of apoptosis, proliferation, invasion, and migration of tumour cells were evaluated. The co-action between microRNA-27b and engrailed-2 was detected by a luciferase reporter system. Finally, the interaction between microRNA-27b and engrailed-2 was further verified in vivo.Results: The study found that the expression level of microRNA-27b is lower in bladder cancer tissues and cells than that in neighbouring ordinary tissues, whereas the opposite outcome was observed regarding the expression level of engrailed-2. Furthermore, microRNA-27b expression level is not significantly linked to the age of patients with bladder cancer; however, it is significantly associated with the clinicopathological grade of bladder cancer. Notably, engrailed-2 is negatively regulated by microRNA-27b. Transfection with microRNA-27b was associated with a significant reduction in the activity of bladder cancer cells and promoted apoptosis, while engrailed-2 restoration effectively reversed the above effects of microRNA-27b on bladder cancer in vitro and in vivo.Conclusions: In conclusion, engrailed-2 is engaged in the development and process of bladder cancer through the negative mediation of microRNA-27b; additionally, microRNA-27b/engrailed-2 could form a signalling pathway with a significant effect on the process of bladder cancer.

Super-Enhancer LncRNA LINC00162 Promotes Progression of Bladder Cancer.

SummaryDue to the lack of effective early diagnostic measures and treatment methods, bladder cancer has become a malignant tumor that seriously threatens people's lives and health. Here, we reported that LINC00162, a super-enhancer long noncoding RNA, was highly expressed in bladder cancer cells and tissues. And LINC00162 was negatively correlated with neighboring PTTG1IP expression. Knocking down LINC00162 expression can inhibit the proliferative activity of bladder cancer cells and the growth of transplanted tumors in vivo, while knocking down the expression of PTTG1IP could restore the proliferative activity of bladder cancer cells. In addition, both LINC00162 and PTTG1IP were found to be able to bind to THRAP3, a transcription-related protein. And THRAP3 can regulate PTTG1IP expression. Finally, we demonstrated a mechanism that LINC00162 could regulate PTTG1IP expression through binding THRAP3. This study provided a potential target molecule for clinical treatment of bladder cancer.

LncRNA ZNF503-AS1 acts as a tumor suppressor in bladder cancer by up-regulating Ca2+ concentration via transcription factor GATA6.

Ca2+ homeostasis plays a pivotal role in regulating proliferation and apoptosis during cancer development. This study intended to examine the potential tumor-suppressing role of ZNF503 antisense RNA 1 (ZNF503-AS1) in bladder cancer, which may be implicated in the regulation of Ca2+ homeostasis. Differentially expressed long non-coding RNAs (lncRNAs) related to bladder cancer were identified using microarray analysis, followed by the verification of transcription factors to which they bind. The relationship between ZNF503-AS1, GATA6 and SLC8A1 was assessed using dual luciferase reporter, RIP and ChIP assays. The expression levels of ZNF503-AS1, GATA6 and SLC8A1 were modulated to examine their effects on the tumorigenic potential, intracellular Ca2+ concentration and Ca2+-ATPase activity in bladder cancer cells. The in vivo tumorigenic ability was validated in nude mice. Microarray-based expression profile analysis of the GEO GSE61615 dataset revealed that the expression of ZNF503-AS1 was decreased in bladder cancer. Subsequently, we found that ZNF503-AS1 can bind to the transcription factor GATA6 to up-regulate the expression of SLC8A1. ZNF503-AS1 and SLC8A1 were found to be down-regulated in both primary bladder cancer tissues and cells. Exogenous overexpression of ZNF503-AS1 or SLC8A1 attenuated bladder cancer cell proliferation, invasion and migration, but promoted their apoptosis, accompanied by decreased Ca2+-ATPase activities and increased intracellular Ca2+ concentrations. Additional in vivo experiments validated the inhibitory effect of ZNF503-AS1 overexpression on the tumorigenic capacity of bladder cancer cells in nude mice. ZNF503-AS1 can recruit transcription factor GATA6 to up-regulate SLC8A1 expression, thereby increasing the intracellular Ca2+ concentration and repressing the proliferation, invasion and migration, and enhancing the apoptosis of bladder cancer cells.

Metformin Augments Panobinostat's Anti-Bladder Cancer Activity by Activating AMP-Activated Protein Kinase

Panobinostat, a histone deacetylase inhibitor, induces histone acetylation and acts against cancer but attenuates its anticancer activity by activating the mammalian target of rapamycin (mTOR) pathway. AMP-activated protein kinase (AMPK) is a cellular energy sensor that reportedly inhibits the mTOR pathway. The antidiabetic drug metformin is also a potent AMPK activator and we investigated whether it augmented panobinostat's antineoplastic activity in bladder cancer cells (UMUC3, J82, T24 and MBT-2). Metformin enhanced panobinostat-induced apoptosis and the combination inhibited the growth of bladder cancer cells cooperatively in vitro and in vivo. As expected, metformin increased the phosphorylation of AMPK and decreased the panobinostat-caused phosphorylation of S6 ribosomal protein, thus inhibiting the panobinostat-activated mTOR pathway. The AMPK activation was shown to play a pivotal role in the combination's action because the AMPK inhibitor compound C attenuated the combination's anticancer activity. Furthermore, the AMPK activation by metformin enhanced panobinostat-induced histone and non-histone acetylation. This acetylation was especially remarkable in the proteins in the detergent-insoluble fraction, which would be expected if the combination also induced endoplasmic reticulum stress.

MiR-411 suppresses the development of bladder cancer by regulating ZnT1.

BackgroundAt present, the molecular genetics of the development and progression of bladder cancer are still unclear. In recent years, the pathological relevance and significance of microRNAs (miRNAs) in bladder cancer have attracted increasing attention.MethodsThe expressions of miR-411 and zinc transporter 1 (ZnT1) in bladder cancer were determined by western blot and real-time PCR. Biological software, luciferase reporter gene, Western blot and real-time PCR were used to determine the regulatory effect of miR-411 on ZnT1. MTT and transwell were used to confirm the regulatory effect of miR-411 on bladder cancer cells. MTT and transwell were used to find how miR-411 modulated the biological activity of bladder cancer cells by regulating ZnT1.ResultsThe expression of miR-411 was low in bladder cancer and was negatively correlated with ZnT1. MiR-411 can inhibit the activity and the expression of ZnT1. MiR-411 can inhibit the growth and metastasis of bladder cancer cells. MiR-411 inhibited the growth and metastasis of bladder cancer cells by targeting ZnT1.ConclusionThe miR-411 target ZnT1 may provide a potential therapeutic target for the treatment of bladder cancer.

IN VITRO Anticancer Activity and Physicochemical Properties of SOLANUM LYCOCARPUM Alkaloidic Extract Loaded in Natural Lipid-Based Nanoparticles

Bladder cancer (BC) is the 9th most common cancer in the worldwide. BC's treatment is still a challenging goal. Thereby, the screening of natural compounds as solasonine (SS) and solamargine (SM) that exhibit antitumor activity is interesting to BC therapy. This work aimed to develop natural lipid-based nanoparticles (NLN) as carrier system of S. lycocarpum's fruits alkaloidic extract (AE), rich in SS and SM, and to evaluate its antitumor activity in bladder cancer cells. The NLN-AE exhibited 56 nm of diameter, low polydispersity index with shelf life estimated of 12 months, measured by LUMiSizer®. NLN-AE showed low recrystallization index with high encapsulation efficiency of SS (86%) and SM (89%). A sustained release profile was verified after 36 h. The free AE exhibited antitumor activity in bladder cancer cells. The cell viability decreased 5.4 fold when AE was encapsulated in NLN. Thus, NLN-AE has higher anti-proliferative activity than the free AE.

LncRNA GAS6‐AS2 promotes bladder cancer proliferation and metastasis via GAS6‐AS2/miR‐298/CDK9 axis

Long noncoding RNAs (lncRNAs) have been proved to play important roles in carcinogenesis and development of numerous cancers, but their biological functions in bladder cancer remain largely unknown. In this study, a novel lncRNA termed GAS6‐AS2 were primary identified, and its roles as well as mechanisms in regulating proliferation and metastasis of bladder cancer cells were investigated. Clinically, GAS6‐AS2 was significantly up‐regulated in bladder cancer tissues and positively correlated with tumour stages and poor prognosis. Moreover, expression of GAS6‐AS2 was also increased in bladder cancer cells compared with normal bladder cells. Further investigating the roles of GAS6‐AS2, we found GAS6‐AS2 regulated proliferation and proliferative activity of bladder cancer cells via inducing G1 phase arrest. What's more, we found that GAS6‐AS2 contributed to metastatic abilities of cells. In mechanism, GAS6‐AS2 could function as a competitive endogenous RNA (ceRNA) via direct sponging miR‐298, which further regulating the expression of CDK9. Finally, we also proved that GAS6‐AS2 knockdown suppressed tumour growth and metastasis in vivo. In conclusion, our study proved that GAS6‐AS2 could function as a ceRNA and promote the proliferation and metastasis of bladder cancer cells, which provided a novel prognostic marker for bladder cancer patients in clinic.

Metformin augments panobinostat’s antineoplastic activity in bladder cancer cells by activating AMP-activated protein kinase

Metformin augments panobinostat’s antineoplastic activity in bladder cancer cells by activating AMP-activated protein kinase

Androgen receptor activity modulates responses to cisplatin treatment in bladder cancer.

Cisplatin (CDDP)-based combination chemotherapy remains the mainstream treatment for advanced bladder cancer. However, its efficacy is often limited due to the development of resistance for which underlying mechanisms are poorly understood. Meanwhile, emerging evidence has indicated the involvement of androgen-mediated androgen receptor (AR) signals in bladder cancer progression. In this study, we aimed to investigate whether AR signals have an impact on sensitivity to CDDP in bladder cancer cells. UMUC3-control-short hairpin RNA (shRNA) cells with endogenous AR and AR-negative 647V/5637 cells stably expressing AR were significantly more resistant to CDDP treatment at its pharmacological concentrations, compared with UMUC3-AR-shRNA and 647V-vector/5637-vector control cells, respectively. A synthetic androgen R1881 significantly reduced CDDP sensitivity in UMUC3, 647V-AR, or 5637-AR cells, and the addition of an anti-androgen hydroxyflutamide inhibited the effect of R1881. In these AR-positive cells, R1881 treatment also induced the expression levels of NF-κB, which is known to involve CDDP resistance, and its phosphorylated form, as well as nuclear translocation of NF-κB. In CDDP-resistant bladder cancer sublines established following long-term culture with CDDP, the expression levels of AR as well as NF-κB and phospho-NF-κB were considerably elevated, compared with respective control sublines. In bladder cancer specimens, there was a strong trend to correlate between AR positivity and chemoresistance. These results suggest that AR activation correlates with CDDP resistance presumably via modulating NF-κB activity in bladder cancer cells. Targeting AR during chemotherapy may thus be a useful strategy to overcome CDDP resistance in patients with AR-positive bladder cancer.

CDODA-Me decreases specificity protein transcription factors and induces apoptosis in bladder cancer cells through induction of reactive oxygen species

ObjectiveThe objective is to determine whether methyl 2-cyano-3,11-dioxo-18b-olean-1,12-dien-30-oate (CDODA-Me) has therapeutic potential in bladder cancer. We investigated the effects of CDODA-Me on the growth and survival of bladder cancer cells, and expression of specificity protein (Sp) transcription factors that regulate genes associated with cancer cell proliferation and survival. MethodsJ82, RT4P, and 253JB-V bladder cancer cell lines were treated with vehicle alone or with CDODA-Me with or without the antioxidant l-glutathione. Cell viability and DNA fragmentation were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and propidium iodide-fluorescence-activated cell sorting (FACS) analysis, respectively. Intracellular reactive oxygen species (ROS) were measured by 2′,7′-dichlorofluorescin diacetate–FACS analysis. We assessed CDODA’s effects on the levels of Sp and Sp-regulated proteins and induction of apoptosis in bladder cancer cells by Western blotting. We also assessed the anticancer effects of CDODA-Me in nude mice bearing RT4v6 bladder cancer. Results3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and FACS analysis revealed that CDODA-Me inhibited the proliferation and survival of the 3 bladder cancer cell lines in a dose-dependent manner. FACS analysis also indicated that CDODA-Me–induced intracellular ROS, and Western blot analysis indicated that CDODA-Me decreased levels of Sp and Sp-regulated proteins and induced apoptosis in a dose-dependent and time-dependent manner. l-Glutathione attenuated CDODA-Me’s down-regulation of Sp and Sp-regulated proteins. Compared with the control treatment, CDODA-Me substantially inhibited tumor growth in vivo. ConclusionsCDODA-Me has antineoplastic activity in bladder cancer cells by inducing ROS, which down-regulate Sp and Sp-regulated proteins. Thus, CDODA-Me has therapeutic potential in bladder cancer, and additional studies of the agent’s efficacy and mode of action are warranted.

MP45-02 CDODA-ME DECREASES SPECIFICITY PROTEIN TRANSCRIPTION FACTORS AND INDUCES APOPTOSIS IN BLADDER CANCER CELLS THROUGH INDUCTION OF REACTIVE OXYGEN SPECIES

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I1 Apr 2016MP45-02 CDODA-ME DECREASES SPECIFICITY PROTEIN TRANSCRIPTION FACTORS AND INDUCES APOPTOSIS IN BLADDER CANCER CELLS THROUGH INDUCTION OF REACTIVE OXYGEN SPECIES Hisashi Takeuchi, Rikiya Taoka, Goodwin G Jinesh, Chinedu O Mmeje, Stephen Safe, Teiichiro Aoyagi, Masaaki Tachibana, and Ashish M Kamat Hisashi TakeuchiHisashi Takeuchi More articles by this author , Rikiya TaokaRikiya Taoka More articles by this author , Goodwin G JineshGoodwin G Jinesh More articles by this author , Chinedu O MmejeChinedu O Mmeje More articles by this author , Stephen SafeStephen Safe More articles by this author , Teiichiro AoyagiTeiichiro Aoyagi More articles by this author , Masaaki TachibanaMasaaki Tachibana More articles by this author , and Ashish M KamatAshish M Kamat More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.279AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES To determine whether CDODA-Me has therapeutic potential in bladder cancer we investigated the effects of CDODA-Me on the growth and survival of bladder cancer cells, and on these cell’s expression of specificity protein (Sp) transcription factors. METHODS J82, RT4P, and 253JB-V bladder cancer cell lines were treated with vehicle alone or with CDODA-Me with or without the antioxidant GSH. Cell viability and DNA fragmentation were measured by MTT assay and FACS analysis, respectively. Intracellular reactive oxygen species (ROS) were measured by FACS analysis. We assessed CDODA-Me’s effects on the levels of Sp and Sp-regulated proteins and induction of apoptosis in bladder cancer cells by Western blotting. We also assessed the anticancer effects of CDODA-Me in nude mice bearing RT4v6 bladder cancer. RESULTS MTT assay and FACS analysis revealed that CDODA-Me inhibited the proliferation and survival of the 3 bladder cancer cell lines in a dose-dependent manner. FACS analysis also indicated that CDODA-Me induced intracellular ROS, and Western blot analysis indicated that CDODA-Me decreased levels of Sp and Sp-regulated proteins and induced apoptosis in a dose-dependent manner. GSH attenuated CDODA-Me’s down-regulation of Sp and Sp-regulated proteins. Compared with the control treatment, CDODA-Me substantially inhibited tumor growth in vivo. CONCLUSIONS CDODA-Me has antineoplastic activity in bladder cancer cells by inducing ROS, which down-regulate Sp and Sp-regulated proteins. Thus, CDODA-Me has therapeutic potential in bladder cancer. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e607-e608 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Hisashi Takeuchi More articles by this author Rikiya Taoka More articles by this author Goodwin G Jinesh More articles by this author Chinedu O Mmeje More articles by this author Stephen Safe More articles by this author Teiichiro Aoyagi More articles by this author Masaaki Tachibana More articles by this author Ashish M Kamat More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Trichlorobenzene-substituted azaaryl compounds as novel FGFR inhibitors exhibiting potent antitumor activity in bladder cancer cells in vitro and in vivo.

In the present study, we examined the antitumor activity of a series of trichlorobenzene-substituted azaaryl compounds and identified MPT0L145 as a novel FGFR inhibitor with better selectivity for FGFR1, 2 and 3. It was preferentially effective in FGFR-activated cancer cells, including bladder cancer cell lines expressing FGFR3-TACC3 fusion proteins (RT-112, RT-4). MPT0L145 decreased the phosphorylation of FGFR1, FGFR3 and their downstream proteins (FRS2, ERK and Akt). Mechanistically, cDNA microarray analysis revealed that MPT0L145 decreased genes associated cell cycle progression, and increased genes associated with autophagy pathway. Accordingly, the data revealed that MPT0L145 induced G0/G1 cell cycle arrest and decreased protein levels of cyclin E. Moreover, we provided the evidence that autophagy contributes to FGFR inhibitor-related cell death. Finally, MPT0L145 exhibited comparable antitumor activity to cisplatin with better safety in a RT-112 xenograft model. Taken together, these findings support the utility of MPT0L145 as a novel FGFR inhibitor, providing a strong rationale for further evaluation of this compound as a therapeutic agent for bladder cancers.

Synthetic miRNA sponges driven by mutant hTERT promoter selectively inhibit the progression of bladder cancer.

The mutant promoter of human telomerase reverse transcriptase (hTERT) shows high transcriptional activity in bladder cancer cells. Some up-regulated microRNAs (miRNAs) are reported as oncogenic factors in bladder cancer. Previous studies report that miRNAs can be inhibited by base-pairing interactions. The purpose of this study is to construct a synthetic device driven by mutant hTERT promoter to suppress four up-regulated miRNAs and to verify its effects on phenotypes of bladder cancer cells and human normal cells. Tandem bulged miRNA binding sites targeting oncogenic miRNAs were inserted into the 3' untranslated region (3' UTR) of mutant hTERT promoter-driven Renilla luciferase gene to construct a synthetic tumor-specific device, miRNA sponges. A negative control was generated by using tandem repeated sequences without targeting any known miRNA. Bladder cancer cells (T24, 5637, UM-UC-3) and human fiber cells (HFC) were transfected with devices. Various functional assays were used to detect the effects of this device. The activity of the mutant hTERT promoter detected by luciferase assay was about three times as large as the wild-type hTERT promoter in bladder cancer cells, while it could not be measured in HFC. Other assays indicated that the synthetic device can significantly inhibit cell growth, decrease motility, and induce apoptosis in bladder cancer cells but not in HFC. A synthetic biology platform is employed to construct tumor-specific miRNA sponges that can be used to target oncogenic miRNAs to inhibit the progression of bladder cancer cells without affecting normal cells.

Abstract 607: Targeting PIM1 kinase enhances 1,25-dihydroxyvitamin D3-mediated anti-tumor activity in bladder cancer cells

Abstract Bladder cancer is the 5th most common in the United States. 30% of cases are advanced stage requiring multimodality therapy at diagnosis and many which are first diagnosed when superficial will progress to more advanced disease. High recurrence and progression rate and drug resistance highlight the need for new therapeutic strategies to improve clinical outcomes. 1,25-dihydroxyvitamin D3 (1,25D3), the most active vitamin D metabolite, has demonstrated broad spectrum antitumor activities in vitro and in vivo. In addition, 1,25D3 potentiates the antitumor effects of common chemotherapeutic agents in several cancer cell types including bladder cancer. 24-hydroxylase, encoded by the CYP24A1 gene, plays a key role in degrading 1,25D3. Increased expression of CYP24A1 has been found in several human tumors. Several studies reveal that the protein kinase signaling pathways including PIM1 are involved in the regulation of CYP24A1 expression. The PIM kinases are a family of serine/threonine kinases (PIM-1, PIM-2, PIM-3) that have been associated with tumorigenesis and drug resistance. PIM1 has been extensively studied in lymphoma and upregulation of PIM kinase expression has been reported in many malignancies. We analyzed mRNA expression of CYP24A1 and PIM1 in 17 bladder cancer cell lines by qRT-PCR. We found that CYP24A1 expression is increased in 12 out of 17 bladder cancer cell lines and PIM1 expression is increased in 8 out of 17 bladder cancer cell lines compared to expression in a benign bladder cancer cell line. Therefore, we hypothesized that inhibition of PIM kinase activity would suppress CYP24A1 expression and thus enhancing 1,25D3-mediated anti-tumor activity. Using the PIM1 inhibitor, JP11646, we found that inhibition of PIM1 kinase activity reduces CYP24A1 expression at transcriptional level in bladder cancer cells RT112 and RT112D21 which express high level of PIM1 and CYP24A1. We further evaluated the efficacy of the PIM1 inhibitor alone and in combination with 1,25D3. Inhibition of PIM1 kinase activity by JP11646 reduces cell growth in a panel of bladder cancer cell lines in a dose-dependent manner as measured by MTT assays. MTT assays detected a significant inhibition of cell proliferation by 50% compared to the control when cells were treated with 128 nM of JP11646 for 72 h in 6 of bladder cancer cell lines tested. Furthermore, inhibition of PIM1 kinase activity enhances 1,25D3-mediated inhibitory effect on cell growth in bladder cancer cells RT112 and RT112D21. This study provides the first evidence that inhibition of PIM kinase activity may be an attractive therapeutic strategy to facilitate the optimization of antitumor therapy of vitamin D in bladder cancer. This study was supported by NIH/NCI grants 5R01CA067267 and 5R01CA095045 Citation Format: Wei Luo, Carmen M. Baldino, Justin Caserta, Yingyu Ma, Candace Johnson, Donald L. Trump. Targeting PIM1 kinase enhances 1,25-dihydroxyvitamin D3-mediated anti-tumor activity in bladder cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 607. doi:10.1158/1538-7445.AM2014-607