L-type amino acid transporter 1 (LAT1) plays a role in transporting essential amino acids including leucine, which regulates the mTOR signaling pathway. Here, we studied the expression profile and functional role of LAT1 in bladder cancer. Furthermore, the pharmacological activity of JPH203, a specific inhibitor of LAT1, was studied in bladder cancer. LAT1 expression in bladder cancer cells was higher than that in normal cells. SiLAT1 and JPH203 suppressed cell proliferative and migratory and invasive abilities in bladder cancer cells. JPH203 inhibited leucine uptake by > 90%. RNA-seq analysis identified insulin-like growth factor-binding protein-5 (IGFBP-5) as a downstream target of JPH203. JPH203 inhibited phosphorylation of MAPK / Erk, AKT, p70S6K and 4EBP-1. Multivariate analysis revealed that high LAT1 expression was found as an independent prognostic factor for overall survival (HR3.46 P = 0.0204). Patients with high LAT1 and IGFBP-5 expression had significantly shorter overall survival periods than those with low expression (P = 0.0005). High LAT1 was related to the high Grade, pathological T stage, LDH, and NLR. Collectively, LAT1 significantly contributed to bladder cancer progression. Targeting LAT1 by JPH203 may represent a novel therapeutic option in bladder cancer treatment.Bladder cancer (BC) is the ninth most common malignant tumour worldwide, with 430 000 patients newly diagnosed and 165 000 deaths annually 1 . The pathological type of BC is mainly urothelial cancer ( > 90%) and approximately 70% of patients had non-muscle-invasive BC at diagnosis 2 . These patients have a favorable prognosis with transurethral resection and subsequent intravesical injection therapy, whereas the survival rate of patients with locally advanced and metastatic BC is poor 3 . For metastatic BC patients, platinum-based systematic chemotherapy is the classical treatment, while immunotherapy targeting programmed cell death ligand 1 (PD-L1) blocking antibody was recently approved in Japan 4 . However, drug resistance will occur, and the survival benefit of these agents is not adequate. Their limited efficacy is due to side effects and challenges of drug resistance, leading to treatment failure and require additional treatment options 5 . Therefore, more effective and less toxic therapeutic strategies are needed for the treatment of metastatic BC. Additionally, there are presently no useful diagnostic markers for BC. The urine cytology test is a non-invasive examination, but its sensitivity remains low. Cystoscopy is an essential diagnostic tool but is invasive for patients 5 . Thus, a novel therapeutic approach and biomarker candidates for BC remain a major issue.
The mammalian carboxylesterases (CESs) comprise a multigene family which gene products play important roles in biotransformation of ester- or amide-type prodrugs. Since expression level of CESs may affect the pharmacokinetic behavior of prodrugs in vivo, it is important to understand the transcriptional regulation mechanism of the CES genes. However, little is known about the gene structure and transcriptional regulation of the mammalian CES genes. In the present study, to investigate the transcriptional regulation of the promoter region of the CES1 and CES2 genes were isolated from mouse, rat and human genomic DNA by PCR amplification. A TATA box was not found the transcriptional start site of all CES promoter. These CES promoters share several common binding sites for transcription factors among the same CES families, suggesting that the orthologous CES genes have evolutionally conserved transcriptional regulatory mechanisms. The result of present study suggested that the mammalian CES promoters were at least partly conserved among the same CES families, and some of the transcription factors may play similar roles in transcriptional regulation of the human and murine CES genes.
Brain microvascular endothelial cells (BMEC), together with astrocytes and pericytes, form the blood−brain barrier (BBB) that strictly restricts drug penetration into the brain. Therefore, in central nervous system drug development, the establishment of an in vitro human BBB model for use in studies estimating the in vivo human BBB permeability of drug candidates has long been awaited. The current study developed and characterized a human immortalized cellbased BBB triculture model, termed the "hiBBB" model. To set up the hiBBB model, human immortalized BMEC (HBMEC/ci18) were cocultured with human immortalized astrocytes (HASTR/ci35) and brain pericytes (HBPC/ci37) in a transwell system. The trans-endothelial electrical resistance of the hiBBB model was 134.4 ± 5.5 (Ω × cm 2 ), and the efflux ratios of rhodamine123 and dantrolene were 1.72 ± 0.11 and 1.72 ± 0.45, respectively, suggesting that the hiBBB model possesses essential cellular junction and efflux transporter functions. In BBB permeability assays, the mean value of the permeability coefficients (P e ) of BBB permeable compounds (propranolol, pyrilamine, memantine, and diphenhydramine) was 960 × 10 −6 cm/s, which was clearly distinguishable from that of BBB nonpermeable compounds (sodium fluorescein and Lucifer yellow, 18 × 10 −6 cm/s). Collectively, this study successfully developed the hiBBB model, which exhibits essential BBB functionality. Taking into consideration the high availability of the immortalized cells used in the hiBBB model, our results are expected to become an initial step toward the establishment of a useful human BBB model to investigate drug penetration into the human brain.
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