The LAT1 inhibitor JPH203 suppresses the growth of castration-resistant prostate cancer through a CD24-mediated mechanism.

L-type amino acid transporter 1 (LAT1) is specifically expressed in many malignancies, contributes to the transport of essential amino acids, such as leucine, and regulates the mammalian target of rapamycin (mTOR) signaling pathway. We investigated the expression profile and functional role of LAT1 in prostate cancer using JPH203, a specific inhibitor of LAT1. LAT1 was highly expressed in castration-resistant prostate cancer (CRPC) cells, including C4-2 and PC-3 cells, but its expression level was low in castration-sensitive LNCaP cells. JPH203 significantly inhibited [14C] leucine uptake in CRPC cells but had no effect in LNCaP cells. JPH203 inhibited the proliferation, migration, and invasion of CRPC cells but not of LNCaP cells. In C4-2 cells, Cluster of differentiation (CD) 24 was identified by RNA sequencing as a novel downstream target of JPH203. CD24 was downregulated in a JPH203 concentration-dependent manner and suppressed activation of the Wnt/ß-catenin signaling pathway. Furthermore, an in vivo study showed that JPH203 inhibited the proliferation of C4-2 cells in a castration environment. The results of this study indicate that JPH203 may exert its antitumor effect in CRPC cells via mTOR and CD24.

Upregulation of ATF4 mediates the cellular adaptation to pharmacologic inhibition of amino acid transporter LAT1 in pancreatic ductal adenocarcinoma cells.

L-type amino acid transporter 1 (LAT1) is recognized as a promising target for cancer therapy; however, the cellular adaptive response to its pharmacological inhibition remains largely unexplored. This study examined the adaptive response to LAT1 inhibition using nanvuranlat, a high-affinity LAT1 inhibitor. Proteomic analysis revealed the activation of a stress-induced transcription factor ATF4 following LAT1 inhibition, aligning with the known cellular responses to amino acid deprivation. This activation was linked to the GCN2-eIF2α pathway which regulates translation initiation. Our results show that ATF4 upregulation counteracts the suppressive effect of nanvuranlat on cell proliferation in pancreatic ductal adenocarcinoma cell lines, suggesting a role for ATF4 in cellular adaptation to LAT1 inhibition. Importantly, dual targeting of LAT1 and ATF4 exhibited more substantial anti-proliferative effects in vitro than individual treatments. This study underscores the potential of combining LAT1 and ATF4 inhibition as a therapeutic strategy in cancer treatment.

Identification of tumor-suppressive miRNAs that target amino acid transporter LAT1 and exhibit anti-proliferative effects on cholangiocarcinoma cells.

Amino acid transporter LAT1 is highly upregulated in various cancer types, including cholangiocarcinoma (CHOL), and contributes to the rapid proliferation of cancer cells and disease progression. However, the molecular mechanisms underlying the pathological upregulation of LAT1 remain largely unknown. This study pursued the possibility of miRNA-mediated regulation of the LAT1 expression in CHOL cells. Using online target prediction methods, we extracted five candidate miRNAs commonly predicted to regulate the LAT1 expression. Three of them, miR-194-5p, miR-122-5p, and miR-126-3p, were significantly downregulated in CHOL cancer compared to normal tissues. Correlation analysis revealed weak-to-moderate negative correlations between the expression of these miRNAs and LAT1 mRNA in CHOL cancer tissues. We selected miR-194-5p and miR-122-5p for further analyses and found that both miRNAs functionally target 3'UTR of LAT1 mRNA by a luciferase-based reporter assay. Transfection of the miRNA mimics significantly suppressed the LAT1 expression at mRNA and protein levels and inhibited the proliferation of CHOL cells, with a trend of affecting intracellular amino acids and amino acid-related signaling pathways. This study indicates that the decreased expression of these LAT1-targeting tumor-suppressive miRNAs contributes to the upregulation of LAT1 and the proliferation of CHOL cells, highlighting their potential for developing novel cancer therapeutics and diagnostics.

Inhibition of amino acid transporter LAT1 in cancer cells suppresses G0/G1-S transition by downregulating cyclin D1 via p38 MAPK activation.

L-type amino acid transporter 1 (LAT1, SLC7A5) is upregulated in various cancers and associated with disease progression. Nanvuranlat (Nanv; JPH203, KYT-0353), a selective LAT1 inhibitor, suppresses the uptake of large neutral amino acids required for rapid growth and proliferation of cancer cells. Previous studies have suggested that the inhibition of LAT1 by Nanv induces the cell cycle arrest at G0/G1 phase, although the underlying mechanisms remain unclear. Using pancreatic cancer cells arrested at the restriction check point (R) by serum deprivation, we found that the Nanv drastically suppresses the G0/G1-S transition after release. This blockade of the cell cycle progression was accompanied by a sustained activation of p38 mitogen-activated protein kinase (MAPK) and subsequent phosphorylation-dependent proteasomal degradation of cyclin D1. Isoform-specific knockdown of p38 MAPK revealed the predominant contribution of p38α. Proteasome inhibitors restored the cyclin D1 amount and released the cell cycle arrest caused by Nanv. The increased phosphorylation of p38 MAPK and the decrease of cyclin D1 were recapitulated in xenograft tumor models treated with Nanv. This study contributes to delineating the pharmacological activities of LAT1 inhibitors as anti-cancer agents and provides significant insights into the molecular basis of the amino acid-dependent cell cycle checkpoint at G0/G1 phase.

Structure-activity characteristics of phenylalanine analogs selectively transported by L-type amino acid transporter 1 (LAT1).

L-type amino acid transporter 1 (LAT1) is a transmembrane protein responsible for transporting large neutral amino acids. While numerous LAT1-targeted compound delivery for the brain and tumors have been investigated, their LAT1 selectivity often remains ambiguous despite high LAT1 affinity. This study assessed the LAT1 selectivity of phenylalanine (Phe) analogs, focusing on their structure-activity characteristics. We discovered that 2-iodo-L-phenylalanine (2-I-Phe), with an iodine substituent at position 2 in the benzene ring, markedly improves LAT1 affinity and selectivity compared to parent amino acid Phe, albeit at the cost of reduced transport velocity. L-Phenylglycine (Phg), one carbon shorter than Phe, was found to be a substrate for LAT1 with a lower affinity, exhibiting a low level of selectivity for LAT1 equivalent to Phe. Notably, (R)-2-amino-1,2,3,4-tetrahydro-2-naphthoic acid (bicyclic-Phe), with an α-methylene moiety akin to the α-methyl group in α-methyl-L-phenylalanine (α-methyl-Phe), a known LAT1-selective compound, showed similar LAT1 transport maximal velocity to α-methyl-Phe, but with higher LAT1 affinity and selectivity. In vivo studies revealed tumor-specific accumulation of bicyclic-Phe, underscoring the importance of LAT1-selectivity in targeted delivery. These findings emphasize the potential of bicyclic-Phe as a promising LAT1-selective component, providing a basis for the development of LAT1-targeting compounds based on its structural framework.

Transcriptomic Analysis Provides Insights into Candidate Genes and Molecular Pathways Involved in Growth of Mytilus coruscus Larvae.

Growth is a fundamental aspect of aquaculture breeding programs, pivotal for successful cultivation. Understanding the mechanisms that govern growth and development differences across various stages can significantly boost seedling production of economically valuable species, thereby enhancing aquaculture efficiency and advancing the aquaculture industry. Mytilus coruscus, a commercially vital marine bivalve, underscores this importance. To decipher the intricate molecular mechanisms dictating growth and developmental disparities in marine shellfish, we conducted transcriptome sequencing and meticulously analyzed gene expression variations and molecular pathways linked to growth traits in M. coruscus. This study delved into the molecular and gene expression variations across five larval development stages, with a specific focus on scrutinizing the differential expression patterns of growth-associated genes using RNA sequencing and quantitative real-time PCR analysis. A substantial number of genes-36,044 differentially expressed genes (DEGs)-exhibited significant differential expression between consecutive developmental stages. These DEGs were then categorized into multiple pathways (Q value < 0.05), including crucial pathways such as the spliceosome, vascular smooth muscle contraction, DNA replication, and apoptosis, among others. In addition, we identified two pivotal signaling pathways-the Hedgehog (Hh) signaling pathway and the TGF-beta (TGF-ß) signaling pathway-associated with the growth and development of M. coruscus larvae. Ten key growth-related genes were pinpointed, each playing crucial roles in molecular function and the regulation of growth traits in M. coruscus. These genes and pathways associated with growth provide deep insights into the molecular basis of physiological adaptation, metabolic processes, and growth variability in marine bivalves.

L-type amino acid transporter 1 inhibitor JPH203 prevents the growth of cabazitaxel-resistant prostate cancer by inhibiting cyclin-dependent kinase activity.

L-type amino acid transporter 1 (LAT1, SLC7A5) is an amino acid transporter expressed in various carcinomas, and it is postulated to play an important role in the proliferation of cancer cells through the uptake of essential amino acids. Cabazitaxel is a widely used anticancer drug for treating castration-resistant prostate cancer (CRPC); however, its effectiveness is lost when cancer cells acquire drug resistance. In this study, we investigated the expression of LAT1 and the effects of a LAT1-specific inhibitor, JPH203, in cabazitaxel-resistant prostate cancer cells. LAT1 was more highly expressed in the cabazitaxel-resistant strains than in the normal strains. Administration of JPH203 inhibited the growth, migration, and invasive ability of cabazitaxel-resistant strains in vitro. Phosphoproteomics using liquid chromatography-mass spectrometry to comprehensively investigate changes in phosphorylation due to JPH203 administration revealed that cell cycle-related pathways were affected by JPH203, and that JPH203 significantly reduced the kinase activity of cyclin-dependent kinases 1 and 2. Moreover, JPH203 inhibited the proliferation of cabazitaxel-resistant cells in vivo. Taken together, the present study results suggest that LAT1 might be a valuable therapeutic target in cabazitaxel-resistant prostate cancer.

Nicotinamide mononucleotide pretreatment improves long-term isoflurane anesthesia-induced cognitive impairment in mice.

Postoperative cognitive dysfunction (POCD) is characterized by impaired cognitive function following general anesthesia and surgery. Oxidative stress is a significant pathophysiological manifestation underlying POCD. Previous studies have reported that the decline of nicotinamide adenine dinucleotide (NAD+) -dependent sirtuin 1 (SIRT1) contributes to the activation of oxidative stress. In this study, we investigated whether pretreatment of nicotinamide mononucleotide (NMN), an NAD+ intermediate, improves oxidative stress and cognitive function in POCD. The animal model of POCD was established in C57BL/6 J mice through 6 h isoflurane anesthesia-induced cognitive impairment. Mice were intraperitoneally injected with NMN for 7 days prior to anesthesia, after which oxidative stress and cognitive function were assessed. The level of oxidative stress was determined using flow cytometry analysis and assey kits. The fear condition test and the Y-maze test were utilized to evaluate contextual and spatial memory. Our results showed that cognitive impairment and increased oxidative stress were observed in POCD mice, as well as downregulation of NAD+ levels and related protein expressions of SIRT1 and nicotinamide phosphoribosyltransferase (NAMPT) in the hippocampus. And NMN supplementation could effectively prevent the decline of NAD+ and related proteins, and reduce oxidative stress and cognitive disorders after POCD. Mechanistically, the findings suggested that protection on cognitive function mediated by NMN pretreatment in POCD mice may be regulated by NAD+-SIRT1 signaling pathway. This study indicated that NMN preconditioning reduced oxidative stress damage and alleviated cognitive impairment in POCD mice.

Long-term sevoflurane exposure relieves stress-enhanced fear learning and anxiety in PTSD mice.

Objectives: Post-traumatic stress disorder (PTSD) is characterized by recurrent episodes of severe anxiety after exposure to traumatic events. It is believed that these episodes are triggered at least in part by environmental stimuli associated with the precipitating trauma through classical conditioning, termed conditioned fear. However, traditional methods of conditioned fear memory extinction are frequently ineffective for PTSD treatment due to the contribution of non-associative sensitization caused by trauma. Anesthetics have shown promise for treating various psychiatric diseases such as depression. Methods: In this study, we examined if the inhaled anesthetic sevoflurane can suppress stress-enhanced fear learning (SEFL) in PTSD model mice. Model mice exposed to 2.4% sevoflurane for 6 h exhibited reduced freezing time and behavioral anxiety compared to sham-treated model mice. To explore the underlying mechanisms, we evaluated the regional expression levels of glucocorticoid receptors (GRs), cannabinoid CB1 receptors (CB1Rs), D1 dopamine receptors (D1Rs), and D2 dopamine receptors (D2Rs). Results: We verified that both GR and CB1R were significantly upregulated in the hippocampus, amygdaloid nucleus, and prefrontal cortex (PFC) of model mice, while D1R and D2R were downregulated. All of these expression changes were partially normalized in the PFC by 6 h but not with 2 h sevoflurane exposure. Conclusions: These results showed that sevoflurane exposure following traumatic events may be an effective treatment for PTSD.

Inhibition of cancer-type amino acid transporter LAT1 suppresses B16-F10 melanoma metastasis in mouse models.

Metastasis is the leading cause of mortality in cancer patients. L-type amino acid transporter 1 (LAT1, SLC7A5) is a Na+-independent neutral amino acid transporter highly expressed in various cancers to support their growth. Although high LAT1 expression is closely associated with cancer metastasis, its role in this process remains unclear. This study aimed to investigate the effect of LAT1 inhibition on cancer metastasis using B16-F10 melanoma mouse models. Our results demonstrated that nanvuranlat (JPH203), a high-affinity LAT1-selective inhibitor, suppressed B16-F10 cell proliferation, migration, and invasion. Similarly, LAT1 knockdown reduced cell proliferation, migration, and invasion. LAT1 inhibitors and LAT1 knockdown diminished B16-F10 lung metastasis in a lung metastasis model. Furthermore, nanvuranlat and LAT1 knockdown suppressed lung, spleen, and lymph node metastasis in an orthotopic metastasis model. We discovered that the LAT1 inhibitor reduced the cell surface expression of integrin αvß3. Our findings revealed that the downregulation of the mTOR signaling pathway, induced by LAT1 inhibitors, decreased the expression of integrin αvß3, contributing to the suppression of metastasis. These results highlight the critical role of LAT1 in cancer metastasis and suggest that LAT1 inhibition may serve as a potential target for anti-metastasis cancer therapy.

Socioeconomic status may affect association of vegetable intake with risk of ischemic cardio-cerebral vascular disease: a Mendelian randomization study.

Background: Previous studies found that increasing vegetable intake benefits are reduced after adjustment for socioeconomic factors. Using genetic variation as an instrumental variable for vegetable intake and socioeconomic status, we investigated the relationship between vegetable intake and ischemic cardio-cerebral vascular diseases and focused on whether socioeconomic status was a possible confounder. Methods: From three independent genome-wide association studies, we extracted instrumental variables reflecting raw and cooked vegetable intake, which were used to perform Mendelian randomization analysis. To evaluate the effects of socioeconomic factors on vegetable intake, univariate and multivariate Mendelian randomization analyses were performed using single nucleotide polymorphisms representing education attainment and household income reported in the literature. We also performed outlier assessment and a series of sensitivity analyses to confirm the results. Results: Genetically predicted raw and cooked vegetable intake were not associated with any ischemic cardio-cerebral vascular diseases and lipid components after Bonferroni correction. Univariate Mendelian randomized analysis revealed that raw vegetable intake was positively correlated with education attainment (ß = 0.04, p = 0.029) and household income (ß = 0.07, p < 0.001). Multivariate Mendelian randomized model showed a positive correlation between household income and raw vegetable intake (ß = 0.06, p = 0.004). Socioeconomic status was closely associated with eating habits and lifestyle related to the risk of cardiovascular diseases. Conclusion: Genetically determined raw and cooked vegetable intake was not associated with significant benefits in terms of ischemic cardio-cerebral vascular diseases while genetically determined socioeconomic status may have an impact on vegetable intake. Socioeconomic status, which was closely associated with other eating habits and lifestyle, may affect the association between vegetable intake and ischemic cardio-cerebral vascular diseases.

Combination effects of amino acid transporter LAT1 inhibitor nanvuranlat and cytotoxic anticancer drug gemcitabine on pancreatic and biliary tract cancer cells.

BACKGROUND: Cytotoxic anticancer drugs widely used in cancer chemotherapy have some limitations, such as the development of side effects and drug resistance. Furthermore, monotherapy is often less effective against heterogeneous cancer tissues. Combination therapies of cytotoxic anticancer drugs with molecularly targeted drugs have been pursued to solve such fundamental problems. Nanvuranlat (JPH203 or KYT-0353), an inhibitor for L-type amino acid transporter 1 (LAT1; SLC7A5), has novel mechanisms of action to suppress the cancer cell proliferation and tumor growth by inhibiting the transport of large neutral amino acids into cancer cells. This study investigated the potential of the combined use of nanvuranlat and cytotoxic anticancer drugs. METHODS: The combination effects of cytotoxic anticancer drugs and nanvuranlat on cell growth were examined by a water-soluble tetrazolium salt assay in two-dimensional cultures of pancreatic and biliary tract cancer cell lines. To elucidate the pharmacological mechanisms underlying the combination of gemcitabine and nanvuranlat, we investigated apoptotic cell death and cell cycle by flow cytometry. The phosphorylation levels of amino acid-related signaling pathways were analyzed by Western blot. Furthermore, growth inhibition was examined in cancer cell spheroids. RESULTS: All the tested seven types of cytotoxic anticancer drugs combined with nanvuranlat significantly inhibited the cell growth of pancreatic cancer MIA PaCa-2 cells compared to their single treatment. Among them, the combined effects of gemcitabine and nanvuranlat were relatively high and confirmed in multiple pancreatic and biliary tract cell lines in two-dimensional cultures. The growth inhibitory effects were suggested to be additive but not synergistic under the tested conditions. Gemcitabine generally induced cell cycle arrest at the S phase and apoptotic cell death, while nanvuranlat induced cell cycle arrest at the G0/G1 phase and affected amino acid-related mTORC1 and GAAC signaling pathways. In combination, each anticancer drug basically exerted its own pharmacological activities, although gemcitabine more strongly influenced the cell cycle than nanvuranlat. The combination effects of growth inhibition were also verified in cancer cell spheroids. CONCLUSIONS: Our study demonstrates the potential of first-in-class LAT1 inhibitor nanvuranlat as a concomitant drug with cytotoxic anticancer drugs, especially gemcitabine, on pancreatic and biliary tract cancers.

The Complete Mitochondrial Genome of Mytilisepta virgata (Mollusca: Bivalvia), Novel Gene Rearrangements, and the Phylogenetic Relationships of Mytilidae.

The circular mitochondrial genome of Mytilisepta virgata spans 14,713 bp, which contains 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, and 22 transfer RNA genes. Analysis of the 13 PCGs reveals that the mitochondrial gene arrangement of Mytilisepta is relatively conserved at the genus level. The location of the atp8 gene in Mytilisepta keenae differs from that of other species. However, compared with the putative molluscan ancestral gene order, M. virgata exhibits a high level of rearrangement. We constructed phylogenetic trees based on concatenated 12 PCGs from Mytilidae. As a result, we found that M. virgata is in the same clade as other Mytilisepta spp. The result of estimated divergence times revealed that M. virgata and M. keenae diverged around the early Paleogene period, although the oldest Mytilisepta fossil was from the late or upper Eocene period. Our results provide robust statistical evidence for a sister-group relationship within Mytilida. The findings not only confirm previous results, but also provide valuable insights into the evolutionary history of Mytilidae.

Insights into the Deep Phylogeny and Novel Gene Rearrangement of Mytiloidea from Complete Mitochondrial Genome.

The extant marine mussels which belong to the Mytiloidea are widespread species inhabiting mostly coastal waters, with some distributed in the deep sea. To clarify the classification systems and phylogenetic relationships range from genus to family level within Mytiloidea, new sequence was used in a phylogenetic analysis including all the available Mytiloidea mitochondrial genomes. In this study, the complete mitochondrial genome of Vignadula atrata is 15,624 bp in length and contains 12 protein-coding genes (PCGs, atp8 is absent), two ribosomal RNA genes, and 22 transfer RNA genes. Phylogenetic analysis based on 12 PCGs showed that it has a close relationship to Bathymodiolus. The analysis of gene rearrangements in the Pteriomorphia showed that the arrangements are highly variable across species, novel gene rearrangements were found within Mytiloidea. The V. atrata mitogenome was provided in detail, with notes on the sequence and a key to the species of Vignadula. This study provides a perspective on the taxonomic histories of the marine mussels and refines the unclear relationship between the origin and evolution of species in Mytiloidea within Bivalvia.

Safety evaluation of FAD2 RNAi transgenic Brassica napus L. based on microbial diversity and metabonomic analysis.

Oleic acid desaturase (FAD2) is the key enzyme that produces polyunsaturated fatty acids in rapeseed (Brassica napus L), which is one of the main oil crops. RNA interference (RNAi) is an emerging technique that provides new opportunities for the generation of new traits in plants. To increase oleic acid content and reduce linoleic and linolenic acid content in rapeseed, we constructed an ihpRNA plant expression vector of the FAD2 gene and obtained transgenic plants for multiple generations by stable inheritance. In this study, third-generation transgenic plants (T3), seventh-generation transgenic plants (T7), and wild-type plants (WT) were used. The differences in microbial community diversity between transgenic plants and wild-type plants and the up- and downregulation of rhizosphere metabolite contents were investigated. In conclusion, the results showed that the soil microbial community structure was stable, the general microbial community structure was not changed by the transgenic rhizosphere exudates, and no significant harmful root exudate of transgenic rapeseed on the environment was found through the microbial community and metabolomics analysis. This work may provide an understanding of the impact of RNAi on plant metabolites and a safety evaluation method for transgenic plants and a reference for rapeseed breeding.

Mild acute stress prevents the memory impairment induced by long-term isoflurane anesthesia.

Objectives: Long-term isoflurane anesthesia exposure could result in postoperative cognitive dysfunction (POCD). Preoperative stress is also reported to be a risk factor of POCD. However, it is unknown whether acute stress could impair memory after long-term isoflurane anesthesia. Methods: In this study, we categorized the mice with acute stress into mild (30 min restraint stress), moderate (60 min restraint stress), and severe (120 min restraint stress) stress groups and then we used Open-Field Test (OFT) to detect whether different scales of acute restraint stress successfully induced acute stress in mice. The memory performance of mice was measured using contextual and cued memory test, and the brain-derived neurotrophic factor protein levels of hippocampus was detected by Western blot. Results: We verified that mild stress has pro-cognitive effect, but severe stress has amnestic effect. Moreover, we found that mild and moderate other than severe acute stress could partially attenuate the memory impairment induced by long-term isoflurane anesthesia. Conclusion: Mild and moderate acute stress could partially attenuate the memory impairment induced by long-term isoflurane anesthesia.

Comprehensive Analysis of Glutamate Receptor-like Genes in Rice (Oryza sativa L.): Genome-Wide Identification, Characteristics, Evolution, Chromatin Accessibility, gcHap Diversity, Population Variation and Expression Analysis.

Glutamate receptors (GLR) are widely present in animals and plants, playing essential roles in regulating plant growth, development and stress response. At present, most studies of GLRs in plants are focused on Arabidopsis thaliana, while there have been few studies on rice. In this study, we identified 26 OsGLR genes in rice (Oryza sativa L.). Then, we analyzed the chromosomal location, physical and chemical properties, subcellular location, transmembrane (TM) helices, signal peptides, three-dimensional (3D) structure, cis-acting elements, evolution, chromatin accessibility, population variation, gene-coding sequence haplotype (gcHap) and gene expression under multiple abiotic stress and hormone treatments. The results showed that out of the 26 OsGLR genes, ten genes had the TM domain, signal peptides and similar 3D structures. Most OsGLRs exhibited high tissue specificity in expression under drought stress. In addition, several OsGLR genes were specifically responsive to certain hormones. The favorable gcHap of many OsGLR genes in modern varieties showed obvious differentiation between Xian/indica and Geng/japonica subspecies. This study, for the first time, comprehensively analyzes the OsGLR genes in rice, and provides an important reference for further research on their molecular function.

Phosphoproteomics revealed cellular signals immediately responding to disruption of cancer amino acid homeostasis induced by inhibition of L-type amino acid transporter 1.

BACKGROUND: Cancer-upregulated L-type amino acid transporter 1 (LAT1; SLC7A5) supplies essential amino acids to cancer cells. LAT1 substrates are not only needed for cancer rapid growth, but involved in cellular signaling. LAT1 has been proposed as a potential target for cancer treatment-its inhibitor, JPH203, is currently in clinical trials and targets biliary tract cancer (BTC). Here, we revealed to what extent LAT1 inhibitor affects intracellular amino acid content and what kind of cellular signals are directly triggered by LAT1 inhibition. METHODS: Liquid chromatography assay combined with o-phthalaldehyde- and 9-fluorenyl-methylchloroformate-based derivatization revealed changes in intracellular amino acid levels induced by LAT1 inhibition with JPH203 treatment in three BTC cell lines. Tandem mass tag-based quantitative phosphoproteomics characterized the effect of JPH203 treatment on BTC cells, and suggested key regulators in LAT1-inhibited cells. We further studied one of the key regulators, CK2 protein kinase, by using Western blot, enzymatic activity assay, and co-immunoprecipitation. We evaluated anticancer effects of combination of JPH203 with CK2 inhibitor using cell growth and would healing assay. RESULTS: JPH203 treatment decreased intracellular levels of LAT1 substrates including essential amino acids of three BTC cell lines, immediately and drastically. We also found levels of some of these amino acids were partially recovered after longer-time treatment. Therefore, we performed phosphoproteomics with short-time JPH203 treatment prior to the cellular compensatory response, and revealed hundreds of differentially phosphorylated sites. Commonly downregulated phosphorylation sites were found on proteins involved in the cell cycle and RNA splicing. Our phosphoproteomics also suggested key regulators immediately responding to LAT1 inhibition. Focusing on one of these regulators, protein kinase CK2, we revealed LAT1 inhibition decreased phosphorylation of CK2 substrate without changing CK2 enzymatic activity. Furthermore, LAT1 inhibition abolished interaction between CK2 and its regulatory protein NOLC1, which suggests regulatory mechanism of CK2 substrate protein specificity controlled by LAT1 inhibition. Moreover, we revealed that the combination of JPH203 with CK2 inhibitor resulted in the enhanced inhibition of proliferation and migration of BTC cells. CONCLUSION: This study provides new perspectives on LAT1-dependent cellular processes and a rationale for therapeutics targeting reprogrammed cancer metabolism.

Inhibition of the integrated stress response reverses oxidative stress damage-induced postoperative cognitive dysfunction.

Postoperative cognitive dysfunction (POCD) is a common complication following anesthesia and surgery that might lead to a decline in learning and memory. Oxidative stress damage is one of the pathogenic mechanisms underlying POCD. Recent studies had shown that the integrated stress response (ISR) is closely related to oxidative stress. The core response of the ISR is phosphorylation of eIF2α. Various cellular stress stimuli trigger activation of eIF2α kinases, thus causing phosphorylation of eIF2α. ISR is associated with many neurodegenerative diseases; however, the relationship between POCD and ISR has not been defined. In the present study, the tibias in 4-month-old male C57BL/6 mice were fractured under isoflurane anesthesia to establish the POCD animal model. Cognitive function was assessed by fear conditioning tests and the Y-maze from 3 to 14 days post-surgery. Western blot was used to determine the levels of PeIF2α, eIF2α, ATF4, GADD34, CHOP, BDNF, proBDNF, and p-NR2B expression. The levels of reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) were measured to determine oxidative stress in hippocampal tissues. After tibial fracture surgery in mice, the hippocampus had increased levels of PeIF2α, ATF4, GADD34, and CHOP protein, ROS-positive cells, and average fluorescence intensity, SOD activity was decreased, and the MDA level was increased. The ISR inhibitor, ISRIB, reduced the levels of PeIF2α, ATF4, GADD34, and CHOP protein, and alleviated oxidative stress in the hippocampus of POCD mice. Moreover, ISRIB ameliorated cognitive dysfunction in POCD mice. Our findings suggested that targeting ISR may represent an effective approach to combat POCD.

Pharmacologic inhibition of LAT1 predominantly suppresses transport of large neutral amino acids and downregulates global translation in cancer cells.

L-type amino acid transporter 1 (LAT1; SLC7A5), which preferentially transports large neutral amino acids, is highly upregulated in various cancers. LAT1 supplies cancer cells with amino acids as substrates for enhanced biosynthetic and bioenergetic reactions and stimulates signalling networks involved in the regulation of survival, growth and proliferation. LAT1 inhibitors show anti-cancer effects and a representative compound, JPH203, is under clinical evaluation. However, pharmacological impacts of LAT1 inhibition on the cellular amino acid transport and the translational activity in cancer cells that are conceptually pivotal for its anti-proliferative effect have not been elucidated yet. Here, we demonstrated that JPH203 drastically inhibits the transport of all the large neutral amino acids in pancreatic ductal adenocarcinoma cells. The inhibitory effects of JPH203 were observed even in competition with high concentrations of amino acids in a cell culture medium. The analyses of the nutrient-sensing mTORC1 and GAAC pathways and the protein synthesis activity revealed that JPH203 downregulates the global translation. This study demonstrates a predominant contribution of LAT1 to the transport of large neutral amino acids in cancer cells and the suppression of protein synthesis by JPH203 supposed to underly its broad anti-proliferative effects across various types of cancer cells.