Preconditioning-Induced Facilitation of Lactate Release from Astrocytes Is Essential for Brain Ischemic Tolerance.

A sublethal ischemic episode [termed preconditioning (PC)] protects neurons in the brain against a subsequent severe ischemic injury. This phenomenon is known as brain ischemic tolerance and has received much attention from researchers because of its robust neuroprotective effects. We have previously reported that PC activates astrocytes and subsequently upregulates P2X7 receptors, thereby leading to ischemic tolerance. However, the downstream signals of P2X7 receptors that are responsible for PC-induced ischemic tolerance remain unknown. Here, we show that PC-induced P2X7 receptor-mediated lactate release from astrocytes has an indispensable role in this event. Using a transient focal cerebral ischemia model caused by middle cerebral artery occlusion, extracellular lactate levels during severe ischemia were significantly increased in mice who experienced PC; this increase was dependent on P2X7 receptors. In addition, the intracerebroventricular injection of lactate protected against cerebral ischemic injury. In in vitro experiments, although stimulation of astrocytes with the P2X7 receptor agonist BzATP had no effect on the protein levels of monocarboxylate transporter (MCT) 1 and MCT4 (which are responsible for lactate release from astrocytes), BzATP induced the plasma membrane translocation of these MCTs via their chaperone CD147. Importantly, CD147 was increased in activated astrocytes after PC, and CD147-blocking antibody abolished the PC-induced facilitation of astrocytic lactate release and ischemic tolerance. Taken together, our findings suggest that astrocytes induce ischemic tolerance via P2X7 receptor-mediated lactate release.

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.

Cryo-EM structure of P-glycoprotein bound to triple elacridar inhibitor molecules.

P-glycoprotein (P-gp) is an ATP-binding cassette transporter known for its roles in expelling xenobiotic compounds from cells and contributing to cellular drug resistance through multidrug efflux. This mechanism is particularly problematic in cancer cells, where it diminishes the therapeutic efficacy of anticancer drugs. P-gp inhibitors, such as elacridar, have been developed to circumvent the decrease in drug efficacy due to P-gp efflux. An earlier study reported the cryo-EM structure of human P-gp-Fab (MRK-16) complex bound by two elacridar molecules, at a resolution of 3.6 Å. In this study, we have obtained a higher resolution (2.5 Å) structure of the P-gp- Fab (UIC2) complex bound by three elacridar molecules. This finding, which exposes a larger space for compound-binding sites than previously acknowledged, has significant implications for the development of more selective inhibitors and enhances our understanding of the compound recognition mechanism of P-gp.

Copy Number Gain in Androgen Receptors Predicts the Poor Prognosis in Japanese Castration-resistant Prostate Cancer.

BACKGROUND/AIM: The prognostic significance of androgen receptor amplification (AR amp) in cell-free DNA (cfDNA) was studied in Japanese patients with castration-resistant prostate cancer (CRPC). PATIENTS AND METHODS: A total of 120 serum samples were obtained from 38 patients with CRPC. Serum cfDNA was purified and the AR copy number was determined. Factors associated with progression-free survival (PFS) and overall survival (OS) were statistically investigated. RESULTS: The number of patients administered enzalutamide (Enza)/abiraterone (Abi)/docetaxel (DTX) was 33/25/11, respectively. The median PSA was 16.5 ng/ml. Thirty patients (79%) had bone metastases and three patients (7.9%) had lung metastases. The median follow-up was 655 days. The median initial AR copy number was 1.27 (1.10-11.50); an AR copy number of 1.27 or higher was defined as an AR-amp. Regarding PFS, the presence of AR-amp, Gleason score (GS), and ALP were significant factors in univariate analysis. In multivariate analysis, AR amplification was an independent prognostic factor (hazard ratio=7.7, p=0.0035). For OS, PSA and AR-amp were significant factors. In multivariate analysis, AR-amp (hazard ratio=4.65, p=0.0188) was the only independent prognostic factor. CONCLUSION: AR-amp was associated with high nadir PSA and low iPSA/PSA ratio. AR-amp was significantly associated with poor prognosis in Japanese patients with CRPC.

Exercise training outcomes in patients with chronic heart failure with reduced ejection fraction depend on patient background.

Background: The aim of this study was to identify significant factors affecting the effectiveness of exercise training using information of the HF-ACTION (Heart Failure: A Controlled Trial Investigating Outcomes of Exercise Training) study. Methods: Background factors influencing the effect of exercise training were comprehensively surveyed for 2,130 patients by multivariable Cox regression analysis with the stepwise variable selection, and only significant factors were selected that were statistically distinguished from dummy noise factors using the Boruta method. Results: The analysis suggested that the use of beta-blockers, pulse pressure, hemoglobin level, electrocardiography findings, body mass index, and history of stroke at baseline potentially influenced the exercise effect on all-cause death (AD). Therefore, a hypothetical score to estimate the effect of exercise training was constructed based on the analysis. The analysis suggested that the score is useful in identifying patients for whom exercise training may be significantly effective in reducing all-caused death and hospitalization (ADH) as well as AD. Such a subpopulation accounted for approximately 40% of the overall study population. On the other hand, in approximately 45% of patients, the effect of exercise was unclear on either AD or ADH. In the remaining 15% of patients, it was estimated that the effect of exercise might be unclear for ADH and potentially rather increase AD. Conclusions: This study is the first analysis to comprehensively evaluate the effects of various factors on the outcome of exercise training in chronic heart failure, underscoring the need to carefully consider the patient's background before recommending exercise training. However, it should be noted that exercise training can improve many outcomes in a wide variety of diseases. Therefore, given the limitations involved in post-hoc analyses of a single clinical trial, the characteristics of patients to whom the results of this analysis can be applied need attention, and also further research is necessary on the relationship between the degree of exercise and the outcomes. A new clinical trial would be needed to confirm the factors detected and the appropriateness of the score.

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.

Central administered xenin induced Fos expression in nesfatin-1 neurons in rats.

Xenin is a 25-amino acid peptide identified in human gastric mucosa, which is widely expressed in peripheral and central tissues. It is known that the central or peripheral administration of xenin decreases food intake in rodents. Nesfatin-1/NUCB2 (nesfatin-1) has been identified as an anorexic neuropeptide, it is often found co-localized with many peptides in the central nervous system. After the intracerebroventricular administration of xenin on nesfain-1-like immunoreactivity (LI) neurons, we examined its effects on food intake and water intake in rats. As a result, Fos-LI neurons were observed in the organum vasculosum of the laminae terminalis (OVLT), the median preoptic nucleus (MnPO), the subfornical organ (SFO), the supraoptic nucleus (SON), the paraventricular nucleus (PVN), the arcuate nucleus (Arc), the lateral hypothalamic area (LHA), the central amygdaloid nucleus (CAN), the dorsal raphe nucleus (DR), the locus coeruleus (LC), the area postrema (AP) and the nucleus of the solitary tract (NTS). After the administration, the number of Fos-LI neurons was significantly increased in the LC and the OVLT, the MnPO, the SFO, the SON, the PVN, the Arc, the LHA, the CAN, the DR, the AP and the NTS, compared with the control group. After the administration of xenin, we conducted double immunohistochemistry for Fos and nesfatin-1, and found that the number of nesfatin-1-LI neurons expressing Fos were significantly increased in the SON, the PVN, the Arc, the LHA, the CAN, the DR, the AP and the NTS, compared with the control group. The pretreatment of nesfatin-1 antisense significantly attenuated this xenin-induced feeding suppression, while that of nesfatin-1 missense showed no improvement. These results indicate that central administered xenin may have anorexia effects associated with activated central nesfatin-1 neurons.

Dysuricemia-A New Concept Encompassing Hyperuricemia and Hypouricemia.

The importance of uric acid, the final metabolite of purines excreted by the kidneys and intestines, was not previously recognized, except for its role in forming crystals in the joints and causing gout. However, recent evidence implies that uric acid is not a biologically inactive substance and may exert a wide range of effects, including antioxidant, neurostimulatory, proinflammatory, and innate immune activities. Notably, uric acid has two contradictory properties: antioxidant and oxidative ones. In this review, we present the concept of "dysuricemia", a condition in which deviation from the appropriate range of uric acid in the living body results in disease. This concept encompasses both hyperuricemia and hypouricemia. This review draws comparisons between the biologically biphasic positive and negative effects of uric acid and discusses the impact of such effects on various diseases.

Machine-learning predicts time-series prognosis factors in metastatic prostate cancer patients treated with androgen deprivation therapy.

Machine learning technology is expected to support diagnosis and prognosis prediction in medicine. We used machine learning to construct a new prognostic prediction model for prostate cancer patients based on longitudinal data obtained from age at diagnosis, peripheral blood and urine tests of 340 prostate cancer patients. Random survival forest (RSF) and survival tree were used for machine learning. In the time-series prognostic prediction model for metastatic prostate cancer patients, the RSF model showed better prediction accuracy than the conventional Cox proportional hazards model for almost all time periods of progression-free survival (PFS), overall survival (OS) and cancer-specific survival (CSS). Based on the RSF model, we created a clinically applicable prognostic prediction model using survival trees for OS and CSS by combining the values of lactate dehydrogenase (LDH) before starting treatment and alkaline phosphatase (ALP) at 120 days after treatment. Machine learning provides useful information for predicting the prognosis of metastatic prostate cancer prior to treatment intervention by considering the nonlinear and combined impacts of multiple features. The addition of data after the start of treatment would allow for more precise prognostic risk assessment of patients and would be beneficial for subsequent treatment selection.

Contribution of the L-Type Amino Acid Transporter Family in the Diagnosis and Treatment of Prostate Cancer.

The L-type amino acid transporter (LAT) family contains four members, LAT1~4, which are important amino acid transporters. They mainly transport specific amino acids through cell membranes, provide nutrients to cells, and are involved in a variety of metabolic pathways. They regulate the mTOR signaling pathway which has been found to be strongly linked to cancer in recent years. However, in the field of prostate cancer (PCa), the LAT family is still in the nascent stage of research, and the importance of LATs in the diagnosis and treatment of prostate cancer is still unknown. Therefore, this article aims to report the role of LATs in prostate cancer and their clinical significance and application. LATs promote the progression of prostate cancer by increasing amino acid uptake, activating the mammalian target of rapamycin (mTOR) pathway and downstream signals, mediating castration-resistance, promoting tumor angiogenesis, and enhancing chemotherapy resistance. The importance of LATs as diagnostic and therapeutic targets for prostate cancer was emphasized and the latest research results were introduced. In addition, we introduced selective LAT1 inhibitors, including JPH203 and OKY034, which showed excellent inhibitory effects on the proliferation of various tumor cells. This is the future direction of amino acid transporter targeting therapy drugs.

Interaction of buspirone and its major metabolites with human organic cation transporters.

Buspirone, a cationic drug, is an anxiolytic and antidepressant drug. However, whether buspirone and its metabolites are interacted with organic cationic transporter remains uncertain. In this study, we examined the interaction of buspirone and its major metabolites 1-(2-pyrimidinyl)piperazine (1-PP) and 6-hydroxybuspirone (6'-OH-Bu) with hOCTs using human hepatocellular carcinoma (HepG2), human colorectal adenocarcinoma (Caco-2) cells, and S2 cells expressing OCT1 (S2hOCT1), 2 (S2hOCT2), or 3 (S2hOCT3). Coadministration of buspirone and fluorescent 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+ ) was examined using HepG2 cells, and [3 H]-1-methyl-4-phenylpyridinium (MPP+ ) transport was assessed in S2 cell overexpressing hOCTs. The results showed that ASP+ transport was suppressed by buspirone with an IC50 of 26.3 ± 2.9 µM without any cytotoxic effects in HepG2 expressing hOCTs cells. Consistently, buspirone strongly inhibited [3 H]-MPP+ uptake by S2hOCT1, S2hOCT2, and S2hOCT3 cells with an IC50s of 89.0 ± 1.3 µM, 43.7 ± 7.5 µM, and 20.4 ± 1.0 µM, respectively. Nonetheless, 6'-OH-Bu and 1-PP caused weak or no inhibition on ASP+ and [3 H]-MPP+ transport. These findings suggest the potential interaction of buspirone with organic cation drugs that are handled by hOCT3. However, further clinical relevance is needed to support these findings for preventing drug-drug interaction in patients who take prescribed drugs together with buspirone.

Effects of a novel hepatitis B anti-viral drug E-CFCP in renal organic acid transporters.

Currently, the emergence of drug resistance is an important issue in the treatment of hepatitis B virus (HBV). Recently, our collaborating group developed a novel long-acting anti-HBV drug, E-CFCP. However, until this study, the effects of E-CFCP in the kidney have remained unclarified. Using cell viability and uptake assays, we examined the effects of E-CFCP on the function of renal organic anion transporters (OATs). No cytotoxicity was shown related to the E-CFCP in the renal OATs in either assay. Thus, this study suggested that E-CFCP may be a novel, excellent candidate drug for the treatment of drug-resistant HBV.

Targeting L-type amino acid transporter 1 in urological malignancy: Current status and future perspective.

Amino acid transporters are responsible for the uptake of amino acids, critical for cell proliferation. L-type amino acid transporters play a major role in the uptake of essential amino acids. L-type amino acid transporter 1 (LAT1) exerts its functional properties by forming a dimer with 4F2hc. Utilizing this cancer-specificity, research on diagnostic imaging and therapeutic agents for malignant tumors targeting LAT1 progresses in various fields. In hormone-sensitive prostate cancer, the up-regulation of L-type amino acid transporter 3 (LAT3) through the androgen receptor (AR) has been identified. On the other hand, in castration-resistant prostate cancer, the negative regulation of LAT1 through AR has been determined. Furthermore, 4F2hc: a binding partner of LAT1, was identified as the specific downstream target of Androgen Receptor Splice Variant 7: AR-V7. LAT1 has been suggested to contribute to acquiring castration resistance in prostate cancer, making LAT1 a completely different therapeutic target from anti-androgens and taxanes. Increased expression of LAT1 has also been found in renal and bladder cancers, suggesting a contribution to acquiring malignancy and progression. In Japan, clinical trials of LAT1 inhibitors for solid tumors are in progress, and clinical applications are now underway. This article will summarize the relationship between LAT1 and urological malignancies.

Cystine-dependent antiporters buffer against excess intracellular reactive sulfur species-induced stress.

Reactive sulfur species (RSS) play a role in redox homeostasis; however, adaptive cell responses to excessive intracellular RSS are not well understood. Therefore, in this study, we generated transgenic (Tg) mice overexpressing cystathionine gamma-lyase (CSE) to produce excessive RSS. Contrary to expectations, tissue concentrations of RSS, such as cysteine persulfide (CysSSH), were comparable in both wild-type and CSE Tg mice, but the plasma concentrations of CysSSH were significantly higher in CSE Tg mice than in wild-type mice. This export of surplus intracellular RSS was also observed in primary hepatocytes of CSE Tg mice. Exposure of primary hepatocytes to the RSS generator sodium tetrasulfide (Na2S4) resulted in an initial increase in the intracellular concentration of RSS, which later returned to basal levels after export into the extracellular space. Interestingly, among all amino acids, cystine (CysSSCys) was found to be essential for CysSSH export from primary mouse hepatocytes, HepG2 cells, and HEK293 cells during Na2S4 exposure, suggesting that the cystine/glutamate transporter (SLC7A11) contributes, at least partially, to CysSSH export. We established HepG2 cell lines with knockout and overexpression of SLC7A11 and used them to confirm SLC7A11 as the predominant antiporter of CysSSCys and CysSSH. We observed that the poor efflux of excess CysSSH from the cell enhanced cellular stresses induced by Na2S4 exposure, such as polysulfidation of intracellular proteins, mitochondrial damage, and cytotoxicity. These results suggest the presence of a cellular response to excess intracellular RSS that involves the extracellular efflux of excess CysSSH by a cystine-dependent transporter to maintain intracellular redox homeostasis.

P2X7 Receptors in Astrocytes: A Switch for Ischemic Tolerance.

A sub-lethal ischemic episode (preconditioning [PC]) protects neurons against a subsequent lethal ischemic injury. This phenomenon is known as ischemic tolerance. PC itself does not cause brain damage, but affects glial responses, especially astrocytes, and transforms them into an ischemia-resistant phenotype. P2X7 receptors (P2X7Rs) in astrocytes play essential roles in PC. Although P2X7Rs trigger inflammatory and toxic responses, PC-induced P2X7Rs in astrocytes function as a switch to protect the brain against ischemia. In this review, we focus on P2X7Rs and summarize recent developments on how astrocytes control P2X7Rs and what molecular mechanisms they use to induce ischemic tolerance.

LAT1-specific inhibitor ameliorates severe autoimmune arthritis in SKG mouse.

L-type amino acid transporter 1 (LAT1, slc7a5) supplies large neutral amino acids to highly proliferative cells. LAT1 is an attractive therapeutic target for treating overactive T cell-mediated immune disorders due to its high expression in activated T cells, but not in resting T cells. Here, we demonstrate that LAT1 plays a crucial role in T helper (Th) 17-mediated autoimmune arthritis in SKG mice, an animal model of human rheumatoid arthritis (RA). Administration of JPH203, a LAT1-specific inhibitor, suppressed mannan-induced joint swelling, synoviocyte proliferation and inflammatory cell infiltration in SKG mice. A diminished metabolic reprogramming, including a decrease in oxidative phosphorylation that regulates Hif-1α expression and subsequent control of glycolysis enzymes, was involved in the downregulation of Th17 differentiation by LAT1 inhibition. Moreover, publicly released database analysis revealed facilitated expression of LAT1 in T cells with cytotoxic features in patients with RA. Our results demonstrate the essential contribution of LAT1 to the development of RA, proposing a potential therapeutic approach targeting amino acid transporters for treating hypersensitive immune diseases.

Effects of 1,5-anhydro-D-glucitol on insulin secretion both in in vitro and ex vivo pancreatic preparations.

Organ bath experiments are conventionally used to investigate the physiological actions and effects of hormones and drugs on organ responses. We developed an experimental method to reproduce insulin secretion from isolated rat pancreas preparations, to investigate substances that promote insulin secretion ex vivo. 1,5-anhydro-D-glucitol (1,5-AG) is found in foods, and exists in humans and rodents; however, whether 1,5-AG stimulates insulin secretion remains unclear. This study aimed to assess the effects of short-term 1,5-AG stimulation on insulin secretion in both ex vivo and in INS-1E (rat-derived) cells in vitro. Our results indicated that 1,5-AG had no potency to increase the proportion of insulin outflow both in ex vivo and in vitro experiments. Insulin outflow significantly increased upon stimulation with 10 µM glimepiride, a member of the sulfonylurea class of drugs, ex vivo. Glucose-stimulated insulin secretion was observed not only in INS-1E cells but also in rat pancreatic preparations. Our findings demonstrated that short-term exposure to 1,5-AG had no effect on insulin secretion in rats.

Hypouricemia and Urate Transporters.

Hypouricemia is recognized as a rare disorder, defined as a serum uric acid level of 2.0 mg/dL or less. Hypouricemia is divided into an overexcretion type and an underproduction type. The former typical disease is xanthinuria, and the latter is renal hypouricemia (RHUC). The frequency of nephrogenic hypouricemia due to a deficiency of URAT1 is high in Japan, accounting for most asymptomatic and persistent cases of hypouricemia. RHUC results in a high risk of exercise-induced acute kidney injury and urolithiasis. It is vital to promote research on RHUC, as this will lead not only to the elucidation of its pathophysiology but also to the development of new treatments for gout and hyperuricemia.

Contribution of LAT1-4F2hc in Urological Cancers via Toll-like Receptor and Other Vital Pathways.

Tumor cells are known for their ability to proliferate. Nutrients are essential for rapidly growing tumor cells. In particular, essential amino acids are essential for tumor cell growth. Tumor cell growth nutrition requires the regulation of membrane transport proteins. Nutritional processes require amino acid uptake across the cell membrane. Leucine, one of the essential amino acids, has recently been found to be closely associated with cancer, which activate mTOR signaling pathway. The transport of leucine into cells requires an L-type amino acid transporter protein 1, LAT1 (SLC7A5), which requires the 4F2 cell surface antigen heavy chain (4F2hc, SLC3A2) to form a heterodimeric amino acid transporter protein complex. Recent evidence identified 4F2hc as a specific downstream target of the androgen receptor splice variant 7 (AR-V7). We stressed the importance of the LAT1-4F2hc complex as a diagnostic and therapeutic target in urological cancers in this review, which covered the recent achievements in research on the involvement of the LAT1-4F2hc complex in urinary system tumors. In addition, JPH203, which is a selective LAT1 inhibitor, has shown excellent inhibitory effects on the proliferation in a variety of tumor cells. The current phase I clinical trials of JPH203 in patients with biliary tract cancer have also achieved good results, which is the future research direction for LAT1 targeted therapy drugs.

L-type amino acid transporter 1 as a target for inflammatory disease and cancer immunotherapy.

Ingestion of amino acids is fundamental for cellular activity. Amino acids are important components for protein synthesis but are also crucial for intracellular metabolic reactions and signal transduction. Following activation, immune cells induce metabolic reprogramming to generate adequate energy and constitutive substances. Hence, the delivery of amino acids by transporters is necessary for the progression of metabolic rewiring. In this review, we discuss how amino acids and their transporters regulate immune cell functions, with emphasis on LAT1, a transporter of large neutral amino acids. Furthermore, we explore the possibility of targeting amino acid transporters to improve immune disorders and cancer immune therapies.