The melatonin-MT1 receptor axis modulates tumor growth in PTEN-mutated gliomas.

More than 40% of glioma patients have tumors that harbor PTEN (phosphatase and tensin homologue deleted on chromosome ten) mutations; this disease is associated with poor therapeutic resistance and outcome. Such mutations are linked to increased cell survival and growth, decreased apoptosis, and drug resistance; thus, new therapeutic strategies focusing on inhibiting glioma tumorigenesis and progression are urgently needed. Melatonin, an indolamine produced and secreted predominantly by the pineal gland, mediates a variety of physiological functions and possesses antioxidant and antitumor properties. Here, we analyzed the relationship between PTEN and the inhibitory effect of melatonin in primary human glioma cells and cultured glioma cell lines. The results showed that melatonin can inhibit glioma cell growth both in culture and in vivo. This inhibition was associated with PTEN levels, which significantly correlated with the expression level of MT1 in patients. In fact, c-fos-mediated MT1 was shown to be a key modulator of the effect of melatonin on gliomas that harbor wild type PTEN. Taken together, these data suggest that melatonin-MT1 receptor complexes represent a potential target for the treatment of glioma.

Proton pump inhibitors have pH-dependent effects on the thermostability of the carboxyl-terminal domain of voltage-gated proton channel Hv1.

The voltage-gated proton channel Hv1 is highly selective for H+ and is activated by membrane depolarization and pH gradient. An increased external and decreased internal pH opens the Hv1 channel. The intracellular C-terminal domain of Hv1 is responsible for channel dimerization, cooperative, and thermosensitive gating. Here, we found that proton pump inhibitors (PPIs) interact with the C-terminal domain of human Hv1. The interaction between PPIs and the C-terminal domain, which is pH-dependent, lowered the thermal and structural stability of the protein at pH 4, but enhanced the thermal and structural stability at pH 8. Furthermore, we investigated in vitro the interaction of PPIs with the C-terminal domain of Hv1 by fluorescence and micro-Raman spectra. Fluorescence quenching measurements revealed that the interaction between the C-terminal domain and PPIs is a mainly hydrophobic interaction. The micro-Raman spectra showed that PPIs did not form stable disulfide bonds with the unique thiol group within this domain (Cys249 residue). The preferential interaction of PPIs with the inactive form of Hv1 stabilizes the high pH inactive state of the C-terminal domain, indicating a mechanism by which PPIs might act explicitly on the stabilization of a closed state of the proton channel.

A novel splice variant of supervillin, SV5, promotes carcinoma cell proliferation and cell migration.

Supervillin is an actin-associated protein that regulates actin dynamics by interacting with Myosin II, F-actin, and Cortactin to promote cell contractility and cell motility. Two splicing variants of human Supervillin (SV1 and SV4) have been reported in non-muscle cells; SV1 lacks 3 exons present in the larger isoform SV4. SV2, also called archvillin, is present in striated muscle; SV3, also called smooth muscle archvillin or SmAV, was cloned from smooth muscle. In the present study, we identify a novel splicing variant of Supervillin (SV5). SV5 contains a new splicing pattern. In the mouse tissues and cell lines examined, SV5 was predominantly expressed in skeletal and cardiac muscles and in proliferating cells, but was virtually undetectable in most normal tissues. Using RNAi and rescue experiments, we show here that SV5 displays altered functional properties in cancer cells, and regulates cell proliferation and cell migration.

The voltage-gated proton channel Hv1 is expressed in pancreatic islet ß-cells and regulates insulin secretion.

The voltage-gated proton channel Hv1 is a potent acid extruder that participates in the extrusion of the intracellular acid. Here, we showed for the first time, Hv1 is highly expressed in mouse and human pancreatic islet ß-cells, as well as ß-cell lines. Imaging studies demonstrated that Hv1 resides in insulin-containing granules in ß-cells. Knockdown of Hv1 with RNA interference significantly reduces glucose- and K(+)-induced insulin secretion in isolated islets and INS-1 (832/13) ß-cells and has an impairment on glucose- and K(+)-induced intracellular Ca(2+) homeostasis. Our data demonstrated that the expression of Hv1 in pancreatic islet ß-cells regulates insulin secretion through regulating Ca(2+) homeostasis.

Lansoprazole induces apoptosis of breast cancer cells through inhibition of intracellular proton extrusion.

The increased glycolysis and proton secretion in tumors is proposed to contribute to the proliferation and invasion of cancer cells during the process of tumorigenesis and metastasis. Here, treatment of human breast cancer cells with proton pump inhibitor (PPI) lansoprazole (LPZ) induces cell apoptosis in a dose-dependent manner. In the implantation of the MDA-MB-231 xenografts in nude mice, administration of LPZ significantly inhibits tumorigenesis and induces large-scale apopotosis of tumor cells. LPZ markedly inhibits intracellular proton extrusion, induces an increase in intracellular ATP level, lysosomal alkalinization and accumulation of reactive oxygen species (ROS) in breast cancer cells. The ROS scavenger N-acetyl-l-cysteine (NAC) and diphenyleneiodonium (DPI), a specific pharmacological inhibitor of NADPH oxidases (NOX), significantly abolish LPZ-induced ROS accumulation in breast cancer cells. Our results suggested that LPZ may be used as a new therapeutic drug for breast tumor.

Dissecting prostate Cancer: Single-Cell insight into Macrophage Diversity, molecular Prognosticators, and the role of Peptidylprolyl Isomerase F.

BACKGROUND: Prostate cancer remains a prominent challenge in oncology, with advanced stages showing poor prognosis. The tumor microenvironment (TME), and particularly tumor-associated macrophages (TAMs), plays a crucial role in disease progression. This study explores the single-cell transcriptomics of prostate cancer, determines macrophage heterogeneity, identifies prognostic gene markers, and assesses the role of PPIF in TAMs. METHODS: Single-cell RNA sequencing data from the GEO database (GSE176031) and transcriptome data from the TCGA were processed to characterize cell populations and identify prognostic genes in prostate cancer. Macrophage subpopulations were examined through clustering, followed by gene set scoring based on migration, activation, and proliferation. PPIF expression in macrophages was investigated using multiplex immunofluorescence staining on matched prostate cancer and adjacent non-tumoral tissues. RESULTS: The single-cell analysis identified 9,178 cells, categorized into 10 principal cell types, with macrophages constituting a significant part of the immune microenvironment. Four macrophage subgroups demonstrated distinct functional pathways: phagocytic, immune-regulatory, and proliferative. A total of 39 genes correlated with prostate cancer prognosis were identified, of which 10 carried the most significant prognostic information. Peptidylprolyl Isomerase F (PPIF) expression was significantly higher in TAMs from tumor tissue than normal tissue, indicating its potential regulatory role in the immune microenvironment. CONCLUSION: The intricate cellular architecture of the prostate cancer TME has been elucidated, with a focus on macrophage heterogeneity and functional specialization. Prognostic genes, including PPIF, were associated with survival outcomes, providing potential therapeutic targets. PPIF's prominent expression in TAMs may serve as a lever in cancer progression, warranting further investigation as a biomarker and a molecule of interest for therapeutic targeting within the prostate cancer milieu.

Subchronic co-exposure of polystyrene nanoplastics and 3-BHA significantly aggravated the reproductive toxicity of ovaries and uterus in female mice.

Both nanoplastics (NPs) and 3-tert-butyl-4-hydroxyanisole (3-BHA) are environmental contaminants that can bio-accumulate through the food chain. However, the combined effects of which on mammalian female reproductive system remain unclear. Here, the female ICR-CD1 mice were used to evaluate the damage effects of ovaries and uterus after NPs and 3-BHA co-treatment for 35 days. Firstly, co-exposure significantly reduced the body weight and organ index of ovaries and uterus in mice. Secondly, combined effects of NPs and 3-BHA exacerbated the histopathological abnormalities to the ovaries and uterus and decreased female sex hormones such as FSH and LH while increased antioxidant activities including CAT and GSH-Px. Moreover, the apoptotic genes, inflammatory cytokines and the key reproductive development genes such as FSTL1 were significantly up-regulated under co-exposure conditions. Thirdly, through transcriptional and bioinformatics analysis, immunofluorescence and western blotting assays, together with molecular docking simulation, we determined that co-exposure up-regulated the FSTL1, TGF-ß and p-Smad1/5/9 but down-regulated the expression of BMP4. Finally, the pharmacological rescue experiments further demonstrated that co-exposure of NPs and 3-BHA mainly exacerbated the female reproductive toxicity through FSTL1-mediated BMP4/TGF-ß/SMAD signaling pathway. Taken together, our studies provided the theoretical basis of new environmental pollutants on the reproductive health in female mammals.

Zn(2+) induces apoptosis in human highly metastatic SHG-44 glioma cells, through inhibiting activity of the voltage-gated proton channel Hv1.

In contrast to the voltage-gated K(+) channels, the voltage-gated proton channel Hv1 contains a voltage-sensor domain but lacks a pore domain. Here, we showed that Hv1 is expressed in the highly metastatic glioma cell SHG-44, but lowly in the poorly metastatic glioma cell U-251. Inhibition of Hv1 activity by 140µM zinc chloride induces apoptosis in the human highly metastatic glioma cells. Zn(2+) ions markedly inhibit proton secretion, and reduce the gelatinase activity in the highly metastatic glioma cells. In vivo, the glioma tumor sizes of the implantation of the SHG-44 xenografts in nude mice that were injected zinc chloride solution, were dramatically smaller than that in the controlled groups. The results demonstrated that the inhibition of Hv1 activity via Zn(2+) ions can effectively retard the cancer growth and suppress the cancer metastasis by the decrease of proton extrusion and the down-regulation of gelatinase activity. Our results suggest that Zn(2+) ions may be used as a potential anti-glioma drug for glioma therapy.

Adverse Effects of Prenatal Exposure to Oxidized Black Carbon Particles on the Reproductive System of Male Mice.

Ambient black carbon (BC), a main constituent of atmospheric particulate matter (PM), is a primary particle that is mainly generated by the incomplete combustion of fossil fuel and biomass burning. BC has been identified as a potential health risk via exposure. However, the adverse effects of exposure to BC on the male reproductive system remain unclear. In the present study, we explored the effects of maternal exposure to oxidized black carbon (OBC) during pregnancy on testicular development and steroid synthesis in male offspring. Pregnant mice were exposed to OBC (467 µg/kg BW) or nanopure water (as control) by intratracheal instillation from gestation day (GD) 4 to GD 16.5 (every other day). We examined the testicular histology, daily sperm production, serum testosterone, and mRNA expression of hormone synthesis process-related factors of male offspring at postnatal day (PND) 35 and PND 84. Histological examinations exhibited abnormal seminiferous tubules with degenerative changes and low cellular adhesion in testes of OBC-exposed mice at PND 35 and PND 84. Consistent with the decrease in daily sperm production, the serum testosterone level of male offspring of OBC-exposed mice also decreased significantly. Correspondingly, mRNA expression levels of hormone-synthesis-related genes (i.e., StAR, P450scc, P450c17, and 17ß-HSD) were markedly down-regulated in male offspring of PND 35 and PND 84, respectively. In brief, these results suggest that prenatal exposure has detrimental effects on mouse spermatogenesis in adult offspring.

MiR-1281 is involved in depression disorder and the antidepressant effects of Kai-Xin-San by targeting ADCY1 and DVL1.

Kai-Xin-San (KXS) is a Chinese medicine formulation that is commonly used to treat depression caused by dual deficiencies in the heart and spleen. Recent studies indicated that miRNAs were involved in the pathophysiology of depression. However, there have been few studies on the mechanism underlying the miRNAs directly mediating antidepressant at clinical level, especially in nature drugs and TCM compound. In this study, we identified circulating miRNAs defferentially expressed among the depression patients (DPs), DPs who underwent 8weeks of KXS treatment and health controls (HCs). A total of 45 miRNAs (17 were up-regulated and 28 were down-regulated) were significantly differentially expressed among three groups. Subsequently, qRT-PCR was used to verify 10 differentially expressed candidate miRNAs in more serum samples, and the results showed that 6 miRNAs (miR-1281, miR-365a-3p, miR-2861, miR-16-5p, miR-1202 and miR-451a) were consistent with the results of microarray. Among them, miR-1281, was the novel dynamically altered and appeared to be specifically related to depression and antidepressant effects of KXS. MicroRNA-gene-pathway-net analysis showed that miR-1281-regulated genes are mostly key nodes in the classical signaling pathway related to depression. Additionally, our data suggest that ADCY1 and DVL1 were the targets of miR-1281. Thus, based on the discovery of miRNA expression profiles in vivo, our findings suggest a new role for miR-1281 related to depression and demonstrated in vitro that KXS may activate cAMP/PKA/ERK/CREB and Wnt/ß-catenin signal transduction pathways by down-regulating miR-1281 that targets ADCY1 and DVL1 to achieve its role in neuronal cell protection.

Molecular subtypes of m6A RNA methylation modification patterns and their clinical significance in clear cell renal cell carcinoma.

Background: In this study, we sought to investigate the association between N6-methyladenosine (m6A) RNA methylation-modification patterns and patient prognosis in clear cell renal cell carcinoma (ccRCC) and construct a ccRCC molecular signature according to expressions of m6A-related genes. Methods: First, the clinical data and the transcriptomes of 530 patients with ccRCC were downloaded from The Cancer Genome Atlas (TCGA). The expression patterns of m6A-related genes were extracted, and the differences in m6A-modification patterns between normal and tumor renal tissues were analyzed. To explore the prognostic role of m6A-modification patterns a in ccRCC, the molecular subtypes of ccRCC were identified based on the expression patterns of the m6A-related genes, and survival rates in patients with the different subtypes were compared. According to expressions of m6A-related genes and clinical prognosis data, a prognostic molecular signature was constructed using least absolute shrinkage and selector operation (LASSO)-Cox regression analysis. Results: Among the 13 m6A -related genes identified in this study, 8 (YTHDC1, YTHDF2, HNRNPC, METTL14, ZC3H13, FTO, YTHDC2, and YTHDF1) showed significant expression differences between normal and tumor renal tissues. The molecular subtypes of ccRCC identified according to their expression of the 13 m6A-related genes were associated with differential clinical outcomes. Conclusions: Following TCGA data-mining, different molecular subtypes of ccRCC based on m6A RNA methylation patterns were found to have different prognoses. The molecular signature constructed according to the expression patterns of m6A-related genes could predict patient prognosis in ccRCC. We believe m6A RNA methylation modification is a potential therapeutic target and may play a crucial role in ccRCC.

Knockdown of Toe1 causes developmental arrest during the morula-to-blastocyst transition in mice.

The target of EGR1 protein 1 (TOE1) is evolutionarily conserved from Caenorhabditis elegans to mammals, which plays a critical role in the maturation of a variety of small nuclear RNAs. Mutation in human TOE1 has been reported to cause pontocerebellar hypoplasia type 7, a severe neurodegenerative syndrome. However, the role of TOE1 in early embryonic development remains unclear. Herein, we found that Toe1 mRNA and protein were expressed in mouse preimplantation embryos. Silencing Toe1 by siRNA led to morula-to-blastocyst transition failure. This developmental arrest can be rescued by Toe1 mRNA microinjection. EdU incorporation assay showed a defect in blastomere proliferation within developmentally arrested embryos. Further studies revealed that Toe1 knockdown caused increased signals for γH2AX and micronuclei, indicative of sustained DNA damage. Moreover, mRNA levels of cell cycle inhibitor p21 were significantly upregulated in Toe1 knockdown embryos before developmental arrest. Together, these results suggest that TOE1 is indispensable for mouse early embryo development potentially through maintaining genomic integrity. Our findings provide further insight into the role of TOE1 in mouse preimplantation embryonic development.

Muscarinic acetylcholine receptors regulate inflammatory responses through arginases 1/2 in zebrafish.

Muscarinic acetylcholine receptors (mAChRs) are widely expressed in various effector cells and have been proved to play vital roles in smooth muscle contraction and digestive secretion. However, there are relatively few literatures revealing the roles of mAChRs in inflammatory processes, and its underlying regulatory mechanisms have not been elucidated. Taking the advantages of live imaging of zebrafish, we found that inhibition of mAChRs resulted in increased neutrophils recruitment and proinflammatory cytokines expression, whereas activation of mAChRs led to opposite outcome. Subsequently, we found that mAChRs regulated the expression of arginases (args), and pharmacological intervention of args level could reverse the effects of mAChRs on neutrophils migration and cytokines expression, suggesting that args are important downstream proteins of mAChRs that mediate the regulation of inflammatory response. In this study, we identified args as novel downstream proteins of mAChRs in inflammatory responses, providing additional evidence for system immune regulation of cholinergic receptors.

Effects of Repetitive Transcranial Magnetic Stimulation on Cognitive Function in Patients With Stress-Related Depression: A Randomized Double-Blind fMRI and 1H-MRS Study.

Objectives: To reveal the effects of repetitive transcranial magnetic stimulation (rTMS) on the improvement of cognitive function in patients with stress-related depression, and to enrich the neural mechanism(s) underlying rTMS so as to improve cognitive function in patients with stress-related depression. Methods: We conducted a randomized, double-blind, placebo-controlled study of rTMS in patients with stress-related depression who were 18-40 years of age. Patients were randomly allocated to either a sham or experimental group in a 1:1 ratio. A 10-session rTMS protocol was used with 10-Hz stimulation over the left dorsolateral prefrontal cortex (DLPFC). Clinical assessments (HAMD, HAMA, DASS, MoCA), neuropsychologic (Stroop, WCST), and resting state fMRI and 1H-MRS assessments were executed at two time points-baseline and after the 10th rTMS session. Results: rTMS relieved the mental symptoms of patients in both groups. The MoCA score of patients in the experimental group increased; the number of correct answers increased significantly in Stroop testing, and the number of errors and omissions decreased significantly; the number of persistent errors decreased significantly; and the time used to complete the test decreased to an even greater extent in the WCST experimental group. The ReHo value in the lingual gyrus of the right hemisphere and the cuneus of the left and right hemispheres in the experimental group decreased after treatment. The DC value in the left and right hemispheric cuneus and postcentral gyrus of the left hemisphere in the experimental group diminished after treatment. The functional connections of these brain regions also changed as the Cho and NAA/Cr of the left DLPFC changed, with alterations related to the improvement in cognitive function. The level of choline (Cho) in the left DLPFC of the experimental group was significantly lower than that of the control group, and the level of N-acetylaspartate/creatine (NAA/Cr) in the left DLPFC of the control group was significantly higher than that of the experimental group. These changes were related to the overall improvement in cognitive function. Conclusions: Ten-Hz rTMS over the left DLPFC improved the cognitive function of patients with stress-related depression. The governing mechanism for this phenomenon may be via rTMS effects on multiple visual-related brain regions and their functional connections, and on the somatosensory cortex and its functional connection with visual and auditory cortex, reducing the level of Cho and stabilizing the level of NAA/Cr in the left DLPFC.

Supervillin promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma in hypoxia via activation of the RhoA/ROCK-ERK/p38 pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world and metastasis is the leading cause of death associated with HCC. Hypoxia triggers the epithelial-mesenchymal transition (EMT) of cancer cells, which enhances their malignant character and elevates metastatic risk. Supervillin associates tightly with the membrane and cytoskeleton, promoting cell motility, invasiveness, and cell survival. However, the roles of supervillin in HCC metastasis remain unclear. METHODS: Tissue microarray technology was used to immunohistochemically stain for supervillin antibody in 173 HCC tissue specimens and expression levels correlated with the clinicopathological variables. Tumor cell motility and invasiveness, as well as changes in the mRNA expression levels of genes associated with cancer cell EMT, were investigated. The relationship between supervillin and Rho GTPases was examined using Co-IP and GST pull-down. RESULTS: Hypoxia-induced upregulation of supervillin promoted cancer cell migration and invasion via the activation of the ERK/p38 pathway downstream of RhoA/ROCK signaling. Furthermore, supervillin regulated the expression of EMT genes during hypoxia and accelerated the metastasis of HCC in vivo. CONCLUSIONS: Hypoxia-induced increase in supervillin expression is a significant and independent predictor of cancer metastasis, which leads to poor survival in HCC patients. Our results suggest that supervillin may be a candidate prognostic factor for HCC and a valuable target for therapy.

EZH2 Palmitoylation Mediated by ZDHHC5 in p53-Mutant Glioma Drives Malignant Development and Progression.

Gliomas with mutant p53 occurring in 30% of glioma patients exhibit therapeutic resistance and poor outcomes. In this study, we identify a novel mechanism through which mutant p53 drives cancer cell survival and malignant growth. We documented overexpression of the zinc finger protein ZDHHC5 in glioma compared with normal brain tissue and that this event tightly correlated with p53 mutations. Mechanistic investigations revealed that mutant p53 transcriptionally upregulated ZDHHC5 along with the nuclear transcription factor NF-Y. These events contributed to the development of glioma by promoting the self-renewal capacity and tumorigenicity of glioma stem-like cells, by altering the palmitoylation and phosphorylation status of the tumor suppressor EZH2. Taken together, our work highlighted ZDHHC5 as a candidate therapeutic target for management of p53-mutated gliomas. Cancer Res; 77(18); 4998-5010. ©2017 AACR.

Melatonin attenuates hypoxia-induced epithelial-mesenchymal transition and cell aggressive via Smad7/ CCL20 in glioma.

Tumor recurrence in gliomas is partly attributed to increased epithelial-mesenchymal transition (EMT) and enhanced tumor cell dissemination in the adjacent brain parenchyma. Thus, exploring effective strategies for against EMT-like changes in glioma invasion and recurrence will be important for glioma treatment. In this study, we investigated the roles of melatonin in hypoxia-induced EMT suppression, and found that melatonin could significantly suppress the release of the cytokine, CCL20, from cancer cells and antagonize glioma cell metastasis and invasion under hypoxic stress in glioma cells. Furthermore, our findings show that melatonin deregulates Smad7 expression to suppress TGFß/Smad-mediated increase in CCL20 transcript levels and CCL20-induced EMT occurrence, suggesting a potential anti-EMT therapeutic role for melatonin in malignant transformation in gliomas.

Human voltage-gated proton channel hv1: a new potential biomarker for diagnosis and prognosis of colorectal cancer.

Solid tumors exist in a hypoxic microenvironment, and possess high-glycolytic metabolites. To avoid the acidosis, tumor cells must exhibit a dynamic cytosolic pH regulation mechanism(s). The voltage-gated proton channel Hv1 mediates NADPH oxidase function by compensating cellular loss of electrons with protons. Here, we showed for the first time, that Hv1 expression is increased in colorectal tumor tissues and cell lines, associated with poor prognosis. Immunohistochemistry showed that Hv1 is strongly expressed in adenocarcinomas but not or lowly expressed in normal colorectal or hyperplastic polyps. Hv1 expression in colorectal cancer is significantly associated with the tumor size, tumor classification, lymph node status, clinical stage and p53 status. High Hv1 expression is associated significantly with shorter overall and recurrence-free survival. Furthermore, real-time RT-PCR and immunocytochemistry showed that Hv1 is highly expressed in colorectal cancer cell lines, SW620, HT29, LS174T and Colo205, but not in SW480. Inhibitions of Hv1 expression and activity in the highly metastatic SW620 cells by small interfering RNA (siRNA) and Zn(2+) respectively, markedly decrease the cell invasion and migration, restraint proton extrusion and the intracellular pH recovery. Our results suggest that Hv1 may be used as a potential biomarker for diagnosis and prognosis of colorectal carcinoma, and a potential target for anticancer drugs in colorectal cancer therapy.