A positive feedback loop between PLD1 and NF-κB signaling promotes tumorigenesis of nasopharyngeal carcinoma.

Phospholipase D (PLD) lipid-signaling enzyme superfamily has been widely implicated in various human malignancies, but its role and underlying mechanism remain unclear in nasopharyngeal carcinoma (NPC). Here, we analyze the expressions of 6 PLD family members between 87 NPC and 10 control samples through transcriptome analysis. Our findings reveal a notable upregulation of PLD1 in both NPC tumors and cell lines, correlating with worse disease-free and overall survival in NPC patients. Functional assays further elucidate the oncogenic role of PLD1, demonstrating its pivotal promotion of critical tumorigenic processes such as cell proliferation and migration in vitro, as well as tumor growth in vivo. Notably, our study uncovers a positive feedback loop between PLD1 and the NF-κB signaling pathway to render NPC progression. Specifically, PLD1 enhances NF-κB activity by facilitating the phosphorylation and nuclear translocation of RELA, which in turn binds to the promoter of PLD1, augmenting its expression. Moreover, RELA overexpression markedly rescues the inhibitory effects in PLD1-depleted NPC cells. Importantly, the application of the PLD1 inhibitor, VU0155069, substantially inhibits NPC tumorigenesis in a patient-derived xenograft model. Together, our findings identify PLD1/NF-κB signaling as a positive feedback loop with promising therapeutic and prognostic potential in NPC.

Comparative single-cell analysis reveals heterogeneous immune landscapes in adenocarcinoma of the esophagogastric junction and gastric adenocarcinoma.

Adenocarcinoma of the esophagogastric junction (AEG) is a type of tumor that arises at the anatomical junction of the esophagus and stomach. Although AEG is commonly classified as a subtype of gastric adenocarcinoma (GAC), the tumor microenvironment (TME) of AEG remains poorly understood. To address this issue, we conducted single-cell RNA sequencing (scRNA-seq) on tumor and adjacent normal tissues from four AEG patients and performed integrated analysis with publicly available GAC single-cell datasets. Our study for the first time comprehensively deciphered the TME landscape of AEG, where heterogeneous AEG malignant cells were identified with diverse biological functions and intrinsic malignant nature. We also depicted transcriptional signatures and T cell receptor (TCR) repertoires for T cell subclusters, revealing enhanced exhaustion and reduced clone expansion along the developmental trajectory of tumor-infiltrating T cells within AEG. Notably, we observed prominent enrichment of tumorigenic cancer-associated fibroblasts (CAFs) in the AEG TME compared to GAC. These CAFs played a critical regulatory role in the intercellular communication network with other cell types in the AEG TME. Furthermore, we identified that the accumulation of CAFs in AEG might be induced by malignant cells through FGF-FGFR axes. Our findings provide a comprehensive depiction of the AEG TME, which underlies potential therapeutic targets for AEG patient treatment.

Single-Cell Analysis Reveals Malignant Cells Reshape the Cellular Landscape and Foster an Immunosuppressive Microenvironment of Extranodal NK/T-Cell Lymphoma.

Extranodal natural killer/T-cell lymphoma (NKTCL) is an aggressive type of lymphoma associated with Epstein-Barr virus (EBV) and characterized by heterogeneous tumor behaviors. To better understand the origins of the heterogeneity, this study utilizes single-cell RNA sequencing (scRNA-seq) analysis to profile the tumor microenvironment (TME) of NKTCL at the single-cell level. Together with in vitro and in vivo models, the study identifies a subset of LMP1+ malignant NK cells contributing to the tumorigenesis and development of heterogeneous malignant cells in NKTCL. Furthermore, malignant NK cells interact with various immunocytes via chemokines and their receptors, secrete substantial DPP4 that impairs the chemotaxis of immunocytes and regulates their infiltration. They also exhibit an immunosuppressive effect on T cells, which is further boosted by LMP1. Moreover, high transcription of EBV-encoded genes and low infiltration of tumor-associated macrophages (TAMs) are favorable prognostic indicators for NKTCL in multiple patient cohorts. This study for the first time deciphers the heterogeneous composition of NKTCL TME at single-cell resolution, highlighting the crucial role of malignant NK cells with EBV-encoded LMP1 in reshaping the cellular landscape and fostering an immunosuppressive microenvironment. These findings provide insights into understanding the pathogenic mechanisms of NKTCL and developing novel therapeutic strategies against NKTCL.

SRSF3/AMOTL1 splicing axis promotes the tumorigenesis of nasopharyngeal carcinoma through regulating the nucleus translocation of YAP1.

Dysregulation of serine/arginine splicing factors (SRSFs) and abnormal alternative splicing (AS) have been widely implicated in various cancers but scarcely investigated in nasopharyngeal carcinoma (NPC). Here we examine the expression of 12 classical SRSFs between 87 NPC and 10 control samples, revealing a significant upregulation of SRSF3 and its association with worse prognosis in NPC. Functional assays demonstrate that SRSF3 exerts an oncogenic function in NPC progression. Transcriptome analysis reveals 1,934 SRSF3-regulated AS events in genes related to cell cycle and mRNA metabolism. Among these events, we verify the generation of a long isoform of AMOTL1 (AMOTL1-L) through a direct bond of the SRSF3 RRM domain with the exon 12 of AMOTL1 to promote exon inclusion. Functional studies also reveal that AMOTL1-L promotes the proliferation and migration of NPC cells, while AMOTL1-S does not. Furthermore, overexpression of AMOTL1-L, but not -S, significantly rescues the inhibitory effects of SRSF3 knockdown. Additionally, compared with AMOTL1-S, AMOTL1-L has a localization preference in the intracellular than the cell membrane, leading to a more robust interaction with YAP1 to promote nucleus translocation. Our findings identify SRSF3/AMOTL1 as a novel alternative splicing axis with pivotal roles in NPC development, which could serve as promising prognostic biomarkers and therapeutic targets for NPC.

Icariside II Attenuates Vascular Remodeling Via Wnt7b/CCND1 Axis.

ABSTRACT: Angioplasty often fails due to the abnormal proliferation of vascular smooth muscle cells (VSMCs). Success rates of angioplasty may increase following the administration of an agent that effectively ameliorates aberrant vascular remodeling. Icariside II (ICS-II) is a natural flavonol glycoside extract from the Chinese herbal medicine Epimedii that possesses several medicinal qualities that are beneficial in humans. Nevertheless, the role of ICS-II in addressing aberrant vascular remodeling have yet to be clarified. The current investigation studies the molecular effects of ICS-Ⅱ on balloon-inflicted neointimal hyperplasia in rats in vivo and on platelet-derived growth factor-induced vascular proliferation in primary rat aortic smooth muscle cells (VSMCs) in vitro. ICS-II was found to be as effective as rapamycin, the positive control used in this study. ICS-II inhibited neointimal formation in injured rat carotid arteries and notably reduced the expression of Wnt7b. ICS-Ⅱ significantly counteracted platelet-derived growth factor-induced VSMCs proliferation. Cell cycle analysis showed that ICS-II triggered cell cycle arrest during the G1/S transition. Western blot analysis further indicated that this cell cycle arrest was likely through Wnt7b suppression that led to CCND1 inhibition. In conclusion, our findings demonstrate that ICS-II possesses significant antiproliferative qualities that counteracts aberrant vascular neointimal hyperplasia. This phenomenon most likely occurs due to the suppression of the Wnt7b/CCND1 axis.

Prototype of a monolithic cavity-based ultrastable optical reference for space applications.

We present a compact, monolithic optical reference for the frequency stabilized laser of future inter-satellite laser interferometer missions. A prototype based on the integration of a high-finesse cavity and associate optics has been designed to be space compatible while maintaining sufficient stability. The prototype has then been developed with a space-qualified bonding technique, and an in situ multi-degree-of-freedom alignment method. The performances of the optical reference have been studied by beat note analysis with another frequency stabilized laser, and the preliminary results are in agreement with the potential requirements of future space missions.

Sildenafil improves right ventricular remodelling in monocrotaline-induced rats by decreasing myocardial apoptosis and activating peroxisome proliferator-activated receptors.

OBJECTIVES: To assess the effect of sildenafil on monocrotaline-induced right ventricular (RV) remodeling and investigate the possible mechanism. METHODS: Rats were subcutaneously injected with monocrotaline to establish an RV remodeling model and then administered sildenafil (25 mg/kg) from days 1 to 28. After 28 days of administration, the RV systolic pressure and the RV hypertrophy index (RVHI) were measured. The morphology of the right ventricle was observed by H&E staining. The ultrastructure of the right ventricle was observed using a transmission electron microscope. The myocardial apoptosis of the right ventricle was evaluated by TUNEL staining. The protein expression of apoptosis-related proteins and PPARs were examined by western blotting. KEY FINDINGS: The results indicated that sildenafil decreased the RV systolic pressure and RVHI, and improved the microstructure and ultrastructure of the right ventricle in monocrotaline-induced rats. In addition, sildenafil suppressed myocardial apoptosis and promoted the protein expression of PPARs of the right ventricle in monocrotaline-induced rats. CONCLUSION: Sildenafil inhibits RV remodeling in monocrotaline-induced rats, which might be partially mediated by reducing myocardial apoptosis and activating PPARs.

Tumour heterogeneity and intercellular networks of nasopharyngeal carcinoma at single cell resolution.

The heterogeneous nature of tumour microenvironment (TME) underlying diverse treatment responses remains unclear in nasopharyngeal carcinoma (NPC). Here, we profile 176,447 cells from 10 NPC tumour-blood pairs, using single-cell transcriptome coupled with T cell receptor sequencing. Our analyses reveal 53 cell subtypes, including tumour-infiltrating CD8+ T, regulatory T (Treg), and dendritic cells (DCs), as well as malignant cells with different Epstein-Barr virus infection status. Trajectory analyses reveal exhausted CD8+ T and immune-suppressive TNFRSF4+ Treg cells in tumours might derive from peripheral CX3CR1+CD8+ T and naïve Treg cells, respectively. Moreover, we identify immune-regulatory and tolerogenic LAMP3+ DCs. Noteworthily, we observe intensive inter-cell interactions among LAMP3+ DCs, Treg, exhausted CD8+ T, and malignant cells, suggesting potential cross-talks to foster an immune-suppressive niche for the TME. Collectively, our study uncovers the heterogeneity and interacting molecules of the TME in NPC at single-cell resolution, which provide insights into the mechanisms underlying NPC progression and the development of precise therapies for NPC.

Single-cell transcriptome profiling of an adult human cell atlas of 15 major organs.

BACKGROUND: As core units of organ tissues, cells of various types play their harmonious rhythms to maintain the homeostasis of the human body. It is essential to identify the characteristics of cells in human organs and their regulatory networks for understanding the biological mechanisms related to health and disease. However, a systematic and comprehensive single-cell transcriptional profile across multiple organs of a normal human adult is missing. RESULTS: We perform single-cell transcriptomes of 84,363 cells derived from 15 tissue organs of one adult donor and generate an adult human cell atlas. The adult human cell atlas depicts 252 subtypes of cells, including major cell types such as T, B, myeloid, epithelial, and stromal cells, as well as novel COCH+ fibroblasts and FibSmo cells, each of which is distinguished by multiple marker genes and transcriptional profiles. These collectively contribute to the heterogeneity of major human organs. Moreover, T cell and B cell receptor repertoire comparisons and trajectory analyses reveal direct clonal sharing of T and B cells with various developmental states among different tissues. Furthermore, novel cell markers, transcription factors, and ligand-receptor pairs are identified with potential functional regulations in maintaining the homeostasis of human cells among tissues. CONCLUSIONS: The adult human cell atlas reveals the inter- and intra-organ heterogeneity of cell characteristics and provides a useful resource in uncovering key events during the development of human diseases in the context of the heterogeneity of cells and organs.

Identification and expression profiling analysis of ascorbate peroxidase gene family in Actinidia chinensis (Hongyang).

Ascorbate peroxidase (APX) is one of the important antioxidant enzymes in the active oxygen metabolism pathway of plants and animals, especially it is the key enzyme to clear H2O2 in chloroplast and the main enzyme of vitamin C metabolism. However, knowledge about APX gene family members and their evolutionary and functional characteristics in kiwifruit is limited. In this study, we identified 13 members of the APX gene family in the kiwifruit (cultivar: Hongyang) genome according the APX proteins conserved domain of Arabidopsis thaliana. Phylogenetic analysis by maximum likelihood split these 13 genes into four groups. The APX gene family members were distributed on nine chromosomes (Nos. 4, 5, 11, 13, 20, 21, 23, 25, 28). Most of the encoded hydrophilic and lipid-soluble enzymes were predicted to be located in the cytoplasm, nucleus and chloroplast. Among them, AcAPX4, AcAPX5, AcAPX8, AcAPX12 were transmembrane proteins, and AcAPX8 and AcAPX12 had the same transmembrane domain. The gene structure analysis showed that AcAPXs were composed of 4-22 introns, except that AcAPX10 was intron-free. Multiple expectation maximization for motif elicitation program (MEME) analyzed 13 APX protein sequences of Actinidia chinensis and identified 10 conserved motifs ranging in length from 15 to 50 amino acid residues. Additionally, the predicted secondary structures of the main motifs consisted of α-helix and random coils. The gene expression of fruits in different growth stages and bagging treatment were determined by qRT-PCR. The results showed that 8 AcAPXs had the highest expression levels during the color turning period and only the gene expression of AcAPX3 was consistent with the ascorbic acid content; five AcAPXs were consistent with the ascorbic acid content after bagging. Our data provided evolutionary and functional information of AcAPX gene family members and revealed the gene expression of different members in different growth stages and bagging treatments These results may be useful for future studies of the structures and functions of AcAPX family members.

Osthole inhibits cell proliferation by regulating the TGF-ß1/Smad/p38 signaling pathways in pulmonary arterial smooth muscle cells.

Pulmonary artery smooth muscle cell (PASMC) proliferation contributes to pulmonary vascular remodeling, which ultimately leads to pulmonary arterial hypertension (PAH). Osthole has been previously shown to inhibit tumor cell growth. Our previous experiments demonstrated that osthole could prevent monocrotaline-induced PAH and pulmonary artery remodeling in rats and that its effects might be associated with inhibiting PASMC proliferation. However, the exact mechanism remains unclear. In this study, we observed the inhibitory effect of osthole on platelet-derived growth factor (PDGF)-BB-induced rat PASMC growth, cell cycle progression and proliferating cell nuclear antigen (PCNA) expression, as measured by CCK-8 assay, flow cytometric analysis and western blotting, respectively. We also detected the expression and activities of the cell cycle regulators cyclin D1/CDK4, cyclin E1/CDK2, p53, p27 and p21 and the TGF-ß1/Smad/p38 signaling pathways in rat PASMCs by western blotting. Our results show that osthole effectively suppressed PDGF-BB-stimulated proliferation, PCNA protein expression, and cell cycle progression in rat PASMCs in vitro. We further demonstrated that treatment with osthole significantly induced cell cycle arrest at the G0/G1 phase in PASMCs, which was supported by the finding that osthole significantly decreased cyclin D1/CDK4 and cyclin E1/CDK2 protein levels and increased p53, p27 and p21 protein levels. These effects may partly be attributed to the downregulation of TGF-ß1/Smad/p38 signaling pathway activation. Our findings suggest that osthole is a potential therapeutic candidate that warrants further investigation regarding its potential use for the treatment of PAH.

Osthole Alleviates Neointimal Hyperplasia in Balloon-Induced Arterial Wall Injury by Suppressing Vascular Smooth Muscle Cell Proliferation and Downregulating Cyclin D1/CDK4 and Cyclin E1/CDK2 Expression.

Percutaneous coronary intervention (PCI) is the most widely used therapy for treating ischemic heart disease. However, intimal hyperplasia and restenosis usually occur within months after angioplasty. Modern pharmacological researchers have proven that osthole, the major active coumarin of Cnidium monnieri (L.) Cusson, exerts potent antiproliferative effects in lung cancer cells, the human laryngeal cancer cell line RK33 and TE671 medulloblastoma cells, and its mechanism of action is related to cell cycle arrest. The goal of the present study was to observe the effect of osthole on vascular smooth muscle cell (VSMC) proliferation using platelet-derived growth factor-BB (PDGF-BB)-stimulated VSMCs isolated from rats and vascular balloon injury as models to further elucidate the molecular mechanisms underlying this activity. We detected the relative number of VSMCs by the MTT assay and EdU staining and examined cell cycle progression by flow cytometry. To more deeply probe the mechanisms, the protein expression levels of PCNA, the cyclin D1/CDK4 complex and the cyclin E1/CDK2 complex in balloon-treated rat carotid arteries and the mRNA and protein expression levels of the cyclin D1/CDK4 and cyclin E1/CDK2 complexes in VSMCs were detected by real-time RT-PCR and western blotting. The data showed that osthole significantly inhibited the proliferation of VSMCs induced by PDGF-BB. Furthermore, osthole caused apparent VSMC cycle arrest early in G0/G1 phase and decreased the expression of cyclin D1/CDK4 and cyclin E1/CDK2. Our results demonstrate that osthole can significantly inhibit PDGF-BB-induced VSMC proliferation and that its regulatory effects on cell cycle progression and proliferation may be related to the downregulation of cyclin D1/CDK4 and cyclin E1/CDK2 expression as well as the prevention of cell cycle progression from G0/G1 phase to S phase. The abovementioned mechanism may be responsible for the alleviation of neointimal hyperplasia in balloon-induced arterial wall injury by osthole.

Ginsenoside Re inhibits PDGF-BB-induced VSMC proliferation via the eNOS/NO/cGMP pathway.

Ginsenoside Re (GS-Re), which is a major monomeric member of the ginseng trialcohol saponin family, is one of the main active components of ginseng and plays an important role in protecting the cardiovascular system. Here, we report a novel function by which GS-Re regulates the eNOS/NO/cGMP pathway, which affects the platelet-derived growth factor-BB (PDGF-BB)-induced proliferation of vascular smooth muscle cells (VSMCs). GS-Re inhibited PDGF-BB-induced VSMC proliferation in a concentration-dependent manner without cytotoxicity, and the endothelial nitric oxide synthase (eNOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) antagonized the antiproliferative effect of GS-Re. The flow cytometry analysis suggested that GS-Re regulates VSMC proliferation by influencing the cell cycle transition from G0/G1 to S phase and decreasing the expression of G0/G1-specific regulatory proteins, including proliferating cell nuclear antigen (PCNA), cyclin D1, and CDK4, in PDGF-BB-treated VSMCs, consequently upregulating the protein expression of p21. After GS-Re treatment, the levels of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) and the phos-eNOS Ser1177/eNOS protein ratio were obviously increased. In addition, treatment with L-NAME blocked the eNOS/NO/cGMP signaling pathway, and the protein levels of PCNA, cyclin D1, and CDK4 were markedly increased in GS-Re-treated VSMCs, while p21 expression was decreased in PDGF-BB-induced VSMCs. Overall, these findings reveal that GS-Re can inhibit the proliferation of VSMCs through G0/G1 cell cycle arrest, which is closely related to eNOS/NO/cGMP pathway activation. The present results provide basic pharmacological evidence of the potential prevention and treatment of cardiovascular diseases by GS-Re.

Osthole inhibits intimal hyperplasia by regulating the NF-κB and TGF-ß1/Smad2 signalling pathways in the rat carotid artery after balloon injury.

Osthole (7-methoxy-8-isopentenoxy-coumarin), a compound extracted from Cnidiummonnieri (L.) Cusson seeds, has been found to exhibit potent therapeutic effects in cancer due to its ability to inhibit inflammation and cell proliferation. However, its effects on arterial wall hypertrophy-related diseases remain unclear. Therefore, in this study, we aimed to investigate the effects of Osthole on intimal hyperplasia in a rat model of carotid artery balloon injury. We established the balloon-induced carotid artery injury rat model in male Sprague-Dawley rats, after which we administered Osthole (20mg/kg/day or 40mg/kg/day) or volume-matched normal saline orally by gavage for 14 consecutive days. Intimal hyperplasia and the degree of vascular smooth muscle cell proliferation were then evaluated by histopathological examination of the changes in the carotid artery, as well as by examination of proliferating cell nuclear antigen (PCNA) expression. Tumour necrosis factor-ɑ (TNF-α), interleukin-1ß (IL-1ß), transforming growth factor-beta (TGF-ß1) and PCNA mRNA expression levels were examined by real-time RT-PCR, while nuclear factor-κB (NF-κB (p65)), IκB-α, TGF-ß1 and phospho-Smad2 (p-Smad2) protein expression levels were analysed by immunohistochemistry or western blot analysis. We found that Osthole significantly attenuated neointimal thickness and decreased the elevations in PCNA protein expression induced by balloon injury. Moreover, Osthole down-regulated the pro-inflammatory factors TNF-α and IL-1ß and NF-κB (p65), whose expression had been upregulated after balloon injury. Moreover, IκB-α protein expression levels increased following Osthole treatment. In addition, the elevations in TGF-ß1 and p-Smad2 protein expression induced by balloon injury were both significantly attenuated by Osthole administration. We concluded that Osthole significantly inhibited neointimal hyperplasia in balloon-induced rat carotid artery injury and that the mechanism by which this occurs may involve NF-κB, IL-1ß and TNF-ɑ down-regulation, which alleviates the inflammatory response, and TGF-ß1/Smad2 signalling pathway inhibition.

Icariin prevents hypertension-induced cardiomyocyte apoptosis through the mitochondrial apoptotic pathway.

A prior study demonstrated that icariin (ICA) could repress angiotensin II-induced apoptosis in H9c2 cells. The activation of mitochondrial apoptotic pathways may play a crucial role in this phenomenon. In this study, we explored the potential protective roles of ICA in apoptosis in cardiomyocytes, cardiac remodelling, and the underlying mechanisms with regard to the mitochondrial apoptotic pathway in rats with spontaneous hypertension. The oral administration of ICA (20 and 40mg/kg/d) inhibited cardiomyocyte apoptosis and ameliorated left heart ventricle remodelling and abnormal mitochondria. ICA also decreased the blood pressure of model rats. ICA treatment increased the expression of Bcl-2 and decreased the expression of p53, Bax, Bok and cleaved caspase 3 in model rats, which suggests the potential mechanism underlying this effect. In summary, ICA inhibits the apoptosis of cardiomyocytes and ameliorates cardiac remodelling. The potential mechanism may relate to the inhibition of the mitochondrial apoptotic pathway.

Lipid Composition of Native Milk Fat Globules by Confocal Raman Microscopy.

Native fat globules composed mainly of triglycerides are secreted as droplets of variable size. The size of fat globules affects the form of fat in dairy products and final functionality, which depends mainly on the composition of the globules and membrane. However, the relation between the composition and size of fat globules has not been studied in detail. In this study, differences in the lipid content and fatty acid composition related to the size of native fat globules were investigated using confocal Raman spectroscopy, which offers the possibility of acquisition and analysis of the Raman signal without disruption of a single fat globule in natural suspension. The results showed small fat globules (SFG) had a higher ratio of band intensities at 2885/2850 cm⁻¹, indicating SFG tend to have a triglyceride core in a fluid state with a milk fat globule membrane in a crystalline state. In addition, the SFG had a higher level of unsaturation compared to large fat globules, shown by a lower ratio at 1655/1443 cm⁻¹. Using cream with selected SFG would allow a harder and more costly churning process but lead to a softer butter.

Selective amino acid restriction targets mitochondria to induce apoptosis of androgen-independent prostate cancer cells.

Relative specific amino acid dependency is one of the metabolic abnormalities of cancer cells, and restriction of specific amino acids induces apoptosis of prostate cancer cells. This study shows that restriction of tyrosine and phenylalanine (Tyr/Phe), glutamine (Gln), or methionine (Met), modulates Raf and Akt survival pathways and affects the function of mitochondria in DU145 and PC3, in vitro. These three restrictions inhibit energy production (ATP synthesis) and induce generation of reactive oxygen species (ROS). Restriction of Tyr/Phe or Met in DU145 and Met in PC3 reduces mitochondrial membrane potential (DeltaPsim) and induces caspase-dependent and -independent apoptosis. In DU145, Tyr/Phe or Met restriction reduces activity of Akt, mitochondrial distribution of phosphorylated Raf and apoptosis inducing factor (AIF), and increases mitochondrial distribution of Bak. Mitochondrial Bcl-XL is increased in Tyr/Phe-restricted but decreased in Met-restricted cells. Under Tyr/Phe or Met restriction, reduced mitochondrial Raf does not inactivate the pro-apoptotic function of Bak. Tyr/Phe restriction also inhibits Bcl-2 and Met restriction inhibits Bcl-XL in mitochondria. These comprehensive actions damage the integrity of the mitochondria and induce apoptosis of DU145. In PC3, apoptosis induced by Met restriction was not associated with alterations in intracellular distribution of Raf, Bcl-2 family proteins, or AIF. All of the amino acid restrictions inhibited Akt activity in this cell line. We conclude that specific amino acid restriction differentially interferes with homeostasis/balance between the Raf and Akt survival pathways and with the interaction of Raf and Bcl-2 family proteins in mitochondria to induce apoptosis of DU145 and PC3 cells.

Specific amino acid restriction inhibits attachment and spreading of human melanoma via modulation of the integrin/focal adhesion kinase pathway and actin cytoskeleton remodeling.

We had previously found that selective restriction of amino acids inhibits invasion of human A375 melanoma. Integrins, cell surface receptors for the components of extracellular matrix (ECM), are activated during cell adhesion and spreading, and initiate signaling pathways that control growth and invasion of tumor cells. We examined the effect of tyrosine (Tyr) and phenylalanine (Phe), methionine (Met) or glutamine (Gln) restriction on attachment and spreading of A375 and MeWo melanoma cell lines on fibronectin and laminin. In A375 cells, restriction of Tyr/Phe or Met inhibited attachment to and spreading on laminin and fibronectin, inhibited alpha3 and alpha4 integrin expression, and inhibited accumulation of FAK-Tyr397 and F-actin at leading edges of cell protrusions. Tyr/Phe restriction also inhibited attachment-induced autophosporylation of FAK-Tyr397. In MeWo cells, the order of inhibition by amino acid restriction on cell attachment and spreading was as follows: Gln > Tyr/Phe > Met. Restriction of Gln reduced alpha5 integrin expression. All amino acid restrictions similarly inhibited phosphorylation of FAK-Tyr397, FAK-Tyr577, FAK-Tyr861 and paxillin-Tyr31. Gln restriction exhibited the strongest inhibition of actin cytoskeleton remodeling during the cell spreading. The present study reveals that specific amino acid restriction inhibits attachment and spreading of melanoma via inhibition of specific integrin expression, inhibition of integrin-mediated FAK phosphorylation, and modulation of actin cytoskeleton remodeling. These data provide additional understanding of the mechanism by which specific amino acid restriction controls invasion and migration of melanoma.

Specific amino acid dependency regulates invasiveness and viability of androgen-independent prostate cancer cells.

Androgen-independent prostate cancer is resistant to therapy and is often metastatic. Here we studied the effect of deprivation of tyrosine and phenylalanine (Tyr/Phe), glutamine (Gln), or methionine (Met), in vitro on human DU145 and PC3 androgen-independent prostate cancer cells, and on nontumorigenic human infant foreskin fibroblasts and human prostate epithelial cells. Deprivation of the amino acids similarly inhibited growth of DU145 and PC3 cells, arresting the cell cycle at G0/G1. Met and Tyr/Phe deprivation induces apoptosis in DU145, but only Met deprivation induces apoptosis in PC3 cells. The growth of normal cells is inhibited, but no apoptosis is induced by amino acid deprivation. Tyr/Phe deprivation inhibits expression and phosphorylation of focal adhesion kinase (FAK) and extracellular-regulated kinase (ERK) in DU145 but not PC3 or normal cells. Met deprivation inhibits phosphorylation but not protein expression of FAK and ERK in PC3. Therefore, apoptosis of DU145 and PC3 cells by amino acid restriction is FAK and ERK dependent. Tyr/Phe and Met deprivation inhibits invasion of DU145 and PC3, but Gln deprivation only inhibits invasion of DU145 cells. This indicates that the inhibition of invasion is not dependent on induction of apoptosis. The inhibition of invasion by Tyr/Phe restriction in DU145 and Met restriction in PC3 is consistent with the inhibition on FAK/ERK signaling. The inhibition of Tyr/Phe restriction in PC3 and Gln restriction in DU145 is not associated with inhibition of FAK/ERK. This indicates that FAK/ERK-dependent and independent pathways are modulated by specific amino acid restriction. This study shows the potential for specific amino acid restriction to treat prostate cancer.

Activation of caspases and cleavage of Bid are required for tyrosine and phenylalanine deficiency-induced apoptosis of human A375 melanoma cells.

Deprivation of tyrosine (Tyr) and phenylalanine (Phe) inhibits growth and induces programmed cell death (apoptosis) of human A375 melanoma cells. Herein, we found that activation of caspases and release of mitochondrial cytochrome c are required for this process. Culturing A375 cells in Tyr/Phe-free medium, containing 10% dialyzed fetal bovine serum, results in activation of caspase-3-like activity. This is accompanied by decreased cell viability and increased apoptosis. Tyr/Phe deprivation also stimulates proteolytic cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP). Western blot analysis showed that caspases 3, 7, 8, and 9 are activated by deprivation of Tyr/Phe. Tyr/Phe deprivation decreases mitochondrial membrane potential, induces cleavage of Bid, increases translocation of Bax from the cytosol to mitochondria, and results in release of cytochrome c from the mitochondria to the cytosol. Apoptosis due to Tyr/Phe deprivation is almost completely inhibited by the broad-spectrum cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z.VAD.fmk). This inhibitor suppresses the cleavage of Bid, the release of cytochrome c from the mitochondria to the cytosol, and the cleavage of PARP. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, does not suppress the activation of caspase 8 but suppresses release of cytochrome c, activation of caspase 9, and induction of apoptosis. These results indicate that activation of caspases, cleavage of Bid, and mitochondrial release of cytochrome c are required for apoptosis induced by Tyr/Phe deprivation.