Stabilized COre gene and Pathway Election uncovers pan-cancer shared pathways and a cancer-specific driver.

Approaches systematically characterizing interactions via transcriptomic data usually follow two systems: (i) coexpression network analyses focusing on correlations between genes and (ii) linear regressions (usually regularized) to select multiple genes jointly. Both suffer from the problem of stability: A slight change of parameterization or dataset could lead to marked alterations of outcomes. Here, we propose Stabilized COre gene and Pathway Election (SCOPE), a tool integrating bootstrapped least absolute shrinkage and selection operator and coexpression analysis, leading to robust outcomes insensitive to variations in data. By applying SCOPE to six cancer expression datasets (BRCA, COAD, KIRC, LUAD, PRAD, and THCA) in The Cancer Genome Atlas, we identified core genes capturing interaction effects in crucial pan-cancer pathways related to genome instability and DNA damage response. Moreover, we highlighted the pivotal role of CD63 as an oncogenic driver and a potential therapeutic target in kidney cancer. SCOPE enables stabilized investigations toward complex interactions using transcriptome data.

Beneficial Effects of Oleosomes Fused with Human Fibroblast Growth Factor 1 on Wound Healing via the Promotion of Angiogenesis.

In our previous study, human fibroblast growth factor 1 was successfully fused with oleosomes, energy-storing organelles of seeds, which are considered to be excellent "expression carriers" for substances with a convenient purification process. The present work aimed to explore the beneficial effects of oleosomes fused with human fibroblast growth factor 1 (OLAF) on wound healing. The data showed marked improvements in terms of the angiogenesis, vascular integrity, collagen and inflammation on the wound sites of rats with a full-thickness skin defect. Moreover, the positive role of OLAF in promoting angiogenesis and its possible pathways were clarified in vivo and in vitro. The results showed that the number, length and branches of the blood vessels of the chick embryo chorioallantoic membrane were markedly increased after OLAF treatment. Meanwhile, the in vitro results also revealed that 100 ng/mL OLAF exhibited a promoting effect on the proliferation, migration and tube formation of human umbilical vein endothelial cells. In addition, the potential of OLAF to improve wound angiogenesis was demonstrated to be associated with an up-regulated PI3K/Akt pathway by transcriptome sequencing analysis and the introduction of a PI3K/Akt pathway inhibitor (LY294002). These findings suggest that OLAF has many prospects in the development of drugs for wound healing.

Jujuboside B Reverse CUMS-Promoted Tumor Progression via Blocking PI3K/Akt and MAPK/ERK and Dephosphorylating CREB Signaling.

Background: Jujuboside B (JUB) is a saponins isolated from the seeds of Zizyphi jujuba var. spinosi, which is used to treat mental illness and is reported recently to induce cancer cell apoptosis. As our previous research showed chronic stress promoted tumor growth, this work aims to investigate whether JUB exert antitumor effect in addition to its antidepressant effect and possible mechanism. Methods: 56 female C57BL/6 mice were grouped into 7 groups: A (blank control), B (tumor-bearing control), C (tumor-bearing + JUB), D (CUMS control), E (CUMS + JUB), F (tumor-bearing + CUMS), and G (tumor-bearing + CUMS + JUB). Groups C, E, G, B, D, and F were administered, respectively, with JUB (40 mg/kg/day) or vehicle for 2 weeks. Serum 5-HT, Trp (tryptophane), inflammatory cytokines TNF-α, IL-4, -6, and -10 levels were detected by ELISA. The tumors in groups B and F were isolated for RNA-seq sequencing. Protein and mRNA expression of Bax, Bcl-2, p-PI3K, p-Akt, p-MAPK, p-ERK, and p-CREB in tumor tissues were detected. In vitro, A549 cells were stimulated with JUB (60 µmol/L), in which proliferation rate and colony formation rate were detected. The PI3K/Akt and, MAPK/ERK pathway were measured. Results: Chronic stress successfully induced the depression-like phenotype (group D vs. A) and promoted tumor growth (group B vs. F). JUB significantly ameliorated the depression-like phenotype and increased 5-HT, Trp levels (group D vs. E), and reversing CUMS-induced tumor progression. Meanwhile, JUB decreased inflammatory cytokine levels. Chronic stress upregulated the phosphorylation levels of PI3K/Akt/MAPK/ERK/CREB; JUB reversed this regulation. JUB significantly inhibited cell viability, colony formation rate, and downregulated the phosphorylation levels of PI3K/Akt/MAPK/ERK/CREB in vitro. Conclusions: JUB reverses CUMS-promoted tumor progression in tumor-bearing mice with depression-like phenotype. JUB exerts the dual beneficial effect on tumor growth and depression-like phenotype by blocking the signal transduction pathway of PI3K/Akt, MAPK/ERK, and dephosphorylating the downstream signaling regulator CREB.

Sex differences in peripheral monoamine transmitter and related hormone levels in chronic stress mice with a depression-like phenotype.

Backgrounds: Chronic stress could induce depression-like phenotype in animal models. Previous data showed that sex differences exist after chronic stress model establishment, however, the detailed information about the difference of blood biochemical indexes is not clear. In this study, we aim to supply comparison of monoamine transmitters and related hormone markers in serum between male and female depressed mice, and in order to better understand the sex difference in transmitters and hormone levels in depression occurrence and development. Methods: Sixty C57BL/6 mice (both male and female) were divided into two groups by gender. Same gender mice were then divided randomly into the non-treated control group and chronic stress group which was exposed to 8 weeks of chronic unpredictable mild stress (CUMS). Depression-like behavior was assessed with open-field test and sucrose preference test. Blood sample was collected and monoamine transmitter and related hormone in serum were measured by ELISA. Results: The depression-like phenotype mice model was established successfully after 8 weeks of chronic stress. The locomotion activity scores in male stressed mice declined more than that in female stressed mice, while the exploratory behavior scores in female stressed mice declined more than that in male stressed mice. Compared to non-treated control group mice, mice in the chronic stress group in response to stress showed greater declines in monoamine transmitters (5-HT, dopamine, norepinephrine) and sex hormones (androgen, estrogen, oxytocin and prolactin), while stress hormones (adrenaline, corticosterone and ACTH) were significantly increased. The decrease of norepinephrine, androgen and estrogen in female stressed mice was greater than in male stressed mice, whereas the 5-HT and oxytocin in male stressed mice decreased more than in female stressed mice, and the corticosterone in male stressed mice increased more than in female stressed mice. Conclusion: Sex differences of monoamine transmitter and related hormone levels in serum occurred in chronic stress induced depression-like phenotype mice model. It may provide a useful reference to guide precise antidepressant treatment in different gender population in clinical care.

Raptinal Induces Gasdermin E-Dependent Pyroptosis in Naïve and Therapy-Resistant Melanoma.

Lack of response and acquired resistance continue to be limitations of targeted and immune-based therapies. Pyroptosis is an inflammatory form of cell death characterized by the release of inflammatory damage-associated molecular patterns (DAMP) and cytokines via gasdermin (GSDM) protein pores in the plasma membrane. Induction of pyroptosis has implications for treatment strategies in both therapy-responsive, as well as resistance forms of melanoma. We show that the caspase-3 activator, raptinal, induces pyroptosis in both human and mouse melanoma cell line models and delays tumor growth in vivo. Release of DAMPs and inflammatory cytokines was dependent on caspase activity and GSDME expression. Furthermore, raptinal stimulated pyroptosis in melanoma models that have acquired resistance to BRAF and MEK inhibitor therapy. These findings add support to efforts to induce pyroptosis in both the treatment-naïve and resistant settings. IMPLICATIONS: Raptinal can rapidly induce pyroptosis in naïve and BRAFi plus MEKi-resistant melanoma, which may be beneficial for patients who have developed acquired resistance to targeted therapies.

Cisplatin-Induced Anorexia and Pica Behavior in Rats Enhanced by Chronic Stress Pretreatment.

Background: Chemotherapy-induced nausea and vomiting severely impairs the treatment and prognosis of cancer patients. Depressive mood disorder might aggravate nausea and vomiting in cancer patients; however, the role of neurotransmitters and receptors involved in the mediation of emesis and nausea is still not well elaborated. Methods: The study was carried out based on the chronic unpredictable mild stress-induced depression-like phenotype rat model and cisplatin-induced pica rat model establishment. Forty male Sprague-Dawley rats were randomized into the non-treated control group and the chronic stress group, which were exposed to 8 weeks of stress. Each group was then sub-divided into vehicle subgroups (n = 10) and cisplatin subgroups (n = 10) which were given cisplatin to induce pica behavior. Kaolin and food intake were recorded after administration. The medulla oblongata and ileum tissues were obtained. Neurotransmitters involved in the mediation of emesis and nausea (5-HT, DA, SP, and AEA) were detected using an ELISA kit. Vomit-related receptors (5-HT3R, DA2R, NK1R, and CB1R) in tissues were assayed for mRNA and protein expression by RT-qPCR and Western blotting. Results: Behavioral test and sucrose preference validated that depression-like phenotype rat models were established successfully. The kaolin consumption test confirmed that chronic stress pretreatment aggravated anorexia and pica behavior. Vomiting-related molecules' data showed that chronic stress exposure increased 5-HT and SP levels in the medulla oblongata. Vomiting-related receptor expression data showed that chronic stress pretreatment upregulated 5-HT3R, DA2R, and NK1R expressions and downregulated the CB1R expression in the medulla oblongata. However, chronic stress pretreatment downregulated 5-HT3R, DA2R, and NK1R expressions and upregulated the CB1R expression in the ileum. Conclusion: Chronic stress pretreatment aggravates anorexia and vomiting progress, which might be via altering neurotransmitters and receptors involved in the mediation of emesis and the nausea level and expression in the central nervous system.

A Genome-Wide Screen Identifies PDPK1 as a Target to Enhance the Efficacy of MEK1/2 Inhibitors in NRAS Mutant Melanoma.

Melanomas frequently harbor activating NRAS mutations. However, limited advance has been made in developing targeted therapy options for patients with NRAS mutant melanoma. MEK inhibitors (MEKi) show modest efficacy in the clinic and their actions need to be optimized. In this study, we performed a genome-wide CRISPR-Cas9-based screen and demonstrated that loss of phosphoinositide-dependent kinase-1 (PDPK1) enhances the efficacy of MEKi. The synergistic effects of PDPK1 loss and MEKi was validated in NRAS mutant melanoma cell lines using pharmacologic and molecular approaches. Combined PDPK1 inhibitors (PDPK1i) with MEKi suppressed NRAS mutant xenograft growth and induced gasdermin E-associated pyroptosis. In an immune-competent allograft model, PDPK1i+MEKi increased the ratio of intratumoral CD8+ T cells, delayed tumor growth, and prolonged survival; the combination treatment was less effective against tumors in immune-deficient mice. These data suggest PDPK1i+MEKi as an efficient immunostimulatory strategy against NRAS mutant melanoma. SIGNIFICANCE: Targeting PDPK1 stimulates antitumor immunity and sensitizes NRAS mutant melanoma to MEK inhibition, providing rationale for the clinical development of a combinatorial approach for treating patients with melanoma.

Targeting SOX10-deficient cells to reduce the dormant-invasive phenotype state in melanoma.

Cellular plasticity contributes to intra-tumoral heterogeneity and phenotype switching, which enable adaptation to metastatic microenvironments and resistance to therapies. Mechanisms underlying tumor cell plasticity remain poorly understood. SOX10, a neural crest lineage transcription factor, is heterogeneously expressed in melanomas. Loss of SOX10 reduces proliferation, leads to invasive properties, including the expression of mesenchymal genes and extracellular matrix, and promotes tolerance to BRAF and/or MEK inhibitors. We identify the class of cellular inhibitor of apoptosis protein-1/2 (cIAP1/2) inhibitors as inducing cell death selectively in SOX10-deficient cells. Targeted therapy selects for SOX10 knockout cells underscoring their drug tolerant properties. Combining cIAP1/2 inhibitor with BRAF/MEK inhibitors delays the onset of acquired resistance in melanomas in vivo. These data suggest that SOX10 mediates phenotypic switching in cutaneous melanoma to produce a targeted inhibitor tolerant state that is likely a prelude to the acquisition of resistance. Furthermore, we provide a therapeutic strategy to selectively eliminate SOX10-deficient cells.

Chronic Stress-Induced Gene Changes In Vitro and In Vivo: Potential Biomarkers Associated With Depression and Cancer Based on circRNA- and lncRNA-Associated ceRNA Networks.

Circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs) have been considered as biomarkers or regulators in many diseases. However, the exact role of circRNA- or lncRNA-mediated competing endogenous RNA (ceRNA) networks in the modulation of depression pathogenesis-relevant processes is not clear. In this study, we profiled whole transcriptome in depression patients' blood samples via microarray analysis. As a result, a total of 340 circRNAs, 398 lncRNAs, 206 miRNAs, and 92 mRNAs were differentially expressed between the depression and control groups. Then, we constructed ceRNA networks according to the differentially expressed genes (DEGs). Using bioinformatics analysis, 89 pairs of circRNA-ceRNA and 49 pairs of lncRNA-ceRNA networks were obtained. Since depression is a broad and heterogeneous condition that is known as promoter for many chronic diseases including cancer, so we further dug out 28 circRNAs, 61 lncRNAs, 26 miRNAs, and 29 mRNAs that are associated with cancer. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the DEGs were significantly enriched in cancer-related signaling pathways such as MAPK, Wnt, IL-17, Ras, and PI3K-Akt. Genes involved in the above pathways such as S100A9, GATA2, SRFP5, SLC45A3, NTRK1, FRZB, has_circ_0014221, has_circ_0014220, and has_circ_0087100 were dysregulated in various cancer cell lines by stress hormones induced. HDC, GATA2, SLC45A3, and NTRK1 were downregulated in tumor-bearing mice subjected to chronic unpredictable mild stress (CUMS). LncRNA-mediated ceRNA network validation showed that overexpression of miR-4530 declined HDC level. Our findings highlight the potential circRNA- and lncRNA-mediated ceRNA regulatory mechanisms in the pathogenesis of depression and as potential biomarkers in depression cancer comorbidity through the pathways of IL-17 or histidine metabolism.

PBRM1 suppresses tumor growth as a novel p53 acetylation reader.

The reader(s) for acetylated tumor antigen p53 remains elusive. Here, PBRM1 (polybromo-1) is identified as a reader for acetylated lysine382 on p53 through its bromodomain 4 (BD4). PBRM1 BD4 mutants fail to support p53 transcriptional activity and suppress tumor growth. Thus PBRM1 suppresses tumor growth through p53 in kidney cancer.

Mutant BRAF and MEK Inhibitors Regulate the Tumor Immune Microenvironment via Pyroptosis.

Combinations of BRAF inhibitors and MEK inhibitors (BRAFi + MEKi) are FDA-approved to treat BRAF V600E/K-mutant melanoma. Efficacy of BRAFi + MEKi associates with cancer cell death and alterations in the tumor immune microenvironment; however, the links are poorly understood. We show that BRAFi + MEKi caused durable melanoma regression in an immune-mediated manner. BRAFi + MEKi treatment promoted cleavage of gasdermin E (GSDME) and release of HMGB1, markers of pyroptotic cell death. GSDME-deficient melanoma showed defective HMGB1 release, reduced tumor-associated T cell and activated dendritic cell infiltrates in response to BRAFi + MEKi, and more frequent tumor regrowth after drug removal. Importantly, BRAFi + MEKi-resistant disease lacked pyroptosis markers and showed decreased intratumoral T-cell infiltration but was sensitive to pyroptosis-inducing chemotherapy. These data implicate BRAFi + MEKi-induced pyroptosis in antitumor immune responses and highlight new therapeutic strategies for resistant melanoma. SIGNIFICANCE: Targeted inhibitors and immune checkpoint agents have advanced the care of patients with melanoma; however, detailed knowledge of the intersection between these two research areas is lacking. We describe a molecular mechanism of targeted inhibitor regulation of an immune-stimulatory form of cell death and provide a proof-of-principle salvage therapy concept for inhibitor-resistant melanoma.See related commentary by Smalley, p. 176.This article is highlighted in the In This Issue feature, p. 161.

PBRM1 acts as a p53 lysine-acetylation reader to suppress renal tumor growth.

p53 acetylation is indispensable for its transcriptional activity and tumor suppressive function. However, the identity of reader protein(s) for p53 acetylation remains elusive. PBRM1, the second most highly mutated tumor suppressor gene in kidney cancer, encodes PBRM1. Here, we identify PBRM1 as a reader for p53 acetylation on lysine 382 (K382Ac) through its bromodomain 4 (BD4). Notably, mutations on key residues of BD4 disrupt recognition of p53 K382Ac. The mutation in BD4 also reduces p53 binding to promoters of target genes such as CDKN1A (p21). Consequently, the PBRM1 BD4 mutant fails to fully support p53 transcriptional activity and is defective as a tumor suppressor. We also find that expressions of PBRM1 and p21 correlate with each other in human kidney cancer samples. Our findings uncover a tumor suppressive mechanism of PBRM1 in kidney cancer and provide a mechanistic insight into the crosstalk between p53 and SWI/SNF complexes.

High affinity binding of H3K14ac through collaboration of bromodomains 2, 4 and 5 is critical for the molecular and tumor suppressor functions of PBRM1.

Polybromo-1 (PBRM1) is an important tumor suppressor in kidney cancer. It contains six tandem bromodomains (BDs), which are specialized structures that recognize acetyl-lysine residues. While BD2 has been found to bind acetylated histone H3 lysine 14 (H3K14ac), it is not known whether other BDs collaborate with BD2 to generate strong binding to H3K14ac, and the importance of H3K14ac recognition for the molecular and tumor suppressor function of PBRM1 is also unknown. We discovered that full-length PBRM1, but not its individual BDs, strongly binds H3K14ac. BDs 2, 4, and 5 were found to collaborate to facilitate strong binding to H3K14ac. Quantitative measurement of the interactions between purified BD proteins and H3K14ac or nonacetylated peptides confirmed the tight and specific association of the former. Interestingly, while the structural integrity of BD4 was found to be required for H3K14ac recognition, the conserved acetyl-lysine binding site of BD4 was not. Furthermore, simultaneous point mutations in BDs 2, 4, and 5 prevented recognition of H3K14ac, altered promoter binding and gene expression, and caused PBRM1 to relocalize to the cytoplasm. In contrast, tumor-derived point mutations in BD2 alone lowered PBRM1's affinity to H3K14ac and also disrupted promoter binding and gene expression without altering cellular localization. Finally, overexpression of PBRM1 variants containing point mutations in BDs 2, 4, and 5 or BD2 alone failed to suppress tumor growth in a xenograft model. Taken together, our study demonstrates that BDs 2, 4, and 5 of PBRM1 collaborate to generate high affinity to H3K14ac and tether PBRM1 to chromatin. Mutations in BD2 alone weaken these interactions, and this is sufficient to abolish its molecular and tumor suppressor functions.

Multiple tumor suppressors regulate a HIF-dependent negative feedback loop via ISGF3 in human clear cell renal cancer.

Whereas VHL inactivation is a primary event in clear cell renal cell carcinoma (ccRCC), the precise mechanism(s) of how this interacts with the secondary mutations in tumor suppressor genes, including PBRM1, KDM5C/JARID1C, SETD2, and/or BAP1, remains unclear. Gene expression analyses reveal that VHL, PBRM1, or KDM5C share a common regulation of interferon response expression signature. Loss of HIF2α, PBRM1, or KDM5C in VHL-/-cells reduces the expression of interferon stimulated gene factor 3 (ISGF3), a transcription factor that regulates the interferon signature. Moreover, loss of SETD2 or BAP1 also reduces the ISGF3 level. Finally, ISGF3 is strongly tumor-suppressive in a xenograft model as its loss significantly enhances tumor growth. Conversely, reactivation of ISGF3 retards tumor growth by PBRM1-deficient ccRCC cells. Thus after VHL inactivation, HIF induces ISGF3, which is reversed by the loss of secondary tumor suppressors, suggesting that this is a key negative feedback loop in ccRCC.

The structure and regulation of Cullin 2 based E3 ubiquitin ligases and their biological functions.

BACKGROUND: Cullin-RING E3 ubiquitin ligase complexes play a central role in targeting cellular proteins for ubiquitination-dependent protein turnover through 26S proteasome. Cullin-2 is a member of the Cullin family, and it serves as a scaffold protein for Elongin B and C, Rbx1 and various substrate recognition receptors to form E3 ubiquitin ligases. MAIN BODY OF THE ABSTRACT: First, the composition, structure and the regulation of Cullin-2 based E3 ubiquitin ligases were introduced. Then the targets, the biological functions of complexes that use VHL, Lrr-1, Fem1b, Prame, Zyg-11, BAF250, Rack1 as substrate targeting subunits were described, and their involvement in diseases was discussed. A small molecule inhibitor of Cullins as a potential anti-cancer drug was introduced. Furthermore, proteins with VHL box that might bind to Cullin-2 were described. Finally, how different viral proteins form E3 ubiquitin ligase complexes with Cullin-2 to counter host viral defense were explained. CONCLUSIONS: Cullin-2 based E3 ubiquitin ligases, using many different substrate recognition receptors, recognize a number of substrates and regulate their protein stability. These complexes play critical roles in biological processes and diseases such as cancer, germline differentiation and viral defense. Through the better understanding of their biology, we can devise and develop new therapeutic strategies to treat cancers, inherited diseases and viral infections.

Loss of 4E-BP1 function induces EMT and promotes cancer cell migration and invasion via cap-dependent translational activation of snail.

The cap-dependent translation is frequently deregulated in a variety of cancers associated with tumor progression. However, the molecular basis of the translation activation for metastatic progression of cancer remains largely elusive. Here, we demonstrate that activation of cap-dependent translation by silencing the translational repressor 4E-BP1 causes cancer epithelial cells to undergo epithelial-mesenchymal transition (EMT), which is associated with selective upregulation of the EMT inducer Snail followed by repression of E-cadherin expression and promotion of cell migratory and invasive capabilities as well as metastasis. Conversely, inhibition of cap-dependent translation by a dominant active mutant 4E-BP1 effectively downregulates Snail expression and suppresses cell migration and invasion. Furthermore, dephosphorylation of 4E-BP1 by mTORC1 inhibition or directly targeting the translation initiation also profoundly attenuates Snail expression and cell motility, whereas knockdown of 4E-BP1 or overexpression of Snail significantly rescues the inhibitory effects. Importantly, 4E-BP1-regulated Snail expression is not associated with its changes in the level of transcription or protein stability. Together, these findings indicate a novel role of 4E-BP1 in the regulation of EMT and cell motility through translational control of Snail expression and activity, and suggest that targeting cap-dependent translation may provide a promising approach for blocking Snail-mediated metastatic potential of cancer.

Expression, purification and immunogenic characterization of hepatitis C virus recombinant E1E2 protein expressed by Pichia pastoris yeast.

Development of an effective vaccine may be the key in the control of hepatitis C virus (HCV) infection. Recent studies have shown that HCV envelope proteins can induce broadly neutralizing antibodies against conserved domain for HCV binding to the cellular receptors. So HCV envelope proteins are considered as the major HCV vaccine candidate. In this study, we used Pichia pastoris yeast to express truncated HCV E1E2 protein, which consists of E1 residues 187-346 and E2 residues 381-699. The yeast can produce high level of recombinant HCV E1E2 protein. The protein has complex glycosylation and can bind to CD81, the putative HCV receptor. Moreover, the purified protein can efficiently induce anti-E1E2 antibodies in rabbits, which are able to neutralize two kinds of HCV pseudotype particles derived from HCV genotype 1a and 1b, as well as HCV virions derived from HCV genotype 2a. These findings indicate that the recombinant E1E2 glycoprotein is effective in inducing broadly neutralizing antibodies, and is a potent HCV vaccine candidate.

Roles of ezrin in the growth and invasiveness of esophageal squamous carcinoma cells.

Ezrin, which crosslinks the cytoskeleton and plasma membrane, is involved in the growth and metastatic potential of cancer cells. Ezrin expression in esophageal squamous cell carcinoma (ESCC) was described recently, but its roles and the underlying mechanism(s) remain unclear. In our study, we first showed that ezrin in ESCC cell is expressed in the nucleus as well as in the cytoplasm and plasma membrane. Then, by using RNAi, we revealed that interference of ezrin expression suppressed the growth, adhesion and invasiveness of ESCC cells. Tumorigenesis experiments revealed that ezrin may directly regulate tumor formation in vivo. To explore the molecular mechanisms through which ezrin contributes to the proliferation and invasiveness of ESCC cells, we used cDNA microarrays to analyze ezrin knockdown cells and the control cells; of 39,000 genes examined, 297 were differentially expressed upon ezrin knockdown, including some proliferation- and invasiveness-related genes such as ATF3, CTGF and CYR61. Furthermore, pathway analysis showed that ezrin knockdown led to decreased activation of the TGF-beta and MAPK pathways, and ezrin-mediated cell invasiveness alteration was dependent on the activation of these pathways. Finally, immunohistochemical staining on 80 ESCC specimens and 50 normal esophageal mucosae revealed that the expression levels of 3 altered genes involved in the regulation of cell proliferation and tumor metastasis, including CTGF, CYR61 and ATF3, were altered in ESCCs, and their expression pattern correlated with ezrin expression. Taken together, we propose that ezrin might function in the growth and invasiveness of ESCC cells through the MAPK and TGF-beta pathways.

Inhibitory effects of four plants flavonoids extracts on fatty acid synthase.

Fatty acid synthase (FAS) had been found overexpress and hyperactive in most cancers. Pharmacological inhibitors of FAS activity preferentially repress cancer cell proliferation and induce cancer cell apoptosis without affecting nonmalignant fibroblasts. These made FAS an excellent drug target for cancer therapy. The activity of FAS in 11 different kinds of cancer cells, including esophageal carcinoma (EC109, EC8712, H5E973), gastric carcinoma (N87, BGC823), lung carcinoma (A549, 95-D), hepatoma (HepG2), uterine cervix cancer (HeLa) and leukaemia (K562, U937) were compared using spectrophotometric method. We selected the cell line with the highest FAS activity as cell model to study the inhibitory effect of the flavonoids extracts on FAS. Four plants including Canavium album Raeuseh leaves, Bombax ceiba Linn, Brassica albograbra Bailey, and Citrus reticulata Blanco were selected for extracting flavonoids. The results showed significantly different FAS activity among different cancer cells. The FAS activity is the lowest in gastric cancer cell N87 (15.91 ± 3.61 U/mg protein) and the highest in lung cancer cell A549 (127.36 ± 10.14 U/mg protein). The cancer cell A549 was used as cell model to test the inhibitory effort of flavonoids extracts on FAS. Results showed that the flavonoids extracts of Citrus reticulata Blanco and Canavium album Raeuseh leaves have higher inhibitory effect on FAS activity compared with the universally used FAS inhibitor C75 and both extracts inhibit cancer cell proliferation when added to cultured cancer cells. These studies provided a good cell model for testing the inhibitory effect on FAS activity and suggested that Citrus reticulata Blanco rind and Canavium album Raeuseh leaves are good biomaterials for separating and purifying natural flavonoids FAS inhibitors and exploring their inhibitory mechanisms.