A Comprehensive Review on the Benefits and Problems of Curcumin with Respect to Human Health.

Curcumin is the most important active component in turmeric extracts. Curcumin, a natural monomer from plants has received a considerable attention as a dietary supplement, exhibiting evident activity in a wide range of human pathological conditions. In general, curcumin is beneficial to human health, demonstrating pharmacological activities of anti-inflammation and antioxidation, as well as antitumor and immune regulation activities. Curcumin also presents therapeutic potential in neurodegenerative, cardiovascular and cerebrovascular diseases. In this review article, we summarize the advancements made in recent years with respect to curcumin as a biologically active agent in malignant tumors, Alzheimer's disease (AD), hematological diseases and viral infectious diseases. We also focus on problems associated with curcumin from basic research to clinical translation, such as its low solubility, leading to poor bioavailability, as well as the controversy surrounding the association between curcumin purity and effect. Through a review and summary of the clinical research on curcumin and case reports of adverse effects, we found that the clinical transformation of curcumin is not successful, and excessive intake of curcumin may have adverse effects on the kidneys, heart, liver, blood and immune system, which leads us to warn that curcumin has a long way to go from basic research to application transformation.

Curcumin Nicotinate Selectively Induces Cancer Cell Apoptosis and Cycle Arrest through a P53-Mediated Mechanism.

Curcumin is an anticancer agent, but adverse effects and low bioavailability are its main drawbacks, which drives efforts in chemical modifications of curcumin. This study evaluated antiproliferative activity and cancer cell selectivity of a curcumin derivative, curcumin nicotinate (CN), in which two niacin molecules were introduced. Our data showed that CN effectively inhibited proliferation and clonogenic growth of colon (HCT116), breast (MCF-7) and nasopharyngeal (CNE2, 5-8F and 6-10B) cancer cells with IC50 at 27.7 µM, 73.4 µM, 64.7 µM, 46.3 µM, and 31.2 µM, respectively. In cancer cells, CN induced apoptosis and cell cycle arrest at G2/M phase through a p53-mediated mechanism, where p53 was activated, p21 and pro-apoptotic proteins Bid and Bak were upregulated, and PARP was cleaved. In non-transformed human mammary epithelial cells MCF10A, CN at 50 µM had no cytotoxicity and p53 was not activated, but curcumin at 12.5 µM activated p53 and p21 and inhibited MCF10A cell growth. These data suggest that CN inhibits cell growth and proliferation through p53-mediated apoptosis and cell cycle arrest with cancer cell selectivity.

[Inhibition of Yiqi Jiedu formula on proliferation of nasopharyngeal carcinoma cells by MAPK/ERK signaling pathway].

To study the effect of aqueous extracts of Yiqi Jiedu formula (YQ) on the proliferation of CNE2 cells in human nasopharyngeal carcinoma, and investigate its mechanism to provide a new theoretical basis for the clinical application of YQ. CNE2 cells were treated with different concentrations (0.125, 0.25, 0.5, 0.25 g·L⁻¹) of YQ, positive control medicine (cisplatin 4.0 mg·L⁻¹), inhibitor PD98059 (50 µmol·L⁻¹), activator isoproterenol hydrochloride (20 µmol·L⁻¹), activator isoproterenol hydrochloride (ISO)+YQ 0.5 g·L⁻¹. Then cell labeling by using real-time analyzer (RTCA) and CCK 8 method were used to detect cell proliferation activity, and the half inhibitory concentration (IC50) was calculated. The cell cycle distribution was detected by fluorescence double dye flow cytometry PI staining, and Western blot method was used to detect the expression levels of related protein and MAPK/ERK signaling pathway. The results of RTCA and CCK-8 test showed that as compared with the control group, YQ group could effectively inhibit the proliferation of CNE2 cells (P<0.01), with a dose and time dependence, and 48 h IC50 value was 0.5 g·L⁻¹. The results of cell cycle showed that after 48 h of water extract treatment, the cell cycle was significantly changed, the proportion of G0/G1 was reduced, the ratio of G2/M increased, and the cell cycle was in G2/M period (P<0.01). Western blot results showed that after 48 h treatment with different concentrations of aqueous extract, cell cycle-related proteins cyclinD1, cyclinD3 and CDK2 expression levels were down-regulated; MAPK/ERK signaling pathway related protein p-c-Raf, p-MEK, p-ERK1/2 expression level significantly lower as compared with the control group (P<0.05). After adding activator and inhibitor in MAPK/ERK signaling pathway on this basis, the results showed that after adding activator ISO, cell proliferation was significantly higher than that in the Control group; the cycle related proteins cyclinD1, cyclinD3, and CDK2 expression levels were increased; at the same time, key protein p-c-Raf, p-MEK, p-ERK1/2 expression levels in the signal pathways were relatively increased. While after the addition of inhibitor PD98059, the cell proliferation was significantly lower than that in the Control group, and the expression level of corresponding protein was decreased, which was significantly different from the Control group (P<0.05). So YQ could block cell cycle and inhibit the proliferation of CNE2 cells mainly by reducing the expression of MAPK/ERK signaling pathway key protein p-c-Raf, p-MEK and p-ERK1/2.

Apoptotic death of cancer stem cells for cancer therapy.

Cancer stem cells (CSCs) play crucial roles in tumor progression, chemo- and radiotherapy resistance, and recurrence. Recent studies on CSCs have advanced understanding of molecular oncology and development of novel therapeutic strategies. This review article updates the hypothesis and paradigm of CSCs with a focus on major signaling pathways and effectors that regulate CSC apoptosis. Selective CSC apoptotic inducers are introduced and their therapeutic potentials are discussed. These include synthetic and natural compounds, antibodies and recombinant proteins, and oligonucleotides.

Unraveling the mechanism of Yiqi Jiedu formula against nasopharyngeal carcinoma: An investigation integrating network pharmacology, serum pharmacochemistry, and metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Yiqi Jiedu formula (YQJDF), rooted in the traditional Chinese medicinal principle of "tonifying qi and detoxifying", is remarkably efficacious in the clinical treatment of nasopharyngeal carcinoma (NPC). Previous studies have shed light on some of its anti-NPC effects and mechanisms, but the responsible pharmacological substances and their precise mechanisms of action remain unclear. AIM OF THE STUDY: The purpose of this study was to identify components of YQJDF that entered the bloodstream and to investigate their mechanisms of action against NPC through network pharmacology and serum metabolomics. MATERIAL AND METHODS: Components of YQJDF in serum were identified using liquid chromatography-tandem mass spectrometry. With these serum species as the focus of our research, network pharmacology analysis was used to identify active compounds and target genes that might mediate the efficacy of YQJDF in the treatment of NPC. Following establishment of an NPC xenograft model in nude mice, a non-targeted metabolomics approach was adopted to identify significant serum metabolites and metabolic pathways influenced by YQJDF. RESULTS: Thirty-six components of YQJDF were identified, primarily consisting of alkaloids, phenylpropanoids, and flavonoids. Notably, pathways such as PI3K/AKT, factors associated with Epstein-Barr virus infection, IL-17 signaling, and lipid metabolism, were highlighted as potential therapeutic targets of YQJDF during NPC treatment. Additionally, our findings suggested that YQJDF modified the metabolism of arginine and proline in the serum of mice bearing nasopharyngeal tumor grafts. CONCLUSIONS: This study identified the primary active components of YQJDF, highlighting its holistic role in the treatment of NPC through multiple targets and pathways. Furthermore, our findings provided a roadmap for future research into the mechanism of YQJDF in the therapy of NPC, setting the stage for its clinical application.

Analysis of active components and molecular mechanism of action of Rubia cordifolia L. in the treatment of nasopharyngeal carcinoma based on network pharmacology and experimental verification.

The aim of this study is to explore the active components and potential molecular mechanism of action of Rubia cordifolia L. against nasopharyngeal carcinoma (NPC). We used network pharmacology, molecular docking, and bioinformatics analysis to identify the active components and their role against NPC. The experimental verification was detected by MTT, AnnexinV-FITC/PI double fluorescence staining and Western blotting method. Network pharmacology identified that mollugin is one of the most effective components inRubia cordifolia L. Important NPC targets included HSP90AA1, CDK1, EGFR, PIK3CA, MAPK14, and CDK2. Molecular docking revealed considerable binding activity of mollugin with either of the 6 important NPC targets. Bioinformatics analysis showed that these 6 important targets were mutated in NPC, and the expression of HSP90AA1, PIK3CA, and CDK2 in cancer tissues was significantly different from that in normal tissues. MTT detection and AnnexinV-FITC/PI double fluorescence staining showed that mollugin inhibited the proliferation and induced apoptosis of NPC cells. Western blotting indicated that the molecular mechanism of mollugin against NPC was related to the regulation of the expression of Survivin and XIAP. This study predicted and partially verified the pharmacological and molecular mechanism of action of Rubia cordifolia L. against NPC. Mollugin was identified as a potential active ingredient against NPC. These results prove the reliability of network pharmacology approaches and provide a basis for further research and application of Rubia cordifolia L. against NPC.

Anti-Nasopharyngeal carcinoma mechanism of sanguinarine based on network pharmacology and molecular docking.

BACKGROUND: The purpose of this study was to investigate the mechanism of sanguinarine (SAN) against nasopharyngeal carcinoma (NPC) by means of network pharmacology, molecular docking technique, and experimental verification. METHODS: The SAN action targets were predicted using the Swiss Target Prediction database, the related NPC targets were determined using the GEO database, and the intersection of drug and disease pathway targets were considered to be the potential targets of SAN against NPC. The target-protein interaction network map was constructed using the STRING database, and the core target genes of SAN against NPC were obtained via topological network analysis. "R" language gene ontology (GO) function and Kyoto encyclopedia of genes and genome (KEGG) pathway enrichment analyses were used to dock the core target genes with SAN with the help of AutodockVina. Cell proliferation was detected using MTT and xCELLigence real-time cell analysis. Apoptosis was identified via Hoechst 33342 staining, JC-1 mitochondrial membrane staining, and annexin V-FITC/PI double fluorescence staining, while protein expression was quantified using western blotting. RESULTS: A total of 95 SAN against NPC targets were obtained using target intersection, and 8 core targets were obtained by topological analysis and included EGFR, TP53, F2, FN1, PLAU, MMP9, SERPINE1, and CDK1. Gene ontology enrichment analysis identified 530 items, and 42 items were obtained by Kyoto encyclopedia of genes and genome pathway enrichment analysis and were mainly related to the PI3K/AKT, MAPK, and p53 signaling pathways. Molecular docking results showed that SAN had good binding activity to the core target. SAN inhibited the proliferation of NPC cells, induced apoptosis, reduced the expression levels of survivin and Bcl2, and increased the expression levels of Bax and cleaved caspase-8. It also decreased the expression levels of the key proteins p-c-Raf, p-MEK, and p-ERK1/2 in the MAPK/ERK signaling pathway in NPC cells. CONCLUSION: SAN inhibits the proliferation and induces the apoptosis of NPC cells through the MAPK/ERK signaling pathway.

Analysis of the Molecular Mechanism of Evodia rutaecarpa Fruit in the Treatment of Nasopharyngeal Carcinoma Using Network Pharmacology and Molecular Docking.

Background: Nasopharyngeal carcinoma (NPC), a neoplasm of the head and neck, has high incidence and mortality rates in East and Southeast Asia. Evodia rutaecarpa is a tree native to Korea and China, and its fruit (hereafter referred to as Evodia) exhibits remarkable antitumour properties. However, little is known about its mechanism of action in NPC. In this study, we employed network pharmacology to identify targets of active Evodia compounds in nasopharyngeal carcinoma and generate an interaction network. Methods: The active ingredients of Evodia and targets in NPC were obtained from multiple databases, and an interaction network was constructed via the Cytoscape and STRING databases. The key biological processes and signalling pathways were predicted using Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes pathway enrichment analyses. Molecular docking technology was used to identify the affinity and activity of target genes, and The Cancer Genome Atlas and Human Protein Atlas databases were used to analyse differential expression. Cell Counting Kit-8 (CCK-8) and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) dual-fluorescence staining were used for experimental verification. Results: Active Evodia compounds included quercetin, isorhamnetin, and evodiamine, and important NPC targets included MAPK14, AKT1, RELA, MAPK1, JUN, and p53, which were enriched in lipid and atherosclerosis signalling pathways. Additionally, we verified the high affinity and activity of the active compounds through molecular docking, and the target proteins were verified using immunohistochemistry and differential expression analyses. Furthermore, CCK-8 assays and Annexin V-FITC/PI dual-fluorescence staining showed that isorhamnetin inhibited the proliferation of NPC cells and induced apoptosis. Conclusion: Our results identified the molecular mechanisms of Evodia and demonstrated its ability to alter the proliferation and apoptosis of NPC cells through multiple targets and pathways, thereby providing evidence for the clinical application of Evodia.

Luteolin Isolated from Polygonum cuspidatum Is a Potential Compound against Nasopharyngeal Carcinoma.

Introduction: To reveal the mechanisms by which luteolin, the major bioactive component of the Traditional Chinese Medicine (TCM) Polygonum cuspidatum, inhibits proliferation and promotes apoptosis in nasopharyngeal carcinoma (NPC) CNE2 cells. Methods: Based on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), bioactive compounds of P. cuspidatum, potential target genes and NPC disease targets of TCMSP were screened, relationship networks were constructed using these potential targets of NPC, and Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. The predicted compounds, targets and pathways were corroborated using in vitro experiments, such as MTT, Cytation™ 5 real-time cell monitoring, cell cycle detection, Annexin V-FITC/PI double staining, Hoechst 33342 staining, and mitochondrial membrane potential (ΔΨm) detection. Results: The results showed that 10 bioactive compounds (OB ≥30% and DL ≥0.18), 157 potential target genes from P. cuspidatum, and 56 common targets related to NPC were found. These included important bioactive compounds such as luteolin, quercetin, and beta-sitosterol. Key common targets included EGFR, MYC, AKT1, CASP3, CCND1, ERBB2, and common targets were enriched for the PI3K-AKT, JAK/STAT, MAPK, and C-type lectin receptor signaling pathways. The binding energy of luteolin for six common targets was less than -5.0 kcal·mol-1. After luteolin (20 µM, and 40 µM) treatment to CNE2 cells for 36 h, cell survival rates decreased, accompanied by cell morphology changes, inhibition of the cell cycle at G2/M phase, and an induction of apoptosis. The expression of the cell proliferation related protein PCNA, the antiapoptosis protein XIAP, and the PI3K-AKT pathway diagram related proteins p-ERK1/2, ERK1/2, AKT, and PI3K, all decreased. Conclusion: Luteolin derived from P. cuspidatum inhibited the proliferation of NPC CNE2 cells and promoted cell apoptosis through the PI3K-AKT signal pathway.

Isoimperatorin Induces Apoptosis of Nasopharyngeal Carcinoma Cells via the MAPK/ERK1/2 Signaling Pathway.

OBJECTIVE: To investigate the effect of isoimperatorin on nasopharyngeal carcinoma CNE2 cell apoptosis and the role of the MAPK/ERK1/2 signaling pathway in inducing apoptosis. METHODS: Real-time cellular analysis technology (RTCA) and MTT were used to detect cell proliferation; Annexin V-FITC/PI dual-fluorescence flow cytometry analysis, Hoechst 33342 staining, and mitochondrial membrane potential detection kit were used to detect cell apoptosis; western blot was used to detect protein expression. RESULTS: Different concentrations of isoimperatorin (10 µM, 20 µM, 20 µM, 20 µM, 20 µM, 20 µM, 20 µM, 20 . CONCLUSION: Isoimperatorin can induce nasopharyngeal carcinoma CNE2 cell apoptosis through the MAPK/ERK1/2 signaling pathway.

MicroRNA-188 regulates aging-associated metabolic phenotype.

With the increasing aging population, aging-associated diseases are becoming epidemic worldwide, including aging-associated metabolic dysfunction. However, the underlying mechanisms are poorly understood. In the present study, we aimed to investigate the role of microRNA miR-188 in the aging-associated metabolic phenotype. The results showed that the expression of miR-188 increased gradually in brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) of mice during aging. MiR-188 knockout mice were resistant to the aging-associated metabolic phenotype and had higher energy expenditure. Meanwhile, adipose tissue-specific miR-188 transgenic mice displayed the opposite phenotype. Mechanistically, we identified the thermogenic-related gene Prdm16 (encoding PR domain containing 16) as the direct target of miR-188. Notably, inhibition of miR-188 expression in BAT and iWAT of aged mice by tail vein injection of antagomiR-188 ameliorated aging-associated metabolic dysfunction significantly. Taken together, our findings suggested that miR-188 plays an important role in the regulation of the aging-associated metabolic phenotype, and targeting miR-188 could be an effective strategy to prevent aging-associated metabolic dysfunction.

The interaction of KCTD1 with transcription factor AP-2alpha inhibits its transactivation.

AP-2 is a transcription factor implicated in mammalian development, cell proliferation, apoptosis, and carcinogenesis. To identify potential AP-2alpha-interacting partners, a yeast two-hybrid screen was performed in human brain cDNA library. One of the identified clones encodes potassium channel tetramerization domain-containing 1 (KCTD1). We demonstrated the novel KCTD1-AP-2alpha interaction in vitro by GST pull-down assays and in vivo by co-immunoprecipitation assays and mapped the interaction domains to the N-termini of both proteins. In addition, we observed that the two proteins were completely co-localized in the nuclei of mammalian cells. Transient transfection assays using four promoters containing AP-2-binding sites confirmed that KCTD1 significantly repressed AP-2alpha-mediated transactivation through the BTB domain, whereas KCTD1 siRNA strongly relieved KCTD1-mediated repression of AP-2alpha transcriptional activity, and other BTB domain proteins such as PDIP1, KCTD10, and TNFAIP1 did not markedly inhibit the transcriptional activity of AP-2alpha, suggesting that KCTD1 specifically acts as a negative regulator of AP-2alpha. Finally, we found that KCTD1 interacted with three major members of the AP-2 family and inhibited their transcriptional activities. Taken together, our results indicate the novel function of KCTD1 as the transcriptional repressor for AP-2 family, especially for AP-2alpha.

Bone Marrow Mesenchymal Stem Cells-Derived Exosomal MiR-29b-3p Regulates Aging-Associated Insulin Resistance.

Insulin resistance is the major pathological characteristic of type 2 diabetes, and the elderly often develop insulin resistance. However, the deep-seated mechanisms for aging-related insulin resistance remain unclear. Here, we showed that nanosized exosomes released by bone marrow mesenchymal stem cells (BM-MSCs) of aged mice could be taken up by adipocytes, myocytes, and hepatocytes, resulting in insulin resistance both in vivo and in vitro. Using microRNA (miRNA) array assays, we found that the amount of miR-29b-3p was dramatically increased in exosomes released by BM-MSCs of aged mice. Mechanistically, SIRT1 (sirtuin 1) was identified to function as the downstream target of exosomal miR-29b-3p in regulating insulin resistance. Notably, utilizing an aptamer-mediated nanocomplex delivery system that down-regulated the level of miR-29b-3p in BM-MSCs-derived exosomes significantly ameliorated the insulin resistance of aged mice. Meanwhile, BM-MSCs-specific overexpression of miR-29b-3p induced insulin resistance in young mice. Taken together, these findings suggested that BM-MSCs-derived exosomal miR-29b-3p could modulate aging-related insulin resistance, which may serve as a potential therapeutic target for aging-associated insulin resistance.

MicroRNA-34a Suppresses Autophagy in Alveolar Type II Epithelial Cells in Acute Lung Injury by Inhibiting FoxO3 Expression.

Excessive autophagic activity of alveolar type II epithelial (AT-II) cells is one of the main causes of acute lung injury (ALI); however, the underlying molecular mechanism remains to be determined. The microRNAs (miRNAs) are involved with autophagy in many diseases. The objective of this study was therefore to investigate the relationship between the miRNA expression and the autophagic activity of the AT-II cells in the pathogenesis of ALI and its molecular mechanism. A mouse model of ALI and AT-II cell injury was induced using lipopolysaccharide (LPS) in vivo and in vitro, and the expression of miR-34a and the autophagy-related proteins LC3 II/I and p62 were determined. Moreover, the autophagic activity was investigated after miR-34a overexpression and inhibition. The effects of miR-34a on its target gene, FoxO3, in regulating autophagic activity in AT-II cells were also determined. LPS induced autophagic activity and increased the expression of miR-34a in lung tissues and in AT-II cells. The in vitro results showed that the upregulation of miR-34a suppressed, whereas the inhibition of miR-34a promoted, autophagy in AT-II cells. Moreover, miR-34a could directly bind to the 3'-untranslated region of the autophagy-related gene, FoxO3, to decrease its expression. In addition, the knockdown of FoxO3 expression inhibited the autophagic activity in AT-II cells. Together, this study suggested that miR-34a might suppress the excessive autophagic activity in AT-II cells via targeting FoxO3 to reduce the damage of LPS-induced ALI.

Overexpression of AKR1B10 in nasopharyngeal carcinoma as a potential biomarker.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is one of the most common cancers in Southern China. Aldo-keto reductase 1B10 (AKR1B10) is upregulated in multiple tumors and plays an oncogenic role. OBJECTIVE: To examine the expression of AKR1B10 at mRNA and protein levels in nasopharyngeal tumors and correlate its expression with clinicopathological parameters. METHODS: A tissue microarray, paraffin blocks, and frozen surgical nasopharyngeal samples were procured. Western blot and immunohistochemistry were used to estimate AKR1B10 protein expression, and mRNA levels were detected by real time RT-PCR. RESULTS: We found that AKR1B10 expression was increased in malignant tissues compared to the normal tissues (p= 0.000). In NPC tissues, AKR1B10 expression appeared high specifically in squamous cell carcinoma, but low in basal cell carcinoma, adenoid cystic carcinoma, adenocarcinoma and undifferentiated carcinoma (p= 0.000). AKR1B10 expression also demonstrated correlation with tumor differentiation, with a high level in well and moderately differentiated but a low level in poorly differentiated carcinoma (p= 0.000). AKR1B10 was also upregulated in hyperplasia and benign tumors (p= 0.000), and demonstrated a specific nuclear distribution in these non-cancerous diseases. CONCLUSIONS: AKR1B10 is overexpressed in nasopharyngeal hyperplasia, benign tumors, and carcinomas, being a potential new biomarker.

Eps8 regulates cellular proliferation and migration of breast cancer.

The role of Eps8 in human breast cancer was studied, and we found that Eps8 was overexpressed in >60% of human breast cancer samples compared with adjacent normal breast tissues by immunohistochemical analysis. Eps8 was highly expressed in the highly invasive breast cancer cell line MDA-MB­231 compared with the weakly invasive breast cancer cell lines MCF7 and MDA-MB­468. MCF7 cell line stably expressing Eps8 was established by G418 screening, and the ectopic expression of Eps8 enhanced MCF7 breast cancer cell growth and survival as assessed by MTT analysis, cell viability and liquid colony formation, whereas the lentiviral expression of Eps8 shRNA in MDA-MB­231 cells resulted in a significant reduction in cellular growth and proliferation in vitro and in vivo. Furthermore, Eps8 knockdown inhibited breast cancer cell migration in wound healing assays, decreased the number and size of EGF-induced filopodia and increased the sensitivity of breast cancer cells to cisplatin analyzed by MTT assays. Eps8 knockdown decreased the levels of phosphorylated extracellular signal-regulated protein kinase (ERK) and MMP9 but increased p53. Moreover, Eps8 knockdown suppressed a partial EMT-like transition and showed a significant increase in E-cadherin and decrease in N-cadherin and vimentin. These results suggest that Eps8 is overexpressed in human breast cancers, possibly by regulating ERK signaling, MMP9, p53 and EMT-like transition to affect breast cancer cell growth, migration and invasion. Therefore, Eps8 might represent a novel potential target in human breast cancer therapy.

[DNA polymerase delta-interacting protein 1 enhances GFP protein degradation].

OBJECTIVE: To prove whether the over-expression of DNA polymerase delta-interacting protein 1 (PDIP1) protein can promote the degradation of exogenous GFP protein. METHODS: The plasmids of pEGFP-C3 and pCMV-myc-PDIP1 were co-transfected into HEK293 cells. The fluorescence intensity of the transfected cells was analyzed using fluorescence microscope. The expressions of GFP and PDIP1 were measured by Western blotting. Then, the expressions of GFP and PDIP1 were again detected in the cells which were treated by ammonium chloride or MG132, respectively. RESULTS: Compared with the ones transfected with pEGFP-C3 along, green fluorescence intensity of HEK293 cells transfected with the plasmids of pEGFP-C3 and pCMV-myc-PDIP1 was lower, and GFP level decreased as the expression of PDIP1 increased. The decreased GFP was significantly alleviated by the proteasome inhibitor MG132 treatment, but was not significantly affected by ammonium chloride. CONCLUSION: Over-expression of PDIP1 can promote the degradation of exogenous GFP, and the degradation might be caused via proteasome pathway. Our results suggest that PDIP1 might have a hand in the degradation of some proteins in cells.

Transcription factor AP-2α regulates acute myeloid leukemia cell proliferation by influencing Hoxa gene expression.

Transcription factor AP-2α mediates transcription of a number of genes implicated in mammalian development, cell proliferation and carcinogenesis. In the current study, we identified Hoxa7, Hoxa9 and Hox cofactor Meis1 as AP-2α target genes, which are involved in myeloid leukemogenesis. Luciferase reporter assays revealed that overexpression of AP-2α activated transcription activities of Hoxa7, Hoxa9 and Meis1, whereas siRNA of AP-2α inhibited their transcription activities. We found that AP-2 binding sites in regulatory regions of three genes activated their transcription by mutant analysis and AP-2α could interact with AP-2 binding sites in vivo by chromatin immunoprecipitation (ChIP). Further results showed that the AP-2α shRNA efficiently inhibited mRNA and protein levels of Hoxa7, Hoxa9 and Meis1 in AML cell lines U937 and HL60. Moreover, decreased expression of AP-2α resulted in a significant reduction in the growth and proliferation of AML cells in vitro. Remarkably, AP-2α knockdown leukemia cells exhibit decreased tumorigenicity in vivo compared with controls. Finally, AP-2α and target genes in clinical acute myeloid leukemia samples of M5b subtype revealed variable expression levels and broadly paralleled expression. These data support a role of AP-2α in mediating the expression of Hoxa genes in acute myeloid leukemia to influence the proliferation and cell survival.

Eps8 promotes cellular growth of human malignant gliomas.

Eps8 was initially identified as a substrate of the epidermal growth factor receptor. Overexpression of Eps8 leads to increased mitogenic signaling and malignant transformation. However, little is known concerning the importance of Eps8 in human gliomas. In this study, we found that Eps8 was overexpressed in 56.6% of human gliomas (WHO grades III and IV) compared with adjacent normal brain tissues by immunohistochemical analysis. The U251 human glioma cell line stably expressing Eps8 was established by G418 screening, and the ectopic expression of Eps8 enhanced U251 glioma cell growth and survival by cell survival, MTT and liquid colony formation assays. By contrast, the lentiviral expression of Eps8 siRNA in SHG-44 cells resulted in a significant reduction in cellular growth and proliferation. Furthermore, Eps8 modulated the levels of phosphorylated extracellular signal-regulated protein kinase (ERK), phosphorylated serine-threonine protein kinase Akt and ß-catenin expression in glioma cell lines and tissues. These results suggest that Eps8 is overexpressed in human gliomas, and affects glioma cell growth possibly by regulating ERK and Akt/ß-catenin signaling. Therefore, Eps8 may represent a novel potential target in human glioma therapy.

Human intersectin 2 (ITSN2) binds to Eps8 protein and enhances its degradation.

Participates in actin remodeling through Rac and receptor endocytosis via Rab5. Here, we used yeast two-hybrid system with Eps8 as bait to screen a human brain cDNA library. ITSN2 was identified as the novel binding factor of Eps8. The interaction between ITSN2 and Eps8 was demonstrated by the in vivo co-immunoprecipitation and colocalization assays and the in vitro GST pull-down assays. Furthermore, we mapped the interaction domains to the region between amino acids 260-306 of Eps8 and the coiled-coil domain of ITSN2. In addition, protein stability assays and immunofluorescence analysis showed ITSN2 overexpression induced the degradation of Eps8 proteins, which was markedly alleviated with the lysosome inhibitor NH4Cl treatment. Taken together, our results suggested ITSN2 interacts with Eps8 and stimulates the degradation of Eps8 proteins.