G3BP1 promotes human breast cancer cell proliferation through coordinating with GSK-3ß and stabilizing ß-catenin.

Ras-GTPase activating SH3 domain-binding protein 1 (G3BP1) is a multifunctional binding protein involved in the development of a variety of human cancers. However, the role of G3BP1 in breast cancer progression remains largely unknown. In this study, we report that G3BP1 is upregulated and correlated with poor prognosis in breast cancer. Overexpression of G3BP1 promotes breast cancer cell proliferation by stimulating ß-catenin signaling, which upregulates a number of proliferation-related genes. We further show that G3BP1 improves the stability of ß-catenin by inhibiting its ubiquitin-proteasome degradation rather than affecting the transcription of ß-catenin. Mechanistically, elevated G3BP1 interacts with and inactivates GSK-3ß to suppress ß-catenin phosphorylation and degradation. Disturbing the G3BP1-GSK-3ß interaction accelerates the degradation of ß-catenin, impairing the proliferative capacity of breast cancer cells. Our study demonstrates that the regulatory mechanism of the G3BP1/GSK-3ß/ß-catenin axis may be a potential therapeutic target for breast cancer.

A novel biphenyl compound IMB-S7 ameliorates hepatic fibrosis in BDL rats by suppressing Sp1-mediated integrin αv expression.

Chronic tissue injury with fibrosis results in the disruption of tissue architecture, organ dysfunction, and eventual organ failure. Therefore, the development of effective antifibrotic drugs is urgently required. IMB-S7 is novel biphenyl compound derived from bifendate (biphenyldicarboxylate) that is used for the treatment of chronic hepatitis in China. In the current study we investigated the potential of IMB-S7 as an antihepatic fibrosis agent. In bile duct ligation (BDL) rat model, oral administration of IMB-S7 (400 mg· kg-1· d-1, for 14 days) significantly ameliorated BDL-induced liver necrosis, bile duct proliferation, and collagen accumulation. We then showed that IMB-S7 treatment markedly suppressed the TGF-ß/Smad pathway in human hepatic stellate cell line LX2 and mouse primary HSCs, as well as in liver samples of BDL rats, thus inhibiting the transcription of most fibrogenesis-associated genes, including TGF-ß1, COL1A1, and ACTA2. Furthermore, IMB-S7 treatment significantly suppressed the expression of integrin αv at the mRNA and protein levels in TGF-ß-treated LX2 cells and liver samples of BDL rats. Using integrin αv overexpression and silencing, we demonstrated that integrin αv activity correlated positively with the activation of TGF-ß/Smad pathway. Based on dual luciferase assay and DNA affinity precipitation assay, we revealed that IMB-S7 inactivated integrin αv through competitively inhibiting the binding of Sp1, a transcription factor, to the integrin αv (ITGAV) promoter (-173/-163 bp). These results suggest that IMB-S7 inhibits HSCs activation and liver fibrosis through Sp1-integrin αv signaling, and IMB-S7 may be a promising candidate to combat hepatic fibrosis in the future.

The novel quinolizidine derivate IMB-HDC inhibits STAT5a phosphorylation at 694 and 780 and promotes DNA breakage and cell apoptosis via blocking STAT5a nuclear translocation.

Sophoridine is a quinolizidine natural product and the exploration of its derivatives has been carried out, and the potent anticancer compound IMB-HDC was acquired. Although previous studies have revealed that some sophoridine derivatives could induce DNA breakage, the underlying mechanisms of inhibition of DNA damage repair (ATR inactivation) and the apoptosis independent of p53, have not been elucidated. Our research reveals a novel DNA response mechanism different from general DNA-damaging agents, and that sophoridine derivate inhibits the phosphorylation of Tyr694 and Ser780 of STAT5a to induce the lessened shuttle from the cytoplasm to the nucleus, and leads to the decreased nuclear STAT5a and subsequently inhibits the expression of STAT5a target gene RAD51 that contributes to the checkpoint activation, thus inhibiting ATR activation. Meanwhile, IMB-HDC that induced the diminished expression of STAT5a target gene contributes to proliferation and leads to apoptosis. More importantly, we give the first evidence that promoting the effect of Tyr694 phosphorylation on nuclear location and subsequent STAT5a target gene transcription depends on Ser780 increased or unchanged phosphorylation and was not correlated with Ser726 phosphorylation.

Different transcriptome profiles between human retinoblastoma Y79 cells and an etoposide-resistant subline reveal a chemoresistance mechanism.

BACKGROUND: Retinoblastoma (RB) is the most frequent pediatric retinal tumor. In the present study, to elucidate chemoresistance mechanisms and identify potential biomarkers in RB, we utilized RNA sequencing (RNAseq) technological platforms to reveal transcriptome profiles and identify any differentially expressed genes (DEGs) between an etoposide drug-resistant subline (Y79/EDR) and parental Y79 cells. METHODS: To test whether Y79/EDR cells showed resistance to antineoplastic agents for RB, we treated the cells with etoposide, carboplatin and vincristine and analyzed them with a Cell Counting Kit-8 (CCK-8). Y79/EDR and parental Y79 cells were used for RNAseq and bioinformatics analysis to enable a genome-wide review of DEGs between the two lines using the DESeq R package (1.10.1). Then, DEG enrichment in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was analyzed with KOBAS software. Next, real-time quantitative reverse transcription polymerase chain reaction (real time QRT-PCR) and cytotoxicity assays were performed to experimentally and functionally validate the identified candidate biomarkers. RESULTS: Y79/EDR cells showed resistance to etoposide, carboplatin and vincristine at different concentrations. In total, 524 transcripts were differentially expressed in Y79/EDR cells based on analysis of fragments per kilobase of transcript per million fragments mapped (FPKM); among these, 57 genes were downregulated and 467 genes were upregulated in Y79/EDR cells compared to parental Y79 cells. We selected candidate DEGs, including ARHGAP9, HIST1H4H, RELN, DDIT4, HK2, STC1 and PFKFB4, for mRNA expression validation with real time QRT-PCR assays and found that the expression levels determined by real time QRT-PCR were consistent with the RNAseq data. Further studies involving downregulation of ARHGAP9 with a specific siRNA showed that ARHGAP9 altered the cellular sensitivity of Y79 cells to etoposide and carboplatin. CONCLUSION: Our initial findings provided a genomic view of the transcription profiles of etoposide-induced acquired resistance in RB. Follow-up studies indicated that ARHGAP9 might be a chemoresistance biomarker in RB, providing insight into potential therapeutic targets for overcoming acquired chemoresistance in RB. These findings can aid in understanding and overcoming chemoresistance during treatment of RB in the clinic.

A novel ASBT inhibitor, IMB17-15, repressed nonalcoholic fatty liver disease development in high-fat diet-fed Syrian golden hamsters.

The manipulation of bile acid (BA) homeostasis by blocking the ileal apical Na+-dependent bile salt transporter (ASBT/SLC10A2) may have therapeutic effects in nonalcoholic fatty liver disease. We developed a novel ASBT inhibitor, an N-(3,4-o-dichlorophenyl)-2-(3-trifluoromethoxy) benzamide derivative referred to as IMB17-15, and investigated its therapeutic effects and the molecular mechanisms underlying the effects. Syrian golden hamsters were challenged with high-fat diet (HFD) to induce NAFLD and were subsequently administered 400 mg/kg IMB17-15 by gavage daily for 21 days. Serum, liver, and fecal samples were collected for further analysis. Plasma concentration-time profiles of IMB17-15 were also constructed. The human hepatocyte cell line HL-7702 was treated with Oleic acid (OA) with or without IMB17-15. Western blotting and real-time PCR were used to study the molecular mechanisms of IMB17-15. We found that IMB17-15 inhibited ASBT and subsequently suppressed ileal farnesoid X receptor (FXR) and FXR-activated fibroblast growth factor15/19 (FGF15/19) expression, which reduced the hepatic phosphorylated extracellular regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) levels and upregulated the cholesterol 7α-hydroxylase (CYP7A1) activity. Additionally, IMB17-15 stimulated adenosine monophosphate (AMP)-activated protein kinase (AMPKα) phosphorylation and enhanced peroxisome proliferator activated receptor α (PPARα) expression and thus promoted triglyceride (TG) oxidation and high-density lipoprotein cholesterol (HDL-c) metabolism through an ASBT-independent mechanism. In conclusion, a novel ASBT inhibitor known as IMB17-15 protected hamsters against HFD-induced NFALD by manipulating BA and lipid homeostasis. IMB17-15 also reduced lipid deposition in human hepatic cell lines, indicating that it may be useful as a therapy for NAFLD patients.

The Recombinant and Reconstituted Novel Albumin-Lidamycin Conjugate Shows Lasting Tumor Imaging and Intensively Enhanced Therapeutic Efficacy.

Depending on increasing extracellular protein utilization and altering metabolic programs, cancer cells could proliferate and survive without restricion by ingesting human serum albumin (HSA) to serve as nutritional amino acids. Here, we hypothesize that the consumption of albumin by cancer cells could be utilized as an efficient approach to targeted drug delivery. Lidamycin (LDM), an antitumor antibiotic with extremely potent cytotoxicity to cultured cancer cells, consists of an apoprotein (LDP) and an active enediyne chromophore (AE). In the present study, a novel albumin-lidamycin conjugate was prepared by DNA recombination and molecular reconstitution. Results show that the IC50 values of albumin-lidamycin conjugate (HSA-LDP-AE) for a variety of tested cancer cells were at subnanomolar levels. At tolerated doses, the albumin-lidamycin conjugate significantly inhibited the growth of lung carcinoma PG-BE1 xenografts by 97.8%. The therapeutic efficacy of the albumin-lidamycin conjugate was much stronger than that of free lidamycin. Meanwhile, the images of albumin-lidamycin conjugate showed obvious and lasting tumor localization and fluorescence enrichment and there was no detectable signal in nontumor locations. Taken together, albumin-lidamycin conjugate, a new format of lidamycin, could be a promising antitumor therapeutic agent and albumin-integration might be a feasible approach to targeted antitumor drug delivery.

EBP50 suppresses the proliferation of MCF-7 human breast cancer cells via promoting Beclin-1/p62-mediated lysosomal degradation of c-Myc.

c-Myc, a key activator of cell proliferation and angiogenesis, promotes the development and progression of breast cancer. Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) is a multifunctional scaffold protein that suppresses the proliferation of breast cancer cells. In this study we investigated whether the cancer-suppressing effects of EBP50 resulted from its regulation of c-Myc signaling in human breast cancer MCF-7 cells in vitro and in vivo. We first found a significant correlation between EBP50 and c-Myc expression levels in breast cancer tissue, and demonstrated that EBP50 suppressed cell proliferation through decreasing the expression of c-Myc and its downstream proteins cyclin A, E and Cdc25A in MCF-7 cells. We further showed that EBP50 did not regulate c-Myc mRNA expression, but it promoted the degradation of c-Myc through the autophagic lysosomal pathway. Moreover, EBP50 promoted integration between c-Myc and p62, an autophagic cargo protein, triggering the autophagic lysosomal degradation of c-Myc. In EBP50-silenced MCF-7 cells, activation of autophagy by Beclin-1 promoted the degradation of c-Myc and inhibited cell proliferation. These results demonstrate that the EBP50/Beclin-1/p62/c-Myc signaling pathway plays a role in the proliferation in MCF-7 breast cancer cells: EBP50 stimulates the autophagic lysosomal degradation of c-Myc, thereby inhibits the proliferation of MCF-7 cells. Based on our results, promoting the lysosomal degradation of c-Myc might be a promising new strategy for treating breast cancer.

D-chiro-inositol effectively attenuates cholestasis in bile duct ligated rats by improving bile acid secretion and attenuating oxidative stress.

Cholestatic liver diseases are important causes of liver cirrhosis and liver transplantation, but few drugs are available for treatment. D-chiro-inositol (DCI), an isomer of inositol found in many Leguminosae plants and in animal viscera, is used clinically for the treatment of polycystic ovary syndrome (PCOS) and diabetes mellitus. In this study, we investigated whether DCI exerted an anti-cholestatic effect and its underlying mechanisms. A cholestatic rat model was established via bile duct ligation (BDL). After the surgery, the rats were given DCI (150 mg·kg-1·d-1) in drinking water for 2 weeks. Oral administration of DCI significantly decreased the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and attenuated bile duct proliferation, parenchymal necrosis and fibrosis in BDL rats. Furthermore, DCI treatment significantly increased the serum and bile levels of total bile acid (TBA), and decreased TBA levels in the liver. Moreover, DCI treatment significantly increased expression of the genes encoding bile acid transporters BSEP (Abcb11) and MRP2 (Abcc2) in liver tissues. DCI treatment also markedly decreased hepatic CD68 and NF-kappaB (NF-κB) levels, significantly decreased the serum and hepatic MDA levels, markedly increased superoxide dismutase activity in both serum and liver tissues. Using whole-genome oligonucleotide microarray, we revealed that DCI treatment altered the expression profiles of oxidation reduction-related genes in liver tissues. Collectively, DCI effectively attenuates BDL-induced hepatic bile acid accumulation and decreases the severity of injury and fibrosis by improving bile acid secretion, repressing inflammation and decreasing oxidative stress. The results suggest that DCI might be beneficial for patients with cholestatic disorders.

Recent progression in the utilization of autophagy-regulating nature compound as anti-liver fibrosis agents.

Hepatic fibrosis is a wound-healing response to chronic liver injury caused by various pathogenesis, such as hepatitis virus infection, drugs toxicity and autoimmune imbalances. Autophagy, a cellular process degrading damaged organelles or aggregative proteins, participates in multiple human diseases including hepatic fibrosis. However, the precise role of autophagy in the pathogenesis of hepatic fibrosis is yet to be elucidated. Accumulated evidences indicate that several nature compounds exhibit anti-fibrotic potential through modulating autophagy activity. For a better understanding of the relationships among autophagy, hepatic fibrosis, and autophagy-regulating nature compounds, this review highlights the recent advancement of nature compounds treating hepatic fibrosis through regulating autophagy.

[Advances in studies of ileal apical sodium-dependent bile acid transporter].

Bile acids play critical roles in the regulation of metabolism and absorption of lipids. The ileal apical sodium-dependent bile acid transporter (ASBT) located at the enterocyte brush border is responsible for the reuptake of bile acids and the maintenance of bile acid homeostasis. Recently, a number of investigations have been made concerning the regulation and control of ASBT and the relationship between ASBT and intestinal inflammation, tumorigenesis, diabetes mellitus and hyperlipemia, which suggests ASBT as a potential therapeutic target of these diseases. In this review, advances in the study of above-mentioned issues were summarized.

[The regulatory role of autophagy in tumor process].

Autophagy is a classical regulatory mechanism of energy metabolism and self-update system in the maintenance of the intracellular homeostasis and cell development. Autophagy has been recently found to play a role in tumor development. Autophagy regulates tumor formation, proliferation, metastasis, and metabolism. At the same time, the anticancer drugs formed with autophagic mediators have been used in the treatment, which suggested that improving autophagy activity to inhibit tumor has become a new way for cancer treatment of cancer patients. This article gives an overview of the regulatory mechanism of autophagy, the relationship between autophagy and tumor, and tumor therapy by targeting autophagy.

Lidamycin inhibits tumor initiating cells of hepatocellular carcinoma Huh7 through GSK3ß/ß-catenin pathway.

Recently, tumor initiating cells are considered as the central role of tumorigenicity in hepatocellular carcinoma. Enediyne anticancer antibiotic lidamycin with great potential antitumor activity is currently evaluated in Phase II clinical trials. In this study, we evaluated the effect of lidamycin on tumor initiating cells of hepatocellular carcinoma Huh7 and identified the potential mechanism. Flow cytometry analysis and sorting assay, surface marker assay, sphere formation assay, and aldefluor assay were used to evaluate the effect of lidamycin on Huh7 tumor initiating cells in vitro. To investigate the potential mechanism, the activity of GSK3ß/ß-catenin pathway was detected by Western blot and T cell factors transcriptional activity assay. Subcutaneous tumor model in nude mice was used to observe in vivo effect of lidamycin on Huh7 cells. Lidamycin decreased the proportion of EpCAM+ cells and the expression of EpCAM protein. Lidamycin inhibited sphere formation of sorted EpCAM+ cells in 7 d, and of parental cells in three serial passages. The population of aldehyde dehydrogenase-positive cells was reduced by lidamycin. In addition, lidamycin restrained tumor volume and incidence in vivo. Lidamycin activated GSK3ß, and degraded the activity of ß-catenin. Consequently, transcriptional activity of ß-catenin/T cell factors was decreased. In brief, these results suggest that lidamycin suppressed Huh7 tumor initiating cells via GSK3ß/ß-catenin pathway. These findings reveal the potential mechanism of lidamycin on tumor initiating cells and the benefit for further clinical evaluation.

The anti-fibrotic effects of epigallocatechin-3-gallate in bile duct-ligated cholestatic rats and human hepatic stellate LX-2 cells are mediated by the PI3K/Akt/Smad pathway.

AIM: (-)-Epigallocatechin-3-gallate (EGCG) is one of the most abundant polyphenols in green tea with strong antioxidant activity and various therapeutic effects. In this study, we investigated the anti-fibrotic effects of EGCG and underlying mechanisms in bile duct-ligated (BDL) rats and a liver fibrosis model in vitro. METHODS: BDL rats were treated with EGCG (25 mg·kg(-1)·d(-1), po) for 14 d, and then the serum, bile and liver samples were collected. Liver fibrosis was assessed by serum, urine and bile biochemistry analyses and morphological studies of liver tissues. TGF-ß1-stimulated human hepatic stellate LX-2 cells were used as a liver fibrosis model in vitro. The expression of liver fibrogenic genes and signaling proteins in the PI3K/Akt/Smad pathway was examined using Western blotting and/or real-time PCR. RESULTS: In BDL rats, EGCG treatment significantly ameliorates liver necrosis, inflammation and fibrosis, and suppressed expression of the genes associated with liver inflammation and fibrogenesis, including TNF-α, IL-1ß, TGF-ß1, MMP-9, α-SMA, and COL1A1. In LX-2 cells, application of EGCG (10, 25 µmol/L) dose-dependently suppressed TGF-ß1-stimulated expression of COL1A1, MMP-2, MMP-9, TGF-ß1, TIMP1, and α-SMA. Furthermore, EGCG significantly suppressed the phosphorylation of Smad2/3 and Akt in the livers of BDL rats and in TGF-ß1-stimulated LX-2 cells. Application of LY294002, a specific inhibitor of PI3K, produced similar effects as EGCG did in TGF-ß1-stimulated LX-2 cells, but co-application of EGCG and LY294002 did not produce additive effects. CONCLUSION: EGCG exerts anti-fibrotic effects in BDL rats and TGF-ß1-stimulated LX-2 cells in vitro via inhibiting the PI3K/Akt/Smad pathway.

[The TGF-ß signaling pathway induced EMT in breast cancer].

Epithelial-mesenchymal transition (EMT) refers to tne transition during which epithelial cells undergo the loss of apical-basal polarity, acquisition of migration capability and transformation into mesenchymal cells. EMT induces breast cancer in situ to developing into metastasis and associates with the drug resistence. The multiple elements including signal pathways, transcriptional factors and downstream genes orchestrate the transition. Among them, the transforming growth factor ß (TGF-ß) signaling pathway plays a key role in the regulation of EMT in breast cancer. And this paper reviews the development of TGF-ß signaling pathway induced EMT in breast cancer.

[Establishment and application of a high-throughput drug screening model based on COL1A1 promoter for anti-liver fibrosis].

For screening the potential drugs as anti-liver fibrosis candidates, we established a high- throughput drug screening cell model based on COL1A1 promoter. The activity of COL1A1 promoter and luciferase reporter gene can be elevated by TGF-ß1, and inhibited by candidate drugs. We constructed a recombined plasmid with COL1A1 promoter and luciferase reporter gene pGL4.17, the activity of COL1A1 promoter was reflected by fluorescence intensity. COL1A1 promoter activity was detected by Dual-Luciferase Reporter Assay System, it came that the relative luciferase activity of COL1A1 promoter was 15.98 times higher than that of control group induced by TGF-ß1, showing the recombined plasmid could be used in cell model. The recombined plasmid was transfected into human hepatic stellate cells LX2, detected the effect of potential drugs, and obtained a stable expression system through stable transfection and monoclonal cell culture. A sample which could reduce COL1A1 promoter activity signally by our cell model, decreased collagen I mRNA and protein expression detected by real-time RT-PCR and Western blotting. It indicates this novel cell model can be used in high-throughput drug screening of potential anti-liver fibrosis drugs.

[Potential targets for anti-liver fibrosis].

Liver fibrosis is a pathological process of the excessive accumulation of extracellular matrix, especially collagen al (I) in liver. Ultimately, hepatic fibrosis leads to cirrhosis or hepatic failure. Liver fibrosis and early cirrhosis can be reversed, thus control of the development of liver fibrosis is very important for preventive treatment of cirrhosis and hepatic failure. This is a review of potential targets for anti-hepatic fibrosis based on plenty of publications, including TGF-ß1 and integrin α(v) and so on, aimed at providing novel therapeutic targets in liver fibrosis.

[The synergistic effect of lidamycin and rituximab on human B cell lymphoma].

This study aimed to investigate the synergistic effect of lidamycin (LDM) and rituximab on human B cell lymphoma Ramos cells. Cell proliferation was measured using MTS assay, cell apoptosis was analyzed by Annexin V-FITC/PI assay, the expression of apoptosis related proteins was analyzed by Western blotting, and the in vivo lymphoma inhibition was verified using BALB/c mice inoculated via tail vein using Ramos cells which stably expressed pEGFP-N1 plasmid. The results showed that, after the pretreatment with rituximab for 48 h, rituximab and LDM showed significantly synergistic effects on cell proliferation. Cells in combined treatment group had a higher apoptosis rate than that in LDM treatment group. Compared with the LDM treatment group, the expression of apoptosis-related proteins such as Cleaved caspase-3, Cleaved caspase-7, Cleaved caspase-9 and Cleaved PARP in combined treatment groups increased, and expression of cIAP-2 and Bcl-2 decreased. The result of in vivo experiment showed that, in the combined treatment group, the survival time of BALB/c mice was significantly longer than the mice in control group and LDM treatment group, and the degree of tumor accumulation and metastasis to lymph nodes and spleen was lower.

[Inhibitions of SphK1 inhibitor SKI II on cell cycle progression and cell invasion of hepatoma HepG2 cells].

Sphingosine kinase 1 (SphK1) plays critical roles in cell biological functions. Here we investigated the effects of SphK1 inhibitor SKI II on hepatoma HepG2 cell cycle progression and invasion. Cell survival was determined by SRB assay, cell cycle progression was assayed by flow cytometry, the ability of cell invasion was measured by Matrigel-Transwell assay and protein expression was detected by Western blotting. The results showed that SKI II markedly inhibited HepG2 cell survival in a dose-dependent manner, induced G1 phase arrest in HepG2 cell and inhibited cell invasion. SKI II markedly decreased the expressions of G1-phase-related proteins CDK2, CDK4 and Cdc2 and the levels of cell invasion-associated proteins MMP2 and MMP9. The results showed that SKI II inhibited cell cycle progression and cell invasion, implying SphK1 as a potential target for hepatoma treatment.

Arylsulfonylamino-benzanilides as inhibitors of the apical sodium-dependent bile salt transporter (SLC10A2).

The apical sodium-dependent bile salt transporter (ASBT) plays a pivotal role in maintaining bile acid homeostasis. Inhibition of ASBT would reduce bile acid pool size and lower cholesterol levels. In this report, a series of novel arylsulfonylaminobenzanilides were designed and synthesized as potential inhibitors of ASBT. Most of them demonstrated great potency against ASBT's bile acid transport activity. In particular, compound 5g2 inhibited ASBT activity with an IC50 value of 0.11 µM. These compounds represent potential cholesterol-lowering drugs.

[Sphingosine kinase 1 and tumor].

Sphingolipids as an important regulator play a critical role in the cell biological functions. Among them, ceramide (Cer) and sphingosine (Sph) induce apoptosis and inhibit cell proliferation; on the contrary sphingosine 1-phosphate (S1P) promotes cell survival and proliferation. The balance between ceramide/sphingosine and S1P forms a so-called "sphingolipid-rheostat", which decides the cell fate. Sphingosine kinases, which catalyze the phosphorylation of sphingosine to S1P, are critical regulators of this balance. Here, we review the role of sphingosine kinase 1 (SphK1) in regulating fundamental biological processes and tumorigenesis and the potential of SphK1 as a new target for cancer therapeutics.