Search

CARM1 is a protein arginine methyltransferase and acts as a transcriptional coactivator regulating multiple biological processes. Aberrant expression of CARM1 has been related to the progression of multiple types of cancers, and therefore CARM1 was considered as a promising drug target. In the present work, we report the structure-based discovery of a series of N1-(3-(pyrimidin-2-yl)benzyl)ethane-1,2-diamines as potent CARM1 inhibitors, in which compound 43 displays high potency and selectivity. With the advantage of excellent tissue distribution, compound 43 demonstrated good in vivo efficacy for solid tumors. Furthermore, from the detailed immuno-oncology study with MC38 C57BL/6J xenograft model, we confirmed that this chemical probe 43 has profound effects in tumor immunity, which paves the way for future studies on the modulation of arginine post-translational modification that could be utilized in solid tumor treatment and cancer immunotherapy.

Overexpression, point mutations or translocations of protein lysine methyltransferase NSD2 was occurred in many types of cancer cells. Therefore, it was recognized as onco-protein and considered as a promising anticancer drug target. NSD2 consists of a SET catalytic domain and two PWWP domains binding to methylated histone proteins. Here, we reported our efforts to develop a series of NSD2-PWWP1 inhibitors, and further structure-based optimization resulted a potent inhibitor 38, which has the high selectivity towards NSD2-PWWP1 domain. The detailed biological evaluation revealed that compound 38 can bind to NSD2-PWWP1 and then affect the expression of genes regulated by NSD2. The current discovery will provide a useful chemical probe to the future research in understanding the specific regulation mode of NSD2 by PWWP1 recognition, and pave the way to develop potential drugs targeting NSD2 protein.

Overexpression, point mutations or translocations of protein lysine methyltransferase NSD2 was occurred in many types of cancer cells. Therefore, it was recognized as onco-protein and considered as a promising anticancer drug target. NSD2 consists of a SET catalytic domain and two PWWP domains binding to methylated histone proteins. Here, we reported our efforts to develop a series of NSD2 PWWP1 inhibitors, and further structure-based optimization resulted a potent inhibitor 38, which has the high selectivity towards NSD2 PWWP1 domain. The detailed biological evaluation revealed that compound 38 can bind to NSD2-PWWP1 and then affect the expression of genes regulated by NSD2. We believe that the current discovery will provide a useful chemical probe to the future research in understanding the specific regulation mode of NSD2 by PWWP1 recognition.

WD40 is a ubiquitous domain presented in at least 361 human proteins and acts as scaffold to form protein complexes. Among them, WDR5 protein is an important mediator in several protein complexes to exert its functions in histone modification and chromatin remodeling. Therefore, it was considered as a promising epigenetic target involving in anti-cancer drug development. In view of the protein–protein interaction nature of WDR5, we initialized a campaign to discover new peptide-mimic inhibitors of WDR5. In current study, we utilized the phage display technique and screened with a disulfide-based cyclic peptide phage library. Five rounds of biopanning were performed and isolated clones were sequenced. By analyzing the sequences, total five peptides were synthesized for binding assay. The four peptides are shown to have the moderate binding affinity. Finally, the detailed binding interactions were revealed by solving a WDR5-peptide cocrystal structure.

PRMT4 is a type I protein arginine methyltransferase and plays important roles in various cellular processes. Overexpression of PRMT4 has been found to be involved in several types of cancers. Selective and in vivo effective PRMT4 inhibitors are needed for demonstrating PRMT4 as a promising therapeutic target. On the basis of compound 6, a weak dual PRMT4/6 inhibitor, we constructed a tetrahydroisoquinoline scaffold through a cut-and-sew scaffold hopping strategy. The subsequent SAR optimization efforts employed structure-based approach led to the identification of a novel PRMT4 inhibitor 49. Compound 49 exhibited prominently high potency and selectivity, moderate pharmacokinetic profiles, and good antitumor efficacy in acute myeloid leukemia xenograft model via oral administration, thus demonstrating this compound as a useful pharmacological tool for further target validation and drug development in cancer therapy.

Myocardial infarction (MI) is a major cause of death and disability worldwide. In the last decade, mesenchymal stem cells (MSCs) based cell therapy has emerged as a promising therapeutic strategy. Although great advance have been made using MSCs to treat MI, the low viability of transplanted MSCs severely limits the efficiency of MSCs therapy. Here, we show evidence that ex vivo pre-treatment with melatonin, an endogenous hormone with newly found anti-oxidative activity, could improve survival and function of adipose tissue derived MSCs (ADSCs) in vitro as well as in vivo. ADSCs with 5 μM melatonin pre-treatment for 24 hrs showed increased expression of the antioxidant enzyme catalase and Cu/Zn superoxide dismutase (SOD-1), as well as pro-angiogenic and mitogenic factors like insulin-like growth factor 1, basic fibroblast growth factor, hepatocyte growth factor (HGF), epidermal growth factor. Furthermore, melatonin pre-treatment protected MSCs from reactive oxygen species (ROS) induced apoptosis both directly by promoting anti-apoptosis kinases like p-Akt as well as blocking caspase cascade, and indirectly by restoring the ROS impaired cell adhesion. Using a rat model of MI, we found that melatonin pre-treatment enhanced the viability of engrafted ADSCs, and promoted their therapeutic potency. Hopefully, our results may shed light on the design of more effective therapeutic strategies treating MI by MSCs in clinic.

Induced pluripotent stem cell (iPSC) provides a promising seeding cell for regenerative medicine. However, iPSC has the potential to form teratomas after transplantation. Therefore, it is necessary to evaluate the tumorigenic risks of iPSC and all its differentiated derivates prior to use in a clinical setting. Here, murine iPSCs were transduced with dual reporter gene consisting of monomeric red fluorescent protein (mRFP) and firefly luciferase (Fluc). Undifferentiated iPSCs, iPSC derivates from induced differentiation (iPSC-derivates), iPSC-derivated cardiomyocyte (iPSC-CMs) were subcutaneously injected into the back of nude mice. Non-invasive bioluminescence imaging (BLI) was longitudinally performed at day 1, 7, 14 and 28 after transplantation to track the survival and proliferation of transplanted cells. At day 28, mice were killed and grafts were explanted to detect teratoma formation. The results demonstrated that transplanted iPSCs, iPSC-derivates and iPSC-CMs survived in receipts. Both iPSCs and iPSC-derivates proliferated dramatically after transplantation, while only slight increase in BLI signals was observed in iPSC-CM transplanted mice. At day 28, teratomas were detected in both iPSCs and iPSC-derivates transplanted mice, but not in iPSC-CM transplanted ones. In vitro study showed the long-term existence of pluripotent cells during iPSC differentiation. Furthermore, when these cells were passaged in feeder layers as undifferentiated iPSCs, they would recover iPSC-like colonies, indicating the cause for differentiated iPSC's tumourigenicity. Our study indicates that exclusion of tumorigenic cells by screening in addition to lineage-specific differentiation is necessary prior to therapeutic use of iPSCs.

Recognizing acetyllysine of histone is a vital process of epigenetic regulation that is mediated by a protein module called bromodomain. To contribute novel scaffolds for developing into bromodomain inhibitors, we utilize a fragment-based drug discovery approach. By successively applying docking and X-ray crystallography, we were able to identify 9 fragment hits from diffracting more than 60 crystals. In the present work, we described four of them and carried out the integrated lead optimization for fragment 8, which bears a 2-thiazolidinone core. After several rounds of structure guided modifications, we assessed the druggability of 2-thiazolidinone by modulating in vitro pharmacokinetic studies and cellular activity assay. The results showed that two potent compounds of 2-thiazolidinones have good metabolic stability. Also, the cellular assay confirmed the activities of 2-thiazolidinones. Together, we hope the identified 2-thiazolidinone chemotype and other fragment hits described herein can stimulate researchers to develop more diversified bromodomain inhibitors.

In this study, the effects of forsterite and clinoenstatite powder extracts on the proliferation and osteogenic differentiation of rat adipose-derived stem cells (ASCs) were investigated and compared with the β-tricalcium phosphate (β-TCP) powder extracts. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and live-dead staining were performed to evaluate the viability and proliferation of rat ASCs. Osteogenic differentiation of rat ASCs were assayed by alkaline phosphatase (ALP) staining and ALP activity test. The expression of osteogenic marker genes (alkaline phosphatase (ALP), runt related transcription factor 2 (Runx2), collagen type Iα1 (Col1α1), secreted phosphoprotein1 (Spp1, osteopontin), integrin binding sialoprotein (Ibsp), bone gla protein (Bglap)) were detected by real-time polymerase chain reaction (PCR). The MTT assay and the live-dead staining showed that all the three ceramics possessed good cytocompatibility with rat ASCs. Furthermore, forsterite and clinoenstatite promoted the proliferation of rat ASCs compared with β-TCP. The results of the ALP activity test and the real-time PCR demonstrated that forsterite and clinoenstatite promoted the osteogenic differentiation of rat ASCs. These results suggested that forsterite and clinoenstatite are bioactive ceramics that may be used for preparation of bone tissue engineering (BTE) scaffolds. forsterite, clinoenstatite, adipose-derived stem cells, osteogenesis, bone, bone tissue engineering

Telocyte (TC) as a special stromal cell exists in mammary gland and might play an important role in the balance of epithelium-stroma of mammary gland. Considering that different types of breast interstitial cells influence the development and progression of breast cancer, TCs may have its distinct role in this process. We here studied the roles of TCs in the self-assembly of reconstituted breast cancer tissue. We co-cultured primary isolated TCs and other breast stromal cells with breast cancer EMT-6 cells in collagen/Matrigel scaffolds to reconstitute breast cancer tissue in vitro. Using histology methods, we investigated the immunohistochemical characteristics and potential functions of TCs in reconstituted breast cancer tissue. TCs in primary mammary gland stromal cells with long and thin overlapping cytoplasmic processes, expressed c-kit/CD117, CD34 and vimentin in reconstitute breast cancer tissue. The transmission electron microscopy showed that the telocyte-like cells closely communicated with breast cancer cells as well as other stromal cells, and might serve as a bridge that directly linked the adjacent cells through membrane-to-membrane contact. Compared with cancer tissue sheets of EMT-6 alone, PCNA proliferation index analysis and TUNEL assay showed that TCs and other breast stromal cells facilitated the formation of typical nest structure, promoted the proliferation of breast cancer cells, and inhibited their apoptosis. In conclusion, we successfully reconstituted breast cancer tissue in vitro, and it seems to be attractive that TCs had potential functions in self-assembly of EMT-6/stromal cells reconstituted breast cancer tissue.

Metastasis is a major cause of death from malignant diseases, and the underlying mechanisms are still largely not known. A detailed probe into the factors which may regulate tumour invasion and metastasis contributes to novel anti-metastatic therapies. We previously identified a novel metastasis-associated gene 1 (mag-1) by means of metastatic phenotype cloning. Then we characterized the gene expression profile of mag-1 and showed that it promoted cell migration, adhesion and invasion in vitro. Importantly, the disruption of mag-1 via RNA interference not only inhibited cellular metastatic behaviours but also significantly reduced tumour weight and restrained mouse breast cancer cells to metastasize to lungs in spontaneous metastatic assay in vivo. Furthermore, we proved that mag-1 integrates dual regulating mechanisms through the stabilization of HIF-1α and the activation of mTOR signalling pathway. We also found that mag-1-induced metastatic promotion could be abrogated by mTOR specific inhibitor, rapamycin. Taken together, the findings identified a direct role that mag-1 played in metastasis and implicated its function in cellular adaptation to tumour microenvironment.

Purpose: Microencapsulated hepatocytes might solve immunological rejection, broadening a new perspective for the treatment of fulminant hepatic failure (FHF). However, the transplantation of microcapsulated hepatocytes is limited by low cell viability Nevertheless, the co-microencapsulation of hepatocytes and human umbilical vein endothelial cells (HUVECs) may make the treatment of FHF more promising. Methods: We prepared the microcapsules using the high-voltage electrostatic droplet spray method, transplanted the empty microcapsules, isolated hepatocytes, microcapsulated hepatocytes, and co-microencapsulated hepatocytes and HUVEC intraperitoneally into rat models of FHF induced by D-aminogalactose (D-gal). After 1, 3, and 7 days, and 2, 3, and 4 weeks posttransplantation, we calculated the mortality and assessed alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (ALB) levels in the serum of the model; evaluated the integrality and recovery of microcapsules; and stained with hematoxylin and eosin (H&E) the recovered microcapsules as well as the liver of the FHF rats. Results: Hepatocyte-specific functions, including the levels of ALT, AST, and ALB in the serum of the co-microencapsulation group, were significantly better than those in the other groups (pConclusion: The microcapsules have good biocompatibility and immunoprotection to protect the hepatocytes from immunological rejection. Cotransplantation of the microencapsulated hepatocytes and HUVECs could decrease mortality rates and improve liver function in FHF.

One challenge of cellular cardiomyoplasty for myocardial infarction (MI) is how to improve MI microenvironment to facilitate stem cell engraftment, survival and homing for myocardial repair. The application of injectable hydrogels is an effective strategy. However, it has not been thoroughly investigated on the role of the injectable scaffolds, in improving MI microenvironment, providing space and guidance for cell survival, engraftment and homing. We explored an injectable chitosan hydrogel for stem cell delivery into ischemic heart and investigated the beneficial effects and mechanisms of the hydrogel. In vitro, H(2)O(2)-treatment was used to mimic reactive oxygen species (ROS) microenvironment. The influence of ROS and protection of chitosan components on adipose-derived mesenchymal stem cells (ADSCs) was analyzed too. In vivo, MI was induced by the left anterior descending artery ligation in SD rats. PBS, chitosan hydrogel, ADSC/PBS and ADSC/chitosan hydrogel were injected into the border of infracted hearts respectively. Multi-techniques were used to assess the beneficial effects of chitosan hydrogel after transplantation. We observed that ROS generated by ischemia would impair ADSC adhesion molecules, including integrin-related adhesion molecules integrin αV and β1, focal adhesion-related molecules p-FAK and p-Src, and corresponding ligands of host myocardium ICAM1 and VCAM1. Chitosan hydrogel could rescue these molecules through ROS scavenging and recruit key chemokine for stem cell homing, such as SDF-1. The results suggest that chitosan hydrogel could improve MI microenvironment, enhance stem cell engraftment, survival and homing in ischemic heart through ROS scavenging and chemokine recruitment, contributing to myocardial repair.

To tissue engineer a kidney is a formidable task because of the complex cell composition and structures in the kidney. This study reconstructed renal tissues using mixed renal cells in collagen/Matrigel® scaffolds in vitro. Neonatal rat renal cells were seeded in collagen I supplemented with Matrigel in a casting mold that could exert static stretch when the renal constructs contracted. During in vitro culture, the renal constructs were observed under microscope and analyzed by histological and immunofluorescent examinations. Results showed that the mixed renal cells reconstituted renal tubular and glomeruli-like structures with different appearances at varying developmental stages. Tubular structures were formed by CK18-positive cells with similar appearances lining the surrounding hollow centres. The glomeruli-like structures were tufts of cell aggregates containing Flk-1-positive cells. These results show that neonatal rat renal cells self-assembled into engineered renal tissues containing both tubules and glomeruli-like structures when cultured in 3D collagen/Matrigel scaffold in vitro.

The present study was carried out to investigate the lipid-lowering effect of luteolin by using a cell model of steatosis induced by palmitate. Incubation of HepG2 cells with palmitate markedly increased lipid accumulation (Oil Red O staining), the genes involved in lipogenesis, including fatty acid synthase (FAS) and its upstream regulator sterol regulatory element binding protein 1c (SREBP-1c), and reactive oxygen species (ROS) production. Luteolin enhanced the phosphorylation of AMP-activated protein kinase α (AMPKα) and its primary downstream targeting enzyme, acetyl-CoA carboxylase (ACC), up-regulated gene expression of carnitine palmitoyl transferase 1 (CPT-1), which is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation, and down-regulated SREBP-1c and FAS mRNA levels in the absence and presence of palmitate. In addition, luteolin significantly decreased ROS production and ameliorated lipid accumulation in HepG2 cells caused by palmitate. Furthermore, intracellular triglyceride (TG) measurement indicated that the luteolin-mediated reduction of enhanced TG caused by palmitate was blocked by pretreatment with the AMPK inhibitor, compound C. The results suggested that the lipid-lowering effect of luteolin might be partially mediated by the up-regulation of CPT-1 and down-regulation of SREBP-1c and FAS gene expression, possibly by activation of the AMPK signaling pathway, and partially might be through its antioxidative actions.

Baicalin, one of the major flavonoids in Scutellaria baicalensis, possesses antioxidant and anti-inflammatory properties. However, the effects of baicalin on metabolic disorders and hepatic steatosis have not been investigated.Body weight was examined in high-fat diet (HFD)-fed rats with or without baicalin treatment. At the end of the experiment, serum biochemical parameters, liver histology and lipid profile were analyzed to assess whether the animals were suffering from metabolic disorders or hepatic steatosis. In the liver, the phosphorylation of AMP activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) and the gene expression of some enzymes involved in lipogenesis were examined. The effects of baicalin on the phosphorylation of AMPK and lipid accumulation induced by high glucose in human hepatoma HepG2 cells were also examined.Baicalin (80 mg/kg) administered ip for 16 weeks suppressed body weight gain in HFD-fed rats. Weight reduction was accompanied by the reduction of visceral fat mass. Baicalin significantly decreased the elevated serum cholesterol, free fatty acid and insulin concentrations caused by the HFD. Baicalin also suppressed systemic inflammation by reducing the serum level of tumor necrosis factor alpha. Baicalin reduced hepatic lipid accumulation, enhanced the phosphorylation of AMPK and ACC and down-regulated genes involved in lipogenesis, including fatty acid synthase and its upstream regulator SREBP-1c. In HepG2 cells, baicalin (5 and 10 micromol/L) increased the phosphorylation of AMPK and decreased lipid accumulation following the addition of high glucose.Our study suggests that baicalin might have beneficial effects on the development of hepatic steatosis and obesity-related disorders by targeting the hepatic AMPK.

Estrogen receptor alpha (ERalpha) plays an important role in the development and progression of breast cancer, and recent studies showed that ERalpha expression is associated with resistance to hormonal therapy. Therefore, a number of studies have explored ways to deplete ERalpha from breast cancer cells as a new therapy especially for hormone-refractory breast cancer. We reported here that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, effectively depletes ERalpha in breast cancer MCF-7 cells. However, the intrinsic mechanisms by which SAHA decreases ERalpha levels are not clear. Our present data demonstrated that both inhibition of ERalpha mRNA level and promotion of ERalpha degradation by the proteasome contribute to SAHA-induced ERalpha depletion, indicating that SAHA may exert its effects through transcriptional and posttranslational mechanisms. Furthermore, the decrease of ERalpha protein level in MCF-7 cells after SAHA treatment is mainly the result of its rapid degradation by the ubiquitin-proteasome pathway rather than transcriptional inhibition. In addition, we showed that inactivation of the heat shock protein-90 (Hsp90) is involved in SAHA-induced ERalpha degradation, and ubiquitin ligase CHIP (C-terminal Hsc70 interacting protein) enhances SAHA-induced ERalpha degradation. SAHA-induced ERalpha depletion is paralleled with reduction of transcriptional activity of ERalpha and SAHA is able to effectively inhibit cell proliferation and induce apoptosis of MCF-7 cells. Taken together, our results revealed a mechanism for SAHA-induced ERalpha degradation and indicated that SAHA is a suitable pharmacological agent for depletion of ERalpha and a potential choice for breast cancer expressing high ERalpha.

To construct a recombinant expression vector pDC315/hSPRY2 carrying human SPRY2 (hSPRY2) gene, and transfect the recombinant plasmid into human embryonic kidney HEK293T cells to express the target protein, and thus explore preliminarily the effect of hSPRY2 on the proliferation and survival of HEK293T cells.The recombinant adenoviral vector expressing hSPRY2 was constructed and transfected into HEK293T cells in vitro. The expression of hSPRY2 target protein in the transfected cells was identified by Western blot analysis. The effect of hSPRY2 on the proliferation and survival of HEK293T cells in a serum-free condition was tested using CCK-8 assay.The recombinant expression vector pDC315/hSPRY2 was constructed, and the target protein hSPRY2 was expressed in the transfected HEK293T cells. The proliferation and survival rates of HEK293T cells transfected with pDC315/hSPRY2 were significantly higher than those transfected with pDC315/EGFP (P0.05).The recombinant expression vector for hSPRY2 was successfully constructed and its expression was demonstrated in the transfected HEK293T cells. Over-expression of SPRY2 promoted the proliferation and survival of HEK293T cells.

To construct a recombinant adeno-associated virus vector carrying the fusion product of extracellular domain of human tumor necrosis factor receptor (hTNFR) with human IgG Fc fragment, and detect the expression and biological activity of hTNFR-Fc.The recombinant hTNFR-Fc fusion gene was cloned into adeno-associated virus (AAV) vector, which was used to transfect human embryo kidney cells (HEK293T) in vitro. The expression of the target protein hTNFR-Fc in the supernatants of the transfected HEK293T cells was identified by Western blot analysis. TNF-α inhibition assay in vitro was performed using TNF-α-sensitive mouse fibrosarcoma L929 cells to determine the neutralizing capacity of hTNFR-Fc against human or rat TNF-α. The binding activity of hTNFR-Fc to human or rat TNF-α was identified by ELISA.The recombinant plasmid pAAV/hTNFR-Fc was constructed and the target protein of hTNFR-Fc was expressed in the supernatants of the transfected HEK293T cells. It could effectively bind to human TNF-α and neutralize its cytotoxicity to L929 cells. The hTNFR-Fc protein also exerted the binding and neutralization activities to rat TNF-α to some extent.The study constructed hTNFR-Fc fusion gene and verified its secretory expression in AAV vector, as well as identified its biological activities, which lay the foundations for future viral package and animal experiments.

To investigate the influence of electroporation on the immunogenicity of the DNA vaccine pVAX- tG250FcGB.The DNA vaccine pVAX-tG250FcGB was constructed by inserting the coding gene of tG250 fusion genes into the expression vector pVAX. The DNA vaccine was delivered in BALB/c mouse by electroporation or intramuscular injection, and the induced antigen specific immune responses were compared.The vaccine delivered by electroporation and intramuscular injection both induced immune responses in BALB/c mouse, but electroporation produced an obviously stronger effect than intramuscular injection.Electroporation-mediated DNA vaccine delivery can produce strong immune response in mice and is an effective means for studying the immunogenic effect of DNA vaccine pVAX-tG250FcGB.

Cell migration is an early-stage and critical step for cancer metastasis. The most common approach to monitor this process is wound-healing assay. However, this traditional method has some unavoidable limitations. We observed that simply scratching the monolayer of cultured cells might cause local cell damage around the injury line. The cells along the scratched border seemed to be irritated and exhibited abnormal distribution of cytoskeleton reassembly with protruding "cell islands" and "pseudopodia" during wound healing, which might potentially affect the assessment of cell migration behavior. Herein, we applied a microfluidic device that mechanically constrained cells seeded in a designed pattern inside microchannels, and monitored cell movement in a way of mimicking the natural microenvironment of cancerous tissues. We illustrated the capacity of this simple method to probe cellular migration behaviors and to screen some biological active agents that reflected in their influence on cellular motility.

Cell migration is an early-stage and critical step for cancer metastasis. The most common approach to monitor this process is wound-healing assay. However, this traditional method has some unavoidable limitations. We observed that simply scratching the monolayer of cultured cells might cause local cell damage around the injury line. The cells along the scratched border seemed to be irritated and exhibited abnormal distribution of cytoskeleton reassembly with protruding "cell islands" and "pseudopodia" during wound healing, which might potentially affect the assessment of cell migration behavior. Herein, we applied a microfluidic device that mechanically constrained cells seeded in a designed pattern inside microchannels, and monitored cell movement in a way of mimicking the natural microenvironment of cancerous tissues. We illustrated the capacity of this simple method to probe cellular migration behaviors and to screen some biological active agents that reflected in their influence on cellular motility.

Background Multiple myeloma (MM) is a B-cell malignancy that is largely incurable and is characterized by the accumulation of malignant plasma cells in the bone marrow. Apigenin, a common flavonoid, has been reported to suppress proliferation in a wide variety of solid tumors and hematological cancers; however its mechanism is not well understood and its effect on MM cells has not been determined. Results In this study, we investigated the effects of apigenin on MM cell lines and on primary MM cells. Cell viability assays demonstrated that apigenin exhibited cytotoxicity against both MM cell lines and primary MM cells but not against normal peripheral blood mononuclear cells. Together, kinase assays, immunoprecipitation and western blot analysis showed that apigenin inhibited CK2 kinase activity, decreased phosphorylation of Cdc37, disassociated the Hsp90/Cdc37/client complex and induced the degradation of multiple kinase clients, including RIP1, Src, Raf-1, Cdk4 and AKT. By depleting these kinases, apigenin suppressed both constitutive and inducible activation of STAT3, ERK, AKT and NF-κB. The treatment also downregulated the expression of the antiapoptotic proteins Mcl-1, Bcl-2, Bcl-xL, XIAP and Survivin, which ultimately induced apoptosis in MM cells. In addition, apigenin had a greater effects in depleting Hsp90 clients when used in combination with the Hsp90 inhibitor geldanamycin and the histone deacetylase inhibitor vorinostat. Conclusions Our results suggest that the primary mechanisms by which apigenin kill MM cells is by targeting the trinity of CK2-Cdc37-Hsp90, and this observation reveals the therapeutic potential of apigenin in treating multiple myeloma.

To isolate and identify the genes related to cancer metastasis by comparison of two cell strains with different metastasis potentials subcloned from human lung giant cell carcinoma cell line.Suppression subtractive hybridization (SSH) was used to compare the levels of gene expression between the two cell strains and SSH library was constructed. After screening the library by gene chip, the expressed sequence tags (ESTs) with different expressing level were sequenced and blasted with GenBank.Seventy-nine genes were obtained that were expressed much higher in PLA-801D than in PLA-801C, including two full-length cDNA. GenBank Accession numbers of the two cDNA, named MAG-1 and MAG-2, were BC006236 and BC002420, the 8.5 kb MAG-1 gene was composed of four exons and located on the chromosome of 4q21. The MAG-2 gene, which was made up by 9 exons, had a length of 5.2 kb and its location was 2q35. Both sequences had open reading frames (ORF) and promoters before the theoretical transcription start points. Using special software, the secondary structure of theoretical products of the two cDNAs was prognosticated, α-helix was the main proportion, but β-pleated sheet and random coil were also included.The expression of MAG-1 and MAG-2 has significant differences in these two cell strains, so they might impact tumor metastasis in some ways that are still uncharted.

Previous studies have shown that histone deacetylase inhibitors (HDACis) can kill cancer cells. In addition, HDACis can induce mitotic catastrophe in cancer cells due to insufficient localization of chromosomal passenger complex (CPC) to the centromere. However, the mechanisms behind these phenomena remain unclear. In this study, we found that a HDACi, FK228, affected multiple epigenetic modification characteristics of the centromere, including enhanced acetylation of histone H3 lysine 9 (H3K9), decreased trimethylation of H3K9, and decreased phosphorylation of histone H3 serine 10 (H3S10) and centromere protein A (CENP-A). These epigenetic changes implied that H3K9 hyperacetylation inhibits the CPC recruitment, induces impaired centromere assembly and function, and eventually leads to aberrant mitosis. These data suggested that hypoacetylation of histone in the pericentromere is the most important landmark for recruiting CPC and leading to the mitotic catastrophe in HDACi-induced killing of cancer cells.

Both tumor proliferation and metastasis are multistep processes with many genes involved. A novel gene MAG-2, which may have close correlation with lung cancer metastasis, was identified in our lab through approach of suppression subtractive hybridization using lung cancer cell strains with same origin but different metastatic potential as models. The objective of our works presented here is to investigate the proliferation related effects of MAG-2 on human lung-giant-cell line PLA801, and to explore the underlying molecular mechanism.Sense and anti-sense expressing vector of MAG-2 were constructed and transfected into PLA801C and D cell respectively and the change of expressing levels of target gene in stable transfected cell strains was assayed by RT-PCR. The alteration in terms of proliferation and cytoplasm Ca(2+) of the stable transfected cell strains and vector controls were tested by the methods of MTT and Fluo-3-Am staining. The proteins levels of p53 and mRNA levels of PCNA of different cell strains were analyzed by Western blot and RT-PCR.The stable transfected cell strains of CMAG2+, DMAG2- and their vector controls Cvector and Dvector were obtained. The MAG-2 mRNA levels of CMAG2+ and DMPAG2- were 7 and 0.6 times higher or lower comparing those of vector controls. MAG-2 could increase the mRNA level of PCNA and down-regulated the p53 protein in PLA801C strain. In consistent with PCNA's mRNA elevating, the proportion of S-phase cell in the mass was also increased. But the significant difference was not observed on MTT assay between CMAG2+ and its control, the reason might be due to the mutated p53, which had strong proliferation promoting effects, was also down-regulated by MAG-2's over-expression in PLA801C, and the levels of cytoplasm calcium in CMAG2+ had 1.05 times more than that of vector control. As to the PLA801D strain, the decreased expression of MAG-2 result in lower level of cytoplasm calcium (0.64 time of control's) and the cell proliferation was also restrained by means of MTT assay.MAG-2 has taken part in the proliferation regulation of lung cancer cells PLA801, and the mechanism of its proliferation-related function maybe lie in its effects on cytoplasmic free calcium.

Tumor metastasis is a multistep process with many genes involved in. A novel gene MAG-1, identified by suppression subtractive hybridization from lung cancer cells was found to be associated with tumor metastasis. The aims of this work are to investigate the metastasis related effects of MAG-1 on human lunggiant-cell line PLA801, and to compare the expression rate of MAG-1 in cancer tissue from lung cancer patients with different metastatic status.Sense and anti-sense expressing vectors of MAG-1 were constructed and transfected into PLA801C and PLA801D respectively. Colony forming, adherence assay, MTT assay and Transwell experiments were used to evaluate the alterations of clone forming, cell-matrix adherence, proliferating and invasion of the stable transfected cell strains. Western blot was employed to detect the proteins levels of CD44, and MMP-2 in cell strains and mRNA state of MAG-1 in lung cancer tissue from patients with or without pathological metastasis were also analyzed by RTPCR.MAG-1 could increase cell-ECM (Extracellular Matrix) adhesive capacity, promote invasion, enhance cell proliferation and had no effects on clone forming ability of PLA801 cells in soft agar. MAG-1 was also found have positive effects on the protein level of CD44 and MMP-2 in PLA801 cells, and the detection rate of MAG-1 mRNA was much higher in cancer tissue from metastatic patients (7/12) than that in non-metastatic patients (2/12).MAG-1 could promote lung cancer metastasis and might be a metastasis associated gene of lung cancer.

To tissue engineer a kidney is a formidable task because of the complex cell composition and structures in the kidney. This study reconstructed renal tissues using mixed renal cells in collagen/Matrigel® scaffolds in vitro. Neonatal rat renal cells were seeded in collagen I supplemented with Matrigel in a casting mold that could exert static stretch when the renal constructs contracted. During in vitro culture, the renal constructs were observed under microscope and analyzed by histological and immunofluorescent examinations. Results showed that the mixed renal cells reconstituted renal tubular and glomeruli-like structures with different appearances at varying developmental stages. Tubular structures were formed by CK18-positive cells with similar appearances lining the surrounding hollow centres. The glomeruli-like structures were tufts of cell aggregates containing Flk-1-positive cells. These results show that neonatal rat renal cells self-assembled into engineered renal tissues containing both tubules and glomeruli-like structures when cultured in 3D collagen/Matrigel scaffold in vitro.

In this paper, we propose a novel out-of-core volume rendering algorithm for large time-varying fields. Exploring temporal and spatial coherences has been an important direction for speeding up the rendering of time-varying data. Previously, there were techniques that hierarchically partition both the time and space domains into a data structure so as to re-use some results from the previous time step in multiresolution rendering; however, it has not been studied on which domain should be partitioned first to obtain a better re-use rate. We address this open question, and show both theoretically and experimentally that partitioning the time domain first is better. We call the resulting structure (a binary time tree as the primary structure and an octree as the secondary structure) the space-partitioning time (SPT) tree. Typically, our SPT-tree rendering has a higher level of details, a higher re-use rate, and runs faster. In addition, we devise a novel cut-finding algorithm to facilitate efficient out-of-core volume rendering using our SPT tree, we develop a novel out-of-core preprocessing algorithm to build our SPT tree I/O-efficiently, and we propose modified error metrics with a theoretical guarantee of a monotonicity property that is desirable for the tree search. The experiments on datasets as large as 25GB using a PC with only 2GB of RAM demonstrated the efficacy of our new approach.

In this paper we propose a novel {\em out-of-core} technique for{\em progressive} lossless compression and {\em selective}decompression of 3D triangle meshes larger than main memory. Most existing compression methods, in order to optimize compression ratios, only allow {\em sequential} decompression. We develop an integrated approach that resolves the issue of so-called {\emprefix dependency} to support {\em selective} decompression, and in addition enables I/O-efficient compression, while maintaining high compression ratios. Our decompression scheme initially provides a global context of the entire mesh at a coarse resolution, and allows the user to select different {\em regions of interest} to further decompress/refine to {\bf different}levels of details, to facilitate out-of-core multiresolution rendering for interactive visual inspection. We present experimental results which show that we achieve fast compression/decompression times and low memory footprints, with compression ratios comparable to current out-of-core {\em single resolution} methods.

Depsipeptide FR901228 (FK228) is a new kind of histone deacetylase inhibitors (HDACi) that induces growth arrest and cell death in a variety of tumor cells. Though its effects on oncogene expression and degradation have been documented, the detailed mechanism of FK228-induced cytotoxicity is still undefined. In this study, a differential proteomic analysis was performed to identify proteins associated with FK228-induced cytotoxicity in human lung cancer cells. Two-dimensional gel electrophoresis (2-DE) revealed a distinct protein profile of H322 cells in response to FK228 treatment, and 45 protein spots with significant alteration were screened. In total, 27 proteins were identified by mass spectrometry and involved in signal transduction, transcriptional regulation, metabolism, cytoskeletal organization, and protein folding, synthesis and degradation, consistent with multiple effects of FK228 on tumor cells. Notably, a novel target protein, thioredoxin reductase (TrxR), was identified, which is downregulated in FK228-sensitive cancer cells, but upregulated in resistant cells. The expression level of TrxR was negatively correlated with ROS accumulation, DNA damage and apoptosis, implicating TrxR in FK228-induced apoptosis and HDACi sensitivity in cancer cells. Thus, proteomic analysis provides new information about target proteins important for FK228-induced cytotoxicity in cancer cells.

Some pan-histone-deacetylase (HDAC) inhibitors have recently been reported to exert their anti-leukemia effect by inhibiting the activity of class IIB HDAC6, which is the deacetylase of Hsp90 and alpha-tubulin, thereby leading to hyperacetylation of Hsp90, disruption of its chaperone function and apoptosis. In this study, we compared the effect of a class I HDAC inhibitor FK228 with the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on the Hsp90 chaperone function of K562 cells. We demonstrated that, although having a weaker inhibitory effect on HDAC6, FK228 mediated a similar disruption of Hsp90 chaperone function compared to SAHA. Unlike SAHA, FK228 did not mediate hyperacetylation of Hsp90, instead the acetylation of Hsp70 was increased and Bcr-Abl was increasingly associated with Hsp70 rather than Hsp90, forming an unstable complex that promotes Bcr-Abl degradation. These results indicated that FK228 may disrupt the function of Hsp90 indirectly through acetylation of Hsp70 and inhibition of its function.

This paper presents a new watermarking technique in the spatial domain expressly devised for color images, which is based on transmit diversity techniques. The RGB tones of color images are considered as three independent slowly fading channels. Modulated by different pseudo-random sequences and interleavers, the same watermark in different forms is put into three color channels simultaneously. This scenario makes up the weakness in single color channel schemes that a single color channel is prone to severe fading. HVS characteristics of the three color channels are separately considered. We use two extraction methods: one is by comparing the energy strength of the three channels, the other is by accumulating the energy of three channels. Experimental results demonstrate the unmistakable advantage of our new approach over algorithms operating on single color channels. In contrast to the present spatial watermarking schemes, this algorithm is considerably robust to many intentional and unintentional attacks.

Whether differentiation of induced pluripotent stem cells (iPSCs) in ischemic myocardium enhances their immunogenicity, thereby increasing their chance for rejection, is unclear. Here, we dynamically demonstrated the immunogenicity and rejection of iPSCs in ischemic myocardium using bioluminescent imaging (BLI). Murine iPSCs were transduced with a tri-fusion (TF) reporter gene consisting of firefly luciferase-red fluorescent protein-truncated thymidine kinase (fluc-mrfp-tTK). Ascorbic acid (Vc) were used to induce iPSCs to differentiate into cardiomyocytes (CM). iPSCs and iPS-CMs were intramyocardially injected into immunocompetent or immunosuppressed allogenic murine with myocardial infarction. BLI was performed to track transplanted cells. Immune cell infiltration was evaluated by immunohistochemistry. Syngeneic iPSCs were also injected and evaluated. The results demonstrated that undifferentiated iPSCs survived and proliferated in allogenic immunocompetent recipients early post-transplantation, accompanying with mild immune cell infiltration. With in vivo differentiation, a progressive immune cell infiltration could be detected. While transplantation of allogenic iPSC-CMs were observed an acute rejection from receipts. In immune-suppressed recipients, the proliferation of iPSCs could be maintained and intramyocardial teratomas were formed. Transplantation of syngeneic iPSCs and iPSC-CMs were also observed progressive immune cell infiltration. This study demonstrated that iPSC immunogenicity increases with in vivo differentiation, which will increase their chance for rejection in iPSC-based therapy.

AMP-activated protein kinase (AMPK) plays an important role in mediating energy metabolism and is controlled mainly by two upstream kinases, LKB1 or Ca(2+)/calmodulin-dependent protein kinase kinase-β (CaMKKβ). Previously, we found that baicalin, one of the major flavonoids in a traditional Chinese herb medicine, Scutellaria baicalensis, protects against the development of hepatic steatosis in rats feeding with a high-fat diet by the activation of AMPK, but, the underlying mechanism for AMPK activation is unknown. Here we show that in two LKB1-deficient cells, HeLa and A549 cells, baicalin activates AMPK by α Thr-172 phosphorylation and subsequent phosphorylation of its downstream target, acetyl CoA carboxylase, at Ser-79, to a similar degree as does in HepG2 cells (that express LKB1). Pharmacologic inhibition of CaMKKβ by its selective inhibitor STO-609 markedly inhibits baicalin-induced AMPK activation in both HeLa and HepG2 cells, indicating that CaMKKβ is the responsible AMPK kinase. We also show that treatment of baicalin causes a larger increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), although the maximal level of [Ca(2+)](i) is lower in HepG2 cells compared to HeLa cells. Chelation of intracellular free Ca(2+) by EDTA and EGTA, or depletion of intracellular Ca(2+) stores by the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin abrogates baicalin-induced activation of AMPK in HeLa cells. Neither cellular ATP nor the production of reactive oxygen species is altered by baicalin. Finally, in HeLa cells, baicalin treatment no longer decreases intracellular lipid accumulation caused by oleic acid after inhibition of CaMKKβ by STO-609. These results demonstrate that a potential Ca(2+)/CaMKKβ dependent pathway is involved in the activation of AMPK by baicalin and suggest that CaMKKβ likely acts as an upstream kinase of AMPK in response to baicalin.