Klotho was originally characterized as an aging suppressor gene that predisposed Klotho-deficient mice to premature aging-like syndrome. Although Klotho was recently reported to exhibit tumor suppressive properties during various malignant transformations, the functional role and molecular mechanism of Klotho in hepatocarcinogenesis remains poorly understood. In our present study, immunohistochemical Klotho staining levels in a clinical follow-up of 52 hepatoma patients were significantly associated with liver cirrhosis, tumor multiplicity and venous invasion. The overall survival rate of hepatoma patients with high Klotho expression was significantly lower than those patients with low Klotho expression. Moreover, Klotho overexpression increased cellular migration, anchorage-independent growth, and anoikis resistance in hepatoma cells. Klotho overexpression elevated p21-activated kinase 1 (PAK1) expression and shRNA-mediated PAK1 knockdown and kinase activity inhibition with kinase dead mutant PAK1 K299R coexpression or allosteric inhibitor IPA3 treatment reversed anoikis resistance in Klotho-overexpressed hepatoma cells. More importantly, the pivotal significance of upregulated VEGFR2 protein levels mediated by Klotho expression was confirmed by VEGFR2 inhibitor Axitinib and blocking antibody treatment in hepatoma cells. Axitinib treatment sensitized anoikis was reversed by constitutive active mutant PAK1 T423E coexpression in Klotho-overexpressed hepatoma cells. Conversely, knockdown of Klotho reduced VEGFR2/PAK1 dependent anoikis resistance, which could be reversed by PAK1 T423E. These results revealed a novel oncogenic function of Klotho in promoting anoikis resistance via activating VEGFR2/PAK1 signaling, thus facilitating tumor migration and invasion during hepatoma progression, which could provide a putative molecular mechanism for tumor metastasis. Primary liver cancer, which consists predominantly of hepatocellular carcinoma (HCC), ranks as the fifth most prevalent malignancy and the third leading cause of cancer mortality worldwide Metastasis requires that tumor cells detach from the primary matrix or cell-cell anchors that control tissue architecture Klotho is recently identified as a senescence-suppressing gene In the present study, we found for the first time that high immunohistochemical Klotho staining levels were significantly associated with liver cirrhosis, tumor multiplicity, venous invasion, and poor overall survival in a clinical follow-up of 52 hepatoma patients. Furthermore, Klotho expression conferred hepatoma cells with resistance to anoikis via activating VEGFR2/PAK1 signaling, which could be reversed by inhibition with PAK1 allosteric inhibitor IPA3 or VEGFR2 inhibitor Axitinib administration in Klotho-overexpressed hepatoma cells. These results identified Klotho/VEGFR2/PAK1 as a crucial molecular pathway underlying resistance to anoikis for hepatoma cells, which could open a new avenue for molecular targeting therapeutic intervention with anoikis resistance in HCC patients with metastasis.Ethical approval was granted by the Ethics Committee of the Fudan University. Written informed consent was obtained for the acquisition and use of patient tissue samples and anonymized clinical data.2 incubator at 37°C. Fifty-two pairs of human hepatoma and peripheral nontumor tissues after surgical resection were collected from HCC patients in Nantong Tumor Hospital  from 2003 to 2005. Use of human tissues with informed consent was approved by the Institutional Review Board of Fudan University.One immortalized liver cell line (L02) and five hepatoma cell lines  were cultured in DMEM supplemented with 10% FBS in 5% COExpression plasmid encoding Klotho was kindly provided by Dr. Ci-Di Chen . The plasmids containing wild type PAK1, kinase dead mutant PAK1 K299R, and constitutive active mutant PAK1 T423E were constructed as described previously Transient and stable transfections with various plasmids were performed as previously described A total of 52 pairs of HCC samples were H&E stained and immunohistochemically analyzed as described previously Protein extraction from cultured cells and Western blot analysis were carried out as previously described In vitro anoikis assay was performed with cell culture on poly-HEMA-coated 6-well tissue culture plates for 48 hours as described previously Flow cytometry analysis for VEGFR2 expression on hepatoma cells was performed with anti-VEGFR2-Percp  as described previously t test. The correlation between Klotho expression and clinicopathological features was assessed using the t test or Fisher's exact test with SPSS version 17.0 statistical software package . Survival was calculated from the date of surgery to date of death or last follow-up. Survival curves were estimated using the Kaplan-Meier method, and log-rank test was used to compute differences between the curves. Differences were considered significant at values of P<0.05.Experimental data were presented as mean±SEM of at least three independent replicates through analyzing with GraphPad Prism 5  and assessing comparisons between different groups by the Student's To investigate the clinical significance of Klotho in hepatocarcinogenesis, we first evaluated immunohistochemical Klotho staining intensity in tumor specimens from 52 patients with HCC and classified these hepatoma samples into four categories: negative staining intensity (grade 0) for 1 case, weak staining intensity (grade I) for 10 cases, moderate staining intensity (grade II) for 18 cases, and strong staining intensity (grade III) for 23 cases . Taken tP = 0.023), tumor multiplicity (P = 0.007), and venous invasion (P = 0.021) of HCC patients, whereas it was not associated with age, gender, HBsAg, tumor size, microsatellite nodules, tumor encapsulation, intrahepatic metastasis,tumor stage, or BCLC stage. Moreover, Kaplan-Meier survival analysis indicated that the overall survival rate of hepatoma patients with high Klotho expression was significantly lower than those with low Klotho expression (P = 0.03)  . These dTo address the potential function of Klotho expression in HCC progression, we selected two hepatoma cell lines (Huh7 and HepG2) with low Klotho expression to explore functional alterations after Klotho overexpression . Cell miin vitro anoikis assay for Huh7 and HepG2 cells was performed in the presence or absence of Klotho overexpression. Elevated caspase 3/7 activities after detachment in these two hepatoma cells were significantly rescued by Klotho expression  in HCC tumor tissues , a bone-derived hormone that regulates the phosphate, calcium and vitamin D balance, and defects in either Klotho or FGF23 cause phosphate retention and a premature-ageing syndrome in mice Klotho is identified as a potential tumor suppressor and an inhibitor of the IGF-1 pathway and activator of the FGF pathway in human breast cancer Since anoikis resistance is prerequisite for hepatoma progression and metastasis, resistance of hepatoma cells to anoikis due to Klotho expression might be the crucial functional role of Klotho in HCC development and progression. Consistent with our previous studies indicating the pivotal significance of PAK1-induced anoikis resistance in HBV-associated hepatocarcinogenesis, our current data also indentify VEGFR2/PAK1 activation as downstream effector of Klotho expression in anoikis resistance of hepatoma cells. Besides, we have also provide evidence that VEGFR2/PAK1 inhibition with VEGFR2 inhibitor Axitinib, or anti-VEGFR2 blocking antibody, or PAK1 allosteric inhibitor IPA3 administration could significantly blunt anoikis resistance in Klotho-overexpressed hepatoma cells. These data suggest that Klotho/VEGFR2/PAK1 signal inhibition with VEGFR2 inhibitor Axitinib, or anti-VEGFR2 blocking antibody, or PAK1 allosteric inhibitor IPA3 administration might be used as potential therapeutic interventions with progression and metastasis of HCC patients, which awaits further clinical investigations. Although we have identified that Klotho expression confers hepatoma cells with resistance to anoikis through VEGFR2/PAK1 activation, the underlying molecular mechanism for Klotho-mediated VEGFR2/PAK1 activation remains still obscure and needs further exploration.As a multikinase inhibitor targeting Raf, VEGFR and PDGFR signals, Sorafenib has been used as a first-line therapeutic drug and shown clinical efficiency for advanced-stage HCC patients Figure S1Klotho expression positively correlates with PAK1 in tumor tissues from patients with HCC. (A) Representative photomicrographs of immunohistochemical Klotho staining in human HCC tissues: Grade 0 for negative staining intensity, Grade I for weak staining intensity, Grade II for moderate staining intensity, and Grade III for strong staining intensity . (B) A significant positive correlation between Klotho and PAK1 was shown .(TIF)Click here for additional data file.


We have previously demonstrated that an improved affinity TCR generated against NYESO-1 has shown remarkable efficacy in clinical studies in multiple myeloma (NCT01352286). The same TCR is also being tested in synovial sarcoma (NCT01343043), melanoma (NCT01350401) and ovarian (NCT01567891) clinical trials. We will extend these clinical studies to include hepatocellular carcinoma shortly. In this present study, we have generated a panel of improved-affinity TCRs for a widely expressed hepatic carcinoma-specific HLA-A2-restricted peptide antigen AFP158-166. This TCR panel demonstrates stepwise increases in affinity from wildtype to approximately 500-fold greater binding to cognate antigen. These affinity-enhanced TCRs were screened for efficacy and specificity by a variety of in vitro assays, such as interferon gamma ELISpot, Incucyte™ cytotoxicity assays for visualizing caspase 3/7 activity, Luminex™ multiple cytokine secretion assays and 3D microtissue models against a variety of hepatocellular carcinoma cell lines and normal primary cell types. This TCR panel demonstrated a window of activity whereby moderately increased affinity TCRs showed maximal response to antigen positive hepatocellular carcinoma cell lines HepG2, HuH6 and JHH5-A2, whilst showing no response to primary normal hepatocytes or primary cells from other organ systems. Greater increases in TCR affinity led to off-target activity, demonstrated as responses to primary normal target cells as well as antigen positive cell lines. This data has led to selection of a lead candidate for full pre-clinical assessment that demonstrates vastly improved efficacy against hepatocellular carcinoma cell lines, yet retains specificity by absence of response to primary normal tissue. This TCR may be suitable for further clinical studies in hepatocellular carcinoma.


E ring of the quinolizidine moiety of cryptopleurine analogs changed the potency and the selective inhibitory effect on NF-κB, AP-1, and CRE signaling pathways. Functional cryptopleurine analogs showed potent inhibition of NF-κB signaling pathway in both HepG2 and HEK-293 cell lines. The E ring structure analogs also differed in suppression of protein translation, and expression of cyclin D1. Our results showed that DCB-3503 or Rac-cryptopleurine could be a scaffold for modification to yield compounds with different mechanisms of action.Tylophorine analogs exhibit a broad range of pharmacological activities, including anti-cancer, anti-inflammatory, anti-autoimmune, and anti-virus effects. Structure-activity relationship study of different structure tylophorine analogs can provide further understanding of their biological activity. Modifications on the  Asclepiadaceae and Moracea family. Their claimed medical uses include the treatment of cancer, lupus, and inflammation in vivo activity.Tylophorine alkaloids are natural products originally identified in the E ring) or quinolizidine (six-member E ring) moiety. Previous SAR study has found that cryptopleurine analogs with quinolizidine moiety . This group of compounds shares a common pentacyclic structure with the phenanthrene ring conjugated with the indolizidine (five-member e moiety  were morE ring) is about 5 to 10 fold more potent than analogs with the indolizidine moiety (five-member E ring) (DCB-3500 and DCB-3503) Our previous work demonstrated that Rac-cryptopleurine with the quinolizidine moiety , replacing the six-membered E-ring with a seven-membered E-ring (YXM-83) or with a five-membered N-cyclopropylpyrrolidinyl E-ring (YXM-93), caused the loss of cytotoxicity towards HepG2 and Huh-7 cell lines. Together with our previous report, these newly synthesized compounds showed similar spectrum of potency against A549, DU145, KB, KBvin, SKBR3 cell lines to HepG2 and Huh 7 cell lines as we reported in the present study 50) against KB, KBvin, and Huh 7 cell line. These results provided important information that the structure requirements for the most potent cytotoxic analogs for different cell lines appear to be the same.The cytotoxicity of these compounds against human hepatoma cell lines (HepG2 and Huh-7) is shown in 50 value is shown in NF-κB plays important role in controlling inflammation, cancer cell survival and death, and formation of chemoresistance 50 concentration against NF-κB pathway ; however, YXM-139 and 140 did not inhibit translation under the same condition at 500 nM and 250 nM, respectively  tail in Retic lysate ectively . YXM-109ectively . This suE-ring have less potency against the growth of HepG2 and Huh-7 cell lines, and some of those are no longer functional analogs. E-ring altered the cytotoxicity of cryptopleurine ananlogs, and the chirality of the hydroxyl group is critical determination factor in the cytotoxicity. Cryptopleurine analogs with 6-member E-ring are more potent than 7-member E-ring analogs (E-ring with R14a-(R)-hydrogen>five member E-ring with R13a-(R)-hydrogen>five member E-ring with R13a-(S)-hydrogen>>six member E-ring with R14a-(R)-hydrogen. The results we obtained suggest that the E ring size may be a critical determination factor for the interaction of cryptopleurine analogs to their molecular targets, and molecular target of cryptopleurine analogs may not be identical. Changing structure of compound may not only be reflected in potency, but also determined the mode of action. Since the biochemical determinants of the primary target(s) of these compounds may vary, the selectivity against different signaling pathways could be cell type specific. This was demonstrated by the comparative study of HepG2 and HEK-293 cell lines. Their potency against HepG2 cell growth correlates well with the inhibitory activity against protein synthesis and TNF/NF-κB pathway. Tylophorine or Rac-cryptopleurine could be a good scaffold for synthesis of biological active compound with diverse action. We are in the process of evaluating the in vivo antitumor activity of selected cryptopleurine analogs with R15 hydroxyl group.Tylophorine analogs with introduction of different moieties (e.g. N or O) in the DCB-3503 (NSC-716802) was synthesized in Dr. D. C. Baker’s laboratory . Crytopleurine analogs were synthesized in Dr. K. H. Lee’s laboratory . Cell culture media, fetal bovine serum (FBS) were purchased from Invitrogen . All chemicals except otherwise noted were purchased from Sigma-Aldrich .2 at 37°C.Cell lines were obtained from the American Type Culture Collection (ATCC). HepG2 cells were maintained in RPMI 1640 medium supplemented with 10% FBS. HepG2 stable cell lines harboring NF-κB, AP-1, CRE response elements in pGL4 vector (Promega) were maintained in the presence of 0.8 mg/ml G418. Huh-7 and HEK-293 cells were maintained in DMEM containing 4.5 g/l glucose supplemented with 10% FBS. All cell lines were maintained in a humidified incubator with an atmosphere of 95% air and 5% CO50 was defined as the concentration of drug that inhibited cell growth by 50% after continuous drug exposure for 72 hours Ten thousands cells/well were plated in 24-well plates. After overnight incubation, cells were treated with drugs for 72 hours. Cells were fixed and stained with 0.5% methylene blue in 50% ethanol for 2 hours at room temperature, followed by washing with tap water to remove excess color. Plates were dried and then resuspended in 1% sarkosyl and incubate for 3 hours at room temperature. Cell growth was quantitated based on the amount of methylene blue adsorbed into cellular proteins measured by spectrophotometer (Molecular Devices) at 595 nm. IC50 was defined as the concentration of drug that inhibited stimulator-triggered luciferase reporter activation by 50% after continuous drug exposure for 4 hours.HepG2 and HEK-293 cell lines stably harboring NF-κB, AP-1, and CRE response elements in pGL4.0 luciferase vector (Promega) were used for the signaling pathway reporter assay Western blot analysis was done using primary antibodies against Cox 2 , iNOS (Abcam), cyclin D1 (Santa Cruz Biotechnology), and β-actin (Sigma-Aldrich) at optimal dilution 35S]-methionine/cysteine (PerkinElmer) for 30 minutes before harvest. Incorporation of [35S]-methionine/cysteine was determined by scintillation counter.The incorporation assay was done followed by the protocol described previously in vitro transcription was reported previously in vitro transcription. Capped luciferase mRNA was generated by mMESSAGE mMACHINE high yield capped RNA transcription kit containing T3 RNA polymerase (Ambion). The in vitro transcribed mRNAs were purified by MEGAclear kit (Ambion); and the integrity of mRNA was examined by Bioanalyzer . The purified mRNAs were used for in vitro translation experiments.The detailed protocol for In vitro translation was performed by Retic Lysate IVT™ (Ambion) as described previously in vitro translation mixtures containing 50 ng/µl T3 luciferase mRNA was incubated for 90 minutes at 30°C. Translation products of T3 luciferase were measured by luciferase assay.p<0.05.Data were analyzed by ANOVA and the Bonferroni multiple comparision test by GraphPad Prism 5 software. The difference was considered to be statistically significant when Figure S1Dose-cell viability curves for DCB-3503 and cryptopleurine analogs in HepG2 (a) and Huh-7 (b) cell lines. Graphs were simplified by showing the mean value from three independent experiments.(TIF)Click here for additional data file.Figure S2Dose-luciferase activity curves for DCB-3503 and cryptopleurine analogs against NF-κB (a), AP-1 (b), and CRE (c) signaling pathways in HepG2 cell line. Graphs were simplified by showing the mean value from three independent experiments.(TIF)Click here for additional data file.Figure S3Dose-luciferase activity curves for DCB-3503 and cryptopleurine analogs against NF-κB (a), AP-1 (b), and CRE (c) signaling pathways in HEK-293 cell line. Graphs were simplified by showing the mean value from three independent experiments.(TIF)Click here for additional data file.Figure S4Schematic description of the SAR of cryptopleurine analogs with modification on the E-ring in HEK-293 cell line.(TIFF)Click here for additional data file.


Trigona laeviceps, is one potential interesting source that is widely available and cultivatable (as bee hives) in Thailand.Cancers are some of the leading causes of human deaths worldwide and their relative importance continues to increase. Since an increasing proportion of cancer patients are acquiring resistance to traditional chemotherapeutic agents, it is necessary to search for new compounds that provide suitable specific antiproliferative affects that can be developed as anticancer agents. Propolis from the stingless bee, 2Cl2 and hexane. After solvent removal by evaporation, each extract was solvated in DMSO and assayed for antiproliferative activity against five cancer  and two normal (HS27 fibroblast and CH-liver) cell lines using the MTT assay. The cell viability (%) and IC50 values were calculated.Propolis (90 g) was initially extracted by 95% (v/v) ethanol and then solvent partitioned by sequential extractions of the crude ethanolic extract with 40% (v/v) MeOH, CHin vitro antiproliferative activity against the five tested cancer cell lines and the lowest cytotoxicity against the two normal cell lines. Further fractionation of the hexane fraction by quick column chromatography using eight solvents of increasing polarity for elution revealed the two fractions eluted with 30% and 100% (v/v) CH2Cl2 in hexane  had a higher anti-proliferative activity. Further fractionation by size exclusion chromatography lead to four fractions for each of 30DCM and 100DCM, with the highest antiproliferative activity on cancer but not normal cell lines being observed in fraction# 3 of 30DCM .The hexane extract provided the highest T. laeviceps propolis was found to contain compound(s) with antiproliferative activity in vitro on cancer but not normal cell lines in tissue culture. The more enriched propolis fractions typically revealed a higher antiproliferative activity . Overall, propolis from Thailand may have the potential to serve as a template for future anticancer-drug development. Cancers are some of the major fatal diseases to humans. Chemotherapy is one of the most widely used approaches for the treatment of many cancers, but the long-term use of chemotherapy can lead to drug resistance via several different mechanisms, such as gene mutation, DNA methylation and histone modification. These resistance mechanisms have been reported to play important roles in the resistance of cancers to chemotherapeutic agents . Thus, pPropolis, one of the economic natural products from bees, is an interesting source for several bioactivities, such as antimicrobial as well as anti-cancer. Although an animal product it is largely plant based in its chemical origins. It is a sticky resin and varies in colour, including brown, green and red amongst others, based upon the plant exudates that the bees have selectively collected from flower buds, leaf buds and tree barks. These plant resins are mixed with waxes and other bee excretions, including enzymes , to formApis mellifera in the Preveza region of Northwest Greece was mainly from Pinaceae [Hyptis di Varicata and Baccharis dracunculifolia [A. mellifera in Brazil, Chili and Myanmar presented a different in vitro cytotoxicity activity against the PANC-1 human pancreatic cancer cells in a nutrient-deprived medium [The chemical composition of each type of propolis and its associated bioactivities mainly depend on the macro- and micro-geographical regions, due to the differences in the plant resin compositions or available plant species ,13, and Pinaceae  whilst iulifolia ,16, suggd medium .in vitro antiproliferative effect on human melanoma, C6 glioma and HL-60 cell lines in tissue culture.Although propolis is typically a complex mixture of diverse compounds from plant resins, volatiles, pollen and animal enzymes, etc, numbering over 300 characterized components, the reported bioactivities associated with propolis have been found in both the crude and the purified extracts ,19, suggA. mellifera propolis in Chili, along with a considerable number of flavonoid compounds, such as p-cumaric acid and ferulic acid, were purified and found to display an in vitro antiproliferative effect on the KB, DU-145 and Caco-2 cell lines in tissue culture [Caffeic acid phenethyl ester (CAPE), the main component from  culture .Given that the main active components found to date are plant derived flavonoids, it would seem likely to be better to preserve the polyphenolic fraction of propolis after purification.Trigona laeviceps Smith (Hymenoptera: Apoidae) was used in this research since these bees can be commercially cultivated in a sustainable and potentially ecologically friendly manner in artificial hives , and can provide a lot of propolis per hive. In addition, the bees are widely distributed throughout Thailand. Although Umthong et al. reported that a high cytotoxicity on the SW620 colon cancer cell line was obtained from the crude extract of propolis harvested from T. laeviceps in Thailand [T. laeviceps propolis from Samut Songkram, central Thailand, using a bioassay-guided isolation procedure. Solvent partitioning of the propolis, based upon solvent polarity, was performed and human cell lines derived from five different types of tissue cancers were used to screen for any in vitro antiproliferative affect in comparison to the two normal cell lines.Propolis from the stingless bee Thailand , it is nT. laeviceps was collected from an apiary in the Samut Songkram province, central Thailand, and was kept in the dark at 4°C until use.Propolis of et al. [The method of propolis extraction followed that reported by Najafi et al. . Briefly2O to increase the partition coefficient (solubility) of polar compounds, and then extracted four times with an equal volume of CH2Cl2 as per hexane above, except that following phase separation the CH2Cl2 phase containing the less polar compounds was the lower phase. The pooled CH2Cl2 phases and the residual 40% (v/v) methanol phase were separately evaporated to remove the respective solvent in a rotary evaporator (40°C), and the residues were each weighed to give the yield of the CH2Cl2 and methanolic extracts, respectively.The ethanolic extract of the propolis was dissolved in 80% (v/v) methanol until it was not sticky and then an equal volume of hexane was added, stirred (15 mins) and then allowed to phase separate in a separating funnel. The upper hexane phase containing the non-polar compounds was harvested and kept whilst the 80% (v/v) methanol phase containing polar compounds was extracted three more times with hexane in the same manner. The four hexane extracts were pooled, the solvent evaporated in a rotary evaporator (40°C), and the sticky liquid residue weighed to give the yield of the hexane extract. The 80% (v/v) methanol phase was then mixed with an equal volume of H2Cl2 until they were not sticky and were then mixed with silica gel, evaporated to dryness and loaded on top of the silica gel containing column. The column was then eluted by various solvents  of different polarities starting from the least polar solvent and increasing, that is from 0:1, 1:9, 3:7, 1:1, 7:3 and 1:0 (v/v) of CH2Cl2: hexane and then followed by 5% and 10% (v/v) MeOH in CH2Cl2, respectively. A reduced pressure was used in order to enable the flow rate of the solvent through the column to be obtained, and fractions were collected. Each fraction was solvent evaporated in a rotary evaporator (40°C), and the residue weighed before being dissolved in DMSO to the required concentrations to assay for antiproliferative activity as detailed below.Silica gel was packed into a glass funnel  connected to a vacuum pump. The selected fractions (see Bioassay guided selection below) were mixed with CH2Cl2 until it was not sticky and was loaded on top of the column. The column was then eluted with 10 ml of a 1:1 (v/v) ratio of MeOH: CH2Cl2, collecting 2 ml fractions. The four fractions obtained were then solvent evaporated by a rotary evaporator (40°C), the residue weighed and then dissolved in DMSO to the required concentrations ready to assay for any selective antiproliferation activity as detailed below.Further partial purification of each selected fraction was performed by size exclusion chromatography using a Sephadex LH-20 column . Each selected fraction was dissolved in a 1:1 (v/v) ratio of MeOH: CH2Cl2: hexane. When the mobile phase had almost reached the top of the plate, the samples were visualized under U.V. light (254 nm). Alternatively, the gel plate was sprayed by a 5% (v/v) H2SO4/0.03% (w/v) α-naphtal methanolic solution, dried in an oven or hot plate and visualized under U.V. light (350 nm).One-dimensional thin layer chromatography (TLC) was used in order to group the obtained components and to determine the purity of fractions. A silica gel plate (10 cm in height) was used as the stationary phase, and the respective extracts or fractions were spotted at the starting line at 0.5 - 1 cm intervals. The mobile phase solvents used (one per TLC plate) were 1:0, 3:1 and 1:1 (v/v) ratio of CHin vitro antiproliferative activity on five selected cancer and two normal cell lines in tissue culture using the MTT assay as detailed below. The extract which provided the best selective antiproliferative activity, that is the highest activity on the cancer cell lines but not on the normal cell lines, as determined by comparison of the IC50 values, was selected for further partial purification by quick column silica chromatography. In the same way, each fraction obtained from the quick column chromatography was likewise assayed for selective antiproliferative activity on the cell lines and this was used to select fractions for further size exclusion chromatography (see above).Each of the three crude solvent partitioned extracts  of propolis, obtained as detailed above, were evaluated for their in vitro antiproliferative bioactivity were derived from colon (SW620), breast (BT474), hepatic (Hep-G2), lung (Chago), and stomach (Kato-III) tissue cancers. The two normal cell lines used were of liver (CH-liver) and fibroblast (HS-27) origins and were used as comparative controls to check for selective specificity towards cancer cells rather than all dividing cells. All cell lines were cultured in RPMI medium containing 5% (v/v) fetal calf serum (complete medium) at 37°C in a humidified air atmosphere containing 5% (v/v) CO2.The five selected cancer cell lines for screening for the 2 for 24 h in order to let the cells attach to the bottom of each well. The cultured cells were then treated with the tested propolis extract (triplicate wells per condition) by the addition of 2 μl of serial dilutions of the propolis extract dissolved in DMSO to give a final concentration of 100, 50, 25, 12.5, 6.25 and 3.125 μg/ml. In addition, 2 μl of DMSO alone was added to another set of cells as the solvent control. The cells were then cultured as above for another 48 h prior to the addition of 10 μl of a 5 mg/ml solution of 3--2, 5-diphenyltetrazolium bromide (MTT)into each well. The incubation was continued for another 4 h before the media was removed. A mixture of DMSO (150 μl) and glycine (25 μl) was added to each well and mixed to ensure cell lysis and dissolving of the formasan crystals, before the absorbance at 540 nm was measured. Three replications of each experiment were performed and the half maximal inhibitory concentration (IC50) of each extract was calculated as detailed below.Cultured cells  in 200 μl complete media were transferred into each well of a flat 96 well plate and then incubated at 37°C in a humidified air atmosphere enriched with 5% (v/v) COThe obtained absorbance at 540 nm was used to determine the percentage of cell survival assuming that 100% survival was obtained from the solvent only control and that no differences in metabolic activity existed between surviving cells under the different conditions. Under these assumptions, the percentage survival of the treated cancer and normal cultured cells was calculated according to the formula below:50 value from the concentration that could provide a 50% cell survival.The mean (± 1 standard deviation (SD)) cell survival (%) was plotted against the corresponding propolis extract concentration and the best fit line was used to derive the estimated ICT. laeviceps propolis a yield of 18 g (20%) of crude ethanol extract was obtained as a sticky brownish to dark brown resin with a distinctive smell. When assayed in vitro on the five cancer and two normal cell lines, using the MTT assay, the IC50 (μg/ml) values for the five cancer cell lines ranged from 1.9-fold lower (SW620 and BT474) to only 1.04-fold lower (Hep-G2) than that for the control HS27 cell line (Table 50 (μg/ml) values compared to the control CH-liver cell line. Over all, it indicates that the crude ethanolic extract of T. laeviceps propolis contained some antiproliferative activity, but with the overlapping variation within the cancer and normal cell lines the existence of any selective specificity for cancer cells was less clear.From 90 g of 2Cl2 extract showed no significant antiproliferative bioactivity on four of the five cancer cell lines tested  methanol, sequential solvent partitioning with three different polarity solvents was used to further fractionate the propolis extracts. For each of the three solvent extractions the new fractions obtained gave different appearances Table , but thed Figure , whilst d Figure .2Cl2 revealed essentially no antiproliferative activity  values, the fraction obtained from the 1:9 (v/v) ratio of CH2Cl2: hexane revealed a general antiproliferative effect and was not specific to the cancer cell lines. The pure hexane eluted fraction revealed a low cytotoxicity to the control CH-liver cells and a high cytotoxicity on four of the cancer cell lines, the exception being the Chago cell line (66.9 μg/ml compared to 66.2 μg/ml for the control CH-liver cell line). In contrast, the fractions eluted from the 3:9, 1:1 and 7:3 (v/v) ratios of CH2Cl2: hexane revealed a significant antiproliferation activity on all five cancer cell lines but only a slight affect on the control CH-liver cell line. From these three fractions that which eluted from the 3:7 (v/v) ratio of CH2Cl2: hexane (30DCM) was selected for further enrichment by size exclusion chromatography. In addition, the fraction that eluted in pure CH2Cl2 (100DCM) was selected for further enrichment. Although the 100DCM extract displayed a high antiproliferation activity on the control normal cell lines, it displayed a very high activity against all five cancer cell lines, and so was selected in case the two activities could be segregated by further enrichment.Given that the hexane partitioned fraction showed the greatest antiproliferative effect on the five tested cancer cell lines Figure , it was in vitro antiproliferation of cancer cell lines, and so were selected for size exclusion chromatography. For both 30DCM and 100DCM, after size exclusion chromatography, four positive fractions (F1 - F4) were obtained with varying appearances  between the inhibition of cancer and control cell line proliferation were close  values for the antiproliferation activity between the five cancer cell lines (< 25) and the two normal cell lines (> 30) was evident  cell lines, and thus showed a good selective antiproliferation activity. That it eluted from a 3:7 (v/v) ratio of CH2Cl2: hexane is still consistent with the notion that the active compounds are of low polarity. The physical appearance of the 30DCM fraction as a brown solid (Table 50 (μg/ml) value for the CH-liver control cell line was increased, whilst that for the five cancer cell lines was significantly decreased . Overall, the bioassay-guided isolation would appear to be suitable to use in order to obtain the purified active compounds Figure , as repo Croatia . In the (Trigona laeviceps) from Thailand was tested for antiproliferative activity. Five cancer cell lines  and two normal cell lines (CH-liver cells and HS27 fibroblast cells) were selected for this purpose. The crude ethanolic extract displayed a good antiprolferative activity. For example, the IC50 was 19.9 and 29.1 μg/ml for the SW620 cancer cell line and CH-liver cells, respectively. After solvent partitioning, the hexane fractions revealed the highest antiproliferative activity against the five cancer cell lines and the lowest cytotoxic activity on the normal cell lines. For example, IC50 values of 16.4 and 32.4 μg/ml for the SW620 cancer cell line and CH-liver cells, respectively. The hexane fraction part was, therefore, purified in the next step by quick column and size exclusion chromatography. Considering the IC50, it was obvious that the more purified fractions were, the higher the antiproliferative activity was achieved. Two fractions that eluted with 30% and 100% (v/v) CH2Cl2 in hexane  are potential sources of new antiproliferative compounds. Thus, T. laeviceps propolis from Thailand contains some bioactive compounds that are not only effective in antiproliferative activity on cancer cell lines, but also nontoxic to normal cell lines. In the future, it is possible that the bioactive compounds could serve as a template for anticancer-drug developing program.Propolis of the stingless bee The authors declare that they have no competing interests.SU collected the samples, prepared the extracts and carried out the experiments. PP contributed to the study design, analyzed and interpreted the data. SP carried out some of the experiments. CC designed and supervised the experiments and contributed in drafting the manuscript. All authors read the manuscript, contributed in correcting it and approved its final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/11/37/prepub


It demonstrated that the uptake of Zn2+ by nZVI was efficient. With the solids concentration of 1 g/L nZVI, more than 85% of Zn2+ could be removed within 2 h. The pH value and dissolved oxygen (DO) were the important factors of Zn2+ removal by nZVI. The DO enhanced the removal efficiency of Zn2+. Under the oxygen-contained condition, oxygen corrosion gave the nZVI surface a shell of iron (oxy)hydroxide, which could show high adsorption affinity. The removal efficiency of Zn2+ increased with the increasing of the pH. Acidic condition reduced the removal efficiency of Zn2+ by nZVI because the existing H+ inhibited the formation of iron (oxy)hydroxide. Adsorption and co-precipitation were the most likely mechanism of Zn2+ removal by nZVI. The FeOOH-shell could enhance the adsorption efficiency of nZVI. The removal efficiency and selectivity of nZVI particles for Zn2+ were higher than Cd2+. Furthermore, a continuous flow reactor for engineering application of nZVI was designed and exhibited high removal efficiency for Zn2+.Application of zero-valent iron nanoparticles (nZVI) for Zn Zinc is one of the trace elements closely related to human health. It is essential for living organisms Nanoscale zero-valent iron (nZVI) has been investigated as a new tool for the reduction of contaminated water and soil for more than 10 years, and the technology has been applied in many countries worldwide. The nZVI has been proven as a highly effective technology for the removal or degradation of various chemical pollutants, such as β-lactam and nitroimidazole based antibiotics 2+ and Cd2+ which have the standard electrode potentials (E0) for reduction to a metallic state that are very close to, or more negative than Fe0 (−0.44 V). For metal ions such as Hg2+ and Cu2+ whose E0 are much more positive than that of Fe0, removal of metal ions is mainly realized via surface-mediated reductive precipitation in comparison. While metal cations are only slightly more electropositive than Fe0, the removal is mainly realized via the adsorption with partial chemical reduction The determined contaminant removal pathways of nZVI include adsorption, complexation, (co)precipitation and surface-mediated chemical reduction 2+ by nZVI was investigated based on the operation conditions, including nZVI solids loading, pH value and dissolved oxygen (DO). The X-ray Photoelectron Spectroscopy (XPS) of nZVI was performed to detect the valence of zinc and iron to determine whether chemical reaction happened. Furthermore, a continuous flow reactor was designed and applied to remove Zn2+ for evaluating the engineering application of nZVI.In this study, the removal mechanism of Zn2), analytic grade cadmium acetate (Cd[CH3COO]2•2H2O), sodium borohydride  and ferric chloride anhydrous (FeCl3) were purchased from Aladin . Hydrochloric acid (HCl), sodium hydroxide (NaOH), nitric acid (HNO3) and anhydrous ethanol (C2H5OH) were obtained from Sinopharm Chemical Reagent Shanghai Co., Ltd. . All chemicals were used without further purification.Zinc chloride . Microporous membranes (0.22 µm×50 mm) were obtained from CNW (Germany).4, 0.5 M) and ferric chloride anhydrous  with the volume ratio of 1∶1 were vigorously reacted. Then the generated jet-black nZVI particles were collected through vacuum filtration and respectively washed with deionized water and anhydrous ethanol for three times. Finally, fresh nZVI particles were stored in anhydrous ethanol solution at 4°C in order to avoid oxidization prior to use.The nZVI was synthesized according to the method of liquid-phase reduction of ferric trichloride by sodium borohydride Samples of nZVI were prepared by depositing a few droplets of ethanol-diluted nZVI solution onto a carbon-coated transmission electron microscopy (TEM) grid in an oxygen-limiting chamber. But the samples were exposed to air transitorily during transfer from the oxygen-limiting chamber to the microscope. The high-resolution TEM observation was performed using a JEOL JEM 2011 HR-TEM operated at 200 kV with an INCA EDS system.The specific surface area of nZVI was measured by BET analysis.The nZVI particles were dried in a refrigerated drying chamber and then kept under seal at 4°C for X-ray photoelectron spectroscopy (XPS) measurement and X-ray diffraction (XRD) measurement. The XPS spectra were obtained with a Perkin Elmer PHI 5000 ESCA System under Al Kα radiation at 1486.6 eV to study the conversion of the element contents and valence states on nZVI surface. The XRD was carried out on a Bruker X-ray D8 Advance diffraction instrument (Cu Kα) and the diffraction angle (2θ) from10 to 90° was scanned.2 was prepared with deionized water. Uptake reactions were initiated by the addition of nZVI particles into 100 mL aliquots of Zn stock solution. The nZVI loading concentration in the solution was 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 and 2.0 g/L, respectively, at a zinc ion concentration of 100 mg/L. After mixing, the reactors were continuously shaken for 2 hours on an orbital shaker. The optimum loading of nZVI was obtained by comparing the results of the above experiments. All the experiments were performed in triplicate.A 100 mg/L stock solution of ZnCl2+ removal by nZVI was investigated under the oxygen-limiting and oxygen-contained conditions with the optimum nZVI loading. The oxygen-limiting condition was established by flowing nitrogen over the solution. The initial solution pH value was controlled at 5. Reaction time was 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 and 100 min, respectively. All the experiments were performed in triplicate.The effect of oxygen on Zn2+ removal by nZVI, the initial solution pH was adjusted from 3 to 5 with the initial Zn2+ concentration at 100 mg/L by small amounts of HCl or NaOH solution. Then water samples with different pH values were applied to 1 g/L nZVI. All the experiments were performed in triplicate.To investigate the effect of solution pH on the Zn2+ removal by nZVI, three different water samples were used. Sample 1 contained 100 mg/L Zn2+ solution. Sample 2 contained 100 mg/L mixture of Zn2+ Cd2+. Sample 3 contained 100 mg/L Cd2+ solution. The uptake experiments were conducted with the optimum loading of nZVI for 2 h. All the experiments were performed in triplicate.To investigate the effect of cadmium on Zn3 before analysis. Zinc and iron in the sample were determined by inductively coupled plasma optical emission spectrometry .All solution samples were filtered with 0.22 µm membrane acidified with 4% ultrahigh purity HNO2+ by nZVI . The experimental data were expressed as mean±standard deviation (SD).One-way ANOVA was performed to assess the removal efficiency of Zn2+ in 2 h, appeared a single particle composed of a dense core surrounded by a thin amorphous shell, which indicated that the reaction occurred on the surface of nZVI, and that a core-shell structure was formed during the reaction. As shown in Three kinds of nZVI particles were analyzed by transmission electron microscopy (TEM). The fresh nZVI particles were shown in 2/g, which was much higher than that of ZVI, 0.048 m2/g The specific surface area of the nZVI sample was measured by BET analysis. The analysis results indicated that the specific surface area of the nZVI sample was 18.9887 mThe XPS spectra were also obtained to study the elements on the surface of nZVI. As shown in 2+ was increased with the increase of nZVI loading. The added Zn2+ was completely removed under the nZVI loading of 0.8 g/L or higher. When nZVI loading was higher than 1.0 g/L, the removal efficiency of Zn2+ remained about 99%. Higher loading of nZVI could provide more surface area, which enhanced the Zn2+ removal efficiency by nZVI. Thus, we proposed that 1 g/L nZVI was the optimum solids concentration of nZVI required for complete removal of 100 mg/L Zn2+ under the examined experimental conditions. And the concentration ratio was adopted in the subsequent experiments.The uptake experiments were conducted with 0.1 to 2 g/L nZVI at a zinc ion concentration of 100 mg/L, respectively for 2 h. As shown in 2+ by nZVI had been proved by Weile Yan 0 (Zn2+/Zn) is −0.76 v, while the standard reduction potential of iron E0 (Fe2+/Fe) is −0.44 v. The ionization tendency of zinc is higher than iron. So Zn2+ removal by nZVI is likely not caused by the surface-mediated chemical reduction.The high removal efficiency of Zn2+ removal was examined to further study the removal mechanism on the surface of nZVI particles. The freshly prepared 1.0 g/L iron particles were injected into the solution with a zinc ion concentration of 100 mg/L in two 3-neck flasks. One of the flasks had an oxygen-limiting condition with the DO concentration below 0.5 mg/L, but the other one had an oxygen-contained condition with the DO concentration above 5.0 mg/L. As shown in 2+ under oxygen-limiting condition was up to 40%, but a higher removal extent of 80% appeared under oxygen-contained condition. Under both conditions, the Zn2+ removal extent increased to 25% in the first 30 min. After that, the removal trend became different. Under oxygen-limiting condition, another 15% of Zn2+ removal extent was achieved. Under oxygen-contained condition, the removal extent reached 80%. Meanwhile, the range of oxidation-reduction potential (ORP) under the two conditions presented different processes. Under the oxygen-limiting condition, the value of ORP dropped rapidly, and then came to a gentle decline in the residual contact time. In contrast, under the oxygen-contained condition, the value of ORP decreased in the first 30 min, remained unchanged for the next 20 min, and then decreased in the residual contact time. The change processes of ORP indicated that complex redox reactions occurred. The initial drop of ORP was likely caused by the consumption of dissolved oxygen during the oxidation of the Fe (0). The slower decline of ORP under oxygen-contained condition was caused by the supply of oxygen from atmosphere. The above results declared that DO was one of the important factors for the removal of Zn2+ by nZVI. As mentioned above, Zn2+ removal by nZVI is likely not caused by the surface-mediated chemical reduction. The result that nZVI particles were corroded and oxidized by DO might be interpreted as follows: the particles were covered by iron (oxy) hydroxide on the surface and the zinc ion was adsorbed on iron (oxy) hydroxide.The effect of dissolved oxygen (DO) on ZnUnder the oxygen-limiting condition, the predominant electron receptors should be water and the corrosion reaction could occur as follows Under the oxygen-contained condition, the corrosion reaction could occur as follows 3 and FeOOH. Nano/mico goethite has been proved to be efficient with variable capabilities in the removal of five metal ions including Zn2+ from aqueous solution 2+ removal extent under the oxygen-limiting condition could be found in 3 was restricted by the oxygen supply, thus leading to the low removal efficiency.Heavy metal could be adsorbed by Fe(OH)The result that nZVI particles was corroded and oxidized into ferric ion by DO might be interpreted as follows: the particles were covered by iron hydroxide precipitation on their surface and the zinc ion was adsorbed on and/or co-precipitation on iron hydroxide.2+ removal by nZVI. The freshly prepared 1.0 g/L iron particles were injected into the solution with a zinc ion concentration of 100 mg/L within 2 h. Uptake results at various pH conditions were shown in 2+ rose gradually with an increase in pH. The similar effect of pH value has been elucidated by Kishimoto 2+ removal.The pH is an important factor for Zn+ inhibited the formation of iron (oxy)hydroxide, resulting in the low removal extent of Zn2+ by nZVI.With the change in pH value, iron was corroded by acid and oxygen. During the corrosion of iron by acid, 2+ on the surface.0(Zn2+/Zn) is −0.76 v, while the standard reduction potential of iron E0(Fe2+/Fe) is −0.44 v. Therefore, it is not true that Zn2+ removal by nZVI is achieved due to the higher ionization tendency of zinc than that of iron. The cementation is usually effective under acidic pH without DO 2+ was lower under acid condition. Accordingly, the high removal extent of Zn2+ by nZVI might be caused by adsorption and co-precipitation, which was proved by the effects of DO and pH as mentioned above. The formation of FeOOH on the surface of nZVI could be the main factor of Zn2+ removal because of its high adsorption affinity for aqueous solutes The heavy metal removal by nZVI generally involves redox, cementation, adsorption and precipitation. The standard reduction potential of zinc E3+, OH− and O2− could be found 2+ removal by nZVI hydroxide, resulting in the low removal extent of Zn2+ by nZVI.The nZVI is the core-shell structure: a single particle composed of a dense core surrounded by a thin amorphous shell exhibiting markedly less density than the interior core. As shown in 2+ and Cd2+ by nZVI were shown in 2+ was much higher than that of Cd2+. Simultaneous removals of Zn2+ and Cd2+ by nZVI were both lower than individual removal. The Zn2+ removal extent was 0.8% lower in sample 2 than sample 1. And the Cd2+ removal efficiency by nZVI was 2.4% lower in mixed contaminants of sample 2 than sample 3. Hardiljeet’s research has proved that Cd2+ removal by nZVI was chemisorption 2+ and Cd2+ on nZVI. There could be the same adsorption sites for both Zn2+ and Cd2+. The removal efficiency and selectivity of nZVI particles for Zn2+ were higher than Cd2+.Simultaneous and individual removals of Zn2+ by nZVI was demonstrated by jar-test. The removal extent of 100 mg/L Zn2+could reach 85% by 1 g/L nZVI in 2 h, which should be adopted during reactor design and parameter control, such as influent flow, velocity, etc. The contact time of nZVI and Zn2+should be 2 h or more and the nZVI solids concentration should be no less than 1 g/L when the concentration of Zn2+ was 100 mg/L. The results of Zn2+ removal in continuous flow reactor were shown in The high removal efficiency of Zn2+ by nZVI was efficient. With the solids concentration of 1 g/L nZVI, more than 85% of Zn2+ could be removed within 2 h. The pH value and DO were the important factors of Zn2+ removal by nZVI. The DO enhanced the removal efficiency of Zn2+. Under the oxygen-contained condition, oxygen corrosion gave the nZVI surface a shell of iron (oxy)hydroxide, and the removal efficiency reached 80%, which could show high adsorption affinity. In contrast, the removal efficiency of Zn2+ was only 40% under oxygen-limiting condition. The removal efficiency of Zn2+ increased with the increasing of the pH. Acidic condition reduced the removal efficiency of Zn2+ by nZVI because the existing H+ inhibited the formation of iron (oxy)hydroxide. The higher removal efficiency was observed in the experiment with an initial pH value of 5. Adsorption and co-precipitation were the most likely mechanism of Zn2+ removal by nZVI. The FeOOH-shell could enhance the adsorption efficiency of nZVI. The removal extent of Zn2+ was much higher than that of Cd2+. The removal efficiency and selectivity of nZVI particles for Zn2+ were higher than Cd2+. Furthermore, a continuous flow reactor for engineering application of nZVI was designed and exhibited high removal efficiency for Zn2+. The maximum removal efficiency was up to more than 95%, and the removal efficiency was steady after a rapid increase in the first 30 min.This study demonstrated that the uptake of Zn


The neural transcription factor SOX11 has been described as a prognostic marker in epithelial ovarian cancers (EOC), however its role in individual histological subtypes and tumour grade requires further clarification. Furthermore, methylation-dependent silencing of SOX11 has been reported for B cell lymphomas and indicates that epigenetic drugs may be used to re-express this tumour suppressor, but information on SOX11 promoter methylation in EOC is still lacking.2 test in a cohort of 154 cases of primary invasive EOC. Kaplan-Meier analysis and the log rank test were applied to evaluate ovarian cancer-specific survival (OCSS) and overall survival (OS) in strata, according to SOX11 expression. Also, the methylation status of the SOX11 promoter was determined by sodium bisulfite sequencing and methylation specific PCR (MSP). Furthermore, the effect of ectopic overexpression of SOX11 on proliferation was studied through [3H]-thymidine incorporation.SOX11 expression and clinicopathological data was compared using χSOX11 expression was associated with an improved survival of patients with high grade EOC, although not independent of stage. Further analyses of EOC cell lines showed that SOX11 mRNA and protein were expressed in two of five cell lines, correlating with promoter methylation status. Demethylation was successfully performed using 5'-Aza-2'deoxycytidine (5-Aza-dC) resulting in SOX11 mRNA and protein expression in a previously negative EOC cell line. Furthermore, overexpression of SOX11 in EOC cell lines confirmed the growth regulatory role of SOX11.SOX11 is a functionally associated protein in EOC with prognostic value for high-grade tumours. Re-expression of SOX11 in EOC indicates a potential use of epigenetic drugs to affect cellular growth in SOX11-negative tumours. EOC is a heterogeneous disease compromising many histological subtypes including clear cell, mucinous, endometrioid and serous carcinoma, which are sub-classified into high- and low-grade . Differein vitro models for functional analyses, EOC cell lines were screened for SOX11 expression and promoter methylation was assessed in both positive and negative cell lines. To verify that methylation is a key event in silencing SOX11, 5-Aza-dC treatment was used to re-express SOX11 in an in vitro model of EOC. Furthermore, the tumour suppressor function of SOX11, as previously reported for B cell lymphomas -thymidine incorporation. 15000 cells were seeded in triplicates onto 96-well plates, left for a couple of hours for the cells to attach, then [3H]-thymidine  was added and the cells were incubated for 8-16 hours. Cells were then frozen and finally thawed and harvested in a Tomtec Harvester 96  and analysed with a 1450 MICROBETA liquid scintillation counter . Transfection efficiency was 40-90%, measured as GFP positive control cells in flow cytometry (data not shown). Both mRNA and protein extracts were analysed by qRT-PCR and western blot to confirm an overexpression of SOX11. Furthermore, the proliferation of the cells was assessed by thymidine incorporation. The level of overexpression at mRNA level varied between a 100-fold (A2780) to a 50000-fold (A2780-CP7). For most cell lines the increase in mRNA was also translated into an overexpression of the SOX11 protein. However, the 1000-fold increase of mRNA in OVCAR-3 only resulted in a limited increase of SOX11 protein Figure . Using lin vitro models could not be performed as the majority of these are poorly characterized. Some of the cell lines we used have previously been xenografted into nude mice to analyse the resulting pathologies [SOX11 has previously been identified as a prognostic antigen in EOC, where SOX11 expression was associated with a prolonged recurrence free survival . In thishologies , but no As previously shown for B cell lymphoma cell lines , the proOf interest, the clinical outcome of patients afflicted by EOC has been associated to differential expression of cell cycle regulatory proteins including Cyclin D1, Cyclin E, p16-INK and E2F transcription factors . We havein vitro confirmed a growth regulatory role for SOX11 in EOC.In the present study, SOX11 was demonstrated to be of prognostic value for high grade EOC, which could have a clear clinical value. The possibility to re-express SOX11 indicates a potential use of epigenetic drugs to affect cell growth through common cell regulatory pathways, controlled by SOX11, and other tumour suppressors that are silenced in EOC. Furthermore, functional investigations A patent has been filed on the use of SOX11 as a diagnostic and prognostic antigen in EOC.SS performed experimental work, including overexpression studies, analysis of the cell lines and took active part in writing the manuscript. EG performed experimental work in relation to promoter methylation analysis and took active part in writing the manuscript. DJB was responsible for the design of the automated SOX11 classification, interpretation of the data and involved in the completion of the manuscript. WMG and ER were involved in the design of the algorithm for automated IHC classification. FR performed experimental work in relation to overexpression of SOX11. KJ provided the tumour material and clinical data and was involved in the completion of the manuscript. CB was involved in the design of the study and the completion of the manuscript. SE was responsible for the design of the study, interpretation of data and writing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/405/prepub


The Hedgehog Signaling Pathway is one of signaling pathways that are very important to embryonic development. The participation of inhibitors in the Hedgehog Signal Pathway can control cell growth and death, and searching novel inhibitors to the functioning of the pathway are in a great demand. As the matter of fact, effective inhibitors could provide efficient therapies for a wide range of malignancies, and targeting such pathway in cells represents a promising new paradigm for cell growth and death control. Current research mainly focuses on the syntheses of the inhibitors of cyclopamine derivatives, which bind specifically to the Smo protein, and can be used for cancer therapy. While quantitatively structure-activity relationship (QSAR) studies have been performed for these compounds among different cell lines, none of them have achieved acceptable results in the prediction of activity values of new compounds. In this study, we proposed a novel collaborative QSAR model for inhibitors of the Hedgehog Signaling Pathway by integration the information from multiple cell lines. Such a model is expected to substantially improve the QSAR ability from single cell lines, and provide useful clues in developing clinically effective inhibitors and modifications of parent lead compounds for target on the Hedgehog Signaling Pathway.In this study, we have presented: (1) a collaborative QSAR model, which is used to integrate information among multiple cell lines to boost the QSAR results, rather than only a single cell line QSAR modeling. Our experiments have shown that the performance of our model is significantly better than single cell line QSAR methods; and (2) an efficient feature selection strategy under such collaborative environment, which can derive the commonly important features related to the entire given cell lines, while simultaneously showing their specific contributions to a specific cell-line. Based on feature selection results, we have proposed several possible chemical modifications to improve the inhibitor affinity towards multiple targets in the Hedgehog Signaling Pathway.Our model with the feature selection strategy presented here is efficient, robust, and flexible, and can be easily extended to model large-scale multiple cell line/QSAR data. The data and scripts for collaborative QSAR modeling are available in the Additional file  The Hedgehog Signaling Pathway plays an important role in regulating embryonic development in vertebrates, and it is highly conserved from flies to humans 2]3]4][3][4]2][[4][3][4][3][4]2]. The misThe Hedgehog Signal Pathway is composed of four important components: Sonic Hedgehog, Patched, Smoothened and Gli transcription factors  Figure . The funThe causal relationship between the activation of Hedgehog Signaling Pathway and oncogenesis has driven cancer researchers in the direction of finding specific inhibitors of hedgehog signaling, since this will provide efficient therapies to a wide range of malignancies ,2. To daRecently, our partners have synthesized 93 cyclopamine derivatives and their activities were tested against four different cell lines  respectively . Based o(1) In our previous QSAR study, for specific cell lines, the activities were categorized into a binary classification under a naïve Bayesian model, and we obtained relatively acceptable QSAR results. However, no matter what kinds of statistical models or 2D descriptors were tested, low testing correlation coefficients were found when numeric activities were used. This may be due to the inherent noise existed in experimental activity measurement, or the relatively small number of training data used for a specific cell line. Due to our compound data tested against multiple cell lines to evaluate their activities, we hypotheses that such information can be integrated to improve the QSAR results rather than only a single cell line QSAR modeling. Such investigation will be extremely useful for the scenario that a small number of compound activities are measured under different experimental conditions , and will provide novel insights on the integration of existing information, avoiding repeatable laborious work in drug discovery. In addition, such a study may also lead to novel computational models for integrated QSAR modeling, which is closely related to multi-task QSAR modeling 10], Mul, Mul10],(2) Due to the existence of compound activity data against multiple cell lines, how can we integrate such information to derive more robust and efficient feature selection strategies for compound modification under such “collaborative” multi-cell line environment? That is, can we derive the commonly important features related to the entire given cell lines for compound description, while in the meantime present their specific contributions to a specific cell-line? This issue is closely related to the first one, but tougher to be solved since it needs much more domain knowledge.Inspired by these two problems, we aim to develop an efficient integrated QSAR model for inhibitors of Hedgehog Signal Pathway against multiple cell lines. This type of model has been used for information retrieval in social network, i.e. collaborative filtering 14], and, and14],PKi, as defined in the following Cheng-Prusoff equation  a-27 a25-2As we mentioned above, Erhan etc. proposed a neural network based collaborative QSAR modeling for drug discovery . This isSpecifically, in our multiple cell line QSAR modeling for the inhibitors of Hedgehog Signaling Pathway, we used a 10-cross fold validation schema to test our data from 4 cell lines in this neural network model. The weights from input layer to hidden layer as well as from hidden layer to output layer for the network will be learned through the back propagation (BP) algorithm. Our in-house test indicated that the neural network based collaborative QSAR modeling was comparable to CMF based QSAR modeling, with no surprisingly better than the single QSAR modeling (Results are not shown here).N tuples, N}, where M} is the indicator corresponding to the example M tasks correspond to M different cell lines or drug targets. A critical issue in this collaborative QSAR modeling is to learn a set of sparse functions across these tasks for drug activity regression. This is commonly achieved by learning M linear regressions of the form Neural network can be viewed as a specific form of multi-task learning. Multi-task learning has been developed for those situations where multiple related learning tasks are to be accomplished together. When explicit or hidden interrelationship among the tasks can be exploited 10], mul, mul10],Wjth row of W.whereW can be optimized and calculated by enforcing the joint sparsity across different tasks with adding the different norm of the matrix W to the square loss function, which leads to only a few non-zero rows of W.In the multi-task learning framework, The relationship between collaborative filtering and multi-task learning has been discussed in previous studies . The mulFor the particular data in our multiple cell line QSAR modeling for the inhibitors of Hedgehog Signaling Pathway, it can be seen that the drug samples for all the cell line are totally identical, thus it is unnecessary to use multi-task learning in the collaborative QSAR here. Nevertheless, if non-identical samples for multiple cell line exist, multi-task learning will be a good choice for collaborative QSAR modeling with integrating of different data sources.Proteochemometric modeling (PCM) is presented based on the similarity of a group of ligands and a group of targets, to the extent that PCM models the so-called ligand-target interaction space 29]. Lik. Lik29].In this study, an efficient collaborative QSAR model for inhibitors of Hedgehog Signal Pathway from multiple cell lines was proposed. The model is derived from the area of information retrieval in social network, i.e. collaborative filtering, and its performance is well demonstrated and explained in our study. By applying this elegant computational model, we successfully addressed two issues remained in our previous study, i.e., (1) The information among multiple cell lines can be integrated to boost the QSAR results, rather than single cell line QSAR modeling. Our extensive experiments indicated that the performance is remarkable compared to other single cell line QSAR methods. (2) A novel feature selection strategy under such collaborative environment was proposed, which can be used to derive the commonly important features related to the entire given cell lines, while meantime presenting their specific contributions to a specific cell-line. Based on the results of feature selection, we presented several ways of chemical modifications which will likely improve the compound affinity towards multiple targets in the Hedgehog Signal Pathway simultaneously. In summary, our study provides useful clues for multiple cell line/targets QSAR modeling when the cell line or target information among a related pathway exist. The proposed collaborative model with the feature selection strategy here is efficient, robust, flexible, and can be easily extended to model large-scale multiple cell line/QSAR data.The authors declare that they have no competing interests.QL and JG carried out the designing of the whole computational algorithm and drafted the manuscript. JG and DC were responsible for the algorithm implementation. DC, VZ and RZ were responsible for the algorithm design. QL conceived the study and participated in the design and coordination of the analyses. All authors read and approved the final manuscript.The data and scripts for collaborative QSAR modeling.Click here for fileTable S1. Detailed explanations of each Drug-like index.Click here for file


The synthesized compounds were evaluated for their anticancer activity in NCI60 cell lines. Among the tested compounds, 3f was found to be the most active candidate with the greatest influence on leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, prostate cancer, and breast cancer subpanel cell lines with GI50 values over a range of 0.37–0.67 μM. It is important to note that these derivatives were highly active towards pathogenic isolates of Trichoderma harzianum, Penicillium simplex, Candida albicans, Mucor hiemalis, Aspergillus oryzae, Actynomices sp., A. fumigatus at the level of fluconazole thiopyranothiazoles 1a–c were obtained in the Knoevenagel-hetero-Diels-Alder reaction via isorhodanine (4-thioxo-2-thiazolidinone) and appropriate 2-allyloxy-benzaldehydes interaction in acetic medium at room temperature (1H NMR of the 1a–c trans annulated cycloadducts were formed. The configuration of 1a–c was deduced from the coupling constant (J = 10.5 Hz) between the 5a and 11b protons thiopyranothiazole-2-one 5 was synthesized  for in vitro cell line screening. Anticancer assays were performed according to US NCI protocol, which was described elsewhere thiopyrano–thiopyranothiazoles except 3n and 3p showed significant growth inhibition; the average value of logGI50 ranges from −4.49 to −6.22  caused inhibition of cell growth of leukemia lines CCRF-CEM and HL-60(TB) in submicromolar concentrations .Analyzing the results of a detailed to −6.22 . The higd]thiazoles. Results of our work may be used for planning the directed synthesis of potential anticancer agents.Functionalization of the position N3 of basic heterocycle structure by introduction of acetamide molecular fragments is the most effective direction for anticancer profile optimization of chromenothiopyrano, the percentage growth was calculated at each of the drug concentration levels. Percentage growth inhibition was calculated as: [(Ti − Tz) / (C − Tz)] × 100 for concentrations for which Ti ≥ Tz, [(Ti − Tz) / Tz] × 100 for concentrations for which Ti < Tz. Three dose response parameters were calculated for each compound. Growth inhibition of 50% (GI50) was calculated from [(Ti − Tz)/(C − Tz)] × 100 = 50, which is the drug concentration resulting in a 50% lower net protein increase in the treated cells (measured by SRB staining) as compared to the net protein increase seen in the control cells. The drug concentration resulting in total growth inhibition (TGI) was calculated from Ti = Tz. The LC50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment was calculated from [(Ti − Tz) / Tz] × 100 = -50. Values were calculated for each of these three parameters if the level of activity is reached; however, if the effect was not reached or was exceeded, the value for that parameter was expressed as greater or less than the maximum or minimum concentration tested. The logGI50, logTGI, logLC50 were then determined, defined as the mean of the logs of the individual GI50, TGI, LC50 values. The lowest values are obtained with the most sensitive cell lines.The cytotoxic and/or growth inhibitory effects of the most active selected compounds were tested in vitro against the full panel of about 60 human tumor cell lines at 10-fold dilutions of five concentrations ranging from 10


To determine if a human bladder cancer-specific peptide named PLZ4 can target canine bladder cancer cells.In vivo tumor-specific homing/targeting property and biodistribution of PLZ4 was performed in a mouse xenograft model via tail vein injection and was confirmed with ex vivo imaging.The binding of PLZ4 to five established canine invasive transitional cell carcinoma (TCC) cell lines and to normal canine bladder urothelial cells was determined using the whole cell binding assay and an affinitofluorescence assay. The WST-8 assay was performed to determine whether PLZ4 affected cell viability. in vivo and ex vivo optical imaging study showed that, when linked with the near-infrared fluorescent dye Cy5.5, PLZ4 substantially accumulated at the canine bladder cancer foci in the mouse xenograft model as compared to the control.PLZ4 exhibited high affinity and specific dose-dependent binding to canine bladder TCC cell lines, but not to normal canine urothelial cells. No significant changes in cell viability or proliferation were observed upon incubation with PLZ4. The PLZ4 can specifically bind to canine bladder cancer cells. This suggests that the preclinical studies of PLZ4 as a potential diagnostic and therapeutic agent can be performed in dogs with naturally occurring bladder cancer, and that PLZ4 can possibly be developed in the management of canine bladder cancer. Current in vivo ,3. Bladd in vivo . Therefo in vivo .6 cells/ml single-cell suspensions of five different canine invasive TCC cell lines including K9TCC-PU, K9TCC-PU-AxA, K9TCC-PU-In, K9TCC-PU-AxC, and K9TCC-PU-Nk  [To determine the binding of PLZ4 to canine invasive TCC cell lines, a whole cell binding assay was performed  . Human b® 488 conjugate  at 1:1000 dilution per manufacturer's protocol. After washing, the slides were mounted with DAPI-containing medium for nuclear staining (blue), and observed under inverted fluorescence microscope (200 X). All five canine TCC cell lines showed diffuse cell membrane staining  were 21.3 and 10.3 μM, respectively, while the Kd50 value for human 5637 cells was 6.67 μM.To further quantify the binding affinity, K9TCC-PU and K9TCC-PU-In cells were seeded in 96-well plates, fixed with acetone, and incubated with increasing concentrations of PLZ4-biotin followed by SA-HRP. As shown in Figure Ligand binding to cell surface molecules may initiate cell signaling and exert biological effects on cells. Here, we determined the effect of PLZ4 on cell viability and proliferation, as these PLZ4-mediated effects may have potential clinical applications. K9TCC, K9TCC-PU-In and K9TCC-PU-Nk cells were seeded in 96-well plates and incubated without or with various concentrations of PLZ4 for 48 hours. The WST-8 cell proliferation assay was performed per manufacturer's protocol . There were no significant changes in cell proliferation/viability in these three cell lines cultured with different concentrations of PLZ4 when compared to the control cells treated with PBS Figure .in vivo biodistribution/binding specificity of PLZ4 in a mouse model with canine TCC xenografts. The near-infrared fluorescent dye Cy5.5 was used for in vivo and ex vivo imaging. PLZ4-biotin-SA-Cy5.5 (PLZ4-Cy5.5) complex or SA-Cy5.5 dye (control), 100 μl (6 nmol) for each mouse, was injected via tail vein into mice carrying TCC-PU-In xenografts at 0.5-0.8 cm in diameter. Substantial accumulation of signals was observed at the tumor site in the mice injected with PLZ4-Cy5.5 complex in a time-dependent manner with a maximum signal observed at 12 hours  and kidney   Figure . No signin vitro and in vivo  in xenograft models, the local vasculature formation and permeability can be dramatically different from that of naturally occurring cancer that usually take months to years to develop. Here we showed that PLZ4 can bind to canine bladder cancer cells both  Figures  and 3. C Figures . Our finOne major application of PLZ4 is local visualization of bladder cancer during transurethral resection of bladder cancer (TURBT). It is clinically relevant since incomplete resection is seen in up to one third of cases after TURBT that contributes to the high recurrence of bladder cancer after therapy . Fluoresin vivo targeting and biodistribution study showed that PLZ4 specifically concentrated at the tumor sites, but not to other sites including bladder (Figure in vivo studies (Figure in vivo targeting with PLZ4 conjugates.Our r Figure . Therefos Figure , only 6 In summary, the human bladder cancer-specific peptide PLZ4 can bind to canine bladder cancer cells, suggesting that the preclinical studies of PLZ4 can be performed in dogs with naturally occurring bladder TCC.TCC: Transitional cell carcinoma; TURBT: transurethral resection of bladder cancer; SA: streptavidin; SI: Supplement information; ALA: 5-Aminolevulinic acid; MRI: magnetic resonance imaging; PET/SPECT: positron emission tomography/single photon emission computed tomography; PLZ4: the name of the bladder cancer-specific ligand.Bin Li has signed an option with UC Davis to license the patent of PLZ4 for commercial development. We have no other financial competing interests to disclose.in vivo and ex vivo imaging and biodistribution studies, and analyzed the data. LX and BL: Initiated this project, conducted the whole bead cell binding and first affinitofluorescence staining. COR: collected canine tissue and helped in the determination the binding of PLZ4 to normal canine urothelial cells and cells with chronic cystitis. RDVW: design and supervision of the overall project, data analysis and manuscript preparation. CP: principal investigator of this project, contributed to conception and design of the project, manuscript preparation and final approval. All authors read and approved the final manuscript.TL: carried out the affinitofluorescence, binding affinity, and biological effects of PLZ4 on canine TCC cells, data acquisition and analysis, and manuscript preparation. HZ: developed PLZ4 ligands and contributed to the conception and design of the experiments. SW: conducted the TL, HZ, SW, LX and BL are current or former trainees of Dr. Pan's lab. COR is a Veterinary Medical oncologist and a Continuing Lecturer. RDVW is the director of the UC Davis Cancer Center and Professor of Urology. CP received 4 years of residency training in Urology in China, finished residency and fellowship training and is board-certified in Internal Medicine, Hematology and Oncology in US. He also had PhD in Microbiology & Immunology. Currently, he is a physician-scientist and an Assistant Professor at the Division of Hematology/Oncology, Department of Internal Medicine, UC Davis Medical Center.Materials and Methods. A detail description of the materials and methods for this publication.Click here for file


Chinese Hamster Ovary (CHO) cells are the most widely used industrial hosts for the production of recombinant DNA technology drugs . In suchSolute Carriers (SLC) database [There are about 46 a.a. transporter proteins in mammalian cells, the genes of which are presented in Table database . A.a. trTo our knowledge, there is no comprehensive study of a.a. transporters in industrially relevant CHO cells in the literature. To that direction, a.a. transporter genes were profiled during batch culture of three CHO cell lines with varying levels of productivity. In parallel, the intra- and extracellular levels of a.a. were quantified.Three cell lines were kindly donated by Lonza Biologics. GSn8 cell line was transfected with an empty glutamine synthetase (GS) vector. GS35 and GS46 cell lines were both transfected with a GS vector that also carries the heavy and light chains of a chimeric IgG4 antibody. The specific productivity of cell line GS46, quantified by a commercial ELISA kit , is approximately double that of GS35 one.Batch cultures were performed in triplicate in 1L Erlenmeyer flasks with a working volume of 300mL in CD-CHO medium  supplemented with 25 μM MSX . Viable cell concentration was determined daily using the trypan blue dye exclusion method.40 a.a. transporters were studied in all cell lines using real time quantitative reverse transcription polymerase chain reaction on samples from different phases of batch culture. Samples were collected at day 4  and day 6 & day 7 (stationary phase) of the growth curve for all cell lines . Results are reported against the housekeeping gene "actb". Housekeeping genes "vezt" and "hirip3" were also well correlated.The extracellular and intracellular a.a. profiles were monitored daily using high performance liquid chromatography . Intracellular samples were quenched with 0.9% w/v NaCl and extracted with a 50% aqueous acetonitrile solution, as described in .The results Table  reveal tThe majority of the transporters are found to be upregulated at stationary phase for all cell lines, as also presented in Figure -AG, glutamate and aspartate). However, no genes, apart from slc6a6 (taurine and b-Ala), were found to be differentially expressed between high (GS46) and low producer (GS35). We find slc6a6 gene differentially expressed early in cell culture (day 3), which makes us hypothesize that the gene could be a candidate for selection purposes. The overexpression of this gene in CHO cells has been found to significantly enhance growth and productivity [In their majority, genes were found to be upregulated for protein producing cell lines at all time points. Genes whose expression is upregulated significantly (two-fold or higher) in the protein-producers at all time points analyzed were: slc43a2  and slc1a2  can be targeted as genetic engineering targets, since are all found highly upregulated at stationary phase of cell culture. Additionally, transporters slc1a2, slc43a2 are associated with rprotein productivity, since all of them are found to be upregulated for producing cell lines vs the null. Gene slc6a6, carrying taurine and β-alanine, can be associated with high productivity (as also suggested in [


Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies.Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders.in vitro and in vivo after transplantation into regenerating muscle of immunodeficient mice.The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both in vivo the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess  Furthermore, not every mutation can be created and evaluated in murine models, and mechanisms common to aging and dystrophies may differ between mice and humans. Consequently, human primary myoblasts isolated from dystrophic patient biopsies provide the most pertinent experimental models to assess a variety of human genetic mutations in their natural genomic environment. Although in vitro models do not fully recapitulate the in vivo environment, cell-culture systems allow rapid, high-throughput screening of molecules or oligonucleotides, and new strategies can be easily tested prior to validation in animal models, which is a costly and time-consuming process. The main drawbacks of using in vitro primary cultures of human cells derived from muscle biopsies are their purity, their limited proliferative capacity, and the variation in phenotype when amplified in vitro; their phenotype will always be confounded by modifications due to cellular senescence, which will progressively occur during cell amplification [Muscular dystrophies constitute a heterogeneous group of genetic muscle diseases characterized by progressive muscle weakness, wasting and degeneration, some of these features are common to muscle aging ,2. Over fication ,8.human telomerase reverse transcriptase; hTERT) cDNA alone will result in an extension of the lifespan and even immortalization in a variety of cell types, including endothelial cells and fibroblasts [hTERT and cyclin-dependent kinase (CDK)-4 is required to successfully overcome cellular senescence in human myoblasts [INK4a-dependent stress pathway.The two major mechanisms responsible for this replicative cellular senescence seen in human myoblasts are (i) activation of the p16-mediated cellular stress pathway, and (ii) the progressive erosion of telomeres at each cell division until they reach a critical length that will trigger p53 activation and cell-cycle exit ,10. Intrroblasts ,12. Howeyoblasts ; while hIn the present study, our goal was to create a large collection of immortalized human myoblasts isolated from a wide range of neuromuscular disorders , oculopharyngeal muscular dystrophy (OPMD), limb-girdle muscular dystrophy (LGMD2B or dysferlinopathy) and congenital muscular dystrophy (CMD)), which could be used as experimental tools to study these diseases and to develop new therapeutic strategies.n triplet expansion in the poly(A) binding protein nuclear 1 (PABPN1) gene [DMD is the most common childhood muscular dystrophy. It is caused by mutations in the dystrophin gene encoding an essential protein of the muscle membrane cytoskeleton , leadingN1) gene , which aN1) gene  and trafN1) gene .in vitro models that can also be implanted in vivo. This non-exhaustive list of cellular models will provide powerful and valuable tools for the scientific community investigating these pathological conditions and/or their mechanisms. as they overcome the problem of limited proliferation usually present in myoblasts. These models should also be useful in the development of gene or cell therapies and pharmacological strategies for muscular dystrophies, some of which might also be used to combat muscle weakness in the elderly.In this study, we report for the first time that for each of these muscular dystrophies, we were able to produce reliable and stable immortalized cell lines from human myoblasts isolated from biopsies, resulting in robust Muscle biopsies Table  were obtHuman myoblasts were isolated from biopsies and cultivated as described previously  in a grohTERT and Cdk4 cDNA were cloned into different pBABE retroviral vectors containing puromycin and neomycin selection markers, respectively. Infection was carried out as described previously [eviously . TransduGenomic DNA was extracted from each proliferating cell line using a salting-out procedure. Telomere length was determined by using a quantitative (q)PCR method, as previously described ,22. PCR To analyze the expression of myogenic markers in proliferating primary and immortalized cell lines, 1 μg RNA from each cell line was used for the cDNA synthesis  using random hexamer primers. cDNA (1 μl) was used as a template for PCR using N-CAM, MyoD and desmin specific primers. The primer sequences and detailed PCR protocols used are available on request.-/- γC-/- C5-/- mice aged 2 to 3 months were anesthetized with an intraperitoneal injection of ketamine hydrochloride (80 mg/kg) and xylasin (10 mg/kg) (Sigma-Aldrich). To induce severe muscle damage and trigger regeneration, the recipient tibialis anterior (TA) muscles were exposed to cryodamage, and a single injection of immortalized human cells (15 μl of cell suspension containing 2.5 × 105 or 5 × 105 cells in PBS) was administered as described previously [Immunodeficient Rag2eviously . Four weIn vitro and in vivo characterizations were performed by immunolabeling as described previously [eviously -25. AntiCells were seeded in six-well plates and grown in growth medium. Transfection of antisense oligonucleotides (AONs) was performed using 1 μl of transfection reagent  per μg of AONs for 4 hours. The chemistry used for AONs was 2'-O-methyl-phosphorothioates. All transfections were performed with at least two independent duplicates. Cells were changed to differentiation medium before transfection. Typically 24 to 48 hours after transfection, RNA was extracted from the cells using Trizol or Qiagen column kit . 1 μg RNA was used for the cDNA synthesis  with DMD exon-specific primers. cDNA (2 μl) was used as a template for a first PCR reaction. From this first reaction of 25 cycles, 1 μl of the product was removed and used as a template for a second nested PCR of 35 cycles. PCR products were analyzed on 1.5 to 2% agarose gels. The primer sequences and detailed PCR protocols used are available on request.hTERT and CDK-4 cDNA. Co-transduced cells were selected by neomycin and puromycin and then purified using magnetic beads coupled to antibodies directed against the myogenic marker CD56. Following culture at clonal density, individual myogenic clones with extended proliferative lifespans, as compared to the untranduced cells, were isolated from each population. In contrast to the parental populations, which stopped proliferating at various stages of the culture, depending on the type of dystrophy, the selected immortalized clones were still able to proliferate after prolonged amplification in vitro under the same culture conditions  and MyoD Figure .In addition, we also tested their ability to differentiate into myotubes, using immunostaining with MF20 antibody, which recognizes all skeletal-muscle myosin heavy chains (MyHCs). After 5 days in differentiation conditions, all of the immortalized cell lines were able to fuse into myotubes expressing MyHCs Figure .-/- γC-/- C5-/- mice; injected muscles were analyzed 4 weeks after transplantation, which corresponds to a complete fiber regeneration process, using antibodies specific for human lamin A/C  and human spectrin (expressed in differentiated fibers). For each injected clone , mature muscle fibers containing human spectrin protein and human lamin A/C+ nuclei were seen  and spectrin (specific to the human protein) to identify fiber sarcolemma, we investigated if these cell lines could replenish the muscle stem-cell niche , at the periphery of the muscle fiber and beneath the basal lamina. Whereas the vast majority of the lamin A/C-positive nuclei (97%) were found as myonuclei , and responses to potential therapeutic strategies. The relatively short proliferative lifespan of human myoblasts, reduced even more in dystrophic conditions by successive cycles of degeneration/regeneration nescence -28, limiin vitro and in vivo after transplantation into the regenerating muscles of immunodeficient mice. We found that these human myoblast cell lines expressing myogenic markers could colonize the host muscles and form mature fibers, thus providing an ideal model to assess therapeutic strategies in vivo, which is closer to bona fide differentiation of muscle stem cells than the converted fibroblasts described previously [Immortal cell lines have been generated from human skeletal muscle, such as those derived from rhabdomyosarcomas, a rare form of skeletal-muscle tumor. However, these lines often have impaired fusion characteristics and perturbed myogenic programs ,30. Otheeviously . We alsoeviously , but arein vivo implantation of these cells offers the possibility to investigate the consequences of defined mutations on cellular behavior in vivo, particularly with regard to their regenerative capacity. Finally, the development of therapeutic strategies, whether these strategies imply gene, cell or pharmacological therapy involving high-throughput screening, is facilitated using these tools before assessment in animal models. As an example, we have described a rapid screen of a range of AONs in an exon-skipping strategy for DMD. In conclusion, the co-transduction of hTERT and CDK4 generates human myoblasts immortal in culture that maintain myogenic potential both in vitro and in vivo, offering new cell paradigms for pathophysiological studies and novel therapeutic strategies.These human myoblast lines represent a powerful tool to assess signaling and/or functional deregulations in neuromuscular diseases, particularly those in which these mechanisms have not yet been clearly elucidated  or those with features common to muscle aging, such as atrophy or muscle wasting. We have described only a subset of the cell lines produced; we have now generated more than 35 cell lines with various mutations covering a range of 14 different pathologies, as well as cell lines from control subjects of various ages. The AONs: antisense oligonucleotides; CDK-4: cyclin-dependent kinase 4; CMD: congenital muscular dystrophy; DMD: Duchenne muscular dystrophy; DMEM: Dulbecco's modified Eagle's medium; FCS: fetal calf serum; FSHD: facioscapulohumeral muscular dystrophy; hTERT: human telomerase reverse transcriptase; LGMD2B: limb-girdle muscular dystrophy type 2b; N-CAM: neural cell adhesion molecule; OPMD: oculopharyngeal muscular dystrophy; PBS: phosphate-buffered serum; PCR: polymerase chain reaction.The authors declare that they have no competing interests.in vitro and in vivo experiments, analyzed data, and wrote the manuscript. AW, PKK, SM, JK, and AA provided technical support. JDS provided the immunodeficient Rag2-/- γC-/- C5-/- mice for the in vivo experiments. JLSG, FM, SP, SS, NL, SB, and TV provided biopsies, and WEW provided DNA constructs. TV and AA discussed the results and gave expert advice. GBB and VM provided conceptual input and supervision. and wrote the manuscript. All authors read and approved the final manuscript.KM, CT, AB, EN, and SC designed and performed the


Half-maximal inhibitory concentration (IC50) of the different chemotherapeutic agents was obtained from an experimentally derived dose-response curve. Relative potency of the drugs was estimated according to Parallel Line assay. This study demonstrated that the selected generic oncology products tested had similar efficacy compared with the originator. Both products showed comparable results as shown both in vitro cytotoxicity assay and statistical analysis. In vitro cell-based cytotoxicity assay promises to be a useful, reliable and rapid method for demonstrating chemotherapeutic drug activity.Chemotherapy is the main treatment modality for certain types of cancer. It is important to monitor and ensure that these chemotherapeutic drugs are potent and effective prior to patient administration. The objective of the study is to evaluate the cytotoxic activity and potency of selected commercially available generic anticancer drugs in comparison with originator using various human cancer cell lines in an  In vitro cell-based assays have been developed to rapidly determine the cytotoxic activity of several compounds. Cell-based assays are also useful in identifying variations in susceptibility of different target cells to several chemotherapeutic agents  with Earle’s salt and nonessential amino acids, supplemented with 5% heat-inactivated fetal calf serum (FCS), 1 mM sodium pyruvate, and 2 mM L-glutamate. NCI-H2126 cells were grown on HITES medium supplemented with 5% fetal bovine, 0.005 mg/mL insulin, 0.01 mg/mL transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM beta-estradiol, 10 mM HEPES, and 2 mM L-glutamine. SKOV-3 cells were grown on McCoy’s 5a medium supplemented with 10% fetal bovine serum. PC-3 cells were grown on F12-K medium supplemented with 10% fetal bovine serum. All cells were grown at 37°C in 95% air with the addition of 5% COMTT Assay. The details of this assay have been described previously . The cytotoxicity index was determined using the untreated cells as negative control. The percentage of cytotoxicity was calculated using the background-corrected absorbance follows  was determined by plotting triplicate data points over a concentration range and calculating values using regression analysis of PRISM program.The ICPLA version 1.2.06)  , 6. Calc , 6. Calin vitro evaluation of the cytotoxic potency of drugs. In the present study we applied the MTT test to evaluate the potency of selected commercially available generic anticancer drugs in comparison with originator using various human cancer cell lines in an in vitro cell-based assay.Metabolic activity can be evaluated by measuring the activity of a mitochondrial enzyme succinate dehydrogenase using MTT test. MTT is designed for the quantification of cytotoxic index in cell population using 96 well plate format. This test is widely used in the The cytotoxic response of the different cell lines to different generic oncology products versus the originator is shown in Figure PLA version 1.2.06) and both were found to be comparably cytotoxic and potent. A value of 1 (± 0.2) means that the relative potency of the tested product is almost the same as that of the competitor product  for measuring cellular proliferation (cell growth) , 8. The 50 is the concentration of the tested drug able to cause the death of 50% of the cells and can be predictive of the degree of cytotoxic effect. The lower the value, the more cytotoxic is the substance. Figure 50 of some chemotherapeutic drugs against human cancer cell lines.MTT measures cell respiration and the amount of formazan produced is proportional to the number of living cells present in culture. An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by the drug. ICin vitro drug response testing is that it can provide the knowledge of the relative efficacy of the various agents used in standard therapy before an empiric in vivo trial. Cell-based assays may help in the selection of chemotherapeutic drugs with the greatest likelihood for clinical effectiveness, and in the exclusion of ineffective therapy. This can lead to improved disease management, response, survival and use of financial resources. This study demonstrated that selected generic oncology products tested has similar efficacy compared with originator. Both products showed comparable results as proven by both in vitro cytotoxicity assay and statistical analysis.The MTT-based assay relies upon the cellular reduction of tetrazolium salts to their intensely colored formazans. The test is easy to perform in hematological malignancies and is adaptable for high throughput of samples, although there are some minor limitations in its application resulting from metabolic interference. This class of assay is highly accurate for predicting drug resistance, whereas its predictive value for drug sensitivity depends on the type of disease and drug or drug combination used . They ha