S3 Cells Research Articles

Induction of apoptosis in S180 tumour bearing mice by polysaccharide from Lentinus edodes via mitochondria apoptotic pathway

JLNT, a purified polysaccharide with the structure of β-(1→3)-D-glucose main chains and β-(1→6)-D-glucose side chains and with an average molecular weight of 605.4 kDa, was extracted and isolated from the fruitbodies of Lentinus edodes, which has immunoregulatory activity and anti-tumour effects. In the present study, we found that JLNT possessed direct anti-tumour effects on MCF-7 and S180 cells and apoptosis -inducing activity in S180 tumour bearing mice. Therefore, we investigated the mechanisms of JLNT-induced apoptosis. Results showed that JLNT increased the expression of p53, enhanced the Bax/Bcl-2 ratios by up-regulating Bax and down-regulating Bcl-2, promoted the expression of Smac, leading to the activation of caspase-3 and finally the initiation of cell apoptosis in S180 tumour bearing mice. Taken together, these findings provide the experimental evidence that JLNT could induce apoptosis of S180 cells via mitochondria pathways.

Mechanistic basis of a combination d-penicillamine and platinum drugs synergistically inhibits tumor growth in oxaliplatin-resistant human cervical cancer cells in vitro and in vivo

The platinum-based regimen is the front-line treatment of chemotherapy. However, development of platinum resistance often causes therapeutic failure in this disease. We previously have generated an oxaliplatin-resistant subline, named S3, from human cervical carcinoma SiHa cells, and its resistant phenotype was well-characterized. In the present study, we aimed to identify the novel therapeutic strategy by combining copper chelator d-penicillamine with oxaliplatin, and to elucidate the underlying mechanisms for overcoming oxaliplatin resistance. As the result, d-penicillamine exerted synergistic killing effects only in S3 cells when combined with oxaliplatin and cisplatin by using Chou–Talalay method. Further study showed that the amounts of platinum DNA adduct formed were positively correlated to the percentage of cell death in S3 cells when co-treated d-penicillamine with oxaliplatin and cisplatin. d-penicillamine promoted copper influx transporter hCtr1 expression through upregulation of Sp1. Sp1 overexpression induced p53 translocation from nucleus to cytosol and caused p53 degradation through ubiquitination, which subsequently suppressed the expression of the copper efflux transporter ATP7A. Importantly, co-treatment of cisplatin with d-penicillamine enhanced oxaliplatin-elicited antitumor effect in the oxalipatin-resistant S3 xenograft tumors, but not found in SiHa xenograft model. Notably, Mice received d-penicillamine alone or in combination of d-penicillamine ad oxalipatin, increased hCtrl protein level in S3 xenograft tumor, however, the protein level of ATP7A was decreased. Taken together, this study provides insight into that the co-manipulation of hCtrl and ATP7A by d-penicillamine could increase the therapeutic efficacy of platinum drugs in oxaliplatin resistant tumors, especially in resistant phenotype with downexpression of hCtrl and overexpression of ATP7A.

Nanosecond Pulsed Electric Fields (nsPEFs) Induce Socs1 and Socs3 but not Socs2 Gene Expressions in Hela S3 Cells

Nanosecond Pulsed Electric Fields (nsPEFs) is one of bioelectric technologies applied widely in a number of sciences. nsPEFs cause some biological responses and known to play a role as a novel cancer therapy. However, the nsPEFs molecular mechanisms have not been fully elucidated. This study determines the effects of nsPEFs in socs (Suppressor of Cytokine Signaling) genes which are target genes of JAK/STAT signaling pathway. Through a negative feedback mechanism, SOCS proteins can suppress both cytokine signal transduction and overgrowth factor, so the cell growth is controlled. In cervix cancer, the presence of E6 and E7 HPV's oncoprotein is associated with methylation and inactivation of socs1 and socs3 genes. This mechanism is related to the increase of STAT expression and cancer prognostic. In this research, nsPEFs as much as 20 kV/cm for 80 ns was exposed over HeLa S3 cells in 4 mm cuvette. Socs1, socs2 and socs3 gene expressions were analyzed using real time PCR SYBR green and reverse transcription PCR (RT-PCR). This study shows that at 20 and 30 shots, nsPEFs significantly increase socs1 and socs3 but not socs2 gene expression. Effect of nsPEFs on socs1 and socs3 gene expression pattern is influenced by duration of post exposure incubation and each cell activity on internal cell condition. This research provides a new cancer therapy target for nsPEFs.Keywords: Bioelectric, gene expression, nsPEFs, shot, socs gene

The Impact of Leukocyte Dose during Autologous Stem Cell Transplant on Lymphocyte Recovery in Lymphoma Patients

s / Biol Blood Marrow Transplant 21 (2015) S147eS170 S162 sequence based typing (SBT) and LuminexTMsequence-specific oligonucleotide primed PCR (PCR-SSO). Both methods showed a homozygous phenotype for HLA class I loci (A, B, C) and heterozygosity for HLA class II alleles (DRB1,DQB1). After consolidation, increasing minimal residual disease (MRD) measured by a close NPM1, monitoring was observed in September 2013 and the patient was scheduled for an allogeneic stem cell transplantation. No blasts were present in the peripheral blood of the patient at the time point of molecular relapse. To initiate search for an unrelated donor a confirmatory patient HLA-typing was performed. Due to poor S2 DNA quality a new blood sample (S3) was ordered and used for secondary typing. Whereas HLA class II results were consistent, SBT and PCR-SSO showed discrepant class I typing results when compared to S1, with heterozygosity for all three loci. Consultation of the transplantation center revealed that S1 + S2 were taken while the patient had 84% blasts in the peripheral blood. Heterozygous patient HLA status was finally confirmed by typing of a saliva sample (S4) with SBT and SSO-PCR, respectively. According to the information about S1 (primary diagnosis, peripheral blasts), S3 (increasing MRD, no peripheral blasts) and S4 (blast free material) we assumed that in S1 cells with somatic HLA class I phenotype were highly outnumbered by blasts bearing a complete class I LOH. In regard to this casewestrongly recommend confirmatory typing to be carried out on blast free samples only, or by using a highly sensitive PCR-SSP in case of a substantial amount of blasts in the blood. This should help to prevent the otherwise potentially fatal selection of a highly mismatched donor. As a consequence we established a routinely feedback on the peripheral blast status of the sample on our test request form. IMMUNE RECONSTITUTION

The synthesis and antitumor activity of twelve galloyl glucosides.

Twelve galloyl glucosides 1–12, showing diverse substitution patterns with two or three galloyl groups, were synthesized using commercially available, low-cost d-glucose and gallic acid as starting materials. Among them, three compounds, methyl 3,6-di-O-galloyl-α-d-glucopyranoside (9), ethyl 2,3-di-O-galloyl-α-d-glucopyranoside (11) and ethyl 2,3-di-O-galloyl-β-d-glucopyranoside (12), are new compounds and other six, 1,6-di-O-galloyl-β-d-glucopyranose (1), 1,4,6-tri-O-galloyl-β-d-glucopyranose (2), 1,2-di-O-galloyl-β-d-glucopyranose (3), 1,3-di-O-galloyl-β-d-glucopyranose (4), 1,2,3-tri-O-galloyl-α-d-glucopyranose (6) and methyl 3,4,6-tri-O-galloyl-α-d-glucopyranoside (10), were synthesized for the first time in the present study. In in vitro MTT assay, 1–12 inhibited human cancer K562, HL-60 and HeLa cells with inhibition rates ranging from 64.2% to 92.9% at 100 μg/mL, and their IC50 values were determined to be varied in 17.2–124.7 μM on the tested three human cancer cell lines. In addition, compounds 1–12 inhibited murine sarcoma S180 cells with inhibition rates ranging from 38.7% to 52.8% at 100 μg/mL in the in vitro MTT assay, and in vivo antitumor activity of 1 and 2 was also detected in murine sarcoma S180 tumor-bearing Kunming mice using taxol as positive control.

Toll-like receptor 4 mediates the antitumor host response induced by Ganoderma atrum polysaccharide.

The aim of this study is to investigate the role of Toll-like receptor (TLR) 4 in Ganoderma atrum polysaccharide (PSG-1)-induced antitumor activity. In vitro, the apoptosis rate of S-180 cells was increased in PSG-1-induced peritoneal macrophage derived from C3H/HeN (wild-type) mice, but not from C3H/HeJ (TLR4-deficient) mice. In the S-180 tumor model, phagocytosis, NO and ROS release, phosphorylation of MAPKs and Akt, and expression of NF-κB were increased by PSG-1 in peritoneal macrophage derived from C3H/HeN mice. Furthermore, PSG-1 elevated Th1 cytokine production and enhanced the cytotoxic activity of CTL and NK cells in C3H/HeN mice. In addition, PSG-1 decreased the tumor weight and increased the apoptosis rate and caspase-3 and caspase-9 activities of tumor derived from the C3H/HeN mice. However, none of these activities were observed in C3H/HeJ mice. In summary, these findings demonstrated that the antitumor activity of PSG-1 is mediated by TLR4.

Combination of CD157 and FLAER to Detect Peripheral Blood Eosinophils by Multiparameter Flow Cytometry.

The identification of eosinophils by flow cytometry is difficult because most of the surface antigens expressed by eosinophils are shared with neutrophils. Some methods have been proposed, generally based on differential light scatter properties, enhanced autofluorescence, lack of CD16 or selective positivity of CD52. Such methods, however, show several limitations. In the present study we report a novel method based on the analysis of glycosylphosphatidylinositol (GPI)-linked molecules. The combination of CD157 and FLAER was used, since FLAER recognizes all GPI-linked molecules, while CD157 is absent on the membrane of eosinophils and expressed by neutrophils. Peripheral blood samples from normal subjects and patients with variable percentages of eosinophils (n = 31), and without any evidence for circulating immature myeloid cells, were stained with the combination of FLAER-Alexa Fluor and CD157-PE. A FascCanto II cytometer was used. Granulocytes were gated after CD33 staining and eosinophils were identified as CD157(-)/FLAER(+) events. Neutrophils were identified as CD157(+)/FLAER(+) events. The percentages of eosinophils detected by this method showed a very significant correlation both with automated counting and with manual counting (r = 0.981 and 0.989, respectively). Sorting assays were carried out by a S3 Cell Sorter: cytospins obtained from CD157(-)/FLAER(+) events consisted of 100% eosinophils, while samples from CD157(+)/FLAER(+) events were represented only by neutrophils. In conclusion, this method shows high sensitivity and specificity in order to distinguish eosinophils from neutrophils by flow cytometry. However, since CD157 is gradually up-regulated throughout bone marrow myeloid maturation, our method cannot be applied to cases characterized by immature myeloid cells.

Role of 2,3-cis Structure of (-)-Epicatechin-3,5-O-digallate in Inhibition of HeLa S3 Cell Proliferation

Flavan-3-ol, which is primarily found in tea, is able to inhibit the proliferation of the human cancer cell line HeLa S3; in this study, we investigate the importance of the 2,3-cis structure in this inhibition. We synthesized six (−)-epicatechin and (+)-catechin analogs modified with a galloyl moiety at either the 3-hydroxyl, 5-hydroxyl, or 3,5-dihydroxyl positions. We then investigated their biological activity, DPPH radical scavenging activity and inhibitory activity on HeLa S3 cell proliferation. Among the six compounds, (−)-epicatechin-3,5-O-digallate showed the strongest inhibitory activity on HeLa S3 cell proliferation, whereas (+)-catechin-3,5-O-digallate was not active. In addition, there is no relation among the cell proliferation inhibitory activity and DPPH radical scavenging activity. Furthermore non-specific BSA binding ability of synthesized compounds was demonstrated. Improved photoaffinity beads method revealed that there is no difference between (−)-epicatechin-3,5-O-digallate and (+)-catechin-3,5-O-digallate on the non-specific BSA absorption. These data indicated that the 2,3-cis structure of flavan-3-ol is essential for the inhibition of HeLa S3 cell proliferation

Middle-Down and Chemical Proteomic Approaches to Reveal Histone H4 Modification Dynamics in Cell Cycle: Label-Free Semi-Quantification of Histone Tail Peptide Modifications Including Phosphorylation and Highly Sensitive Capture of Histone PTM Binding Proteins Using Photo-Reactive Crosslinkers.

Mass spectrometric proteomics is an effective approach for identifying and quantifying histone post-translational modifications (PTMs) and their binding proteins, especially in the cases of methylation and acetylation. However, another vital PTM, phosphorylation, tends to be poorly quantified because it is easily lost and inefficiently ionized. In addition, PTM binding proteins for phosphorylation are sometimes resistant to identification because of their variable binding affinities. Here, we present our efforts to improve the sensitivity of detection of histone H4 tail peptide phosphorylated at serine 1 (H4S1ph) and our successful identification of an H4S1ph binder candidate by means of a chemical proteomics approach. Our nanoLC-MS/MS system permitted semi-quantitative label-free analysis of histone H4 PTM dynamics of cell cycle-synchronized HeLa S3 cells, including phosphorylation, methylation, and acetylation. We show that H4S1ph abundance on nascent histone H4 unmethylated at lysine 20 (H4K20me0) peaks from late S-phase to M-phase. We also attempted to characterize effects of phosphorylation at H4S1 on protein-protein interactions. Specially synthesized photoaffinity bait peptides specifically captured 14-3-3 proteins as novel H4S1ph binding partners, whose interaction was otherwise undetectable by conventional peptide pull-down experiments. This is the first report that analyzes dynamics of PTM pattern on the whole histone H4 tail during cell cycle and enables the identification of PTM binders with low affinities using high-resolution mass spectrometry and photo-affinity bait peptides.

BPIC: A novel anti-tumor lead capable of inhibiting inflammation and scavenging free radicals

Inflammation has a critical role in the tumor progression, free radical damage can worse the status of patients in cancer condition. The anti-cancer agents capable of inhibiting inflammation and scavenging free radicals attract a lot of our interest. Aimed at the discovery of such anti-tumor agent, a novel intercalator, benzyl 1-[4-hydroxy-3-(methoxycarbonyl)-phenyl-9H-pyrido[3,4-b]indole-3-carboxylate (BPIC) was presented. The docking investigation of BPIC and doxorubicin towards the DNA (PDB ID: 1NAB) gave equal score and similar feature. The anti-proliferation assay of 8 cancer cells identified S180 cells had equal sensitivity to BPIC and doxorubicin. The anti-tumor assay defined the efficacy of BPIC been 2 folds higher than that of doxorubicin. At 1μmol/kg of dose BPIC effectively inhibited xylene-induced ear edema and decreased the plasma TNF-α and IL-8 of the mice. BPIC scavenged OH, O2− and NO free radicals in a concentration dependent manner and NO free radicals had the highest sensitivity. BPIC could be a novel anti-tumor lead capable of simultaneously inhibiting inflammation and scavenging free radicals.

Activation of the Erythroid Transcriptional Program in Vivo Requires a Transient Shortening of S Phase, Regulated By the Cyclin-Dependent-Kinase Inhibitor p57KIP2

We characterized a rapid commitment switch in mouse fetal liver cells in vivo, that activates the GATA-1 –dependent erythroid transcriptional program as well as other key erythroid differentiation milestones including the reconfiguration of chromatin at erythroid gene loci, and the onset of erythropoietin dependence (1,2). Our published work shows that this switch takes place in early S phase of the last CFU-e generation, as cells transition from flow cytometric Subset 0 (S0, Lin-CD71medium) to Subset 1 (S1, Lin-CD71high). This S0/S1 commitment switch requires S phase progression, and unexpectedly, is associated with a 50% increase in the rate at which S phase cells synthesize DNA (1,2). This latter observation suggests that the duration of S phase is altered during the commitment switch, becoming shorter and faster in S phase cells in S1, compared with S phase cells in S0. We also found that the accelerated DNA synthesis in S phase cells in S1 is essential for an unusual process of genome-wide DNA demethylation, which is rate- limiting for erythroid gene transcription (2). While it is well documented that growth factors may promote shorter cell cycles, this has been considered to be largely the result of their ability to promote the transition from G1 to S phase, resulting in a shorter G1 phase. By contrast, relatively little is known of how S phase duration is modulated during cell fate decisions and differentiation. Here we determined directly the lengths of S phase in CFUe cells and during subsequent erythroid differentiation in the fetal liver in vivo; and identified the cyclin-dependent kinase (CDK) inhibitor, p57KIP2, as a key regulator of S phase duration at the S0/S1 commitment switch.To measure S phase duration, we injected pregnant female mice sequentially with two nucleotide analogs: first, with BrdU, and 2 hours later, with EdU. Fetal livers were harvested shortly following the second injection. Cells that were in S phase during the time of the first injection, but have left S phase by the time of the second injection, were measured as the BrdU+EdU- fraction. This approach allowed us to determine that S phase duration in S0 cells is 7.5 hours, transiently falling to under 4 hours in S1 cells, before resuming a slower pace in more differentiated, Ter119high erythroblasts.We identified the cyclin-dependent kinase (CDK) inhibitor, p57KIP2, as a novel negative regulator S phase DNA synthesis rate. p57KIP2 is expressed in S phase cells in S0, prior to the commitment switch, and is rapidly downregulated (>30 fold) during the switch (1). Here we found that its exogenous over-expression in S0 cells prevented S phase from becoming shorter and faster in S1.We therefore proceeded to investigate p57KIP2-null mice, found to die perinatally with a range of developmental abnormalities; their erythropoietic system was not investigated (3,4). We found that mouse embryos deficient for p57KIP2 are variably pale and/or anemic. Their fetal liver S0 cells showed premature shortening of S phase prior to the commitment switch, as deduced from an elevated DNA synthesis rate in S phase cells of p57KIP2+/- or p57KIP2-/- S0 cells (reaching 83% of the DNA synthesis rate in S1 cells in the same fetal livers; p<0.007; n=12 p57KIP2-/- embryos and 18 p57KIP2+/- embryos), compared with the intra-S phase DNA synthesis rate of S0 cells in wild-type littermates (where DNA synthesis reaches only 64% of the DNA synthesis rate in S1 cells, n=28; see Figure). The premature shortening of S phase in S0 of p57-deficient embryos was associated with increased cell death, as measured by Annexin V, and with reduced differentiation. The latter was deduced from the significantly increased frequencies of earlier erythroid progenitors in the S0 subset (increased by 27% in p57KIP2+/- embryos, p<0.0008; and by 34% in p57KIP2-/- embryos, p<0.025) and a corresponding decrease in later, Ter119+ cells.We conclude that the efficient activation of the erythroid differentiation program at the S0/S1 commitment switch requires transient shortening of S phase. S phase becomes shorter and faster, most likely as a result of increased S phase CDK activation, when p57KIP2 expression is rapidly down-regulated at the S0/S1 transition.

CB-08 * KIF3A IS ESSENTIAL FOR CILIOGENESIS, CILIA FUNCTION AND PROMOTES GLIOBLASTOMA PROGRESSION

Despite recent findings that cilia transduce diverse signaling pathways affecting cell proliferation, migration and survival, little is known about the influence of cilia or cilia-associated proteins in glioblastoma multiforme (GBM). We recently showed that primary cilia project from subsets of cells in GBM patient biopsies and derived cell lines. To determine if cilia contribute to GBM growth, we blocked ciliogenesis using a lentivirus expressing a dominant negative form of KIF3A, an essential ciliogenesis protein. We generated stable GBM cell lines (L0 and S3; representing different molecular subclasses) whereby dnKIF3A+ cells exhibited virtual complete loss of cilia compared to controls (confirmed by immunostaining and EM). Canonically, secreted Sonic hedgehog (SHH) ligand binds and activates receptor signaling cascades (e.g., smoothened (SMO)) within cilia to promote normal cell proliferation and tumor cell growth in specific developmental and pathological contexts, respectively. To examine the role of SHH in GBM proliferation, we exposed control and dnKIF3A+ L0 and S3 cells to saline or recombinant SHH. We found the number of L0 control cells significantly increased after SHH compared to saline, an effect blocked by pretreatment with cyclopamine (SMO inhibitor). However, SHH did not increase the number of L0 dnKIF3A+ cells. Interestingly, SHH exposure had no effect on S3 control cell numbers, despite observations that SHH signaling components (SMO and Gli3) were recruited to their cilia in response to SHH. This suggests GBM cilia are SHH-responsive but the downstream consequences of ciliary signaling may differ between cell lines. Notably, mice intracranially xenografted with L0 cells expressing dnKIF3A survived significantly longer than mice receiving control cells, and retained the loss of cilia phenotype in the tumors. Collectively, these data suggest KIF3A promotes GBM tumor progression, but the extent to which the effects are mediated by cilia and the heterogeneity of SHH-dependence across GBM subtypes requires further investigation.

Regulation of Greatwall kinase by protein stabilization and nuclear localization

Greatwall (Gwl) functions as an essential mitotic kinase by antagonizing protein phosphatase 2A. In this study we identified Hsp90, Cdc37 and members of the importin α and β families as the major binding partners of Gwl. Both Hsp90/Cdc37 chaperone and importin complexes associated with the N-terminal kinase domain of Gwl, whereas an intact glycine-rich loop at the N-terminus of Gwl was essential for binding of Hsp90/Cdc37 but not importins. We found that Hsp90 inhibition led to destabilization of Gwl, a mechanism that may partially contribute to the emerging role of Hsp90 in cell cycle progression and the anti-proliferative potential of Hsp90 inhibition. Moreover, in agreement with its importin association, Gwl exhibited nuclear localization in interphase Xenopus S3 cells, and dynamic nucleocytoplasmic distribution during mitosis. We identified KR456/457 as the locus of importin binding and the functional NLS of Gwl. Mutation of this site resulted in exclusion of Gwl from the nucleus. Finally, we showed that the Gwl nuclear localization is indispensable for the biochemical function of Gwl in promoting mitotic entry.

Coencapsulation of epirubicin and metformin in PEGylated liposomes inhibits the recurrence of murine sarcoma S180 existing CD133+ cancer stem-like cells

Cancer stem cells (CSCs), also known as tumor-initiating cells, which constitute a subpopulation of tumor cells, are key drivers of tumorigenesis and potential recurrence of cancer. The CSC theory has brought new opportunities as well as challenges to the development of sophisticated drug delivery systems for treating cancer. In the present study, CD133+ cells were sorted from S180 cell lines by magnetic activated cell sorting and a fraction (approximately 1.01%) of CD133+ cells with higher proliferative potential and stronger tumorigenicity in vivo compared with CD133− cells was identified. Furthermore, a procedure for the coencapsulation of epirubicin (EPI) and metformin (MET) was developed with the primary goal of eradicating the bulk population of CD133− cells and the rare population of CD133+ cancer stem-like cells, thus ultimately preventing tumor relapse. The inhibitory effect of free MET was more potent in CD133+ cells than in CD133− cells; in addition, EPI- and MET-coencapsulated liposomes exhibited increased cytotoxicity against CD133+ cells compared with liposomal EPI alone. Meanwhile, tumors in KM mice were completely eliminated upon multiple intravenous injections of liposomal EPI and MET, and tumors virtually eliminated in the experimental period, which could be attributed to the arrest of CD133+ cells in the G0/G1 phase. The coencapsulation of an anti-CSC agent with conventional chemotherapy drugs in liposomes may be a promising drug delivery strategy for fighting cancer and eradicating tumor stem cells.

Compositions and anti-tumor activity of Pyropolyporus fomentarius petroleum ether fraction in vitro and in vivo.

The chemical compositions and anti-tumor activities of the petroleum ether fraction (PE), from mushroom Pyropolyporus fomentarius, were studied. Upon gas chromatography–mass spectrometry (GC–MS) analysis, nine major constituents were identified in the fraction. In vitro, the PE showed cytotoxic activity against murine sarcoma S180 (S180) cells in a dose- and time-dependent manner, and the cytotoxic effects were associated with apoptosis. The mitochondrial membrane potential loss and the intracellular ROS generation were greatly increased in the Pyropolyporus fomentarius PE treated group, suggesting cell apoptosis, induced by the PE in S180 cells, might be mitochondria dependent and ROS mediated. Consistent with in vitro findings, the in vivo study showed that the Pyropolyporus fomentarius PE was also effective in inhibiting the tumor growth induced by S180 cells and had lower immune organ toxicity. We found that the Pyropolyporus fomentarius PE has significant anti-tumor activity and great potential in screening anti-tumor drugs.

Antitumor effects and immune regulation activities of a purified polysaccharide extracted from Juglan regia

A water-soluble polysaccharide, named as JRP1, was extracted and fractioned from the epicarp of immature fruit of Juglans mandshurica Maxim. The determination of the monosaccharide composition in JRP1 with gas chromatography (GC) showed that JRP1 was composed of Gal (43.1%), Glu (23.6%), Ara (16.2%), Rha (9.8%) and Fru (7.3%). The results in vitro showed that 25, 50 and 100μg/mL of JRP1 could present a significant inhibition on the growth of S180 cells, and furthermore, a significant improvement on the proliferation ability of lymphocytes and the phagocytic activity of macrophages. The results in vivo showed that compared with those in the control group, the inhibition rates of different doses of JRP1 on S180 cells in the tumor-bearing mice were 35.3%, 40.6% and 48.1%, respectively, and serum immune cytokine levels such as IL-2, TNF-α and IFN-γ were significantly improved. Our results confirm that JRP1 has the activities of effective antitumor and immunomodulatory function.

Abstract 3753: Copper-chelator exerts synergistic interaction with platinum drugs through modulating copper transporters in oxaliplatin-resistant human gastric cancer cell

Abstract Although platinum-based chemotherapy drugs including cisplatin, carboplatin, and oxaliplatin are commonly used in the treatment of human solid tumor, patients ultimately develop drug-resistance and result in treatment failure. Combination copper-chelator with platinum drugs has been reported to successfully treat a subset of cisplatin- resistant ovarian cancer patients. However, the underlying mechanisms of action remain unknown. We have previously established an oxaliplatin-resistant subclone from human gastric adenocarcinoma TSGH cells, S3. The mechanisms responsible for oxaliplatin resistance of S3 cells included down-regulation of hCtr1 and over-expression of ATP7A. In contrast to displaying additive to antagonistic interaction with platinum drugs in parental, TSGH cells, our studies showed that copper-chelator, D-penicillamine, exerts synergistic interaction with platinum drugs through increasing platinum- DNA adduct formations in S3 cells. D-penicillamine promotes copper uptake transporter hCtr1 expression via activation of transcription factor Sp1. In addition, Sp1 overexpression promotes p53 translocation from nucleus to cytosol and binds to ubiquitin, and finally causes p53 degradation, which further suppressed the expression of copper efflux transporter, ATP7A. Combination of D-penicillamine and oxaliplatin significantly inhibited S3 cell growth in animal model. Immunohistochemical analysis showed that up-regulation of hCtr1 and down-regulation of ATP7A were found in both D-penicillamine- and D-penicillamine/ Oxalipatin- treated S3 tumors, which were consistence to in vitro results. In conclusion, our results showed that D-penicillamine increases hCtr1 expression through regulating Sp1 level. Notably, D-penicillamine could down-regulate ATP7A expression through regulation of p53. Our finding demonstrated a new treatment strategy for cancer patient resistant to oxaliplatin treatment with low expression of hCtr1 and overexpression of ATP7A in tumor tissues. (This work was partially supported by the following grants: Department of Health, Taiwan DOH99-TD-C-111-004, National Health Research Institutes CA-101-PP-22, National Research Program for Biopharmaceuticals 100CAP015-5 and National Science Council 102-2325-B-400 -007) Citation Format: Szu-Jung Chen, Jang-Yang Chang, Ching-Chuan Kuo, Hsin-Yi Pan. Copper-chelator exerts synergistic interaction with platinum drugs through modulating copper transporters in oxaliplatin-resistant human gastric cancer cell. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3753. doi:10.1158/1538-7445.AM2014-3753

Abstract 5435: Molecular subclasses of human hepatocellular carcinoma predict sensitivity to fibroblast growth factor receptor inhibition

Abstract Background: A recent gene expression classification of hepatocellular carcinoma (HCC) includes two poor survival subclasses, termed S1 and S2. S1 cells are more locally invasive, while S2 cells express E-cadherin and secrete AFP. As the expression of fibroblast growth factor receptors (FGFRs) differs between the S1 and S2 gene signatures, this study investigated whether molecular subclasses of HCC could predict sensitivity to FGFR inhibition. Methods: The effects of AZD4547 and BGJ398, two small molecule inhibitors of FGFR1-4, were assessed in four S1 and five S2 human HCC cell lines. Western blotting was performed for FGFR1-4 and phosphorylated p42/44 as a marker of MAPK signaling. Small interfering RNA (siRNA) transfection was used to assess the effects of individual FGFR knockdown. Results: Cell proliferation was inhibited at least two-fold more in S2 than in S1 cell lines at all doses tested above 1 uM of BGJ398 and AZD4547 (p&amp;lt;0.002). BGJ398 also decreased MAPK signaling in S2 but not in S1 cell lines. In xenografts from the S2 cell line HuH-7, BGJ398 reduced tumor volume by 70% over 14 days and this effect was associated with decreased MAPK signaling and a 36% decline in the number of Ki67 positive cells. BGJ398 had no effect on xenografts from the S1 cell line SK-Hep. All cell lines expressed FGFR1 and FGFR2, but only S2 cell lines expressed FGFR3 and FGFR4. FGFR4 siRNA decreased proliferation by 59 ± 12% in all five S2 cell lines (p&amp;lt;0.00001), a significantly greater decrease than seen with knockdown of FGFR1, FGFR2 or FGFR3. FGFR4 knockdown also decreased MAPK signaling in S2 cell lines. Conclusions: The S2 molecular subclass of HCC is sensitive to FGFR inhibition. FGFR4-MAPK signaling plays an important role in driving proliferation of S2 cells. This classification system may help identify those patients who are most likely to benefit from inhibition of the FGFR pathway. Citation Format: Benjamin Schmidt, Lan Wei, Danielle K. DePeralta, Yujin Hoshida, Michael Lanuti, Kenneth K. Tanabe, Bryan C. Fuchs. Molecular subclasses of human hepatocellular carcinoma predict sensitivity to fibroblast growth factor receptor inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5435. doi:10.1158/1538-7445.AM2014-5435

Abstract 3548: Study of the tumor suppressive machinery of arsenic trioxide-induced glioblastoma multiforme inhibition via microRNA-182-Sestrin2 regulatory circuit

Abstract Glioblastoma multiforme (GBM) is classified the fourth grade of human glioma that indicates the most malignant brain tumor with poor five-year survival (5%). Accumulated evidence showed that GBM cells were resistant to various therapeutic strategies. Recently, use of arsenic trioxide (ATO) to induce apoptosis and to increase the radiosensitivity was reported for the benefit in treating GBMs. However, the responsible signaling cascades are still remain investigation. MicroRNA, a category of RNA interference, regulates its downstream genes post transcriptionally by binding to the 3′untraslated region (UTR) of its targets. In this study, we used S1 and S1R1 GBM cells which were derived from the primary and the recurrent lesion respectively. Firstly, we identified that an oncogenic microRNA, miR-182, was down-regulated after the treatment of ATO in both GBM cells. In addition, one of the miR-182 targeted coding genes, Sestrin 2 (SESN2), plays an important role in relieving intracellular oxidative stress and participates in TP53 tumor suppressive pathway. Use of luciferase-based reporter assay confirmed that miR-182 directly bound to SESN2 3′UTR and interrupted the further translation. Intriguingly, the basal level of miR-182 in the primary S1 GBM was significantly lower than the recurrent S1R1 GBM, supporting the oncogenic evidence in the previous studies. The counteracted SESN2 expression was also determined by real-time qPCR and western blot. We then hypothesized that ATO may retarded the growth of GBM through a miR-182-SESN2 related regulatory axis. Overexpression of miR-182 in S1 GBM resulted in a decrease of SESN2 and insensitive to ATO treatment. On the opposite, inhibition of miR-182 in S1R1 GBM dramatically retarded the growth rate and sensitized the cells to ATO. Moreover, pretreatment of an antioxidant, N-acetyl-cysteine (NAC), rescued the miR-182 suppression, suggesting that ATO-induced oxidative stress may be dominant in this responsive axis. Concordant expression of miR-182/96/182 cassette was revealed, indicating the transcriptional regulation was unveiled. Further investigation on the detailed machinery still remain elucidating. Based on our findings, we displayed that ATO-induced oxidative stress triggered a tumor suppressive response through a miR-182-SESN2 dependent machinery. Citation Format: Liang-Ting Lin, Shih-Hwa Chiou, Yi-Jang Lee. Study of the tumor suppressive machinery of arsenic trioxide-induced glioblastoma multiforme inhibition via microRNA-182-Sestrin2 regulatory circuit. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3548. doi:10.1158/1538-7445.AM2014-3548

Abstract 2326: Understand the telomerase-dependent transcriptional regulation in cancer cells

Abstract We and others have previously demonstrated that modulation of telomerase activity induces rapid and defined changes in genome-wide gene expression profiles in telomerase-positive cancer cells in culture, which point to the possibility of telomerase's involvement in gene regulation. Previous results also indicated the direct involvement of telomerase catalytic subunit-hTERT in transcriptional regulation through its interaction with Brg1 as well as NFkB. Such transcriptional function of hTERT is not dependent on its reverse transcriptase activity, suggesting a novel telomere-independent function of telomerase that promote stem cell proliferation and survival via activation of key genes regulating cell proliferation and survival. To further explore the potential role of hTERT in direct regulation of genome-wide gene expression in cancer cells, we have engineered cancer cells expressing 3xFlag-tagged hTERT from endogenous loci using AAV-mediated recombination. These genetically engineered cells allow the reliable detection and immunopurification of endogenous and functional hTERT in cancer cells. Such unique tools provide the key to identify potential transcriptional targets regulated by hTERT directly in vivo. Using ChIP-seq, we have mapped the genome-wide targets of potential hTERT binding sites in both HeLa S3 cells and HCT116 cells. Citation Format: TingDong Yan, Chang-Ching Liu, Lai-Fong Poon, Xiaoran Chai, Melissa Jane Fullwood, Sujoy Ghosh, Shang Li. Understand the telomerase-dependent transcriptional regulation in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2326. doi:10.1158/1538-7445.AM2014-2326