Cancerous inhibitor of PP2A (CIP2A) is a recently characterized oncoprotein, which promotes cancer cell proliferation. But the role of CIP2A in lung cancer progression is still not well understood.The expression level of CIP2A in lung cancer tissues was examined by immunohistochemistry. CIP2A-associated cell proliferation was performed by knock down or overexpression of CIP2A in lung cancer cells. Phospho-array was used to screen kinase candidates related to expression change of CIP2A. Western-blot and luciferase reporter assay were used to validate phospho-array results.Overexpression of CIP2A in lung cancer not only triggers immune response in lung cancer patients but also promotes lung cancer cell proliferation. By phospho-array, several kinase candidates were identified, one of which is c-Jun activated kinases (JNK). The knock down of CIP2A decreased JNK phosphorylation, and the phosphorylation of downstream transcriptional factors, ATF2 and c-Jun, whose transcriptional activity were decreased as well. Furthermore, the expression level of CIP2A also affected the phosphorylation of the upstream kinase of JNK, MKK4/MKK7. At last, treatment with JNK inhibitor partially abolished CIP2A-induced cell proliferation.CIP2A is a tumor-associated autoantigen in lung cancer, which promote lung cancer proliferation partially through MKK4/7-JNK signaling pathway. Lung cancer is one of the major causes of cancer-related deaths both in men and women throughout the world. The 5 year survival rate of lung cancer is as low as 15 % , 3. HoweBlood proteins have always been attractive targets for biomarkers in many different types of diseases including cancer. The expression change of blood proteins is associated with the progression of diseases , 5. AutoCIP2A was initially identified as a cancer-associated autoantigen in gastric and liver cancer. But the function of CIP2A was largely unknown due to the lack of homology of CIP2A to any other proteins . Until 2During the search of novel tumor-associated autoantigens in lung cancer patients, we found that CIP2A was overexpressed in lung cancer and elicited autoimmune response in lung cancer patients. The depletion and overexpression of CIP2A via genetic methods decreased and increased cancer cell proliferation, respectively. To define the function of CIP2A in cell proliferation, phospho-array containing key phosphorylation molecules in cancer was used. Our studies showed that CIP2A positively regulates the phosphorylation of AKT, p38 kinase and c-Jun activated kinase (JNK). The further analysis of the JNK pathway showed that CIP2A is a positive regulator of mitogen-activated kinase 4 (MKK4)-JNK signaling pathway in response to growth factors. Inhibition of JNK activation by JNK inhibitor partially abolished CIP2A-induced cell proliferation. Therefore, our study proposed a novel function of CIP2A in cancer progression.2. A549 is cultured with the same conditions as for other cell lines, except that it is cultured in F12K medium (Invitrogen). The immortalized cell line BEAS-2B was purchased from ATCC and was cultured in LHC-9 medium (Invitrogen). HBEC3 cell was generally provided by Dr. Jerry W. Shay . This cell line was cultured in Keratinocyte-SFM supplemented with 50 μg/mL bovine pituitary extract and 5 ng/mL epidermal growth factor (Gibco) at 37 °C on porcine gelatin-coated tissue dishes as previously described . Twenty micrograms of protein were separated in 10 % SDS-PAGE gels and wet transferred to nitrocellulous membrane , then blocked for 1 h at room temperature in TBS-T  buffer containing 5 % nonfat milk. Membranes were then incubated overnight at 4 °C or 1 h at room temperature with the respective primary antibodies: CIP2A (1:500), pJNK , pATF2 (1:1000), phospho-c-Jun (1:1000) and actin . Anti-mouse or anti-rabbit secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology) was used to visualize the stained bands with an enhanced chemiluminescence visualization kit (Santa Cruz Biotechnology).shRNA1: 5’-AAACTTCTCTCAACATACTAGC-3’shRNA2: 5’-TTTCTGATTCAACTTGCTGCG-3’Two CIP2A short hairpin RNAs (knock-down), ligated in pLKO.1 vector and pLOX-KIAA1524 (overexpression plasmid containing the full-length CIP2A) were obtained from Open Biosystems. The mature antisense sequences for the two shRNA we used are as following:Lentivirus was produced by co-transfection of pLKO.1 control (ligated with scramble sequence) or other pLKO.1-derived vector, or pLOC-KIAA1524 with pMD2.G, and pCMV-VSVG into HEK293T packaging cell lines. The supernatants containing lentvirus of HEK293T were harvested at 36 and 72 h post transfection. Supernatant were pooled, centrifuged to remove cells and then filtered through 0.45 μm low protein binding filter. Cells were plated in monolayer at different densities and infected with lentivirus constructs using 8 ng/mL polybrene. The stable cell lines were selected in the presence of 1 μg/ml puromycin (knock down) or 10ug/ml blastidinin (overexpression) for 2 weeks.Human phospho-kinase array were purchased from R&D system. This array contained 43 antibodies, which were pre-spotted in duplicate on nitrocellulose membranes. Before incubating with cell lysates, membranes were incubated with blocking buffer for 1 h at room temperature. Cell lysates (400 μg protein) were mixed with a cocktail of biotinylated detection antibodies. The mixture was then incubated with the array overnight at 4 °C. After the incubation, membranes were washed three times with washing buffer. Streptavidin-horseradish peroxidase and chemiluminescent detection reagents were subsequently added. Chemiluminescence was detected in darkroom with the same protocol for Western blot. Densitometry was measured with NIH Image J software version 1.45 to quantify the average pixel density/spotted area after normalization to the background controls using plug-in function, protein array. Data are representation of two independent experiments.t test was used. Chi-square analysis was used for categorical outcomes. Both of these tests were performed in PRISM software . All tests of statistical significance were two-sided. P values less than 0.01 and 0.05 were considered to be statistically significant.For comparisons between two groups, Student To identify novel tumor-associated antigens in lung cancer patients, we screened the presence of autoantibodies in 102 sera from lung cancer patients and 82 sera from normal human individuals. As shown in Table To confirm this 90 kDa protein is p90/CIP2A as previously identified, we applied immune-depletion and Western-blot analysis. Five randomly chosen 90 kDa autoantibody positive sera were used as the primary antibody to probe the recombinant CIP2A protein by Western-blot. All of these five sera contained autoantibodies against the CIP2A. The recombinant CIP2A was used to immune-deplete the autoantibodies in each sera, which were used for Western-blot analysis. As shown in Fig. To gain a better view of the immune response triggered by CIP2A in lung cancer patients, we used ELISA to measure the frequency of autoantibodies against CIP2A in additional 105 sera from lung cancer patients. Among the 105 samples, 27 sera (25.7 %) contained autoantibodies against CIP2A. While a much lower frequency (4.9 %) of anti-CIP2A positive sera were found in normal human sera  by using monoclonal CIP2A antibody. The CIP2A positive tissue is defined by having a score ranging from 1–6 Fig. . As demoTo examine the role of CIP2A in lung cancer, we first established the expression profile of CIP2A in two immortalized lung cell lines (BEAS-2B and HBEC3), six lung cancer cell lines including four non-small cell lung cancer cell lines , and two small cell lung cancer cell lines (H69 and H146). Interestingly, one of the immortalized cell lines, BEAS-2B, did express CIP2A while another cell line HBEC3 was negative of CIP2A expression. In contrast to the immortalized cell lines, all of these six lung cancer cell lines are expressing endogenous CIP2A with distinct expression level Fig. . H1299 aTo assess whether CIP2A could affect lung cancer cell growth, we either depleted CIP2A or increased CIP2A by ectopic expression of the recombinant CIP2A. The expression of CIP2A in different cell lines was evaluated by Western-blot Fig. . Down-reTo identify proteins involving in CIP2A-mediated cell proliferation, we performed human phospho-kinase array, which include 43 representing phosphorylation events of kinases in cell in response to environmental change, to identify the possible molecules associated with the expression change of CIP2A. Total proteins extracted from H460 cells transfected with vector control (H460+ vector) or from H460 overexpressing CIP2A (H460 + CIP2A) were used as antigen to react with the phospho-antibody array, which was subsequently detected with phospho-antibody cocktails Fig. . SeveralJNK belongs to the family of mitogen-activated protein family (MAPK), which responds to stress stimuli like UV radiation, cytokines, heat shock and osmotic shock. Activated JNK is associated with increased cell proliferation, cell migration and cell invasion in non-small cell lung cancer. Therefore, it is possible that CIP2A may regulate lung cancer progression in a JNK signaling-dependent manner. To validate our finding that CIP2A regulates JNK phosphorylation in lung cancer cells, we treated the cells with EGF to activate the growth factor-induced JNK activation for 15 mins Fig. . ConsistTo test whether the transcriptional activity of the downstream molecule of JNK, we measured the AP-1 transcription activity using luciferase reporter assay. The overexpression of CIP2A increased the AP-1 transcriptional activity to at least 2.3 folds while the depletion CIP2A in H838 reduced of AP-1 transcriptional activity around 30–35 % of the control group Fig. .JNK pathway is frequently activated in lung cancer during cell transformation and lung cancer cell progression. Therefore, it is possible that CIP2A may promote cell proliferation in a JNK-dependent manner. We treated CIP2A-overexpressing H460 with different concentrations of JNK inhibitor, SP610025. After 48 h, cell proliferation was measured with MTT assay. As shown in Fig. The relationship between the immune system and tumorigenesis is not only intrinsically important, but also fundamental to the design of future cancer therapies. The fact that proteins involve in tumorigenesis and cancer progression can be sensed by host’s immune system promoted researchers to profile proteins with immunogenic ability during the onset and progression of diseases . Our labDuring our evaluation of CIP2A expression in different lung cancer cell lines, one interesting observation is the expression of CIP2A in immortalized cell line BEAS-2B but not in another immortalized cell line HBEC3. Although many studies have demonstrated the overexpression of CIP2A in cancer cell lines, few studies had examined the expression in immortalized cell line. The expression difference between the pre-malignant cell lines may be due to the different genetic background. The BEAS-2B was immortalized with chimera viral protein while HBEC3 cell line was immortalized by inactivating only an individual protein, p16. The viral chimera protein may activate the transcription CIP2A but not by p16-mediated pathway. This is also consistent with the study that the expression of CIP2A does not statistically correlated with the expression of p53. In addition, the expression of CIP2A in immortalized cell line is an alternative evidence that CIP2A can be present in pre-malignant tissue as reported previously that CIP2A was present in pre-malignant tissue in the mouse studies as well as the in the patients’ tissues of head and neck cancer . Taken tAlthough abundant evidences had shown that CIP2A is overexpressed in many types of cancer, the role of this protein in cancer progression is still unknown. In our study, we found that CIP2A overexpression is correlated with increased phosphorylation of JNK. JNK bears both of tumor-promoting and tumor-suppressive function depending on the types and genetic contexts of the tumor cells . AlthougOur phospho-antibody array has found the phosphorylation change of several other signaling molecules like AKT, GSK3beta and p38. The role of CIP2A in promoting AKT phosphorylation in a stress-dependent and growth-factor-dependent way has been well established. CIP2A in promoting AKT-mediated signaling through the effect on AKT-associated PP2A phosphatase activity has been confirmed in hepatocellular carcinoma, breast cancer and lung cancer cell lines. For GSK3beta and p38, we actually did not find overexpression or knockdown of CIP2A has direct effect on the phosphorylation of these two proteins by Western blot. And our results that CIP2A does not affect the protein level of beta-catenin indirectly supported that CIP2A does not regulate the phosphorylation of GSK3beta. Although MKK4/MKK7 activates both JNK and p38, we here only showed CIP2A regulates JNK phosphorylation.Our study of CIP2A-mediated cell proliferation partially through priming JNK signaling for cell proliferation rather than cell apoptosis expanded the understanding of oncogenic function of CIP2A in cancer progression. An important aspect in our study is that JNK is only partially contribute to CIP2A-mediated cell proliferation indicating CIP2A has multi-targets, which synergistically to promote cell proliferation. The previous findings that CIP2A physically interacted with c-myc to increase stability, and bind with Plk1 and NEK2 to extend cell division are the possible reasons that inhibition of JNK pathway only partially rescued CIP2A-associated cell proliferation , 20, 25.In this study, we mainly demonstrated that CIP2A is overexpressed in lung cancer and mount immune response in a high percentage of lung cancer patients. And the overexpressed CIP2A in lung cancer could enhance JNK signaling through the phosphorylation of MKK4/MKK7-JNK-c-Jun pathway. Our data could have two impacts, CIP2A can be a candidate biomarker for cancer diagnosis and CIP2A has novel function in JNK signaling pathway in cancer progression.


Ampullary cancer is a relatively rare form of cancer and usually treated by pancreatoduodenectomy, followed by adjuvant therapy. The intestinal subtype is associated with markedly improved prognosis after resection. At present, only few cell lines are available for in vitro studies of ampullary cancer and they have not been collectively characterized.We characterize five ampullary cancer cell lines by subtype maker expression, epithelial-mesenchymal transition (EMT) features, growth and invasion, drug sensitivity and response to cancer-associated fibroblast conditioned medium (CAF-CM).On the basis of EMT features, subtype marker expression, growth, invasion and drug sensitivity three types of cell lines could be distinguished: mesenchymal-like, pancreatobiliary-like and intestinal-like. Heterogeneous effects from the cell lines in response to CAF-CM, such as different growth rates, induction of EMT markers as well as suppression of intestinal differentiation markers were observed. In addition, proteomic analysis showed a clear difference in intestinal-like cell line from other cell lines.Most of the available AMPAC cell lines seem to reflect a poorly differentiated pancreatobiliary or mesenchymal-like phenotype, which is consistent to their origin. We suggest that the most appropriate cell line model for intestinal-like AMPAC is the SNU869, while others seem to reflect aggressive AMPAC subtypes.The online version of this article (doi:10.1186/s12885-016-2193-5) contains supplementary material, which is available to authorized users. Posing a challenge to clinical and pathological assessment and diagnosis, ampullary adenocarcinoma (AMPAC) involves a wide range of histological differentiation as well as complex anatomic localization , 2. BesiHistological differentiation of AMPAC has a strong impact on patient prognosis, as intestinal differentiation is associated with improved survival in comparison to the pancreatobiliary subtype , 7. In pThe cellular plasticity phenomenon of epithelial-mesenchymal transition (EMT) has become an extensively studied biological concept to explain local tumor invasion and metastasis . RecentlWith recent large scale screening methods to demonstrate relevant correlations between the in vitro characteristics in of cell lines and clinical tumor biology –22, therEthics board approval was obtained from the institutional ethics board of the University of Freiburg (ref 13/11). Written informed consent was obtained from patients for the use of tumor tissue for cell culture experiments. For retrospective histopathologic study, patients who underwent surgical treatment for AMPAC were identified from an in-house curated clinical database. A standard pathology protocol was applied for pancreatoduodenectomy specimens and clinicopathologic case review were performed as previously described . Only cawww.tm4.org) [Data collection and statistical analyses were performed operating with IBM SPSS Version 21  and MedCalc Version 14 software . Precise scaling for the different variables were expressed as median (and range), categorical parameters (cross-tabulation and percentages), and survival data (Kaplan-Meier method). For statistical testing, Spearman rank correlation and log rank test were employed. For hierarchical clustering and generation of heat-map images, Multi Experiment Viewer   was usedtm4.org)  and heathttp://cell.brc.riken.jp/en/rcb, Japan) and the Korean Cell Line Bank  [www.atcc.org). All cells were cultured at 37 °C in 5 % CO2 atmosphere in DMEM high glucose medium with GlutamaX (Life Technologies #10566-032) containing 10 % FBS (Life Technologies Standard FBS #10500-064).Ampullary carcinoma cell lines  were obtained from Prof. Frazier  , Prof. Sh Korea) . PANC1 c2 cell culture flasks to 70 % confluence and cryopreserved at -80 °C in standard freezing medium containing DMSO. Only CAF up to passage six were used.Cancer associated fibroblasts (CAF) were isolated by Bachem’s outgrowth method  from a hFor generation of CAF conditioned medium (CAF-CM), CAFs were grown until 70 % confluence, before switching to fresh medium (DMEM 10 % FCS) and incubated for 72 h. CAF-CM was then removed from the CAFs, centrifuged at 1000 rpm for 5 min, sterile filtered (0.22 μm), and transferred to AMPAC cells, which were previously seeded in standard culture flasks for one day prior and washed with PBS. Fresh CAF-CM was added every three days. AMPAC cells were cultured in CAF-conditioned medium for 5–7 days until 70 % confluence and CAF-CM treated and control cells were harvested by cell scraper after three times rinsing with PBS, centrifuged and pelleted at 1000 rpm for 5 min.Immunofluorescence of cultured cells and real time PCR for measurement of mRNA expression was performed as previously described . Primer 12COH2 formaldehyde  while samples from fibroblast conditioned medium were isotopically labeled using 40 mM 13COD2 formaldehyle  [2PO4 and 25 % (v/v) acetonitrile (pH 2.7), and buffer B consisted of 5 mM KH2PO, 1 M KCl, and 25 % acetonitrile (pH 2.7). Peptides were eluted in a linear gradient with increasing concentration of buffer B. Resulting fractions were collected, desalted using self-packed C18 STAGE tips  [Cell pellets from harvesting were lysed using buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 % (v/v) Triton X-100 in the presence of protease inhibitors protease inhibitors -trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, 1 mM phenylmethanesulfonyl fluoride). Lysates were kept on ice for 30 min with intermittent inverting to ensure proper lysis. Lysates were centrifuged for 10 min at 16 000 × g and 4 °C. Protein concentration of lysates was determined using bicinchoninic acid assay. Proteins were precipitated using ice-cold acetone and trypsinized . Cysteine residues were reduced and alkylated, followed by dimethylation of peptide N-termini and lysine residues. Samples from control treated cells were isotopically labeled using 40 mM MA, USA) , 36, botMN, USA) , and anaSamples were analyzed on an Orbitrap XL  or an Orbitrap Q-Exactive plus (Thermo Scientific) mass spectrometer. The Orbitrap XL was coupled to an Ultimate3000 micro pump (Thermo Scientific). Buffer A was 0.5 % (v/v) acetic acid, buffer B 0.5 % (v/v) acetic acid in 80 % acetonitrile (HPLC grade). Liquid phases were applied at a flow rate of 300 nl/min with an increasing gradient of organic solvent for peptide separation. Reprosil-Pur 120 ODS-3 (Dr. Maisch) was used to pack column tips of 75 μm inner diameter and 11 cm length. The MS was operated in data dependent mode and each MS scan was followed by a maximum of five MS/MS scans. The Q-Exactive plus mass spectrometer was coupled to an Easy nanoLC 1000 (Thermo Scientific) with a flow rate of 300 nl/min. Buffer A was 0.5 % formic acid, and buffer B was 0.5 % (v/v) formic acid in acetonitrile . A gradient of increasing organic proportion was used for peptide separation (5–40 % (v/v) acetonitrile in 80 min). The analytical column was an Acclaim PepMap column (Thermo Scientific), 2 μm particle size, 100 Å pore size, length 150 mm, inner diameter 50 μm. The mass spectrometer operated in data dependent acquisition mode with a top ten method at a mass range of 300–2000 Da.12COH2 formaldehyde, +28.03 Da; 13COD2 formaldehyde, +34.06 Da). For relative peptide and protein quantification, XPRESS [2 transformed, resulting in fold-change values. Proteins were considered as being differentially regulated if the proteins are consistently expressed minimum three out of five cell lines with a fold-change value of greater than 0.58 . The list of affected proteins was submitted to STRING database version 9.1 [www.qiagen.com/ingenuity) was further used to derive and visualize biological themes that were significantly associated to affected proteins identified from mass spectrometry analyses.LC-MS/MS data were analyzed using X! Tandem (Version 2013.09.01)  in conju, XPRESS  was used, XPRESS  via the sion 9.1 . Predictn = 39 patients  treated by pancreatoduodenectomy for AMPAC from 2001 to 2011. Median tumor size was 20 mm, about half of tumors were of T1/T2 stage and 59 % had locoregional lymph node metastasis, with a lymph node ratio of 0.10 or more in 44 %. Most tumors displayed low grading (78 % G1 or G2), microscopic lymphangiosis was common (49 %), perineural invasion found in only 33 %, and hemangiosis rare (5 %). There were only three cases with positive surgical resection margin (Table Clinicopathologic review resulted in the inclusion of p = 0.000). Biological factors that showed significant prognostic impact (p < 0.05) include intestinal subtype, lymph node radio and lymphangiosis. Correlation analysis between these prognostic factors disclosed a negative correlation between intestinal subtype and lymph node ratio, and a positive correlation of lymphangiosis with lymph node ratio  Fig.  Fig. 2a. This haCAFs were isolated from a human ampullary cancer explant using Bachem’s outgrowth method . Cell tyThe ampullary adenocarcinoma cell lines MDA-AMP7, AVC1, RCB1280, SNU478 and SNU869 were grown in standard culture medium and expression of KRT7, KRT20, CDX2, E-Cadherin and ZEB1 mRNA was measured by real-time PCR. To investigate the heterogeneous expression pattern, we employed unsupervised hierarchical clustering for analysis. Results evidently indicate that three types of expression patterns Fig. : Cell liTo compare the gene expression pattern of cell lines to the expression pattern in the human AMPAC tumor samples, maximum gene expression was defined as 100 % and relative expression values calculated on a linear scale for each gene. Upon analysis of the relative expression datasets of tumors and cell lines, unsupervised hierarchical clustering revealed two main clusters; intestinal type tumors and pancreatobiliary, respectively Fig. . Poorly Following above-mentioned observations, we hypothesized that cell invasion in vitro would reflect the clinical tumor aggressiveness. Baseline cell growth assessed by MTT assay demonstrated that SNU869 cells were the slowest growing among all AMPAC cell lines Fig. . Also thGiven the clinical association of high CAF activation grade in the more aggressive tumors with pancreatobiliary differentiation and EMT features, we further hypothesized that CAF could induce EMT and invasion, as well as subtype shift in cancer cells. To examine the paracrine effect of CAF, ampullary cancer cells were treated with conditioned medium from CAF (CAF-CM) and subjected to growth assay as well as matrigel transmigration assay with CAFs serving as attractant Fig.  and c.In the intestinal-like cell line SNU869, ZEB1 levels remained unchanged but E-Cadherin expression levels were reduced by approximately 50 %, while cell growth and invasion increased by about 50 % with CAF-CM treatment Figs.  and 6b. Cell growth was slightly enhanced by CAF-CM in the mesenchymal-like cell lines except for the already fast-growing MDA-AMP7 Fig.  and b. A12COH2 formaldehyde (light) or 13COD2 formaldehyde (heavy) was used for relative quantitation.To investigate the influence of CAF on the AMPAC cancer cell proteome, we performed quantitative shotgun proteomics of AMPAC cells treated with CAF-CM versus controls under standard culture conditions, respectively. Quantitative proteome comparison was performed for cell lysates of all five AMPAC cell lines. Stable isotope labeling with either 2 of CAF-treated/untreated ratio) show a near-normal distribution for all five replicates comparing CAF-conditioned medium treated and non-treated cells  is either greater than 0.58 or less than -0.58, corresponding to a change in abundance exceeding 50 %. Mass spectrometry analysis showed that 1 184 proteins were significantly affected in MDA-AMP7; 1 323 in AVC1; 876 in RCB1280; 1 603 in SNU487; and 745 in SNU869. A total of 345 proteins were commonly affected in these four AMPAC cell lines  . Further 87 proteins were commonly affected in three cell lines with adequate correlation . Collectively, a total of 432 proteins were differentially affected more than two-fold in at least three cell lines (n ≥ 3); 208 up-regulated , and 224 down-regulated . Additional data is listed in Additional file Proteins were considered as being differentially abundant upon cultivation in CAF-conditioned medium if the corresponding Fc-value  revealed that several pathways including cellular proliferation and metabolism, cell death, and protein synthesis were significantly affected   between the mesenchymal-like cell lines MDA-AMP7, AVC1, RCB1280 and the pancreatobiliary-like SNU487. In contrast, the more intestinal SNU869 showed regulation in the opposite manner in many identified biological themes.Ingenuity pathway analysis was used to elucidate biological themes being induced or repressed in AMPAC cells upon cultivation in CAF-conditioned medium. Comparison of the expression of all affected proteins (with pre-defined log01) Fig. . HierarcThe twenty most relevant biological themes affecting tumorigenesis and cancer progression of AMPAC cells upon the treatment with fibroblast conditioned medium are illustrated in Fig. in vitro experimental data on AMPAC is absent. Hence, our aim is to establish an experimental platform for the study of AMPAC biology.AMPAC is a relatively rare tumor comprising about 15–20 % of all adenocarcinomas resected by pancreatoduodenectomy , 46–48. Clinical data from patients treated at our center confirmed that intestinal subtype is a main prognostic factor and was significantly associated with KRT 20 and CDX2 expression and KRT7 negativity. A novel aspect was a significant association between intestinal differentiation and reduced features of EMT . We were able to obtain and grow one cell line from each of the five reported patients  under the same standard culture conditions. Basic characterization on the basis of markers for subtype and EMT revealed remarkable heterogeneity among these cell lines. However, hierarchical clustering analyses combining tumors and cell lines based on expression pattern suggested three main pattern: mesenchymal-like, PB, and INT differentiation. Interestingly, these observations also reflect the origin of the cell lines: two out of the three mesenchymal-like cell lines were isolated from metastases, where theoretically increased EMT features would be postulated. The AVC1 cells were derived from a moderately differentiated primary tumor, but displayed a loss of differentiation markers and partial EMT. The pancreatobiliary-like cell line SNU478 was derived from an AMPAC with signet ring cell features, which is a rare and aggressive tumor . Loss ofAmong the five cell lines, only one intestinal-like cell line (SNU869) was found, which is not surprising given that it was previously suggested that only the most aggressive tumor cells can be grown as cell lines in standard culture conditions . NotablyCancer associated fibroblasts (CAF) are known to promote tumorigenesis, invasion and metastasis , 59. AddTreatment of AMPAC cells with CAF-CM from CAF isolated from a human AMPAC had several effects. In general, cellular growth and invasion were increased, ZEB1 expression increased and/or E-Cadherin expression decreased in the cell lines where these genes were expressed. With regard to AMPAC subtype marker expression, decrease of the intestinal markers KRT20 and CDX2 were observed. The cell line derived from signet ring cell AMPAC (SNU478) however displayed decreased cellular invasion, and increased CDX2 and KRT7 genes expression. The undifferentiated MDA-AMP7 cell line showed a slight decrease compared to the very high ZEB1 baseline expression. These findings finding in which CAF-conditioned medium enhanced AMPAC cell growth and EMT features while suppressing intestinal markers and increasing KRT7 are well correlated with clinical data from histopathology and patient survival. It is also known that some cancer cell lines display a loss of differentiation markers and E-Cadherin in standard culture , 64, 65.Proteomic analysis revealed parallel regulation shifts in the mesenchymal- and pancreatobiliary-like cell lines. Pathophysiological pathways of cellular proliferation and survival, activation of the cytoskeleton and intracellular transport were up-regulated, whereas pathways of cell apoptosis, protein translation, oxidative, amino acid and lipid metabolism were down-regulated. These findings suggest a supportive role of CAF in AMPAC cell survival, proliferation and motility. Of note, the intestinal-like cell line SNU869 showed many of these regulation processes to respond in an opposite manner, suggesting CAF response to be differentiation-dependent in AMPAC. In addition, KRT20, CDX 2, KRT 7, ZEB 1 and CDH 1 were not consistently identified in all the cell lines (Additional file A major drawback of this study is that classification of cell lines relies on in vitro parameters and correlation with an independent clinical dataset from tumors. However, no information on the subtype of the tumors that the cell lines were derived from, nor patient follow-up, is available for validation of conclusions drawn. Up to now, this remains a general problem of virtually all standard cancer cell lines. Even the most advanced large scale cell line panel characterization studies have not been validated using this approach –22. NeveOn the basis of clustering for EMT features and subtype marker expression, most of the available AMPAC cell lines seem to reflect a poorly differentiated pancreatobiliary or mesenchymal-like phenotype, which is consistent with their state of origin from moderate to poorly differentiated primary tumors or metastases. Only SNU869, which is derived from a well-differentiated primary tumor, displays an intestinal phenotype and characteristically segregated from the other cell lines specifically in basic marker expression, drug sensitivity as well as proteomic CAF-response. Based on these findings, we therefore suggest that SNU869 is presently the most appropriate cell-based model for intestinal-like AMPAC, while others seem to reflect aggressive and metastatic AMPAC subtypes.


To study the efficacy of novel rhenium compounds to treat triple node negative breast cancer.PC1-6) were synthesized and triple node negative breast cancer cell lines HTB-132 and Balb/c mouse kidney cell lines were treated with each of them for 48 hours. The results were analyzed by a common trypan blue cell death assay system and statistically analyzed.Six (6) novel rhenium pentycarbanato compounds 10 with the corresponding α-diimine ligands in 1-pentanol.The compounds were characterized spectroscopically.The novel rhenium compounds were synthesized from one-pot reactions of ReThe cell lines were cultured by standard cell culture procedure and treated with each of the six compounds in DMSO for 48 hours with a negative control and a DMSO vehicular control along with a cisplatin positive control.The cytotoxicity was evaluated by standard trypan blue assay and the results were statistically analyzed.The trypan blueassay reveals that these compounds have significant cytotoxicity against MDA-MB-468 (HTB-132) triple node negative breast cancer cell lines and are less nephrotoxic than cisplatin.PC 1-6 can potentially find applications in the treatment of highly malignant triple node negative breast cancer.The novel rhenium compounds  Scientists reported success in treating breast cancer with the organic drugs tamoxifen and raloxifene, shrinking advanced tumors with herceptin and treating early cancers with taxol. However, these drugs have severe side effects. For example, tamoxifen causes human endometrial cancer and liver cancer in rats ,2. Numert cancer . Also thWA-2114R  have beeWA-2114R .PC 1-6) are shown in PC 1-6 against triple node negative breast cancer cell lines MDA-MB-468 (HTB-132) and Balb/c mouse kidney cell lines.Several rhenium carbonyl complexes have been reported to be cytotoxic against numerous cancer cell lines ,10. ManyMDA-MB-468 (HTB-132) triple node negative breast cancer cell lineswere obtained from ATCC (USA), cultured inL-15 medium supplemented with 10% Fetal Bovine Serum(FBS), penicillin and streptomycin antibiotics and grownat 37°C inan incubator. The mouse glomerular mesangialcell line was a kind gift from Dr. C. Reilly of Virginia Tech University, USA. These cells were cultured in RPMI-1640 medium supplemented with 10%FBS, penicillin and streptomycin antibiotics and grown at 37°C in a carbon dioxide incubator.PC 1-6: The rhenium compounds PC 1-6 were synthesized according to published procedure (2(CO)10 and the corresponding α-diimines in 1:2 mole ratio was refluxed in 1-pentanol while CO2 was bubbled through the solutions. The α-diimines in PC 1–6 are 2,2′-bipyridyl, 1,10-phenanthroline, 5-methyl-1,10-phenanthroline, 2,9-dimethyl-1,10-phenanthroline, 5, 6-dimethyl-1,10-phenanthrolineand4,7-diphenyl-1,10-phenanthroline,respectively. The reaction was discontinued after 24 hours.After cooling to room temperature, the solid pentylcarbonato complexes PC 1–6 were isolated by filtration.Synthesis of rhenium pentacarbanato compounds rocedure . In briePC1-6 (dissolved in DMSO) along with untreated cells and DMSO vehicular control for 48 hours. Since previous studies with different cancer cell lines [50)dose for these compounds ranged between 2-5 μM, we selected that range as our loading dose. Atrypan blue assay was done to measure cell viability using a standard hemocytometer and light microscope.HTB-132 breast cancer cell lines were grown in six well plates to confluency, the cells were then kept in serum-free medium overnight and then treated with each of drugsll lines  showed 5Each experiment was repeated three times and statistical t test was used to analyze the results with P<0.05.PC 1, PC 4 and PC 6 were characterized through X-ray crystal structure determinations. The X-ray structures of PC 1 and PC 6 were reported earlier . The X-ray structure of PC 4 has been carried out recently and is shown in PC 4 will be published elsewhere.The pentyl carbonato compounds were characterized spectroscopically. AdditioPC 1-6 on MDA-MB-468 (HTB-132) and Balb/c mouse kidney cell lines. All the drugs were cytotoxic to the breast cancer cell lines and found to be less nephrotoxic thancisplatin.PC compounds to be effective anticancer agents with less nephrotoxicity than cisplatin.Anticancer drugs follow a variety of modes (mechanisms) of action.Cisplatin and related compounds produce intrastrand d(GpG) and d(ApG) adducts . DinuclePC-series compounds (drugs) showed effective cytotoxicity against the triple node negative breast cancer cells for 48 hours exposure.Almost all the Triple node negative breast cancer is a fulminant radio-resistant malignant tumor that has almost no known drugs that is an effective chemotherapeutic agent. Further studies are needed with more different node negative breast cancer cell lines and eventually in animal models to come to a conclusion about the real effectiveness of these drugs.Our research demonstrates that the rhenium compounds are effective against triple node negative breast cancer cells. Further studies need to be done regarding the mechanism, nature of cell death and pathway involved in induction of cell death by these compounds.


The development of induced pluripotent stem cells offers the possibility of the scalable manufacture of cellular therapies for regenerative medicine. Moreover, donors can be selected on the basis of major transplant antigen systems to match the widest possible number of recipients worldwide, reducing the likely risk of immunological rejection and the degree of immune suppression or tolerance required. If such cell lines are to be broadly available, there will need to be mutual recognition of common standards across different jurisdictions. Extensive international collaboration will be required around issues such as determination of the optimal homozygous human leukocyte antigens (HLA) panel, donor selection, screening and consent, good manufacturing practice (GMP), standards and quality control and regulatory legislation. The challenges in establishing a global GMP induced pluripotent stem cell (iPSC) haplobank are formidable. We argue that now is the time to attempt to reach international agreement around common standards for GMP iPSC manufacture before the field develops in a fragmented manner. The development of pluripotent stem cells has raised the prospect of the scalable manufacture of cellular therapeutics for a wide range of the degenerative disorders, which currently afflict many people across all countries of the world. However, such cellular therapies will be subject to the same challenges of immunological compatibility with the patient as currently experienced with solid organ and haematopoietic stem cell transplantation. Long-term immune suppression poses a number of risks for patients including general complications such as increased susceptibility to opportunistic infection, neoplasia and cardiovascular disease and specific side effects associated with individual immunosuppressive agents. In broad general terms, the closer the immunological matching between donor (and therefore the cellular product) and recipient, the lower the likely risk of rejection and degree of immunosuppression required. It is therefore advantageous to match as closely as possible for the major transplant antigen systems. The opportunity to pre-select donors for the generation of induced pluripotent stem cell (iPSC) lines opens up an opportunity not possible with human embryonic stem cell lines, which is to create a bank of cell lines specifically chosen to match the widest possible number of recipients worldwide. This will most efficiently be done by recruiting donors who are blood group O and are homozygous for common human leukocyte antigens (HLA). The diversity of HLA types means that it is highly unlikely that any single regional or national bank could contain sufficient cell lines to cover all people within their population base, and therefore, international collaboration between cell banks will be key to equity of access. This is very similar to the situation with haematopoietic stem cell registries and cord blood banks, whereby international collaboration enables access to a much larger pool of HLA-typed potential donors than that provided by individual countries. Such a global iPSC haplobank might provide a non-discriminatory platform for the development of iPSC-derived cellular therapeutics, but in practical terms, two conditions will need to be met: first, the data will have to demonstrate that the manufacturing process is reproducible and that cell lines derived from different donors can be generated to a similar standard; second, international regulators would need to accommodate the general proposition that a cellular product derived from one cell line as a starting material is sufficiently similar to that derived from another  that it can be considered the same product for clinical and regulatory purposes—repeat clinical trials on patients of every different HLA type is clearly impracticable. This requires attention to the following issues:Transplantation of nucleated somatic cells, tissues and complex organs obtained from a randomly selected donor into an immune-competent host is almost invariably recognised and destroyed by the recipient immune system. There is, however, considerable variation in the survival time, and this reflects the degree of genetic disparity between the donor and recipient, correlating most closely with compatibility for ABO blood group and the level of matching for human leukocyte antigens (HLA). In clinical cell, tissue and organ transplantation, it is therefore desirable to match, as closely as possible, the blood group and HLA of the donor and recipient in order to reduce the immunogenic burden and minimise the rejection response.The ideal scenario to achieve immunological compatibility of transplanted tissue is to use autologous cells and tissue obtained directly from the intended recipient, which is not therefore subject to immunological rejection. Clearly, this ideal also applies to transplantation of autologous iPSC-derived cellular therapies but to achieve this on a large scale would require the generation of individualised iPSC lines for many millions of potential beneficiaries worldwide which, at least for the time-being, is not a practicable proposition. A more practical approach to achieve HLA matching is to create a bank of HLA-typed iPSC lines from which cellular therapies can be manufactured which are immune compatible with the largest possible number of potential recipients .HLA polymorphism has evolved from common ancestral genes and developed substantial heterogeneity in response to the enormous variety of environmental infectious agents and the potential for neoplastic transformation. As a result, HLA diversity is highest between populations and ethnic groups that have evolved in different geographical regions of the world. Despite such high levels of genetic diversity, certain HLA alleles and HLA haplotypes are conserved within populations, most likely through natural selection as a result of conferring a survival advantage to environmental challenges within a given geographical region. Therefore, the diversity of HLA polymorphism within populations and ethnic groups is lower, and some alleles and conserved haplotypes occur more frequently. For example, the HLA haplotype: HLA-A*01:01, HLA-B*08:01, HLA-C*07:02, HLA-DRB1*03:01, HLA-DRB3*01:01, HLA-DQA1*05:01 and HLA-DQB1*02:01 is carried by around 12–15 % of white northern Europeans and is also frequent amongst Americans of Hispanic and African ancestries , 5. ThisSimulations of the estimated number of homozygous HLA lines required to provide HLA-A, HLA-B and HLA-DR compatible tissue in various populations worldwide have been undertaken for UK , Japanesuniversal blood donors) and female donors who carry two X-chromosomes and are compatible for minor HY histocompatibility loci. Attention will need to be paid to the practical, legal and ethical issues involved on approaching people who have volunteered to donate blood, cells or tissues for the treatment of individual patients rather than as a starting material for the manufacture of medicinal products. In addition, the nature of the informed consent given by those who choose to donate for this purpose needs to be considered, including the likely scope of testing for infectious agents and genetic abnormalities, the implications of long-term traceability and the circumstances under which they would be informed of findings of potential clinical significance, and the likelihood of commercial development of products derived from their donated material [The identification of volunteers with the desired homozygous HLA types to donate tissue to populate a global iPSC haplobank would require the random screening of many hundreds of thousands of individuals collectively representing the broad range of human genetic diversity. An alternative and readily available source is to identify such individuals amongst the many millions of HLA-typed volunteers that are already registered worldwide on platelet apheresis panels, haematopoietic stem cell donor and cord blood registries, and healthy controls identified in genome-wide association studies. Furthermore, for homozygous HLA types in which there are many hundreds of potential donors, additional desirable criteria may also include selecting volunteer donors who are blood group O  requirements. Review of the HLA types present in populations around the world will allow determination of the optimal homozygous HLA panel required to match each and appropriate balancing to ensure that all ethnic groups are equitably represented.Whilst a primary determinant of donor selection will be blood group and HLA type, a number of other considerations pertain including the general health of the donor her/himself and the risk of transmitting infection, neoplastic or genetic disease to the recipient(s). Routine donor selection and screening processes mitigate these risks in the context of clinical blood, tissue and organ transplantation, but it ought to be borne in mind that iPSC-derived therapies could be used for many patients receiving different kinds of cellular products over a prolonged period of time, at least some of whom are likely to be immunosuppressed. Consideration therefore needs to be given to extended screening of donors either generically or in the context of specific clinical applications.Attention also needs to be given to the retention of traceability between donor(s) and recipient(s) for an extended period of time . In Finally, agreement needs to be reached a propos the nature and extent of informed consent given the long-term retention of traceability and uncertainty over future screening and application of the iPSC lines—in particular the duty of care to the donor should findings arise which are of relevance to her/his health, family or public health.Given the variation in the extent and nature of infectious and genetic risk, organisational infrastructure and cultural norms across the world, achieving consensus on even these donor-related issues may not be straightforward.The use of iPSC-derived cellular therapies in the clinic requires the products to be manufactured under good manufacturing practice (GMP). Globally, the regulations and guidelines for GMP vary to a certain extent depending on country and regulatory environment, but share the common objective of establishing minimum requirements to ensure that products are consistently produced and controlled to quality standards appropriate to their intended use.The full manufacturing chain for a cell therapy product derived from iPSC requires good practices to be applied from the procurement of the donor tissue or cells, through the derivation and expansion of the iPSC line to the manufacture and characterisation of the cellular therapy product itself. In order to protect the health of both the donor and recipient(s), the procurement of starting cells or tissue for iPSC generation needs to comply with the standards set down in national law for blood, cell and tissue donation. A variety of approaches have been developed to effect cellular reprogramming. Commonality of approach or at least a common understanding of the strengths and weaknesses of different approaches would help prevent lack of comparability of different cell lines. Since iPSC lines can proliferate indefinitely in vitro, quality control measures need to be adequately designed to monitor genetic and epigenetic stability through derivation, seed lots and master cell banks over multiple passages. Appropriate control and testing will be crucial to ascertain that the iPSC line has not changed over prolonged storage. The ability of different iPSC lines to differentiate into different cell lineages in the context of standardised culture protocols will need to be assessed if the relative potency of different iPSC lines is to be properly understood. Finally, in addressing the risks from diverse sources of donation, more extensive evaluation of potential contamination with adventitious agents will be required which may require the application of new technologies. For these key scientific features, a common set of evaluation criteria and assays will be required to be applied to seed stocks of the candidate cell lines as discussed below.A number of countries have embarked upon the manufacture of GMP iPSC lines, and several more countries are in the planning phase of such activities (pers comm. haplobank library survey). Manufacturing and up-scaling optimisation of iPSCs are currently global activities, and substantial dedicated public and private funding is invested in this area. As a consequence, many stakeholders are developing in-house GMP protocols and will protect these processes via a patent filing or trade secret strategy. Thus, the IP landscape is another factor to take into account when planning GMP manufacturing of iPSC lines since it is likely that there will be multiple patent holders independent of which manufacturing process is chosen. Several different manufacturing processes are likely to be acceptable to the community as long as an appropriate GMP standard is demonstrated. As discussed above, it is desirable if a minimum GMP standard could be agreed upon globally by the various international and national developers and regulatory bodies to ensure that, in due course, cell lines and relevant therapies can reach as large patient populations as possible in the future.Standardisation in stem cell research and development is clearly very challenging as a result of the complex cell biology and rapid development of the science and technologies involved.Cultures of undifferentiated iPSC lines show a degree of variability in the phenotype of their undifferentiated state as they are passaged in vitro and also appear to respond differently when subjected to the same differentiation protocols •, 15. DeStandardised approaches to culture and characterisation will be important to resolve the real differences between cell lines.To confirm the key attributes of selected candidate cell sources and use a range of analytical techniques to compare them to enable final selection of the cell source of preference.To establish standardised scale-up and analytical systems for the bioprocessing of the cells to make the therapeutic product including stable expansion of undifferentiated cells.To establish standardised assays and reference materials to assess the key safety and efficacy properties of the final therapeutic product.Standards may be written documents providing general points to consider, guidance on technical procedures or even specific protocols required under certain regulatory requirements. They may also be physical preparations used to enable comparison of different cellular preparations . StandarSterility Identity (correct origin)Purity (cellular composition)Potency Stability in vitro (changes that occur on passage or use in different culture environments)At this early stage of development, the standards of most value for iPSC banking for clinical use are the establishment of standards for preparation of seed stocks of early passage iPSC lines which will be based on five attributes as follows:All cell lines should be checked for the common laboratory contaminants. General hazards of bacterial and fungal contamination are screened for using protocols for sterility testing, although it is important to recognise that many species will not grow under the conditions of these tests. One of the commonest of these in cellular therapy culture is mycoplasma which can cause dramatic physiological and genetic changes in cell cultures and for which there are pharmacopoeial standard tests. For other, non-culturable organisms, routine microscopic inspection of culture may be the only means to identify them although 16S rRNA PCR may provide a useful screening method and may need to be used, as discussed above, for the diversity of cells used in haplobank next-generation technologies.Switching or cross-contamination of cell lines is a problem that has dogged the field of cell culture and could have a significant effect on cellular therapy development. In order to assure correct identity of iPSC lines, samples of the original tissue should be retained if possible, and DNA profiling  performed to show that the original tissue and derived cell line are from the same donor. Sharing of identity data amongst researchers and stem cell resource centres is desirable.Phenotypic identity is a complex issue due to the heterogenous populations often present in cell cultures. In addition, these change with time in culture. For undifferentiated pluripotent stem cells, there is some consensus on key phenotypic markers that should be used for banks of undifferentiated pluripotent stem cells •.This is an especially challenging aspect of iPSC characterisation, and the most fundamental aspect of this is the pluripotency of the cell culture which requires a defined set of protocols to show that cells can be directed into the three germline lineages required to generate all cells of the human body. Replication of such testing in different labs is very challenging and will be one of the early tests to require standardisation.As already mentioned, the stability of iPSC in culture is a significant concern for delivery of reproducible cells for therapy. Genomic instability could potentially give rise to transformed and potentially tumourigenic cells, which might persist in preparations of iPSC lines intended for patient therapy. It is unlikely that any cell culture is absolutely genetically stable, and stem cell lines, both iPSC and hESC, show genetic change in vitro culture as do other cell cultures . An impoAn international consensus on the development of seed stocks of human pluripotent stem cell lines has been developed by the International Stem Cell Banking Initiative  including contributors to the haplobank project.A final problem in achieving common global acceptance of GMP iPSC lines is that even if the lines themselves are procured, manufactured and quality controlled in a similar manner, the regulatory environment varies between countries and may treat the lines and cellular therapy products derived therefrom differently. Part of the problem lies in the emergent nature of cell therapy from a background of blood, cell and tissue provision on the one hand and medicinal product manufacture on the other, the complex and rapidly evolving nature of the field and the need to build on and adapt regulatory legislation mainly designed for other purposes.The challenge for the developers of such banks is to meet compliance with the legislation in each of the territories where a product from an internationally accessible haplobank may be used. Whilst there are international guidelines for the quality standards for biotechnology products, there are no such international guidelines for the sourcing of human donor cells; the method of manufacture, testing and quality standards for GMP compliant iPS banks; and subsequent preclinical and clinical testing and market authorisation approval of products derived from such haplobanks.Using the three territories of the EU, US and Japan as examples, there are multiple legislative documents covering the selection of donor material, manufacture, testing and subsequent clinical use which need to be considered. In the EU, there are multinational legislative documents which cover the selection and eligibility of donors , 12, manMandatory donor eligibility and selection criteria applicable in each territory aim to achieve the selection of human tissues and cells of the highest standard from donors who are free from, and pose a low risk of transmission of, relevant communicable diseases. There are many common requirements; however, there are also some differences which must be incorporated when developing the specification and consent for donors who will be recruited to contribute to the bank.Likewise, although striving to achieve the same goal, there are divergent manufacturing standards; for example, there are different pharmacopoeial standards , cleanroom operating, testing and traceability requirements. For example, there is a requirement to store donor-related records for 10 years in Japan and the US, whereas this is extended to 30 years in Europe. Furthermore, there is considerable divergence in the preclinical and clinical requirements of products derived from an induced pluripotent bank.This environment therefore poses a significant challenge for the development of a global haplobank predicated on mutual recognition of iPSC lines and approval of the cellular therapies derived therefrom for clinical trial and market authorisation. Developers of haplobanks, and products derived from these, must take these divergent standards into consideration at an early stage in the development and establishment of the bank.The comparability of master cell banks (MCB) and cellular therapies derived from the various donors selected for the haplobank is arguably the key to the establishment of such a bank. A requirement for preclinical and clinical testing of each of the cell lines as if it were a separate product would negate the utility of a bank and restrict immunological matching of cellular therapies to a minority of potential recipients. The authors argue that, albeit that each iPSC line manufactured from different donors will be unique in some respects, they will each be manufactured and tested according to validated procedures and must meet the set in-process and iPSC line specification. As such, each cell line may be considered as a manufacturing lot of the haplobank. These lots in turn may form the starting material for any number of intermediate and final cell-based medicinal products, with each product type  having its own in-process and release specification. Assuming the extensive characterisation work performed on the MCB and downstream products demonstrates that the starting materials and subsequent clinical products are comparable, and within a set range of inter-batch variability, then it is reasonable to apply this approach in clinical trial. It is hence proposed that the haplobank cell lines could be used interchangeably to produce multiple lots of various products with the safety and efficacy of a product type derived from a number of different haplobank cell lines evaluated in one clinical trial. It is envisaged that a slightly larger number of subjects may be required but that all product lots should be viewed as comparable, and the data generated from each of these lots from the trial could be amalgamated. Early discussions with a number of major regulatory authorities have indicated the potential acceptability of this approach contingent upon the inclusion of adequate data in the dossier.As Bravery remarks in his insightful critique , the cha


Cell lines derived from tumor tissues have been used as a valuable system to study gene regulation and cancer development. Comprehensive characterization of the genetic background of cell lines could provide clues on novel genes responsible for carcinogenesis and help in choosing cell lines for particular studies. Here, we have carried out whole exome and RNA sequencing of commonly used glioblastoma (GBM) cell lines  to unearth single nucleotide variations (SNVs), indels, differential gene expression, gene fusions and RNA editing events. We obtained an average of 41,071 SNVs out of which 1,594 (3.88%) were potentially cancer-specific. The cell lines showed frequent SNVs and indels in some of the genes that are known to be altered in GBM- EGFR, TP53, PTEN, SPTA1 and NF1. Chromatin modifying genes- ATRX, MLL3, MLL4, SETD2 and SRCAP also showed alterations. While no cell line carried IDH1 mutations, five cell lines showed hTERT promoter activating mutations with a concomitant increase in hTERT transcript levels. Five significant gene fusions were found of which NUP93-CYB5B was validated. An average of 18,949 RNA editing events was also obtained. Thus we have generated a comprehensive catalogue of genetic alterations for six GBM cell lines.


The oncogenic BRAF(V600E) mutation is common in melanomas as well as moles. The roles that this mutation plays in the early events in the development of melanoma are poorly understood. This study demonstrates that expression of BRAF(V600E) is not only clastogenic, but synergizes for clastogenesis caused by exposure to ultraviolet radiation in the 300 to 320 nM (UVB) range. Expression of BRAF(V600E) was associated with induction of Chk1 pS280 and a reduction in chromatin remodeling factors BRG1 and BAF180. These alterations in the Chk1 signaling pathway and SWI/SNF chromatin remodeling pathway may contribute to the clastogenesis and UVB sensitivity. These results emphasize the importance of preventing sunburns in children with developing moles. In vitro expression of BRAF(V600E) in normal human cells, including melanocytes, results in a very rapid oncogene-induced growth arrest phenotype that is independent of p53 [The oncogenic BRAFV600E) mutation is a common mutation in many cancers and precancerous lesions making it an important diagnostic marker and treatment target ,2,3. Of 00E mutatt of p53 . Slow reThe Loeb mutator theory of human carcinogenesis holds that the background rate of mutation in human cells is too low to generate the 6–8 independent mutations that are needed to produce a cancer, so an early event in malignant transformation is a mutation  that increases the rate of mutation . PreviouBRAF and NRAS, and engineered to express oncogenic BRAF(V600E) has been described previously [BRAF and NRAS. The DNA damage checkpoint responses have been described previously for both of these melanoma lines [1 checkpoint response due to alterations in p53 signaling. Both have an intact G2 checkpoint response. A melanoma cell line that is wild-type for eviously . Using tma lines ,16,17. NSince expression of oncogenic BRAF causes oncogene-induced growth arrest we tested whether this happened in our cell lines. As shown in Previous reports have suggested that V5-BRAF(V600E) expression may result in chromosomal breaks and a DNA damage signal in cells as measured by comet assay, micronuclei formation, or induction of phosphorylated γH2aX ,11. We eAlthough both cell lines exhibit similar levels of chromosomal aberrations in the absence of V5-BRAF(V600E), the PMWK cell line exhibited a much higher break frequency than the RPMI cell line after induction. This may represent an underlying difference in chromatin architecture. The PMWK cell line was found to be polyploid with most metaphases having about 90 chromosomes while the RPMI cell line was near diploid. The total frequency of the aberrations in the RPMI line did not increase significantly with increasing expression of the oncogenic BRAF. The aberrations are unlikely to be due to an effect of the doxycycline since incubation of RPMI8322 + TetON cells, parents of the RPMI8322 + TetON + V5-BRAF(V600E) cells, with doxycycline did not increase aberrations . This is0 value, or dose to reduce the viable cell population to approximately 37%, was then calculated for each cell line according to published methods [0 of 191 J/m2, presumably due to defective nucleotide excision repair [0 value of 254 J/m2. For subsequent experiments a fluence of UVB was chosen for each cell line that resulted in approximately equal killing.Melanoma progression is linked to DNA damage caused by exposure to ultraviolet radiation . Consist methods . As prevn repair . PMWK wa0, ½D0, or D0 fluences of UVB 24 h after seeding the cells as described in the Experimental section. Metaphases were prepared 24 h after exposure, 48 h total culture time. The baseline clastogenesis resulting from UVB exposure in the absence of V5-BRAF(V600E) is shown in The melanoma cells were exposed to 0, ¼D0 of UVB could not be accurately scored. This dose of UVB in the presence of oncogenic BRAF resulted in very few scorable metaphases with every chromosome in those few metaphases containing multiple aberrations. At ¼D0 and ½D0 both lines exhibited a similar pattern of damage with the PMWK line now also showing exchange aberrations.To determine if expression of oncogenic BRAF had an effect on UVB clastogenesis, cells cultured in an amount of doxycycline  on the PMWK and RPMI8322 cell lines. At the lowest fluence of UVB both cell lines exhibited a similar induction of aberrations. Combining the ¼D0 of UVB with oncogenic BRAF expression, the resulting induction of aberrations appears to be additive for both cell lines. This is seen in 0 in cells expressing oncogenic BRAF resulted in a fold-increase in the aberrations that was 3.6 and 7.6 times greater than that induced by doxycycline alone for the PMWK and RPMI8322 cells respectively  in the V5-BRAF(V600E) expressing RPMI and PMWK cells exposed to a Described . InductiThe exchange aberrations and breaks observed after induction of V5-BRAF(V600E) and the increased sensitivity to UVB suggested that misregulation of BRCA1 might be a possible cause. BRCA1 is required for efficient repair of UV damage  as well BRCA1 is known to exhibit cell cycle dependent expression ,26. To tWhile it did not appear likely that the amount or location of BRCA1, or alterations in NER, were responsible for the oncogenic BRAF-induced clastogenesis or UVB sensitivity, another possibility was our previously published observation that expression of oncogenic BRAF attenuates the DNA damage-induced G2 checkpoint response . Chk1 isChk1 S280 is phosphorylated by AKT1  and p90RA final possibility for explaining the clastogenesis induced by the oncogenic BRAF and the synergy with UVB exposure is alteration of the SWI/SNF chromatin remodeling complex. As shown in The synergistic induction of chromosomal aberrations by UVB in cells expressing oncogenic BRAF suggests that developing moles are sensitive targets for UVB-induced chromosomal damage. As melanocytic nevi commonly develop during childhood and most nevi express oncogenic BRAF, strategies for prevention of melanoma must emphasize protecting children against damaging sunburns. This study also suggests possible implications in other cancers such as colon cancer where the oncogenic BRAF mutation has been reported in 70% of the tumors exhibiting the highest level of microsatellite instability . This maA V5-BRAF(V600E) expression vector was constructed by cloning a synthetic V5-BRAF(V600E) DNA cassette into the pRetro-X-Advanced  vector. Vector construction and sequence verification of the vector was done by Blue Heron Bio .2 atmosphere. Packaging of retroviral vectors and transduction of cells was done as previously described [Melanoma cell lines RPMI8322 and PMWK were grown in DMEM high glucose  supplemented with 10% Benchmark fetal calf serum , and 200 mM L-glutamine (Life Technologies) at 37 °C in a 5% COescribed . The celThe ability of the oncogenic V5-BRAF(V600E) to reduce clonogenic survival was assessed as follows. Cells were trypsonized to achieve a single cell suspension. The viable cell count was determined by trypan blue exclusion and counting cells in a heamocytometer. Three hundred viable cells were seeded per 10 cm diameter dish in triplicate in 10 mL of media containing various amounts of doxycycline. The media was changed on day seven, maintaining the doxycycline. On day 14 the colonies were fixed and stained with a solution of 40% methanol and 0.05% crystal violet.2. By keeping the lids on the dishes all wavelengths less than about 300 nM were filtered out resulting in a UVB profile more similar to that found in sunlight [0 value were done as previously published [UVB cytotoxicity was determined by seeding 300 viable cells per dish as above. Twenty-four hours after seeding the media was removed and the cells exposed to various fluences of UVB with the lids on the dishes in an open cabinet to the output from two high intensity broad range UVB lamps  at a fluence of 3.13 J/s/msunlight . The UVBublished .® (Life Technologies) according to manufactures protocol. Cells were fractionated into cytoplasmic and chromatin fractions by previously published methods [Whole cell extracts were prepared by solubilizing cell pellets in urea buffer  for 30 min on ice prior to protein quantification. Proteins were quantified using a Qubit methods . Primary®  unless noted otherwise.The primary antibodies were detected using fluorescently labeled anti-mouse and anti-rabbit secondary antibodies at a dilution of 1:10,000 and obtained from Li-Cor . The proteins were visualized by scanning the blots on a Li-Cor OdysseyExpression of oncogenic BRAF(V600E) was clastogenic in two human melanoma cell lines. The melanoma cell lines provided an opportunity to study mitotic chromosome structure after induction of the oncogene but prior to cessation of cell division. Equivalent studies were done with a TERT-expressing, immortal human melanocyte line but no mitotic cells could be recovered two days after induction of BRAF(V600E) (results not shown). As BRAF(V600E) attenuated the DNA damage G2 checkpoint  it may bWe have previously reported that expression of oncogenic BRAF attenuates the DNA damage induced G2 checkpoint response . Oncogen


Recent work suggests a role for IAPP aggregation in cardiovascular complications of type-2 diabetes and hints at a possible role in type-1 diabetes. The mechanisms of IAPP amyloid formation in vivo or in vitro are not understood and the mechanisms of IAPP induced β-cell death are not fully defined. Activation of the inflammasome, defects in autophagy, ER stress, generation of reactive oxygen species, membrane disruption, and receptor mediated mechanisms have all been proposed to play a role. Open questions in the field include the relative importance of the various mechanisms of β-cell death, the relevance of reductionist biophysical studies to the situation in vivo, the molecular mechanism of amyloid formation in vitro and in vivo, the factors which trigger amyloid formation in type-2 diabetes, the potential role of IAPP in type-1 diabetes, the development of clinically relevant inhibitors of islet amyloidosis toxicity, and the design of soluble, bioactive variants of IAPP for use as adjuncts to insulin therapy.The hormone islet amyloid polypeptide  plays a role in glucose homeostasis but aggregates to form islet amyloid in type-2 diabetes. Islet amyloid formation contributes to  Hyaline lesions in the pancreas were first described more than 110 years ago by Opie , 3 Figu. IAPP wa in vivo  suitable for use as adjuncts in insulin therapy. It is not possible to cover all topics and all of the recent developments in IAPP research in a limited review and the reader is referred to other articles in this issue for coverage of other topics and for alternative views. We do not discuss efforts at inhibitor design as they are described in other articles in this issue and have been reviewed elsewhere . A numbe in vivo is controversial. The mechanism(s) of IAPP amyloid formation in vivo and in vitro are still not understood nor are the factors which trigger islet amyloidosis in type-2 diabetes (T2D). The nature of the toxic species generated during IAPP amyloid formation is not well defined and the mechanisms of β-cell death are not completely understood. The possible role of IAPP aggregation in the complications of diabetes has yet to be fully defined and the potential role of IAPP in type-1 diabetes remains to be elucidated . The. The7]. sed IAPP –65. in vitro [Mature IAPP is stored in the insulin secretory granule and is found in the halo region of the granule, while insulin is located in the dense core. The concentration of IAPP in the granule is noticeably lower than that of insulin, about 1%-2% of the insulin level, but it is still much higher than that required to promote rapid amyloid formationin vitro , 67. Thuin vitro , 68–70. in vitro –75.In rats, the circulating concentration of IAPP is reported to be on the order of 3 to 5 picomolar and to rise to 15 to 20 picomolar with elevation of blood glucose , 5. HowehIAPP plays a role in maintaining glucose homeostasis, in controlling gastric emptying, and in the suppression of glucagon release –6. The hϕ, Ψ angles in solution, but the level of persistent helical structure is low [α-helical structures in the presence of detergent micelles, but they bind to micelle in different orientations [1–19 inserts more deeply into the nonpolar core of the membrane, while rat IAPP1–19, with its Arg substitution binds near the surface. hIAPP1–19 binds near the surface, similar to rat IAPP1–19, at acidic pH when His-18 is protonated, indicating that the net charge of residue 18 is important in controlling the orientation [α-helical structure [Monomeric hIAPP does not fold to a compact globular structure and can be classified as an intrinsically disordered protein, but it is not a random coil. NMR chemical shift studies have shown that the region encompassing residues 5–20 of rIAPP and hIAPP transiently sample helical e is low , 90. More is low . NMR base is low –94. hIAPntations , 95. hIAentation , 95. Memtructure –95. hIAPtructure . The rolβ” architecture with the interstrand hydrogen bonds oriented parallel to the long axis of the fibril and the β-strands oriented perpendicular to the long axis. The parallel, in-register, β-sheet structure of amyloids generates effectively infinite arrays of stacked identical residues and this has important energetic consequences. The in-register arrangement implies that there can be significant electrostatic interactions in amyloids. In hIAPP, Arg-11 and His-18 are in the structured core of the fibril, or immediately adjacent to it, arguing that they will make net unfavorable electrostatic contributions to the stability of the fibril. Calculations performed at the level of the linearized Poisson-Boltzmann (PB) approach show that Arg-11 makes significant unfavorable electrostatic interactions but that His-18 does not do so when its side chain is neutral. In this case, the desolvation penalty paid by His-18 is overcome by specific interactions with the imidazole ring [β structure. Consistent with this hypothesis, experimental studies argue that the His-18 to Arg substitution contributes to the inability of rIAPP to form amyloid [Amyloid fibrils form a so-called “cross-ole ring . His-18  amyloid . A numbe amyloid , 97–99.  amyloid , 98.β-sheet core.Linearized PB calculations may not be rigorously valid for a strongly coupled system and thus the quantitative details of the analysis should be interpreted with caution. The problem of electrostatic interactions in amyloids has not been studied in detail and seems ripe for further investigation. The pattern of like charges in amyloid fibrils is reminiscent of other systems with repetitive arrangements of charges such as DNA and approaches that have been used to analyze the energetics of DNA should be applicable to amyloids. Unfavorable electrostatic interactions are also likely to arise from the N-terminus of IAPP. Lys-1 is the region of highest charge density in the polypeptide and is expected to make unfavorable electrostatic interactions in the amyloid fibril, even though that region is not part of the well-ordered The importance of electrostatic interactions in hIAPP amyloid is also reflected in the dependence of the kinetics of hIAPP amyloid formation on ionic strength and on the type of salt. In particular, the rate amyloid formation is significantly accelerated with increasing salt and the effects depend on the choice of anion. An excellent correlation with the anion selectivity series was observed at low and moderate salt concentrations, strongly arguing that anion binding plays a role in the effects . A corolThe parallel in-register structure also leads to networks of interactions involving polar uncharged residues. In the fragment model, Ser-28 and Ser-29 are involved in a steric zipper and make extensive hydrogen bonding interactions . Ser-29 Islet amyloid contains the heparan sulfate proteoglycan (HSPG) perlecan, apolipoprotein E (apoE), and serum amyloid P component (SAP) , 103 in  in vivo amyloid fibers are long-lived structures that present HSPG binding sites, or because HSPGs directly promote amyloid formation, but it is well documented that the glycosaminoglycan (GAG) chains of HSPGs catalyze hIAPP amyloid formation in vitro [ in vivo. Overexpression of heparanase in a double transgenic mouse model that overexpresses hIAPP reduced the amyloid load, while inhibition of GAG synthesis reduced hIAPP amyloid deposition in cultured islets [hIAPP is cationic and ionic interactions facilitate its binding to negatively charged membranes, negatively charged biopolymers, and negatively charged surfaces. It is not known if perlecan is associated with islet amyloid becausein vitro , 104. Thd islets , 106. in vivo are still mysterious, but one model postulates binding of the Npro-hIAPP processing intermediate to the GAG chains of perlecan [ in vitro and the resulting fibrils can seed amyloid formation by fully processed mature hIAPP [The factors that trigger islet amyloid formationperlecan , 62, 64. in vitro and more highly charged systems have a larger effect for experiments conducted at high peptide to lipid ratios [β-cells [β-cell is also very different from most model systems. In addition, β-cell membranes contain cholesterol and the β-cell plasma membrane is asymmetric with the anionic lipids localized on the inner leaflet. These considerations naturally lead to the question of how well model membranes recapitulate the situation in vivo. Model membranes made up of the phospholipids found in β-cell membranes, but lacking cholesterol, also accelerate hIAPP amyloid formation, as do anionic model membranes that are capable of forming lipid rafts [Anionic model membranes promote hIAPP amyloid formationd ratios . The mecd ratios , 107–109[β-cells . The phoid rafts –112. Theid rafts . in vivo is controversial and there are conflicting reports in the literature. Amyloid deposits detected in T2D appear to be extracellular and initial studies with transgenic mice were consistent with extracellular deposition. However other studies with rodent models in which IAPP is overexpressed are consistent with an intracellular origin [β-cell toxicity and islet amyloid formation. Two complimentary sets of reagents were employed: one that inhibited IAPP secretion, but maintained the level of production of IAPP and a second that increased IAPP secretion but did not increase the amount of IAPP produced. Inhibiting IAPP secretion reduced amyloid formation, but increasing secretion increased toxicity and amyloid formation [The initiation site for islet amyloid formationr origin , 114. Itormation . The resormation . Clarifyβ-cell dysfunction and death and the loss of β-cell mass are key features of T2D [β-cell dysfunction and the decline in β-cell mass are attributed to several factors, including glucolipotoxicity, inflammation, accumulation of cholesterol, and islet amyloid formation [Interest in islet amyloid has undergone resurgence due to the realization that s of T2D , 118. β-ormation –122.β-cells and occurs before the recurrence of hyperglycemia. Porcine IAPP is far less amyloidogenic than hIAPP and the prevention of amyloid formation by transplantation of porcine islets prolongs islet graft survival [Islet amyloid deposition is also a key factor contributing to the failure of islet cell transplants. Islet amyloid has been detected in transplanted human islets in a patient that suffered islet graft failure and has been shown to form rapidly after transplantation of human islets into nude mice , 123–125survival . Recent survival , 127.A range of mechanisms have been proposed to account for the toxic effects of amyloidosis, but the exact causes of cell death are still not completely defined. In some cases, amyloid fibril deposits disrupt tissue and can lead to organ failure, but, in most cases, activation of overlapping cellular mechanisms and downstream signaling pathways are believed to lead to toxicity. These include receptor-mediated mechanisms and non-receptor-mediated processes.ER stress, defects in autophagy, the enhanced production of proinflammatory cytokines, mitochondrial membrane damage, permeabilization of cell membranes, activation of Calpain-2, receptor-mediated mechanisms linked to oxidative stress, and the activation of cell death signaling pathways have all been proposed to contribute to IAPP toxicity –147. Herβ-cells has been reported to lead to impaired autophagy [β-cell apoptosis, while stimulation of autophagy protected against IAPP toxicity [Defects in autophagy play a role in the toxicity of other amyloidogenic proteins. Upregulation of autophagy is a common protective response to the accumulation of toxic amyloidogenic aggregates in degenerative disease. However, autophagocytosis and lysosomal degradation of amyloidogenic polypeptides are not always entirely successful and the resulting accumulation of amyloidogenic aggregates can lead to autophagy-mediated lysosomal dysfunction and cell death. Overexpression of hIAPP in utophagy , 146. Intoxicity , 146.β-cell death by hIAPP aggregates. ProIAPP and partially processed proIAPP may be one of deleterious species in cases where toxicity arises from intracellular aggregates since proIAPP miss-processing has been shown to occur in diabetes and posttranslational modification is completed in the Golgi and insulin secretory granules [ in vivo is controversial. Transgenic mouse studies using mice that overexpress hIAPP led to the proposal that ER stress is a key contributor to hIAPP induced β-cell dysfunction and exogenously added hIAPP has been reported to induce ER stress [Defects in ER stress, endoplasmic reticulum associated protein degradation (ERAD), and the unfolded protein response (UPR) have all been reported to induce granules , 7, 8. TR stress , 141. OnR stress .β-cell dysfunction by triggering a localized inflammatory response by stimulating inflammasome activity [β and IL-18 by cleaving their proforms. IL-1β is thought to play a direct role in hIAPP induced β-cell death and dysfunction [Chronic inflammation may be an important contributing factor to amyloidosis toxicity and it is frequently observed in local and systemic amyloidosis. hIAPP aggregates can contribute to activity , 137. Infunction , 137.β-cell apoptosis are not yet fully elucidated [β-cell apoptosis in islets and in cultured cells exposed to high concentrations of hIAPP and is upregulated in response to amyloid formation by endogenous hIAPP [β-cells from hIAPP induced toxicity and inhibition of caspase-3 in vivo protects β-cells from hIAPP induced β-cell apoptosis [Amyloid formation by hIAPP induces apoptosis in cell culture and in isolated human islets, but the pathways that lead to IAPP induced ucidated –134. Theus hIAPP . Interacpoptosis , 134.β-cell membrane [IAPP toxicity has also been proposed to result from the perturbation of membrane integrity , 148. Thmembrane  and usuamembrane , 148. Inmembrane , 148. Momembrane –112. in vitro biophysical studies using model membranes and in vivo toxicity is not clear and caution needs to be employed when extrapolating from biophysical studies that utilize simple model membranes to the in vivo environment. For example, variants of hIAPP that do not induce β-cell death in vivo and are not toxic in vitro can disrupt standard model membranes in vitro and can do so as effectively as hIAPP [The correlation betweenas hIAPP . It is aas hIAPP .β-structure is not required to disrupt membranes [Mechanistic studies of IAPP induced model membrane disruption are an active area of research and a variety of models have been proposed. Some studies provide evidence for a detergent or carpeting mechanism while others have argued for a pore-like mechanism. The process of fiber growth at the membrane surface can contribute to membrane disruption in some cases, while other studies have shown that formation of embranes , 151–156embranes , 158. MuThe cell is an inherently crowded environment, containing numerous proteins and other macromolecules as well as osmolytes, and there is no guarantee that a protein will behave the same in dilute solution and in a crowded environment. The effects of molecular crowding and osmolytes on the stability and folding of globular proteins are well studied and are still an active area of research. Early work focused on inert crowders and the role of excluded volume effects, but more recent efforts have been directed at better mimics of the cellular environment and consideration of interactions beyond excluded volume –163. IndCoadministration of IAPP with insulin helps to normalize fluctuating glucose levels to a greater degree than is possible with insulin alone; however the extreme tendency of IAPP to aggregate and its amyloidogencity and toxicity prevent its direct use as an adjunct to insulin therapy –174. A n in vivo; defining the nature of the toxic species; elucidating the mechanisms of islet amyloid formation in vivo and in vitro; understanding the mechanisms of β-cell death; defining the mechanisms of hIAPP clearance in vivo and the role such processes may play in IAPP toxicity. Experimental challenges include relating reductionist biophysical experiments to the situation in vivo and understanding the connection between mouse models that highly overexpress hIAPP and human β-cell physiology. Although not discussed in this review, the development of inhibitors of hIAPP toxicity is also an area that warrants further effort. There are no clinically approved inhibitors of IAPP toxicity and very few, if any, effective “drug-like” inhibitors of IAPP amyloid formation have been reported in the literature.Impressive progress has been made in studies of amyloid formation by hIAPP, but important challenges remain. These include identifying the initiation site(s) of amyloid formation


In the present work, a chemical investigation of irciniid sponges from Tunisian coasts led to the purification of three known FTAs and three known PHQs. The in vitro growth inhibitory properties of the six purified compounds have been evaluated in the same experiment in a panel of five human and one murine cancer cell lines displaying various levels of sensitivity to proapoptotic stimuli. Surprisingly, FTAs and PHQs elicited distinct profiles of growth inhibitory-responses, differing by one to two orders of magnitude in favor of the PHQs in all cell lines. The obtained comparative results are discussed in the light of a better selection of drug candidates from natural sources.Marine sponges of the Irciniidae family contain both bioactive furanosesterterpene tetronic acids (FTAs) and prenylated hydroquinones (PHQs). Both classes of compounds are known for their anti-inflammatory, antioxidant, and antimicrobial properties and known to display growth inhibitory effects against various human tumor cell lines. However, the different experimental conditions of the reported This report, in particular, focuses on compounds isolated from sponges of the genera de novo biosynthesized by the sponges themselves, a panel of bioactivities with pharmaceutical potential, including cytotoxic, anti-inflammatory, antioxidant, and antimicrobial properties, have been attributed to both classes of compounds D + 27.5 ; and [α]D + 16.5 ]  , 41.2O extract (846.6 mg) of S. spinosulus from Monastir was fractionated on silica gel column eluted with a gradient solvent system ranging from 100% to 20% of light petroleum ether in Et2O affording a fraction (57.4 mg) containing compound 4. An aliquot (15.2 mg) of this fraction was further purified on preparative TLC  obtaining 12 mg of pure compound 4 D + 28.0 ; and [α]D + 17.5 ] and 3 D − 21.5 ; and [α]D − 10.5 ].An aliquot (2 g) of Etin A (2) , 42 and  (2) [3) , 44, res2O extract (61.6 mg) from S. foetidus was subjected to silica gel column eluted with a gradient solvent system ranging from 100% to 0% light petroleum ether in diethyl ether affording pure compound 6 (4.6 mg) [The Et(4.6 mg) .http://dx.doi.org/10.1155/2016/5318176) with the literature values [All isolated compounds were identified by comparison of their spectral data . The overall growth level of the human cancer cell lines was determined using a colorimetric MTT  assay as detailed previously [ 1–6 were dissolved in DMSO and redissolved in the cell culture media at a final concentration of 0.1%, which induces no observable toxic effects on cells. Six replicates of each experimental condition were performed. Thus, this procedure enables the concentration of compounds 1–6 that decreased by 50% the growth of each cell line (GI50 concentration) after having cultured it with the compound of interest for 72 h (the GI50 index in μM) to be determined.The cells were cultured in RPMI  medium supplemented with 10% heat inactivated foetal calf serum (Lonza). All culture media were supplemented with 4 mM glutamine, 100 eviously –32. Comp1), sarcotin A (2), and variabilin (3), along with the known PHQs 4–6  also tests for a given compound 10 μM as its higher potential growth inhibitory concentration. Consequently, if we refer to these criteria, among the studied compounds only compound 6 behaves actually as an inhibitor of cancer cell growth independently of the levels of sensitivity to proapoptotic stimuli of the various cancer cell lines under study.Before proceeding to an interpretation of the data, we need to define when the compounds can be considered remarkably active with respect to the effects they produce on cancer cells. The GI 4 and 5 showed a very similar pattern of growth inhibition with the most sensitive cell lines being MCF-7, SKMEL-28, and B16F10, while their growth inhibitory activity against the remaining three cell lines was about one magnitude weaker. This evidence seems to reflect the strong structural similarities among hepta- and octaprenylhydroquinones 4 and 5, which just differ by one isoprene unit  studies are necessary for accurate identification of the structural features required for the activity of compound 6, our finding suggests that the presence of a hydroxyl group on the chain of 6 could be related to its enhanced growth inhibitory activity on cancer cells as compared to the nonhydroxylated 4 and 5. This finding, however, contrasts with earlier findings indicating less efficiency of 6 compared to 4 and 5 when tested against the chronic myelogenous leukemia (CML) cell line K562 [ 6 (and to a lesser extent compounds 4 and 5) markedly inhibits cancer cell growth and that (ii) the 6-induced growth inhibition in cancer cells is independent of the levels of resistance of the cancer cells (at least the models under study) to proapoptotic stimuli.On the other hand, compoundsene unit . In addiine K562 . This apine K562  used oneine K562 . The dat Sarcotragus, our study evidenced higher in vitro growth inhibitory effects of PHQs on cancer cell lines, compared to FTAs. In addition, our results suggest, in spite of previous evidence reported in the literature, that compound 6 is the most active among the tested PHQs, inducing growth inhibition in cancer cells independent of their levels of resistance to proapoptotic stimuli. Consequently, 6 clearly deserves further in-depth analyses of its anticancer mechanism of action, toward its possible use to treat metastatic cancer.By comparing different metabolites isolated from sponges belonging to the genus1–6, acquired using instruments and methods as described in the Materials and Methods section of the paper.The Supplementary Material includes NMR and mass spectra of compounds