A serious limitation of numerous antitumor drugs is the incapacity to penetrate solid tumors. However, addition of an RGD fragment to peptide drugs might solve this problem. In this study, we explored whether the introduction of a permeability-enhancing sequence, such as iRGD (CRGDK/RGPD/EC) fragments, would enhance the activity of thymosin alpha 1 (Tα1). The modified Tα1 (Tα1-iRGD) was successfully expressed and purified, and the in vitro assay showed that Tα1-iRGD presented a similar activity as Tα1 in promoting proliferation of mouse splenocytes. Meanwhile, cell adhesion analysis revealed that Tα1-iRGD exhibited more specific and greater binding with tumor cells compared with Tα1. Furthermore, the iRGD fragment evidently enhanced the basal ability of Tα1 to inhibit proliferation of cancer cells in vitro, particularly of mouse melanoma cell line B16F10 and human lung cancer cell line H460. Our findings indicated that the addition of an iRGD fragment increased the anti-proliferative activity of Tα1 against cancer cells by improving the ability of Tα1 to penetrate the tumor cells. This study highlighted the important roles of an iRGD sequence in the therapeutic strategy of Tα1-iRGD. Thus, Tα1-iRGD could be a novel drug candidate for cancer treatment. Thymosin alpha 1  is a 28-amino acid peptide that was first isolated from the thymus gland in mammals by Goldestein in 1977 The more powerful tumor-penetrating peptide iRGD (CRGDK/RGPD/EC) was identified recently based on the mechanism of RGD 4 DNA ligase was purchased from Promega (USA).B16F10 mouse melanoma cell, human gastric cancer cell line BGC-823, human lung cancer cell line H460, and human colon cancer cells line HT-29 were available in our institute. Our institute purchased the cell lines from American Type Cell Culture . Concanavalin A (ConA) was purchased from sigma-aldrich Company (USA). Paclitaxel . Enterokinase was obtained from New England Biolabs, USA. Nickel-chelating column was purchased from GE Healthcare Company. All the restriction enzymes were purchased from Takara Biotechnology  Co., Ltd., and TEscherichia coli BL21 (DE3) harboring positive recombinant plasmid was grown at 37°C on Luria-Bertani medium containing 100 µg/ml of ampicillin at 37°C. Lactose (5 mM) was added to the medium for 4 h to induce protein expression, when the absorbance at 600 nm reached 0.6. Then, the cells were harvested by centrifugation , suspended in ice-cold 10 mM Tris and 1 mM EDTA at pH 7.6, and disrupted by sonication. The supernatant of the cell lysate was further purified by a nickel-chelating affinity resin according to the manufacturer’s instruction, and the eluted fraction was analyzed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The fusion proteins were digested by enterokinase at 23°C for 16 h. The target peptides were obtained by loading the nickel-chelating affinity column with equilibration buffer. The eluted peptides were collected, desalinated by gel filtration chromatography, and confirmed by mass spectroscopy.The synthetic genes coded for Tα1 and Tα1 -iRGD were digested with KpnI and HindIII and inserted into pET32a, separately. After the calcium chloride transformation, the positive clones were selected based on ampicillin resistance, which was further confirmed by DNA sequencing. 6/mL) was prepared in RPMI 1640 containing 10% FBS  after teasing through a sterilized autoclaved mesh. The adjusted cells were placed in a 96-well microtiter plate and added with 200 µL aliquots per well. The samples were incubated with or without recombinant Tα1 or Tα1-iRGD at 37°C in an atmosphere containing 5% CO2 and 95% humidity. ConA (5 µg/mL) and RPMI 1640 were set as positive and negative control, respectively. After 68 h, cells were counted by the 3--2, 5-diphenyltetrazolium bromide (MTT) assay T − AN)/AN ×100% .All experimental procedures using animals used in the our study were performed in strict accordance with the recommendations from the Guide for the Care and Use of Laboratory Animals, which is promulgated by the United States National Institute of Health, and was approved by Jiangsu Provincial Experimental Animal Management Committee under Contract 2012(su)-0035. Spleen lymphocyte proliferation experiments were performed as previously described 4/100 µL) were added to the pre-coated plate. After 1h of incubation at 37°C, the plates were washed twice with isotonic buffer saline to remove unbound cells. Cells bound to the wells were fixed, stained by 0.5% crystal violet staining buffer, and photographed. Finally, 10% acetic acid (100 µL per well) was added to each well to extract the crystal violet. The remaining adherent B16F10 melanoma Cells were determined by a microplate spectrophotometer at 595 nm. The relative cell adhesion (%) was calculated using the equation (AT − AC)/AC × 100% .Cell attachment experiments were conducted as previously described N − AT)/AN × 100% . Other cells such as human gastric cancer cell line BGC-823, human lung cancer cell line H460, and human colon cancer cell line HT-29, were treated with the same method.B16F10 melanoma cell in the logarithmic growth phase were dispersed in 0.25% trypsin with RPMI 1640 complete medium. Cell suspension (50 000 cells/mL) was plated into a 96- well culture plate (0.1 mL/well), which was inoculated for 8 h in 37°C. The cells were incubated with different concentrations of recombinant Tα1 or Tα1-iRGD at 37°C. Paclitaxel (0.0125 µmol/mL) was set as a positive control, whereas RPMI 1640 was set as a negative control. After 36 h, cells were counted by MTT assay The structure of Tα1-iRGD was modeled using the DS|Built homology model  based on template (PDB file 2L9I) E.Coli BL21 (DE3), the fusion proteins were purified by a nickel-chelating affinity resin respectively, and then confirmed by SDS-PAGE (Tα1 and Tα1-iRGD genes were cloned in the frame between KpnI and HindIII restriction sites of the pET32a plasmid, which produced fusion proteins partnered with Trx-A separately . Most ofSDS-PAGE . Then, tSDS-PAGE . MeanwhiSDS-PAGE . These rDifferent concentrations of recombinant Tα1 or Tα1-iRGD were added to spleen lymphocyte to test their proliferation activity. As shown in The cell attachment of Tα1-iRGD and Tα1 to a melanoma cell line was further evaluated to determine whether the addition of the iRGD motif to Tα1 enhances its binding to the tumor cell or not. PBS wells were used as negative control. As shown in Tumor cell proliferation was analyzed by MTT assay to determine whether the modification of Tα1 affected its biological activity. When human gastric cancer cell line BGC-823 was treated with Tα1 or Tα1-iRGD at doses that ranged from 0.03125 µmol/mL to 0.5000 µmol/mL , Tα1 exhFurthermore, when human lung cancer cell line H460 was treated with Tα1 or Tα1-iRGD at doses that ranged from 0.03125 µmol/mL to 0.5000 µmol/mL , Tα1-iRGMoreover, when human colon cancer cell line HT-29 was treated with Tα1 or Tα1-iRGD at doses that ranged from 0.03125 µmol/mL to 0.5000 µmol/mL , Tα1-iRGThe 3D structures of Tα1 and Tα1-iRGD were compared using computational modeling to determine how the addition of the iRGD motif improves the efficacy of Tα1 to arrest tumor cell growth. However, no obvious differences can be observed from the modeling . AlthougTα1 has already been approved for the treatment of Hepatitis B and C in several countries E. coli. Cell attachment assay, spleen cell proliferation experiments and tumor cell proliferation experiments revealed that Tα1-iRGD has higher antitumor activity than Tα1. In the melanoma cell attachment assay, we found that Tα1-iRGD has higher attachment activity than Tα1. In the cancer cell inhibition assay, we also found that Tα1-iRGD has a better anti-proliferation activity in several cancer cell lines than Tα1, particularly in mouse melanoma cell line B16F10 and human lung cancer cell line H460. With the aid of the iRGD, Tα1-iRGD directly inhibited the proliferation of human lung cancer cell line H460 and mouse melanoma cells line B16F10 at very low concentrations in contrast to the slight inhibition exhibited by Tα1.In this study, both Tα1 and Tα1-iRGD were successfully expressed and purified by bioengineering methods in Previous publications have reported the effectiveness of Tα1 in clinical trials of non-small cell lung cancer In this study, we tried to connect Tα1 and tumor-homing. We expressed and evaluated the effectiveness of Tα1- iRGD in vitro for the first time. The research on Tα1- iRGD fusion peptide provided a new effective way of maximizing the effectiveness of Tα1 in treating solid tumors. The iRGD Tα1 fusion peptide could be an attractive strategy for designing novel therapeutics against tumors. We are currently studying the activity of Tα1- iRGD on cancer targeting, and the pharmacokinetic and pharmacodynamic aspects of Tα1- iRGD will be further investigated to guarantee better clinical application.


Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis.Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so  Rhabdomyosarcoma (RMS) is a malignancy that arises from skeletal muscle precursors . It is tIn vitro cell-line research and in vivo xenograft models aid in the basic and preclinical effort to identify potential new treatments. If shown promising, these agents can then be taken into human clinical trials, and compared to standard of care agents. The Pediatric Preclinical Testing Panel (PPTP) is an initiative formed by the National Cancer Institute, working to further characterize and validate available cell lines in multiple kinds of pediatric cancer, including RMS so that preclinical evaluations of new chemotherapeutic agents can be tested . It was originally developed by Dr. Hajime Hosoi and can be grown in DMEM with 10% FBS .in vitro on a lethally irradiated layer of mouse fibroblast 3T3 cells; the cell line was later tested and found to contain only human cells. While the tumor biopsy was positive for desmin staining, the HX170c cell line was almost completely negative for this marker when cultured in vitro, except when grown to almost complete confluence. Interestingly, HX170c subcutaneous mouse xenografts regained desmin positivity and also appeared histologically similar to the initial tumor biopsy. HX170c cells are near diploid (50 ± 6)  .TP53 gene  and is derived from a bone marrow metastasis. It is grown in DMEM with 10% FBS .MYC oncogene  in Memphis, TN directly from a 16-year-old male with an infra-renal lymph node . Of note, it has no relation to the Rh6 xenograft listed in . When evOf historical and nomenclature import, all of the “RH” cell lines (sometimes denoted “SJRH”) were generated at SJCRH and can be obtained from a representative of that institution, or from Dr. Peter Houghton at Nationwide Children’s Hospital, Columbus, OH, USA who has maintained these lines. However, during the years in which human RMS cell lines were first being established, two separate laboratories were deriving and naming the cell lines independently. Cell lines established in the laboratory of Dr. Peter Houghton were generated by directly transplanting tumor into mice to establish xenografts, and originally labeled “Rh.” Cell lines established in the laboratory of Dr. Edwin Douglass (and subsequently Dr. David Shapiro) were generated directly from patient samples and labeled “RH.” The upper or lower case designations for these cell line names have since been interchanged in the literature and are no longer preserved.RH12 was derived from a xenograft created from a buttock tumor in a previously untreated patient. The original tumor and corresponding xenograft have an embryonal histology based on their spindled appearances . ChromosThe RH14 xenograft was derived from an inguinal tumor in a patient previously treated with vincristine, dactinomycin, and cyclophosphamide, with complete response and no evidence of disease for 2.5 years, then recurrence. It had an embryonal histology based on histologic appearance of the original tumor and the xenograft, both of which showed differentiation and cross striations . In evalRH36 has an embryonal histology  and was derived from a paratesticular relapse in a 15-year-old male . The PPTRMS559 cells were established in the laboratory of Dr. Jonathan A. Fletcher, Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA , 45. TheMYCN and RAS genes showed no amplification or rearrangement, nor mutations of the 12th, 13th, or 61st codons of KRAS, HRAS, or NRAS. TP53 pathway gene analysis showed no mutation of the TP53 gene, but did show upregulation of MDM2  and t . It is iTTC442, also occasionally called A91 in the literature, has an embryonal histology and is grown in RPMI-1640 with 10–20% FBS. This cell line was originally obtained from the laboratory of Dr. Tim Triche of Children’s Hospital Los Angeles (CHLA) , 53.TTC516, also known as A995 in the literature, has an embryonal histology and is grown in RPMI-1640 with 10–20% FBS. This cell line was originally obtained from the laboratory of Dr. Tim Triche of CHLA , 53.PAX7-FOXO1 fusion gene . CW9019 has a stable reciprocal translocation of t, expresses the resulting PAX7-FOXO1 fusion protein and is grown in DMEM with 10% FBS . Unlike ion gene .D-RHA1 has an alveolar histology and was originally described in , having KFR was derived from a bone marrow metastasis of a 13-year-old female and designated RMS based on positive staining for vimentin and desmin. Alveolar histology was assigned based on morphology and upon identification of the t translocation, expressing the resulting PAX3-FOXO1 (formerly known as PAX3-FKHR) fusion protein . AlthougRH5 has an alveolar histology and has been cited in a study of the role of PDGFR-A .RH10 was derived from a 15-year-old female with a perineal tumor at relapse; she had previously received extensive chemotherapy with vincristine, cyclophosphamide, dactinomycin, and doxorubicin. RH10 was first established as a xenograft. The resulting cell line is hyperdiploid and cytogenetic analysis revealed t . AlthougPAX-FOXO1 fusion.The RH18 cell line was generated from a xenograft derived from a tumor from a previously untreated 2-year-old patient with a perineal RMS assigned as embryonal histology. Interestingly, there was a small amount of alveolar histology in the tumor  . At the RH18 cell-line chromosome number ranges from 70 to 80 per cell, and cytogenetic abnormalities include del(1)(p22), der(3)t, del(6)(q24), and der(22)t . In the The RH18 cell line also harbors amplification of the 12q13–15 region (including MDM2) , althougWhen tumor volumes were measured following injections of various chemotherapy agents, RH18 showed growth inhibition with vincristine, cyclophosphamide, dactinomycin, doxorubicin, L-PAM, DTIC, cis-DDP, and mitomycin-C. No tumor growth inhibition was seen with bleomycin or busulfan . When evin vitro shows that between passages 59–63, doubling time progressively increases until growth stops completely, and the cells appear to senesce. Further, these cells are multinucleated and show morphologies comparable to normal myoblasts  translocation . Evaluatyoblasts . When teyoblasts .There exists a sub-line of RH28, denoted RH28/LPAM, which was generated from RH28 cells in xenografts exposed to increasing doses of melphalan. RH28/LPAM cells are available for use and do not differentiate or stop proliferating with time . There aTP53. The RH30 cell line was derived from the same patient bone marrow sample maintained in culture in the Douglass laboratory. The RH30 cell line expresses the t translocation  translocation . RH41 alP53 gene . RH41 waThe cell line designated RH4 was developed from the same patient by another laboratory at SJCRH , in which the tumor specimen was placed directly into tissue culture. These cells designated RH4 possess the t translocation  and haveRH65 was derived from 18-year-old female with a relapsed RMS; it has an alveolar histology based on the original tumor .PAX7-FOXO1 fusion gene and MYCN were found amplified  and was derived from the bone marrow metastasis of a 16-year-old male who relapsed 5 months after multi-agent chemotherapy and radiation . ChromosSMARCB1 (SNF5) mutation, consistent with its classification as a rhabdoid line (A204 was previously (but no longer) listed in the ATCC catalog as an RMS cell line. However in 1998, A204 cells were found to lack expression of myogenin, MyoD, or desmin, suggesting that either they were not of RMS origin or that with passaging in culture there was down-regulation of myogenic genes . These ooid line .EWS-FLI1 fusion gene, indicating the cell line is derived from a Ewing sarcoma  listed in the ATCC catalog as an RMS cell line. However, like A204 cells, in 1998 these cells were found to not express myogenic markers . In 2003 sarcoma .EWS-FLI1 fusion characteristic of the Ewing family of tumors . RH1 has no relation to the Rh1 xenograft described in . In 1998f tumors . In retrf tumors .While all above mentioned studies propagated RMS cell lines as adherent cells or xenografts, Walter et al. recently described a method of culturing rhabdospheres in suspension . In thisin vitro or transplanted into mice as xenografts. One possibility is that tumors can be contaminated by murine stromal cells, thereby potentially affecting the outcome of the study. To combat this problem, analysis for species-specific LDH isoenzymes, as well as evaluating for acrocentric mouse chromosomes, can be performed. For example, Hazelton et al. cultured rhabdomyoblasts from the cell lines RH10, RH18, and RH28 and found human specific isoenzymes of LDH present in the lines, without evidence of murine LDH. In this same study, metaphase spreads were analyzed for acrocentric mouse chromosomes, and none of the three cell lines evaluated showed murine cells , the cell lines resembled the other human RMS specimens . FurtherA third problem is the situation in which two cell lines with different names are apparently from the same tumor, such as RH36 and Birch, RH30 and RMS13. This situation can represent two different nomenclatures or possibly contamination of one cell line with the other. In addition, although RH3 and RH28 appear genetically identical by fusion breakpoint and STR analysis , 81, it A fourth problem is unintentional cross-contamination between cell lines. It is estimated that 15–35% of all cell lines are of a different parental origin than assumed, and this results in the misidentification of cell lines and ultimately the misinterpretation of scientific data –84. For http://www.aacrjournals.org/site/AuthServCtr/cell_line_auth.xhtml). On the other hand, it is important to acknowledge the limitations of STR in cell line authentication. For example, in a study of four human leukemia cell lines grown under variable culture conditions, STR profiles changed in two of the four cell lines due to subclone outgrowth  to standard agents. These previously treated cell lines would therefore be optimal for preclinical testing of new agents proposed for use in relapsed or refractory disease. These further progressed tumors may also be easier to establish in culture than primary tumors. It is necessary to ensure that cell lines used in preclinical trials closely resemble the tumors from which they were derived. While some data is currently available that validates several lines, this work is ongoing and will surely continue to develop as the use of cell lines and RMS xenografts increase in pediatric oncology research.When examining RMS cell lines as a whole, there is a surprising lack of cell lines derived from primary, untreated RMS tumors. Indeed, the majority of both embryonal and alveolar RMS cell lines are established from tumor relapses or distant metastases. Cells that arise from relapses or metastases (in addition to any adaptations as the result of radiation and/or chemotherapy given to the patient) have genetic changes distinct from the primary tumor, and may be easier to establish in culture than cells from primary tumors. Therefore, applicability of these lines in preclinical studies examining RMS primary tumor biology is questionable. This gap in our understanding of RMS reveals a significant challenge to researchers that routinely use RMS cell lines for preclinical research. How can we make significant strides to identify therapeutic targets when the reagents available do not accurately represent the biology of the primary tumor? Has RMS research been hindered by an excess of false negative hits in pharmacologic and genetic screens due to these cell-line adaptations?In summary, it will be important for RMS scientists, as a community, to critically evaluate the cell lines we use, and identify approaches to generate improved reagents for future research. Ideally, human cell lines would be established from primary, untreated RMS tumor biopsies. Then, any cell lines generated from subsequent tumor relapse or metastasis could be paired with the primary cell line as a “matched set.” To our knowledge there are no matched set cell lines publicly available to RMS researchers. When available, such a resource would support the study of the genetic and biologic changes in the primary tumor, as well as the changes leading to recurrence or metastasis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Primula auriculata demonstrated the highest cytotoxicity with IC50 of 25.79, 35.79 and 43.34 μg.mL−1 against MCF7, HepG2 and HT- 29 cells, respectively. For some of the plants, their traditional use was correlated with the cytotoxic results, whereas for others the results may support the non-cytotoxicity of species used traditionally as natural remedies. The cytotoxic species could be considered as potential of anticancer compounds. Medicinal plants have been investigated for possible anti-cancer effects. The aim of the present study was to examine the cytotoxic activity of several medicinal plants on different tumor cell lines. 11 selected plant species which have been used in folkloric prescriptions were collected from different sites of Hamedan district of Iran. The methanolic extracts of the plants were prepared and their cytotoxic effects on four human cancer cell lines  and one normal cell line  were examined using the MTT assay. Three of these were exhibited antiproliferative activity against one or more of the cell lines. The extract from  Cancer is one of the main causes of death all over the world. The world health organization (WHO) estimates that 84 million people would die of cancer between 2005 and 2015  was separated, shade dried and grinded into powder using mortar and pestle at room temperature. Then extracted by maceration with methanol for 72 h. The supernatants were filtered and evaporated under vacuum by means of a rotary evaporator to obtain crude methanolic extracts.Cytotoxic assayCell lines and culture mediumThe following cancer cell lines were used for this study: A549 (human lung adenocarcinoma), MCF7 (human breast adenocarcinoma), MDBK (bovine kidney cells), HepG2 (hepatocellular carcinoma) and HT-29 (human colon carcinoma). Cells were obtained from National Cell Bank of Iran .MCF7 and HT-29 were cultured in Dulbecco’s modified eagle medium  with respectively 5% and 10% bovine serum  while other three cell lines were cultured in RPMI 1640 medium (Sigma) with 10% FBS to maintain the desired growth. All cell lines were treated with 1% penicillin-streptomycin (Sigma) in a humidified atmosphere with 5% Co2 at 37 °C throughout the assay.MTT assayCell viability was quantified by an MTT colorimetric assay  . The cel8.5× 103 for MDBK cells, 7.5 × 103 for MCF7, 15 × 103 for HepG2, 9 × 103 for A549 cells, and 5 × 103 for HT-29 cells and incubated at 37 °C. After 24 h of incubation, when cells reached more than 80% confluence, the medium was removed and the cells were treated with fresh medium containing various concentrations of plant extracts to be tested. Control cells were supplemented with 0.05% DMSO (v/v) vehicle. After 24 h, the supernatants were removed and a fresh medium containing MTT (0.5 mg mL-1) was added to each well at the time of incubation. After 4 h incubation, the supplement was carefully removed, and the remaining formazan crystals were dissolved in DMSO. The plates were shaken for 20 min. The absorbance of each well was measured on an enzyme-linked immunosorbent assay reader (TECAN) at the wavelength of 570 nm.in-vitro experiments were carried out on two microplates with at least three parallel wells. The antitumor agent 5-FU was used as a positive control in all cell lines. The dose–response curves of the compounds were fitted by means of the computer program GraphPad Prism 6.0 , and IC50 values (the concentration at which the cell proliferation is 50% of the untreated control) were calculated. All In order to evaluate the cytotoxic effect of 11 plant extracts that are used in Hamedan district of Iran, an antiproliferative assay on four human cancer cell lines  and one normal cell line  was performed. Cytotoxicity activity (IC50) of the eleven plant extracts was shown in Extracts with IC50 > 100 μg.mL-1 in MTT assay were considered inactive.Centaurea aucheri, Centaurea pseudoscabiosa subsp pseudoscabiosa and Primula auriculata was expected to show more cytotoxic activity against the tumor cells, whereas others were not cytotoxic against any of the cell lines tested.Our study describes investigations into the anticancer potential of 11 so far not studied Iranian medicinal plants by screening for cytotoxic activity against normal bovine kidney and four human cancer cell lines. All plant extracts showed no toxicity against normal bovine kidney, but methanolic extract of Centaurea comprises about 500 species, which are predominantly distributed around the Mediterranean area and in west Asia . White powders that were produced by plant inflorescences named Tootia have been used traditionally for eye infectious diseases  have been screened against human lung, liver, colon and breast cancer cell lines and one normal cell line. This study provides an important basis for further investigation into the isolation, characterization and mechanism of cytotoxic compounds from some of the screened Iranian medicinal plants. Thus, these plants could be used as a source for new lead structures in drug design to combat cancer.This is the first time that methanolic extracts from the 11 listed Iranian plants (


The questions addressed were (i), which changes occur in cell growth and metabolism upon A1AT production and (ii), what are specific cellular properties distinguishing the producer clone AGE1.HN.AAT from the parental cell population.The human designer cell line AGE1.HNications . For furCultivations of the human neuronal cell line AGE1.HN®  were carried out in shake flasks . Extracellular metabolites were quantified using different HPLC methods ,4. A1AT Model for A1AT production was set up using the Kyoto Encyclopedia of Genes and Genomes (http://www.kegg.com) pathway database for Homo sapiens.Fluxes were estimated using standard methods ,5 by incCell growth was similar in the first 5 days. After 5 days AGE1.HN.AAT cells were growing less than the parental cell line and the viability dropped faster. After 2 days dry weight was slightly higher in the parental cell line. A1AT production was low in the first 2 days and then increasing up to a final concentration of ~0.4 g/l in the end of the cultivation. The total cell mass was increasing similarly in the first 4 days. After 4 days AGE1.HN.AAT cell mass remained constant whereas the extracellular protein concentration was increasing. This was different in the AGE1.HN parental cells where the cell mass was still increasing after 4 days and the increase in total extracellular protein mass was less. Intracellular protein concentration was decreasing in AGE1.HN.AAT after 4 days. Specific cellular proteins might be degraded upon nutrient deprivation to maintain the production of the recombinant glycoprotein A1AT. This was clearly different in the parental cell line. Fractions of the biomass constituents RNA, lipid and phosphatidylcholine were increased in AGE1.HN.AAT. The total mass of the analyzed components in the end of the cultivation was similar in the cultivations of both cell lines being around 2.5 g/l. The final A1AT concentration which was 0.4 g/l was ~30 % of the total protein in the culture.Similar time courses for most extracellular metabolites were observed. Glycine and glutamate production was higher in AGE1.HN.AAT whereas uptake of arginine and aspartate as well as alanine production were lower. Glutamine was consumed in the cultivations of both cell lines at ~4 days which resulted also in reduced growth. Especially the increase in glycine and glutamate production points to differences in C1 metabolism and cellular nucleotide demand.The differences that can be seen in the metabolism upon production of the glycoprotein were explained by using an appropriate model of the anabolic demand needed to produce active A1AT. The whole cellular production process of a glycoprotein includes its expression (transcription), synthesis of the amino acid chain (translation), posttranslational modification in endoplasmic reticulum (ER) and Golgi apparatus (Golgi) and secretion of the mature protein  were similar. Differences were observed in C1 and glutamate metabolism including changes in activities of several transaminases. The observed changes in metabolic flux reflect the anabolic differences between producer and parental cells.® cell line.This improved understanding of the metabolism and cellular changes during glycoprotein production supports the identification of targets for further improvement of (i) cell line, e.g., by genetic modifications and (ii) cultivation process, e.g., by improved feeding strategies. The presented data indicate that nucleotide and lipid metabolism might be interesting targets for further engineering of the AGE1.HN


The mouse squamous epithelial cell line HCC7 appears incorrectly throughout the article. The correct cell line is the mouse squamous carcinoma cell line SCC7.


Primary effusion lymphoma (PEL) is a rare KSHV/HHV8-associated high-grade non-Hodgkin's lymphoma (NHL) of B-cell origin, characterized by serous effusions in body cavities. Most patients are HIV-infected men with severe immunosuppression and other HHV8-associated diseases such as Kaposi's sarcoma (KS). The prognosis for those infected is poor, with a median survival of less than 6 months in most cohorts. Sustained complete remission is rare. High-dose chemotherapy regimens are used to improve remission rate and survival. The aim of the present study was to compare the drug sensitivity pattern of the available primary effusion (body cavity based) lymphoma-derived cell lines in order to find additional, potentially effective drugs that are not included in current chemotherapy treatment protocols.We have analyzed 11 cell lines against 27 frequently used cytostatic drugs in short term (3 days) survival assays using automated high throughput confocal microscopy.in vitro used and clinically achieved drug concentration, Vinorelbine, Paclitaxel, Epirubicin and Daunorubicin were the most effective drugs.All cell lines showed a distinct, individual drug sensitivity pattern. Considering the We suggest that inclusion of the above drugs into PEL chemotherapy protocols may be justified. The heterogeneity in the drug response pattern however indicated that assay-guided individualized therapy might be required to optimize therapeutic response. Human herpesvirus 8 (HHV8) or Kaposi sarcoma herpesvirus (KSHV) is the probable causative agent of two distinct lymphoproliferative disorders: primary effusion lymphoma (PEL) and the plasma cell variant of multicentric Castleman disease (MCD) in addition to Kaposi sarcoma (KS) .Primary effusion lymphoma (PEL), or alternatively: body cavity lymphoma is a non-Hodgkin's lymphoma (NHL) of B-cell origin that develops predominantly in the serous body cavities . The lymConversely, PEL cells generally express various markers associated with activation, including CD30 (approximately 75% of cases), CD38, CD71 and the epithelial membrane antigen. Moreover, PEL cells express several plasma cell markers including CD138, VS38c and MUM-1/IRF4 [The prognosis of PEL is poor, as the median survival in the previously published series does not exceed 3 months -10.Given its rarity, however, there are very few longitudinal observational series of patients with PEL and no large prospective trials have ever defined optimal treatment strategies .Prior to the introduction of antiretroviral therapy, the therapeutic results were unsatisfactory in cohorts of HIV+ patients, despite the use of aggressive polychemotherapy regimens including anthracyclines. The significant improvement in the prognosis of AIDS-related lymphomas observed in the antiretroviral therapy era also applies to the PEL setting.In addition, the routine use of growth factors, such as the granulocyte colony-stimulating factor (G-CSF), to avoid prolonged periods of neutropenia resulting from chemotherapy is standard practice for all AIDS-related lymphoma (ARL) patients.Despite the improvement in therapeutical strategies during the last few years, there is no evidence of a cure for PEL patients with conventional systemic chemotherapy addressed to aggressive NHL. The suggested benefit of high-dose Methotrexate in association with CHOP -like regimens is negatively balanced by the hampered toxicity of Methotrexate in the presence of serous effusions .Novel approaches for body cavity lymphoma therapy outside traditional chemotherapy have been suggested as well . These iin vitro [Another approach may be to target NF-κB through the use of proteasome inhibition with drugs, such as Bortezomib that induces apoptosis of PEL cell lines in vitro .In the present study we have investigated 11 different primary effusion (body cavity based) lymphoma-derived cell lines to compare the drug sensitivity pattern, in order to find new potentially successful chemotherapy agents, that are not used in current treatment protocols.The following primary effusion (body cavity based) lymphoma-derived cell lines were used in the present study. CRO-AP/2, CRO-AP/5, CRO-AP/6, BC-2, BC-3 were established from pleural effusion, CRO-AP/3, HBL-6, BC-3, BCBL-1, JSC-1 were established from ascites fluid and BCP-1 from peripheral blood.2. Cultures were fed twice weekly with the above-mentioned medium; maintained at ca. 0.5 × 106 cells/ml. All cell lines were examined daily in their culture vessels under an inverted microscope. Absence of mycoplasma contamination was routinely assessed using staining with Hoechst 33258.Body-cavity cell lines were cultured in IMDM (Sigma), supplemented with 20% heat-inactivated (at 56°C for 45 min) fetal calf serum , 100 IU/ml penicillin (Sigma), 100 μg/ml streptomycin (Sigma) and 2 mM L-glutamine (Sigma). Cell lines were grown at 37°C in the presence of 5% COIn vitro drug resistance of body-cavity cell lines were assessed using a 3-day cell culture on microtiter plates. 27 drugs  values of the individual drugs. For this comparison Quotient of Area Under Curve values (QAUC72 hr) were determined by the following formula:To calculate the relationship between the in vivo AUC72 hr corresponds to the area under curve value achieved in patients under a 72 hours period. The in vivo AUC72 hr was established from the clinical dose and half-time using the standard trapezoidal rule calculation. The in vivo AUC72 hr data is summarized in the seventh column of Table in vivo halftime data are available at the Swedish pharmacological website http://fass.se. A QAUC72 hr value higher than 1 indicates that the in vitro drug concentration is higher than the one used in the clinical practice. If this value is 1, it means that the in vitro concentration corresponds to the clinically achieved in vivo concentration.The in vitro survival assays. The clinical origin and viral status of the individual lines is summarized in Figure We have tested the drug sensitivity patterns of the body cavity lymphoma lines in short term, The summarized cell survival data is shown in Figure Most of the lines were sensitive for sixteen of the 27 drugs where sensitivity was defined as less than 50% mean survival at any of the drug dilutions (effective drugs). If more than half of the cells were alive even at the highest concentration than the drug was considered to be ineffective.We found that the sixteen effective drugs against body-cavity lymphoma were the following in the order of effectiveness: Dactinomycin, Chlorambucil, Paclitaxel, Vinorelbine, Epirubicin, Docetaxel, Daunorubicin, Vinblastin, Doxorubicin, Etoposide, Gemcitabine, Asparaginase, Fluorouracil, Topotecan, Vincristine and Methotrexate.Most body-cavity lymphoma lines were resistant to Oxaliplatin, Bleomycin, 6-Mercaptopurine, Hydroxyurea, Cladribine, Carboplatin, Bortezomib, Cytosine-arabinosid, Prednisolone, Mitomycin and Fludarabin. Although the Oxaliplatin, Cisplatin and Prednisolone drugs were not effective against any of the body-cavity lymphoma lines these drugs show concentration-dependent growth-inhibitory effect on other cell lines or primary tumors in parallel experiments (data not shown) at the same concentration as used in this paper .In order to identify possible co-segregation of the sensitivity patterns of the individual drugs as well as to systematically compare all the lines with each other, we have carried out unsupervised two-dimensional hierarchical clustering of the simplified drug sensitivity data using the Cluster 3.0 program for MacOS X. The results were visualized using the program TreeView . The senin vivo concentrations. In order to analyze the data in relation to the pharmacokinetic behavior of the given drugs we have plotted the mean survival values as the function of the Quotient of the Area Under Curve (QAUC72 hr) values of the particular drugs. The QAUC72 hr values of a drug were created by dividing the calculated in vitro AUC72 hr values by the in vivo achievable AUC values . As shown in Figure 72 hr (close to 1).Absolute drug sensitivity values have relatively little clinical relevance if they are not correlated with clinically achievable 72 hr axis shows that Daunorubicin, Epirubicin, Paclitaxel and Vinorelbine were the most effective drugs . Moreover most body cavity lymphoma lines were sensitive to these drugs. Importantly, Doxorubicin, the only antracyclin drug that is currently included in chemotherapy protocols against body cavity lymphomas showed a rather heterogeneous effect. Two of the eleven lines were highly sensitive for Doxorubicin whereas two were completely resistant at the maximum drug concentration that we could reach in the current assay (QAUC = 0.11). The two lines (JSC-1 and BC-3) were, in general, the least sensitive for chemotherapeutic drugs, however were still sensitive for Daunorubicin, Epirubicin and Vinorelbine.Plotting the mean cell survival for each individual drug against a common QAUCin vitro assay. The calculation of the QAUC72 hr value for the corresponding Dactinomycin concentration however revealed that the concentration that was required for the high killing effect is higher than the levels that are realistically achievable in a patient.Dactinomycin showed the highest killing efficiency in the present 72 hr values, a relative increase in the number of surviving cells was observed as compared to non-treated controls. The survival was above 100% in case of all the 11 lines suggesting that low dose Carboplatin protected from spontaneous cell death.When treating the body-cavity lymphoma cells with Carboplatin at low QAUCIn vitro growing cell lines are the closest model systems available today for studying the biological features of body cavity lymphomas. The cell lines that were used in the present study represent a variety of different origin. The investigation included cell lines established from ascites fluid, pleural effusion or from the peripheral blood of PEL patients. Despite their different origin, the body-cavity lymphoma lines showed a remarkably similar sensitivity pattern for a number of drugs. Only one cell line was highly resistant for most of the drugs (JSC1) whereas two cell lines (BC1 and BCBL-1) showed increased overall sensitivity to most of the drugs.The presented data suggests that, for a number of cytostatic drugs the body cavity lymphoma cell lines share a common cytotoxic drug sensitivity profile. These profiles show no obvious correlation with the biological or clinical features of the lymphomas. Clustering of the drug sensitivity data revealed that the profiles are independent of the EBV status, anatomical localization of the lesion, the age the patient or the rapidity of the progression of the disease. The two cell lines (BCP-1 and BC-3) that arouse from HIV negative patients showed relatively low drug sensitivity.The current treatment alternative is a combination of Methotrexate with CHOP -regimes. The present data showed that the cell lines exhibit varying sensitivity to Methotrexate and Vincristine and are completely resistant to Prednisolone.in vivo.Cyclophosphamide or Ifosphamide were not tested on the body cavity lymphoma lines, because both of these compounds are prodrugs that have to be converted into active metabolites by the liver in vitro [It has been reported that the proteasome inhibitor Bortezomib induces apoptosis of the cell lines BCBL-1 and BCP-1 in vitro . In this72 hr values. Epirubicin required tenfold lower concentration than the in vivo achievable concentration to kill more than 80% of the cells for most of the lines. Epirubicin is primarily used against breast and ovarian cancer, gastric cancer, lung cancer and lymphomas, but has not yet been tested against body cavity lymphomas [In the present study body cavity lymphoma lines showed considerable sensitivity for anti-microtubule drugs and anthracyclins. Importantly all lines were sensitive to Epirubicin and Vinorelbine even at low QAUCymphomas .In summary, the analysis of drug sensitivity profiles of the available body cavity lymphoma lines against 27 commonly used drugs revealed considerable heterogeneity in drug response. Four drugs, namely, Daunorubicin, Epirubicin, Paclitaxel and Vinorelbine showed uniformly high efficiency on the cell lines. These drugs are not yet included in the current chemotherapy protocols of body cavity lymphomas. The heterogeneity of drug response also suggests that optimal care of the lymphoma patients would include the determination of drug sensitivity patterns of the primary tumor samples and that these patients would benefit from assay guided individualized therapy.We suggest that inclusion of the above drugs into PEL chemotherapy protocols may be justified. The heterogeneity in the drug response pattern however indicated that assay guided individualized therapy might be required to optimize therapeutic response.The authors declare that they have no competing interests.in vitro drug sensitivity assays. EF was responsible for measuring the plates using the automated laser confocal fluorescent microscope. LS and EF wrote the computer programs QuantCapture 4.0 and QuantCount 5.0. LM and HS analysed and interpreted the data. SE made comparable the in vitro results with the in vivo data. JK, LG, AC together with the other authors have been involved in the planning of the experimental details, and the drafting and critical reading of the manuscript. All authors read and approved the final manuscript.The project was conceived and designed by LS. The experiments were mainly carried out and/or coordinated by ÖR. LLK, AG and LM took part in cell culturing, and preparation of the microtiter plates for The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/441/prepub


Eliciting broadly neutralizing antibodies (bNAbs) to HIV Env through immunization has been problematic.To better understand the requirements for activation of B cells producing bNAbs, we generated a series of cell lines and transgenic mice expressing such antibodies or selected germline-reverted versions as B cell surface receptors. The bNAb cell lines included those that recognize the CD4 binding site , the MPER of gp41 (4E10), and additional glycan-dependent sites on the trimer . Different Env-containing antigens and virions were tested for the ability to stimulate bNAb cell lines.Mouse strains expressing germline or mutated forms of 4E10 and b12 bNAb Ig genes were generated by gene targeting to the physiological loci. These “knock-in” mice were studied for their B cell development, and responses to HIV immunogens.Many HIV Env antigen preparations, notably including infection-competent pseudovirions, were poorly recognized by high affinity bNAb-expressing cells, as measured by calcium flux assay. However, other antigen forms were highly stimulatory: in particular, soluble gp140 foldon trimers and a multimerized, scaffolded epitope protein.4E10, but not b12 knock-in mice showed signs of abortive B cell development. b12 H mice had gp120-binding cells and responded well in vivo to gp140 trimers.Analysis of bNAb cell line activation suggested that HIV is difficult to recognize by B cells, probably because of the low density of surface proteins. Based on these results, soluble gp140 trimers or epitope scaffolds might offer more promise as vaccine candidates. In knock-in mice, primary 4E10 B cell precursors appeared to be negatively selected, whereas b12 B cells were normal and readily stimulated with gp140 trimers.


It has been recently demonstrated that substance P (SP) and neurokinin-1 (NK-1) receptor antagonists induce cell proliferation and cell inhibition in human melanoma cells, respectively. However, the antitumor action of the NK-1 receptor antagonist L-732,138 on such cells is unknown. The aim of this study was to demonstrate an antitumor action of L-732,138 against three human melanoma cell lines . We found that L-732,138 elicits cell growth inhibition in a concentration dependent manner in the melanoma cells studied. Moreover, L-732,138 blocks SP mitogen stimulation. The specific antitumor action of L-732,138 occurred through the NK-1 receptor and melanoma cell death was by apoptosis. These findings indicate that the NK-1 receptor antagonist L-732,138 could be a new antitumor agent in the treatment of human melanoma. Melanoma is a neoplasm originating from melanocytes, which are derived from the neural crest. It represents 1% of cancers and it accounts for approximately 65% of skin cancer deaths. Melanoma now accounts for approximately 4% of all cancers diagnosed in the United States [in situ, atypical (dysplastic) nevi, and spindle and epithelioid cell (Spitz) nevi has been described, but it was not detected in any acquired benign melanocytic nevi [in vivo and of the proliferation of cultured endothelial cells in vitro, and that SP also induces neoangiogenesis [Substance P (SP) is an undecapeptide that belongs to the tachykinin family of peptides. It is known that SP, neurokinin A (NKA), neuropeptide K and neuropeptide Gamma (the two latter elongated forms of NKA) are derived from the preprotachykinin A gene, whereas neurokinin B (NKB) is derived from the preprotachykinin B gene. The biological actions of SP, NKA and NKB are mediated by three receptors, named neurokinin (NK)-1, NK-2 and NK-3; the NK-1 receptor showing preferential affinity for SP. After binding to the NK-1 receptor, SP regulates many biological functions  as well tic nevi . Moreovetic nevi . It has tic nevi ,10,11 antic nevi ,18,19,20ogenesis . Moreoveogenesis . in vitro [The NK-1 receptor antagonist L-733,060 (a piperidine derivative) showed a high affinity for the human NK-1 receptor in vitro . We havein vitro  and agaiin vitro ,16,19,20in vitro  and exerin vitro . Additioin vitro . It is kin vitro  and it hin vitro . In addiin vitro ,18,19,282, according to the manufacturer’s instruction. We used COLO 858 (ICLC Interlab Cell Line Collection-CBA-Genova), MEL HO and COLO 679 (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen) human melanoma cell lines. These cell lines were incubated at 37 ºC in a humidified atmosphere of 95% air/5% CO50, different concentrations (10 to 100 µM) of L-732,138 were evaluated. SP, acetate salt , was dissolved in distilled water and different concentrations of SP  were used. The most mitogenic nanomolar SP concentration for each cell line was incubated 1 h before the addition of L-732,138.The NK-1 receptor antagonist N-acetyl-L-tryptophan 3, 5-bis (trifluoromethyl) benzyl ester, molecular weight 472.39  , was dissolved in distilled water containing 0.2% dimethylsulphoxide (DMSO) before sample treatment. In order to determine the IC4 cells/0.1 mL), control wells with DMSO, control wells treated with L-732,138 and control wells treated with the most effective SP concentration and L-732,138. The plates were inoculated with L-732,138 (10-100 µM for tumor cell lines) and were incubated for the first doubling time specific for each tumor cell line. Moreover, control wells were treated with different concentrations of SP. For the proliferation assay, 20 µL of the MTS reagent was added to each well 90 min before reading the samples on a multiscanner microplate reader  at 492 nm. Each experimental condition was assayed in duplicate and all experiments were performed at least three times.Cell proliferation was evaluated using the tetrazolium compound 3--5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), according to the manufacturer’s instructions . Cell numbers were quantified using a Coulter counter. The plate included blank wells (0 cells/0.1 mL), control wells . The homogeneity of the variance was tested using the Levene test. If the variances were homogeneous, the data were analyzed by using the one-way ANOVA test with Bonferroni’s correction for multiple comparisons. For data sets with non-homogeneous variances, the ANOVA test with T3 Dunnett posthoc analysis was applied. The criterion for significance was In order to determine whether apoptosis was induced by the NK-1 receptor antagonist L-732,138, DAPI staining was performed. Briefly, after treatment with L-732,138 for their first doubling time approximately, the cells were fixed in 4% paraformaldehyde. Following a second wash in PBS, cells were incubated in 1 μg/mL DAPI solution (Sigma-Aldrich) for 30 min in the dark. The cells were then observed under a fluorescence microscope . Apoptotic cells were defined by the chromatin condensation and nuclear fragmentation. We counted the number of apoptotic cells. In each case, the count was repeated in three different slides. Finally, in each slide, we counted the number of apoptotic cells located in five different sequentially fields. 50 and IC100 growth inhibition concentration of L-732,138 on melanoma cell lines are shown in After the administration of increasing concentrations of L-732,138, we observed growth inhibition in all the melanoma cell lines studied . Moreove50 µM of L-732,138 and nM of SP was lower than that observed with different doses of SP alone for the melanoma cell lines studied here. These results indicate that L-732,138 blocks SP mitogen stimulation. Moreover, L-732,138-induced growth inhibition was partially reversed by the administration of the most mitogenic nanomolar dose of exogenous SP. These data indicate the specificity of the NK-1 receptor activation in the growth of the melanoma cell lines by SP, since an increase in the cellular concentration was observed in the cell lines studied with respect to the values found when the antagonist was administered alone % apoptotic cells for the three melanoma cell lines, whereas at IC100 concentration we found 51.4 ± 4.5 (SD)% apoptotic cells. Conversely, in control melanoma cells  we observed a mean of 0.1 ± 0.2 (SD)% apoptotic cells for the three melanoma cell lines. After administration of L-732,138, a large number of apoptotic cells were found in COLO 858, MEL HO and COLO 679 melanoma cell lines . In DAPIin vitro studies carried out in lung cancer [in vitro and in vivo, the growth of small cell lung cancer [in vivo the growth of glioma and breast carcinoma cells [We have demonstrated for the first time the antitumor action of the NK-1 receptor antagonist L-732,138 in human melanoma COLO 858, MEL HO and COLO 679 cell lines. We have also demonstrated that the cell death observed in this study was due to a specific toxic effect of L-732,138 and not to a non-specific action of this drug. In our competition experiments, exogenous SP cell proliferation was partially reversed by the administration of L-732,138, suggesting the specificity of the NK-1 receptor blocking by L-732,138. Moreover, it has been reported that the above-mentioned melanoma cell lines express several isoforms of the NK-1 receptor [g cancer , rhabdomg cancer , neuroblg cancer ,19,20,28g cancer ,28. In ama cells ,31. This suggests that NK-1 receptor antagonists block the NK-1 receptors expressed by tumor cells and that the structurally very different molecules piperidine L-733,060), L-tryptophan L-732,138) and morpholine (aprepitant) exert the same antitumor action (these molecules only have their specificity for the NK-1 receptor in common). In this sense, it seems that the antitumor action of the NK-1 receptor antagonists is related to stereochemical features and no to the chemical structure of the molecules. In sum, the data suggest the possibility of a common mechanism for cancer cell proliferation mediated by SP and NK-1 receptors. Were this the case, it would mean that NK-1 receptor antagonists L-732,138 could inhibit a large number of tumor cell types in which both SP and NK-1 receptors are expressed [,138 and ,060, L-tin vivo, and on the proliferation of cultured endothelial cells in vitro, as well as it has been demonstrated that NK-1 receptor agonists also induced neoangiogenesis probably through induction of endothelial cell proliferation, and this response is blocked specifically by NK-1 receptor antagonists [via a dual mechanism that involves both antiproliferative and antiangiogenic properties [Moreover, it has been reported the effect of SP, a main mediator of neurogenic inflammation, on the growth of capillary vessels agonists . Furtheragonists ,40. Alsoagonists . In the agonists . In factagonists . Additiooperties . Moreoveoperties . These d50 for fibroblast cells was approximately three times more than the IC50 of the tumor cells [All the data mentioned above suggest that the treatment of melanoma cell lines (expressing NK-1 receptors) with the NK-1 receptor antagonist L-732,138 could improve melanoma treatment, because it exerts an antitumor action through three mechanisms: (1) An antiproliferative effect due to the inhibition of tumor cell growth, inducing cell death by apoptosis; (2) An inhibition of angiogenesis in the tumor mass; and (3) An inhibition of the migration of tumor cells (invasion and metastasis). Moreover, it has been reported that a NK-1 receptor antagonist (the drug aprepitant) was used in a placebo-controlled trial in patients with depression, showing that the safety and the tolerability of this drug were generally similar to placebo . Recentlor cells . Moreoveor cells , analgesor cells , anxyolior cells , antidepor cells , antiemeor cells , hepatopor cells , and neuor cells . We describe for the first time the antitumor action of the NK-1 receptor antagonist L-732,138 on MEL HO, COLO 858 and COLO 679 human malignant melanoma cell lines. Also, we have demonstrated that the antitumor action of L-732,138 is through the NK-1 receptor, and finally, that L-732,138 induces apoptosis in melanoma cell lines. All these observations suggest that the NK-1 receptor antagonist L-732,138 could be a novel and promising antitumor agent in the treatment of human melanoma.


Bisphenol A (BPA) is a chemical compound widely used in manufacturing plastic products. Recent epidemiological studies suggest BPA exposure is positively associated with the incidence of type 2 diabetes mellitus (T2DM), however the mechanisms underlying this link remain unclear. Human islet amyloid polypeptide (hIAPP) is a hormone synthesized and secreted by the pancreatic β-cells. Misfolding of hIAPP into toxic oligomers and mature fibrils can disrupt cell membrane and lead to β-cell death, which is regarded as one of the causative factors of T2DM. To test whether there are any connections between BPA exposure and hIAPP misfolding, we investigated the effects of BPA on hIAPP aggregation using thioflavin-T based fluorescence, transmission electronic microscopy, circular dichroism, dynamic light scattering, size-exclusion chromatography,fluorescence-dye leakage assay in an artificial micelle system and the generation of reactive oxygen species in INS-1 cells. We demonstrated that BPA not only dose-dependently promotes the aggregation of hIAPP and enhances the membrane disruption effects of hIAPP, but also promotes the extent of hIAPP aggregation related oxidative stress. Taken together, our results suggest that BPA exposure increased T2DM risk may involve the exacerbated toxic aggregation of hIAPP. Diabetes is a panepidemic endocrine disease, with approximately 285 million diagnosed patients worldwide Bisphenol A BPA;  is a comRecent epidemiological evidence suggests that a concentration dependent correlation exists between BPA exposure and the occurrence of diabetes. BPA levels have been found significantly higher in both diagnosed diabetic and borderline diabetic patients than those of non-diabetic subjects It is well recognized that environmental factors, including multiple metal ions, polyphenols, fatty acids and certain natural products of small molecule size, can affect the toxic misfolding of hIAPP and may cause diabetes Synthetic hIAPP (1–37) was obtained from Genscript Inc. . Bisphenol A was obtained from Aladdin-reagent . Carboxyfluorescein, thioflavin-T (ThT), 2-Oleoyl-1-palmitoyl-sn-glycerol-3-phospho-rac (1-glycerol) sodium salt (POPG) and hexafluoroisopropanol (HFIP) were purchased from Sigma-Aldrich . INS-1 cells were obtained from the China Center for Type Culture Collection (CCTCC). All other chemicals were of the highest grade available.For all experiments, hIAPP was freshly dissolved in HFIP and vigorously sonicated for 2 min to homogenize the sample. After a short-spin, the solution was diluted to desired concentration in 25 mM sodium phosphate buffer (pH 7.4) containing 50 mM NaCl, and a final HFIP concentration of 1%. Freshly prepared BPA stock solution was then immediately added to desired concentrations,thoroughly mixed and ready for further analysis. The whole preparation process is strictly limited to 5 min.2. Freshly dissolved hIAPP was diluted to a final concentration of 15 µM,. Spectra were obtained from 260 to 190 nm with a 2 nm bandwidth, 1 s response time, 50 nm/min scanning speed and a 1 mm pathlength. Each sample was measured at least three times and the spectra were averaged to give the final result. Spectra of PBS buffer containing corresponding concentrations of BPA were measured as the baselines. The final spectra were obtained by subtracting corresponding baseline spectrum from sample spectrum, which were further converted to mean residue ellipticity [θ] and were analyzed with the software CDPro using the CONTINLL algorithm as previously described CD spectra were obtained with a JASCO-810 spectropolarimeter at 25°C under a constant flow of NFreshly prepared hIAPP solution (15 µM) was incubated at 25°C for amyloid formation in the presence of different molar ratios of BPA. ThT fluorescence assays were preformed on a Hitachi FL-2700 fluorometer to detect the formation of amyloid at designated time points. The final assay solution contains 25 mM PBS (pH 7.4), 50 mM NaCl and 20 µM thioflavin-T The TEM was performed as previously described 2, and samples were dried under vacuum to remove residual chloroform. Multilamellar vesicles were made by mixing dry POPG films with 25 mM PBS (pH 7.4) containing 40 mM carboxyfluorescein. PD-10 columns  were then used to remove nonencapsulated carboxyfluorescein as previously described POPG was dissolved in chloroform at a concentration of 10 mg/mL. Chloroform was then removed under a stream of NThe SEC analysis was performed on a Tosoh TSK GW2000 column . hIAPP was freshly prepared to a final concentration of 30 µM,mixed with different amounts of BPA, and were immediately injected into a Hitachi L-2000 HPLC system, and the column was eluted with a 20% acetonitrile containing 0.003% TFA at a flow rate of 0.3 ml/min as previously described Dynamic light scattering was performed by using a zeta pals potential analyzer . 30 µM hIAPP was measured in a 200 µl cuvette incubated at 37°C with a scattering angle of 90°. The starting time for the very first sample scan was marked as time zero. All of the samples were scanned for three times (4 min/scan) and the mean particle size was recorded and analyzed by the multimodal size distribution (MSD) software.4 cells/well and incubated at 37°C in 5% CO2 atmosphere for 24 h. The medium was then replaced with fresh medium containing hIAPP (5 µM) and varied amounts of BPA for 24 h further incubation. Cells treated with BPA or PBS were used as the controls. For MTT assay, cells were co-incubated with 10 µl MTT (5 mg/ml) per well for 4 h. 100 µl formazan buffer was then added to each well and the absorbance was measured at 570 nm Pancreatic INS-1 cells were cultured in 1640 medium containing 10% FBS, 1% sodiumpyruvat, 1% penicillin-streptomycin solution and 50 µM β-mecaptoethanol. And cells were plated in 96-well plates at a density of 5×102DCFDA  for 20 min at 37°C. The cells were then washed twice with PBS and the levels of ROS were detected by a flow cytometer  with an excitation wavelength of 485 nm and an emission wavelength of 530 nm INS-1 cells were seeded into 6-well plates and treated with or without 10 µM hIAPP and different ratios of BPA for 12 h. The harvested cells were washed by PBS and incubated in 1640 medium containing 10 µM carboxy-HThe Kruskal-Wallis test and the Mann-Whitney test were used to evaluate statistical significance. All results were expressed as the mean ± SD. Difference was considered statistically significant at P<0.05.The transition from random coil structure to predominant β-structure is characteristic of hIAPP amyloid formation Further CDPro deconvolution analysis confirmed the formation of β-structures . CD dataThe existence of β-sheet-rich structure is characteristic of amyloid fibrils Morphology of hIAPP aggregates was observed by TEM. After incubating for 2 h, TEM images of hIAPP with or without 0.5-fold BPA showed no sign of aggregation; scattered short fibrils or protofibrils were found only after 4 h incubation . ClassicThe oligomerization status of hIAPP was analyzed using size-exclusion chromatography (SEC). Freshly dissolved hIAPP showed a monomer peak eluting at 25 min with a calculated MW of 3.9 kD . The samDynamic light scattering (DLS) measurements were then performed to monitor particle size distributions during the aggregation of hIAPP. Gradually increased particle sizes were observed during incubation. The average diameter of native hIAPP monomers was 0.1 nm , the hydThe cytotoxic process of hIAPP fibrillation has been shown to decrease the viability of pancreatic INS-1 cells hIAPP oligomers and mature fibrils have been reported to disrupt β-cell membranes and release intracellular contents, which results in β-cell apoptosis and eventually diabetes It has been well documented that amyloid formation is also associated with the generation of toxic ROS hIAPP has a strong tendency to form toxic oligomers and fibrils that lead to pancreatic β-cell apoptosis and eventually the onset of T2DM It is well established that the toxic hIAPP oligomers can disrupt the islet β-cell membrane and lead to permeabilization. In MTT study, we observed the cytotoxicity of hIAPP on INS-1 cells rose sharply with the addition of BPA . The obsFurther analysis was conducted to distinguish the direct molecular toxic effects of BPA on live cells from its interaction with hIAPP. Dye leakage assays were performed to monitor exclusively the membrane disruption property of hIAPP in the presence of BPA. The hIAPP oligomers have been proven to bind and penetrate membranes more efficiently than monomers and are regarded as an important causative factor to β-cell death Oxidative stress induced cytotoxicity was another mechanism underlying amyloid-related β-cell apoptosis besides direct membrane disruption. Amyloid formation has been reported to associate with ROS generation in vitro. Therefore the system we used may be considered as a model that simulates physiological interactions at accelerating rates. Future biophysical study with novel experimental methods which can tackle hIAPP and BPA interaction at physiological conditions will be important.The influence on human health of BPA exposure is regarded as an accumulative process because of its widespread penetration in daily life In summary, our data provide the evidence that BPA exposure concentration-dependently accelerates the toxic amyloid formation, exacerbates the toxic membrane disruption of hIAPP and promotes the levels of toxic ROS generated by hIAPP in vitro. Our study suggest that in addition to direct biological effect, long-term BPA exposure may also have adverse effects on hIAPP amyloid formation that eventually contribute to the onset of T2DM. The results may provide a new angle on how BPA exposure influences the risk of diabetes from hIAPP aggregation related pathogenesis. Moreover, since BPA also possess other important biological effects including estrogen-like function, it will be interesting to explore the effect of BPA exposure on physiological functions of hIAPP, for example, hIAPP secretion and hIAPP related insulin resistance. In addition to hIAPP, a great variety of amyloidogenic proteins such as amyloid β peptide and α-synuclein, are known to form extracellular amyloid deposits that induce human diseases including Alzheimer's disease and Parkinson's disease Table S1Secondary structure compositions (in %) of hIAPP incubated with different ratios of BPA. The spectra were calculated with the algorithm CONTINLL provided by the CDPro package using SDP42 as the reference set.(DOC)Click here for additional data file.Table S2Amyloidogenic properties of hIAPP incubated with different ratios of BPA.(DOC)Click here for additional data file.Table S3Synergistic effects of BPA and hIAPP.(DOC)Click here for additional data file.Figure S1Aggregation analysis of hIAPP co-incubated with BPA and corresponding BPA control. (A) ThT-fluorescence profile over 24 h incubation. hIAPP concentration was 15 µM. (B) Size-exclusion gel filtration profile. (C) TEM images of samples incubated for 4 h.(DOC)Click here for additional data file.Figure S2Percentages of secondary structure contents of hIAPP in the presence of different molar ratios of BPA as calculated by the CONTINLL algorithm. (A) hIAPP; (B) hIAPP with equal BPA; (C) hIAPP with 5-fold molar BPA.(DOC)Click here for additional data file.


However, the expression of MMP-1 mRNA in the disseminated nodules was significantly increased in the KD cells compared to the SC cells derived from the two parental cell lines (p<0.01). An immunohisto-chemical study further demonstrated that there was stronger staining for MMP-1 in the MKN74-KD in comparison to MKN74-SC cells. Loss of HIF-1α may contribute to the development of aggressive peritoneal dissemination via the upregulation of MMP-1 in gastric cancer cells.This study was performed to clarify the role of hypoxia-inducible factor-1 α (HIF-1α) in the development of peritoneal dissemination in a xenograft mouse model of gastric cancer. HIF-1α knockdown (KD) and control (SC) gastric cancer cells, which were established using the MKN45 and MKN74 cell lines, were studied. The two paired cell lines were directly inoculated into the peritoneal cavity of nude mice. The number and the weight of disseminated nodules were compared between tumors generated from the KD and SC cells. In addition, the molecular mechanism was addressed through analysis of the expression levels of metastasis-related genes. The MKN45-KD cell line demonstrated significantly greater numbers of disseminated nodules and formed a larger tumor mass than the MKN45-SC cell line (p<0.05). MKN74-KD cells also tended to induce a greater number of nodules and to produce those with a heavier weight than the SC cells. An  Peritoneal metastasis is a common type of metastasis, and occurs in 34% of gastric cancer patients with recurrence, even following curative resection of the primary tumor ,2. PeritHypoxia is a hallmark of solid tumor formation, and is an independent prognostic factor in a diverse range of malignant tumors . HypoxiaWe previously established two gastric cancer cell lines with HIF-1α knockdown (KD), MKN45-KD and MKN74-KD . MKN45 was derived from a poorly differentiated adenocarcinoma and MKN74 was derived from a moderately differentiated adenocarcinoma. To analyze the role of HIF-1α in peritoneal dissemination, we established two HIF-1α knockdown sublines  and control sublines  as reported in a previous study (6) were suspended in 100 μl of PBS, and injected on day 0 into the abdominal cavity. Five mice per group were injected with the various cell lines. All of the mice were sacrificed on day 28, and then the number of disseminated nodules and their total weight were measured.The animal protocols were approved by the Animal Care Committee of Saga University. Female athymic BALB/cA Jcl mice  were obtained from Nihon Crea Co. . They were maintained under specific pathogen-free conditions and were given γ-irradiated food and autoclaved water. We examined the peritoneal dissemination using the four gastric cancer cell lines, MKN45-SC and -KD, and MKN74-SC and -KD. To generate the xenograft model, cancer cells , converted to cDNA using a ReverTra Ace reverse transcription reaction kit  and subjected to real-time quantitative reverse transcription (RT)-PCR using Light Cycler FastStart DNA Master™ SYBR Green I kit and a Light Cycler™ instrument system (Roche)  . The mRNin vitro adhesion assay was carried out using the CytoSelect 48-well cell adhesion assay extracellular matrix (ECM) array  according to the manufacturer’s instructions. Another adhesion assay using the monolayer mesothelium was performed as described below. The mesothelial cell line, MeT-5A (CRL-9444), was purchased from the American Type Culture Collection . A total of 2x104 MeT-5A cells were added in 100 μl of medium to each well of a 96-well plate. The plate was incubated at 37°C under normoxic conditions overnight. Then a mesothelial monolayer was established. Each well was washed with PBS to remove non-adherent cells, then 2x104 cells of each gastric cancer cell line were added to 100 μl of medium to each well. The plate was then incubated at 37°C under normoxic conditions for 5 h. Each well was washed with PBS to remove non-adherent cells, and then the MTT assay was performed using a Cell-Titer 96™ nonradioactive cell proliferation assay kit . The adhesion index of the mesothelium was calculated using the following formula: Adhesion index = (Adherent cells in well)/(Seeded cells in well).To quantify the tumor cell adhesion to the extracellular matrix and the monolayer mesothelium, an adhesion assay was performed. The The immunohistochemical staining of MMP-1 and MMP-11 was performed according to a previous report . The antThe statistical analysis was carried out using the SPSS 1.5 statistical software package for Windows (SPSS Japan Inc.) A P-value <0.05 was considered to be indicative of statistical significance.All four gastric cancer cell lines, MKN45-SC, -KD, MKN74-SC, -KD, resulted in peritoneal dissemination in nude mice . The differences in the development of peritoneal dissemination between the SC and KD cell lines were compared using the MKN45 and MKN74 cells. Regarding the MKN45 cell lines, the HIF-1α KD cell line resulted in significantly greater numbers of disseminated nodules and a heavier weight of nodules than the SC cell lines  and hypoxic (KD/SC ratio: 1.96) conditions . In the The mRNA expression level of MMP-1 in the disseminated nodules in nude mice was analyzed. The expression was significantly increased in the nodules derived from the KD sublines compared to those from the SC sublines for the MKN45 and MKN74 cell lines . HoweverPeritoneal metastasis is the most common cause of tumor progression in gastric cancer. When the dissemination of multiple peritoneal nodules are obvious, curative surgery with a standard gastrectomy cannot be achieved. In addition, peritoneal metastasis exhibits resistance against various chemotherapeutic drugs, and causes massive ascites and intestinal obstruction. A previous study reported that 50–60% of gastric cancer patients with serosal invasion following curative resection eventually developed peritoneal metastasis  are believed to mediate a number of physiological and pathological processes, such as the degradation of the extracellular matrix, tissue remodeling, inflammation, tumor invasion and metastasis. The present study focused on the expression of MMP 1, 7 and 11, since several studies previously reported the relationships between the MMP members and invasion or dissemination in gastric cancer –21. For et al  reportedet al ,23. Takeet al reported that hypoxic stress accelerated cancer invasion by upregulating the expression levels of MMP-7 and -14 by a HIF-1α-independent pathway (et al demonstrated that treatment with HIF-1α siRNA resulted in the downregulation of MMP-2 mRNA under hypoxic conditions in all of the glioma cell lines examined (Knocking down HIF-1α, which leads to MMP-1 upregulation, might contribute to the degradation of the extracellular matrix of the peritoneum, allowing the invasion of cancer cells and the formation of peritoneal metastasis. However, contrary to this result, Miyoshi  pathway . Fujiwarexamined . ConsideIn conclusion, the present study demonstrates for the first time that the loss of HIF-1α contributes to the development of aggressive peritoneal dissemination by upregulating MMP-1 in gastric cancer cell lines. Therefore, HIF-1α expression might be suppressed during the development of peritoneal dissemination from primary gastric cancer.


In cancer research, cell lines are used to explore the molecular basis of the disease as a substitute to tissue biopsies. Breast cancer in particular is a very heterogeneous type of cancer, and different subgroups of cell lines have been established according to their genomic profiles and tumor characteristics. We applied GCMS metabolite profiling to five selected breast cancer cell lines and found this heterogeneity reflected on the metabolite level as well. Metabolite profiles of MCF-7 cells belonging to the luminal gene cluster proved to be more different from those of the basal A cell line JIMT-1 and the basal B cell lines MDA-MB-231, MDA-MB-435, and MDA-MB-436 with only slight differences in the intracellular metabolite pattern. Lactate release into the cultivation medium as an indicator of glycolytic activity was correlated to the metabolite profiles and physiological characteristics of each cell line. In conclusion, pantothenic acid, beta-alanine and glycerophosphoglycerol appeared to be related to the glycolytic activity designated through high lactate release. Other physiological parameters coinciding with glycolytic activity were high glyoxalase 1 (Glo1) and lactate dehydrogenase (LDH) enzyme activity as well as cell migration as an additional important characteristic contributing to the aggressiveness of tumor cells. Metabolite profiles of the cell lines are comparatively discussed with respect to known biomarkers of cancer progression. It was shown to be distinguishable, for example, according to cell differentiation grade, histological origin, or gene expression profile ,2,3. Recet al.  and Enge [et al.  compared [et al.  comparedholipids ,9. Metabholipids .et al. [Differential assessment of metabolite profiles from tumor cell lines could be an important tool in cancer research, but is not widely applied. Recent comparative studies of breast cancer cell lines using other methods reported a large variance regarding primary energy metabolism . The glyet al.  showed tet al. [The glycolytic activity of tumor cells has been connected to changes in mRNA expression of enzymes related to glycolysis such as lactate dehydrogenase (LDH)  and glyoet al.  showed tet al. .Based on this, we aimed to differentially assess the known constituents of the molecular phenotype of cancer in cell lines used for cancer research with respect to their known tumor characteristics displayed in In summary, we present a comprehensive characterization of five widely used breast cancer cell lines, namely MCF-7, MDA-MB-231, MDA-MB-435, MDA-MB-436, and JIMT-1, using biochemical and physiological methods. Within this study, metabolite profiles were related to physiological characteristics of these cell lines such as cell migration and selected enzyme activities. Metabolite patterns were comparatively discussed addressing (i) differences in genetic subgroups of the cell lines , (ii) me®-Mark, Rotiphorese Gel 30 SDS Ultra-Pure and TRIS were from Carl Roth , Coomassie Brilliant Blue R250, N,N,N’,N’-tetramethylethylenediamine (TEMED), 3,3-diaminobenzidine-4-hydrochloride (DAB) and β-mercaptoethanol were from Serva ; RPMI 1640 medium (cat. no. 21875-034), Dulbecco’s modified Eagle medium (DMEM) supplemented with 4.5 g glucose/L (cat. no. 41966-029), Opti-MEM®, fetal calf serum (FCS) (cat. no. 10500-064), glutamine (cat. no. 25030-024), penicillin/streptomycin  (cat. no. 15140-122) and trypsin/ethylenediamine-tetraacetate (EDTA) were from Life Technologies , hematoxylin was from Merck , milk powder from Heirler Cenovis , bovine serum albumin from PAA Laboratories . Tumor cell lines used were the human breast cancer cell lines MCF-7 (DSMZ ACC115), MDA-MB-231 (ATCC HTB-26), MDA-MB-435 (DSMZ ACC 65), MDA-MB-436 (ATCC HTB-130), and JIMT-1 . N-methyl-N-trifluoroacetamide (MSTFA) was purchased from Macherey-Nagel , pyridine and methoxyamine hydrochloride were from Fluka , methanol was from VWR , and trypan blue and phosphate-buffered saline (PBS) from Seromed . All other chemicals were from Sigma-Aldrich  and all additional cell culture material from Greiner Bio-One .Reduced glutathione (GSH), protein marker Roti2 using an incubator . Except for JIMT-1, each cell medium contained penicillin/streptomycin . Cells were grown in T25 or T75 cell culture flasks, or in 24-well plates depending on the experimental conditions.MCF-7 and JIMT-1 cells were cultured in RPMI 1640 medium, MDA-MB-231, MDA-MB-435 and MDA-MB-436 cells were cultured in DMEM. Medium was always supplemented with 10% fetal calf serum (FCS) and 2 mL glutamine (2 mM). All cells were grown at 37 °C in a humidified atmosphere containing 5% COEnzymatic cell disruption by trypsinization, and direct extraction of cells from 24-Well plates was carried out according to .Cell count and viability was determined by diluting the cells 1:1 with trypan blue (0.4% w/v in PBS) and counting in a Neubauer counting chamber using a light microscope .et al. [™ according to the manufacturer’s instruction. Briefly, cells were seeded in 6-well plates and cultured over night until 50%–70% confluence. Cultivation medium was replaced by Opti-MEM and cells were transfected immediately with a mixture of 4 µg DNA in 1 mL Opti-MEM and 6 µL TurboFect™ incubated previously for 20 min at room temperature. Cells were incubated 5 h before Opti-MEM medium was replaced by normal cultivation medium. After two days, cells were harvested by trypsinization, split to about 20% confluence and positively transfected cells were selected using Zeocin (250 µg/mL) and EGFP fluorescence. Glo1 protein content and Glo1 enzyme activity were tested in the resulting MCF-7 siGlo1 mutants and mock vector transfected cells.Human GLO1 specific shRNA was designed as 63-mer containing a hairpin-loop and cloned into pSuper vector with the H1 RNA polymerase promoter. The vector described in van de Wetering et al.  with an Methanolic extraction of metabolites from cell samples, derivatization and GCMS analysis were performed as described previously . MetabolWe created two datasets with n = 6 each addressing the issue of biological variance as well as fast turnover of metabolites enhancing the robustness of our results. Thus, we used one trypsinized sample batch (sample set 1) that was consecutively harvested over a time period of 6 weeks (one replicate per week for each cell line). All analyses were performed within each replicate set from the same cell pool . The second sample batch (sample set 2) was directly extracted from the well plate. In this case, analysis of all parameters from the same cell pool was not feasible but extraction allowed for faster quenching of cellular metabolism.An aliquot of cells from each cell line was seeded into 24-well plates. After 24 h, the medium was changed, the pH value of the control medium and the pH-value in the cultivation medium of cells at 48 and 72 h, respectively, was determined using a pH-electrode . The pH decrease in the cell medium at each time point was calculated for each cell line and expressed as the mean difference (n = 6).2O2 as substrate for HPR reaction, or by chemiluminescence  according to the manufacturer’s instructions.Cytosolic protein extracts were prepared as described in  and protLDH activity measurement was carried out as described previously , Glo1 ac® reagent. RNA concentration of the pellet resuspended in 50 µL DEPC water (0.1% v/v diethylpyrocarbonate) was determined at 260 and 280 nm using a nanodrop spectrometer ND 1000 . Reverse transcription of RNA from the different cell lines was performed using GoScriptTM Reverse Transcription System  according to the manufacturer’s instructions and 1 µg RNA for each sample. Reaction was performed in a thermal cycler PTC 200  for 1 h at 42 °C and 10 min at 70 °C. cDNA was stored at −80 °C until further use. Semi-quantitative PCR for Glo1 and β-actin was performed using GoTaq® Flexi DNA Polymerase  according to the manufacturer’s instructions using 5 µL cDNA per sample. The PCR program for Glo1 was as follows: denaturation 5 min at 95 °C, 20 cycles 30 s at 95 °C, 1 min at 68 °C, 1 min at 72 °C and, finally, 5 min at 72 °C using 5'-AGGGCATCAATCAGCTCAAC-3' as forward primer sequence (fw) and 5-CCAAGAGCACAATGGTCAAA-3 as reverse primer sequence (rv), β-actin primer were 5'-AGAAAATCTGGCACCACACC-3' (fw) and 5'-CTCCTTAATGTCACGCACGA-3' (rv). 20 µL PCR product and 8 µl of a 100 bp DNA ladder reference standard  were loaded onto separate lanes on a 1% agarose gel containing ethidium bromide and electrophoresis was run at 100 V for 30 min. GLO1 knockdown in MCF-7 siGlo1 cells was confirmed by qRT-PCR according to the method described in Lindner et al. [Total RNA of different breast cancer cell lines was isolated using TRIzolr et al. . GLO1 mRr et al. .Cell migration was assessed using the Boyden chambers assay according to  using a Peak identification was performed as described before , using aMetabolite profiles of the five breast cancer cell lines  with different origin and different molecular characteristics  were comp < 0.05; mean ratio > 2) a principal component analysis (PCA) was performed, shown in All identified metabolites were assessed for significant differences between the replicates of any two cell lines of the data set with Student’s t-test. Using the metabolites with significant differences between at least two cell lines (The metabolite profile of MCF-7 cells exhibited the largest difference to the other tested cell lines (illustrated exemplarily for sample set 1). All other cell lines did not separate from each other. The higher variation observed for MCF-7 replicates may be due to the stronger attachment of MCF-7 cells to the cultivation flasks, resulting in a higher variation of the sampling process in comparison to the other cell lines.Kyoto Encyclopedia of Genes and Genomes (KEGG) [All identified analytes that were significantly different between MCF-7 and all other cell lines (Student’s t-test) with the same intensity pattern  in both sample batches, are listed in the online resource see the . We founs (KEGG) . Among ts (KEGG) .de novo synthesis in tumor cells with early metastasis [Based on the number of listed metabolites in tastasis . Higher tastasis  and incret al. [N-acetyl aspartate, lactate, and cystathione increased the most in the tumor cells. However, according to our results cystathione was highest in MCF-7 although MCF-7 is the least tumorigenic cell line. Notably, differences among tumor cell lines according to their tumor characteristics are not necessarily in agreement with a comparison of malignant vs. non-malignant cells. Yang et al. [myo-inositol to be increased in the malignant cell line, which was also higher in the more tumorigenic cell lines in our analysis. We further found that the relative intensities of two fatty alcohols (C16 and C18) were much higher in MCF-7 and observed different relative intensities of several amino acids and pantothenic acid (PA) compared with the other cell lines  and 4-aminobutyric acid . The difference between basal A and basal B was suggested to correspond to the epithelial-mesenchymal transition (EMT) . Gene clFinally, our data address the issue of the biological variance within datasets and independent replicate measurements in metabolomics research. This becomes obvious when comparing thep-values for sample sets 1 and 2, in which all replicates were harvested the same day; lower p-values were found with sample set 2. In sample set 1, some of the listed metabolites were not detected in enough samples or the differences were not significant. Also, the relative intensities of one metabolite in the five cell lines were sometimes different in sample set 1 and sample set 2. Biological variance was shown before to be about twice the intraday variance . Thus, ip < 0.05) indicating possible changes in energy metabolism. The relative intensities of these metabolites are listed for all cell lines compared to MCF-7 as a reference in online resource  the investigated cell lines may have different metabolic fluxes through glycolysis, which is (ii) not necessarily reflected by concentrations of the intracellular glycolytic metabolites.We assessed activities of enzymes related to glycolysis to further investigate the differential pattern of glycolytic flux among the cell lines, suggested by differential lactate concentration in the medium. In addition to hints on higher activity of PK observed with higher glycolytic activity (data not shown), LDH and Glo1 enzyme activities exhibited a similar pattern compared to the observed lactate release C. LDH cap < 0.05) as indicated in p = 0.023, We additionally determined Glo1 activity as an enzyme whose activity is assumed to be related to the glycolytic shift of tumor cells . The glyet al. [This suggests that Glo1 activity may indeed be enhanced in relation to glycolytic activity in agreement with the assumption that enhanced Glo1 activity is needed for detoxification from toxic by-products of this process. Upregulation of Glo1 in cancer cells was shown previously in various cancer tissues including breast cancer ,44,45. Bet al.  found anet al. , a correp = 0.006, n = 15). These results suggest differential regulation of Glo1 on mRNA expression rather than on protein level between the cell lines.The relation of LDH and glycolysis and the regulation of LDH as one of the enzymes controlling the switch between glycolysis and oxidative phosphorylation  is well Beside its relation to glycolytic activity , lactateet al. [Cell migration was previously found to be related to the glycolytic flux of tumor cells . Recentlet al.  found a et al.  and in Het al. .To confirm the observation that Glo1 activity was indeed positively correlated to cell migration, we measured cell migration of a previously created stably transfected Glo1 knockdown clone  using MCF-7 as wild-type cells (MCF-7 wt). Cell migration, lactate release and LDH activity were also significantly decreased in siGlo1 cells while no difference could be observed in a mock transfected control (data not shown). Taken together, we found a comparably high glycolytic flux combined with high cell migration in the three cell lines MDA-MB-231, MDA-MB-435 and JIMT-1, while MCF-7 and MDA-MB-436 showed low lactic acid release and also lower cell migration.Knowing that Glo1 and LDH activity as well as cell migration coincide with lactate release, we wanted to test whether relative intensities of any intracellular metabolites would also correlate with the glycolytic activity in the cell lines. Therefore, Pearson correlation coefficients and significance levels were calculated for both sample sets between all detected metabolites and lactate release, Glo1 and LDH activity and cell migration. Beta-alanine is a direct precursor of PA which is needed for the synthesis of coenzyme A (CoA). CoA acts as an acyl group carrier to form acetyl-CoA and other related compounds as a means to transport carbon atoms within the cell. Further, in the TCA cycle, CoA is important for pyruvate to enter as acetyl-CoA, and for α-ketoglutarate to be transformed to succinyl-CoA. CoA is involved in the biosynthesis of many important compounds such as fatty acids, cholesterol, and acetylcholine.et al. [de novo to continually provide lipids for membrane production; synthesized fatty acids are used for energy production through β-oxidation and for lipid modification of proteins [According to KEGG pathways, PA and GPG are not in the direct “neighborhood” of glycolysis or the TCA cycle. Slyshenkov and colleagues  showed pet al.  showed tproteins .Thus, in summary we suggest higher concentrations of extracellular lactate, beta-alanine and PA and enhanced LDH and Glo1 activity to be a response to enhanced aerobic glycolysis in cancer cells, while GPG concentration may be associated with enhanced cell migration. We found that this was reflected in comparisons of tumor cell lines with different aggressiveness. Cancer cell lines exhibiting different intensities of one or the other parameter can therefore be a valuable resource to study the interconnection and, potentially, the regulation of these parameters.Non-targeted metabolite analysis identified significant differences between the breast cancer cell lines under investigation. The metabolite profile of MCF-7 as the only luminal-type and less invasive cell line, differed the most from the four other cell lines tested. Our results emphasize the potential of metabolite profiling as a non-targeted approach for classification of known cancer subtypes. We further show that a higher abundance of metabolites in a tumor cell line compared to a non-tumor cell line is not necessarily reproduced comparing highly tumorigenic cell lines to low tumorigenic ones, though it is highly suggestive. Concluding from this, metabolites with different concentrations in comparison of a non-tumorigenic with a tumorigenic cell line could also be specific for the used cell line only without general relevance as tumor markers. Within this context, the genetic subtype of the cancer cell line might be a particularly critical parameter.Considering our results on metabolic changes with known relevance to tumorigenesis, we found enhanced glycolytic activity as indicated by lactate release accompanied by increased LDH and Glo1 enzyme activity. In addition, glycolytic activity in the cancer cell lines was associated with their migration behavior. To our knowledge, this is the first report on a relation between LDH, Glo1 activity and cell migration in human breast cancer cell lines. We found higher Glo1 and LDH activity to be associated with enhanced glycolytic activity and cell migration and, therefore, with tumor aggressiveness.By correlating metabolite profiles to lactate release, Glo1 and LDH activity, and cell migration, we identified beta-alanine, pantothenic acid and glycerophosphoglycerol as potentially important metabolites of malignancy. These particular metabolites have not been associated to glycolytic activity or cancer cell migration in any previous study.