Cell banking of initial outgrowths from newly derived human embryonic stem cells (hESCs) requires an efficient freezing method. Vitrification is used for the preservation of gametes and early embryos in assisted reproduction techniques (ART). Moreover, vitrification was applied for cryopreservation of hESCs using open pulled straws.To derive and characterize new hESC lines and then use Cryotech and Cryowin tools for their vitrification.Human ESC lines were generated in a microdrop culture system using mouse embryonic fibroblasts (MEFs) as the feeder layer; this was later scaled up using both MEFs and Yazd human foreskin fibroblasts batch 8 (YhFF#8). To bank the cell lines, master cell banks of 100 Cryotech and Cryowin tools were produced for each individual cell line using the vitrification method; flasks of hESC lines were also cryopreserved using a conventional slow-freezing method.OCT4/POU5F1, NANOG, and SOX2) and markers such as SSEA4, TRA-1-60, and TRA-2-49. Their in vitro capacity to differentiate into germ layers and germ cells using embryoid body (EB) formation and monolayer culture was assessed by screening the expression of differentiation-associated genes. The chromosomal constitution of each hESC line was assessed by G-banding karyotyping.The pluripotency of cell lines was assessed by their expression of pluripotency-associated genes (Cryotech and Cryowin tools used to vitrify new hESCs at an early stage of derivation is an efficient method of preserving hESCs. Using immunosurgery (2), mouse ESCs (mESCs) were first derived in 1981 . Later, other groups made unsuccessful attempts to generate human ESCs (hESCs) (5), followed by generation of ESCs in primates , and eventually, in 1998, hESCs were derived (8). This coincided with the advent of their putative counterpart, human embryonic germ cells (hEGCs) (9). Typical hESC morphology is an initial characteristic that helps in the derivation process. The morphology of hESCs is very similar to pluripotent embryonal carcinoma cells (ECCs), with a high ratio of nuclei to cytoplasm, pale nuclei, and prominent nucleoli (10). Four major factors play a critical role in the hESC derivation process. (a) The source and stage of pre-implantation embryos from cleavage (11) to blastocyst (8). Recently, it was shown that embryos obtained following in vitro embryoid body (EB) formation or by in vivo teratocarcinoma formation, to investigate further differentiation potential (21). The chromosome content of the cell line is another issue that can be evaluated by G-binding or the CGH-array method (22). One of the challenges in banking any cell type is the method of freezing used. The use of an ideal method for cryopreservation can improve the survival rate and proliferative capacity of post-thawed hESCs (23). Studies have demonstrated that fewer than 5% of hESCs survived an equilibrium slow-freezing procedure using 10% dimethylsulphoxide (DMSO) in fetal calf serum; in contrast, high viability among hESCs was reported when using a vitrification procedure for the cell lines using an “open pulled-straw” method with a small volume of cells (13). Vitrification is a state-of-the-art method used for the freezing of a small number of cells, including gametes and embryos, and is used for the cryopreservation of hESCs using an open pulled-straw method (13). Vitrification is also a good choice of method to use shortly after the derivation of hESCs that are in urgent need of cell line preservation (23).Following their initial derivation, hESCs must be cryopreserved and expanded for further characterization of specific gene and marker expression to assess their undifferentiated status (13). In addition, their capacity to differentiate into the three germ layers  and germ cells, to demonstrate their pluripotency, should be evaluated, either by Here, we report the vitrification of new outgrowths to save newly derived hESC lines (Yazd1-3) using Cryotech and Cryowin tools. Whole, zona-free blastocysts were cultured on an MEF feeder layer in microdrop culture. The purpose of this study was first to derive and characterize new hESC lines and then using Cryotech and Cryowin tools for their vitrification .Chemicals were purchased from Sigma Aldrich . Culture media and supplements were purchased from Invitrogen and Gibco (UK), unless otherwise stated.The vitrified donated embryos (n = 10) were warmed as described elsewhere (24) and cultured in a microdrop system with G series medium  plus 5% human serum albumin (Vitrolife) till getting to the blastocyst stage. The fresh donated embryos were cultured in the same culture medium for in vitro blastocyst development.et al., unpublished data). MEFs and YhFFs#8 were mitotically inactivated using Mitomycin-C (by adding 2 mg Mitomycin-C to 200 ml DMEM/10% FBS) for 2 and 4 hrs, respectively. After washing three times with Dulbecco's phosphate buffered saline (D-PBS), cells were detached using 1 x trypsin/EDTA, centrifuged at 1000 rpm for 5 minutes, and then re-plated in 50 µl drops onto dishes  at a concentration of 3,500 cells per 50 µl and covered with mineral oil . The following day the microdrop medium of feeders was replaced with KOSR/HES medium  and left for 2 hrs for conditioning prior to use. Each dish was plated with seven microdrops of feeder layer.MEFs were derived from Naval Medical Research Institute (NMRI) mouse embryos according to ethical guidelines relating to animals and cultured as described elsewhere (25). Briefly, 13 days after the appearance of the vaginal plug, fetuses were recovered from the uterus and their heads, spinal cords, and livers were removed. Following enzymatic and mechanical treatment, the resulting cell suspension was transferred to a T25 tissue culture flask containing Dulbecco's Modified Eagle's Medium (DMEM), 10% fetal bovine serum (FBS), and antibiotics, then incubated at 37°C in 5% COTo obtain intact zona-free blastocysts, and based on the grade of each individual blastocyst, embryos were either cultured to fully hatched blastocyst stage or were treated with pronase  to lyse the zona pellucida. The intact zona-free blastocysts were plated onto the microdrops of MEF feeders. Yazd1 cells were initially generated using VitroHES culture medium (ready-to-use from Vitrolife), Yazd2 cells were derived and expanded in KOSR/HES medium, while for Yazd3 cells were, the blastocysts were initially cultured in DMEM/10% FBS overnight, and the following day the medium was exchanged for KOSR/HES medium. Small modifications to the culture conditions for the derivation of each individual Yazd hESC line (Yazd1-3) are shown in Table I.All three cell lines were derived using intact embryo co-culture on a microdrop of MEF feeders. All lines were later expanded on both MEFs  and Yazd HFFs (YhFF#8). Yazd1 cells were generated from a fresh embryo, which was cultured to the blastocyst stage (grade 3CB) in VitroHES medium. Yazd2 cells were derived from a frozen-warmed embryo, which was allowed to develop to the early cavity stage (grade 1) on KOSR/HES medium. Yazd3 cells were generated from an embryo which lost its zona during the freezing and thawing process and was allowed to develop to the blastocyst stage (grade 2). All cell lines were expanded using KOSR/HES medium. Yazd1 and Yazd2 hESC lines contained normal karyotypes ; however, the Yazd3 karyotype was initially a triploid 69, XXY.To establish the Yazd1-3 hESC lines following initial hESC outgrowth formation, the cells were mechanically passaged using a mouth pipette and pulled Pasteur pipettes. To detach the colonies, they were first treated with collagenase type IV (1 mg/ml) at 37°C in 5% CO-3 hESC lines, according to the scale of the culture. Colonies within the microdrops for master cell-banking were cryopreserved using Cryotech and Cryowin tools  for the vitrification method (13). For larger scale cultures, such as central well dishes or tissue culture flasks , at later passages colonies were frozen using a slow-freezing solution consisting of 10% DMSO in FBS. In summary, for vitrification, small pieces  of hESC colonies were scraped off and placed into a small drop of KOSR/HES medium. Then, colonies were transferred into homemade vitrification solutions 1 and 2 (VS1 and VS2) containing ethylene glycol and DMSO, first at 10%, then at 20%, respectively, in basal medium . Small colonies were drawn onto the Cryotech and Cryowin tools, plunged into liquid nitrogen, and then transferred to long cryovials, which were sealed with a cap located in the cryocane and placed in a liquid nitrogen storage container. For thawing, a Cryotech or Cryowin was taken from the liquid nitrogen container tank and then placed into a drop of KOSR/HES medium. The cells were washed five times by transferring them through more drops before being placed onto microdrops containing feeders in KOSR/HES medium and incubated at 37°C at 5% COTwo different methods were applied for freezing the Yazd1The identification of pluripotency-associated cell markers was carried out using IF localization, as previously described (26). In summary, Yazd1-3 hESC colonies were washed with PBS containing 1% FBS before being fixed in cooled methanol  and differentiated colonies in the monolayer culture system (after 7 days) were collected. The resulting pellets were suspended in 1 mL TRI reagent (Sigma) and total RNA was extracted according to the standard protocol supplied by the manufacturer. Total RNA was treated with DNase I  to remove genomic DNA. First-strand cDNA synthesis was performed using the Superscript II reverse transcriptase system . PCR was performed using the prepared cDNA and primers for different genes (Table III) by Platinum Blue PCR Super Mix  in triplicate. PCR cycling conditions were as follows: 5min at 95°C for initial denaturation, followed by 40 cycles of 45 sec at 95°C, 45 sec at 59°C-65°C (see Table III), and 45 sec at 72°C.To investigate the chromosomal content of Yazd1-3 hESC lines, the karyotype of ten cells in metaphase of each Yazd cell line was determined by a standard G-banding procedure. Briefly, cells were cultured in flasks without feeder for 5 to 6 days to form mesenchymal-like cells (27). Following treatment with colchicine (10 µg/ml), harvested Yazd hESC-derived cells were stained using a standard G-banding technique. G-bandings were analyzed under light microscopy  using applied spectral imaging software.In vitro differentiation of Yazd1-3 hESC lines was performed as previously described (13), using EB formation and gene expression profile assessment after 4, 7, and 14 days for mesoderm, endoderm, ectoderm, and germ-cell development. The same was done for spontaneously differentiation in the monolayer culture with DMEM/10% FBS (after 7 days).Sixteen fresh (n = 6) and frozen (n = 10) supernumerary embryos were donated for research by infertile couples after they had provided full, written, informed consent according to the Shahid Sadoughi University of Medical Sciences Ethical Committee (ethics committee reference number: IR.SSU.REC.1394.104). For research purposes, all embryos were coded to ensure patient anonymity.in vitro, the zona pellucida was lysed as described above, and plated onto a microdrop of MEFs  in KOSR/HES medium. The first outgrowths after two and three days were as shown in Figure 3B and C, respectively. As indicated in Figure 3C, a population of cells were overgrown, resembling putative hEGC clusters (28), which were distinguished from these areas after several passages of hESC colonies . To derive Yazd3 cells, a frozen embryo donated for stem cell research was thawed. During thawing the zona pellucida was destroyed and the zona-free embryo was allowed to develop to blastocyst stage (grade 2) in vitro, plated onto a microdrop of MEFs , and then incubated overnight in DMEM/10% FBS. On the following day attachment of the embryo onto the feeder layer and the medium was replaced with KOSR/HES. The first outgrowths after two and five days were as shown in Figure 4B and C, respectively. Following passaging, as previously described (13) and explained above, for Yazd2 cells a compact area  was distinguished from other parts of the colony from which, in later passages, typical hESC colonies were generated . Summary information of modifications to the techniques used for derivation of Yazd1-3 hESC lines is shown in Table I.In the current study, sixteen fresh (n = 6) and frozen (n = 10) donated surplus human embryos were used to generate Yazd1-3 hESC lines using a microdrop culture system (13)  and MEFs as feeder layer cells. The pre-implantation human embryos  were used as described above following pronase treatment (for Yazd1 and Yazd2) in a 50 μl microdrop co-culture system . The Yazd1 cell line was produced from a fresh 3CB full blastocyst donated for stem cell research , which was plated onto a microdrop of MEFs . After two days, the outgrowth of cells was observed . Following eight passages, colonies with a typical hESC morphology were obtained . The Yazd1 cell line was derived and initially expanded using VitroHES (Vitrolife) culture medium. For the generation of the Yazd2 cell line, a frozen-warmed embryo, which had been donated for stem cell research was developed to the early blastocyst stage (grade 1) in vitro (13).All Yazd cell lines (1-3) were initially derived and expanded on MEFs . Later, YhFFs#8  and Matrigel  were applied to maintain undifferentiated Yazd hESCs in culture. Half of the KOSR/HES medium was replaced with fresh medium each day. Non-adherent petri-dishes with EB medium were used to produce EBs  to induce spontaneous differentiation NANOG, SOX2, and OCT4/POU5F1  determined by RT-PCR and contained normal 46, XX karyotypes . Yazd2 hESCs (passage number 8+38+10) displayed expression of pluripotency-associated markers such as SSEA4 , TRA-1-60 , and TRA-2-49  and genes such as NANOG, SOX2, and OCT4/POU5F1 , as assessed by IF and RT-PCR, respectively. The initial karyotype of Yazd2 hESCs was normal, 46, XY, as revealed by a G-binding procedure . The undifferentiated Yazd3 hESC colonies (passage number 68) were characterized by SSEA4 , TRA-1-60 , and TRA-2-49  using IF, and NANOG, SOX2, and OCT4/POU5F1 using RT-PCR . Yazd3 was an abnormal hESC line with a karyotype of 69, XXY in its early passages .Yazd1 hESCs (passage number 7+5+10) showed positive expression of pluripotency associated markers, SSEA4 , TRA-1-60 , and TRA-2-49 , as assessed using an IF technique. Undifferentiated Yazd1 cells expressed the pluripotency genes in vitro using different EB formations  and monolayer cultures , with assessment of the expression of specific genes for the three germ layers and germ cells. Differentiated cells expressed specific genes of the three embryonic germ layers  and germ cells (VASA and DAZL).The pluripotent differentiation capacity of the first three Yazd hESC lines (Yazd1-3) was proven This study reports the derivation of Yazd1-3 hESC lines, initially using an MEF feeder layer in a microdrop culture system, and the banking of these cell lines using Cryotech and Cryowin tools for vitrification, which has started since 2008. Later Yazd hESCs were expanded on YhFF#8 while establishment and characterization.NANOG, SOX2, and OCT4/POU5F1. Furthermore, the differentiation potential of the cell lines in EB formation and monolayer culture was determined using RT-PCR for the three embryonic germ layers and germ-cell-specific genes.Following the generation of Shef lines using the microdrop culture system, we have applied this method for the derivation of new Yazd hESC lines (13). In recent studies, the efficiency of hESC derivation was reported to be more than 50% (29). In this study, however, three new hESC lines (Yazd1-3) were generated from sixteen fresh and frozen embryos, representing a success rate of 18.75%. This difference could be due either to the source of the embryos or the culture conditions, which in our case involved conventional KOSR/HES medium with MEFs as a feeder layer. The success rate for hESC generation from fresh embryos in this study was about 16.66%, whereas it was 20% for hESC lines derived from frozen embryos. As far as we know, this is the first report to describe the vitrification of hESCs using Cryotech and Cryowin tools; however, no comparisons were made between the efficiency of this method with the conventional open pulled straws method. This new method is applicable for the preservation of early outgrowths in early passages of pluripotent stem cells, and in our experience: hESCs. The new Yazd1-3 hESC lines were characterized by specific pluripotency markers, such as SSEA-4, TRA-1-60, and TRA-2-49, and pluripotency genes, including The use of a microdrop culture system was first reported in 1994 for the culturing of bovine ESC (30). In an open culture system, secreted growth factors may be lost or diluted, whereas it has been demonstrated that a combination of a co-culture and a microdrop system had the highest rates of blastocyst formation and hatching in comparison with an open co-culture system (31). Wakayama and colleagues (32) derived mESC lines using an efficient micro-scale method. Single blastomeres were co-cultured with MEFs in individual wells of 96-well plates. Following that, Aflatoonian and co-workers generated hESC lines using a microdrop culture system (13).In assisted reproductive techniques (ART), a laser is commonly used to assist with different processes, such as assisted hatching (33) and blastomere and trophectoderm (TE) biopsies (34). In addition, laser-assisted technology has been used for ICM biopsies in mice  and humans . In the present study, no ICM isolation methods, such as immunosurgery and laser-assisted biopsy, were used. The Yazd1-2 hESC lines were derived from blastocyst and early cavity embryos respectively, which were extruded from the zona pellucida by pronase treatment. It should be noted; however, that pronase is derived from bacteria and is therefore not suitable for the derivation of clinical grade hESCs in the future (14). The Yazd3 hESC line was derived from a zona-free embryo following the thawing process. While the ICM is known to be the usual source for hESC derivation, the development of hESCs from single blastomeres of cleavage- and morula-stage embryos  has also been reported, but these methods have not been used routinely.To avoid animal contamination following the co-culture of hESCs with MEFs, xeno-free derivation of hESC lines can be achieved by using human feeders including HFFs (18), HFGFs (13), human endometrial-derived fibroblasts (19), hESC-derived feeder (EDF), and human placental fibroblast feeders (43). In this regard, hESCs derived fibroblast-like cells conditioned medium demonstrated to support undifferentiated hESC proliferation under feeder-free conditions (44).Herein, MEFs were used for the initial derivation of Yazd1-3 hESC lines, followed by expansion of the cell lines on MEFs and YhFFs#8. Mesenchymal stem/stromal cells have been derived from Yazd hESC lines (27), although hESC-derived MSCs have yet to be used as feeders.The Yazd cell lines, which were characterized as described above and have been used during workshops (45), are available for other research groups and companies to use, according to Yazd Reproductive Sciences Institute rules and laws, as the owner of the cell lines.New hESC lines (Yazd1-3) were derived on microdrops of an MEF feeder layer, without immunosurgery, using whole blastocyst culture. Following expansion of the cell lines, YhFFs#8 were used as a xeno-free condition with KOSR/HES. This study is the first report of Cryotech and Cryowin tools being used for the vitrification of hESCs in the very early stages of derivation. Further studies are in progress to evaluate the efficiency of this method compared with other techniques and tools.The authors declare no conflicts of interest.


Merkel cell carcinoma (MCC) is an aggressive neuroendocrine tumour of the skin with growing incidence. To better understand the biology of this malignant disease, immortalized cell lines are used in research for in vitro experiments. However, a comprehensive quantitative proteome analysis of these cell lines has not been performed so far.Stable isotope labelling by amino acids in cell culture (SILAC) was applied to six MCC cell lines . Following tryptic digest of labelled proteins, peptides were analysed by mass spectrometry. Proteome patterns of MCC cell lines were compared to the proteome profile of an immortalized keratinocyte cell line (HaCaT).In total, 142 proteins were upregulated and 43 proteins were downregulated. Altered proteins included mitoferrin-1, histone H2A type 1-H, protein-arginine deiminase type-6, heterogeneous nuclear ribonucleoproteins A2/B1, protein SLX4IP and clathrin light chain B. Furthermore, several proteins of the histone family and their variants were highly abundant in MCC cell lines.The results of this study present a new protein map of MCC and provide deeper insights in the biology of MCC. Data are available via ProteomeXchange with identifier PXD008181. Merkel cell carcinoma (MCC) is a rare malignant tumour of the skin with neuroendocrine differentiation , 2 and gA genomic profiling study of patients with MCC revealed that the most frequent abnormalities are related to the TP53 gene and the cell cycle pathway. Further abnormalities were found in the PI3K/AKT/mTOR pathway and DNA repair genes . However2 in a humidified incubator. For SILAC labelling cells were grown in SILAC Media supplemented either with 13C6l-Lysine-2HCl (heavy) and l-Arginine-HCl (light) or with l-Lysine-2HCl (light) and l-Arginine-HCl (light) . Cells were cultured for at least ten cell doublings. Three biological replicates were cultured per each cell line, and these were merged into one sample upon cell lysis for further treatment. The labelling efficiency was estimated using the method described by Rappsilber et al. [The human Merkel cell carcinoma cell lines BroLi, MKL-1, MKL-2, PeTa, WaGa, and MCC13 were a kind gift of Prof. Houben . HaCaT, ®-Total Protein Extraction Kit” containing TM buffer  according to manufacturer’s manual. Protein precipitation was performed using modified Wessel–Fluegge method as described elsewhere [MCC cells were lysed using the “Chemiconlsewhere –17. Lyse® PepMap100 . Both columns were operated at 60 °C in the column compartment as described earlier [Methanol, was purchased from Merck , 98% formic acid, acetonitrile, trifluoroacetic acid were purchased from Sigma-Aldrich . HPLC grade water was prepared using an in-house Milli-Q plus device from Millipore , and trifluoroethanol was purchased from Alfa Aesar . Reverse phase separation of tryptic peptides was conducted on a nanoRSLC UltiMate3000  HPLC system consisting of an autosampler, thermal compartment and pumping module. PepMap C18 trap-column  was used for sample loading and desalting. The analytical column used for peptide separation was a 75 µm ID × 50 cm length Acclaim earlier . A totalAll raw data were converted into Mascot “mgf” files by using Bruker’s Data Analysis and these files were searched against the Swissprot database (version of November 2016) of human proteins using ProteinScape V 3.1.5 474  and Mascot V2.6 . Protein quantitation was performed using WARP-LC V1.3.136 .All samples were analysed as technical triplicates to ensure statistical sound data and avoid artefacts due to variations in ionization efficiency.Detailed information on separation gradient, the MS settings, and the data search and quantitation can be found in supplemental information . Proteins in MCC cell lines with H/L ratios of > 1.5 or < 0.5 where considered as significantly differently abundant. To put differential protein abundance into biologic context, Cytoscape  in combination with ClueGO/CluePedia (a Cytoscape plug-in) was used with default parameters except for following: Database Gene Ontology Biological Process, levels between 4 and 13 and GO Fusion set on true . p-valueThe SILAC method was used in six MCC cell lines to determine quantitative changes of proteins at the proteome level. Proteins detected in MCC cells were compared to the reference cell line HaCaT Fig. . We chosIn order to visualize the similarity and the difference between the particular cell lines, a heat map was created showing all quantified proteins. A list of proteins (overlapping and specific) for each sample is provided as Additional file Next, the protein profile of MCC cell lines was compared with the reference cell line HaCaT. Proteins present specifically in the MCC cells were determined. Then a Venn diagram was constructed. Since BroLi was the cell line with the least number of proteins and the difference between HaCaT and BroLi was small, the cell line BroLi has been omitted. Figure It is of crucial importance and of highest interest to identify and quantify biological processes involved in the biology of cancer. Term Enrichment Analysis using ClueGO showed that multiple pathways where affected by differentially represented proteins Fig. . CellulaIn total, 317 dysregulated (i.e. up- and downregulated) proteins with significance threshold of p < 0.05 were identified. Proteins altered > 1.5-fold were considered as upregulated and proteins altered < 0.5-fold were considered as downregulated. Based on these criteria, 142 proteins were identified as upregulated and 43 proteins were downregulated. The differently abundant proteins and their ratios are shown in Tables Bioinformatic analysis revealed that different cell lines have individual protein profiles. None of the dysregulated proteins was present in all tested cell lines at the same time. However, a high occurrence of histone variants was detected in all cell lines except in BroLi. In more detail, only three upregulated and two downregulated proteins were identified in the BroLi cell line. BroLi cell line is a very slowly growing cell line with a doubling time of 5 days  and thisFor the BroLi cell line, mitoferrin-1 was found to be 9.64-fold upregulated compared to HaCaT cell line. Mitoferrin-1 is a protein involved in the mitochondrial iron transport and storage . As ironIn MKL-2 cells, protein-arginine deiminase type-6 was upregulated 9.47-fold compared to the control cell line making it the most differently regulated protein for this cell line. This protein is an enzyme involved in post-translational modifications, which can have substantial effects on the structure and function of proteins. Citrullination is one such post-translational modification being catalysed by the family of protein arginine deiminase (PADs) enzymes. Five isoenzymes (PAD1-4 and 6) are known and they were identified in different types of tissue . An overFurthermore, we identified the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) to be the most upregulated protein in the PeTa cell line. The hnRNPs are a group of proteins binding to RNA and playing a role in mRNA processing . So far,In the WaGa cell line, SLX4IP (SLX4 interacting protein) was the protein showing the highest upregulation. SLX4 is a DNA repair protein and it coordinates structure-specific endonucleases  but its Finally, clathrin light chain B was the protein with the highest upregulation in the MCC13 cell line. Clathrin light chain B is a part of the clathrin protein, which is the main component of vesicles involved in intracellular transport. Recently, it was reported that clathrin light chains promote cell migration and therefore may play a role in cancer metastasis .In the PeTa cell line various histones and their variants were found to be dysregulated compared to the control cell line HaCaT. Histones are substantial components for the packaging of the DNA in the chromosomes. The smallest packaging units are nucleosomes consisting of DNA wrapped around a histone octamer. A histone octamer in turn consists of two copies of each of the core histones: H2A, H2B, H3, and H4, being the smallest units. The linker histone H1 holds the nucleosome together and is the fifth member of the histone protein family . Beside In the current study, proteins from all five major histone families with 15 different subfamily members were differently abundant in MCC cell lines compared to control samples. In particular, H2A1H and H2B1O were found to be overexpressed in MKL-1 and H2B1H and H2B2E were upregulated in MKL-2. Most of the highly abundant histone variants were found in the cell line PeTa: H2A1B, H2AJ, H1.4, H3.3, H1.5, H2B1N, and H2B2E. Furthermore, H2B1O and H2B1D were identified to be upregulated in WaGa, and H2B1C, H2B1K, H2B2F and H2AJ in MCC13. The role of histone variants in the development of carcinomas has been discussed and described in a number of publications –46. As dAnother interesting group of proteins that were identified as dyregulated in several MCC cell lines were the heat shock proteins (HSPs). It is a group of proteins that inhibit the unfolding or denaturation of cellular proteins and therefore being known as molecular chaperones whose expression is induced by stress. The major groups are classified according to their sizes and imply HSP10, HSP27, HSP40, HSP60, HSP70, and HSP90. Recent studies have shown that HSPs are highly expressed in many malignant tumours and due to their important role in cell proliferation and differentiation they are involved in carcinogenesis and metastasis , 49. In Beside HSP70, we found HSP60 and co-chaperone HSP10 to be overexpressed in the tested MCC cell lines. Actively produced by cancer cells, HSP60 exhibits a protective effect against cell stressors like chemotherapeutics. In particular, HSP60 stabilizes the anti-apoptotic protein survivin, a protein over-expressed in most human tumours, and therefore it inhibits apoptosis. Furthermore, HSP60 builds a complex with p53, which leads to the loss of the pro-apoptotic function of p53 and this process again results in inhibition of apoptosis .In conclusion, this work provides an additional insight in the biology of Merkel cell carcinoma. Multiple dysregulated proteins from various pathways were identified. The most abundant proteins were mitoferrin-1, histone H2A type 1-H, protein-arginine deiminase type-6, heterogeneous nuclear ribonucleoproteins A2/B1, protein SLX4IP and clathrin light chain B. Furthermore, the family of histone variants was frequently upregulated. In overall, each Merkel cell carcinoma cell line has its own distinct proteomic profile. This may be due to the biological heterogeneity of MCC. In this study we could demonstrate for the first time the similarities and differences between commonly used MCC cell lines.Current analysis can be significantly improved by: (a) using multidimensional separation approach for fractionation of tryptic peptides and (b) using a more sensitive mass spectrometer. We are aware of this facts and new analysis of these samples are currently being processed. However, taking into consideration that these data are the very first describing differences of putative Merkel cells we are confident that they can provide valuable help for researchers addressing this condition.Additional file 1: Additional methods information.Additional file 2: Table S1. Shows overlapping and specific proteins for each cell line.Additional file 3. Table S2. Lists all proteins that are shown in the Venn diagram (Fig.


Pancreatic cancer (PC) is the fourth most common cause of cancer death. Combination therapies with classical chemotherapeutic agents improved treatment of advanced PC at the cost of a relevant toxicity, but the 5-year survival rate remains below 5%. Consequently, new therapeutic options for this disease are urgently needed. In this study, we explored the effect of two repurposed drug candidates nelfinavir and nitroxoline, approved for non-anticancer human use, in PC cell lines. Nelfinavir and nitroxoline were tested as single agents, or in combinations with or without erlotinib, a targeted drug approved for PC treatment.The effects of the drugs on the viability of AsPC-1, Capan-2 and BxPC-3 PC cell lines were assessed by MTT. The impact of the treatments on cell cycle distribution and apoptosis was analyzed by flow cytometry. The effects of treatments on proteins relevant in cell cycle regulation and apoptosis were evaluated by western blot. Self-renewal capacity of PC cell lines after drug treatments was assessed using a clonogenic assay.When used as single agents, nelfinavir and nitroxoline decreased viability, affected cell cycle and reduced the expression of relevant cell cycle proteins. The effects on apoptosis were variable among PC cell lines. Moreover, these agents drastically impaired clonogenic activity of the three PC cell lines. Combinations of nelfinavir and nitroxoline, with or without erlotinib, resulted in dose- and cell-dependent synergistic effects on cell viability. These effects were paralleled by cell cycle alterations and more consistent apoptosis induction as compared to single agents. Treatments with drug combinations induced drastic impairment of clonogenic activity in the three cell lines.This study shows that two non-antitumor drugs, nelfinavir and nitroxoline, as single agents or in combination have antitumor effects that appear comparable, or in some case more pronounced than those of erlotinib in three PC cell lines. Our results support repurposing of these approved drugs as single agents or in combination for PC treatment.The online version of this article (10.1186/s13046-018-0904-2) contains supplementary material, which is available to authorized users. Pancreatic cancer (PC) remains the fourth most common cause of cancer death due to poor survival rate and rapid fatality after diagnosis . PC is tIdeally, combination therapies should include non-toxic drugs that act synergistically to kill tumor cells in a selective way. Several natural or synthetic compounds have been explored as single agents or in combination with standard chemotherapy in preclinical models of PC –7. CurreThere is a growing body of evidence that a number of non-anticancer drugs already approved for disparate human diseases have anticancer properties , 11. TheIn this study, we evaluated the effects of two non-anticancer drugs, nelfinavir and nitroxoline, as single agents and/or in combinations with or without erlotinib, in PC cell lines. The three drugs are already approved for human use, have a relatively low toxicity and their combination has not been tested before. Nelfinavir is a competitive inhibitor of HIV aspartyl protease used in combination with other antiretroviral drugs to treat patients with HIV infection . Chemora3--2,5-diphenyl-2H-tetrazolium bromide (MTT), crystal violet, RNAse, propidium iodide (PI), RIPA Buffer  containing 1 mM PMSF and protease inhibitor cocktail were obtained from Sigma . Mouse monoclonal anti-cyclin D3 antibody and rabbit polyclonal PARP antibody were purchased from Cell Signaling Technology, Inc. . Goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP, anti-cyclin B1 antibodies were obtained from Santa Cruz Biotechnology, Inc. . Monoclonal anti-β-actin antibody was obtained from Sigma .KRAS mutations, while BxPC-3 and AsPC-1 are TP53 mutated. Cells were cultured in RPMI 1640, supplemented with 10% FBS at 37 °C, 5% CO2. Nelfinavir mesylate and erlotinib HCl (OSI-744) were obtained from Selleckchem . 8-hydroxy-5-nitroquinoline (nitroxoline) was purchased from Sigma .Human pancreatic cancer (PC) cell lines AsPC-1 and Capan-2 were purchased from Cell Lines Service . PC cell line BxPC-3 was purchased from American Type Culture Collection . The three cell lines are known to express the EGFR protein , 21. AsP3 cells/well) and were treated the following day for 48 h with erlotinib, nelfinavir or nitroxoline as single agents, or with combinations of the drugs at various concentrations as indicated. Then, the MTT solution was added to each well and incubated at 37 °C for at least 3 h, until a purple precipitate was visible. In order to dissolve formazan crystals, the culture medium was replaced with dimethyl sulfoxide . Absorbance of each well was quantified at 540 and 690 nm, using a Synergy H1 microplate reader .Cell viability was tested by MTT assay . Briefly, cells were seeded in 96-well plates . The CI was calculated by CompuSyn software, based on the Chou-Talalay equation [ICequation : a CI < 6 cells per experimental condition were harvested, fixed in 70% cold ethanol and kept at 4 °C overnight. Cells were then resuspended in 50 μg/ml PI  and 200 μg/ml RNAse  as previously described [5cells) was performed using a FACScanto II flow cytometer . Data were analyzed with FlowJo software v8.8.6  and FCS Express 5 Software .Approximately 0.5 × 10escribed . Cell cyCells were collected and lysed in RIPA buffer supplemented with protease inhibitor cocktail. Protein concentrations were determined by the BCA Protein Assay  and 30 μg were subjected to electrophoresis followed by immunoblotting. The membranes were blocked in 5% nonfat dry milk for one hour at room temperature and incubated with the appropriate primary antibodies. Then the membranes were incubated with either anti-rabbit or anti-mouse HRP-conjugated secondary antibodies. The blots were revealed by chemiluminescence using the SuperSignal West Pico Chemiluminescence Substrate  according to the manufacturer’s instructions. β-actin was used as loading control.5 events were collected. Viable cells were Annexin-Vneg, while apoptotic cells were Annexin-Vpos.To assess apoptosis, BD Pharmingen™ APC Annexin V and 7-AAD viability staining solutions  were used according to the manufacturer’s instructions. Cells were resuspended in a specific binding buffer and stained using 7-AAD for dead cell exclusion and Annexin V antibody for 30 min at 4 °C in the dark. Subsequently, the samples were washed and analyzed on a FACScanto II flow cytometer . For each sample, at least 103 cells/well) and following cell attachment they were exposed for 48 h to erlotinib, nelfinavir, nitroxoline or their combinations as indicated. Then, after medium refreshment, the plates were incubated at 37 °C with 5% CO2, until cells in the control vehicle formed colonies consisting of at least 30 cells (3–4 days). Colonies were fixed with 70% methanol and stained with 0.5% crystal violet, then rinsed with tap water, dried and counted.Clonogenic assay was performed essentially as previously described . PC cellp-value ≤0.05 was considered statistically significant.Statistical analyses were performed using GraphPad Prism version 5.01 software . Comparisons of mean values were performed by an unpaired Student’s t-test. Multiple comparisons were performed by one-way ANOVA followed by Dunnett’s test. A 50 values  comparable to those obtained with erlotinib (22.8 μM). In Capan-2, nitroxoline had an IC50 value (16.9 μM) lower than those obtained with nelfinavir and erlotinib . Conversely, in BxPC-3 the IC50 of erlotinib (10.9 μM) was lower as compared to nelfinavir and nitroxoline .We analyzed by MTT the effect of erlotinib, nelfinavir and nitroxoline on the viability of three PC cell lines. The three drugs significantly affected cell viability in a dose-dependent manner, with distinct sensitivities for the three cell lines  . Combination therapies with classical chemotherapeutic agents improved treatment of advanced PC at the cost of a relevant toxicity, but the 5-year survival rate remains below 5%. Consequently, new approaches for the treatment of this lethal disease are urgently needed. In the present study, we analyzed the effect of nitroxoline and nelfinavir, two already approved non-anticancer drugs with low toxicity. These repurposed drug candidates were tested in PC cell lines as single agents, or in different combinations with or without erlotinib, a molecule already approved for PC treatment.50 values comparable, or in some case lower than those obtained with erlotinib. Conversely, in BxPC-3 the IC50 of erlotinib was lower as compared to repurposed drugs. Indeed, the IC50 of erlotinib in BxPC-3 was also lower than the IC50 observed with the same drug in AsPC-1 and Capan-2. These different effects of the drugs on the viability of the three PC cell lines were possibly related to their different genetic background. It has been previously shown that nitroxoline and nelfinavir affected cell cycle in different cancer cell lines [The effect of nitroxoline and nelfinavir on viability was analyzed in three PC cell lines AsPC-1, Capan-2 and BxPC-3 that display different genetic profiles. Interestingly, in AsPC-1 and Capan-2 these non-toxic drugs had ICll lines , 25–27. Considering that there is a substantial lack of studies combining repurposed drugs in PC and that these drug combinations might have a greater antitumor effect as compared to single agents, we determined the effect of nelfinavir, nitroxoline and erlotinib combinations on PC cells. As observed with single agents, the effects were distinct in the three PC cell lines. Overall, nelfinavir and nitroxoline in combination had a more pronounced effect on viability than single agents and this effect was more consistent at high concentrations of the drugs. Notably, the addition of erlotinib to the drug combinations in most cases did not increase the effects on cell viability. At low concentrations, only the combination of nelfinavir and nitroxoline in Capan-2 was assessed as deriving from synergistic drug interactions by CompuSyn. Conversely, at high concentrations the combinations of nelfinavir and nitroxoline, with or without erlotinib, were assessed as synergistic by CompuSyn both in Capan-2 and BxPC-3. It is interesting to note that also in AsPC-1 the combinations at high drug concentrations had enhanced effects as compared to single agents, resulting in a lower residual cell viability as compared to Capan-2 and BxPC-3 with the same drug combinations. Nevertheless, in AsPC-1 drug interactions were not assessed synergistic by CompuSyn analyses. Flow cytometry and immunoblot analyses showed that the reduced viability observed in PC cell lines with drug combinations was related to an interference with cell cycle progression and was associated to a reduction of cyclin D3 and cyclin B1 protein expression, which was more marked and homogeneous across the three cell lines at high drug concentrations. The results of flow cytometry and immunoblot analyses indicated also that apoptosis contributed to the reduced viability observed after treatment with the drug combinations. In particular, the effects of drug combinations on apoptosis across the three PC cell lines were more consistent than the effects observed with single drugs, while the addition of erlotinib to the combinations did not substantially affect apoptosis. Drug combinations had also drastic effects on clonogenic ability, indicating that these combinations had a strong impact on cell self-renewal capacity in PC cell, although it should be noted that also nitroxoline as single agent had comparable strong effects. Overall, the impact of nitroxoline as single agent on clonogenicity, as well as the corresponding effect of drug combinations was more pronounced than the effects of the same treatments on viability, as assessed by MTT.An interesting issue that is currently being investigated is how repurposed drugs exert their anticancer actions. Disparate off-target anticancer effects have been proposed and new targets are emerging for each drug. In particular, nitroxoline was shown to inhibit MetAP2, SIRT1, SIRT2 and was also shown to bind and inhibit cathepsin B, although the relevance of the latter target has been questioned because of the relatively high concentrations of the drug necessary to induce this effect . NelfinaOur results indicate a remarkable antitumor activity of nelfinavir and nitroxoline in PC cells. Nelfinavir and nitroxoline when used as single agents decreased viability, induced apoptosis, affected the expression of relevant cell cycle proteins, drastically compromised clonogenic activity with distinct effects in the three PC cell lines tested. These effects were enhanced by combined treatments with nelfinavir and nitroxoline. To our knowledge, this is the first study providing evidence that these human approved, non-anticancer drugs, as single agents or in combination, affect several crucial biological processes in different PC cell lines. In the experimental conditions tested in the present study the antitumor activity of these repurposed drugs appears comparable or superior to erlotinib, a targeted agent approved for PC treatment. Therefore, the results obtained with nelfinavir and nitroxoline in PC cell lines suggest that these drugs could be effectively repurposed, as single agents or in combination, for the treatment of the poorly responsive pancreatic cancer.Additional file 1:Table S1. Effects of erlotinib, nelfinavir and nitroxoline as single agents versus drug combinations on cell viability. (XLSX 12 kb)Additional file 2:Table S2. Mean percentages of cells identified as sub-G1 population. (XLSX 11 kb)Additional file 3:Figure S1. Representative images of clonogenic assays. (TIF 13253 kb)


C. mangga against six human cancer cell lines, namely the hormone-dependent breast cell line (MCF-7), nasopharyngeal epidermoid cell line (KB), lung cell line (A549), cervical cell line (Ca Ski), colon cell lines (HCT 116 and HT-29), and one non-cancer human fibroblast cell line (MRC-5) were conducted using an in-vitro neutral red cytotoxicity assay. The crude methanol and fractionated extracts (hexane and ethyl acetate) displayed good cytotoxic effects against MCF-7, KB, A549, Ca Ski and HT-29 cell lines, but exerted no damage on the MRC-5 line. Chemical investigation from the hexane and ethyl acetate fractions resulted in the isolation of seven pure compounds, namely (E)-labda-8(17),12-dien-15,16-dial (1), (E)-15,16-bisnor-labda-8(17),11-dien-13-on (2), zerumin A (3), β-sitosterol, curcumin, demethoxycurcumin and bis-demethoxycurcumin. Compounds 1 and 3 exhibited high cytotoxic effects against all six selected cancer cell lines, while compounds 2 showed no anti-proliferative activity on the tested cell lines. Compound 1 also demonstrated strong cytotoxicity against the normal cell line MRC-5. This paper reports for the first time the cytotoxic activities of C. mangga extracts on KB, A549, Ca Ski, HT-29 and MRC-5, and the occurrence of compound 2 and 3 in C. mangga.Investigations on the cytotoxic effects of the crude methanol and fractionated extracts   Cancer is one of the leading causes of deaths in Malaysia. It is the fourth leading cause of death after diseases of the circulatory and respiratory systems and motor vehicle accidents, and responsible for 10.3% of medically certified deaths. In 2002, about 26,089 cases of cancer were diagnosed in Peninsular Malaysia and an estimated 10,656 cases were not reported. As many as 30,000 new cases are reported yearly [i.e., proliferation, deregulation of apoptosis, angiogenesis, invasion and metastasis), but also synergize with chemotherapy and radiotherapy [Natural products provide a wide variety of phytochemicals which have been shown to have a broad spectrum of activity across multiple signalling pathways. Natural products not only disrupt aberrant signalling pathways leading to cancer , nasopharyngeal epidermoid cell line (KB), lung cell line (A549), cervical cell line (Ca Ski), colon cell line (HCT 116), colon cell line (HT-29) and one non-cancer human fibroblast cell line (MRC-5), using an in-vitro neutral red cytotoxicity assay. We also identified the components in the active hexane and ethyl acetate fractions of C. mangga rhizomes. The chemical investigation led to the isolation of seven compounds, namely (E)-labda-8(17),12-dien-15,16-dial (1), (E)-15,16-bisnorlabda-8(17),11-dien-13-one (2), zerumin A (3), β-sitosterol, curcumin, demethoxycurcumin and bisdemethoxycurcumin. Compounds 2 and 3 are reported for the first time in this plant. To our knowledge, there is no report on the cytotoxic activities of C. mangga on the above mentioned human cancer cell lines, except for the MCF-7 and HCT 116 cell lines [In the present study, we investigated the cytotoxic effects of the crude and fractionated extracts of  line A54, cervicall lines .C. mangga are summarized in The results of preliminary cytotoxic screening of the methanol and fractionated extracts of 50 values ranging from 22.0 to 36.8 μg/mL. The ethyl acetate fraction only showed good activity against HT-29, with IC50 18.5 μg/mL, whilst it showed mild inhibitory activity against the other investigated cell lines. The hexane fraction of C. mangga exhibited good activities against all the cell lines tested (except HCT 116) with IC50 values of 8.1, 15.4, 17.4, 11.4 and 17.9 μg/mL against the MCF-7, KB, A549, Ca Ski and HT-29 cell lines, respectively. Interestingly, the hexane fraction showed no adverse effect on the non-cancer human fibroblast cell line MRC-5 (IC50 >100 μg/mL). If this also occurs in vivo, the hexane extract would be considered safe for human consumption and its non-toxic effect on the MRC-5 cell line merits consideration for further toxicity and clinical studies. It was also observed that the crude methanol and the ethyl acetate extracts caused no damage to the normal human fibroblast cell line. The water extract did not show any cytotoxicity against any of the tested human cancer cell lines (IC50 >100 μg/mL). As the hexane fraction demonstrated good inhibitory activity and the ethyl acetate fraction exhibited mild activity, further chemical investigation was focussed on these two fractions. This led to the isolation of compounds 1-3.The methanol extract exhibited mild cytotoxic effects against all selected human cancer cell lines, with ICE)-labda-8(17),12-dien-15,16-dial (1), (E)-15,16-bisnorlabda-8(17),11-dien-13-one (2) and β-sitosterol. The ethyl acetate fraction afforded zerumin A (3) and the curcuminoids. Compounds 2 and 3 are reported for the first time in this plant. All chemical components . The cytotoxic effect shown by the hexane fraction may be due to the presence of compound 1 which comprises of 1.5% (w/w) in this fraction, whilst compound 3 and the curcuminoids may be responsible for the observed activity of the ethyl acetate fraction.The isolated compounds were further tested on the selected cancer cell lines as the hexane fraction showed strong cytotoxic effect against MCF-7, KB, A549, Ca Ski and HT-29 cells, whilst the ethyl acetate fraction exhibited moderate effects on KB, A549, HCT 116 and HT-29 cells. 50 values from 0.05 to 0.58 μg/mL), but also on the human normal cell line . Due to its high toxicity on human normal cell lines, the continuous use of doxorubicin can cause major adverse effects to human health [In the present study, doxorubicin, used clinically for the treatment of a great variety of cancer diseases, was the positive control ,17,18. Hn health . Even thNMR analysis: 1H- and 13C-NMR spectra were obtained on Bruker Avance III 500 MHz Nuclear Magnetic Resonance spectrophotometers.  GC-MS analysis: GC-MS analysis was performed using an Agilent Technologies 6980N gas chromatograph equipped with a 5975 Mass Selective Detector (70 eV direct inlet); the HP-5ms capillary column (5% phenylmethylsiloxane) with column dimensions 30.0 m × 25 mm × 25 μm was initially set at 150 °C, then the temperature of the oven was increased at 5 °C per minute to 300 °C using helium as carrier gas at flow rate 1 mL/min. The total ion chromatogram obtained was auto integrated by ChemStation and the components were identified by comparison with the supplied mass spectral data database  whenever possible. HPLC analysis: HPLC analysis was performed on a Shimadzu LC system equipped with a Shimadzu LC-10AT VP pump, Shimadzu SCL-10A VP system controller, Shimadzu SPD-M10A VP Photo Diode Array detector, Shimadzu DGU-12A vacuum degasser and Shimadzu LC Solution software. The solvents used for HPLC were of chromatographic grade acetonitrile (J.T. Baker), methanol (J.T. Baker) and ultra-pure water (H2O). The column used was a Chromolith Performance RP-18e (Merck) (100.0 mm × 4.6 mm i.d.) for analytical scale HPLC and semi-preparative column was a Chromolith Performance RP-18e (100.0 mm × 10.0 mm i.d.) for preparative scale separation. In-vitro neutral red cytotoxicity assay: All cancer cell lines were purchased from the American Type Culture Collection , medium like RPMI 1640, McCOY’S 5A, Eagle Minimum Essential Medium (EMEM) from Sigma-Aldrich, USA. The supplements or antibiotics such as foetal bovine serum, penicillin/streptomycin (100 μg/mL), amphotericin B (50 μg/mL), tripsin-EDTA (100X) were purchased from PAA Lab, Austria as well as sodium pyruvate (11 mg/mL) from Sigma-Aldrich, USA. The cells were cultured in a Shel Lab water-jacketed CO2 incubator with 25 cm3 tissue culture flasks , which was observed routinely under inverted microscope  for any contamination and seeded into 96-well flat bottom microtiter plate . The extract or pure compounds were dissolved in dimethyl sulfoxide  from Sigma-Aldrich, USA. The 96-well microtiter plate was agitated rapidly on a microtiter plate incubator shaker (LT Biomax 500) and absorbance of eluted dye measured at 540 nm using a ELISA microplate reader . Cell lines such as MCF-7, A549, Ca Ski and HT-29 were maintained in RPMI 1640 medium, HCT 116 in McCOY’S 5A Medium, KB and MRC-5 cells in Eagle Minimum Essential Medium (EMEM). All of these media were supplemented with 10% foetal bovine serum (FBS), penicillin/streptomycin, amphotericin B and only additional sodium pyruvate in EMEM. The cells were cultured in tissue culture flask in 5% CO2 incubator kept at 37 °C in a humidified atmosphere and observed routinely under inverted microscope from any contaminations. Each fresh medium was replaced every 2 or 3 days until cell confluence was achieved and the cells were detached by using tripsin-EDTA. C. mangga were obtained from Yogjakarta, Indonesia in July 2006. A voucher specimen (voucher number HI 1331) was deposited in the Herbarium of the Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.The authenticated rhizomes of C. mangga (1.0 kg) were soaked in methanol  for three days at room temperature. The solvent-containing extract was then decanted, any traces of water was removed using anhydrous sodium sulphate and evaporated under reduced pressure using a rotary evaporator to give a dark brown crude methanol extract . The crude methanol extract was then extracted with hexane (500 mL) and concentrated in vacuo to obtain a yellowish brown hexane fraction . The hexane insoluble residue was further partitioned using ethyl acetate and water (500 mL:500 mL) to give a dark brown ethyl acetate fraction  and a light brown water fraction . A summary of procedures in the bioassay-guided chemical investigation of C. mangga is shown in The dried, ground rhizomes of 2O) under the following gradient conditions: 0–5 min linear gradient of eluting solvent 45 to 50% ACN; 5–11 min linear gradient of 50 to 65% ACN; 11–21 min linear gradient 65 to 100% ACN; 21–25 min isocratic using 100% ACN; 25–30 min linear gradient solvent 100 to 45% ACN at a flow rate of 1.0 mL/min. Fractions H2 (617.0 mg) and H5 (744.3 mg) were subjected to semi-preparative separation on HPLC using the same method as utilised in the analytical scale separation described above. Fraction H2 gave the following compounds: (E)-labda-8(17),12-dien-15,16-dial  and (E)-15,16-bisnorlabda-8(17),11-dien-13-one . β-Sitosterol  was obtained from fraction H5.The hexane fraction (1.0 g) was subjected to vacuum liquid chromatography  initially eluting with hexane followed by hexane enriched with increasing percentages of acetone and finally with methanol. A total of nine fractions (labelled H1 to H9) were collected. Subsequently, the fractions were subjected to an analytical scale separation on the Shimadzu HPLC system. Each fraction was prepared in 1.0 mg/mL with methanol, filtered and 20 μL was injected into a Chromolith Performance RP-18e analytical column (100.0 mm × 4.6 mm i.d.) and peaks were detected by monitoring the UV absorbance at 214 nm. The mobile phase was a binary eluent of chromatographic grade acetonitrile (ACN) and ultra-pure water . Three curcuminoids were isolated from fraction E8 using preparative thin layer chromatography technique, PTLC : curcumin , demethoxycurcumin  and bis-demethoxycurcumin . All pure compounds obtained were checked by TLC and HPLC to confirm their purity and then subsequently identified through their mass spectral and NMR data which were consistent with published data [One gram (1.0 g) of the ethyl acetate fraction was also subjected to the same VLC setup as for the hexane fraction and a total of eight fractions (labelled as E1 to E8) were collected. The fractions were then prepared and subjected to the same analytical HPLC condition as described for the hexane fraction. Two fractions, E7 (874.4 mg) and E8 (380.0 mg), were subjected to semi-preparative HPLC separation using the same method as utilised in the analytical scale separation. Fraction E7 afforded a colourless oil, zerumin A (hed data ,14.in-vitro cytotoxicity activity was performed using the neutral red cytotoxicity assay based on the initial protocol described by Borenfreund and Puerner, with some modifications [The ications ,19.C. mangga rhizomes are ascribable to the presence of the active compound 1 in the hexane fraction, and compound 3 and the curcuminoids in the ethyl acetate fraction. Even though these compounds are not as effective as doxorubicin against cancer cell lines, in comparison they have low toxicity against human normal cell lines. The present study provides important preliminary data of extracts or isolated compounds with potential anti-neoplastic properties for future work. Since C. mangga is widely used in traditional medicine for the treatment of cancer, the current findings provide scientific validation in the use of the rhizomes for this purpose. As the rhizomes are also widely consumed as salad in food without any known undesirable side effecat, it can be assumed that the plant is safe for consumption at the normal dose as food. Curcuma mangga, is therefore a promising dietary agent that holds great potential for use in chemopreventive and chemotherapeutic strategies. Nevertheless, further investigations are necessary to validate its use and to determine its mode of action.The cytotoxic activities observed for


Cell lines form the cornerstone of cell-based experimentation studies into understanding the underlying mechanisms of normal and disease biology including cancer. However, it is commonly acknowledged that contamination of cell lines is a prevalent problem affecting biomedical science and available methods for cell line authentication suffer from limited access as well as being too daunting and time-consuming for many researchers. Therefore, a new and cost effective approach for authentication and quality control of cell lines is needed.ρ > 0.9). Our benchmarking studies revealed that CeL-ID method can identify a cell line with high accuracy and can be a valuable tool of cell line authentication in biomedical science. Finally, CeL-ID estimates the possible cross contamination using linear mixture model if no perfect match was detected.We have developed a new RNA-seq based approach named CeL-ID for cell line authentication. CeL-ID uses RNA-seq data to identify variants and compare with variant profiles of other cell lines. RNA-seq data for 934 CCLE cell lines downloaded from NCI GDC were used to generate cell line specific variant profiles and pair-wise correlations were calculated using frequencies and depth of coverage values of all the variants. Comparative analysis of variant profiles revealed that variant profiles differ significantly from cell line to cell line whereas identical, synonymous and derivative cell lines share high variant identity and are highly correlated  contains supplementary material, which is available to authorized users. Cell lines are an indispensable component of biomedical research and serve as excellent in vitro model systems in disease biology research including cancer. Cell lines are usually named by the researcher who developed them and till recently were lacking a standard nomenclature protocol –3. This Profiling of short tandem repeats (STRs) across several loci is the most common and standard test for cell line authentication as recommended by the Standards Development Organization Workgroup ASN-0002 of American Type Culture Collection (ATCC) , 2, 9–11With the advent and success of sequencing technologies, more and more researchers are using RNA sequencing to profile large amounts of transcript data to gain new biological insights. Moreover, RNA-seq data is also being used to identify single nucleotide variants in expressed transcripts . It may In this study, we present a RNA-seq based approach for Cell Line Identification (CeL-ID). We identify variants in each cell lines using RNA-seq data followed by pairwise variant profile comparison between cell lines using frequencies and depth of coverage (DP) values. Comparative analysis of variants revealed that variant profiles are unique to each cell line. Our benchmarking studies revealed that CeL-ID method can identify a cell line with high accuracy and can be a valuable tool for cell line authentication in biomedical research. In addition, using linear model regression technique, the approach can also reliably identify possible contaminator if requested. We choose to explore the utility of RNA-seq data in cell line authentication because it is the most commonly used technique among the seq-based methods and also relatively inexpensive, and we also demonstrated the minimum sequence reads requirement for each RNA-seq to maintain the authentication accuracy using a series of subsampling BAM files at 1million up to 50 million reads. With the popularity and accessibility of RNA-seq technology, a significant number of studies anyway involve the use of data from RNA-seq and hence the same can also be used to check the authenticity of the cell line.https://portal.gdc.cancer.gov/). The names of cell lines are used as is listed in NCI GDC archive and are listed in Additional file https://broadinstitute.github.io/picard]), followed by local re-alignment and re-calibration of base quality scores  and it contains RPKM values for all the genes in 1019 cell lines, covering all 935 CCLE RNA-seq set.The Cancer Cell Line Encyclopedia (CCLE) is a collaborative project focused on detailed genomic and pharmacologic characterization of a large panel of human cancer cell lines in order to link genomic patterns with distinct pharmacologic vulnerabilities and to translate cell line integrative genomics into clinic , 20. Genes (GATK ), and fies  whereas the second one has data for eight HCT116 cell lines (GSE101966) , 25. TheMEN1  and in Hd ARID1B  to alignd ARID1B , here toi, j 〉, the variants to be tested areTo assess the confirmation of two cell-lines to be either identical or highly similar in terms of their sequence variation profiles genome-wide or their expression levels, we choose to use Pearson Correlation to evaluate altered allele frequencies (FREQ) across two cell-lines or expression levels, facilitated by the number of non-zero FREQ shared between two cell-lines with at least 10 fold coverage in both cell lines. We choose FREQ, instead of direct counting of altered allele depth (AD), because that majority of altered allele fractions does not change with the expression level, and allele-specific expression may appear in cell lines with certain treatments but hopefully it will be a small proportion over a typically massive number of SNPs under consideration. To be specific, for any two cell lines 〈 di,k and fi,k are the depth of coverage (DP) and altered allele frequency at genomic location k of ith cell line, respectively. Note that we require variant has to exist in at least one cell line with 10 fold coverage. If a gene does not express, all mutations within this gene will not be considered unless its partner cell-line expresses this gene at a sufficient level. Therefore, the expression difference is already embedded in Pearson correlation, V. Similarly, correlations over gene expression levels between two cell lines are evaluated also by Pearson correlation coefficient, with requirement that genes with expression level > 0.1 (RPKM level) in at least one cell line. Hierarchical clustering was performed using MATLAB, using Pearson correlation of FREQ as the distance measure . Similar distribution is also observed in pair-wise correlation from WES samples. To estimate distribution parameters, we removed correlation coefficients less than 0 (unlikely) and greater than 0.8 (most likely due to replicate and derivative cell lines in CCLE collection), therefore it forms a truncated normal density function within an interval , as follows,To determine the significance of a detected correlation coefficient for a given cell line, we generated all pair-wise correlations for 934 RNA samples, and its distribution follows normal distribution a = 0, and b = 0.8. ϕ and Φ are standard normal density and distribution functions, respectively. We chose b = 0.8 as a cut-off threshold since pairs with correlation > 0.8 are derived from same parental lines or with some other biological relevance (see subsection Cell line authentication using variant comparisons in Results Section). Maximum-likelihood estimate (using MATLAB mle function) was employed in this study, and distribution parameters from distribution  for CCLE collection were estimated. For any given correlation coefficient ρi for the test sample against ith sample in CCLE, its p = P(ρ ≥ ρij) = 1 − F, where F is the cumulative distribution function of Eq. p < 0.001, and they are most likely derived from same cell origin if p < 10− 4. Multiple samples are identified as matching cells, we can revise Eq. where we fixed cut-off For gene expression level, the distribution of pair-wise correlation coefficient is more skewed towards 1.0; therefore, it is difficult to separate matching cells from mismatch cells (data not shown).x1 and x2, with unknown proportion q1 and q2, and we denoted the mixture cell as y, or,In addition to authenticate cells, one may also want to know whether or not the processed cells are contaminated by other cells, possibly from CCLE or additional cell lines collected in the lab, along with RNA-seq data. Assuming the test sample is a mixture of cell lines y, x1, x2 are vectors of FREQs from selected variant sites of test mixture sample and CCLE cell lines. Eq. Y = qX, where q = , if more than two cell mixture is hypothesized. To demonstrate the proof-of-concept, our current implementation takes top 200 sites, each direction that has most difference in FREQ comparing two samples . To further simplify the procedure, we also use our CeL-ID to identify the dominant cell, say x1 first. Following the similar studies for de-convoluting cell type proportions [x2, using robust linear model regression method (implemented in MATLAB fitlm function) to estimate q1 and q2, provided q1 + q2 ≤ 1. Slightly different to typical cell-type deconvolution methods, after determining the first contaminator, we can iteratively add other candidates from the entire CCLE collection and perform linear regression, and terminate the process until q value becomes negative or regression fails  is the Gaussian noise we added to q values  were downloaded and bam files were processed to call variants using an in-house pipeline described earlier in the methods section. Additionally, WES data for 326 cell lines available from GDC were also obtained and variants were identified. A total of 1,027,428 of variants were identified across all the cell lines with an average of 27,310 variants per cell line. As shown in Fig. RNA-seq data for 934 cell lines available from the NCI GDC legacy portal (ρ > 0.8) whereas most other pairs show poor correlation  were turned out to be known replicates, subclones, derived from same patients or have been known in the literature to share high SNP identity ; Fig. ρ = 0.97), and G27298.EKVX.1 & G41811.EKVX.1 (ρ = 0.96). Moreover, pair – G20492.HEL_92.1.7.2 & G28844.HEL.3 also identified to be very similar . Majority of cell line pairs rightly show poor correlation  but have different cells of origin and derived from different cancers. These cell lines may just happen to share high variant identity or somewhere during the cell culturing and maintenance cells got contaminated with each other. As expected, correlated cell lines tend to share more common mutations . Notice the difference of correlation coefficients of the best sample and the next-to-best samples are much smaller than those derived from variant profiles.Transcriptome profiles of any given cells are known to change during various treatments, and adapt to their environment as well. For base-line expression data provide through CCLE project, we can see their correlation holds for pair G20492.HEL_92.1.7.2 & G28844.HEL.3  = . Therefore, at L0.001 = 0.609, two samples will be considered similar with p < 0.001, or at L10-6 = 0.686 two samples will be unlikely similar (p < 10− 6). As a comparison, between RNA-seq and WES variant profiles  = , excluding all pair-wise comparison between same cell lines  .We tested the robustness of CeL-ID method by adding noise (Gaussian noise with zero mean) to the allele frequency of variant data for six pairs of cell lines as aforementioned. As evident from the Fig. p-value is much higher, which is due to the removal of common variants, only assess the agreement with variant sites perhaps differentiate MCF7 and KPL-1. Similar results were also obtained for HCT116 cells and are provided in Additional file Moreover, we tested the robustness of CeL-ID method on two independent test sets. First independent test set comprises of 12 RNA-seq datasets for MCF7 cells, which were downloaded from GEO (GSE86316) and represents mRNA expression profiles in MCF7 cells after silencing of MEN1 using small hairpin or chemical inhibition that affected expression profile of selected group of transcripts . The secFurthermore, to test the robustness of the system, effect of sequencing depth on the results was checked. We randomly selected nine cell lines and randomly subsampled it to 1 million (1 M), 2 million (2 M), 5 million (5 M), 10 million (10 M), 25 million (25 M), and 50 million (50 M) reads and ran the pipeline on subsampled subset of reads. As evident from the Fig. x1), and G20469.JHOS-2.2 as contaminant candidate (x2);For example, we select G20492.HEL_92.1.7.2 as test sample , and q2 ~ N. We also tested proportion of 0.70/0.30, as shown in Fig. Generate proportion z = q1 ∙ x1 + q2 ∙ x2 for both FREQ and DP;Following Eq. σnoise = , we perform,4.1.y = z + N only for FREQ, and then reset y to 0 if y < 0, and 100 if y > 100;Following Eq. 4.2.σ = 0.01, we obtained:Calling function CCLE_Identification to identify dominant cell-line, or provide cell line identification. For our particular example selection and for ρ = 0.97, p = 9.762 × 10− 271st match cell-line = G20492.HEL_92.1.7.2, with ρ = 0.90, p = 1.768 × 10− 202nd match cell-line = G28844.HEL.3, with ρ = 0.47, p = 0.5143rd match cell-line = G25242.K-562.3, with For each standard deviation Cell line contamination is a major issue facing biomedical sciences , 9. Huma4.3.q1 and q2. Results for our demonstrated case with ;Calling function CCLE_MixtureEstimate to identify contaminant and The possible mixture is G20469.JHOS-2.2, with proportion q2 = 82.3%, with t-stat = 210.0, p-value 0.000000e+00Note that 2nd matched cell line (HEL.3) is the cell line that has a high correlation with HEL_92.1.7.2 ; andσ from 0 to 6. With the increase of the s, the t stats for each proportion variable estimate decreases  for estimating cell line purity from RNA-seq data. A key advantage of using the CeL-ID method for cell line authentication is that it relies on a complete set of variants from the transcriptome instead of a fixed panel of small numbers of STRs or SNPs, and hence avoids the loss of statistical power caused by allelic dropout that affects STR-based authentication methods , 9–11. TCurrently, STR profiling is the ANSI standard for authenticating cell lines . STR proBenchmarking studies on independent test sets showed that CeL-ID method is precise and robust and can be used as a resource for cell line authentication. Genentech authenticated cell lines contain a consolidated list of 3587 cell lines , of whicIn summary, we have developed a new method called CeL-ID, for cell line authentication using variant profiles derived from RNA-seq data and has shown its robustness. CeL-ID successfully identifies identical, synonymous and derivative cell lines and also estimates about the possible contaminant. We have attempted to provide simple solution to problem associated with cell line authentication and hope this would help in adoption of regular cell line authentication.Additional file 1:List of sample names in CCLE dataset (BG-04-S1.xls). (XLS 92 kb)Additional file 2:Test results on an independent test set of eight HCT116 cells obtained from GSE101966 (BG-04-S2.xls). (XLS 38 kb)


Compound 3, reported in the literature as a synthetic derivative of furoscalarol 2, was for the first time isolated from a natural source. During the separation performed using a silica column in the presence of methanol, 16-methoxy derivatives  were obtained from the unintended reaction of 2. The isolated natural products 3 and 4 and the artifact 5 showed moderate to high cytotoxicity against six human cancer cell lines, whereas compound 6, the C-16 epimer of 5, showed no cytotoxicity at a concentration of 60 μΜ.Three furan-containing scalarane sesterterpenoids ( Scalarispongia is a genus of sponges that was established in the year 2000; previously, it was assigned as Cacospongia scalaris + .Compound 4: a pale yellow amorphous solid; c 0.3, CHCl3); UV λmax (log ε) 255 (2.98) nm; IR (KBr) νmax 3330, 2926, 1733, 1643, 1462, 1370, 1262, 1027 cm−1; 1H and 13C-NMR , see m/z 426.3011 [M + H]+ .Compound 5: a pale yellow amorphous solid; c 0.3, CHCl3); UV λmax (log ε) 218 (4.05) nm; IR (KBr) νmax 2957, 1766, 1463, 1373, 1058 cm−1; 1H- and 13C-NMR , see m/z 443.3129 [M + H]+ , m/z 411.2901 [M-OCH3]+ .Compound 6: a pale yellow amorphous solid; c 0.3, CHCl3); UV λmax (log ε) 219 (4.32) nm; IR (KBr) νmax 2957, 1766, 1463, 1373, 1058 cm−1; 1H- and 13C-NMR , see m/z 443.3129 [M + H]+ , m/z 411.2901 [M-OCH3]+ .Compound 2. The growth inhibition assays were performed as reported previously [50 values were calculated using GraphPad Prism 4.0 software .Six human cancer cell lines, particularly HCT-15 , NCI-H23 , ACHN , MDA-MB-231 , NUGC-3 , and PC-3 , were used in this study. The cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. Cell cultures were incubated at 37 °C under a humidified atmosphere of 5% CO


PCDH9, NSBP1, MCTP1 and SEMA3A genes in the PAC-resistant cell lines. Short-term exposure to PAC led to increased expression of the MDR1 and BCRP genes in the A2780 and W1 cell lines. In the A2780 cell line, we also observed increased expression of the C4orf18 gene and decreased expression of the PCDH9 and SEMA3A genes after PAC treatment. In the W1 cell line, short-term treatment with PAC upregulated the expression of the ALDH1A1 gene, a marker of Cancer stem cells (CSCs). Our results suggest that downregulation of the PCDH9, NSBP1, MCTP1 and SEMA3A genes and upregulation of the MDR1, BCRP, C4orf18 and ALDH1A1 genes may be related to PAC resistance.Development of drug resistance is the main reason for low chemotherapy effectiveness in treating ovarian cancer. Paclitaxel (PAC) is a chemotherapeutic drug used in the treatment of this cancer. We analysed the development of PAC resistance in two ovarian cancer cell lines. Exposure of drug-sensitive cell lines (A2780 and W1) to PAC was used to determine the primary response. An established response was determined in PAC-resistant sublines of the A2780 and W1 cell lines. qRT-PCR was performed to measure the expression levels of specific genes. We observed decreased expression of the  Epithelial ovarian cancer (EOC) is one of the most aggressive tumours of all gynaecological malignancies and is the fifth leading cause of death from gynaecological malignancies. At the time of diagnosis, the majority of patients have advanced intraperitoneal metastatic disease . AlthougTaxus brevifolia. It belongs to the family of antimitotic anticancer agents and blocks mitosis through stabilizing microtubules by binding to β-tubulin subunits. Consequently, PAC blocks cell division and leads to apoptotic cell death . Downregulation of the MCTP1 gene was observed in all PAC-resistant cell lines, although in the A2780PR1 cell line, downregulation of this gene was not statistically significant. In the A2780PR2 cell line, we observed approximately 120-fold downregulation of the MCTP1 gene (p < 0.001) (p < 0.001) and 20-fold (p < 0.01) downregulation of the MCTP1 gene  A. W1PR1 TP1 gene B.SEMA3A gene expression was observed in PAC-resistant cell lines. A 4-fold decrease in the SEMA3A transcript level was observed in the A2780PR1 cell line (p < 0.05) vs. 223-fold in the A2780PR2 cell line (p < 0.01) (SEMA3A transcript level—approximately 850-fold (p < 0.001) (SEMA3A transcript level (p < 0.05) (A different pattern of  < 0.01) A. Even l< 0.001) B. In con < 0.05) A.The second part of our study focused on the early response to PAC treatment. In these experiments, drug-sensitive cell lines A2780 and W1 were treated with low concentrations of PAC (20 ng/mL and 25 ng/mL for A2780) and (15 ng/mL and 20 ng/mL for W1) for 24, 48 and 72 h. Then, changes in gene expression were investigated.MDR1 and BCRP genes encoding the most important drug transporters can also be expressed during the first days of PAC treatment. In the A2780 cell line, we observed a dose- and time-dependent increase in the MDR1 transcript level (p < 0.05 or p < 0.01)  (We were interested in whether  < 0.01) A. In the< 0.001) B.BCRP transcript level was observed at all time points and concentrations (p < 0.05 or p < 0.01) (p < 0.05 or p < 0.01), with the exception of PAC 15 ng/mL after 72 h, for which the increase was close to significant (p = 0.07)  A. Simila = 0.07) B.PCDH9 and SEMA3A genes were investigated to determine if PAC could induce changes in the expression of these genes during the first days of treatment. We did not observe any changes in the expression of these genes in the W1 cell line (not shown). In the A2780 cell line, we observed statistically significant downregulation of the PCDH9 gene after 48 and 72 h of PAC treatment (p < 0.05)  only after 48 h of treatment and was close to significant (p = 0.09) after 72 h of treatment at a PAC concentration of 25 ng/mL  A. Downre25 ng/mL B.C4orf18 gene after PAC treatment was observed in only the A2780 cell line. At all time and concentration points, we observed statistically significant expression, with the highest C4orf18 transcript levels after 72 h of treatment (p < 0.05 and p < 0.01) (Increased expression of the  < 0.01) .ALDH1A1 transcript in the A2780 cell line (not shown). In contrast, we observed an increase in ALDH1A1 mRNA after PAC treatment in W1 cell line. The increase was statistically significant after 24 and 48 h at a PAC concentration of 20 ng/mL and after 48 h at a concentration of 15 ng/mL (p < 0.05 or p < 0.01). After 72 h, the increase was close to significant (p = 0.13 for 15 ng/mL and p = 0.07 for 20 ng/mL) (ALDH1A1 is the most popular marker of cancer stem cells (CSCs), and its expression was previously observed by us in PAC- and TOP-resistant cell lines . Therefo0 ng/mL) .MDR1 and MDR3 genes  at 37 °C in a 5% CO2 atmosphere.In our study, we used two ovarian cancer cell lines: the established ovarian cancer cell line A2780 and the primary ovarian cancer cell line W1. The human ovarian carcinoma A2780 cell line was purchased from ATCC . A2780 sublines that were resistant to PAC [A2780PR1 and A2780PR2 (A2780 paclitaxel resistant)] were generated by exposing A2780 cells to PAC at incrementally increasing concentrations. The human primary ovarian cancer cell line W1 was established using ovarian cancer tissue obtained from an untreated patient. The W1 sublines resistant to PAC [W1PR1 and W1PR2 (W1 paclitaxel resistant)] were obtained by exposing W1 cells to PAC at incrementally increasing concentrations. The final concentrations used for selecting the resistant cells was 1100 ng/mL of PAC and was two-fold higher than the plasma concentrations of PAC two hours after intravenous administration. The increase in resistance according to parental drug-sensitive cell lines were as follows: 146-fold for A2780PR1 vs. A2780; 1202-fold for A2780PR2 vs. A2780; 743-fold for W1PR1 vs. W1 and 967-fold for W1PR2 vs. W1 as described previously . All the6 (A2780 and W1) in 1 mL of medium per well of a 6-well plate. Cell count and viability were determined before the cells were used in the different assays. Viability was determined by the trypan blue exclusion criteria. Cells were harvested and used for RNA isolation 24, 48 and 72 h after treatment.In time course experiments, the A2780 and W1 cell lines were treated with PAC at concentrations of 20 ng/mL and 25 ng/mL for A2780 or 15 ng/mL and 20 ng/mL for W1. The starting cell concentration was 0.4 × 10PCDH9, MCTP1, NSBP1, SEMA3A, C4orf18, ALDH1A1, MDR1 and BCRP gene expression in A2780 and W1 cell lines as well as in the PAC-resistant cell lines were examined. RNA was isolated using a Gene Matrix Universal RNA Purification Kit  as described by the manufacturer’s protocol. Reverse transcription was performed using M-MLV reverse transcriptase  as described in the manufacturer’s protocol using a thermal cycler ; 2 μg of RNA was used for cDNA synthesis. Real-time PCR was performed using a 7900HT Fast Real-Time PCR System , Maxima SYBR Green/ROX qPCR Master Mix  and sequence-specific primers, as indicated in GADPH), β-actin, hypoxanthine-guanine phosphoribosyltransferase 1 (HRPT1) and beta-2-microglobulin (β2M) served as the normalizing genes (geometric mean) against which changes in the expression of examined genes were compared. Gene expression was analysed using the relative quantification (RQ) method. RQ estimates the differences in the level of gene expression against a calibrator . The drug-sensitive cell lines (A2780 and W1) were used as the calibrators. The analysis was conducted by employing the standard formula: RQ = [sample (drug-resistant line) calibrator (drug-sensitive line)]. The graphs were made using SigmaPlot .Changes in For amplification, 12.5 μL of Maxima SYBR Green/ROX qPCR Master Mix , 1 μL of each primer  followed by 45 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. After amplification, melting curve analysis was performed to analyse the melting temperature of the product. The amplification products were also resolved by 3% agarose gel electrophoresis and visualized by ethidium bromide staining.t-test. The statistical analysis was performed using Excel software . The statistical significance of the differences was determined by applying Student’s PCDH9, NSBP1, MCTP1 and SEMA3A genes suggests that downregulation of these genes is important in PAC resistance. Upregulation of the BCRP and C4orf18 genes was characteristic of only the primary response to PAC. The significance of these genes in the resistance to PAC has not been described previously and should be further investigated. We also confirmed the significance of the MDR1 gene in the PAC response. Upregulation of the ALDH1A1 gene suggests the role of CSCs in PAC resistance development in the W1 cell line.In summary, our results show the changes in the expression of new genes in response to PAC treatment in ovarian cancer cell lines. The decreased expression of