Sarcomas are rare mesenchymal malignant tumors with unique biological and clinical features. Given their diversity, heterogeneity, complexity, and rarity, the clinical management of sarcomas is quite challenging. Cell lines have been used as indispensable tools for both basic research and pre-clinical studies. However, empirically, sarcoma cell lines are not readily available. To understand the present status of sarcoma cell lines and identify their current challenges, we systematically reviewed reports on sarcoma cell lines. We searched the cell line database, Cellosaurus, and categorized the sarcoma cell lines according to the WHO classification. We identified the number and availability of sarcoma cell lines with a specific histology. We found 844 sarcoma cell lines in the Cellosaurus database, and 819 of them were named according to the WHO classification. Among the 819 cell lines, 36 multiple and nine single cell lines are available for histology. No cell lines were reported for 133 of the histological subtypes. Among the 844 cell lines, 148 are currently available in public cell banks, with 692 already published. We conclude that there needs to be a larger number of cell lines, with various histological subtypes, to better benefit sarcoma research.  Sarcomas are rare mesenchymal malignant tumors with unique biological and clinical features. Sarcomas are unique malignancies for several reasons. First, sarcomas originate from diverse mesenchymal tissue lineages such as adipose, muscle, fibrous, cartilage, nervous, and vascular tissues, or bone. Since these tissues are distributed throughout the human body, sarcomas can occur in almost all organs. Second, sarcomas are heterogeneous diseases that are pathologically grouped into more than 70 described subtypes . The hisRecent advances in genomic technology using next-generation sequencing have enabled the classification of sarcomas, which did not fit into known specific diagnostic categories . Such clGiven their diversity, heterogeneity, complexity, and rarity, the clinical management of sarcomas is quite challenging. However, there are many successful examples of innovative drugs for sarcomas. For example, a tyrosine kinase inhibitor, imatinib mesylate, was originally developed for the treatment of chronic myelocytic leukemia, which has a unique chromosome translocation; later, imatinib mesylate was repurposed to gastrointestinal stromal tumor (GIST), which is characterized by mutations or overexpression of c-kit and PDGFR ,16,17,18Other drugs include trabectedin, pazopanib, eribulin, olaratumab, and denosumab. Trabectedin, which binds to the minor groove of DNA to cause DNA damage, demonstrated evidence of cytotoxic activity against soft tissue sarcomas ,25,26,27This recent progress in the development of novel anti-cancer drugs suggests improvement in the clinical outcomes of patients with sarcomas in the near future ,47,48. HCell lines have been used as indispensable tools for both basic research and pre-clinical studies. Since animal cell culture became a common laboratory technique in the mid-1900s, it has served as a model for human cancer research. Since cell lines are maintained in artificial tissue culture conditions, there are critical arguments about the utility of cell lines; however, the advantages of other cancer models, such as xenografts  and orgaTo date, different sarcoma cell lines have been developed. These cell lines represent a useful experimental model to examine the hypothesis about the etiology of diseases, to evaluate the molecular mechanisms of cancer progression, and to examine the effect of potent anti-cancer drugs at the cellular and subcellular levels. At the same time, besides the obvious utilities of cell lines in cancer research, researchers may be empirically aware that sarcoma cell lines are not readily available, probably due to the rarity of the disease, and a lack of proper cell lines hinders basic studies and development of effective therapies for sarcomas. In this review, we provide an overview of the current status of reported sarcoma cell lines, and finally discuss what types of sarcoma cell lines need to be established, what system needs to be created to promote sarcoma research using cell lines, and what biological studies need to be performed to improve the present status of sarcoma cell lines.https://web.expasy.org/cellosaurus/) and searched using ontology: human cell lines were searched with the term ‘NCBI_TaxID=9606; ‘Homo sapiens’ in ‘Species of origin’, and sarcoma cell lines were searched with a term that was one of the children of the term ‘NCIt:C3810 Connective and Soft Tissue Neoplasm’ in ‘Disease’. The NCI thesaurus ontology file was downloaded from the website of NCI Thesaurus (https://ncit.nci.nih.gov).All potentially relevant cell lines were identified by searching the Cellosaurus database  ,57. The The following criteria had to be met for a cell line to be included in this review: cell line established from human patients with connective and soft tissue neoplasm, regardless of histology or original sites. Cell lines derived from other cell lines and modified by genes or reagents were considered as duplicates and excluded from the data analysis.https://www.python.org/): cell line name, disease, publications, and cell line collections.The following data were examined for each cell line using Python version 3.6.1  for published sarcoma cell lines. The publications in PubMed were also considered for investigation because these cell lines are available from public cell banks or published in academic journals and were, therefore, somehow assured by the public organizations or research community. In addition, the cell lines and their relevant data were supposed to be easily obtainable.In the Cellosaurus database, the availability of cell lines was examined from the following cell banks: AddexBio, ATCC, BCRC, BCRJ, BEI_Resources, CBA, CCLV, Cell_Biolabs, CLS, Coriell, DGRC, DiscoverX, DSMZ, ECACC, FCDI, ICLC, Imanis, IZSLER, JCRB, KCB, KCLB, KYinno, Millipore, MMRRC, NCBI_Iran, NCI-DTP, NHCDR, NIH-ARP, NISES, RCB (Riken), RSCB, TCB, TKG, TNGB, and WiCell. In addition, we searched the Ximbio website . Those modified cell lines are most common in osteosarcoma (275 cell lines). Among the 275 cell lines, U2OS cell lines are most commonly used as original cell lines for modifications. We found that there are cell lines for which the reported histology did not match that in the WHO classification (Table 3).Among the 819 cell lines originally derived from patients with connective and soft tissue neoplasm, 139 cell lines were available from the public cell banks , while 6Among the 819 cell lines originally derived from patients with connective and soft tissue neoplasm, 674 cell lines were cited in the PubMed database , while 1The cell lines whose establishment was reported in academic journals that are cited in the PubMed database can be useful research resources because the relevant data of cell lines are available from the published papers. Among the 844 original cell lines, there were 692 cell lines that have been published: among them, 108 cell lines are available from public cell banks A Supple. Among tA lack of sarcoma cell lines is empirically noticed in the research community; it is important to know their availability from a practical view point. In this review, we investigated the current status of sarcoma cell lines to reveal what cell lines have to be established to promote sarcoma research. The cell line database, Cellosaurus, used in this study includes more than one hundred thousand cell lines and is frequently updated. Thus, Cellosaurus is an adequate cell line database for investigation.We grouped the cell lines according to the histology of their original tumor. We found that 45 histological subtypes were covered by the currently reported cell lines, while 133 were not. Considering the diversity and complexity of sarcomas, we need more cell lines that represent the different histological subtypes. In addition, we found that multiple cell lines were established for 36 histological subtypes, with a single cell line reported for nine subtypes. During the course of the cell line establishment, clonal selection and expansion may occur, and only limited cell populations may survive under tissue culture conditions. To understand the sustainability of the original characteristics of the established cell lines, the capability of tumor tissue formation and the histology of the formed tumors can be evaluated by xenograft experiments. In addition, patient-to-patient variations are clinically considerable even if they have tumors with the same histology. Therefore, no single cell line can represent the characteristics of whole tumor tissues; we need to use multiple cell lines. In this sense, we also need more cell lines for sarcomas that already have corresponding cell lines.Cell lines are most frequently established from Ewing’s sarcoma and osteosarcoma samples. However, the absolute number of patients with Ewing’s sarcoma and osteosarcoma is small according to medical statistics; undifferentiated pleomorphic sarcoma, liposarcoma, and leiomyosarcoma are more common sarcomas . Thus, tClassification of Tumours of Soft Tissue and Bone [The histological diagnosis of original tumors of cell lines may need to be updated in cases where the name of cell lines did not match the official classification. Among the 844 sarcoma cell lines investigated, 42 were not named according to the 2013 World Health Organization and Bone . The diaUnfortunately, cell lines are not always deposited in cell banks. We found that only 139 of 819 sarcoma cell lines named according to the WHO classification were deposited in public cell banks. Probably, the rest of the cell lines can be provided upon request by researchers. The current cell bank systems may rely on researchers and institutes to undertake the cell line establishment. Establishing novel cell lines costs a considerable amount of resources, such as time and money; furthermore, because cell lines are properties of the institutes to which researchers are affiliated, it may be difficult to deposit all cell lines in public cell banks and share them with other researches. As the establishment of cell lines itself is not necessarily a novel discovery, nor would the publication be in high-impact journals, researchers may not be motivated to establish and share cell lines. A system to motivate cell line establishers and their institutes may be required to improve the availability by depositing cell lines.This systematic review has several limitations. First, although the genetic background and biological characteristics of some but not all cell lines were reported in publications, this review did not summarize those data. In our research, 692 cell lines were reported in previous papers, and 108 of them were deposited in cell banks . AlthougCell lines have been considered a valuable tool for both basic research and pre-clinical studies. The functional significance of genetic products such as mRNA, miRNA, and proteins can be clarified using living cells, and cell lines are an indispensable research resource. In the preclinical evaluation of new drugs, their tumor suppressive effects and mode-of-action are also investigated using cell lines. Although the predictive power of cell lines can be undermined by the selective pressures during the process of establishment and long-term passaging, a great advantage of cell lines is that the examinations can be done in a high-throughput manner with relatively low costs. Patient-derived xenografts (PDXs) may complement the inherent drawbacks of cell lines, because PDXs may retain the microenvironmental conditions of the original tumors. However, the manipulation of PDX requires time-consuming and tremendous efforts, and their unstable molecular backgrounds have been revealed at the genome level. Moreover, because the human stromal components in PDX tumors are replaced with mouse ones after several passes, consistent results may be limited in experiments using PDXs. Taken together, cell lines have a unique utility, and they are indispensable in cancer research.We conclude that (1) more sarcoma cell lines representing the various histological types as well as those established by single cell lines are needed to effectively capture the diversity and complexity of the disease; (2) a system is needed to reward the efforts of researchers who establish and deposit cell lines into public cell banks to promote cell line sharing in the research community, and (3) further investigations are required to determine the critical factors determining the success rate of the cell line establishment and create effective experimental protocols.


Cisplatin is one of the major drugs used in oral cancer treatments, but its usage can be limited by acquired drug resistance. In this study, we established three cisplatin-resistant oral squamous cell carcinoma (OSCC) cell lines and characterized them using cell viability assays, qPCR, Western blotting, FACS, immunofluorescence, and wound healing assays. Three OSCC cell lines  underwent long-term exposure to cisplatin, eventually acquiring resistance to the drug, which was confirmed by an MTT assay. In these three newly established cell lines , overexpression of multidrug resistance (MDR)-related genes was detected by qPCR and Western blotting. The cell lines displayed an increase in the functional activities of breast cancer resistance protein (BCRP) and multidrug resistance protein1 (MDR1) by rhodamine 123 and bodipy FL prazosin accumulation assays. Moreover, the cisplatin-resistant cells underwent morphological changes, from round to spindle-shaped, increased expression of epithelial-to-mesenchymal transition (EMT)-related molecules such as N-cadherin, and showed increased cell migration when compared with the parental cell lines. These results suggest that these newly established cell lines have acquired drug resistance and EMT induction.  Oral cancer is an increasingly global disease ,2, howevCisplatin, a platinum-based drug, is one of the standard first-line chemotherapeutic agents used to treat OSCC . UnfortuEpithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell–cell adhesion, and gain mobility and invasiveness to become mesenchymal stem cells . In mostThe purpose of our study was to establish three cisplatin-resistant human OSCC cell lines, and evaluate their biological characteristics compared to their respective parental cell lines.Three OSCC cell lines, YD-8, YD-9, and YD-38, were continuously exposed to stepwise increasing concentrations of cisplatin over a one-year period . An MTT assay was performed to determine whether the cell lines had developed cisplatin-resistance. MDR1 (>2.4-fold change), BCRP (>7.2-fold change) and MRP3 (>2.5-fold change) were upregulated. In YD-9/CIS cells, MDR1 (>7.5-fold change), MRP4 (>2.7-fold change), and BCRP (>1.6-fold change) were upregulated. In YD-38/CIS cells, BCRP (>3.7-fold change) was upregulated. We confirmed the expression changes in BCRP and MDR1 by Western blot  showed resistance to paclitaxel. In addition, YD-8/CIS, YD-9/CIS, and YD-38/CIS showed low, medium, and no resistance to CKD-602, respectively. None of the cell lines acquired a resistance to 5-fluorouracil. Therefore, our results show that the three cisplatin-resistant cell lines acquired resistance to cisplatin, and the cross-resistance to different chemotherapeutic drugs was cell-specific.BCRP expression was significantly upregulated in YD-8/CIS and YD-38/CIS cells, and MDR1 expression was significantly upregulated in YD-9/CIS cells; these results were confirmed via Western blotting. Therefore, it has been confirmed that long-term exposure of OSCC cell lines to cisplatin increased the expression of the ABC transporter genes, MDR1 and BCRP. MDR1 is a 170 kDa ATP-dependent membrane transporter that is widely distributed throughout the body and pumps many foreign substances out of cells [One of the common mechanisms of drug resistance is through a reduction in their intracellular accumulation, which is associated with overexpression of ABC transporters ,24. Thusof cells . BCRP isof cells .Next, we compared the functional activities of MDR1 and BCRP between the parental and cisplatin-resistant cells using rhodamine 123 and bodipy FL prazosin accumulation assays. Rhodamine 123, a cationic fluorescent dye, is a good MDR1 substrate. Bodipy FL prazosin, a fluorescent derivative of the quinazoline based α-adrenergic antagonist prazosin, is known as a BCRP substrate ,26,27,28Several studies have shown that MDR is associated with EMT ,31. ThusCisplatin (PubChem CID: 84691) was purchased from Choongwae Pharm. C. (Korea) and CKD-602 (PubChem CID: 123773277) was kindly provided by Chongkundang Pharm. Co. . These drugs were dissolved in distilled water at 1 mg/mL. 5-Fluorouracil (PubChem CID: 3385) was obtained from Sigma  and dissolved in DMSO at 20 mg/mL. Paclitaxel (PubChem CID: 36314) was purchased from Hanmi Pharm. Co. . Each aliquot of the stock solutions was stored at −20 °C until use.2. When culturing the resistant cell lines, the final concentration of cisplatin was maintained at 2 μg/mL. Parental OSCC cell lines  were obtained from the Korean Cell Line Bank , and cisplatin-resistant cell lines  were generated in our lab by exposing the original cell lines to increasing concentrations of cisplatin over a period of one year. The cell lines were maintained in RPMI-1640 medium  supplemented with 10% heat-inactivated FBS  and 1% penicillin-streptomycin  at 37 °C in a humidified atmosphere containing 5% CO4 cells per well in 96-well plates and then treated with cisplatin, 5-fluorouracil, paclitaxel, or CKD-602. Cells were incubated with the drugs for 72 h, after which 0.5 mg/mL of MTT reagent  was added to each well. After 2 h, the medium was discarded, the resulting formazan was dissolved in DMSO, and the absorbance at 570 nm was measured using a SpectraMax Plus 384 microplate reader . For cell viability assays, single cell suspensions were seeded at 1 × 10MDR1, MRP1-6, MRP9, and BCRP was analyzed by a qPCR ViiA7 Real-Time PCR System using PowerUp SYBR Green Master Mix . The following primers were used: MDR1, 5′-CACCCGACTTACAGATGATG-3′ (forward) and 5′- GTTGCCATTGACTGAAAGAA-3′ (reverse); MRP1, 5′-GCCGAAGGAGAGATCATC-3′ (forward) and 5′-AACCCGAAAACAAAACAGG-3′ (reverse); MRP2, 5′-CGACCCTTTCAACAACTACTC-3′ (forward) and 5′-CACCAGCCTCTGTCACTTC-3′ (reverse); MRP3, 5′-GTGGGGATCAGACAGAGAT-3′ (forward) and 5′-TATCGGCATCACTGTAAACA-3′ (reverse); MRP4, 5′-GCTCAGGTTGCCTATGTGCT-3′ (forward) and 5′- CGGTTACATTTCCTCCTCCA-3′ (reverse); MRP5, 5′-CAGCCAGTCCTCACATCA-3′ (forward) and 5′-GAAGCCCTCTTGTCTTTTTT-3′ (reverse); MRP6, 5′-AGGAGGCCCGAGCTTAGAC-3′ (forward) and 5′-CCTGCCGTATTGGATGCTGT-3′ (reverse); MRP9, 5′-ATGCGGTTGTCACTGAAG-3′ (forward) and 5′-GTTGCCTCATCCATAATAAGAAT-3′ (reverse); BCRP, 5′-TGACGGTGAGAGAAAACTTAC-3′ (forward) and 5′-TGCCACTTTATCCAGACCT-3′ (reverse); and GAPDH, 5′-AATCCCATCACCATCTTCCA-3′ (forward) and 5′-TGGACTCCACGACGTACTCA-3′ (reverse) [−ΔΔCT method [Total RNA was extracted from the harvested culture cells in 1.5 mL tubes using an RNA-spin Total RNA Extraction Kit . cDNA synthesis was performed using High-Capacity cDNA Reverse Transcription Kits . The expression of reverse) . The relT method . To investigate the expression levels of multidrug resistant- or EMT-related proteins in the parent and resistant cell lines, we performed a Western blot assay. Whole cell lysates (20 μg) were resolved by 10% SDS-PAGE, and proteins were transferred onto a PVDF membrane . The membrane was incubated overnight at 4 °C with the following antibodies: anti-MDR1 from Abcam , anti-BCRP from Santa Cruz Biotechnology , anti-E-cadherin and -N-cadherin purchased from CellSignaling Technology , and anti-β-actin obtained from Sigma . The immunobands were detected using a ChemiDoc Touch imaging system .5 cells per well in a 6-well plate. The next day, cells were treated with rhodamine 123 (1 μg/mL) or prazosin (250 nM) for 30 min. After incubation, cells were washed twice in ice-cold PBS, harvested by centrifugation at 1000 rpm for 5 min, and resuspended in 500 μL of PBS. Accumulation of rhodamine 123 or prazosin in cells was measured using a FACSCalibur flow cytometer . At least 10,000 events were analyzed for each tube.For the rhodamine 123 and bodipy FL prazosin accumulation assays, cells were seeded at 2 × 10E-cadherin and N-cadherin were analyzed by immunofluorescence in parental and cisplatin-resistant cells. Cells were seeded on cover slips in 6-well plates and incubated overnight at 37 °C. The next day, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% NP-40 in PBS for 10 min at room temperature. The cells were blocked with 10% FBS in PBS for 30 min and incubated with either anti-E-cadherin or N-cadherin primary antibody (1:50 dilution) overnight at 4 °C. After this, the cells were washed three times with ice-cold PBS and incubated with Alexa Fluor_488 goat anti-rabbit secondary antibody (1:50 dilution) for 2 h at room temperature. The stained cells were then washed three times with PBS, mounted on slides using fluoroshield mounting medium with DAPI  and observed with an AxioVision 4 imaging system . 5/well) were seeded into a 48-well plate and incubated for 24 h. Confluent cell cultures were scratched using a sterile pipette tip and then washed with PBS. After incubation for 8 h, the cells which migrated into the wound region were counted and captured using an Olympus CKX53 inverted microscope .For the wound healing assays, the cells . A p value of less than 0.01 was considered to be statistically significant.All the data were presented as the mean ± SD of at least three independent experiments. The significance of the difference between the two paired groups was analyzed using a Student’s In conclusion, our results show that we have successfully established three cisplatin-resistant human OSCC cell lines: YD-8/CIS, YD-9/CIS, and YD-38/CIS. These cell lines have highly similar biological characteristics, such as acquired drug resistance and EMT induction, and could be used as ideal models for further studies on the reversal of cisplatin-resistance in OSCC. They can also be used to help clarify the mechanism behind the molecular changes occurring in chemoresistance.


OSF-2 transcript , protein expression in cell lysates and cell culture medium (western blot), and expression of the OSF-2 protein in cell lines (immunofluorescence) were investigated in this study. Increased expression of OSF-2 mRNA was observed in drug-resistant cells and followed by increased protein expression in cell culture media of drug-resistant cell lines. A subpopulation of ALDH1A1-positive cells was noted for W1DR and W1MR cell lines; however, no direct co-expression with OSF-2 was demonstrated. Both drugs induced OSF-2 expression after a short period of exposure of the drug-sensitive cell line to DOX and MTX. The obtained results indicate that OSF-2 expression might be associated with the development of DOX and MTX resistance in the primary serous W1 ovarian cancer cell line.One of the main obstacles to the effective treatment of ovarian cancer patients continues to be the drug resistance of cancer cells. Osteoblast-Specific Factor 2  is a secreted extracellular matrix protein (ECM) expressed in fibroblasts during bone and teeth development. Expression of OSF-2 has been also related to the progression and drug resistance of different tumors. The present study investigated the role of OSF-2 by evaluating its expression in the primary serous ovarian cancer cell line, sensitive (W1) and resistant to doxorubicin (DOX) (W1DR) and methotrexate (MTX) (W1MR). The  Chemoresistance of cancer cells—inherent or developed during treatment—remains an obstacle to the effective treatment of ovarian cancer patients . Hundredwww.ensembl.org, ENSG00000133110) with a molecular mass from 83.5 to 93.5 kDa [Osteoblast-Specific Factor 2 (OSF-2), also known as a Periostin (POSTN), is a secretory glycoprotein expressed as at least six different isoforms , with statistically higher expression in chemoresistant, high-grade serosum and endometrioid tumors than in chemosensitive ones. In vitro experiments proved that OSF-2 stimulated the resistance of ovarian cancer cells to carboplatin and PAC . AnotherTo investigate the role of OSF-2 in drug resistance development, we used a model of ovarian cancer, the most lethal gynaecological malignancy . At the Previously, from the drug-sensitive W1 ovarian cancer cell line, we have developed a series of cell lines resistant to CIS, PAC, DOX, and topotecan (TOP)—drugs used in ovarian cancer chemotherapy, as well as vincristine (VIN) and MTX, which are not used in standard ovarian cancer treatment . These cOSF-2 gene in the following ovarian cancer cell lines was examined: high-grade serous—OVCAR3, PEA1, PEA2; low-grade serous—PEO23; serous—SKOV-3; endometroid adenocarcinoma - A2780 and primary ovarian cancer cell line—W1. The highest OSF-2 gene expression was observed in the A2780 cell line, and was assigned as 1. In comparison, for the W1 cell line, about a 20-fold lower expression level was noted. In the consecutive investigated cell lines, expression levels were much lower. Due to big differences in expression level among the examined cell lines, the results are presented as a logarithmic scale  increased levels of the OSF-2 transcript in both cell lines  in response to DOX treatment with a maximum increase in transcript level of about 3.5-fold after 72 h of treatment (OSF-2 transcript level after 72 h of treatment (p < 0.05) with a maximum increase in transcript level of about 1.7-fold  and MTX (50 and 100 ng/mL) for 24, 48, and 72 h. Then, changes in gene expression were investigated. The dose- and time-dependent increase in reatment A. A simi1.7-fold B was obsPreviously, we observed an ALDH1A1 CSCs-like population in PAC- and TOP-resistant ovarian cancer cell lines ,42. In tPreviously in drug-resistant cell lines, the increased expression of ECM molecules, like COL3A1, COL21A1, and LOX in ALDH1A1-positive cells was observed ,43. TherChemoresistance of cancer cells still remains one of the obstacles to the effective treatment of ovarian cancer patients The cellular mechanisms of resistance are already well-described in the literature. Cancer-tissue-specific mechanisms are still being investigated and need further evaluation. This is important especially from a clinical point of view, as it was hypothesized that the tumor microenvironment is a dominant force in multidrug resistance and low treatment efficacy of cancers . These mUsing the RNA microarray technique, we have analyzed the expression of ECM molecules in ovarian cancer cell lines resistant to CIS, PAC, DOX, TOP, VIN, and MTX. The obtained results found OSF-2 to be overexpressed in cell lines resistant to DOX and MTX . The eleOSF-2 in A2780 endometroid adenocarcinoma [OSF-2 expression [OSF-2 transcript levels in DOX- and MTX-resistant W1 cell lines. Since PCR primers used in our study recognize all of the OSF-2 isoforms, a more detailed western blot analysis was performed. As shown in The expression analysis conducted for the different ovarian cancer cell lines confirmed the very high expression of arcinoma , whereaspression , we founOSF-2 expression after a short period of treatment of a sensitive OC cell line with DOX or MTX. In a similar experiment conducted by Xiao et al., increased expression of periostin in SW480 and HT-29 colon cancer cell lines treated with oxaliplatin and fluorouracil was demonstrated for 24 to 72 h [As previously reported, most studies on the effects of cytotoxic drugs describe the difference in gene expression between drug-sensitive and resistant cell lines. The results of our previous experiments indicate that the expression of some genes changes after a short exposure time of cells to cytotoxic drugs ,52,53. I to 72 h . The resDifferent models of drug resistance development are described in the literature. In the last 10 years, one of the most-studied models of drug resistance is the one based on the theory of CSCs . SeveralOSF-2 expression seems to play a role in drug resistance—not only in vitro, but also in vivo. In two independent ovarian cancer studies, periostin expression in tumor stroma was associated with chemotherapy resistance. In a study by Sung et al., stromal OSF-2 expression was significantly higher in platinum-resistant patients, and correlated with the FIGO stage and tumor recurrence rate after the first treatment. Patients with high OSF-2 expression in stroma also had significantly lower overall survival (OS) and progression free survival (PFS) than those with low expression. In contrast, expression in tumor cells did not correlate with patients’ survival; however, patients with high OSF-2 expression in both compartments had shorter OS and PFS. . In a stTaken together, periostin seems to play a role in drug resistance, tumor growth, and metastasis in many cancers. As metastatic tumors are usually much more resistant to chemotherapy, we can suppose that periostin could also play a role in the drug resistance of metastatic tumors, such as in the case of drug-resistant cell lines. However, we did not find any data comparing resistant profiles of primary and metastatic tumors together with OSF-2 expression.®488 Donkey Anti-Rabbit and Alexa Fluor®594 Donkey Anti-mouse IgG—were obtained from Jackson ImmunoResearch Laboratories . Western blot reagents  were purchased from Biorad .Culture media , fetal bovine serum, antibiotic-antimycotic solution, L-glutamine, sodium private, DAPI mounting medium, and DOX and MTX were purchased from Sigma . Rabbit polyclonal anti-OSF-2 Ab was obtained from Proteintech , rabbit monoclonal anti-ALDH1A1 Ab was purchased from Abcam , mouse monoclonal anti-ALDH1A1 Ab was purchased from ABGENT , and rabbit polyclonal anti-GADPH Ab was purchased from Santa Cruz Biotechnology . Goat policlonal anti-rabbit horseradish peroxidase (HRP)-conjugated Ab was purchased from Cell Signaling . The fluorescent secondary antibodies—Alexa FluorThe human ovarian carcinoma A2780, SKOV-3, and OVCAR-3 were purchased from ATCC . The human ovarian carcinoma PEA1, PEA2, and PEO23 were purchased from Sigma . The human primary ovarian cancer cell line W1 was established from the tumor tissue of an untreated 54-year-old Caucasian female patient diagnosed for serous ovarian adenocarcinoma . Cells grow as a monolayer, and present an epithelial morphology and adherent growth model. The W1 subline, resistant to MTX (W1MR) and DOX (W1DR) were obtained by exposing W1 cells to the MTX or DOX at incrementally increasing concentrations. The final concentrations used for selecting the resistant cells were 28 ng/mL of MTX, and 100 ng/mL of DOX. The increase in resistance according to the parental drug-sensitive cell line W1 was as follows: 138-fold for W1MR vs. W1, and 10.3-fold for W1DR vs. W1 .2.Cells were cultured in Minimum Essential Medium Eagle (MEM) medium (A2780), RPMI-1640 medium (W1), RPMI-1640 medium supplemented with 2mM sodium private , or Dulbecco’s Modified Eagle Medium (DMEM) (SKOV-3) supplemented with 10% fetal bovine serum, 200 mML-glutamine, penicillin (100 units/mL), streptomycin (100 units/mL), and amphotericin B (25 μg/mL) at 37 °C in an environment of 5% COThe total RNA was extracted from cells using a Gene Matrix Universal RNA purification Kit  according to the manufacturer’s protocol. Reverse transcription was performed using the M-MLV reverse transcriptase  and a thermal cycler  as described in the manufacturer’s protocol. The cDNA was amplified from two µg of RNA. Real-time PCR was performed using the 7900 HT Fast Real-Time PCR System , Maxima SYBR Green/ROX qPCR Master Mix , and the sequence-specific primers, as indicated in Ct−ΔΔ (where ΔΔCt = ΔCt of the sample (drug-resistant line) − ΔCt of the calibrator (drug-sensitive line)). The graphs were plotted using Sigma Plot.The relative quantification (RQ) method was used for gene expression analysis, where RQ estimates the difference at the level of gene expression against the calibrator . As a calibrator, the drug-sensitive cell lines (A2780 or W1) were used. The standard formula was applied: RQ = 26 cells/25 μL) were prepared using a RIPA buffer containing a protease inhibitor cocktail , and then, supernatant was collected after centrifugation at 4 °C and 12,000× g for 15 min. A Bradford protein assay system  was used to determine the protein concentrations. The isolation of proteins from culture media was prepared after a 72 h culture of cells in serum-free media. Next, the media was centrifuged at 15,000 rpm for 30 min at RT, and supernatants were placed in Amicon Ultra-15 3K centrifuge filter devices  and centrifuged using a swinging-bucket rotor for 60 min at 4,000× g/RT.Cell lysates . Next, the membrane was incubated with rabbit policlonal anti-OSF-2 or rabbit monoclonal anti-ALDH1A1 antibodies at dilutions of 1:500, followed by incubation with the appropriate HRP-conjugated secondary antibody. The bands detection was analyzed using a chemical luminescence kit  and Hyperfilm ECL . The quality of protein loading was made by reblotting the membrane with a rabbit anti-GAPDH antibody (1:1000) and goat anti-rabbit HRP-conjugated antibody For the western blot analysis, 10 μg protein from each sample were resuspended in 4× loading buffer  and loaded into each well of a 4–20% mini-PROTEAN®488 secondary antibody  for 1 h at room temperature was proceed. In the double immunofluorescence, the cells were incubated with the mixture of two respective green dye-labelled  and red dye-labelled  secondary antibodies for 1 h at room temperature. Finally, cells were washed with PBS and mounted in DAPI mounting medium. The expression analysis and pictures were taken under fluorescence microscope .For immunofluorescence and double immunofluorescence analysis, cells were cultured in 24-well chamber glass slides and grown to a near-confluent state. The standard procedure was performed as follows. The culture medium was replaced by PBS for rinsing, and next by ice-cold acetone/methanol (1:1) for 10 min for fixation and permeabilization. Then, cells were washed with PBS and blocked in 3% BSA for 30 min at room temperature. After that, the incubation with primary antibodies was prepared. For classic immunofluorescence one type of primary antibody at a time was used . For double immunofluorescence purpose the cells were incubated with the mixture of two primary antibodies simultaneously: OSF-2  + ALDH1A1 . After 2 h incubation at room temperature, cells were washed with PBS and incubated with secondary antibodies. For single immunofluorescence, the incubation with green dye-labelled Alexa FluorOSF-2 was measured after a short period of exposure to a citotoxic agent. The drug-sensitive cell line (W1) was cultured in 1 mL of medium at a density of 0.5 × 106 cells per well (six-well plate). Then, the DOX at 15 and 20 ng/mL concentrations or MTX at 50 ng/mL and 100 ng/mL concentrations were added, respectively . The int-test. p < 0.05 was considered to indicate a statistically significant difference.Data are presented as standard error of the mean (SEM) and were analyzed using Student’s To our knowledge, this study is the first to describe the expression analysis of OSF-2 in drug-resistant ovarian cancer cell lines. Our research showed elevated levels of OSF-2 expression in W1 DOX- and MTX-resistant ovarian cancer cell lines and corresponding media. The short exposure time of cells to DOX- or MTX-induced expression of OSF-2 in the drug-sensitive cell line, which may suggest its potential role in the primary resistance of this cell line before other mechanisms of drug resistance appear. An extracellularly secreted OSF-2 protein could be involved in CAM-DR. Thus, we have found that the expression of OSF-2 might be associated with the development of DOX and MTX resistance of the primary serous W1 ovarian cancer cell line.


Endothelial cell senescence is a hallmark of vascular aging that predisposes to vascular disease. We aimed to explore the capacity of the renin–angiotensin system (RAS) heptapeptide angiotensin (Ang)‐(1‐7) to counteract human endothelial cell senescence and to identify intracellular pathways mediating its potential protective action. In human umbilical vein endothelial cell (HUVEC) cultures, Ang II promoted cell senescence, as revealed by the enhancement in senescence‐associated galactosidase  positive staining, total and telomeric DNA damage, adhesion molecule expression, and human mononuclear adhesion to HUVEC monolayers. By activating the G protein‐coupled receptor Mas, Ang‐(1‐7) inhibited the pro‐senescence action of Ang II, but also of a non‐RAS stressor such as the cytokine IL‐1β. Moreover, Ang‐(1‐7) enhanced endothelial klotho levels, while klotho silencing resulted in the loss of the anti‐senescence action of the heptapeptide. Indeed, both Ang‐(1‐7) and recombinant klotho activated the cytoprotective Nrf2/heme oxygenase‐1 (HO‐1) pathway. The HO‐1 inhibitor tin protoporphyrin IX prevented the anti‐senescence action evoked by Ang‐(1‐7) or recombinant klotho. Overall, the present study identifies Ang‐(1‐7) as an anti‐senescence peptide displaying its protective action beyond the RAS by consecutively activating klotho and Nrf2/HO‐1. Ang‐(1‐7) mimetic drugs may thus prove useful to prevent endothelial cell senescence and its related vascular complications. ACE2angiotensin‐converting enzyme 2Ang‐(1‐7)angiotensin‐(1‐7)AngangiotensinAREantioxidant‐regulated elementsBSAbovine serum albuminFCSfetal calf serumHO‐1heme oxygenase‐1ICAM‐1intercellular adhesion molecule‐1IL‐1βinterleukin‐1βNrf2nuclear factor‐erythroid 2‐related factor 2RASrenin–angiotensin systemr‐klothorecombinant klothoSA-β-galsenescence‐associated β‐galactosidaseSASPsenescence-associated secretory phenotypeSn‐PPtin protoporphyrin IXTIFstelomere dysfunction‐induced fociTRF‐1telomere protein telomeric repeat binding factorVCAM‐1vascular cell adhesion molecule‐1γH2AXphosphorylated histone H2AX1Vascular aging is a complex multifaceted process displaying functional and structural alterations that ultimately favor vascular disturbances, including endothelial dysfunction and atherosclerosis  plays a pivotal role in the regulation of cardiovascular homeostasis, in both health and disease. In recent years, a number of studies have been dedicated to decipher the biological actions of novel RAS components, such as angiotensin (Ang)‐(1‐7). This heptapeptide is generated not only from angiotensin II (Ang II) through the action of angiotensin‐converting enzyme 2 (ACE2), but also from angiotensin I (Ang I) via neutral endopeptidase activity  has been regarded as a physiological antagonist of Ang II by opposing its vasoconstrictor, proliferative, hypertrophic, or pro‐inflammatory actions /antioxidant‐regulated element (ARE) system is a major evolutionary conserved cytoprotective system. It is also nowadays considered a powerful modulator of species longevity /Mas receptor axis to antagonize cell senescence induced by Ang II and by other non‐RAS stressors. The participation of klotho and Nrf2/HO‐1 in the protective actions of Ang‐(1‐7) on endothelial cells was also analyzed.22.1The fraction of senescence‐associated β‐galactosidase  cells, used as a marker of cell senescence, was of 2.97 ± 0.23% in control unstimulated human umbilical vein endothelial cell (HUVEC) pre‐senescent cultures. After the exposure to Ang II 100 nM) for 18 hr, the fraction of cells positively stained for SA‐β‐gal was significantly enhanced, and this effect was mitigated by Ang‐(1‐7)  at the sites of injury  in late‐passage (P12) senescent HUVEC, in which the proliferation rate was markedly reduced and the average SA‐βgal+ staining in basal unstimulated conditions was shifted up to 53.52 ± 3.44%  has been proposed as a physiological antagonist of Ang II. However, we next aimed to determine the capacity of the heptapeptide to counteract endothelial senescence induced by other stressors independent of the RAS. The pro‐inflammatory cytokine interleukin (IL)‐1β was chosen based on its growing relevance in human vascular disease Libby, . Similar2.3Senescent endothelial cells undergo a series of changes that result into an activated pro‐inflammatory and secretory phenotype that eventually favors leukocyte recruitment.Consistent with this notion, both Ang II and IL‐1β enhanced the expression of the adhesion molecules, intercellular adhesion molecule (ICAM)‐1 Figure a,d and v2.4To analyze the cytoprotective mechanisms that might be on the basis of the anti‐senescence action of Ang‐(1‐7), we first explored a possible role for klotho. In endothelial cells, human recombinant klotho (r‐klotho) prevented the enhanced SA‐β‐gal+ cell number induced by both Ang II and IL‐1β, without affecting cell senescence by itself Figure a. In fac2.5To gain insight into the common cytoprotective pathways that might be activated by both Ang‐(1‐7) and klotho, we next focused on Nfr2/HO‐1. First, the pharmacological activation of Nrf2 by means of sulforaphane protected endothelial cultures against cell senescence induced by both Ang II and IL‐1β Figure a. MoreovHO‐1 levels were equally enhanced by either Ang‐(1‐7) or r‐klotho Figure e,f. In fGlobally considered, these data identified Nrf2/HO‐1 as key mediators of the cytoprotective effects derived from the consecutive activation of Ang‐(1‐7) and klotho. This concept was further reinforced using a functional ex vivo vascular reactivity assay. In noradrenaline pre‐contracted murine mesenteric microvessels, the vasodilation exerted by Ang‐(1‐7), but also the one induced by r‐klotho, was markedly inhibited by Sn‐PP, while the one exerted by the vasodilator acetylcholine remained unaffected Figure k.3Vascular aging is a main risk factor for developing adverse cardiovascular events /Mas axis, may be a suitable target to delay endothelial cell senescence, one of the key hallmarks of vascular aging that drives to endothelial dysfunction and atherosclerosis.We herein demonstrate that Ang‐(1‐7) attenuates total and telomeric DNA damage, an early senescence‐associated event that signals downstream to provoke growth arrest and senescence , klotho is able to exert nitric oxide‐dependent vasorelaxant actions  as a valuable tool to protect against human endothelial cell senescence through the consecutive activation of klotho and the Nrf2/HO‐1 axis. Over the last years, a notable effort is being made to develop pharmacological activators of the ACE2/Ang‐(1‐7)/Mas axis to fight a number of conditions, including cardiovascular diseases. In light of the present results, these drugs may prove useful to counteract endothelial cell senescence and premature vascular aging, as well as their deleterious complications. Since blocking senescence with Ang‐(1‐7) might promote dysplasia or neoplasia of endothelial cells, or Ang‐(1‐7) may itself exert hypotension or cardiac and renal fibrosis . M199 medium, fetal calf serum (FCS), and trypsin‐EDTA were from Biological Industries . Human r‐klotho and IL‐1β were purchased from Abcam  and Peprotech , respectively. Ang‐(1‐7) and the Mas antagonist A779 were purchased from Bachem . Sn‐PP and sulforaphane were from Frontier Scientific  and LKT Laboratories , respectively. All other reagents were purchased from Sigma  unless otherwise stated.4.22. For experiments, pre‐senescent cells at passages 1–5 were incubated for the indicated time periods with the different test compounds in M199 medium supplemented with 10% FCS, ECGS, heparin and antibiotics. For some experiments, senescent HUVEC were used at passage 12. All the procedures were reviewed and approved by the ethics committee of Universidad Autónoma of Madrid and Hospital Universitario La Paz, respectively, and written informed consent was obtained from all cord donors.Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cords, as previously described  as previously described  were examined by immunofluorescence microscopy as previously described  were then perfused across the endothelial monolayer. In all experiments, leukocyte interactions were determined after 5 min at 0.5 dyn/cm2. Cells interacting with the surface of the endothelium were visualized and recorded  using phase‐contrast microscopy .Mononuclear cells were obtained from buffy coats of healthy donors by Ficoll‐Hypaque density gradient centrifugation, as previously described  according to the manufacturer instructions.After the appropriate treatments and incubation times, supernatants were collected, centrifuged at 900 4.8Klotho, Nrf2, and HO‐1 levels in HUVEC were determined by Western blot as previously described  were mounted on a small‐vessel myograph to measure isometric tension as described before (Peiró et al., 4.12t test and one‐way ANOVA followed by Bonferroni post hoc test or two‐way ANOVA, as appropriate. A p value ≤0.05 was considered statistically significant.Results are expressed as mean ± SEM. Statistical analysis was performed using Student's None declared.C.P., C.F.S.‐F., and T.R. conceived the study. A.S.‐H.L., A.R., L.A.V., S.V., I.V., P.M. R.L., N.P.‐L., and I.S.‐P. performed in vitro and ex vivo experiments, analyzed raw data, performed statistical analysis, and drew figures. J.D.E. and M.J.S. designed and analyzed the DNA damage foci and flow chamber experiments, respectively. C.P., C.F.S.‐F., and T.R. wrote the manuscript. J.L.B., J.D.E., and M.J.S. contributed to the discussion. All authors reviewed and approved the final version of the manuscript. Click here for additional data file.


Fucus vesiculosus, F. serratus, F. distichus subsp. evanescens, Dictyosiphon foeniculaceus, Laminaria digitata, Saccharina latissima) as well as three reference compounds  on tumor  and non-tumor  cell lines. All fucoidans were extracted according to a standardized procedure and tested in a commercially available MTS assay. Cell viability was measured after 24 h incubation with test compounds (1–100 µg/mL). Apart from few exceptions, fucoidans and heparins did not impair cell viability. In contrast, fucoidans significantly increased cell viability of suspension cell lines, but not of adherent cells. Fucoidans slightly increased viability of tumor cells and had no impact on the viability of non-tumor cells. The cell viability of HeLa and ARPE-19 cells negatively correlated with protein content and total phenolic content (TPC) of fucoidans, respectively. In summary, none of the tested fucoidans turned out to be anti-proliferative, rendering them interesting for future studies and applications. Fucoidans extracted from brown algae exert manifold biological activities paving the way for the development of numerous applications including treatments outside tumor therapy such as age-related macular degeneration or tissue engineering. In this study, we investigated the antiproliferative effects of fucoidans extracted from six different algae ( Cancer is one of the leading causes of death in industrial nations and a wide variety of types of cancer exists. Despite enhanced research interest and already existing medical treatments, the mortality rate is still very high, depending on the cancer type. A common complication in cancer patients and one of the leading causes of death is venous thromboembolism (VTE) . The firA potential alternative may be fucose-containing sulfated polysaccharides, the so-called fucoidans, extracted from brown algae. Fucoidans display an attractive array of bioactivities and potential applications including not only cancer inhibition, but also immune modulation, pathogen defense and numerous others  ,10. ManyIn general, one of the pivotal initial in vitro tests is the examination of the influence of potentially active agents such as fucoidans on cell viability. Whereas antiproliferative effects on tumor cell lines are favorable for antitumor therapies, cytotoxicity on non-cancerous cells is a big limitation for any lead compound, including fucoidans. Such toxicity may hamper other potential applications of fucoidans, for example their use in tissue engineering for improvement of bone vascularization during bone repair ,17. AnotSaccharina japonica inhibited the proliferation of human myeloma and T-cell lymphoma cells as well as having cell growth inhibitory effects on SH-SY-5Y (neuroblastoma cells) and MKN-28 (adenocarcinoma cells) , thus including a total of seven tumor cell lines  and two non-cancerous cell lines (ARPE-19 and HaCaT). After incubation for 24 h with the different fucoidans, the cell viability was measured using a commercial MTS assay according to a standardized protocol. As reference compounds three different commercially available substances, namely reference heparin as well as reference enoxaparin (a LMWH) and the commercial Fucus vesiculosus fucoidan, were additionally tested. The tests were performed to obtain comparative information on the suitability of the different fucoidans for further investigations. In the present study, we tested the cell viability effects of six fucoidans on nine different cell lines. The fucoidans were extracted from six different brown algal species by using the same extraction method. Cell viability effects were assessed using one standardized assay enabling direct comparison of the results. The extracted algal species were The details of the extraction procedure of the six fucoidans as well as their basic characteristics namely degree of sulfation (DS), molecular mass, monosaccharide composition, their contents of polyphenols, laminarin and protein as well as their radical-scavenging capacity, three exemplary bioactivities and their fluorescence intensity (FI) increasing effect on the sulfated glycan sensor Polymer-H are described elsewhere (unpublished data until accepted). To compare the effects of the different fucoidans and reference substances on cell viability, we determined their effects at concentrations up to 100 µg/mL  on nine different cell lines in relation to the untreated control (100%) . p < 0.05), heparin only at 50 µg/mL (p < 0.05) and the LMWH enoxaparin only at 100 µg/mL (p < 0.001). A questionable exception was the significant lower cell viability  after treatment with 10 µg/mL enoxaparin, whereas 100 µg/mL increased the cell viability significantly (p < 0.001). The commercial fucoidan had a stimulating effect at 50 and 100 µg/mL (p < 0.05). In the leukemic cell line HL-60, all test compounds apart from the DF fucoidan increased the cell viability a. The efp < 0.01). The other fucoidans and the reference substances showed no significant effect.In the Burkitt lymphoma cell line Raji, no inhibitory but rather a stimulatory effect was observed b. FV andp < 0.01; 10 µg/mL 65.7 ± 17.8%, p < 0.01; 50 µg/mL 68.7 ± 19.2%, p < 0.01; 100 µg/mL 63.6 ± 24.3%, p < 0.05). FE fucoidan decreased the cell viability at 1 µg/mL (p < 0.01). The reference substance heparin showed significant reduction in cell viability at concentrations of 50 and 100 µg/mL .The cervical adenocarcinoma cell line HeLa was one of the four cell lines, in which the compounds partly reduced the cell viability c. This wp < 0.001; 90.9 ± 2.6%, p < 0.001; 87.4 ± 8.7%, p < 0.01; 79.3 ± 14.7%, p < 0.01) . The cell viability of the ARPE-19 cells incubated with the other compounds was comparable to the untreated control, although some small changes turned out to be significant  d. In connificant d.p < 0.001, 87.1 ± 11.9%, p < 0.01) and 100 µg/mL  as well as by FE and SL at 100 µg/mL . Significant increase in the cell viability was observed for FV, LD and SL fucoidans at 10 µg/mL. A trend of increase was further seen with heparin at concentrations of 1–50 µg/mL. Similar to ARPE-19 cells, the uveal melanoma cell line OMM-1 responded differently to the treatment with the compounds, whereby FE and DF led to concentration-dependent effects e. The cep < 0.001, and 100 µg/mL, 105.2 ± 0.9%, p < 0.001) as well as DF at the highest concentration  and LD at the lowest concentration . Of note, slightly significant reduction of cell viability for A-375 was measured only for SL . In the skin melanoma cell line A-375 f a signip < 0.01; 50 µg/mL 108.7 ± 5.4%, p < 0.05; 100 µg/mL, 111.9 ± 3.8%, p < 0.001) and LD , as well as heparin at 100 µg/mL (p < 0.001) showed significantly increased cell viability. Lower but still slightly significant increasing effects on cell viability were observed for fucoidans from FV , FE , SL at the lowest three tested concentrations . A slight, but significant increase in cell viability was also measured for the Sigma fucoidan at 10 µg/mL and 100 µg/mL, p < 0.01; heparin 100 µg/mL, p < 0.05). The colon carcinoma cell line HCT-116 treated with DF . A slightly significant increase in cell viability was however measured for fucoidans from all Fucus species, namely FV with increasing concentration , FS at the two highest test concentrations , FE  as well as LD . The hepatocellular carcinoma cell line Hep G2 showed no significant change of viability after application of almost all reference compounds at all concentrations with one exception: enoxaparin at 1 µg/mL, p < 0.05; 10 µg/mL 91.8 ± 3.0%, p < 0.001; 50 µg/mL 87.2 ± 2.6%, p < 0.001; 100 µg/mL 87.9 ± 3.9%, p < 0.001). The heparins had differential effects: whereas no significant change in HaCaT viability was detected for LMWH, heparin showed a significant decrease at the three highest test concentrations . In the non-cancerous keratinocyte cell line HaCaT, commercial fucoidan lowered the cell viability significantly at all used concentrations  and SL . Notably, DF fucoidan significantly reduced the proliferation at 50 µg/mL .Slight, but significant reduction of cell viability was further found for fucoidan extracts from LD . In the HeLa cell line, the cell viability was significantly decreased with 10 µg/mL DF fucoidan . UFH decreased viability compared to control at 50 and 100 µg/mL . SL fucoidan significantly increased viability  as well as 1 µg/mL Sigma fucoidan . The differences of Raji and HeLa cell viability between 24 h and 72 h incubation time compared to control are minor.In addition to the incubation for 24 h, cell viability was estimated after 72 h for selected cell lines . In geneBesides the comparison of individual effects of fucoidans on the nine cell lines, we aimed to evaluate whether the differently sourced fucoidans show different effects on cell viability as well as whether the cell lines differ in their response to all fucoidans and heparins . Regarding the medians (ranging from 99% to 107%), neither the fucoidans nor the heparins modified the cell viability a. The laThe effect of the fucoidans and heparins on cell viability differed stronger depending on the cell line than from each used fucoidan or heparin b. Concerp < 0.05), which further increased to 11 and 10% at 10 and 50 µg/mL (p < 0.001). Interestingly, the increasing effect of 6% at the highest concentration (100 µg/mL) was not significant , to 123 ± 8% at 50 µg/mL (p < 0.001) and to 120 ± 5% at 100 µg/mL (p < 0.001). The comparison of the suspension cell lines (HL-60 and Raji) with the adherent cell lines  revealed a significant difference as well b. In conBoth comparisons a,b did np < 0.05)  d. ortho-aminomethyl-phenylboronate units, ethylammonium units and dansyl units) is specifically increased in the presence of sulfated glycans. The so-called Polymer-H assay was shown to be an useful parameter for an initial quality screening of fucoidans [In order to get indications of potential causes for the divergent reactions of the different cell lines in response to application of the different fucoidans, we performed correlation analyses between the cell viability at the highest fucoidan concentration (100 µg/mL) and the fucoidan characteristics protein content, total phenolic content (TPC) and fluorescence intensity (FI) increase of the glycan sulfate sensor Polymer H (unpublished data) for each cell line . The fluucoidans ,32  (2 = 0.6278) (2 = 0.7644) by the fucoidans. Additionally, HL-60 viability correlated with fucose content (R2 = 0.8994) and their DS (R2 = 0.9358)  a as well 0.6278) b. In con 0.9358) .Fucus vesiculosus fucoidan from Sigma Aldrich [Fucoidans extracted from brown algae have been described to exhibit antitumor effects ,23,35, b Aldrich ,38,39,40 Aldrich . Consequ Aldrich ,37, whicMoreover, for the cell viability analyses reported in literature, many different assays were used such as WST-1, MTT, Alamar Blue, Annexin V/PI staining, cell cycle analysis, neutral red release assay, trypan blue exclusion assay, which not always deliver similar results ,36,37,41To come to a general statement about the influence of all tested fucoidans and the reference heparins on cell viability, we calculated the overall mean of all cell viability values after incubation with fucoidans (n = 7 × 9 × 4) and heparins (n = 2 × 9 × 4), respectively. The resulting means of 104.1 ± 13.1% and 100.5 ± 10.1% did not significantly differ from the control (data not shown). Additionally, in previous studies we did not find any cytotoxic effect of fucoidans measured by the release of lactate dehydrogenase (LDH) (data not shown). This leads to the conclusion that fucoidans just as heparins are non-toxic. To obtain an indication about longer incubation times, selected cell lines were treated with fucoidans and heparins for 72 h. Here, the effects were very comparable to those after 24 h of incubation, i.e., similar minor stimulating or decreasing effects on cell viability, respectively. Hence, our data show no indication of long term toxicity. However, as demonstrated by the results of this study, certain fucoidans and certain cell lines, respectively, may deviate from this general conclusion.The results of the present study are compared with published studies investigating the effects of fucoidans on the same cell lines. As cancer cell lines are genetically unstable and often differ between laboratories, results obtained with these cell lines can be heterogeneous . TherefoF.v.) for 48 h instead of 24 h, whereby they used neutral red release assay, MTT and WST-8 proliferation assay [In the leukemia cell line HL-60 treated with fucoidans, no reduction but rather an increase of cell viability was detected a. In conon assay ,39,43. Won assay , or crudon assay . After 22O2 did not [Fucus sp. and did not influence the proliferation [The influence of fucoidan purity on cell viability has previously been demonstrated by experiments with the Burkitt lymphoma cell line Raji: Crude Sigma fucoidan exerted an antiproliferative effect on Raji cells, whereas the fraction purified by ion exchange chromatography did not show such an effect . Similar did not . Moreove did not . Like thferation . Saccharina japonica and Ascophyllum nodosum on the HeLa cell line which showed no significant effect on cell viability [Cladosiphon novae-caledoniae inhibited the cell proliferation [Saravana et al. and Jiang et al. tested fucoidan from iability ,21, wherferation ,35. The feration . Ascophyllum nodosum [In line with our data on ARPE-19 cells, Dithmer et al. and Li et al. observed no toxic effect of 100 µg/mL Sigma fucoidan on ARPE-19 cells ,44. Prol nodosum . In EPC  nodosum  which fiIn the OMM-1 cell line, neither pure Sigma fucoidan  nor crudS. cichorioides, F. evanescens and U. pinnatifida on the skin melanoma cell line RPMI-7951 and showed a slight reduction of cell viability after 24 h using MTS assay, which could be due to the higher concentration [To our knowledge, the present study is the first one testing fucoidans on cell viability of A-375 skin melanoma cells, but there are some studies using B16 and RPMI-7951 skin melanoma cells. Similar to our results, Wang et al. found no effect on the cell viability of B16 cells treated with 100 µg/mL Sigma fucoidan (of undefined purity) for 48 h using the cell counting kit-8 (CCK8). This was confirmed by Ale et al. 2011, who tested crude Sigma fucoidan using MTT assay. However, 6–10 times higher concentrations reduced the B16 viability in both studies ,47. Vishntration . Furtherntration .F.v.) on HCT-116 cells [A recent study of Park et al. reported the time- and concentration-dependent effects of crude Sigma fucoidan  did not affect the cell viability up to 50 µg/mL, whereas 100 µg/mL fucoidan reduced it by about 10%, which corresponds to our findings on Sigma fucoidan [00 µg/mL .Overall, the literature confirms that different cell lines react differently to fucoidans regarding cell viability b and theL. japonica, L. cichorioides and F. evanescens, respectively, were required to induce apoptosis on human peripheral blood lymphocytes (PBL) [The viability of the two non-tumor cell lines ARPE-19 and HaCaT turned out to be not modified by the tested fucoidans a. This ies (PBL) . 0 or SubG1 or G1 phase arrest [In contrast to the non-tumor cell lines, the fucoidans slightly increased the viability of the seven tumor cell lines a. Howevee arrest ,28,51. Re arrest . The dise arrest , concente arrest ,41,51,55e arrest ,55. The S. latissima and purified Sigma fucoidan stimulated the cell proliferation measured using a BrdU-based proliferation ELISA, whereas the Sigma fucoidan (other batch than used in the present study) reduced the proliferation [In total, we detected a stimulating effect of the fucoidans on the viability of the two suspension cell lines HL-60 and Raji, whereas the mean viability of the seven adherent cell lines was not modified b. There feration . The stiferation ,56.Regarding the adherent cells, the detection of any proliferation stimulating effect is impeded by the fact that further proliferation of confluent cells is restricted because of the limited growth area. According to the overall mean of the cell viability, the heparins  showed no significant effects (100.5 ± 10.1%). Similar to the fucoidans, there were certain differences depending on the cell line, but even significant effects amounted to less than 22%. Heparin and enoxaparin, respectively, showed no effect on the proliferation on endothelial cells , human pAnalysis of the correlation between the cell viability and various characteristics of the six different fucoidans suggest that highly pure fucoidans do not impair the cell viability. The viability of HeLa cells negatively correlated with the protein content of the fucoidans a and thaDepending on the algae species the purity of the extracted crude fucoidans was shown to differ (manuscript submitted) and this characteristic turned out as the most important one for changes of the viability of the used cell lines. Due to the overall only small effects of the fucoidans on the cell viability, its impact was only observed in those three cell lines with the largest fucoidan-dependent variability. This may be also the reason for missing correlations between the cell viability and other parameters such as the molecular weight (data not shown), although that is generally considered an important parameter for the biological activities of fucoidans ,70,71. v/v) ethanol was applied for defatting of the pulverized algal material. Then, the algal material was extracted using aqueous 2% calcium chloride for 2 h at 85 °C under reflux conditions. The supernatants of the raw extracts were concentrated by evaporation and precipitated with ice-cold ethanol in a final concentration of 60% at 4 °C. The precipitate was centrifuged, dissolved in demineralized water, dialyzed and lyophilized.The algal material was kindly provided by the company Coastal Research & Management GmbH . The used algae species for extraction, their harvest date and place are shown in Fucus vesiculosus) from Sigma Aldrich , heparin (Cat. No. Y0001282) and enoxaparin (Cat. No. E0180000) from EDQM were purchased and solved in Ampuwa water .As reference compounds fucoidan , HL-60 , HCT-116  and Hep G2  were purchased from the German Collection of Microorganisms and Cell Cultures . The HeLa cell line  was a kind gift of Prof. Dr. Clement . ARPE-19 cells  were bought from ATCC . The OMM-1 cell line  was a kind gift of Dr. Sarah Coupland . The human malignant melanoma cell line A375 (skin melanoma) and the immortalized HaCaT cell line (keratinocytes) were purchased from Cell Line Service .The characteristics of the used cell lines are shown in 2.Raji, HL-60 and HeLa cell lines were cultivated in RPMI-1640  with 10% heat inactivated fetal bovine serum (FBS). The ARPE-19 cell line was cultivated in DMEM  with 10% FBS and 1% penicillin/streptomycin and 1% non-essential amino acids. OMM-1 cells were cultivated in RPMI-1640  with 10% FBS and 1% penicillin/streptomycin. HaCaT and Hep G2 cell lines were cultivated in Gibco RPMI-1640  medium supplemented with 10% FBS and 1% penicillin/streptomycin. The A-375 and HCT-116 cell line were cultivated in DMEM  after adding 10% FBS and 1% penicillin/streptomycin. All cell lines were incubated in a humidified incubator at 37 °C and 5% CO® AQueous One Solution Cell Proliferation Assay  was used to determine cell viability. The assay was performed in 96-well microplates in phenol red-free medium ). For HL-60, HeLa and Raji cells 1X Gibco GlutaMAX™-I  was added. The medium for ARPE-19 and OMM-1 was supplemented as described above in cell culture. For OMM-1 cells 2 mM L-glutamine  was added. For Hep G2, HaCaT, A-375 and HCT-116 cell lines the cell viability assay was also performed using Gibco RPMI 1640 medium as described above for cell culture. Extracts in medium were filtered through a 0.2 µm filter before treatment . The Raji (10.000 cells/100 µL) and HL-60 (15.000 cells/100 µL) cells were seeded and treated with fucoidan extracts on the same day. ARPE-19 cells (10.000 cells/100 µL) were seeded and treated at 100% confluence. OMM-1 cells were treated at 80% confluence. HeLa (3000 cells/100 µL), Hep G2, HaCaT, A-375 and HCT-116 cells (each 10.000 cells/100µL) were seeded the day before treatment with fucoidan.The CellTiter 962. The absorbance was measured at 490 nm. Cells were treated with the six different fucoidans as well as with the reference compounds  in four different concentrations  for 24 h. As control, cells treated with 100 µL medium were used. After the incubation time 20 µL of the MTS solution were added to each well and incubated for 1 h at 37 °C, 5% COt-test. A Student’s t-test with p < 0.05 was considered as significant. All bars represent mean and standard deviation.Each experiment was performed in triplicates at least four times. Data of the experiments were analyzed statistically in Microsoft Excel using the mean, standard deviation and Student’s Fucus vesiculosus, F. serratus, F. distichus subsp. evanescens, Dictyosiphon foeniculaceus, Laminaria digitata, and Saccharina latissima) as well as three reference compounds  were tested on nine different human cell lines . The cell viability was determined by the commercially available MTS assay according to a standardized test and analysis protocol for comparability of the results. After 24 h incubation of the cells with the fucoidans (1–100 µg/mL), the overall mean of the cell viability amounted to 104.1 ± 13.1% and was quite similar to that of the heparins (100.5 ± 10.1%), which are known to exhibit no antiproliferative effects. Although some individual cell viability values were significantly reduced compared to the control , no consistent antiproliferative effect was found for any fucoidan. The cell viability medians of the different fucoidans ranged from 99% to 107%, whereby the largest cell line- and concentration-dependent variability was observed for SL (86–158%) and DF (61%–129%). As indicated by the medians of the different cell lines ranging from 94% (HeLa and HaCaT) to 115% (HL-60), the cell lines differently reacted to fucoidans. Whereas cell viability of the adherent cell lines was not influenced by the fucoidans, that of the suspension cell lines  was significantly increased. In contrast to the non-tumor cell lines , the viability of the tumor cell lines was also slightly increased, but this was mainly due to the increased viability of HL-60 and Raji cells. Analyses on the role of the various analytical fucoidan characteristics for their marginally different cellular effects revealed that the effect on the cell viability is primarily dependent on the purity of the fucoidans including co-extracted compounds. The cell viability of HeLa and ARPE-19 cells negatively correlated with protein content and TPC, respectively, whereas that of HL-60 cells positively correlated with FI increase of Polymer H, fucose content and DS.Due to the heterogeneous and partly conflicting data in the literature, the aim of the present study was to investigate the effects of different fucoidans on cell viability and to compare them depending on cell line and type . For this, six fucoidans extracted from different brown algal species .


PIK3A, PTEN, and KRAS. Different gene mutations in endometrial cancer cells have varied responses to anticancer drugs and cancer therapies. The most frequently altered gene in endometrioid endometrial carcinoma tumors is PTEN. PTEN protein has lipid phosphatase and protein phosphatase activity, as well as other functions in the nucleus. Although the tumor-suppressive function of PTEN has mainly been attributed to its lipid phosphatase activity, a role for PTEN protein phosphatase activity in cell cycle regulation has also been suggested. Various tumor type-specific PTEN mutations are well documented. Here, nine endometrioid endometrial cancer cell lines with PIK3A, PTEN, and KRAS gene mutations were treated with docetaxel and radiation. One mutation with a docetaxel drug-resistant effect was a truncated form of PTEN. Among PTEN mutations in endometrial cancer cells, the Y68 frame shift mutation of PTEN constitutes a major mechanism of resistance to docetaxel treatment. The molecular mechanism involves truncation of the 403 amino acid PTEN protein at amino acid 68 by the Y68 frame shift, leading to the loss of PTEN protein phosphatase and lipid phosphatase activities.In this study, we aimed to identify mutations of key genes associated with docetaxel resistance in nine endometrial cancer cell lines. Endometrial cancers are associated with several critical gene mutations, including  Endometrial cancers are divided two types, estrogen dependent (type I) and estrogen independent (type II). Type I is the most common type of endometrial cancer. Type II cancers include clear cell carcinoma, mucinous adenocarcinoma, and papillary serous adenocarcinoma, which are less common types of endometrial adenocarcinomas.Endometrial cancer is one of the most common gynecological malignancies in women worldwide6. However, no standard management modality is available. Adjuvant chemotherapy and radiotherapy in the “sandwich” sequence were adopted to help identify the most effective adjuvant method for patients with advanced disease11.Early stage diseases can have good outcomes through surgery, chemotherapy, radiotherapy or hormonal therapy, while advanced diseases are more likely to recur and require adjuvant chemotherapy and radiotherapy. The combination of chemotherapy and postoperative radiotherapy has been used in the treatment of advanced endometrial cancer14. Currently, chemotherapeutics remains the primary treatment for endometrial cancer. However, a major problem with chemotherapeutics is drug resistance. Therefore, the identification of genetic mechanisms involved in the chemotherapeutic response is critical for predicting the drug response of tumors with gene mutations. We propose that critical mutations of the tumor suppressor gene PTEN may be the major chemotherapeutic resistant factor in the treatment of patients with docetaxel-resistant endometrial cancer.Type I and type II endometrial cancers contain more than 20 gene mutations. Thus, improving our understanding of the disease at the molecular level and finding more effective strategies are importantPIK3A, PTEN, KRAS and FGFR2 might affect adjuvant treatment of endometrial tumors18. Radiation therapy is a key therapeutic strategy for endometrial carcinomas. However, how different gene mutations affect radiation sensitivity and drug responses remains unknown. Currently, treatment for metastatic or recurrent disease is based on the conventional chemotherapy method. Despite the different gene mutations in endometrial cancers, most clinical treatments have not taken this diversity into account20.Frequent mutations in PTEN lead to deregulation of the cell cycle21. PTEN suppresses the progression of the cell cycle through reduced cyclin D1 and increased p27.Gene mutations in PIK3A, PTEN, KRAS and FGFR2 gene mutations and five different mutations of PTEN in endometrioid endometrial carcinoma (EEC) cells to identify the mechanisms of docetaxel chemotherapy and radiation therapy resistance for different mutations in endometrial carcinomas. Cells were exposed to a chemotherapy drug (docetaxel), ionizing radiation (2 Gy) or a combination of both (sandwich method). Drug responses and radiosensitizing effects were evaluated using MTT assays and xCELLigence Real-Time Cell Analysis (RTCA). The effects of treatment with different doses of the chemotherapy drug (docetaxel) were evaluated following exposure to ionizing radiation (2 Gy).Here, we aimed to investigate the roles of We present multiple analyses of MTT assays and xCELLigence RTCA of 9 EEC cell lines treated with docetaxel chemotherapy and radiation. This integrated analysis provides the molecular parameters of different responses of endometrial carcinoma cells with various gene alterations, which may have a direct effect on treatment recommendations for patients. Our analysis also provides references for gene mutation-based clinical practice and novel treatments involving docetaxel chemotherapy and radiation.21.The effects of docetaxel on malignant cell growth were studied in a panel of 9 established human endometrial cancer cell lines. The individuality of each cell line was confirmed by mitochondrial DNA sequencing immediately after receipt from the collaborating research laboratory. Cell lines were passaged for less than 6 months after authentication and SPAC-1-L cell line was confirmed by PCR and sequencing experiments. Ishikawa cells were obtained from the European Collection of Animal Cell Cultures. The established human endometrial carcinoma cell line HEC155 was obtained from the Japanese Health Science Research Resources Bank. The cell line SPAC-1-L was provided by the laboratory of Dr. Y. Hirai from the Department of Gynecology, Cancer Institute Hospital . Dr. A. Santin provided ARK1 (USPC1) and ARK2 (USPC2) cells from the Division of Gynecologic Oncology at the University of Arkansas . The cell lines were cultured in Modified Eagle’s Medium (MEM) supplemented with 10% heat-inactivated FBS, 2 mmol/L glutamine and Antibiotic-Antimycotic Solution 50 for a given drug were nominally assigned the highest concentration screened . Experiments were performed in triplicate for each cell line. The association between a cell line and the drug response was determined using a two-tailed P value less than 0.05, which was considered statistically significant.Cells were plated in 96-well plates at a density of 2,000 cells per well, and untreated control cells were optimized to 85 to 95% confluence at the endpoint of the experiment. After 24 hours, cells were treated with radiation doses (2 Gy) and different concentrations  was used according to the manufacturer’s instructions. Briefly, the working mechanism of the system can be summarized as follows. The xCELLigence System allows continuous measurement and quantification of cell adhesion and proliferation. This system uses 96-well E-plates, and these disposable plates are suitable for single use. On the bottom of the wells, the plates have a base containing gold microelectrodes. There is 70% electrode coverage on the bottom of the wells. The electrical impedance of these sensor electrodes is recorded to monitor the changes in the cells. Changes in electrical impedance are presented with a parameter called the “cell index (CI)”. When there are no cells in the wells, electrode impedance and the CI are zero. After seeding cells, the CI will increase. The increase in the CI correlates with the increase in attached cell numbers. When more cells are attached to the surface of the E-Plate, the CI increases. Additionally, instead of cell numbers, cell viability and the strength of cell adhesion can cause changes in the CIA cell proliferation experiment was performed using an xCELLigence System. When EEC cells reached 80% confluence in a 6-well plate, the cells were washed with PBS. Then, the cells were treated with 0.05% trypsin/EDTA. After 2 min, 5 mL complete medium was added to the wells. The cell resuspension was centrifuged at 400 × g for 5 min. The pellet was resuspended in 5 mL media, and the cells were counted by using a hemocytometer. A total of 5,000 cells per well were seeded in an E-Plate, and the total volume of the wells was adjusted to 100 µL with media. The E-Plate was incubated overnight in a cell culture incubator. Before measuring the cells in the E-Plate, a standard background was measured by adding 50 μL complete medium at 37 °C to the wells. EEC cells were monitored every 15 min for 144 hours. Data are presented as a normalized CI.PIK3A, PTEN and KRAS mutations. Four cell lines  were obtained from patients with type II uterine serous papillary endometrial carcinoma. The MFE319 cell line originates from an endometrial adenosquamous carcinoma. The remaining cell lines are derived from type I EECs24. Among these cell lines, five out of nine (56%) EEC cell lines harbor PTEN gene mutations in different positions . We irradiated each of the 9 EEC cell lines with 0, 1, 2, 4, 6, 8, 11, or 15 Gy of X-rays and measured by MTT. The results showed the irradiation dose of 2 Gy not only partially reduced survived but also reserved a fraction room for docetaxel chemotherapeutics. Therefore the irradiation 2 Gy is an appropriate dose for the studying combination of irradiation and chemotherapeutics of docetaxel to EEC cells. We exposed nine EEC cell lines to different concentrations of the chemotherapy drug docetaxel, radiation (2 Gy) and both treatments for two or five days (mean of at least 3 independent experiments performed in triplicate), followed by MTT assays.PTEN gene mutations are common in EEC cell lines. We investigated the responses of nine EEC cell line panels to docetaxel chemotherapy and radiation to determine docetaxel drug resistance in association with 10-IC90) was identified using a wide range of drug concentrations (0.5–100 ng/ml). Docetaxel inhibited the proliferation of all endometrial cancer cell lines investigated in a concentration-dependent manner. However, the IC50 values significantly varied between individual cell lines with up to a 50-fold difference in the IC50 values and ranged between 1.95 in ARK1 endometrial cells with a PI3K gene mutation and 105.82 in SAPC-1-L cells that harbor a PTEN mutation (E288 frame shift /Y68 frame shift)  were less sensitive to docetaxel with a mean IC50 of 18.2 ng/ml, while ECC1 cells were sensitive to 2 Gy radiation with a relative viability ratio of 0.29. HEC155 cells were less sensitive to docetaxel with an IC50 value of 19.7 ng/ml, but these cells were sensitive to radiation with a relative viability ratio of 0.48 with a 2 Gy irradiation dose.The effective dose range for drug docetaxel  responded to docetaxel with low IC50 values of 5.28 and 7.36 to higher values of 18.21 ng/ml. ISHIKAWA and MEF319 cells were sensitive to docetaxel in a dose-dependent fashion from a low drug concentration of 1 ng/ml  were sensitive to docetaxel with a dose-dependent reduction from a low drug concentration of 1 ng/ml, with a drug IC50 value of 2.63 ng/ml. Additionally, SAPC-1-L cells that harbor a PTEN mutation (E288 frame shift/Y68 frame shift) were resistant to docetaxel, exhibiting a dose-dependent reduction with a high drug IC50 value of 105.8 ng/ml, which suggests that the Y68 frame shift mutation of PTEN may be associated with docetaxel resistance in endometrial cancer cells.Exposure of ECC-1, ISHIKAWA, MFE319, SNG-II and SPAC-1-L human endometrial cancer cells with Because SPAC-1-L cells were highly resistant to docetaxel, we sought other methods (radiation) to more effectively kill these cells. The survival rates of ARK1, ARK2, ECC-1, HEC155, HEC-1-A, ISHIKAWA, SNG-II and SPAC-1-L cells ranged from 0.65 to 0.96 with a 2 Gy dose for two days and from 0.28 to 0.57 with a 2 Gy dose for five days. MFE319 cells were resistant to radiation (2 Gy), with survival rates of 0.98 for two days and 0.75 for five days  EC cell lines harbored PTEN gene mutations in different amino acid change positions (ECC-1/V317 frame shift). The docetaxel IC50 values of the ECC-1, ISHIKAWA, MFE319, and SNG-II cell lines with PTEN mutations were 18.21 ng/ml, 7.36 ng/ml, 5.28 ng/ml and 2.63 ng/ml, respectively. These IC50 values were similar to those of cell lines with other gene mutations . The docetaxel IC50 value of SPAC-1-L with the Y68 frame shift mutation of PTEN was 105.8 ng/ml, which was five times greater than the IC50 values of cell lines with other PTEN mutations and other gene mutations.Due to the loss of the core motif  and C2 tensin-type domain (residues 186–351), the Y68 frame shift leads to the loss of the protein phosphatase and lipid phosphatase activities of in vitro findings demonstrate that loss of PTEN expression with the Y68 frame shift mutation predicts a lack of benefit from docetaxel in patients during endometrial cancer chemotherapy. Therefore, other chemotherapy drugs should be considered instead of docetaxel. This novel potent biomarker might lead to further refinement of therapy for patients with endometrial cancer with a Y68 frame shift mutation of PTEN, facilitating better outcomes, a better toxicity profile, and more effective health care delivery. Due to the relevance of the Y68 frame shift mutation and docetaxel resistance in endometrial cancer, this mutation is a predictive marker worth further preclinical and clinical evaluation.In this study, we found an association between the Y68 frame shift and increased resistance to docetaxel. Our preclinical Since cancer is one of the leading causes of death worldwide, finding better treatments is an urgent need. Although remarkable progress has been made towards understanding the molecular and cellular mechanisms of cancer development and treatment in recent years, the clinical management of cancer remains a challenge. Currently, chemotherapeutics and radiation therapy remain the predominant options for cancer therapy. However, resistance to chemotherapy in cancer is common. Therefore, the identification of gene mutations involved in the chemotherapeutic response is critical for predicting tumor responses and treating patients with drug-resistant cancer with different gene mutations. In this study, we sought to identify genetic mutations associated with resistance to facilitate effective combination therapy.28. Mutations of the RAS gene are also very common, accounting for more than 20% of mutations in human tumors29. Moreover, the fibroblast growth factor receptor (FGFR) pathway is involved in the pathogenesis and progression of endometrial cancer32. These gene mutations hold great promise for more effective treatment strategies for this disease.Therapeutic strategies could be improved according to appropriate biomarkers and patient-specific mutation profiles to achieve more benefits from combination therapies. The PI3K pathway is frequently activated in most EECsMost endometrial carcinomas are diagnosed at an early stage. Nevertheless, 15–20% of these carcinomas recur with limited effects of systemic therapies in metastatic disease. Recent progress in the identification of genetic abnormalities in endometrial cancer has spurred the development of novel strategies for the treatment of patients with endometrial cancer. Novel strategies involving therapies with greater efficacy and reduced toxicity are needed. Currently, mutation-based treatments are not available in the clinic; thus, more systematic approaches to the integration of biomarkers in clinical trials of therapeutic agents are needed for clinical implementation.50 values. Surprisingly, significantly higher IC50 values were associated with a Y68 frame shift mutation of PTEN in SPAC-1-L cells. The role of the Y68 frame shift mutation of PTEN in endometrial cancer has not yet been studied. However, our preclinical findings suggest that the PTEN Y68 frame shift mutation is associated with resistance to docetaxel in endometrial cancer.To date, no studies have investigated the responses of endometrial cancer cells with different gene mutations to docetaxel chemotherapy. Here, we show a concentration-dependent reduction in the viability of nine endometrial cancer cell lines in response to docetaxel. However, the response significantly varied among individual cell lines, with differences of up to 40 times in IC33.PTEN is an essential tumor suppressor gene that antagonizes the PI3K/Akt/mTOR antiapoptotic pathway and is often abnormal in endometrial cancers. Impairment of this tumor suppressor pathway potentially becomes a causal factor for the development of malignancies. Activation of the PI3K/Akt/mTOR pathway is implicated in the pathogenesis of malignancies and the development of resistance to anticancer therapiesPI3K gene mutation are sensitive to docetaxel with an IC50 of 1.95 ng/ml, while ECC-1 cells with a PTEN mutation are nine times more resistant to docetaxel with an IC50 of 18.21 ng/ml  based on the real-time response curves of radiation. When docetaxel concentrations were higher than 1 ng/ml, the drug achieved a greater therapeutic effect than did the combination of drug and radiation , SNG-II (E288 frame shift) and SPAC-1-L (E288 frame shift/Y68 frame shift) cells with a ion Fig. . CompareThe main goal of radiation therapy is to kill cancer cells by destroying their multiplication (cell division) potential. Resistance to chemotherapy and radiation therapy is a major problem facing current cancer research. In the current study, we evaluated the therapeutic effect of docetaxel with or without the addition of radiation  in nine endometrial cancer cell lines.37. Given the known molecular mutations, we hypothesized that EEC cell lines with activating the Y68 frame shift mutation of PTEN are resistant to irradiation therapy compared to EEC cell lines with wild-type PTEN.An antagonistic interaction was observed for docetaxel plus radiation 2 Gy) in ECC-1, HEC155, HEC-1-A, ISHIKAWA, MFE319, SNG-II and SPAC-1-L cells. In Fig.  Gy in EC50 value of 17.3 ng/ml. Since HEC-1-A cells were with PI3K and KRAS mutations and wild type PTEN, they were more sensitive to radiation with a relative viability ratio of 0.28 with a 2 Gy irradiation dose. SPAC-1-L is mutated with the Y68 frame shift mutation of PTEN, and then it is more resistant to radiation. This result supports hypothesis that SPAC-1-L is mutated with the Y68 frame shift mutation of PTEN induces docetaxel resistance in endometrial carcinoma cells.HEC-1-A cells were less sensitive to docetaxel with an ICBased on data from the xCELLigence Real-Time Cell Analyzer System, an antagonistic interaction was observed for docetaxel plus irradiation (2 Gy) in a dose-dependent manner with drug concentrations of 0, 1, 5, and 10 ng/ml in the HEC155 cell line Fig.  for fivePTEN gene led to docetaxel drug resistance in chemotherapy of endometrial cancer cells by destroying the protein phosphatase and lipid phosphatase activities of PTEN. Compared with treatment with docetaxel alone, the combination of docetaxel and X-ray (2 Gy) radiation resulted in a significant decrease in overall cytotoxicity for most endometrial cancer cells. The radiation sensitizing effects of docetaxel on endometrial cancer cells were observed at very low drug levels, far below the levels required for cytotoxic effects. Our results demonstrated that the addition of X-ray radiation interfered with the cytotoxic effects of docetaxel and might provide further proof for the cell cycle-dependent interaction.Our results showed that the Y68 frame shift mutation of the