CopA3 peptide induces permanent cell-cycle arrest in colorectal cancer cells.

Cell-cycle arrest reflects an accumulation of responses to DNA damage that sequentially affects cell growth and division. Herein, we analyzed the effect of the 9-mer dimer defensin-like peptide, CopA3, against colorectal cancer cell growth and proliferation in a dose-dependent manner upon 96 h of treatment. As observed, CopA3 treatment significantly affected cancer cell growth, reduced colony formation ability, increased the number of SA-ß-Gal positive cells, and remarkably reduced Ki67 protein expression. Notably, in HCT-116 cells, CopA3 (5 µM) treatment effectively increased oxidative stress and, as a result, amplified the endogenous ROS, mitochondrial ROS, and NO content in the cells, which further activated the DNA damage response and caused cell-cycle arrest at the G1 phase. The prolonged cell-cycle arrest elevated the release of inflammatory cytokines in the cell supernatant. Nevertheless, mechanistically, NAC treatment effectively reversed the CopA3 effect and significantly reduced the oxidative stress; subsequently rescuing the cells from G1 phase arrest. Overall, CopA3 treatment can inhibit the growth and proliferation of colorectal cancer cells by inducing cell-cycle arrest through the ROS-mediated pathway.

Clonogenic Assays to Detect Cell Fate in Mitotic Catastrophe.

Mitotic catastrophe (MC) is a cell death modality induced by DNA damage that involves the activation of cell cycle checkpoints such as the "DNA structure checkpoint" and "spindle assembly checkpoint" (SAC) leading to aberrant mitosis. Depending on the signal, MC can drive the cell to death or to senescence. The suppression of MC favors aneuploidy. Several cancer therapies, included microtubular poisons and radiations, trigger MC. The clonogenic assay has been used to study the capacity of single cells to proliferate and to generate macroscopic colonies and to evaluate the efficacy of anticancer drugs. Nevertheless, this method cannot analyze MC events. Here, we report an improved technique based on the use of human colon cancer HCT116 stable expressing histone H2B-GFP and DsRed-centrin proteins, allowing to determine the capacity of cells to proliferate, and to determine changes in the nucleus and centrosomes.

The effects of human sera conditioned by high-intensity exercise sessions and training on the tumorigenic potential of cancer cells.

PURPOSE: There is growing evidence of an association between physical activity and a reduced risk of cancer and cancer recurrence. The aim of this study was to assess the effects of exercise-conditioned human serum (HS) effects on the proliferative and tumorigenic potential of triple-negative breast cancer (TNBC) and prostate cancer (PC) cells. Moreover, modulated mechanisms and several physiological factors that can predict exercise effects were investigated. METHODS: Thirty healthy sedentary subjects were recruited for the study. The subjects performed two high-intensity endurance cycling (HIEC) sessions before and after a nine-week period of high-intensity interval training (HIIT). Cell tumorigenic capacity affected by HS collected before (t0), immediately after (t1), 4 h (t2), and 24 h (t3) after the HIEC sessions was evaluated by in vitro three-dimensional colony formation. The modulation of molecular pathways was analyzed by western blotting and qPCR in TNBC and PC cells, and in TNBC xenografts in exercised mice. RESULTS: All of the HIEC-conditioned HS (t1, t2, and t3) markedly impacted the proliferative and the microtumor-forming capacity of both TNBC and PC cell lines, while the HS collected from the subjects at rest did not. Modulation of the Hippo and Wnt/ß-catenin pathways by HIEC-conditioned HS before and after the period of HIIT was shown. Multiple linear regression analysis showed relationships between the effects of HIEC-conditioned HS in PC cells, lactate threshold and VO2max. CONCLUSIONS: These results highlight the potential of HIEC bouts in tumor progression control and the importance of optimizing an approach to identify physiological predictors of the effects of acute exercise in tertiary cancer prevention.

The clonogenic assay: robustness of plating efficiency-based analysis is strongly compromised by cellular cooperation.

BACKGROUND: The clonogenic assay is a versatile and frequently used tool to quantify reproductive cell survival in vitro. Current state-of-the-art analysis relies on plating efficiency-based calculations which assume a linear correlation between the number of cells seeded and the number of colonies counted. The present study was designed to test the validity of this assumption and to evaluate the robustness of clonogenic survival results obtained. METHODS: A panel of 50 established cancer cell lines was used for comprehensive evaluation of the clonogenic assay procedure and data analysis. We assessed the performance of plating efficiency-based calculations and examined the influence of critical experimental parameters, such as cell density seeded, assay volume, incubation time, as well as the cell line-intrinsic factor of cellular cooperation by auto-/paracrine stimulation. Our findings were integrated into a novel mathematical approach for the analysis of clonogenic survival data. RESULTS: For various cell lines, clonogenic growth behavior failed to be adequately described by a constant plating efficiency, since the density of cells seeded severely influenced the extent and the dynamics of clonogenic growth. This strongly impaired the robustness of survival calculations obtained by the current state-of-the-art method using plating efficiency-based normalization. A novel mathematical approach utilizing power regression and interpolation of matched colony numbers at different irradiation doses applied to the same dataset substantially reduced the impact of cell density on survival results. Cellular cooperation was observed to be responsible for the non-linear clonogenic growth behavior of a relevant number of cell lines and the impairment of survival calculations. With 28/50 cell lines of different tumor entities showing moderate to high degrees of cellular cooperation, this phenomenon was found to be unexpectedly common. CONCLUSIONS: Our study reveals that plating efficiency-based analysis of clonogenic survival data is profoundly compromised by cellular cooperation resulting in strongly underestimated assay-intrinsic errors in a relevant proportion of established cancer cell lines. This severely questions the use of plating efficiency-based calculations in studies aiming to achieve more than semiquantitative results. The novel approach presented here accounts for the phenomenon of cellular cooperation and allows the extraction of clonogenic survival results with clearly improved robustness.

Reporting of methodologies used for clonogenic assays to determine radiosensitivity.

Radiotherapy treatment strategies should be personalized based on the radiosensitivity of individual tumors. Clonogenic assays are the gold standard method for in vitro assessment of radiosensitivity. Reproducibility is the critical factor for scientific rigor; however, this is reduced by insufficient reporting of methodologies. In reality, the reporting standards of methodologies pertaining to clonogenic assays remain unclear. To address this, we performed a literature search and qualitative analysis of the reporting of methodologies pertaining to clonogenic assays. A comprehensive literature review identified 1672 papers that report the radiosensitivity of human cancer cells based on clonogenic assays. From the identified papers, important experimental parameters (i.e. number of biological replicates, technical replicates, radiation source and dose rate) were recorded and analyzed. We found that, among the studies, (i) 30.5% did not report biological or technical replicates; (ii) 47.0% did not use biological or technical replicates; (iii) 3.8% did not report the radiation source; and (iv) 32.3% did not report the dose rate. These data suggest that reporting of methodologies pertaining to clonogenic assays in a considerable number of previously published studies is insufficient, thereby threatening reproducibility. This highlights the need to raise awareness of standardization of the methodologies used to conduct clonogenic assays.

Schiefer counter: An alternative method for clonogenic assay evaluation.

INTRODUCTION: Clonogenic assay evaluates the potential of cells to undergo division or generate clones following treatment with a chemical or other agent, thereby allowing the evaluation of cytotoxic and/or antiproliferative effects. Clonogenic assay analysis using traditional methods tends to be time-consuming and yield inconsistent results, whereas results from analyses conducted using automated image processing methods may be misleading or subject to misinterpretation. Thus, the aim of this work was to validate and demonstrate the applicability of a recently developed software. METHODS: Repeatability of measurements was evaluated by comparing results from 10 replicate images from a single well. To evaluate the viability of the software, results were compared with those obtained from manual counting, crystal violet optical density, and up-to-date automated methods. A clonogenic index was experimentally developed using the individual area occupied by colonies, while clone stratification was used to differentiate between antiproliferative and cytotoxic effects. RESULTS: The developed software showed to be a reliable and consistent tool for clonogenic assay evaluation, presenting a repeatability mean error of 0.79% for the number of colonies and 0.89% for the total area of colonies, as well as exhibiting a significant correlation (p < 0.05) with results obtained from widely adopted gold standard methods. The software was also able to detect an appropriate dose-dependent effect as well as a predominant cytotoxic effect of vincristine on MCF-7 cells and calculate the clonogenic index. DISCUSSION: Therefore, this software is adequate for the analysis of clonogenic assay images, differentiating between cytotoxic and antiproliferative trends.

Pulsed low dose-rate irradiation response in isogenic HNSCC cell lines with different radiosensitivity.

Background Management of locoregionally recurrent head and neck squamous cell carcinomas (HNSCC) is challenging due to potential radioresistance. Pulsed low-dose rate (PLDR) irradiation exploits phenomena of increased radiosensitivity, low-dose hyperradiosensitivity (LDHRS), and inverse dose-rate effect. The purpose of this study was to evaluate LDHRS and the effect of PLDR irradiation in isogenic HNSCC cells with different radiosensitivity. Materials and methods Cell survival after different irradiation regimens in isogenic parental FaDu and radioresistant FaDu-RR cells was determined by clonogenic assay; post irradiation cell cycle distribution was studied by flow cytometry; the expression of DNA damage signalling genes was assesed by reverse transcription-quantitative PCR. Results Radioresistant Fadu-RR cells displayed LDHRS and were more sensitive to PLDR irradiation than parental FaDu cells. In both cell lines, cell cycle was arrested in G2/M phase 5 hours after irradiation. It was restored 24 hours after irradiation in parental, but not in the radioresistant cells, which were arrested in G1-phase. DNA damage signalling genes were under-expressed in radioresistant compared to parental cells. Irradiation increased DNA damage signalling gene expression in radioresistant cells, while in parental cells only few genes were under-expressed. Conclusions We demonstrated LDHRS in isogenic radioresistant cells, but not in the parental cells. Survival of LDHRS-positive radioresistant cells after PLDR was significantly reduced. This reduction in cell survival is associated with variations in DNA damage signalling gene expression observed in response to PLDR most likely through different regulation of cell cycle checkpoints.

A Sox2:miR-486-5p Axis Regulates Survival of GBM Cells by Inhibiting Tumor Suppressor Networks.

Glioblastoma multiforme (GBM) and other solid malignancies are heterogeneous and contain subpopulations of tumor cells that exhibit stem-like features. Our recent findings point to a dedifferentiation mechanism by which reprogramming transcription factors Oct4 and Sox2 drive the stem-like phenotype in glioblastoma, in part, by differentially regulating subsets of miRNAs. Currently, the molecular mechanisms by which reprogramming transcription factors and miRNAs coordinate cancer stem cell tumor-propagating capacity are unclear. In this study, we identified miR-486-5p as a Sox2-induced miRNA that targets the tumor suppressor genes PTEN and FoxO1 and regulates the GBM stem-like cells. miR-486-5p associated with the GBM stem cell phenotype and Sox2 expression and was directly induced by Sox2 in glioma cell lines and patient-derived neurospheres. Forced expression of miR-486-5p enhanced the self-renewal capacity of GBM neurospheres, and inhibition of endogenous miR-486-5p activated PTEN and FoxO1 and induced cell death by upregulating proapoptotic protein BIM via a PTEN-dependent mechanism. Furthermore, delivery of miR-486-5p antagomirs to preestablished orthotopic GBM neurosphere-derived xenografts using advanced nanoparticle formulations reduced tumor sizes in vivo and enhanced the cytotoxic response to ionizing radiation. These results define a previously unrecognized and therapeutically targetable Sox2:miR-486-5p axis that enhances the survival of GBM stem cells by repressing tumor suppressor pathways. SIGNIFICANCE: This study identifies a novel axis that links core transcriptional drivers of cancer cell stemness to miR-486-5p-dependent modulation of tumor suppressor genes that feeds back to regulate glioma stem cell survival.

ATM Paradoxically Promotes Oncogenic Transformation via Transcriptional Reprogramming.

The role of the ataxia-telangiectasia-mutated (ATM) gene in human malignancies, especially in solid tumors, remains poorly understood. In the present study, we explored the involvement of ATM in transforming primary human cells into cancer stem cells. We show that ATM plays an unexpected role in facilitating oncogene-induced malignant transformation through transcriptional reprogramming. Exogenous expression of an oncogene cocktail induced a significant amount of DNA double-strand breaks in human fibroblasts that caused persistent activation of ATM, which in turn enabled global transcriptional reprogramming through chromatin relaxation, allowing oncogenic transcription factors to access chromatin. Consistently, deficiencies in ATM significantly attenuated oncogene-induced transformation of human cells. In addition, ATM inhibition significantly reduced tumorigenesis in a mouse model of mammary cancer. ATM and cellular DNA damage response therefore play a previously unknown role in facilitating rather than suppressing oncogene-induced malignant transformation of mammalian cells. SIGNIFICANCE: These findings uncover a novel pro-oncogenic role for ATM and show that contrary to established theory, ATM does not always function as a tumor suppressor; its function is however dependent on cell type.

CTC-Derived Models: A Window into the Seeding Capacity of Circulating Tumor Cells (CTCs).

Metastasis is the main cause of cancer-related death owing to the blood-borne dissemination of circulating tumor cells (CTCs) early in the process. A rare fraction of CTCs harboring a stem cell profile and tumor initiation capacities is thought to possess the clonogenic potential to seed new lesions. The highest plasticity has been generally attributed to CTCs with a partial epithelial-to-mesenchymal transition (EMT) phenotype, demonstrating a large heterogeneity among these cells. Therefore, detection and functional characterization of these subclones may offer insight into mechanisms underlying CTC tumorigenicity and inform on the complex biology behind metastatic spread. Although an in-depth mechanistic investigation is limited by the extremely low CTC count in circulation, significant progress has been made over the past few years to establish relevant systems from patient CTCs. CTC-derived xenograft (CDX) models and CTC-derived ex vivo cultures have emerged as tractable systems to explore tumor-initiating cells (TICs) and uncover new therapeutic targets. Here, we introduce basic knowledge of CTC biology, including CTC clusters and evidence for EMT/cancer stem cell (CSC) hybrid phenotypes. We report and evaluate the CTC-derived models generated to date in different types of cancer and shed a light on challenges and key findings associated with these novel assays.

Fasting Reduces Intestinal Radiotoxicity, Enabling Dose-Escalated Radiation Therapy for Pancreatic Cancer.

PURPOSE: Chemotherapy combined with radiation therapy is the most commonly used approach for treating locally advanced pancreatic cancer. The use of curative doses of radiation in this disease setting is constrained because of the close proximity of the head of the pancreas to the duodenum. The purpose of this study was to determine whether fasting protects the duodenum from high-dose radiation, thereby enabling dose escalation for efficient killing of pancreatic tumor cells. METHODS AND MATERIALS: C57BL/6J mice were either fed or fasted for 24 hours and then exposed to total abdominal radiation at 11.5 Gy. Food intake, body weight, overall health, and survival were monitored. Small intestines were harvested at various time points after radiation, and villi length, crypt depth, and number of crypts per millimeter of intestine were determined. Immunohistochemistry was performed to assess apoptosis and double-strand DNA breaks, and microcolony assays were performed to determine intestinal stem cell regeneration capacity. A syngeneic KPC model of pancreatic cancer was used to determine the effects of fasting on the radiation responses of both pancreatic cancer and host intestinal tissues. RESULTS: We demonstrated that a 24-hour fast in mice improved intestinal stem cell regeneration, as revealed by microcolony assay, and improved host survival of lethal doses of total abdominal irradiation compared with fed controls. Fasting also improved survival of mice with orthotopic pancreatic tumors subjected to lethal abdominal radiation compared with controls with free access to food. Furthermore, fasting did not affect tumor cell killing by radiation therapy and enhanced γ-H2AX staining after radiation therapy, suggesting an additional mild radiosensitizing effect. CONCLUSIONS: These results establish proof of concept for fasting as a dose-escalation strategy, enabling ablative radiation in the treatment of unresectable pancreatic cancer.

miR-873-5p inhibits the progression of colon cancer via repression of tumor suppressor candidate 3/AKT signaling.

BACKGROUND AND AIM: We previously discovered that tumor suppressor candidate 3 (TUSC3) was overexpressed and predicted worse prognosis in colon cancer patients. However, the mechanisms of upregulation of TUSC3 in colon cancer remained unclear. METHODS: MiR-873-5p was predicted and identified as the regulator of TUSC3 via online programs and luciferase reporter assays. The roles of miR-873-5p in regulating colon cancer cell proliferation, colony formation, and invasion were evaluated in vitro. Animal studies were performed to investigate the effects of miR-873-5p on proliferation and lung metastasis. Moreover, the miR-873-5p/TUSC3 related signaling pathway and the prognostic value of combining miR-873-5p and TUSC3 for colon cancer patients were also explored. RESULTS: Here, we identified miR-873-5p as a novel regulator of TUSC3 in colon cancer. Functionally, ectopic expression or silencing of miR-873-5p, respectively, inhibited or promoted colon cancer cells proliferation, colony formation, and invasion, as well as prevented or enhanced the metastasis of colon cancer cells in vitro and in vivo. Molecularly, miR-873-5p functioned as a tumor suppressor by inhibiting the TUSC3/AKT pathway. Overexpression or silencing of TUSC3 could partially reverse the effects of the overexpression or repression of miR-873-5p on colon cancer progression caused by activation of the AKT pathway. Clinically, low miR-873-5p expression predicted poor survival in colon cancer patients, especially combined with high TUSC3 expression. CONCLUSIONS: We identified miR-873-5p as a tumor suppressor, which acts by directly repressing TUSC3 in colon cancer.

Collective radioresistance of T47D breast carcinoma cells is mediated by a Syncytin-1 homologous protein.

It is generally accepted that radiotherapy must target clonogenic cells, i.e., those cells in a tumour that have self-renewing potential. Focussing on isolated clonogenic cells, however, may lead to an underestimate or even to an outright neglect of the importance of biological mechanisms that regulate tumour cell sensitivity to radiation. We develop a new statistical and experimental approach to quantify the effects of radiation on cell populations as a whole. In our experiments, we change the proximity relationships of the cells by culturing them in wells with different shapes, and we find that the radiosensitivity of T47D human breast carcinoma cells in tight clusters is different from that of isolated cells. Molecular analyses show that T47D cells express a Syncytin-1 homologous protein (SyHP). We observe that SyHP translocates to the external surface of the plasma membrane of cells killed by radiation treatment. The data support the fundamental role of SyHP in the formation of intercellular cytoplasmic bridges and in the enhanced radioresistance of surviving cells. We conclude that complex and unexpected biological mechanisms of tumour radioresistance take place at the cell population level. These mechanisms may significantly bias our estimates of the radiosensitivity of breast carcinomas in vivo and thereby affect treatment plans, and they call for further investigations.

Cabazitaxel and silibinin co-encapsulated cationic liposomes for CD44 targeted delivery: A new insight into nanomedicine based combinational chemotherapy for prostate cancer.

Cancer stem cells (CSCs) are the promising targets for cancer chemotherapy that cannot be eliminated by conventional chemotherapy. In this study cationic liposomes of cabazitaxel (CBX) and silibinin (SIL) were prepared with an aim to kill cancer cells and CSCs for prostate cancer. CBX act as cancer cell inhibitor and SIL as CSC inhibitor. Hyaluronic acid (HA), an endogenous anionic polysaccharide was coated on cationic liposomes for targeting CD44 receptors over expressed on CSCs. Liposomes were prepared by ethanol injection method with particle size below 100 nm and entrapment efficiency of more than 90% at 10% w/w drug loading. Liposomes were characterized by dynamic light scattering, transmission electron microscopy, 1H nuclear magnetic resonance and scanning electron microscopy-energy dispersive x-ray spectroscopy. Liposomes were evaluated for their anticancer action in androgen independent human prostate cancer cell lines (PC-3 and DU-145). HA coated liposomes showed potential cytotoxicity over other groups with low IC50, significantly inhibited cell migration and induced apoptosis. Synergistic cytotoxic effect was also observed with HA coated liposomes that resulted in colony formation inhibition and G2/M phase arrest. Proficient cytotoxicity against CD44+ cells (14.87 ± 0.41% in PC-3 and 33.95 ± 0.68% in DU-145 cells) indicated the efficiency of HA coated liposomes towards CSC targeting. Hence, the outcome of this combinational therapy with CD44 targeting indicates the suitability of HA coated CBX and SIL co-loaded liposomes as a potential approach for eradicating prostate cancer and herein might provide a insight for future studies.

Retinoic Acid Receptor α Knockdown Suppresses the Tumorigenicity of Esophageal Carcinoma via Wnt/ß-catenin Pathway.

BACKGROUND: Aberrant expression of retinoic acid receptor α (RARα) was correlated with diverse carcinomas such as acute promyelocytic leukemia and colorectal carcinoma. Nevertheless, the function and mechanism of RARα in esophageal carcinoma (EC) remain unclear. AIM: To investigate the expression of RARα in EC and its effect in the tumorigenesis of EC. METHODS AND RESULTS: In immunohistochemistry study, RARα was overexpressed in human EC tissues, and its overexpression was closely related to the pathological differentiation, lymph node metastasis, and clinical stages in EC patients. Functionally, RARα knockdown suppressed the proliferation and metastasis of EC cells through downregulating the expression of PCNA, Ki67, MMP7, and MMP9, as well as enhanced drug susceptibility of EC cells to 5-fluorouracil and cisplatin. Mechanistically, RARα knockdown inhibited the activity of Wnt/ß-catenin pathway through reducing the phosphorylation level of GSK3ß at Ser-9 and inducing phosphorylation level at Tyr-216, which resulted in downregulation of its downstream targets such as MMP7, MMP9, and P-gP. CONCLUSIONS: Our results demonstrated that RARα knockdown suppressed the tumorigenicity of EC via Wnt/ß-catenin pathway. RARα might be a potential molecular target for EC clinical therapy.

Miniaturization of the Clonogenic Assay Using Confluence Measurement.

The clonogenic assay is a widely used method to study the ability of cells to 'infinitely' produce progeny and is, therefore, used as a tool in tumor biology to measure tumor-initiating capacity and stem cell status. However, the standard protocol of using 6-well plates has several disadvantages. By miniaturizing the assay to a 96-well microplate format, as well as by utilizing the confluence detection function of a multimode reader, we here describe a new and modified protocol that allows comprehensive experimental setups and a non-endpoint, label-free semi-automatic analysis. Comparison of bright field images with confluence images demonstrated robust and reproducible detection of clones by the confluence detection function. Moreover, time-resolved non-endpoint confluence measurement of the same well showed that semi-automatic analysis was suitable for determining the mean size and colony number. By treating cells with an inhibitor of clonogenic growth (PTC-209), we show that our modified protocol is suitable for comprehensive (broad concentration range, addition of technical replicates) concentration- and time-resolved analysis of the effect of substances or treatments on clonogenic growth. In summary, this protocol represents a time- and cost-effective alternative to the commonly used 6-well protocol (with endpoint staining) and also provides additional information about the kinetics of clonogenic growth.

Rabdocoestin B exhibits antitumor activity by inducing G2/M phase arrest and apoptosis in esophageal squamous cell carcinoma.

PURPOSE: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive squamous cell carcinomas and is generally resistant to chemotherapy. In the present study, the cytotoxic activity of Rabdocoestin B (Rabd-B) against ESCC and the underlying mechanisms were investigated. METHODS: The inhibitory effect of Rabd-B on KYSE30 and KYSE450 was evaluated by Cell Counting Kit-8 (CCK8) and colony formation assays in vitro. The cell cycle distribution and apoptosis of cells treated with Rabd-B were determined by flow cytometry. The mechanisms underlying the effects of Rabd-B were systematically examined by Western blot. The in vivo anti-tumor ability of Rabd-B was measured in mouse xenograft models and cisplatin (DDP) was used as positive control. RESULTS: Rabd-B efficiently induced G2/M phase arrest in ESCC cells by upregulating the Chk1/Chk2-Cdc25C axis to inhibit the G2→M transition facilitated by Cdc2/Cyclin B1. Furthermore, Rabd-B suppressed ATM/ATR phosphorylation, thereby inhibiting BRCA1-mediated DNA repair, which resulted in mitotic catastrophe and induced cell apoptosis. Rabd-B also decreased the activity of the Akt and NF-κB survival signaling pathways and ultimately initiated the caspase-9-dependent intrinsic apoptotic pathway in ESCC cells. The apoptosis induced by Rabd-B could be partially reversed by a caspase-9-specific inhibitor (Z-LEHD-FMK) and a pan-caspase inhibitor (Z-VAD-FMK). Moreover, Rabd-B effectively suppressed tumor growth in mouse xenografts which was comparable to that of DDP without significant injuries to the mice. CONCLUSION: Taken together, these findings indicate that Rabd-B is a promising precursor compound that may be useful as a treatment for ESCC and thus warrants further investigation.

Establishment and proteomic characterization of patient-derived clear cell sarcoma xenografts and cell lines.

Clear cell sarcoma (CCS) is an aggressive mesenchymal malignancy characterized by the unique chimeric EWS-ATF1 fusion gene. Patient-derived cancer models are essential tools for the understanding of tumorigenesis and the development of anti-cancer drugs; however, only a limited number of CCS cell lines exist. The objective of this study was to establish patient-derived CCS models. We established patient-derived CCS models from a 43-yr-old female patient. We prepared the patient-derived xenografts (PDXs) from tumor tissues obtained through biopsy or surgery and isolated stable cell lines from PDXs and the original tumor tissue. The presence of gene fusions was examined by RT-PCR, and Sanger sequencing. The established cell lines were characterized by short tandem repeat, viability, colony and spheroid formation, and invasion analyses. Differences in gene enrichment between the primary tumor and cell lines were examined by mass spectrometry and KEGG pathway analysis. The cell lines were maintained for more than 80 passages, and had tumorigenic characteristics such as colony and spheroid formation and invasion. Mass spectrometric proteome analysis demonstrated that the cell lines were enriched for similar but distinct molecular pathways, compared to those in the xenografts and original tumor tissue. Next, tyrosine kinase inhibitors were screened for their suppressive effects on viability. We found that ponatinib, vandetanib, and doxorubicin suppressed the growth of cell lines, and had equivalent IC50 values. Further in-depth investigation and understanding of drug-sensitivity mechanisms will be important for the clinical applications of our cell lines.

Parthenolide reduces gene transcription of prosurvival mediators in U937 cells.

In acute myeloid leukemia (AML) the functional abnormalities of osteopontin (OPN), NF-kB, PI3K/AKT/mTOR/PTEN pathway or ß-catenin have been considered. AIM: To analyze the response of U937 cells to parthenolide (PTL) through the involvement of expression of OPN protein, RelB, AKT1, mTOR, PTEN and ß-catenin genes. MATERIALS AND METHODS: The U937 cells were treated with PTL at concentrations of 4 µM (IC25) or 6 µM (IC50) and with OPN siRNA for MTT assay and colony forming assay. Western blot analysis using antibodies against OPN was performed with lysates of PTL-treated cells. Quantitative real-time polymerase chain reaction was performed using primers for OPN siRNA, RelB, AKT1, mTOR, PTEN and ß-catenin. RESULTS: PTL reduces OPN protein level and down-regulates RelB mRNA in U937 cell line. Suppression of OPN with siRNA increases the cytotoxic effects of PTL. Also, mRNA expression of AKT1, mTOR, PTEN, and ß-catenin decreases with PTL or OPN siRNA. CONCLUSION: Sensitivity of U937 cells to PTL can be associated with the reduction in expression of prosurvival mediators.