Quantitative Proteomic Analysis of 2D and 3D Cultured Colorectal Cancer Cells: Profiling of Tankyrase Inhibitor XAV939-Induced Proteome.
ABSTRACT: Recently there has been a growing interest in three-dimensional (3D) cell culture systems for drug discovery and development. These 3D culture systems better represent the in vivo cellular environment compared to two-dimensional (2D) cell culture, thereby providing more physiologically reliable information on drug screening and testing. Here we present the quantitative profiling of a drug-induced proteome in 2D- and 3D-cultured colorectal cancer SW480 cells using 2D nanoflow liquid chromatography-tandem mass spectrometry (2D-nLC-MS/MS) integrated with isobaric tags for relative and absolute quantitation (iTRAQ). We identified a total of 4854 shared proteins between 2D- and 3D-cultured SW480 cells and 136/247 differentially expressed proteins (up/down-regulated in 3D compared to 2D). These up/down-regulated proteins were mainly involved in energy metabolism, cell growth, and cell-cell interactions. We also investigated the XAV939 (tankyrase inhibitor)-induced proteome to reveal factors involved in the 3D culture-selective growth inhibitory effect of XAV939 on SW480 cells. We identified novel XAV939-induced proteins, including gelsolin (a possible tumor suppressor) and lactate dehydrogenase A (a key enzyme of glycolysis), which were differentially expressed between 2D- and 3D-cultured SW480 cells. These results provide a promising informative protein dataset to determine the effect of XAV939 on the expression levels of proteins involved in SW480 cell growth.
Project description:XAV939, a tankyrase inhibitor, exerts an anticancer effect in 3-dimensional (3D) cultured SW480 cells, however this is not exhibited in 2-dimensional (2D) cultured SW480 cells. In the current study, XAV939 induced a 3.7-fold increase in cellular apoptosis in 3D culture but not in the 2D culture. However, no significant changes were indicated in cell cycle distribution in the 2D or 3D culture. Based on the observation that protein expression, which was associated with the glycolytic pathway, was increased in the 3D culture, the effect of XAV939 on the patterns of glycolytic protein expression was assessed. XAV939 was revealed to decrease lactose dehydrogenase A (LDHA) expression in 3D cultured SW480 cells, but only exerted a small effect in the 2D culture. The coadministration of XAV939 with the LDHA inhibitor FX11 decreased proliferation in 3D cultured SW480 cells compared with the single administration of FX11, while there was no additive effect in the 2D culture. The lactate assay also indicated that XAV939 decreased lactate secretion in the 3D cell culture but not in the 2D culture. These results suggest that XAV939 exerts an anticancer effect through inhibition of LDHA in the 3D culture.
Project description:Tumor models based on cancer cell lines cultured two-dimensionally (2D) on plastic lack histological complexity and functionality compared to the native microenvironment. Xenogenic mouse tumor models display higher complexity but often do not predict human drug responses accurately due to species-specific differences. We present here a three-dimensional (3D) in vitro colon cancer model based on a biological scaffold derived from decellularized porcine jejunum (small intestine submucosa+mucosa, SISmuc). Two different cell lines were used in monoculture or in coculture with primary fibroblasts. After 14 days of culture, we demonstrated a close contact of human Caco2 colon cancer cells with the preserved basement membrane on an ultrastructural level as well as morphological characteristics of a well-differentiated epithelium. To generate a tissue-engineered tumor model, we chose human SW480 colon cancer cells, a reportedly malignant cell line. Malignant characteristics were confirmed in 2D cell culture: SW480 cells showed higher vimentin and lower E-cadherin expression than Caco2 cells. In contrast to Caco2, SW480 cells displayed cancerous characteristics such as delocalized E-cadherin and nuclear location of ?-catenin in a subset of cells. One central drawback of 2D cultures-especially in consideration of drug testing-is their artificially high proliferation. In our 3D tissue-engineered tumor model, both cell lines showed decreased numbers of proliferating cells, thus correlating more precisely with observations of primary colon cancer in all stages (UICC I-IV). Moreover, vimentin decreased in SW480 colon cancer cells, indicating a mesenchymal to epithelial transition process, attributed to metastasis formation. Only SW480 cells cocultured with fibroblasts induced the formation of tumor-like aggregates surrounded by fibroblasts, whereas in Caco2 cocultures, a separate Caco2 cell layer was formed separated from the fibroblast compartment beneath. To foster tissue generation, a bioreactor was constructed for dynamic culture approaches. This induced a close tissue-like association of cultured tumor cells with fibroblasts reflecting tumor biopsies. Therapy with 5-fluorouracil (5-FU) was effective only in 3D coculture. In conclusion, our 3D tumor model reflects human tissue-related tumor characteristics, including lower tumor cell proliferation. It is now available for drug testing in metastatic context-especially for substances targeting tumor-stroma interactions.
Project description:The present study aimed to reveal the key long non-coding RNAs (lncRNAs) and the potential molecular mechanisms of XAV939 treatment in non-small cell lung cancer (NSCLC). The NSCLC cell line, NCI-H1299, was cultured with 10 µM XAV939 for 12 h, and NCI-H1299 cells without XAV939 treatment were used as controls. Following RNA isolation from the two groups, RNA-sequencing was performed to detect transcript expression levels, and differentially-expressed lncRNAs (DE-lncRNAs) and DE-genes (DEGs) were identified between groups and analyzed for their functions and associated pathways. The potential associations between proteins encoded by DEGs were revealed via a protein-protein interaction (PPI) network. Subsequently, the microRNA (miRNA/miR)-mRNA, lncRNA-miRNA and lncRNA-mRNA interactions were explored, followed by competing endogenous RNA (ceRNA) network construction. A total of 396 DEGs and 224 DE-lncRNAs were identified between the XAV939 and control groups. These lncRNAs were mainly enriched in pathways such as 'ferroptosis' [DEG, solute carrier family 7 member 11 (SLC7A11)]. The PPI network consisted of 97 nodes and 112 interactions. Furthermore, a total of 10 noteworthy lncRNAs were revealed in the DE-lncRNA-DEG interaction. Finally, the lncRNA-miRNA-mRNA regulatory association, including MIR503 host gene (MIR503HG)-miR1273c-SRY-box 4 (SOX4), was explored in the current ceRNA network. The downregulation of lncRNA MIR503HG induced by XAV939 may serve an important role in NSCLC suppression via sponging miR-1273c and regulating SOX4 expression. Furthermore, the downregulation of SLC7A11 induced by XAV939 may also inhibit the development of NSCLC via the ferroptosis pathway.
Project description:The demand for novel three-dimensional (3D) cell culture models of adipose tissue has been increasing, and proteomic investigations are important for determining the underlying causes of obesity, type II diabetes, and metabolic disorders. In this study, we performed global quantitative proteomic profiling of three 3D-cultured 3T3-L1 cells (preadipocytes, adipocytes and co-cultured adipocytes with macrophages) and their 2D-cultured counterparts using 2D-nanoLC-ESI-MS/MS with iTRAQ labelling. A total of 2,885 shared proteins from six types of adipose cells were identified and quantified in four replicates. Among them, 48 proteins involved in carbohydrate metabolism (e.g., PDH?, MDH1/2, FH) and the mitochondrial fatty acid beta oxidation pathway (e.g., VLCAD, ACADM, ECHDC1, ALDH6A1) were relatively up-regulated in the 3D co-culture model compared to those in 2D and 3D mono-cultured cells. Conversely, 12 proteins implicated in cellular component organisation (e.g., ANXA1, ANXA2) and the cell cycle (e.g., MCM family proteins) were down-regulated. These quantitative assessments showed that the 3D co-culture system of adipocytes and macrophages led to the development of insulin resistance, thereby providing a promising in vitro obesity model that is more equivalent to the in vivo conditions with respect to the mechanisms underpinning metabolic syndromes and the effect of new medical treatments for metabolic disorders.
Project description:Solid tumours naturally grow in 3D wherein the spatial arrangement of cells affects how they interact with each other. This suggests that 3D cell culture may mimic the natural in vivo setting better than traditional monolayer (2D) cell culture, where cells are grown attached to plastic. Here, using HER2-positive breast cancer cell lines as models (BT474, HCC1954, EFM192A), the effects of culturing cells in 3D using the poly-HEMA method compared to 2D cultures were assessed in terms of cellular viability, response/resistance to anti-cancer drugs, protein expression and enzyme activity. Scanning electron microscopy showed the morphology of cells in 3D to be substantially different to those cultured in 2D. Cell viability in 3D cells was substantially lower than that of cells in 2D cultures, while 3D cultures were more resistant to the effects of HER-targeted (neratinib) and classical chemotherapy (docetaxel) drugs. Expression of proteins involved in cell survival, transporters associated with drug resistance and drug targets were increased in 3D cultures. Finally, activity of drug metabolising enzyme CYP3A4 was substantially increased in 3D compared to 2D cultures. Together this data indicates that the biological information represented by 3D and 2D cell cultures is substantially different i.e. 3D cell cultures demonstrate higher innate resistance to anti-cancer drugs compared to 2D cultures, which may be facilitated by the altered receptor proteins, drug transporters and metabolising enzyme activity. This highlights the importance of considering 3D in addition to 2D culture methods in pre-clinical studies of both newer targeted and more traditional anti-cancer drugs.
Project description:Triple-negative breast cancer (TNBC) accounts for 15% of overall breast cancer. A lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2 receptor) makes TNBC more aggressive and metastatic. Wnt signaling is one of the important pathways in the cellular process; in TNBC it is aberrantly regulated, which leads to the progression and metastasis. In this study, we designed a therapeutic strategy using a combination of a low dose of paclitaxel and a Wnt signaling inhibitor (XAV939), and examined the effect of the paclitaxel-combined XAV939 treatment on diverse breast cancer lines including TNBC cell lines (MDA-MB-231, MDA-MB-468, and BT549) and ER+ve cell lines (MCF-7 and T-47D). The combination treatment of paclitaxel (20 nM) and XAV939 (10 µM) exerted a comparable therapeutic effect on MDA-MB-231, MDA-MB-468, BT549, MCF-7, and T-47D cell lines, relative to paclitaxel with a high dose (200 nM). The paclitaxel-combined XAV939 treatment induced apoptosis by suppressing Bcl-2 and by increasing the cleavage of caspases-3 and PARP. In addition, the in vivo results of the paclitaxel-combined XAV939 treatment in a mice model with the MDA-MB-231 xenograft further confirmed its therapeutic effect. Furthermore, the paclitaxel-combined XAV939 treatment reduced the expression of β-catenin, a key molecule in the Wnt pathway, which led to suppression of the expression of epithelial-mesenchymal transition (EMT) markers and angiogenic proteins both at mRNA and protein levels. The expression level of E-cadherin was raised, which potentially indicates the inhibition of EMT. Importantly, the breast tumor induced by pristane was significantly reduced by the paclitaxel-combined XAV939 treatment. Overall, the paclitaxel-combined XAV939 regimen was found to induce apoptosis and to inhibit Wnt signaling, resulting in the suppression of EMT and angiogenesis. For the first time, we report that our combination approach using a low dose of paclitaxel and XAV939 could be conducive to treating TNBC and an external carcinogen-induced breast cancer.
Project description:Three-dimensional (3D) culture changes cell characteristics and function, suggesting that 3D culture provides a more physiologically relevant environment for cells compared with 2D culture. We investigated the differences in cell functions depending on the culture model in human trophoblast cells (Sw.71).Sw.71 cells were incubated in 2D monolayers or simple 3D spheroids. After incubation, cells were corrected to assess RNA-seq transcriptome or protein expression, and culture medium were corrected to detect cytokines. To clarify the role of actin cytoskeleton, spheroid Sw.71 cells were treated mycalolide B (inhibitor of actin polymerization) in a 3D culture.RNA-seq transcriptome analysis, results revealed that 3D-cultured cells had a different transcriptional profile compared with 2D-cultured cells, especially regarding inflammation-related molecules. Although interleukin-6 (IL-6) mRNA level was higher in 3D-culured cells, its secretion levels were higher in 2D-cultured cells. In addition, the levels of mRNA and protein expression of regnase-1, regulatory RNase of inflammatory cytokine, significantly increased in 3D culture, suggesting post-translational modification of IL-6 mRNA via regnase-1. Treatment with mycalolide B reduced cell-to-cell contact to build 3D formation and increased expression of actin cytoskeleton, resulting in increased IL-6 secretin.Cell dimensionality plays an essential role in governing the spatiotemporal cellular outcomes, including inflammatory cytokine production and its negative regulation associated with regnase-1.
Project description:Background:The small molecule inhibitor XAV939 could inhibit the proliferation and promote the apoptosis of non-small cell lung cancer (NSCLC) cells. This study was conducted to identify the key circular RNAs (circRNAs) and microRNAs (miRNAs) in XAV939-treated NSCLC cells. Methods:After grouping, the NCL-H1299?cells in the treatment group were treated by 10??M XAV939 for 12?h. RNA-sequencing was performed, and then the differentially expressed circRNAs (DE-circRNAs) were analyzed by the edgeR package. Using the clusterprofiler package, enrichment analysis for the hosting genes of the DE-circRNAs was performed. Using Cytoscape software, the miRNA-circRNA regulatory network was built for the disease-associated miRNAs and the DE-circRNAs. The DE-circRNAs that could translate into proteins were predicted using circBank database and IRESfinder tool. Finally, the transcription factor (TF)-circRNA regulatory network was built by Cytoscape software. In addition, A549 and HCC-827 cell treatment with XAV939 were used to verify the relative expression levels of key DE-circRNAs. Results:There were 106 DE-circRNAs (including 61 upregulated circRNAs and 45 downregulated circRNAs) between treatment and control groups. Enrichment analysis for the hosting genes of the DE-circRNAs showed that ATF2 was enriched in the TNF signaling pathway. Disease association analysis indicated that 8 circRNAs (including circ_MDM2_000139, circ_ATF2_001418, circ_CDC25C_002079, and circ_BIRC6_001271) were correlated with NSCLC. In the miRNA-circRNA regulatory network, let-7 family members?circ_MDM2_000139, miR-16-5p/miR-134-5p?circ_ATF2_001418, miR-133b?circ_BIRC6_001271, and miR-221-3p/miR-222-3p?circ_CDC25C_002079 regulatory pairs were involved. A total of 47 DE-circRNAs could translate into proteins. Additionally, circ_MDM2_000139 was targeted by the TF POLR2A. The verification test showed that the relative expression levels of circ_MDM2_000139, circ_CDC25C_002079, circ_ATF2_001418, and circ_DICER1_000834 in A549 and HCC-827 cell treatment with XAV939 were downregulated comparing with the control. Conclusions:Let-7 family members and POLR2A targeting circ_MDM2_000139, miR-16-5p/miR-134-5p targeting circ_ATF2_001418, miR-133b targeting circ_BIRC6_001271, and miR-221-3p/miR-222-3p targeting circ_CDC25C_002079 might be related to the mechanism in the treatment of NSCLC by XAV939.
Project description:Three-dimensional (3D) culture, which can simulate in vivo microenvironments, has been increasingly used to study tumor cell biology. Since most preclinical anti-glioma drug tests still rely on conventional 2D cell culture, we established a collagen scaffold for 3D glioma cell culture. Glioma cells cultured on these 3D scaffolds showed greater degree of dedifferentiation and quiescence than cells in 2D culture. 3D-cultured cells also exhibited enhanced resistance to chemotherapeutic alkylating agents, with a much higher proportion of glioma stem cells and upregulation of O6-methylguanine DNA methyltransferase (MGMT). Importantly, tumor cells in 3D culture showed chemotherapy resistance patterns similar to those observed in glioma patients. Our results suggest that 3D collagen scaffolds are promising in vitro research platforms for screening new anti-glioma therapeutics.
Project description:Background:Mass production of exosomes is a prerequisite for their commercial utilization. This study investigated whether three-dimensional (3D) spheroid culture of mesenchymal stem cells (MSCs) could improve the production efficiency of exosomes and if so, what was the mechanism involved. Methods:We adopted two models of 3D spheroid culture using the hanging-drop (3D-HD) and poly(2-hydroxyethyl methacrylate) (poly-HEMA) coating methods (3D-PH). The efficiency of exosome production from MSCs in the 3D spheroids was compared with that of monolayer culture in various conditions. We then investigated the mechanism of the 3D spheroid culture-induced increase in exosome production. Results:The 3D-HD formed a single larger spheroid, while the 3D-PH formed multiple smaller ones. However, MSCs cultured on both types of spheroids produced significantly more exosomes than those cultured in conventional monolayer culture (2D). We then investigated the cause of the increased exosome production in terms of hypoxia within the 3D spheroids, high cell density, and non-adherent cell morphology. With increasing spheroid size, the efficiency of exosome production was the largest with the least amount of cells in both 3D-HD and 3D-PH. An increase in cell density in 2D culture (2D-H) was less efficient in exosome production than the conventional, lower cell density, 2D culture. Finally, when cells were plated at normal density on the poly-HEMA coated spheroids (3D-N-PH); they formed small aggregates of less than 10 cells and still produced more exosomes than those in the 2D culture when plated at the same density. We also found that the expression of F-actin was markedly reduced in the 3D-N-PH culture. Conclusion:These results suggested that 3D spheroid culture produces more exosomes than 2D culture and the non-adherent round cell morphology itself might be a causative factor. The result of the present study could provide useful information to develop an optimal process for the mass production of exosomes.