dld1 cells  (ATCC)

Journal: Frontiers in Pharmacology

Article Title: Integrated network pharmacology and experimental verification to reveal the mechanisms of curcumin in the treatment of colorectal cancer

doi: 10.3389/fphar.2025.1703562

Figure Lengend Snippet: Venn diagram of curcumin targets, CRC-related genes, and DEGs from curcumin-treated DLD1 cells.

Article Snippet: The human CRC cell line DLD1 (ATCC, United States) was cultured as described and seeded in 96-well plates to reach 80% confluence before treatment.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Integrated network pharmacology and experimental verification to reveal the mechanisms of curcumin in the treatment of colorectal cancer

doi: 10.3389/fphar.2025.1703562

Figure Lengend Snippet: Curcumin promotes ferroptosis and regulates Wnt/β-catenin signaling pathway in vitro . (A) Representative images of transmission electron microscopy observation of mitochondria; curcumin induced reduced mitochondrial volume and increased membrane density in HCT116 cell lines (scale bar: 2 μm and 500 nm); (B) Relative mitochondrial area in HCT116 cell lines. (C) Relative mitochondrial density in HCT116 cell lines. (D–E) Curcumin induced the accumulation of Lipid ROS in CRC cells. Dose-dependent accumulation of Lipid ROS in HCT116 and DLD1 cells treated with curcumin (5–20 μm). (F–G) Curcumin effectively inhibited the expression of SLC7A11 and GPX4, downregulated level of β-catenin and promoted phosphorylation of GSK3β at Ser9. *P < 0.05, **P < 0.01 and ***P < 0.001.

Article Snippet: The human CRC cell line DLD1 (ATCC, United States) was cultured as described and seeded in 96-well plates to reach 80% confluence before treatment.

Techniques: In Vitro, Transmission Assay, Electron Microscopy, Membrane, Expressing, Phospho-proteomics

Journal: Advanced Science

Article Title: m 1 A‐Dependent TRMT6/61A‐ARG2 Axis Drives Protumorigenic Senescence by Remodeling the Tumor Microenvironment

doi: 10.1002/advs.202518536

Figure Lengend Snippet: TRMT6/TRMT61A‐mediated m 1 A modification drives cellular senescence in CRC. (A) Representative immunohistochemistry (IHC) images showing co‐expression patterns of m 1 A, p21, p16, TRMT6, and TRMT61A in CRC tumor tissues. The upper and lower rows displayed the two regions with highly and lowly concurrent staining of three markers, respectively. Scale bars, 100 µm. (B) Pearson's correlation heatmap that visualized the relationships between the IHC scores of intracellular markers, including TRMT6, TRMT61A, m 1 A, p21, and p16 in CRC tumor tissues (NEPDC cohort, n = 58) was shown. The numbers in the boxes represented Pearson correlation coefficients (r), and the color scale indicated the strength of the correlation. (C) The TRMT6/61A‐mediated RNA m 1 A modification depended on the catalytic domains. We constructed the DLD1 and HCT116 cells with stable overexpression of vector (CTRL), wild‐type TRMT6/61A (OE‐WT), and catalytically inactive mutant TRMT6‐R377L/TRMT61A‐D181A (OE‐MUT) (top), and the dot blot analysis showed global RNA m 1 A levels were elevated in OE‐WT cells rather than OE‐MUT cells compared with CTRL cells (bottom). The methylene blue (MB) staining served as a loading control for RNA. (D) Gene set enrichment analysis (GSEA) of differentially expressed genes between OE‐WT and CTRL groups of DLD1 cells based on their RNA‐seq profiles. The plot showed that cellular senescence was active in the cells highly expressing wild‐type TRMT6/61A. NES: Normalized enrichment score. (E) Western blot analysis demonstrating increased protein levels of senescence markers p21 and p16 in the OE‐WT rather than the OE‐MUT group of DLD1 and HCT116 cells compared with the CTRL group cells. Relative densitometry quantification (normalized to GAPDH) is shown below the bands. Blots are representative of three independent biological replicates ( n = 3). (F) Representative images of senescence‐associated β‐galactosidase (SA‐β‐Gal) staining in the three groups of DLD1 and HCT116 cells. The senescent cells were identified by their blue color. Scale bars, 100 µm. (G) Proportions of SA‐β‐Gal‐positive cells in the three groups of DLD1 and HCT116 cells from three independent experiments. The percentage of SA‐β‐Gal‐positive cells was determined by quantifying both positive (blue‐stained) and total cells from three randomly selected high‐power fields (100×) per well using ImageJ software. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human colorectal cancer cell lines DLD1 (RRID: CVCL_0248) and HCT116 (RRID: CVCL_0291) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) in October 2022.

Techniques: Modification, Immunohistochemistry, Expressing, Staining, Construct, Over Expression, Plasmid Preparation, Mutagenesis, Dot Blot, Control, RNA Sequencing, Western Blot, Software

Journal: Advanced Science

Article Title: m 1 A‐Dependent TRMT6/61A‐ARG2 Axis Drives Protumorigenic Senescence by Remodeling the Tumor Microenvironment

doi: 10.1002/advs.202518536

Figure Lengend Snippet: TRMT6/TRMT61A‐mediated m 1 A modification promotes cellular senescence by enhancing the translational efficiency of ARG2 (A) Schematic diagram illustrating the tRNA m 1 A‐seq workflow. (B) Scatter plot of differentially m 1 A‐modified tRNA between DLD1 cells with stable overexpression of vector (CTRL) and wild‐type TRMT6/61A (OE‐WT) based on their tRNA‐m 1 A‐seq profiles. Red dots represented tRNAs with significantly upregulated m 1 A methylation levels (Fold Change > 1 and p < 0.05) in the OE‐WT group. (C) Gene ontology (GO) enrichment analysis of transcripts with upregulated translation efficiency in OE‐WT cells compared to CTRL cells, as determined by ribosome profiling (Ribo‐seq). The enriched biological processes were ranked by p ‐value, and the positive regulation of cellular senescence involving ARG2 and YPEL3 was highlighted. (D) A heatmap illustrating the top 30 genes with the highest increase in translation efficiency in OE‐WT cells, and the senescence‐associated gene, ARG2 , was highlighted. (E) RT‐qPCR analysis of ARG2 and YPEL3 mRNA expression in the CTRL and OE‐WT cells. Data were presented as scatter dot plots showing individual data points from three independent experiments ( n = 3), along with mean ± SD. (F) Western blot analysis showed that the protein levels of ARG2 and phosphorylated mTOR (p‐mTOR) were increased in OE‐WT cells rather than OE‐MUT cells, compared with CTRL cells. Relative densitometry quantification (normalized to GAPDH) is shown below the bands. Blots are representative of three independent biological replicates ( n = 3). (G, H) Overexpression of ARG2 (OE‐ARG2) induced cellular senescence similar to wild‐type TRMT6/61A. (G) Western blot analysis confirmed ARG2 overexpression and mTOR activation. (H) SA‐β‐Gal staining images and quantification. The percentage of positive cells was quantified from three random fields. (I, J) Knockdown of ARG2 rescued TRMT6/61A‐induced senescence. The Western blot showed that ARG2 knockdown reversed the increased levels of p‐mTOR, p21, and p16 in OE‐WT HCT116 and DLD1 cells (I), and the decreased SA‐β‐Gal‐positive cells after ARG2 knockdown in OE‐WT cells with representative images were shown (J). (K) The mTOR pathway is required for TRMT6/61A‐induced senescence. Each group of cells was treated with the mTOR inhibitor rapamycin for 48 h. Representative images and quantification of SA‐β‐Gal staining showed that rapamycin treatment reversed the senescent phenotype in OE‐WT cells. Scale bars, 100 µm. (L) The dual‐luciferase reporter system. A schematic diagram of the reporter constructs is provided in Figure . We constructed reporter plasmids by inserting six‐repeated cognate codons for either Asp (GAC) or Glu (GAG)—decoded by the m 1 A‐hypermethylated tRNA‐Asp‐GTC and tRNA‐Glu‐CTC, respectively—immediately upstream of the firefly luciferase gene. The relative luciferase activity was measured. The enhanced ratio in T6‐OE‐WT cells demonstrated that TRMT6/61A‐mediated m 1 A modification of these tRNAs directly enhanced the translational efficiency of their cognate codons. (M) Schematic diagram of the ARG2 codon‐switch assay. To validate specificity, we engineered a synonymous mutant of ARG2 (ARG2‐MUT) by replacing the key cognate codons (GAC and GAG) with different, synonymous codons not recognized by the m 1 A‐modified tRNAs. aa, amino acid. (N) Western blot analysis comparing the protein expression of wild‐type ARG2 (ARG2‐WT) and the codon‐mutated version (ARG2‐MUT) in CTRL and OE‐WT cells, demonstrating that translation of ARG2‐WT is preferentially enhanced in OE‐WT cells. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human colorectal cancer cell lines DLD1 (RRID: CVCL_0248) and HCT116 (RRID: CVCL_0291) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) in October 2022.

Techniques: Modification, Over Expression, Plasmid Preparation, Methylation, Quantitative RT-PCR, Expressing, Western Blot, Activation Assay, Staining, Knockdown, Luciferase, Construct, Activity Assay, Mutagenesis

Journal: Advanced Science

Article Title: m 1 A‐Dependent TRMT6/61A‐ARG2 Axis Drives Protumorigenic Senescence by Remodeling the Tumor Microenvironment

doi: 10.1002/advs.202518536

Figure Lengend Snippet: Senescent tumor cells reprogram the tumor microenvironment in CRC (A) Uniform manifold approximation and projection (UMAP) visualization of integrated single‐cell transcriptomes from the CRC patients. The cell clusters with annotation of major cell types were shown (left top), and the feature plots showed the expression of TRMT6 , TRMT61A , ARG2, CDKN1A , and MTOR among cell types. (B) Chord diagrams illustrating the predicted intercellular communication network based on the number of interactions (top) and the overall interaction strength (bottom). Tumor cells were sub‐classified into ARG2 + and ARG2 ‐ subsets to reveal their distinct communication patterns. (C) Heatmaps provided an alternative visualization of the data in (B), quantifying the number of interactions (left) and interaction strength (right) between all identified cell populations. (D) Heatmaps visualizing the outgoing and incoming signaling patterns for key SASP‐related pathways, including GDF, HGF, CCL, TGFb, CXCL, IL1, and IGFBP. (e.g., HGF and IGFBP). The analysis identified ARG2 + cancer cells as prominent sources of these signals targeting M2 macrophages and fibroblasts. (E) In vitro validation of SASP gene expression. RT‐qPCR analysis revealed the relative mRNA fold change of a panel of key SASP factors in DLD1 (left) and HCT116 (right) cells. Expression in the TRMT6/61A‐overexpressing group (OE‐WT) is compared to the control group (CTRL). Data were presented as mean ± SD from three independent biological replicates ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human colorectal cancer cell lines DLD1 (RRID: CVCL_0248) and HCT116 (RRID: CVCL_0291) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) in October 2022.

Techniques: Single Cell, Expressing, In Vitro, Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Control

Journal: Advanced Science

Article Title: m 1 A‐Dependent TRMT6/61A‐ARG2 Axis Drives Protumorigenic Senescence by Remodeling the Tumor Microenvironment

doi: 10.1002/advs.202518536

Figure Lengend Snippet: TRMT6/61A‐induced STCs promote the malignant biological behaviors of recipient cancer cells. (A) Schematic diagram illustrating the experimental design. Conditioned medium (CM) was collected from cancer cells with stable overexpression of vector (CTRL) and wild‐type TRMT6/TRMT61A (OE‐WT), respectively. The two groups of CM were subsequently used to culture non‐transfected recipient CRC cells for functional assays. (B, C) The CM from OE‐WT cells enhanced the migration and invasion of recipient CRC cells. Representative images and quantification of transwell invasion (B) and wound‐healing (C) assays for DLD1 and HCT116 cells cultured with the two groups of CM are shown. Scale bars for (B), 100 µm. (D) CM from OE‐WT cells promoted the proliferation of recipient CRC cells. Real‐time cell proliferation curves for DLD1 and HCT116 cells were generated using the IncuCyte system. (E) Western blot analysis showing that CM from OE‐WT cells induced epithelial‐mesenchymal transition (EMT) in recipient DLD1 and HCT116 cells, as indicated by the upregulation of Snail, Slug, and N‐cadherin. Relative densitometry quantification (normalized to GAPDH) is shown below the bands. Blots are representative of three independent biological replicates ( n = 3). (F) TRMT6/61A‐overexpressing cancer cells promoted tumor growth in an in vivo model. The MC38 cells were co‐injected with either MC38‐derived CTRL or OE cells into BALB‐c/Nude mice ( n = 5 per group). The representative images of tumors and quantification of tumor weights were shown. (G) Western blot analysis of tumor lysates from the in vivo experiment (F) confirmed the expression of senescence and EMT markers. (H) Representative IHC images of tumor sections from the xenograft models, showing the abundance of m 1 A and expression of p16. Scale bars, 100 µm. (I) TRMT6/61A enhanced cancer stem cell (CSC)‐like properties. Representative bright‐field images and quantification of tumor spheres formed by DLD1 and HCT116 cells co‐cultured with CM derived from CTRL or OE‐WT cells. Scale bars, 50 µm. (J, K) Expression of stemness markers was upregulated in OE‐WT cells. RT‐qPCR analysis revealed increased NANOG and OCT4 mRNA in OE‐WT cells (J), and the Western blot analysis indicated upregulated EpCAM and CD44 protein levels in OE‐WT cells (K). Relative densitometry quantification (normalized to GAPDH) is shown below the bands. Data are presented as mean ± SD from three independent experiments ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human colorectal cancer cell lines DLD1 (RRID: CVCL_0248) and HCT116 (RRID: CVCL_0291) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) in October 2022.

Techniques: Over Expression, Plasmid Preparation, Transfection, Functional Assay, Migration, Cell Culture, Generated, Western Blot, In Vivo, Injection, Derivative Assay, Expressing, Quantitative RT-PCR

Journal: Advanced Science

Article Title: m 1 A‐Dependent TRMT6/61A‐ARG2 Axis Drives Protumorigenic Senescence by Remodeling the Tumor Microenvironment

doi: 10.1002/advs.202518536

Figure Lengend Snippet: TRMT6/61A‐induced STCs promote M2 macrophage polarization and cancer‐associated fibroblast (CAF) activation. (A) Conditioned medium (CM) derived from the TRMT6/61A‐overexpressing CRC cells (OE‐WT) promoted the migration of THP‐1 monocytes and primary colorectal CAFs compared with that derived from the vector group of CRC cells (CTRL). Representative images (left) and quantification (right) of a transwell migration assay are shown. (B) CM from OE‐WT cells induced the M2‐like polarization of THP‐1 monocytes. Representative flow cytometry plots (left) and quantification (right) showed an increased percentage of CD206‐positive cells after incubation with the indicated OE‐WT‐derived CM. (C, D) RT‐qPCR analysis of M2‐associated cytokine and chemokine expression in THP‐1 monocytes co‐cultured with CM derived from OE‐WT and CTRL groups of DLD1 (C) and HCT116 (D) cells. The expression levels were normalized by the expression in CTRL‐CM‐treated groups. (E) Western blot analysis showing the increased expression of CAF activation markers, including Fibroblast Activation Protein (FAP) and α‐Smooth Muscle Actin (α‐SMA), in CAFs cultured with CM derived from OE‐WT cells. Relative densitometry quantification (normalized to GAPDH) is shown below the bands. Blots are representative of three independent biological replicates ( n = 3). (F) immunofluorescence assay confirmed the increased expression and co‐localization of FAP (green) and α‐SMA (red) in CAFs upon treatment with OE‐WT‐derived CM. Nuclei were counterstained with DAPI (blue). Scale bars, 20 µm. Data were presented as mean ± SD from three independent biological replicates ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human colorectal cancer cell lines DLD1 (RRID: CVCL_0248) and HCT116 (RRID: CVCL_0291) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) in October 2022.

Techniques: Activation Assay, Derivative Assay, Migration, Plasmid Preparation, Transwell Migration Assay, Flow Cytometry, Incubation, Quantitative RT-PCR, Expressing, Cell Culture, Western Blot, Immunofluorescence

Journal: Advanced Science

Article Title: m 1 A‐Dependent TRMT6/61A‐ARG2 Axis Drives Protumorigenic Senescence by Remodeling the Tumor Microenvironment

doi: 10.1002/advs.202518536

Figure Lengend Snippet: NF‐κB/SASP is essential for pro‐tumorigenic effects of senescent CRC cells (A) Western blot analysis showing an activation of the NF‐κB pathway in TRMT6/61A‐overexpressing (OE‐WT) DLD1 and HCT116 cells compared with vector group (CTRL) cells, as indicated by increased levels of phosphorylated p65 (p‐p65). Relative densitometry quantification (normalized to GAPDH) is shown below the bands. Blots were representative of three independent biological replicates ( n = 3). (B) Schematic diagram of the experimental design for the NF‐κB inhibition experiments. CM was collected from OE‐WT cells pre‐treated with or without the NF‐κB inhibitor, BAY11‐7082. (C) RT‐qPCR analysis showing that NF‐κB inhibition abrogated the SASP gene signature in OE‐WT cells. (D, E, F) NF‐κB/SASP inhibition in STCs abolished its pro‐malignant effects on recipient cancer cells. Quantification and representative images were shown for the transwell invasion (D), real‐time cell proliferation (E), and wound‐healing (F) assays. The non‐transfected CRC cells were cultured with the CM derived from CTRL cells, OE‐WT cells, or OE‐WT cells pre‐treated with BAY11‐7082. Scale bars for (F), 100 µm. (G) NF‐κB/SASP inhibition rescued the STC‐induced M2 polarization. The flow cytometry showed that the CM from BAY11‐7082‐treated OE‐WT cells did not increase the proportion of CD206‐positive THP‐1 cells. (H,I) NF‐κB /SASP inhibition prevented STC‐induced activation of cancer‐associated fibroblasts (CAFs). Western blot analysis (H) and immunofluorescent assay (I) showed that CM from BAY11‐7082‐treated OE‐WT cells failed to upregulate the activation markers FAP (green) and α‐SMA (red). Relative densitometry quantification (normalized to GAPDH) is shown below the bands in (H). Nuclei were counterstained with DAPI (blue). Scale bars for (I), 20 µm. Data were presented as mean ± SD from three independent biological replicates ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human colorectal cancer cell lines DLD1 (RRID: CVCL_0248) and HCT116 (RRID: CVCL_0291) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) in October 2022.

Techniques: Western Blot, Activation Assay, Plasmid Preparation, Inhibition, Quantitative RT-PCR, Transfection, Cell Culture, Derivative Assay, Flow Cytometry

Journal: iScience

Article Title: SLC7A11-specific CAR-T cell therapy potently targets colorectal and pancreatic cancer

doi: 10.1016/j.isci.2025.113713

Figure Lengend Snippet: Antibody design and affinity evaluation (A) The procedures for generating murine monoclonal antibody using hybridoma technology. (B) The qPCR experiment confirmed the successful knockdown of SLC7A11 expression levels in DLD1 cells mediated by lentivirus ( n = 3). (C) The specificity of the antibody targeting SLC7A11 was validated using flow cytometry ( n = 4). (D) The affinity between 1A4 and SLC7A11 was confirmed through SPR analysis. (E) The affinity between humanized 1A4 and SLC7A11 was confirmed through SPR analysis. The SLC7A11 expression level data by qPCR were analyzed by t test. The MFI of SLC7A11 data was analyzed by ANOVA test. Data were presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The human colorectal cancer and pancreatic cell lines DLD1 (RRID: CVCL_0248), HT29 (RRID: CVCL_0320), HCT116 (RRID: CVCL_0291), PANC-1 (RRID: CVCL_0480) and MIA PaCa-2 (RRID: CVCL_0428) were obtained from the American Type Culture Collection.

Techniques: Knockdown, Expressing, Flow Cytometry

Journal: iScience

Article Title: SLC7A11-specific CAR-T cell therapy potently targets colorectal and pancreatic cancer

doi: 10.1016/j.isci.2025.113713

Figure Lengend Snippet: The cytotoxicity assays in vitro by CCK8, LDH, and MFI detection (A) The survival rate of target cells after 24 h of co-culture ( n = 5). (B) The lysis rates of target cells after 24 h of co-culture with UTD, MOCK, and CAR-T cells ( n = 3). (C) The co-culture figures under the fluorescence microscope for the Mock group and CART group ( n = 3). Scale bars represent 50 μm. (D andE) The cytotoxic effects of DLD1 cells by measuring the fluorescence intensity after co-culturing ( n = 3). Scale bars represent 50 μm. Data were analyzed by ANOVA test. The CCK8 data and MFI data were presented as mean ± SD. The LDH data were presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The human colorectal cancer and pancreatic cell lines DLD1 (RRID: CVCL_0248), HT29 (RRID: CVCL_0320), HCT116 (RRID: CVCL_0291), PANC-1 (RRID: CVCL_0480) and MIA PaCa-2 (RRID: CVCL_0428) were obtained from the American Type Culture Collection.

Techniques: In Vitro, Co-Culture Assay, Lysis, Fluorescence, Microscopy