Journal: Biomolecules & Therapeutics

Article Title: Combination Therapy with Betulinic Acid and TRAIL Increases ROS-Dependent Cytotoxicity and Inhibits PI3K/Akt Signaling in Human Bladder Cancer Cells

doi: 10.4062/biomolther.2026.035

Figure Lengend Snippet: Betulinic acid (BA) enhances tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity in T24 human bladder cancer cells. T24 cells were treated with the indicated concentrations of BA and TRAIL alone or in combination for 24 h. (A, B) After treatment, cell viability was assessed via 3′-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (C) Morphological changes in the cells treated with BA and TRAIL alone or in combination were observed under an inverted microscope. (D, E) After 4′,6-diamidino-2-phenylindole (DAPI) staining, nuclear morphological changes were observed under a fluorescence microscope (D), and the frequency of cells with chromatin condensation and fragmentation was determined (E). (F, G) Flow cytometry was performed after propidium iodide (PI) staining. Representative histograms (F) and the frequency of cells in the sub-G1 phase (G) are shown. * p <0.05, ** p <0.01, and *** p <0.001 vs. control cells; ### p <0.001 vs. BA-treated cells (n=3).

Article Snippet: T24 human bladder cancer cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured as previously described ( Kim et al. , 2021 ).

Techniques: MTT Assay, Inverted Microscopy, Staining, Fluorescence, Microscopy, Flow Cytometry, Control

Journal: Biomolecules & Therapeutics

Article Title: Combination Therapy with Betulinic Acid and TRAIL Increases ROS-Dependent Cytotoxicity and Inhibits PI3K/Akt Signaling in Human Bladder Cancer Cells

doi: 10.4062/biomolther.2026.035

Figure Lengend Snippet: Combination treatment with BA and TRAIL increases reactive oxygen species (ROS) production and decreases ATP levels in T24 human bladder cancer cells. T24 cells were treated with BA and TRAIL alone or in combination and cultured for 30 min (A-C) or 24 h (D). (A, B) After treatment, the cells were stained with 2′,7′-dichlorofluorescein diacetate (DCF-DA) and analyzed via flow cytometry. Representative histograms (A) and the frequency of DCF-positive cells (B) are shown. (C) Representative fluorescence images of the cells stained with DCF-DA, indicating ROS production, were captured via fluorescence microscopy. (D) Intracellular ATP levels were measured using an ATP assay kit. * p <0.05 and ** p <0.01 vs. control cells; ### p <0.001 vs. BA-treated cells (n=3).

Article Snippet: T24 human bladder cancer cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured as previously described ( Kim et al. , 2021 ).

Techniques: Cell Culture, Staining, Flow Cytometry, Fluorescence, Microscopy, ATP Assay, Control

Journal: Biomolecules & Therapeutics

Article Title: Combination Therapy with Betulinic Acid and TRAIL Increases ROS-Dependent Cytotoxicity and Inhibits PI3K/Akt Signaling in Human Bladder Cancer Cells

doi: 10.4062/biomolther.2026.035

Figure Lengend Snippet: Combination treatment with BA and TRAIL increases mitochondrial damage and induces changes in the expression levels of Bcl-2 family proteins in T24 human bladder cancer cells. T24 cells were treated with BA and TRAIL alone or in combination and cultured for 24 h. (A, B) After treatment, the cells were stained with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetramethylbenzimidazolyl carbocyanine iodide (JC-1) and analyzed via flow cytometry. Representative histograms (A) and the frequency of cells with JC-1 monomers (B), indicating mitochondrial membrane potential (MMP) loss, are shown. * p <0.05 vs. control cells; ### p <0.001 vs. BA-treated cells (n=3). (C, D) Mitochondrial and cytosolic fractions were isolated from the cells (C), total protein was extracted (D), and immunoblotting was performed using antibodies against target proteins. Cytochrome c oxidase (COX IV) and β-actin were used as loading controls for the two fractions, respectively.

Article Snippet: T24 human bladder cancer cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured as previously described ( Kim et al. , 2021 ).

Techniques: Expressing, Cell Culture, Staining, Flow Cytometry, Membrane, Control, Isolation, Western Blot

Journal: Biomolecules & Therapeutics

Article Title: Combination Therapy with Betulinic Acid and TRAIL Increases ROS-Dependent Cytotoxicity and Inhibits PI3K/Akt Signaling in Human Bladder Cancer Cells

doi: 10.4062/biomolther.2026.035

Figure Lengend Snippet: Combination treatment with BA and TRAIL activates the caspase-dependent extrinsic and intrinsic apoptotic pathways in T24 human bladder cancer cells. T24 cells were either directly co-treated with BA and TRAIL or pretreated with z-VAD-fmk for 1 h before co-treatment with BA and TRAIL and cultured for 24 h. (A) Total protein was extracted, and immunoblotting analysis was performed using antibodies against target proteins. (B) Caspase activity was examined using caspase assay kits. (C, D) After DAPI staining, nuclear morphological changes were observed under a fluorescence microscope (C), and the frequency of cells with chromatin condensation and fragmentation was determined (D). (E, F) Flow cytometry was performed after PI staining. Representative histograms (E) and the frequency of cells in the sub-G1 phase (F) are shown. ** p <0.01 and *** p <0.001 vs. control cells; ### p <0.001 vs. BA and TRAIL co-treated cells (n=3).

Article Snippet: T24 human bladder cancer cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured as previously described ( Kim et al. , 2021 ).

Techniques: Cell Culture, Western Blot, Activity Assay, Caspase Assay, Staining, Fluorescence, Microscopy, Flow Cytometry, Control

Journal: Biomolecules & Therapeutics

Article Title: Combination Therapy with Betulinic Acid and TRAIL Increases ROS-Dependent Cytotoxicity and Inhibits PI3K/Akt Signaling in Human Bladder Cancer Cells

doi: 10.4062/biomolther.2026.035

Figure Lengend Snippet: Combination treatment with BA and TRAIL inhibits activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway in T24 human bladder cancer cells. T24 cells were either directly co-treated with BA and TRAIL or pretreated with LY294002 for 1 h before co-treatment with BA and TRAIL and cultured for 24 h. (A) Total cellular proteins were isolated from the cells, and phosphorylation levels of PI3K and Akt were determined via immunoblotting. (B, C) After DAPI staining, nuclear morphological changes were observed under a fluorescence microscope (B), and the frequency of cells with chromatin condensation and fragmentation was determined (C). (D, E) Flow cytometry was performed after PI staining. Representative histograms (D) and the frequency of cells in the sub-G1 phase (E) are shown. (F) Cell viability was assessed via MTT assay. *** p <0.001 vs. control cells; ### p <0.001 vs. BA and TRAIL co-treated cells (n=3).

Article Snippet: T24 human bladder cancer cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured as previously described ( Kim et al. , 2021 ).

Techniques: Activation Assay, Cell Culture, Isolation, Phospho-proteomics, Western Blot, Staining, Fluorescence, Microscopy, Flow Cytometry, MTT Assay, Control

Journal: Biomolecules & Therapeutics

Article Title: Combination Therapy with Betulinic Acid and TRAIL Increases ROS-Dependent Cytotoxicity and Inhibits PI3K/Akt Signaling in Human Bladder Cancer Cells

doi: 10.4062/biomolther.2026.035

Figure Lengend Snippet: Combination treatment with BA and TRAIL increases apoptosis in T24 human bladder cancer cells in an ROS-dependent manner. T24 cells were pretreated with N-acetyl-l-cysteine (NAC) 1 h before combination treatment with BA and TRAIL and cultured for 24 h. (A, B) Total protein was extracted and analyzed via immunoblotting using antibodies against target proteins. β-actin was used as a loading control. (C) Caspase-3 activity was measured using a caspase-3 assay kit. (D, E) After DAPI staining, nuclear morphological changes were observed under a fluorescence microscope (D), and the frequency of cells with chromatin condensation and fragmentation was determined (E). (F, G) Flow cytometry was performed after PI staining. Representative histograms (F) and the frequency of cells in the sub-G1 phase (G) are shown. (H) Cell viability was assessed via MTT assay. *** p <0.001 vs. control cells; ### p <0.001 vs. BA and TRAIL co-treated cells (n=3).

Article Snippet: T24 human bladder cancer cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured as previously described ( Kim et al. , 2021 ).

Techniques: Cell Culture, Western Blot, Control, Activity Assay, Caspase-3 Assay, Staining, Fluorescence, Microscopy, Flow Cytometry, MTT Assay

Journal: Biomolecules & Therapeutics

Article Title: Combination Therapy with Betulinic Acid and TRAIL Increases ROS-Dependent Cytotoxicity and Inhibits PI3K/Akt Signaling in Human Bladder Cancer Cells

doi: 10.4062/biomolther.2026.035

Figure Lengend Snippet: Schematic diagram of cytotoxicity induction in betulinic acid and TRAIL co-treated T24 human bladder cancer cells. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; PI3K, phosphoinositide 3-kinase; NAC, N-acetyl-l-cysteine; MMP, mitochondrial membrane potential; FADD, Fas-associated death domain; tBid, truncation of BH3-interacting domain death agonist; PARP, poly(ADP-ribose) polymerase.

Article Snippet: T24 human bladder cancer cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured as previously described ( Kim et al. , 2021 ).

Techniques: Membrane

Journal: Pharmaceuticals

Article Title: Identification and Functional Analysis of Targets of Dehydrodiisoeugenol in Bladder Cancer Based on Chemoproteomics-Based Profiling

doi: 10.3390/ph19040651

Figure Lengend Snippet: DHE suppresses the proliferation and migration of T24 and 5637 bladder cancer cells. ( A ) The natural source (Myristica fragrans) and chemical structure of DHE. Atom numbering of the 2,3-dihydro-1-benzofuran core is shown for clarity; the stereogenic centers are located at C-2 and C-3. ( B , C ) Dose-response curves of T24 and 5637 cells treated with indicated concentrations of DHE for 48 h, as measured using the CCK-8 assay. ( D , E ) Proliferation curves of T24 and 5637 cells treated with DMSO or DHE (20, 40 μM) over 5 consecutive days. ( F , G ) Representative images and statistical quantification of colony formation assays for T24 ( F ) and 5637 ( G ) cells following DHE treatment. ( H ) Wound healing assays evaluating the migratory ability of T24 and 5637 cells treated with increasing concentrations of DHE (0, 10, 20, 40 μM). Representative micrographs at 0 h and 24 h are shown on the left; quantitative analysis of the area recovery percentage is shown on the right. Data are presented as Mean ± SD ( n = 3). **** p < 0.0001 vs. DMSO group ( t -test or one-way ANOVA).

Article Snippet: Cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), including the T24 cell line (ATCC Cat. No. HTB-4) and the 5637 cell line (ATCC Cat. No. HTB-9), and were cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin (100 U/mL; Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Migration, CCK-8 Assay

Journal: Pharmaceuticals

Article Title: Identification and Functional Analysis of Targets of Dehydrodiisoeugenol in Bladder Cancer Based on Chemoproteomics-Based Profiling

doi: 10.3390/ph19040651

Figure Lengend Snippet: Synthesis and biological validation of a DHE-derived photoaffinity probe. ( A ) Schematic representation of the ABPP (Activity-Based Protein Profiling) workflow. The process includes cell lysis, probe incubation, UV-induced cross-linking, click chemistry-mediated biotinylation, and streptavidin-based enrichment followed by LC-MS/MS or SDS-PAGE analysis. ( B ) Synthetic route of the DHE-Probe. Reaction conditions: (a) diazirine–alkyne linker, (b) K 2 CO 3 , DMF, 65 °C, 16 h. The final probe includes a photo-cross-linker and an alkyne handle for target capturing. ( C , D ) Comparison of the anti-proliferative effects of DMSO, DHE, and DHE-Probe (40 μM) in T24 ( C ) and 5637 ( D ) bladder cancer cells. Cell viability was measured 24 h post-treatment. Data are presented as Mean ± SD ( n = 3). **** p < 0.0001 vs. DMSO group ( t -test or one-way ANOVA).

Article Snippet: Cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), including the T24 cell line (ATCC Cat. No. HTB-4) and the 5637 cell line (ATCC Cat. No. HTB-9), and were cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin (100 U/mL; Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Biomarker Discovery, Derivative Assay, Activity Assay, Lysis, Incubation, Liquid Chromatography with Mass Spectroscopy, SDS Page, Comparison

Journal: Pharmaceuticals

Article Title: Identification and Functional Analysis of Targets of Dehydrodiisoeugenol in Bladder Cancer Based on Chemoproteomics-Based Profiling

doi: 10.3390/ph19040651

Figure Lengend Snippet: Gel-based ABPP validates specific protein labeling by the DHE-Probe. ( A ) Concentration-dependent labeling of the T24 cell proteome. Cell lysates were incubated with indicated concentrations of DHE-Probe (0, 5, 10, 20, 40, 80 μM), followed by UV cross-linking (365 nm) and click chemistry. The left panel shows the labeling profile (fluorescence/chemiluminescence); the right panel shows Coomassie Brilliant Blue (CBB) staining as a loading control. ( B ) Competition assay for binding specificity. Lysates were either treated with the DHE-Probe alone or pre-incubated with excess unlabeled DHE (Competition) prior to probe labeling. The significant reduction in band intensity in the competition lane (middle) confirms the specificity of the interaction. CBB staining (right) indicates equal loading. Molecular weight markers (kDa) are indicated on the left of each gel. Representative images from three independent experiments are shown.

Article Snippet: Cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), including the T24 cell line (ATCC Cat. No. HTB-4) and the 5637 cell line (ATCC Cat. No. HTB-9), and were cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin (100 U/mL; Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Labeling, Concentration Assay, Incubation, Fluorescence, Staining, Control, Competitive Binding Assay, Binding Assay, Molecular Weight