Dataset for Evaluating the Impact of P-50-Mediated SDT in Combination with Chemotherapy on HepG2 and Eca-109 Tumor Cells

Description

Sonodynamic Therapy (SDT) can enhance biofilm permeability, promote drug delivery, and improve bioavailability without significant cell damage. P-50 is a novel nano-sized porphyrin polymer with good water solubility, but its basic research as a sonosensitizer has not been carried out. The study corresponding to this dataset aims to evaluate the effects of P-50-mediated SDT alone and in combination with oxaliplatin and cisplatin on liver cancer (HepG2) and esophageal cancer (Eca-109) cells. The experimental ultrasound parameters were uniformly set to 1.0 MHz frequency, 1.5 W/cm² power, and 30 seconds of treatment duration. The core contents of the dataset include: (1) The uptake efficiency and time dependence of P-50 by HepG2 and Eca-109 cells (focusing on the 4-6 hour time point); (2) The inherent cytotoxicity of P-50 (detected 18 hours after 6 hours of co-incubation), its enhancing effect on SDT-induced cytotoxicity, and the inhibitory effect of the combination of SDT and chemotherapeutic drugs on tumor cell viability; (3) The effects of different treatment groups on tumor cell apoptosis (including late apoptotic characteristics) and the expression of apoptosis-related proteins (Bax, Caspase-9, Caspase-3, Bcl-2); (4) The level of reactive oxygen species (ROS) production in each group (quantification of fluorescence staining intensity); (5) Changes in the drug interaction coefficient and mitochondrial membrane potential (red/green fluorescence intensity ratio) in Eca-109 cells, and differences in calcium ion fluorescence intensity in HepG2 cells. Key data results: (1) Both cell types efficiently uptook P-50, and the uptake amount increased significantly at 4-6 hours; (2) P-50 had no significant effect on the survival of Eca-109 cells, but showed potential toxicity to HepG2 when the concentration was >5 μM. However, 5 μM P-50 could significantly enhance SDT-induced apoptosis, and the anti-tumor effect of the combination with chemotherapy was better than that of single drugs; (3) The combination therapy group had the highest apoptosis rate, accompanied by late apoptotic characteristics, with upregulated expression of pro-apoptotic proteins and downregulated anti-apoptotic protein Bcl-2; (4) The combination group showed the strongest ROS fluorescence, while the chemotherapy alone group only showed weak green fluorescence; (5) In Eca-109 cells, the drug interaction coefficient was <1, and the mitochondrial membrane potential of the combination group was higher than that of other groups; in HepG2 cells, the combination group had the strongest calcium ion fluorescence. The above data indicate that P-50 has low inherent cytotoxicity and can be used as a novel SDT sonosensitizer to induce apoptosis in two types of tumor cells. The therapeutic effect of its combination with chemotherapy is better than that of single drugs, and the mechanism may be related to the massive production of ROS and the activation of the mitochondrial apoptotic pathway after SDT.

Steps to reproduce

Data were collected via standardized in vitro experiments on HepG2 (hepatocellular carcinoma) and Eca-109 (esophageal squamous cell carcinoma) cells, using validated protocols, specific reagents, instruments, and software. Below is a concise breakdown of the workflow, methods, and key tools for reproducibility: 1. Cell Lines & Key Reagents Cell Lines: HepG2 (ATCC), Eca-109 (CCTCC); maintained in complete medium. Reagents: P-50 (provided by Dalian University of Technology, synthesis/assembly per Figure 1[22]), cisplatin (DDP), oxaliplatin (L-OHP), CCK-8 kit, Hoechst 33342, Annexin V-FITC/7-AAD, DCFH-DA, Fluo-4 AM, JC-1, BCA protein assay kit, SDS-PAGE reagents, PVDF membrane (Millipore), primary antibodies (β-actin, Bax, Bcl-2, Caspase-9, Caspase-3; Proteintech), HRP-conjugated secondary antibody, ECL detection kit (GE Healthcare). 2. Experimental Workflow & Methods 2.1 Cell Grouping & Treatment Eca-109: 5 groups (Control, DDP, P-50, SDT, SDT+DDP). SDT used 1 MHz ultrasound (1.5 W/cm², 50% duty cycle, 30s/well). HepG2: 4 groups (Control, L-OHP, SDT, SDT+L-OHP). All groups included drug-free controls and cell-free blanks. 2.2 Data Collection Methods P-50 Uptake: Generated P-50 standard curve (1–20 µM, microplate reader for OD); measured intracellular P-50 (2–8h timepoints) via lysis + OD, normalized to protein (BCA assay). Cell Viability: CCK-8 assay (10% CCK-8 in RPMI-1640; 450 nm OD, microplate reader); calculated survival rate. Drug Interaction: CDI calculation (CDI=AB/(A×B)) using absorbance ratios of combination vs. single treatments. Apoptosis: Nuclear morphology: Hoechst 33342 staining (30min dark incubation; fluorescence microscope imaging). Apoptosis rate: Annexin V-FITC/7-AAD staining (48h post-treatment; flow cytometry). Apoptosis-Related Proteins: Western Blot (cell lysis → BCA quantification → SDS-PAGE → PVDF transfer → blocking → primary/secondary antibody incubation → ECL detection; BioRed software for quantification). Intracellular Molecules: ROS: DCFH-DA (10 µM, 20min incubation; fluorescence microscope). Ca²⁺: Fluo-4 AM (1 µM, 30min incubation + 30min rest; fluorescence microscope). Mitochondrial membrane potential (Eca-109): JC-1 (2 µM, 20min dark incubation; centrifugation → PBS wash → fluorescence microscope). 3. Instruments & Software Instruments: Ultrasound device (1 MHz), microplate reader, fluorescence microscope, flow cytometer, centrifuge, SDS-PAGE system, Western Blot imaging system. Software: GraphPad Prism 9.5 (statistical analysis: one-way ANOVA, significance: ns=P>0.05, *=P<0.05, **=P<0.01, ***=P<0.001), BioRed (Western Blot quantification). 4. Reproducibility Notes Standardize ultrasound parameters, reagent concentrations (e.g., 5 µM P-50, 40 µg/ml DDP), incubation times, and replicate counts (3/ group). Validate P-50 standard curve and BCA protein assay before use; follow antibody incubation protocols (4°C overnight for primaries, 1h RT for secondaries).

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