Dulbecco’s Modified Eagle's Medium (DMEM, HiGlutaXL, AL007G) and Bovine Serum Albumin (BSA) were procured from Himedia Labs, Mumbai, India. Trypsin and Fetal Bovine Serum (FBS) were obtained from Invitrogen, USA. Propidium iodide, RNase, and Dichloro-fluorescein-diacetate (DCFDA) as well as JC-1 dyes were purchased from Sigma, MO, USA. All other unspecified chemicals were sourced from Sigma, MO, USA.
2.2. METHOD
2.2.1. CELL CULTURE
Lung adenocarcinoma cells, A549 were procured from National Centre for Cell Science, Pune-Maharashtra, India. The cell lines are maintained in DMEM with 10% FBS and 1% antibiotic and antimycotic solution in an incubator (95% relative humidity; 5% CO2; 37 ºC).
2.2.2. PULSE GENERATOR
To generate microsecond duration electrical pulse a capacitor C is charged by DC Power Supply (HV4800E, ECIL India) and discharged (through load) with the help of IGBT (SKM800GA176D, SEMIKRON) switch. The equivalent of load here can be assumed as capacitive. Therefore, we require \(C\gg Load Capacitance\) to transfer the energy from the charging capacitor, C to the load capacitor. To fulfil this requirement charging capacitor, C of value 100 µF is selected. IGBT is triggered with a square low voltage trigger pulse generator. To vary the voltage, the capacitor C is charged with a variable DC voltage source (here 100 V-1200 V, 100 V step since the IGBT has a maximum rating of 1200 V). To vary the duration of pulse, variable duration low voltage square pulse generator will be required to trigger the IGBT for the required duration. Figure 1 shows the experimental setup and Fig. 2 shows the electrical circuit diagram (a) and output voltage waveform at 300 V of charging voltage [22].
2.2.3. PULSE ELECTRIC FIELD APPLICATION
The electrical pulse parameters were set to 300 V, a 50µs duration, with options for 5 or 10 pulses at a repetition rate of 1 Hz. These output pulses were utilized to expose cancer cells within a 0.2 cm gap electroporation cuvette (volume of 400 µl or 0.4 cm³), generating an electrical field within the cuvette. The pulse duration of 50 µs remained constant at a repetition rate of 1 Hz. Cancer cell suspensions in complete media were placed in the 0.2 cm electroporation cuvette (BioRad, Inc., Hercules, CA). An output pulse of 300 V created electrical fields of 1.5 kV/cm between the electrodes of the 0.2 cm electroporation cuvette. The current passing through the load was measured using a current shunt (R = 0.005056Ω) and averaged around 15 A at 300 V. The energy delivered to the load per pulse for a 50 µs duration (VIt) was approximately 0.225 J at 300 V, resulting in a total energy exposure of 2.25 J to the cancer cells.
2.2.4. LIGHT MICROSCOPY
A cell suspension containing 1.5 × 105 cells/mL of A549 cells was subjected to different treatments: HV-µsPEF, curcumin, and a combination of both, as illustrated in Fig. 3A. Subsequently, the treated cells were seeded into a 6-well plate. After 48 hours of treatment and seeding, the cells were examined using a light microscope. Images were taken to facilitate a morphology-based analysis of the impacts of the separate pulse and curcumin treatments, as well as the combined effect of the two treatments.
2.2.5. ASSESSMENT OF APOPTOSIS BY SUB-G1 POPULATION ANALYSIS BY FLOW CYTOMETERY
Healthy and exponentially growing culture of cells was washed with PBS and trypsinized to get resuspended in cell culture medium. Further, Cell suspension of 1.5 × 105 cells/mL was treated in a cuvette with HV-µsPEF pulse, curcumin and the combination of the two as shown Fig. 3A and seeded in a 6 well plate. After 72 h, the spent media in each well was collected, adherent cells were washed once with 1X PBS and trypsinized to dislodge the cells and collected in the respective tube. The cells were pelleted at 2500 RPM for 3 min in a table top centrifuge (Remi, Mumbai, India), supernatant was discarded and to the pellet, hypotonic sodium citrate buffer containing 50 µg/mL propidium iodide and 50 µg/mL RNase was added. The cells were incubated for 10 min followed by flow cytometry analysis. Analysis of data after acquisition was carried out by using FlowJo software, (FlowJo LLC, USA).
2.2.6. ASSESSMENT OF CURCUMIN UPTAKE BY FLOW CYTOMETERY
A cell suspension containing 1.5 × 105 cells/mL was subjected to treatment in a cuvette using HV-µsPEF, curcumin, and the combination of both. The treated cells were subsequently seeded in a 6-well plate. Afterward, curcumin uptake was evaluated by measuring the increase in intensity in the FL1 channel (green fluorescence) using flow cytometry analysis. This assessment was conducted 2h post-treatment, with the cells resuspended in PBS during the analysis process.
2.2.7. ASSESSMENT OF REACTIVE OXYGEN SPECIES USING DCFDA ASSAY
Cell suspension of 1.5 × 105 cells/mL was treated in a cuvette with HV-µsPEF, curcumin and the combination of the two and seeded in a 6 well plate. After the indicated time durations, cells were incubated with 10 µM DCFDA in DMEM without FBS for 30 mins in dark. After the incubation, the cells were washed with 1X PBS, trypsinized and collected in a tube and resuspended in 1X PBS for flow cytometry analysis. Analysis of data after acquisition was carried out by using FlowJo software. The change in FL1 (green) fluorescence was analysed with different treatment conditions.
2.2.8. ASSESSMENT OF MITOCHONDRIAL MEMBRANE POTENTIAL (MMP) BY JC1 DYE ASSAY
Cell suspension of 1.5 × 105 cells was treated in a cuvette with HV-µsPEF, curcumin and the combination of the two as shown and seeded in a 6 well plate. After the indicated time durations, cells were incubated with 10 µM JC1 in DMEM without FBS for 30 mins in dark. After the incubation, the cells were washed with 1X PBS, trypsinized and collected in a tube and resuspended in 1X PBS for flow cytometry analysis. Analysis of data after acquisition was carried out by using FlowJo software. The ratio of fluorescence FL1 to FL3 was analysed.
2.2.9. STATISTICAL ANALYSIS
ANOVA test was performed and the mean of each group was compared with every other group mean. Inter-group comparisons are indicated wherever it is applicable. Detailed description on the analysis is provided in the legend section of the Fig.s wherever they apply. Statistical difference between means was assessed using Student's t test, with a P value less than 0.05 considered significant.