2.4. Experiential section 1. Synthesis of compounds
2.4.4.12. Biological Activity Studies 1. MTT-based cytotoxicity assay
Effect of all the compounds (2.1-2.7) on the viability of HeLa and MDA-MB-231 cells were tested by using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. HeLa and MDA-MB-231 cells were seeded in 96-well flat- bottom tissue culture plates, at a density of 105 cells/well (per 100 µL) containing 10% fetal bovine serum (FBS) in Dulbecco's Modified Eagle Medium (DMEM) and incubated at 37°C in 5% CO2. After 16 h, media was discarded, and each well was washed with phosphate-buffered saline (PBS) followed by the addition of the compounds to each well plate with media. After 48 h treatment, 10 µl of MTT solution (5 mg/ml) was added to each well, and the plates were incubated for another 4 h.
MTT containing media was removed from each well, and 100 µL of DMSO was added, dissolving the formazan crystals. The absorbance was recorded on a microplate reader (Multiskan™ GO) at the wavelength of 570 nm. All experiments were performed in triplicate, and the relative cell viability (%) was expressed as a percentage relative to the untreated cells.
2.4.4.12.2. Flow cytometry analysis
The identification of apoptotic cells was investigated by staining the cells with propidium iodide (PI) as described earlier. HeLa and BHK21 cells were seeded on a 6-well plate. Cells were treated with 40 µM of compounds 2.2 (PIT-1) and 2.5 (DM- PIT-1). After 24 hours, cells were harvested and suspended in 0.5 mL of PBS. Cells
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were gently aspirated several times with pipette to obtain a mono-dispersed cell suspension, with minimal cell aggregation. Cells were fixed by adding ice-cold 70%
ethanol dropwise, with constant vortexing and incubated overnight at 4 ºC. On the next day, ethanol-suspended cells were centrifuged for 10 min at 300g and ethanol was decanted thoroughly. The cell pellet was suspended in 3 ml of PBS and centrifuged at 300g for 10 min. Cells were resuspended in 200 µl of staining solution (PBS with 100 µg/mL RNase A and 50 µg/mL Propidium Iodide). The resuspended cells were incubated at 37 °C for 20 min. The samples were then transferred to the flow cytometer and cell fluorescence was measured. The samples were excited at 488 nm and emission were collected at 635/20-nm filter (FL2-A) respectively.
Appropriate gating of cell populations was performed to identify and determine healthy, early apoptotic, late apoptotic and dead cells from the total population of the cells. Cells treated with serum-free medium alone were considered as control and used to draw the initial quadrant.
Figure S2.14. DNA content frequency histograms of HeLa and BHK-21 cells in the absence and presence of compounds (A-F). Cells were treated with 40 µM of 2.2 (PIT- 1) and 2.5 (DM-PIT-1) that induce apoptosis. The cells were stained with PI, according to standard protocols, respectively. Fluorescence of the PI-stained cells was measured using CellQuest software (BD Biosciences). Histograms were plotted by the Flowing Software (Version 2.5.1). The software provided the estimate of the
(A) (B) (C)
(D) (E) (F)
percentage of cells with fractional DNA content (apoptotic cells: Sub G1) and cells in G0/G1, S, and G2/M phases of the cycle.
Table 2.5. The population of cells in the absence and presence of potent compounds.
Compound Cell G0/G1
(% of cells)
S (% of cells)
G2/M (% of cells)
Sub G1 (% of cells)
Control HeLa 60.34 7.59 19.13 4.03
BHK-21 61.48 8.87 15.30 5.91
2.2 (PIT-1)a HeLa 38.27 10.74 10.45 19.3
BHK-21 35.47 4.21 6.65 14.77
2.5 (DM- PIT-1)a
HeLa 35.75 4.64 12.79 4.95
BHK-21 29.49 5.83 5.18 10.18
aCompound concentration = 40 µM.
Figure S2.15. Cell viability of the compounds 2.1–2.7 was measured at a fixed concentration of 25 μM. Cell viability was measured in HeLa, after 48 h of compound treatment (A). Viability of 2.2 (PIT-1) and 2.5 (DM-PIT-1) was measured in BHK-21 cells after incubation for 48 hours (B). The viability of doxorubicin was measured in HeLa and BHK-21 cells after incubation for 48 hours. All MTT assays were performed in triplicates (C).
2.4.4.12.3. Chloride mediated cell death 2.4.4.12.3.1. HBSS assay
MTT assay was repeated using HBSS (Hank’s balanced salt solution) buffer in presence and absence of Cl─ ion. HeLa cells were maintained and prepared according to the above-mentioned procedure. HBSS buffer (with Cl─ and without Cl─) containing 10% FBS replaced cell culture media. The transporters 2.5 (DM-PIT-1) and 2.2 (PIT-1) were added to each well in different concentration and incubated for
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48 h. MTT containing media was removed from each well, and 100 µL of DMSO was added, dissolving the formazan crystals. The absorbance was recorded on a microplate reader (Multiskan™ GO) at the wavelength of 570 nm. All experiments were performed in triplicate, and the relative cell viability (%) was expressed as a percentage relative to the untreated cells. Hank's balanced salt solution with Cl─ ion has the following composition: 136.9 mM NaCl, 5.5 mMKCl, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 0.81 mM MgSO4, 1.25 mM CaCl2, 5.5 mM D-glucose, 4.2 mM NaHCO3 and 10 mM HEPES (pH 7.4) Hank's balanced salt solution without Cl─ ion was prepared using 136.9 mM Na-gluconate, 5.5 mM K-gluconate, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 0.81 mM MgSO4, 1.25 mM Ca-gluconate, 5.5 mM D-glucose, 4.2 mM NaHCO3
and 10 mM HEPES (pH 7.4).
2.4.4.12.2.2. Analysis of mitochondrial membrane potential
Analysis of mitochondrial membrane potential was done using 5,5,6,6’- tetrachloro-1,1’,3,3’ tetraethylbenzimidazoylcarbocyanine iodide (JC-1) dye. HeLa cells were seeded in 6 well plates at density not exceeding 106 cells/cm containing 10% fetal bovine serum (FBS) in Dulbecco's Modified Eagle Medium (DMEM) and incubated at 37°C in 5% CO2. After 16 h, media was discarded and each well was washed with phosphate-buffered saline (PBS) followed by addition of 2.2 (PIT-1) and. 2.5 (DM-PIT-1) to each well at 25 µM and 50 µM concentrations respectively with HBSS buffer containing Cl─. After 12 hours, 2 μM (final concentration) of JC-1 dye was added in each well and incubated at 37°C, 5% CO2 for 15-30 min. Cells were then washed with PBS and observed under fluorescence microscope (FLoid Cell Imaging Station, Thermo Fischer Scientific) for progressive loss of red J-aggregate fluorescence and cytoplasmic diffusion of green monomer fluorescence.
2.4.4.12.3. Immunoblot analysis
Cultured HeLa cells were grown on 6-well plate and treated with different concentrations of 2.5 (DM-PIT-1) and 2.2 (PIT-1). After 24 h, cells were washed with PBS. 100 µl of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) loading buffer was added to each well for immediate cell lysis and increased the viscosity of the sample. The extract was transferred to a microcentrifuge tube and heated at 95°C for 10 min. Total cell protein was separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were blocked in Phosphate-
buffered saline 5% dry milk for 2 h and incubated with primary antibody overnight at 4 ℃ with shaking. The membranes were washed several times with Phosphate- buffered saline/Tween-20 and incubated with horseradish peroxidase (HRP)- labeled secondary antibody for 1 h at room temperature with shaking. Band intensity was detected by ECL detection reagent (Biorad) with β-actin as an internal control.
2.4.4.13. Transportation of Chloride Ion across Giant Unilamellar Vesicles