Cell culture

AML cell line, namely, U937 was obtained from American Type Culture Collection. The cells were maintained in suspension in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) in a humidified incubator at 37°C and 5% CO2. The cells were split every 3 days at a ratio of 1:2. Before any experiment, 10 µL of the cells was mixed with 10 µL of trypan blue and counted using a hemocytometer to check cell viability.

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Drug preparation

TP (5 μM) and andrographolide (1000 μM) stock solutions were prepared by dissolving the compounds in dimethyl sulfoxide (DMSO) and then diluting them with RPMI on the day of treatment. The different working concentrations of the compounds (0.05–50 μM TP and 0.25–25 μM andrographolide) were obtained by serial dilutions of the DMSO stock solutions using RPMI. The concentration of DMSO to which the cells were exposed was always <0.5% (v/v).

Cell proliferation

The cells were plated in a 96-well plate at a density of 1×105 cells/well for 24 h. Different concentrations of TP and andrographolide were applied either separately or in combination. Co-treatment was performed by adding the two compounds simultaneously on cells for 24 h. Pretreatment was performed by exposing the cells to andrographolide for 24 h followed by TP treatment for another 24 h. RPMI alone was added to the control cells. WST-1 (Roche) and XTT (Sigma Aldrich) reagents were used, according to the manufacturer’s guide (10 μL of WST-1 reagent was added to each well while 40 μL of XTT was added to each well), to detect cell proliferation. Cell proliferation was assessed by recording the absorbance at a wavelength of 450 nm (using a Biotek ELx808 ELISA reader), which reflects the amount of formazan dye produced by metabolically active cells.

Cell-cycle analysis

Cells were seeded in six-well plates at a density of 0.5×105 cells/well. After incubation for 24 h and treatment with various compounds separately and in combination, the cells were fixed with ethanol, followed by propidium iodide (PI) staining. Cell DNA content was assessed by flow cytometry using Accuri C6 flow cytometer. The distribution of the cells into their respective cell-cycle phases was based on their DNA content: sub-G0/G1 (Pre-G) cells were <2n, G0/G1 cells were 2n, S cells were >2n but <4n, whereas M phase cells were 4n. Cell death was determined by an increase in the percentage of cells in the pre-G phase as compared to the control.

Apoptosis detection

Cells were plated in six-well plates at a density of 0.5×105 cells/well and incubated for 24 h. After treatment, Annexin V/PI staining was performed to detect and analyze apoptosis using flow cytometry. Cells were collected and centrifuged at 1500 rpm at 4°C for 5 min. The pellet was suspended in 500 µL suspension buffer, in addition to 5 µL Annexin and 5 µL PI (Annexin V–fluorescein isothiocyanate [FITC] Apoptosis Detection Kit, Abcam, Cambridge, UK) and immediately analyzed by the flow cytometer. Annexin V is a Ca2+-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is translocated from the cytoplasmic surface to the outer leaflet of the cell membrane upon apoptosis. The cell membrane is impermeable to PI and hence PI is excluded from living cells. Cells that are stained negative for FITC–Annexin V and negative for PI are considered living cells. Cells that are stained positive for both FITC-Annexin V and PI are either at the end stage of apoptosis, undergoing necrosis, or are already dead.

Western blots

Cells were plated in six-well plates at a density of 106 cells/mL to extract total proteins using the Q-proteome Mammalian Protein kit. Proteins were quantified using Lowry assay. Western blot analysis was done to measure the protein expression of p53, p21, Bax, Bcl2, cytochrome c, PARP, and caspase-3 and -9.

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins were separated on 10% gels and transferred to polyvinylidene difluoride membranes at 0.25 mA for 75 min. The membranes were then blocked with 5% skimmed dry milk in PBS containing 0.05% Tween 20 for a night at 4°C. Membranes were then incubated with primary antibody using anti-β-actin, anti-p53, anti-p21, anti-Bax, anti-bcl2, anti-caspase-3 and anti-caspase-9 antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) at a concentration of 1:500. After washing the membranes for 1 h using 1× PBS with 0.5% Tween 20, they were incubated with secondary antibody (obtained from Promega, Madison, WI, USA) at a concentration of 1:2500 for 1 h at room temperature. The membranes were then washed and the development was done using Western blotting chemiluminescent reagent enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA) He and figures were taken using ChemiDoc XRS+ machine (Bio-Rad, Hercules, CA, USA).

Statistical analysis

All the experiments were carried out in triplicate and each experiment was repeated three times. The results were reported as mean value±SD. The analysis was done using two-way analysis of variance. The level of significance upon comparing control versus treatment was set at P<0.05.