Chemicals & reagents

Dulbecco’s Modified Eagle’s Medium (DMEM)- cell culture media, antibiotic-antimycotic mix, sodium pyruvate, non-essential amino acid mix and stable glutamine were purchased from Himedia (Mumbai, India); fetal bovine serum (FBS) was purchased from (GIBCO, Invitro-gen USA), 3-[4, 5-dimethyltiazol-2-yl]-2.5-diphenyl-tetrazolium bromide (MTT), Dimethylsulfoxide (DMSO), Propidium Iodide, Ribonuclease A, Dithiothreitol (DTT), 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), Ethylene diamine tetra acetic acid (EDTA), Phenyl methyl sulfoxide (PMSF), Crystal-violet, Sodium dodecyl sulphate (SDS) and 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) ( HEPES) were purchased from Sigma-Aldrich (St Louis, MO, USA). The fluorogenic proteasomal peptide substrates Suc-LLVY-AMC (chymotrypsin-like substrate), BOC-Leu-Arg-Arg-AMC (trypsin -like substrate) and Z-Leu-Leu-Glu-AMC (caspase- like substrate) and MG-132 (carbobenzoxy-Leu-Leu-leucinal - a specific inhibitor of the 26S proteasome) were procured from ENZO Life sciences, USA. 20S rabbit proteasome was purchased from Boston Biochem, USA. All other reagents were procured from Qualigens fine chemicals (Mumbai, India). Annexin staining was done using a kit (Annexin V-FITC Apoptosis detection kit; BD Pharmingen, San Jose, CA, USA).

Preparation of curry leaf extracts

Curry leaves were collected from the local area from a single tree. Identity of the curry leaves was confirmed by Dr. B. Pratibha Devi, Professor and Head, Department of Botany, Osmania University, Hyderabad, India. A voucher specimen (voucher no: 068) was deposited in a herbarium at the Department of Botany, Osmania University, Hyderabad, India. The leaves were washed and air dried in shade for 3 weeks. After drying, the leaves were ground to a fine powder using an electric mixer grinder. The leaf powder was extracted with 80% methanol in water by keeping on a vortex mixer for 3-4days. This was followed by centrifugation of the extract at 5000 rpm for 30 min. The supernatant was filtered using a 0.4 μm filter (Millipore). The resultant Methanol:Water extract was stored at −20°C and was used for all our studies. These extracts designated as ‘CLE’, were used in the cell-culture assays at different doses based on their total phenolic content [equivalent to μg of gallic acid (GAE)] measured spectrophotometrically by the Folin-Ciocalteau method.

Total Phenolic Content (TPC)

The total phenolic content of the extract was determined with Folin–Ciocalteau’s reagent using Gallic acid as a standard [35]. Different concentrations of Gallic acid standards (20-100 μg/μl) and CLE samples were taken in glass test tubes and volume was made up to 150 μl with distilled water. 750 μl of 10% Folin’s reagent was added and kept for 5 minutes at room temperature, followed by addition of 750 μl of 6% Na 2 CO 3 and vortexed for 5 minutes. The tubes were then incubated for 90 minutes at room temperature. The absorbance was measured at 725 nm in a Hitachi double beam spectrophotometer. The final concentration of the total polyphenols present in the extract was expressed as μg of G allic A cid E quivalents (GAEs).

Cell lines

MCF-7 and MDA-MB-231 (breast carcinoma cell lines) and WI-38 (normal human lung fibroblasts) were obtained from the National Centre for Cell Sciences, Pune, India. Cells were maintained in DMEM supplemented with 10% FBS, 2 mM Higluta-XL, 100 units/ml penicillin, 100 μg/ml streptomycin and 0.5 ng/ml amphotericin B, 1 mM sodium pyruvate and 1X non-essential amino acid mixture. Cells were maintained and grown in a humidified atmosphere at 37°C and 5% CO 2 . WI-38 cells were grown for no more than 30 passages, as recommended by European Collection of Cell Cultures (ECACC).

Cell viability/proliferation assay

Cell viability was determined by quantification of 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) reduction by mitochondrial dehydrogenases. In brief, 1 × 105 cells/well were plated in a 96-well plate and incubated with different concentrations of the CLE for either 12 h or 24 h or with MG-132 for 24 h. Following this, MTT was added to a final concentration of 100 μg/well and further incubated for 3 h at 37°C. The formazan dye crystals formed were solubilized in DMSO and the plate was incubated at room temperature for 1 h. The absorbance was measured at 595 nm in an ELISA microplate reader (Biotek, New York, USA). All samples were assayed in triplicate in three independent experiments. Absorbance values plotted are the mean from three independent experiments and the results are expressed as percentage of the control, which was considered to be 100%.

Colony formation assay

MCF-7 and MDA-MB-231 cells were plated in duplicate at a density of 1 × 104 and 1 × 103 cells/ml respectively in 6-well plates. Next day, the cells were treated with varying concentrations of CLE. Plates were incubated at 37°C and 5% CO 2 for one week. After a week, the colonies were fixed with 4% formaldehyde for 15mins followed by staining with 0.005% crystal violet. The colonies were photographed with a digital Nikon D90 camera. Three independent experiments were done with each cell line.

Cell cycle analysis and annexin V binding assay

Cell cycle analysis: MCF-7 or MDA-MB-231 or WI-38 cells were plated at a density of 1.5 × 106 cells in a 10 cm dish. Next day, cells were treated with different doses of the CLE. After 24 h incubation, cells were harvested and suspended in 1X-PBS containing 2% FBS. The cells were fixed with 70% ethanol at 4°C for 1 h followed by the addition of propidium iodide (5 μg/ μl) and RNase (10 μg/ μl) and further incubated for 3 h at 4°C. The DNA content was evaluated in a flow cytometer (BD FACS ARIAII, USA). The data was analyzed using Modfit software (BD Biosciences, USA).

Annexin-V staining: MCF-7 or MDA-MB-231 cells were plated at a density of 1.5 × 106 cells in a 10 cm dish. Next day, cells were treated with different doses of the CLE. After 24 h incubation, cells were washed with 1X-PBS and re-suspended in 100μl binding buffer (supplied by the vendor). Cells were stained with Annexin V-FITC and propidium iodide according to the manufacturer’s protocol before analysis by flow cytometry.

Inhibition of purified 20S proteasome activity

Chymotrypsin-like activity of the purified 20S proteasome was measured as follows: In brief, 200 ng of purified 20S proteasome was incubated in 200μl of assay buffer (50 mmol/L Tris–HCl, pH 8.0 containing 0.035% SDS) with or without different concentrations of CLE and 40 μM substrate Suc-Leu-Leu-Val-Tyr-AMC (for chymotrypsin-like activity) and incubated for 2h at 37°C. The free 7-amino-4-methylcoumarin (AMC) liberated was measured fluorimetrically using a multi-mode reader [Spectra Max M5] using an excitation filter (380 nm) and emission filter (460 nm). The data is plotted as mean (+/− standard error) and is expressed as a percentage of the control, which was considered to be 100%. The assay was repeated thrice.

Inhibition of 26S proteasome activity in intact cells

To measure inhibition of the proteasome activity in living tumor cells, MCF-7 or MDA-MB-231 or WI-38 cells were plated at a density of 1 × 104 in a 24 well plate. Next day, cells were treated with or without the CLE at the indicated concentrations. After 24 h of treatment, the media was aspirated out and 500 μl of 1X-PBS was added followed by addition of fluorogenic substrates (20 μM final concentration) specific for the chymotrypsin-like (Ch-L), trypsin-like (T-L) and caspase-like (Cp-L) activities of the 20S proteasome. The plate was then incubated for 2 h at 37°C. 200 μl of the 1X- PBS was then transferred into a black plate and the free 7-amino-4-methylcoumarin (AMC) liberated was measured fluorimetrically in a multi-mode reader [Spectra Max M5] at excitation (380 nm) and emission (460 nm). The results are displayed as mean (+/− standard error) and are expressed as a percentage of the control, which was considered to be 100%. All samples were assayed in triplicate in three independent experiments.

Inhibition of 26S proteasome activity in cell extracts

MCF-7 or MDA-MB-231 cells (1 × 107) were harvested, washed twice in 1X-PBS and re-suspended in 1 ml ATP/DTT lysis buffer [10 mmol/L Tris–HCl (pH 7.8), 5 mmol/L ATP, 0.5 mmol/l DTT, 5 mmol/L MgCl 2 ]. Cells were incubated on ice for 10 minutes, followed by sonication for 15 seconds. The lysate was centrifuged at 2000 rpm for 10 minutes at 4°C. The supernatant enriched in 26S proteasomes is the cell extract which was used for the assay. This was mixed with glycerol (20% final concentration), aliquoted and stored at −80°C, and was stable for at least 1 month. The total protein content of the cell extract was estimated by the Bicinchoninic Acid (BCA) method using a kit (Bangalore Genei, Bangalore, India). The assay was carried out in a total of 200 μl reaction volume containing proteasome extract (50 μg protein), 50 mM EDTA, varying concentrations of the CLE/MG-132 and 50 μM of the proteasomal fluorogenic substrates and incubated for 2 h at 37°C. The amount of free 7-amino-4-methylcoumarin (AMC) liberated was measured fluorimetrically. The results are expressed as mean (+/− standard error) as a percentage of the control, which was considered to be 100%. All samples were assayed in triplicate in three independent experiments.

Statistical analysis

All experiments were performed in triplicates and repeated at least three times and the data are presented as mean+/− SEM. Mean values were compared across concentrations of CLE using non-parametric test of Kruskal-Wallis one-way ANOVA for each cell line using the SPSS statistical software. Differences between groups were considered significant at α (probability) level of </= 0.05.