Extraction of SAP
A. proylei J. cocoons were collected from Uyumpok Tasar Silk Farm, Imphal East, Manipur (24o 57’ 2.7396” N, 94o 2’ 55.2282” E), India. Five grams of fresh cocoon cut (~ 1 cm2 pieces) were added to 100 mL distilled water and subjected to heat treatment at 121°C under pressure for 1hr. The resulting suspension was filtered through Whatmann filter paper No.1 and centrifuged at 21,000g for 30 mins. The process was repeated for 2 times with the same cocoon shell sample. The supernatants obtained were pooled and then lyophilized. The lyophilized SAP powder was stored at -20°C until use.
HPLC analysis of predominant amino acids of SAP:
SAP was subjected to acid hydrolysis by dissolving in 6N HCl in boiling water bath for 24 hrs and mixed every hour for proper hydrolysis. It was then centrifuged at 3500 rpm for 15 mins. The supernatant was filtered and neutralized with 1N NaOH. The filtered solution was then diluted to 1:1000 of the volume with milli-Q water and then analyzed for amino acids in HPLC (Agilent 1100 HP), C18, 4.5 X 150, 5µm column using mobile phase A (20 mM sodium acetate + 0.018% triethyalmine, pH to 7.20 ± 0.05) and mobile phase B (20% of 100 mM sodium acetate + 40% methanol and + 40% acetonitrile, pH 7.20 ± 0.05). The flow rate was maintained at 0.5mL/min and the column temperature was kept at 40̊C and detected at 338nm.
Cell lines and culture conditions
Three human cancer cell lines, namely, lung cancer (A549), cervical cancer (HeLa), and prostate cancer (PC3) were obtained from the National Centre for Cell Science (NCCS), Pune, India. All the cell lines were cultured in RPMI 1640 (Gibco, USA) media with 10% FBS (Gibco, USA) and 1% PenStrep (Gibco, USA) and incubated with 5% CO2 at 37°C.
Cell treatment with SAP
SAP was dissolved in RPMI culture media and centrifuged at 15700g for 30 mins. The supernatant was taken and sterilized through a syringe filter (0.2 µm pore size) for the treatment to the above three cancer cells with different doses (final concentration; 0.17, 0.34, 0.7, 1.4, 2.7, 5.5, and 11µg/µL).
Cell viability assay
Cell viability assay was carried out as per the manufacturer’s protocol provided with MTT assay kit, Vybrant MTT assay Kit (Invitrogen Life Technologies). A549, HeLa or PC3 cells were cultured with a density of 1×104 cells per well in 100 µL RPMI (without phenol red), 10% FBS and incubated with 5% CO2 at 37oC in a 96-well tissue culture plate. Various doses of SAP were treated to the cells in triplicates. After 24 hrs, cell viability was assessed by adding 10μL of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to all the wells followed by incubation at 37oC for 4 hrs. The formazan crystals formed were dissolved by adding 50µL of DMSO after removal of the culture media and further incubated for 10 mins at room temperature. The numbers of viable cells were then quantified by measuring absorbance at 570 nm. The experiment was conducted in triplicates at least three times.
Comet assay was carried out according to the protocol described by Olive and Banath, 2006 . A549, HeLa, or PC3 cells were treated with various doses of SAP for 24hrs and cells were detached by trypsinization. The number of cells was adjusted at 2×104 cells/ml and suspended in PBS. About 400µl of the cell suspension was mixed with 1.2ml of low-melting agarose at 40oC by gentle pipetting followed by pouring onto agarose pre-coated microscope slides. After proper solidification of the agarose, the slides were submerged in a neutral lysis solution containing 2% SDS, 0.5 mg/ml Proteinase K, 0.5M EDTA and incubated at 37oC for 16 hrs in the dark. The slides were then washed three times with a neutral rinse buffer followed by electrophoresis at 0.6 V/cm for 25 mins. Slides were then stained with 10µg/ml propidium iodide (PI) and observed under a fluorescence microscope and photographed. The tail lengths of at least 50 comets in each slide were scored for analysis by Leica Application Suite (LAS, GmBH, Germany).
A549, HeLa or PC3 cells were seeded in a 60 mm culture dish, grown overnight, and treated with various doses of SAP. The cells were lysed using RIPA buffer and the protein concentration of the cell lysate was estimated using the BCA protein assay kit (Thermo Scientific) and 20 µg of proteins were separated by12% SDS-PAGE. After transferring the separated proteins on PVDF membrane, it was blocked with 5% (w/v) skimmed milk in 1X Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 hr at room temperature. The blocked membranes were then incubated with the primary antibodies (1:1000 dilution) with gentle shaking on a rocker at 4°C overnight. The membrane was probed with antibodies anti-PARP, anti-caspase 3 (both total and cleaved), anti-ERK, anti-p38 or anti-JNK antibodies (both total and phosphorylated) (Cell Signalling, USA). The membranes were then washed with 1X TBST three times for 10 mins each with changes of buffer. After washing the membrane, secondary antibodies conjugated with horseradish peroxidase (HRP) were added at 1:1000 dilutions and kept with gentle shaking for 1hr at room temperature. The membranes were re-blotted with an anti-ß actin antibody to normalize the total protein loaded. The blot was then developed using ECL (GE Amersham) and visualized under BioRad Gel Doc.
MAPK protein inhibition analysis
Cells cultured in 96 well plates were pre-treated with specific inhibitors for p38 (SB203580), SAPK/JNK (SP600125) or ERK (FR180205) proteins 1hr prior to SAP treatment. The treated cells were incubated at 37oC for 24 hrs after which cell survival was quantified by MTT assay. Statistically significance between cells treated with SAP and SAP along with specific inhibitors were determined by unpaired t-test. A p-value of less than 0.05 is considered statistically significant and rescued from apoptosis.
Cell cycle analysis
A549, HeLa, or PC3 cells were cultured in a 6-well culture plate, grown overnight, and treated with various doses of SAP. After 12 hrs, cells were harvested after trypsinization and washed with PBS. The cells were then fixed with ice-cold 70% ethanol added dropwise with regular vortexing to avoid cell clumps and kept at 4°C for 30 mins. The fixed cells were washed twice with PBS by centrifuging at 850 g for 5 mins. Then the cells were treated with 50 µg of RNaseA (100µg/mL) and 200 µL of propidium iodide (PI) (50µg/mL) was added. Cell cycle distribution was analyzed in a FACS flow cytometer (BD Biosciences, USA).
Significant variance between groups was performed for all groups with Independent t-test using Graph pad prism 6 Data were expressed as mean±SD. Multiple comparisons among groups were evaluated using Student’s t-test. Difference with p<0.05(95%CI) were considered statistically significant.