Chemicals, reagents and antibodies
The chemicals and reagents used in our experiments were: bafilomycin A1(Baf, Sigma, #19-148) chloroquine (CQ, Sigma, C6628), rapamycin (Sigma, #R8781) cathepsin B substrate, GFP-LC3 (plasmid, CST), and mTORC1 (WT plasmid, addgene #11546), DMSO (#D2650), 2′,7′-dichlorofluorescin diacetate (H2DCFDA) (Sigma, #35845), EDTA (Sigma, #E9884), Propidium iodide (Sigma, #P4170), Insulin-like growth factor (IGF-1, Sigma, #I3769), L-Leucine (Sigma, #L8000), acridine orange dye (Sigma, #235474), LysoTracker™ Red DND-99 (ThermoFisher, #L7528), Phosphate buffer saline (Sigma, PBS, #P5493), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, #M2128), phenylmethylsulfonyl fluoride (PMSF, Sigma, #P7626), Sodium orthovanadate (Sigma, #S6508), annexin V and propidium iodide (PI) (Santa Cruz Biotechnology, #sc-4252), fixer (Sigma, #P7167), developer (Kodak, Sigma, #DA163, #P6557), protease inhibitors cocktail (Sigma, #P8340), PVDF membrane (0.2 µm, BIO-RAD#162-0177), protein assay kit (BIO-RAD, #500-0002), chemiluminescences film (Bioscience, #28906035), protein ladder (BIO-RAD, #161-0374), Amersham™ Hypercassette™ (#RPN11644).
The antibodies used in our experiments were: Cell Signaling Techonology (CST): ATG7 (#8558P), p-mTOR (#2971P, #2974S), m-TOR(#2983S) PARP ( #9542SS), ATG5 (#8540S), p70S6K (#9205, #9234P), p4EBP (#9455P, #2855P), anti-p62 antibody (#5114, #8025S), ATG7 siRNA (#6604), ATG5 siRNA (#6345), Beclin-1 siRNA (#6222), beclin-1antibody (#3495S), Caspase-3 (#9662S) Sigma–Aldrich: Anti-LC3B antibody (#L7543), p62 (#P0067), Monoclonal anti-β-Actin antibody (#A3854), anti Rabbit IgG-peroxidase antibody (A9169) Santa Cruz Biotechnology: cathepsin D antibody (Santa Cruz, SC-6486), cathepsin L antibody (Santa Cruz, SC-6498), TFEB (H-125) SC-48784, LAMP1 (H4A3) SC-2001 Mouse monoclonal.
Cell culture
MIA PaCa-2 cells were obtained from the American Type Culture Collection (ATCC). Cell lines were maintained in DMEM (Sigma, D1152) containing 10% fetal bovine serum (FBS) in a standard cell incubation conditions—humidity around 95%, 5% CO2 at 37 °C.
MTT assay
MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay was used to check cell viability. MTT solution prepared in phosphate buffer saline (PBS) with the concentration (5 mg/mL) was incubated with pancreatic cancer cells treated with SDS-203 for 2 h before termination of the experiment.. Live cells change MTT to formazan crystals which were dissolved in dimethyl sulfoxide (DMSO) giving a purple color solution. Intensity of the color was measured by using a microplate reader (TECAN) with absorbance reading at 570 nm.
Flowcytometry
SDS-203 treated pancreatic cancer cells were incubated with various dyes and the cell population was analyzed using flowcytometry. Briefly, 0.2 × 104 MIA PaCa-2 cells were seeded in 6-well plates and later treated with SDS-203- in time (0, 6, 12, 24 h) or concentration-dependent manner (0, 5, 10, 20, 30 µM). Before termination of the experiment cells were stained with various dyes—Annexin V FITC/PI or DCFDA or Lysotracker red DND in incomplete media for 20 min each. Samples were later washed twice with PBS then trypsinized and resuspended for analysis through flowcytometry (BD FACS Calibur). 10,000 live events /sec were taken for data collection.
Protein isolation, quantification, western blotting and immunoprecipitation
At the end of the designated treatments, cells were lysed by RIPA or M2 lysis buffer and collected for protein estimation, each steps were done at 4 °C. Following that equal amount of protein from each samples were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane (Bio-Rad, 162-0177). Later PVDF membrane was blocked with 5% non-fat milk and cut into strips (based on molecular weight; using Bio-Rad protein standard 10–250 kDa) prior to hybridization with target primary antibodies and HRP+ tagged secondary antibodies. Western signaling was detected by using chemiluminescence Horseradish Peroxidase (HRP) substrates and the signals were captured on X-ray film. The density of the various bands in the western blot was quantified using ImageJ software.
Acridine orange staining
Acid vesicular organelles (AVO) formation is a characteristic feature of autophagy induction. To detect the formation of AVO, acridine orange was used. Accumulated AO in acid compartments gives bright red fluorescence (exc = 488 nm laser). 2 × 104 cells were seeded in 6 well plates and then treated with SDS-203 in a time-dependent manner (0, 6, 12, 24 h). 15 min before termination AO (1 µg/mL) was added, post staining cells were washed with PBS and taken for microscopy. Data were analyzed by using ImageJ software.
Transfection
GFP-LC3 (plasmid), TFEB siRNA and mTORC1 (WT plasmid) were used to transiently transfect pancreatic cancer cells, later SDS-203 was treated to these transfected cells. MIA PaCa-2 cells were seeded in 6 well plates and allowed to grow up to 70% confluency and then incubated with the plasmid along with transfection reagent, (Fugene kit based transfection reagent; Promega FuGENE HD Transfection Reagent #E2311) in an incomplete media for 14 h. Desired concentration of SDS-203 was later treated to the cells and its effect on the expression of the above-mentioned targets was calculated.
Immunofluorescence and confocal microscopy
Expression of various proteins like LC3-II, mTORC1, LAMP1, GFP-LC3 and intensity of lysotracker DND-red or DCFDA dye in pancreatic cancer cells upon SDS-203 treatment were analyzed by using fluorescence microscopy. 0.2 × 104 MIA PaCa-2 cells were allowed to grow on coverslips in 6-well plates, after selected treatment of SDS-203 for particular time points were either incubated with dyes for 15 min before termination or fixed with 4% paraformaldehyde (PFA) for 15 min then permeabilized with (0.02% of Triton-X-100 for 5 min) and later blocked by using blocking buffer (3%BSA + 0.01% Triton-X-100 in PBS for 1 h). Blocked samples were then incubated with primary antibody (mentioned dilution) overnight at 4 °C. Dye treated or GFP-LC3 transfected samples were washed and mounted on glass slides. While antibody incubated samples were washed and incubated with anti-Alexa Fluor secondary antibody for 2 h at room temperature, meanwhile samples were washed 3 times with PBS and mounted on glass slides. Mounted samples were then visualized under confocal microscopy. Ten different fields of each sample were taken and final results were taken as an average mean of each sample. Data interpretation was done by using ImageJ software
Electron microscopy
MIA PaCa-2 cells were seeded in complete DMEM high glucose and treated with 20 μM SDS-203 and rapamycin 200 nM for 24 h, and pelleted down at 1800 rpm for 10 min. Cell pellets were fixed with 2.5% glutaraldehyde at 4 °C for 1 h, then post-fixed with 1% OsO4 for 1 h. Samples were later dehydrated from lower to higher graded ethanol solutions and then embedded in Epon 81239. Semi-thin sections of samples were taken and stained with 1% toluidine aqueous blue at 40 °C and were observed with a Vanox light microscope (Olympus, Tokyo, Japan). Ultrathin sections of 60–90 nm were taken with an LKB ultramicrotome (LKB, Bromma, Sweden), which were collected on copper grids and stained with uranyl acetate and lead citrate. Stained sections were examined with a JEOL JEM 1400CXII electron microscope at 80 kV. To evaluate ultrastructural alterations, ~ 100 cells per sample were examined from two independent experiments.
Measurement of lysosomal enzyme (CTSB-CTSL) activity
Enzyme activity of lysosome was done by a nonradioactive method by using rhodamine-110 following protocol36,37. Rhodamine 110 (R110) is a sensitive and selective probe for assaying proteases in the cell lysate. This dye contains a fluorogenic substrate called Rhodamine. The substrate can be used to measure lysosomal enzyme activity in cell extracts or purified enzyme preparations using a fluorescence microplate reader. Treated cancer cell samples (SDS-203, NH4Cl or SDS-203 + NH4CL) were lysed by M2 buffer 20 mM Tris at pH 7, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 20 mM glycerol phosphate, 1 mM sodium vanadate and protease inhibitor cocktail for protein extraction. Protein estimation was done in order to incubate equal amount of (25 µg) protein from each sample with 50 µM of Rhodamine110, in 100 µL cell-free system buffer (10 mM HEPES–NaOH pH 7.4, 68 mM sucrose 220 mM mannitol, 2 mM NaCl 2.5 mM KH2PO4, 0.5 mM EGTA, 2 mM MgCl2, 5 mM pyruvate, 0.1 mM PMSF and 1 mM dithiothreitol) in 96 well plate for 1 h at 37 °C. Reading was taken at an exc/emi wavelength = 380/460 nm by using fluorometry (Tecan Spectra Fluor Plus). Activity was represented as a percentage of fluorescence intensity compared with the control group.
In vivo tumor mice model
The animals were housed under standard husbandry conditions: 24 ± 2 °C temperature, 15–20 complete fresh air changes per hour and 50–60% relative humidity as per guide for the care and use of laboratory animals. Animals were fed with a standard pellet diet (M/S Ashirwad Industries, Chandigarh, India) and autoclaved water was given ad libitum. Approval of the Institutional Animal Ethics Committee, CSIR-Indian Institute of Integrative Medicine, Jammu was sought for the study and number of animals used in all the experiments. SDS-203 was taken for in vivo anticancer assessment against murine tumor model and NH4Cl was taken as a negative control. Swiss albino mice (18–23 g) under optimum laboratory conditions were injected with Ehrlich Tumor cells (EAC), grafted from 8 to 10 days old ascites tumor-induced Swiss albino mice. On day zero 1 × 107 EAC cells were injected intraperitoneally, later tumor-induced animals were categorized randomly into four test groups, with seven animals per group. The first test group was administrated with normal saline (0.9% i.p.) which act as vehicle control. Another group was treated with SDS-203 (25 mg/kg i.p), and the remaining two groups were treated with or without SDS-203 in presence of NH4Cl (20 mg/kg i.p). Treatment was followed up to nine consecutive days; tumor assessment was done on day 12. Tumor measurements included size, weight and volume, and were taken from different groups. Some tumor samples from each group were frozen for protein extraction, which was later done by using tissue lysis buffer and homogenizer. All animal experimental procedures were carried out following the ethical guidelines for the use of animals in experiments and were conducted in compliance with the Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA) and the ARRIVE guidelines. All experiments were approved by the animal house CSIR IIIM. The use of experimental animals in this study was approved by the Ethics and Institutional Animal Care and Use, Committees of Council of Scientific and Industrial Research-Indian Institute of Integrative Medicine (CSIR-IIIM).
Ethical approval and consent to participate
The use of experimental animals in this study was approved by the Ethics and Institutional Animal Care and Use, Committees of Council of Scientific and Industrial Research-Indian Institute of Integrative Medicine (CSIR-IIIM) following guidelines of the Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA). I confirm that all methods were carried out “in accordance with relevant guidelines and regulations. I confirm that study was carried out in compliance with the ARRIVE guidelines.
Institutional publication number
CSIR-IIIM/IPR/00218.
