Reagents and chemicals
Benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (Z-VAD-FMK), BAPTA-AM were purchased from ALEXIS, (NY, USA). Praesodymium oxide (Pr6O11), cobalt chloride (CoCl2), ammonium persulfate (APS), necrotatin-1, 3-methyladenine (3-MA), wortmannin, pepstatin A, crystal violet, bromophenol blue, Tween 20, EGTA, HEPES, DTT, glycine, DMF, DMSO, Earle’s Balanced Salt Solution (EBSS), bafilomycin A1, chloroquine, staurosporine, rapamycin, nigericin, were purchased from Sigma Aldrich Co (St Louis, MO). Acrylamide/bis-acrylamide (30% solution), Coomassie Plus Protein Assay Reagent and Protease Inhibitor Cocktail were obtained from Pierce, (IL, USA). 10X PBS, 10X SDS, Tris-HCl buffer (pH7.4) were purchased from NUMI Media Preparation Facility (NUS, Singapore). Triton X-100 was obtained from USB, (OH, USA). Torin1 was purchased from Tocris Bioscience (Bristol, UK).
Synthesis and analysis of Pr-MPO
In a small flask Pr6O11 (102.1 mg, 0.1 mmol) was dissolved in HCl (6 M, 10 mL). The solution was neutralized with a solution of NaOH (1 M) to pH = 5. A solution of MPO-Na (282.3 mg, 1.8 mmol, 95% active) in water was added. The solution was stirred for 2 h at 40oC and then was cooled to room temperature to give green microcrystals, which were collected by filtration, washed successively with H2O, EtOH, and dried in vacuo. Pale green prism crystals suitable for X-ray analysis were obtained by recrystallization of the precipitate from dimethyl sulfoxide. Analysis revealed: C, 33.81; H, 3.62; N, 6.28%. Calc. for C19H24N3O5S5 Pr: C, 33.78; H, 3.58; N, 6.22%); IR (cm–1): 1141(s, C S), 1090 (s, N O); UV-vis (DMSO): 290, 346 nm. Crystal Data : M. Formula = C19H24N3O5S5Pr, M W = 675.62; Crystal system : Triclinic, Space group P¯1; Unit cell dimensions (4148 a = D 9:570(2)°A; reflections in full µ range) b = D 9:902(2)°A, c = D 15:743(3)°A, Alpha = D 89:00(3)±, Beta = D85:54(3)±, Gamma = D 62:97(3)± Final R indices [I > 2gI )] R1 = D 0:0352, wR2 = D 0:0706.
Cell lines and cell culture
The human cervical carcinoma Hela cell line was purchased from American Type Culture Collection (ATCC, MD, USA), and cultured in Dulbecco’s Modified Eagle Medium (DMEM, GE Healthcare Life Science, Utah, USA) supplemented with 1% L-glutamine, 1% penicillin/streptomycin (Hyclone, Thermo Fisher Scientific, MA, USA) and 10% fetal bovine serum (FBS, Hyclone, CA, USA). Hela cell line stably expressing a FRET probe fused with a Caspase 3 recognition motif (DEVD-FRET) was a generous gift from Dr. Qian Luo (Nanyang Technological University, Singapore). Human breast cancer cell lines, MDA-MB-231 and MCF-7 were purchased from American Type Culture Collection (ATCC, Rockville, MD) and maintained in Roswell Park Memorial Institute 1640 medium (RPMI, Hyclone, Utah, USA) supplemented with 1% L-glutamine, 1% penicillin/streptomycin and 10% FBS. Mouse embryonic fibroblasts WT MEF and Atg5 KO MEF cell lines were generously granted by Dr. Noboru Mizushima (The University of Tokyo, Graduate School and Faculty of Medicine, Tokyo, Japan) and maintained in DMEM medium supplemented with 1% L-glutamine, 1% penicillin/streptomycin and 10% FBS. Human colorectal carcinoma HCT116 WT, HCT116 p53 KO and HCT116 Bax/Bak DKO cell lines were generously provided by Dr. Bert Vogelstein (The Johns Hopkins University School of Medicine, Baltimore, MD) and maintained in McCoy5A (Gibco® Invitrogen Corporation, Carlsbad, CA) supplemented with 1% L-glutamine and 1% penicillin/streptomycin and 10% FBS. All cell lines were maintained in a humidified incubator at 37 °C with 5% CO2 and propagated according to suppliers’ recommendations.
Xenograft animal model
Tumor inoculation was performed by injecting human prostate cancer cells DU145 subcutaneously into female BALB/c nude mice (n = 5)/group as per the approved guidelines of the Institutional Animal Care and Use Committee (IACUC). When tumors reached a mean volume of 50 mm3, Pr-MPO (5, 10, 20 mg/kg) was administered intraperitoneally 3-times a week for a total duration of 21 days, whilst the control group of mice only received solvent (DMF) injections. Tumor volume and body weight of mice were measured and recorded throughout the duration of the study. At the end of the experiment, mice were euthanized and tumors were collected for comparison between groups.
Crystal violet staining and small interference RNA gene silencing
The cytotoxicity effects of Pr-MPO and various pharmacological reagents or stimuli were determined by staining the treated cells with crystal violet and measuring the viable cell count by correlating with the absorbance values. Hela cells (1 × 105/ well) were seeded in triplicates in a 24 well plate one day prior to drug treatment. At the end point of drug treatment, culture medium was carefully discarded and cells were washed once with 1x PBS, stained with 200 µl of 0.1% crystal violet solution (prepared in 10% methanol) per well and incubated on the shaker for 10 min at room temperature. Subsequently, crystal violet solution was disposed into a cytotoxic waste container and cell plate was gently washed with deionized water until the water no longer ran dark. The plate was drained by inverting a couple of times on a tissue paper, followed by the addition of 500 µl of 1% SDS solution (prepared in 1x PBS) into each well to solubilize the crystals. Absorbance was measured with a TECAN Spectrophotometer (Tecan Trading AG, Switzerland) at 570 nm. Percentage of cell viability was calculated relative to the untreated control cells (presented as 100%). At least three independent experiment were performed in triplicates to obtain statistical significance. For gene silencing, ON-TARGETplus SMARTpool siRNA (pool of 4 single siRNAs) targeting human Atg5, Atg7, RIP1 or RIP3 as well as a scrambled siRNA control were purchased from Dharmacon Technologies (ThermoFisher Scientific, Lafayette, CO, USA). Gene specific siRNA was introduced into cells using DharmaFECT1 reagent (ThermoFisher Scientific, Lafayette, CO, USA) according to the manufacturer’s instructions. Briefly, HeLa cells to be transfected were grown to 40% confluence in 6-well plates on the day of transfection. Each siRNA was diluted with 250µL of Opti-MEM (Gibco, ThermoScientific, MA, USA) to attain a final concentration of 50nM; in another tube 5µL DharmaFECT was mixed with 250µL of Opti-MEM. Both solutions were gently mixed and incubated for 5 min at RT. The two solutions were combined by gentle pipetting and the resultant complex with a total of 500µL was incubated for 20 min at RT. Finally, 500µL of siRNA complex was carefully added into the wells and gently mixed by rocking the plates back and forth.
Colony forming assay
At the end point of drug treatment, Hela cells were washed with 1X PBS, trypsinized and counted. 10,000 cells were re-plated onto a petri-dish containing completed DMEM medium and kept in the incubator to allow colony formation over a period of 14 days. The culture medium was changed periodically. Cells were stained with crystal violet solution to visualize colonies; colony number and size were determined using the image analysis software Image J (http://imagej.nih.gov/ij/) to assess colony forming capabilities of individual sample. Only populations containing more than 20 cells were considered as colony units.
Analysis of cell viability using propidium iodide
Necrotic cells are usually defined by lack of integrity of plasma membrane, which can be labelled with propidium iodide (PI) staining and measured by flow cytometry. 2.5 × 105 Hela cells were harvested by trypsinization after periodic exposure to drugs. After washing twice with cold 1X PBS, cells were stained with 1 µM PI solution (prepared in cold 1x PBS) and filtered before flow cytometry (CyAn ADP, Beckman Coulter, USA). 10,000 events were collected with the excitation set at 488 nm and emission at 610 nm. Data were collected and analysed with WinMDI software (Windows multiple document interface for flow cytometry, Beckman Coulter Inc., Sunnyvale, CA). Dot plots with a gate drawn around a population of dead cells were adopted to present the results.
Detection of Caspase 3 activity
Caspase 3 activity assay was performed with a Hela stable cell line constitutively expressing caspase3-specific FRET biosensor (ECFP-DEVD-EYFP) to track apoptosis induction. Cells were plated onto a 48-well plate and incubated with indicated doses of Pr-MPO for 18 h. After treatment, cells were washed with 1x PBS and subjected to TECAN spectrophotometer for fluorescence readout detection. The final relative fluorescence units (RFU) was normalized against protein concentration of samples. The plotted X-fold decrease in RFU was inversely proportional to the increase in caspase 3 activity.
SDS-PAGE and Western blot analysis
Whole cell lysate was extracted with 0.1% Triton X-100 cell lysis buffer containing protease and phosphatase inhibitors. Protein concentration was quantified and equal amount of protein (30-40 mg) was prepared with 5x Laemmli loading dye and boiled at 100 °C for 5 min. Denatured protein samples were then subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with 8, 10, 12 or 15% (v/v) of acrylamide resolving gel depending on the molecular weight. Kaleidoscope pre-stained standards (Bio-Rad, CA, USA) were used to indicate the molecular sizes of the resolved protein bands. Subsequently, proteins separated on the gel were transferred onto PVDF membrane (Bio-Rad, CA, USA) using the wet transfer method at 350 mA for 1 h in an ice bath. Transferred membrane was then blocked with blocking buffer for 1 h on the shaker. Membrane was washed 3-times with 1x TBST before probing for the target proteins with correspondent primary antibodies at 4 °C overnight. On the following day, membrane was washed 3-times (10 min each time) with 1X TBST to avoid non-specific binding with excess primary antibodies and probed with appropriate HRP-conjugated secondary antibody solutions for 1 h at room temperature. For signal visualization, after three washes, membrane was incubated with Chemiluminescent Plus Western Blot Enhancing Kit (Merk Millipore, Darmstadt, Germany) and developed with X-ray film (Medical X-ray Processor, Kodak, NY, USA). Primary antibodies specific for PARP, Caspase 3, Bax, LC3B, phospho-mTOR, mTOR, phospho-4EBP1, 4EBP1, phospho-S6K, S6K, Atg5 and Atg7 were purchased from Cell Signaling Technology (Danvers, MA); QSTM1/p62, Bcl-2, β-actin and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HRP-conjugated polyclonal secondary antibodies for anti-rabbit and anti-mouse were purchased from Thermo Fischer Scientific (Wilmington, DE, USA).
Transmission electron microscopy
Hela cells were seeded in a 4-well glass bottom chamber and incubated with 5µM Pr-MPO at indicated time points, followed by fixation for 2 h in 0.1 M Sorensen’s Phosphate buffer (Na2HPO4 ∙ 2H2O 35.61 g dissolved in 1 L dH2O, pH 7.4) containing 2.5% glutaraldehyde (EM grade) at room temperature. The specimen was washed 3-times with PBS and incubated in Sorensen’s Phosphate buffer overnight at 4 °C. Specimens were then embedded in plastic, sectioned, and stained with uranyl acetate and lead citrate. Thin sections were imaged on a JEOL 1230 EX transmission electron microscope. Images were acquired by Gatan ES500W Erlangshen camera with 1350 × 1040 pixels.
Live cells immunofluorescence microscopic analysis
HeLa cells (25,000 cells per well) were seeded onto a 4-well glass bottom chamber (Lab-Tek® Chamber Slides, Thermo Scientific Nunc, Wilmington, DE, USA) and transfected with green fluorescent protein conjugated LC3B (LC3B-GFP). 24 h post-transfection, cells were treated with 5 µM Pr-MPO for 3 and 6 h followed by three washes with 1x PBS. Subsequently, cells were fixed with paraformaldehyde (4% w/v) for 30 min and permeabilized with 0.5% Triton X-100 solution for 5 min at room temperature. To remove the remaining fixative thoroughly, cells were washed with 1X PBS for 3-times at each interval of 10 min and subjected to FluoView FV10i confocal microscopy system (Olympus, Hamburg, Germany). The fluorescence of GFP was excited by 488 nm spectral line of the argon-ion laser. Fluorescent images were acquired and analysed with Image J software.
Live-cell confocal imaging for intracellular calcium measurement
Time-lapse confocal microscopy experiment was performed on an inverted Eclipse TE2000-E microscope (Nikon, Melville, NY) equipped with a spinning-disk confocal scan head (CSU-10; Yokogawa, Tokyo, Japan), an autofocusing system (PFS; Nikon), and a temperature/CO2-controlled automated stage. Images were acquired with a Cool SNAP HQ2 charge-coupled device camera (Photometrics, Tucson, AZ) driven by MetaMorph 7.6 (Molecular Devices, Sunnyvale, CA). For short-term imaging (up to 10 min) cells were imaged in complete DMEM medium at 37 °C supplied with 5% CO2.
Fluo4-AM is a cell permeable, green-fluorescent calcium indicator for detecting intracellular calcium. Fluo 4-AM stock solution was prepared with Pluronic F-127 20% (w/v) solution in DMSO to a concentration of 2 mM. BCECF-AM is a cell permeable, red-fluorescent pH sensor for detecting intracellular H+. BCECF-AM stock solution was prepared in DMSO to a concentration of 2 mM. Calcium Green-1 dextran (Potassium Salt, 10,000 MW) is a cell impermeable, green-fluorescent calcium indicator. Stock solution was prepared by dissolving it with dH2O to a final concentration of 10 mg/ml. Texas Red dextran (Potassium Salt, 7,000 MW) is a cell impermeable, calcium-insensitive, red fluorescence probe. Texas Red stock solution was prepared with dH2O to a final concentration of 5 mg/ml. 10 mg/ml stock solution of Oregon Green dextran (10,000 MW), a hydrophilic green fluorescent probe for detecting H+, was prepared with dH2O. MetaMorph time series images (stacks) were analysed with Image J software. Fluorescence intensity within the chosen ROI (the entire cell or several areas within the cells) was analyzed over the whole series of fluorescent images.
Intracellular pH measurement
pH calibration is a crucial determinant ensuring an accurate and quantitative outcome in the detection of intracellular pH. The simplest way to calibrate pH is to employ a series of buffers of different pH values, with which a standard pH calibration curve is generated. To do so, pH calibration buffer comprising 25 mM Na2HPO4 and 25 mM KH2PO4 dissolved in distilled water was prepared. Calibration buffer of a series of pH values ranging from 6.4, 6.8, 7.2, 7.6, 8.0 were adjusted with HCl or NaOH. Hela cells (7,500 cells) were seeded onto 48-well plate 18–24 h before the measurement of intracellular pH. On the day of the experiment, cells in triplicates were incubated with 5 µM Pr-MPO for a short duration of 2, 5–10 min. Treated cells were then washed 3-times with 1X HBSS buffer and loaded with fluorescence pH indicator solution containing 2µM of BCECF-AM at room temperature for 30–45 min. Cells were washed with 1X HBSS buffer thoroughly to remove the remnant dye and the emission fluorescence representative of intracellular H+ concentration was measured using excitation λ of 485 nm and emission λ of 535 nm. Cell samples designated for pH calibration were incubated in buffers of different pH values and followed the same dye loading procedure as mentioned above. Before measurement of emission fluorescence, 10 µM of nigericin was added to equilibrate intracellular pH with the extracellular buffer pH.
GAA activity assay
Treated cells were harvested, washed twice in ice cold PBS and pelleted at 1000 g for 5 min. Cell pellet was resuspended in 150–250 µL Sodium Acetate 0.1 M, pH 4.0 and homogenized by pipetting up and down before leaving on ice for 30 min with two to three quick vortex cycles during the incubation. The solution was then centrifuged for 10 min at 16,000 g, 4 °C to remove all debris. The collected supernatant was used to measure protein concentration in the sample using the BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA). The volume of sample tested (2 to 5 µg of proteins per reaction) was loaded in 96-wells plates and the volume was then adjusted to 48.5 µL before adding 1.5 µL of 4-methylumbelliferyl α-D-glucopyranoside 100 mM. The plate was incubated at 37 °C from 0 to 6 h. Before each measurement, 117 µL of glycine 0.4 M, pH 10.8 was added to stop the reactions and induce fluorescence of 4-methylumbelliferone released by the enzymatic activity of GAA. Fluorescence was measured on a Tecan M200 plate reader (Ex λ of 365 nm; Em λ of 450 nm). A standard curve of known quantities of 4-methylumbelliferone was used to determine enzymatic activity in the samples.
Metabolomics
The pellet of treated cells was resuspended with 300 µL of ice-cold deionized water containing 0.6% formic acid. A 30 µL aliquot was collected for protein content measurement. A volume of 270 µL of acetonitrile was added to the mix and vortexed before storage at -80° C. Samples were shipped and the organic acid panel was measured by Duke-NUS Metabolomics Facility (Duke-NUS Graduate Medical School, Singapore) as per their established protocols.
Measurement of glucose content, NAD+/NADH and lactate
Measurements of total glucose, free glucose and glycogen were performed using the Glycogen Colorimetric/Fluorometric Assay Kit (BioVision, Waltham, MA, USA). Measurement of NAD+/NADH was performed using the NAD+/NADH Quantification Colorimetric Kit (BioVision, Waltham, MA, USA). Measurement of lactate content was performed using the Lactate Colorimetric/Fluorometric Assay Kit (BioVision, Waltham, MA, USA). All measurements were done following the manufacturer’s instructions.
Statistical analysis
Data are presented as mean ± SEM where n corresponds to at least three independent experiments unless stated otherwise. Statistical significance was determined using one-way ANOVA followed by a post hoc Dunnett multiple comparison test, and two-tailed unpaired student’s t-test as indicated in the figure legends with p < 0.05 considered statistically significant.
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