MCF-7, MCF-10A, and T47D cells were a generous gift from Andrea Bild at the University of Utah; MDA-MB-231 cells were obtained directly from the American Type Culture Collection (ATCC). All cell lines were authenticated within six months of manuscript preparation by the ATCC in conjunction with Promega (Madison, WI, USA) using short tandem repeat analysis.
MCF-10A and MCF-7 cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM)/F12 media containing 2.5 mM L-glutamine and 15 mM HEPES buffer (Life Technologies, Grand Island, NY, USA). MDA-MB-231 and T47D cells were cultured with RPMI-1640 medium containing 2.5 mM L-glutamine and 25 mM HEPES buffer (Life Technologies). All cells were cultured at 37°C with 5% CO2. For standard culture conditions, medias were supplemented with 10% fetal bovine serum (FBS) (heat inactivated, HyClone, Logan, UT, USA) penicillin-streptomycin-glutamine, 5.0 μg/mL of insulin-transferrin-selenium-X (ITS-X) (Life Technologies), and 2.5 nM epidermal growth factor (EGF), recombinant human (BD Biosciences, San Jose, CA, USA). In the case of treatment with C-6 or its analogs, low serum medias were used (2% fetal bovine serum).
Reagents and antibodies
Preparation procedures and characterization data for C-6 and its analogs can be found in a supplementary file (Additional file 1). The following primary antibodies were purchased from Cell Signaling (Danvers, MA, USA): CHOP, GRP78, p-EIF2α, pan-EIF2α, p-JNK, and pan-JNK. In addition, the vinculin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA). Actinomycin D (ACTD) and cycloheximide (CHX) were also obtained from Sigma-Aldrich.
Dose response assays
All cells were seeded in clear 96-well plates (Costar, Tewksbury, MA, USA) in 100 μL of their respective media at densities necessary to achieve 90% confluency at the end of the 5-day assay. Dimethyl sulfoxide (DMSO) stocks of the compounds were diluted in their corresponding media containing 2% FBS and an EP Motion 5075 (Eppendorf North America, Hauppauge, NY, USA) liquid handler was utilized to prepare serial dilutions; a vehicle control corresponding to the highest DMSO concentration, which did not exceed 0.2% (v/v), was also prepared. Following an overnight incubation, the media was aspirated and the cells were treated with the serially diluted compounds and vehicle control. Every 48 hours, the drug-containing media was refreshed. Following 5 days of treatment, cell viability was measured using a CellTiter 96 AQueous One Solution Cell Proliferation assay per the manufacturer’s protocol (Promega). Blank-subtracted absorbance values were obtained at 490 nm and normalized to the DMSO vehicle control wells. Normalized values were plotted as an average ± standard deviation of three replicates then analyzed using the dose-response nonlinear curve fitting function with Prism 6.0 (GraphPad Software, San Diego, CA, USA) to establish the EC50.
In preparation for transcriptome sequencing, MCF-7 cells were seeded in triplicate in 10-cm tissue culture-treated plates such that the plate would be 80% confluent at the time of drug treatment. Cells were treated with 10 mL of low serum media containing either 30 μM C-6 or DMSO as a vehicle control for 3 hours. Following the completion of treatment, RNA was isolated using an RNeasy RNA isolation and purification kit (Qiagen, Hilden, Germany) per the manufacturer’s protocol.
Library construction was performed using the Illumina TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA, USA) as described herein. Briefly, total RNA (100 ng to 4 ug) was poly-A selected using poly-T Oligo-attached magnetic beads. Poly-A RNA eluted from the magnetic beads was fragmented and primed with random hexamers in preparation for cDNA synthesis. First-strand reverse transcription was accomplished using Superscript II Reverse Transcriptase (Invitrogen, Waltham, MA, USA). Second-strand cDNA synthesis was accomplished using DNA polymerase I and Rnase H under conditions in which dUTP is substituted for dTTP, yielding blunt-ended cDNA fragments. An A-base was added to the blunt ends in preparation for adapter ligation and to prevent concatemer formation during the ligation step. Adapters containing a T-base overhang were ligated to the A-tailed DNA fragments. Ligated fragments were PCR-amplified (12 to 15 cycles) under conditions that enabled only amplification of the first-strand cDNA product. The PCR-amplified library was purified using Agencourt AMPure XP beads (Beckman Coulter Genomics, Danvers, MA, USA). The concentration of the amplified library was measured with a NanoDrop spectrophotometer and an aliquot of the library was resolved on an Agilent 2200 Tape Station using a D1K or a High Sensitivity D1K assay (Agilent Technologies, Santa Clara, CA, USA) to define the size distribution of the sequencing library. Libraries were adjusted to a concentration of approximately 10 nM and quantitative PCR was performed using the Kapa Library Quantification Kit (Kapa Biosystems, Boston, MA, USA) to calculate the molarity of adapter-ligated library molecules. The concentration was further adjusted following qPCR to prepare the library for sequence analysis on an Illumina HiSeq instrument. Following completion of sequencing, genome alignment was conducted using National Center for Biotechnology Information (NCBI) build GRch37 and differential expression analysis was performed using the RNAseq application , which wraps the DESeq Bioconductor package, and statistical significance was calculated as described by Anders and Huber .
Western blot analyses
For Western blot analyses, cells were lysed in ice-cold radioimmunoprecipitation assay buffer (pH = 8.0, 50 mM 150 mM NaCl, Tris HCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% triton-X-100,) supplemented with protease inhibitor cocktail (Sigma-Aldrich), phosphatase inhibitor cocktail 2 (Sigma-Aldrich), and 1 mM dithiothreitol (DTT) (Sigma-Aldrich). The lysate was sonicated for 30 seconds with a 450 Sonifier (Branson Ultrasonics, Danbury, CT, USA) and then centrifuged at 14,000 RPM for 5 minutes at 4°C. A bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA) was used to measure the protein concentration and the samples were subsequently boiled for 5 minutes in 4x SDS Laemmli buffer. Proteins were separated on SDS polyacrylamide gels and transferred to a Immobilon-FL PVDF membrane (EMD Millipore, Billerica. MA, USA). The blots were blocked in Odyssey Blocking Buffer (LI-COR, Lincoln, NE, USA) for 1 hour at room temperature, stained with the primary antibodies overnight at 4°C, then stained with IR800CW or IR680 anti-mouse or rabbit secondary antibodies (LI-COR) for 1 hour at room temperature. The blots were imaged with the Odyssey Infrared Imaging System (LI-COR).
In tissue culture-treated six-well plates, cells were seeded at 200,000 cells per well in 3 mL of their respective media. After allowing the cells to recover for 48 hours, the media was aspirated and replaced with 4 mL of low serum media containing 30 μM C-6 or DMSO as a vehicle control. For the duration of the time course imaging, cells were maintained at 37°C in a humidified chamber with 5% CO2. Images were acquired using an IX81 microscope (Olympus, Center Valley, PA, USA) running Slide Book 5.0 software (Intelligent Imaging Innovations, Denver, CO, USA). For experiments utilizing MitoTracker Deep Red FM (Life Technologies), cells were drug treated for the appropriate amount of time, then MitoTracker added directly to the well to achieve a final concentration of 100 nM. The plate was then incubated for 10 minutes at 37°C, the dye-containing media discarded and replaced with fresh drug-containing media, and images acquired.
Transmission electron microscopy
MCF-10A and MCF-7 cells were cultured in 10-cm plates in preparation for transmission electron microscopy (TEM) analysis. Following the completion of treatment with 10 mL of either 30 μM C-6 or a matched DMSO vehicle control, the cells were fixed in a pH 7.4 0.1 M sodium cacodylate buffer containing 2.5% glutaraldehyde, 1% paraformaldehyde, 2.4% sucrose, and 8 mM calcium chloride. After fixation, the cells were rinsed in 0.1 M sodium cacodylate buffer and post-fixed in 2% osmium tetroxide also in the 0.1 M sodium cacodylate buffer. Cells were rinsed in type 1 water and en bloc stained in saturated aqueous uranyl acetate. Cells were then dehydrated in a graded ethanol series, transitioned through acetone, infiltrated with Embed 812 and acetone, embedded in fresh Embed 812, and allowed to cure overnight in a 60°C oven. Plastic sections were cut on a Leica (Wetzlar, Germany) ultramicrotome with a diamond knife and placed on copper grids at a thickness of 80 to 100 nm. Sections were contrasted with saturated aqueous uranyl acetate followed by Reynold’s lead citrate. They were then examined on an FEI Tecnai (Hillsboro, OR, USA) T-12 TEM with a LaB6 filament at 120KV. Images were acquired with a Gatan (Pleasanton, CA, USA) Ultrascan 1000 digital camera using Gatan’s digital micrograph.
Cells were cultured in 10-cm plates and following the completion of the appropriate drug treatments, the cells were collected and pelleted, resuspended in 90% MeOH, and flash frozen in liquid nitrogen. Ten microliters of a 0.2 ug/uL solution of D4-succinate was added to each sample as an internal control. All GC-MS analysis was performed with a Waters (Milford, MA, USA) GCT Premier mass spectrometer fitted with an Agilent 6890 gas chromatograph and a Gerstel (Mülheim an der Ruhr, Germany) MPS2 autosampler. Dried samples were suspended in 40 uL of a 40 mg/mL O-methoxylamine hydrochloride (MOX) in pyridine and incubated for 1 hour at 30°C. To the autosampler vials was added 25 uL of this solution. Ten microliters of N-methyl-N-trimethylsilyltrifluoracetamide (MSTFA) was added automatically via the autosampler and incubated for 60 minutes at 37°C with shaking. After incubation, 3 uL of a fatty acid methyl ester standard solution was added via the autosampler then 1 uL of the prepared sample was injected to the gas chromatograph inlet in the split mode with the inlet temperature held at 250°C. Two GC-MS runs were performed, one at a 10:1 split ratio to detect low-level metabolites and a second at 50:1 split ratio to accurately measure high-concentration metabolites, which saturate the detector at the 10:1 split ratio. For the 10:1 split ratio analysis, the gas chromatograph had an initial temperature of 95°C for 1 minute followed by a 40°C/minute ramp to 110°C and a hold time of 2 minutes. This was followed by a second 5°C/minute ramp to 250°C, a third ramp to 350°C, then a final hold time of 3 minutes. For the 50:1 split ratio analysis, the gas chromatograph had an initial temperature of 95°C for 1 minute followed by a 40°C/minute ramp to 110°C and a hold time of 2 minutes. This was followed by a second 25°C/minute ramp to 330°C. A 30 m Phenomex (Torrance, CA, USA) ZB5-5 MSi column with a 5-m-long guard column was employed for chromatographic separation. Helium was used as the carrier gas at 1 mL/minute.
Measurement of oxygen consumption and extracellular acidification rates
Cells were seeded in XF24 V7 culture plates (Seahorse Bioscience, North Billerica, MA, USA) in 300 μL of their respective media. After culturing overnight, the media was aspirated and the cells were treated with 600 μL of media containing 2% FBS and 30 μM C-6 for 3, 6, 12, and 24 hours as well as 0.03% (v/v) DMSO (four wells per condition). The media was aspirated and 600 μL of unbuffered assay media at pH = 7.4, which consists of DMEM supplemented with 2 mM GlutaMax-1 (Life Technologies), 1 mM sodium pyruvate (Sigma-Aldrich), 25 mM glucose (Sigma-Aldrich), and 31.7 mM NaCl was added and the cells were incubated at 37°C for 1 hour. Utilizing the XF24 Extracellular Flux Analyzer (Seahorse Biosciences), the baseline oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was measured. For the mitochondrial stress test, oligomycin A (Oligo, Sigma-Aldrich), which inhibits ATP synthase, was subsequently added to a final concentration of 1 μg/mL followed by addition of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (Sigma-Aldrich) to a concentration of 0.5 μM. Finally, a mixture of myxothiazol (Myx, Sigma-Aldrich) and rotenone (Rot, Sigma-Aldrich) were added to a final concentration of 0.5 μM and 1 μM respectively to inhibit complexes I and III of the electron transport chain.
Measurement of oxidative stress
Oxidative stress was measured by the fluorescent indicator 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Life Technologies). Briefly, cells were seeded in triplicate in tissue culture-treated 15-mm plates at 500,000 cells per plate in 3 mL of their respective media. After allowing the cells to recover for 48 hours, the media was aspirated and replaced with 3 mL of low serum media containing 30 μM C-6 or DMSO as a vehicle control for 3, 6, 12, 24, or 48 hours. Following the completion of treatment, the media was discarded and replaced with 1 mL of Hank’s balanced salt solution (HBSS) containing 30 μM H2DCFDA and the cells incubated for 30 minutes at 37°C. The staining media was then discarded and the cells trypsinized and analyzed by a FACscan flow cytometer (Becton Dickinson, Mountain View, CA, USA) for fluorescence.
Real-time PCR (RT-PCR)
For experiments designed to measure gene expression, real-time PCR (RT-PCR) was conducted using a LightCycler 480 (Roche, Basel, Switzerland). Following drug treatment, RNA was isolated using an RNeasy RNA isolation and purification kit (Qiagen) per the manufacturer’s protocol. Genomic DNA was removed with a DNase digest and 1 μg of RNA was used to synthesize cDNA using a Superscript III Reverse Transcriptase kit from Invitrogen per the manufacturer’s protocol; after an RNase H digestion, RT-PCR was performed in a 5 μL reaction using KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems, Boston, MA, USA). The following primer sets were used: CHOP 5′-AGTCTCTCCTCGGCTTGC-3′ and 5′-ACATCTGGGAGAAAGGTTGTC-3′ . Data were normalized to an internal reference gene (GAPDH 5′-AAATTCCATGGCACCGTC-3′ and 5′-GATGGTGATGGGATTTCCA-3′) and relative gene expression was assessed using the comparative CT method .
Measurement of caspase activity
Caspase activity was measured using the Caspase-Glo assay (Promega) for caspases 3/7, 8, and 9 according to the manufacturer’s protocol.
No human or animal experiments were conducted; ethical approval from an independent review committee was not required for the studies conducted and presented herein.
For in vitro assays, an unpaired Student’s t test was performed using GraphPad Prism 6.0 or Microsoft Excel (Microsoft, Redmond, WA, USA) and P values <0.05 between groups were considered significant.