Informed consent was taken of the healthy donor prior to collection of blood samples from San Diego Blood Bank according to the regulation of the Institutional Review Board and Ethics committee at UC San Diego and maintained strict compliance with the Helsinki Declaration.
FD-895 was prepared through total synthesis. Pladienolide B (sc-391691, Santa Cruz Biotechnology, Santa Cruz, CA), etoposide (E1383, Sigma-Aldrich, St Louis, MO) and cisplatin (479306, Sigma-Aldrich, St Louis, MO) were obtained commercially (Fig. 1). Oligonucleotides were purchased via custom synthesis (Integrated DNA Technologies).
Cell culture methods
The MCF-7 (RRID:CVCL_0031), MDA-MB-468 (RRID:CVCL_0419), HS578T (breast cancer, RRID:CVCL_0332), A2780 (RRID:CVCL_0134), SKOV3 (ovarian cancer, RRID:CVCL_0532), 786-O (renal adenocarcinoma, RRID:CVCL_1051), HeLa (Cervical cancer, RRID:CVCL_0030), and HEK-293 cell lines were obtained from ATCC. MCF7 was cultured in DMEM + 10% FBS + 2mM L-glutamine and Pen/Strep supplemented with 0.01 mg/mL human recombinant insulin. Other cell lines were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 U/mL of penicillin and 100 µg/mL of streptomycin. Additionally, two ovarian cancer cell lines with differential cisplatin sensitivity, OV-2008 (sensitive, RRID:CVCL_0473) and it’s resistant variant C13 were obtained from Prof. Stephen Howell (UC San Diego). To complete the set, a final colon cancer cell line HCT116 (RRID:CVCL_0291) was obtained from the Johns Hopkins School of Medicine, Baltimore, MD. The suspension cell lines including Jeko-1, JVM2, and Mino cell lines were cultured in RPMI-1640 supplemented with 10% FBS along with 1% Pen-Strep. All cell lines were incubated 37°C in an atmosphere of 5% CO2 and routinely monitored for Mycoplasma infections by PCR analyses.
Flow cytometry analyses
Normal primary peripheral blood mononuclear cells (PBMCs) were treated with FD-895 (100 nM to 2.0 µM), and pladienolide B (100 nM to 2.0 µM), for 48 h. Cell viability was determined by flow cytometry after staining with conventional live staining with 40 µM 3,3′dihexyloxacarbocyanine iodide (DiOC6; Life Technologies, Carlsbad, CA) and 15 µM (10 µg/mL) of propidium iodide (PI; Sigma-Aldrich, St Louis, MO). Data were analyzed by using FlowJo software (version 6.4.7; Tree Star). Using this assay, viable cells excluded PI and stained brightly positive for DiOC6 as it targets metabolically active mitochondria of alive cells ,.
Cell proliferation assays
The cell proliferation assays were conducted in adherent cell lines (HCT116, MCF-7, MDA-MB-468, HS578T, OV-2008, A2780, SKOV3, 786-O, HEK-293, and HeLa) by using CellTiter 96 AQueous non-radioactive colorimetric method (G5421, Promega, Madison, WI). Briefly, a total of 3000 cells/well were seeded in a 96-well flat-well plate followed by treatment with FD-895 (100 nM to 2.0 µM), pladienolide B (100 nM to 2.0 µM), cisplatin (1 µM to 30 µM) or etoposide (1 µM to 30 µM) in triplicate for 48 h at 37°C. Following the incubation, 20 µL of CellTiter 96 AQueous solution (Promega, Madison, WI) was added directly to each well. Non-treated cells were considered as the control. After staining, the plates were incubated for an additional 2 h and then read on a 96-well plate reader (Molecular Devices, Sunnyvale, CA). Absorbance readings were record absorbance at 490 nm using empty wells (air) for background collection.
Reverse transcriptase PCR (RT-PCR) analyses
HCT116, MCF-7, MDA-MB-468, HeLa, Jeko-1, JVM-2 and Mino cells (106 cells/well) were treated with 100 nM FD-895, 100 nM pladienolide B, 30 µM cisplatin or 30 µM etoposide for 4 h. RNA isolation was done using mirVana miRNA Isolation Kit (Ambion, Austin, Texas). The 200 ng of RNA was subjected to DNase I (Life Technologies, Carlsbad, CA). The cDNA was prepared by using SuperScript III Reverse Transcriptase Kit (Life Technologies, Carlsbad, CA), and PCR reactions were performed in 20 µL of reaction volume. PCR conditions were 95°C for 3 min; 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 45 s; followed by 72°C for 5 min. PCR products were separated on a 2% agarose gel and stained with ethidium bromide. Details of the primers used for RT-PCR are described in Table-1.
Quantitative reverse transcriptase-PCR (qRT-PCR) analyses
The HeLa cells treated with 100 nM FD-895 or 100 nM pladienolide B for 6 h, 12 h, or 24 h and the RNA isolation and cDNA preparation were done as described above. The amounts of mRNA of LEF1 (Lymphoid enhancer-binding factor-1), FN1 (fibronectin 1), and CCND1 (cyclin D1) genes were determined using Power SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) real-time qRT-PCR using specific primers. PCR was conducted using 5 picomole of each primer and 20 ng of the obtained cDNA. PCR conditions were 50°C for 2 min; 95°C for 10 min; 40 cycles of 94°C for 15 s, and 60°C for 1 min. The mRNA levels were calculated using the 2−ΔΔCT method. GAPDH was used as a control for normalization.
Pathway reporter arrays
Cignal Finder Reporter Array (336821, Qiagen/SABiosciences, Frederick, MD) was used to assess 45 different signaling pathways. HeLa cells were seeded into wells (50,000 cells/well) of the Cignal Finder 96-well plates (CCA-901L, Qiagen, SABiosciences, Frederick, MD) for introducing pathway reporters into cells by reverse transfection according to the manufacturer's protocol. Briefly, reporter DNA constructs in each plate well were re-suspended with 50 µL Opti-MEM and then mixed with 50 µL diluted Lipofectamine 2000 transfection (Life Technologies, Carlsbad, CA) reagent. Cells were suspended in Opti-MEM (Life Technologies, Carlsbad, CA) supplemented with 10% of FBS and 0.1 mM non-essential amino acids at a density of 6 × 105 cells/ml, and then 50 µL of the cell suspension was added into each plate well and mixed with DNA resident in the plate and added transfection reagent. The cells were incubated for 3 h. Following transfection; the cells were treated with vehicle (Opti-MEM) or 100 nM FD-895 for 3 h in Opti-MEM media. Luciferase and renilla expression were determined (Qiagen/SABiosciences corp., Frederick, MD).
Western blot analyses
HeLa cells were treated with 100 nM FD-895 or 100 nM pladienolide B for 12 h, 24 h, and 48 h for β-catenin, LEF1, LRP6 (LDL Receptor Related Protein 6), and phospho-LRP6. The cells were then washed with PBS (2 × 5 mL) and lysed with modified RIPA buffer at 4°C. Untreated cells were used as a control. The whole-cell protein was quantified according to the Bradford method. Lysates in sample buffer (2% Sodium dodecyl sulfate (SDS), 10% glycerol, 80 mM Tris•HCl (pH 6.8), 720 mM β-mercaptoethanol and 0.001% bromophenol blue) were denatured at 95°C for 5 min. Total cellular proteins (30 µg via Bradford analyses) were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) using a 4–20% Criterion Precast Gel (Bio-Rad, Hercules, CA), and the proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). After blocking with 5% bovine serum albumin (BSA) for 1 h in Tris-buffered saline, 0.1% Tween 20 (TBST, 20 mM Tris•HCl, 137 mM NaCl, 0.1% Tween-20 pH 7.6), the membrane was incubated with the following primary antibody overnight at 4°C. The primary antibodies including rabbit mAb anti-LEF1, rabbit anti-Phospho-LRP6 (Ser1490), rabbit anti-LRP6, rabbit mAb anti-β-catenin, and mouse Ab anti-β-actin were obtained from Cell Signaling Technology (Beverly, MA) and used at a dilution of 1:1000. After primary mAb staining and washing thrice with TBST, the membranes were incubated with HRP-labeled anti-rabbit (sc-2030, Santa Cruz Biotechnology) or HRP-labeled anti-mouse (sc-2031, Santa Cruz Biotechnology) secondary antibodies with a dilution of 1:5000 dilutions for 40 min at rt. After incubation, the membrane was washed thrice times with TBST and developed using an enhanced chemiluminescence (ECL) kit (Pierce Thermo Scientific Inc., Rockford, IL).
Gene‑gene interaction networks were predicted and generated with GeneMANIA (Gene Multiple Association Network Integration Algorithm) available at http://genemania.org.
The data presented as mean ± standard deviation (SD). The data was analyzed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA). Multiple groups were compared using Bonferroni correction and p < 0.05 was considered statistically significant.