Source of drug
FC101 in its free base form (MW 292.3) was isolated and purified from Fusarium equiseti (originally supplied by Dr. Xie at the University of Minnesota). This fungus is found exclusively in the arctic latitudes and grown on rice cultures by Dr. Brian Furmanski. FC101 was then crystallized in the presence of phosphoric acid to form the more stable phosphate salt form (MW 390.3). Its purity (>98%) was confirmed by 1H-NMR, 13C-NMR, and UV–vis spectroscopy.
Cell lines and culture
Seven human tumor cell lines were used in this study. HaCat (pre-malignant skin, a gift from Prof. N.E. Fusenig, Deutsches Krebforschungszentrum), SRB12-p9 (malignant skin SCC, a gift from Dr. Reuben Lotan, Univ. of Texas-MD Anderson Cancer Center), MCF-7 (low malignant mammary gland adenocarcinoma), MDA-231 (malignant mammary gland adenocarcinoma), SV-HUC (premalignant bladder), UM-UC14 (malignant bladder, a gift from Dr. H.B. Grossman, Univ. of Texas-MD Anderson Cancer Center), and PC3 (malignant, prostate). MCF-7, MDA-231, SV-HUC and PC3 cells were obtained from the American Type Culture Collection (Manassas, VA). HaCaT cells were cultured in 4x modified Eagle’s medium (MEM) media (1.4 mM Ca2+), supplemented with 5% fetal calf serum (FCS) and 1% Penicillin Streptomycin Solution (Pen-Strep). SRB12-p9, MCF-7, MDA-MB231, and PC3 cells were cultured in Dulbecco’s MEM (DMEM)/F12 medium containing 10% FCS and 1% Pen-Strep. UM-UC14 cells were cultured in 50% DMEM/F12 media and 50% DMEM low glucose media containing 10% FCS and 1% Pen-Strep. SV-HUC cells were cultured in HyQ Ham’s/F-12 media with 7% FCS and 1% Pen-Strep. For passaging, subconfluent cells were incubated with 0.1% trypsin/2 mM EDTA and suspended in media before replating. All cells were cultured at 37°C in the humidified atmosphere of 5% CO2/95% air.
Cell viability was determined by the MTT 3-(4, 5-dimethyltiazol 2-yl)-2,5-diphenyltetraolium bromide assay. Briefly, cells were plated in triplicate wells at varying densities (1,000 cells/well for HaCaT, SRB12-p9, MCF7, MDA-MB231; 1,500 cells/well for MCF7, PC3; 2,000 cells/well for SV-HUC) in 100 μl growth media in 96-well plates and treated with the FC101 at the concentrations indicated in Figure 4. After a range of treatment times a solution of MTT (20 μl of a 12 mM solution in PBS) was added and incubated for 2 hours at 37°C. The cells were washed gently with PBS, and 100 μl of DMSO was then added to the wells followed by mild shaking to dissolve the MTT precipitate. Absorbance at a wavelength of 540 nm was measured for each well using a Wallac Victor3 1420 Multilabel multi-well plate reader (Perkin-Elmer).
Cell viability assays for multi-drug resistant MCF-7/Dox cells
Cell viability was analyzed by quantitation of ATP, an indicator of active cells, using the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI). Briefly, cells (4,000 cells/well) were grown in 96-well plates with 10% FBS RPMI-1640 medium for 24 hr. FC101 was introduced into cells by Lipofectamine 2000 (vehicle control) in Opti-MEM I reduced-serum medium, for a 4 hr incubation. Cells were then incubated with increasing concentrations of FC101 in 5% FBS medium for an additional 72 hr. Cell viability was determined by the measurement of luminescent ATP in a Synergy HT microplate reader, following incubation with CellTiter-Glo reagent. Triplicate experiments were repeated two times.
Cell cycle analysis
The cell cycle profile was determined by cell cycle flow cytometry based on cellular DNA content, using an Epics Profile II cell sorter (Coulter Electronics, Inc.). Cells were treated with FC101 or DMSO vehicle control for the indicated durations, trypsinized, collected and pelleted together with any material floating in the medium. Cells were fixed in cold 70% ethanol, resuspended in PBS at a density of > 106 cells/ml followed by RNase A (1 mg/ml) treatment, addition of propidium iodide (20 μg/ml final concentration) and analysis by flow cytometry. The percentage of cells in different phases of the cell cycle were determined from the raw data using the ModFit V3.2.1 flow cytometry software. Apoptosis was assessed by determining the amount of sub-G1 sized particles.
Western blot analysis
Western blotting was carried out as described previously . Briefly, control and treated cells (0–10 μM) were washed twice with cold PBS and then lysed in the lysis buffer [50 mmol/L Tris, pH 7.2; 150 mmol/L NaCl; 1% sodium deoxycholate; 0.1% SDS; 1% Triton X-100; 10 mM NaF, 1 mM Na3VO4; Protease inhibitor cocktail (1:1000 dilution, Sigma). Lysates were sonicated for 10 seconds and centrifuged at 13,000 X g for 10 minutes at 4°C. Protein concentration was determined using the BCA assay (Protein Assay Kit, Pierce). Protein samples (~40 μg) were electrophoresed on 8–12% SDS-PAGE, transferred to PVDF membrane (Millipore, USA), and probed with respective primary and secondary antibodies. The following primary antibodies were used: p38, phospho-p38 (Thr180/Tyr182), PARP, Bcl-2, Mcl-1, surviving, (all from Santa Cruz Biotechnology), Bcl-xL, BAK, BAX (Biomedia), p-4E-BP1(Thr37/Thr46), caspase 3, cleaved caspase 3, cleaved caspase 8, BAD, phospho-Erk1/2 (Thr202/Tyr204; Cell Signaling), and β-tubulin (Sigma). Goat anti-mouse Ig-G-horseradish peroxidase (HRP), goat anti-mouse IgM-HRP, and goat anti-rabbit IgG-HRP were purchased from Pierce.
For our anti-angiogenic assays we used the murine MS1 microvascular endothelial cell line, selected due to its high VEGFR2 expression and responsiveness to VEGF. Inhibition of MS1 cell proliferation in the presence and absence of VEGF was evaluated using DNA incorporation of BrdU. MS-1 cells were serum starved overnight and the next morning preincubated with various concentrations of FC101 for 30 minutes. Next, 50 ng/ml VEGF or PBS alone (i.e., control) was added to respective wells along with BrdU and incubated for 4 hours. Cells were then fixed and BrdU incorporation measured by ELISA.
In vivo tumorigenicity assay
All animal studies were carried out following the USDA, PHS, and NIH animal use and care guidelines. The animal protocol was approved by the Institutional Animal Care and Use Committee at the Louisiana State University Health Science Center in Shreveport (LSUHSC-S) (Protocol Number, P10-045). SCID Beige mice (CB17/Icr.Cg-PrkdcscidLystbg/Crl) were maintained on AIN76A semi-purified diets (Dyets, Bethlehem, PA). Mice were administered FC101 or PBS by IP injections 5 days per week. Total daily drug intake was 8 mg/kg. On day 3, groups of 8 mice (6–7 week old) were injected subcutaneously (s.c.) in the dorsal region with 1 × 106 SRB12-p9 cells in 0.1 ml PBS. Tumors were measured five times weekly by digital caliper using the formula V = ((W + L)/4))3 × 4/3π, where L = length and W = width. Tumors were harvested and bisected at 25 days post-injection, or at the time of attaining a volume of 2 cm3. Tumors were fixed in formalin for histological analysis.
The differences between the groups of mice in terms of tumor volume or persisting benign cysts was compared using the Mann–Whitney test and differences in latency to tumor formation and survival were calculated by the Log-rank test and Kaplan-Meier Cumulative Survival tests.
Statistical analyses were completed using ANOVA, followed by Fisher's protected least significant difference procedure. A p-value of < 0.05 (2-tail) was considered statistically significant.