186689-07-6 br Both parental EMT P and doxorubicin resistant
Both parental (EMT6/P) and doxorubicin resistant (EMT6/AR1) EMT6 cells were generously provided by Dr. X.Y. Wu (University of Toronto), originally from Dr. Ian F. Tannock at the Ontario Cancer Institute, (Toronto, ON, Canada) and maintained in our laboratory. Non-cancerous fibroblast cells (NIH/3T3) were grown in DMEM media supplemented with 10% fetal calf serum and 1% penicillin/strepto-mycin and grown as above. EMT6/P cells were grown in α-MEM medium supplemented with 10% FBS, 1% penicillin/streptomycin at 37 °C in a humidified incubator with 5% CO2 atmosphere. EMT6/AR-1 cells were grown as above, with the addition of doxorubicin at 1 μg/mL to maintain doxorubicin resistance and permeation glycoprotein (Pgp) overexpression.
2.8. Cell culture cytotoxicity assay
Cells were seeded into 96-well flat-bottomed tissue culture plates at a density of 3000 cells per well and allowed to adhere for 24 h. H8R8 peptides dissolved in DMSO were serially diluted in PBS and then into full medium, and incubated with cells for 72 h. The doses chosen were based on the observed inhibitor concentration to kill 50% of the cells (IC50). Similar DMSO concentration was used as a control. DMSO was used at concentration < 0.5% v/v. After the addition of peptides, cells were allowed to grow for 72 h at 37 °C in a 5% CO2 and 95% air hu-midified incubator. Presto Blue (Life Technology) was added to fresh medium as per the manufacturer's protocol and incubated with cells for Journal of Controlled Release 305 (2019) 210–219
1.5 h. Viable cells are able to reduce the resazurin dye in Presto Blue to a highly red fluorescent resorufin (ex/em 540/590 nm) which can be read by a microplate fluorescent reader (Infinit m200 Pro, Tecan Group Ltd., Switzerland). Each measurement is an average of 3 separate pas-sages of cells. Dose response curves and inhibitor concentration to kill 50% of the cells (IC50) were obtained from Graph Pad Prism version 6.00 for Windows (Graph Pad Software, CA, USA, www.graphpad. com). Relative viability was calculated as the fluoresce intensity of the treated group divided by the fluorescence intensity of a control group.
2.9. Laser scanning confocal microscopy
EMT6/P cells were seeded at a density of 20,000 cells per well in a 8-well Nunc Lab-Tek II chambered cover glass (Thermo Fisher Scientific, MA, USA) and allowed to adhere for 24 h at 37 °C in a 5% CO2 and 95% air humidified incubator. 0.8 μM of fluorescein-modified peptide was added to the cells and incubated for 3 h. The peptide containing media was aspirated off, and 200 nM of MitoTracker Deep Red FM (Thermo Fisher, MA, USA) in full media was added to the cells. After 15 min incubation, the cells were washed 3-times with PBS, and then Hoescht 33,342 nuclei 186689-07-6 dye (Molecular Probes, Inc., Eugene, OR, USA) in PBS was added to the cells. Live cell imaging was done using an Olympus FV1000 confocal microscope equipped with an oil immersion 60× lens. Excitation and emission wavelengths are as fol-lows: Hoescht 33,342 (ex/em: 405/460 nm) fluorescein (ex/em 488/ 520 nm), MitoTracker Deep Red FM, (ex/em: 640/670 nm). Unlabeled control cells were used to set the laser power to avoid fluorescent bleed over between channels.
2.10. Fluorescein-modified peptide uptake in intact cells and isolated mitochondria
The uptake of the VES-H8R8, Str-H8R8 in the mitochondria of intact cells was quantified as previously reported . 5 × 106 cells were seeded in T-25 flasks and allowed to adhere for 24 h at 37 °C in a 5% CO2 and 95% air humidified incubator. Non-toxic concentrations of peptides were used (< 0.8 μM). The EMT6/AR-1 cells were treated the next day with the fluorescein-modified peptides of VES-H8R8, Str-H8R8, or H8R8 (0.8 μM), or free fluorescein as a dye control (0.8 μM). The cells were incubated with peptides or dye control for 3 h in full medium, and then the cells were harvested with trypsin. After centrifugation, mi-tochondria from the pelleted cells were extracted according to the in-structions of the Mitochondria Isolation Kit for cultured cells (Thermo Fisher Scientific, MA, USA). cOmplete Mini, EDTA free protease in-hibitor cocktail was added to reagents A and C of the mitochondrial isolation kit with 1 tablet/10 mL of extraction buffer. A more purified fraction of mitochondria was obtained by centrifuging the post-nuclear supernatant at 3000 ×g. The mitochondria pellet was then suspended in PBS, transferred into a 96-well clear bottom black plate and fluor-escein was quantified (ex/em 490/520 nm) against a standard curve in PBS using a Tecan Plate Reader. The average of three biological repeats was used to measure the amount of fluorescein uptake.
The uptake of the fluorescein-modified peptides of VES-H8R8, Str-H8R8, H8R8, or free fluorescein was also investigated with isolated mitochondria as reported previously . Mitochondria from 1.1 × 108 cells of untreated EMT6/AR-cells were extracted according to the previous section. The mitochondria of approximately 6 × 106 cells were then suspended in mitochondria isolation buffer (10 mM Tris hydrochloride, 0.15 mM MgCl2, 0.25 mM sucrose, 1 mM DTT, pH 6.7, and 1 tablet/10 mL of cOmplete, Mini, EDTA free protease inhibitor cocktail) and fluorescein-modified peptides of VES-H8R8, Str-H8R8, H8R8, or free fluorescein were added at a final concentration of 0.8 μM. The isolated mitochondria were incubated with the peptides or free fluorescein for 1 h at 37 °C, and then washed with PBS three times by centrifuging at 12,000 ×g for 5 min. The isolated mitochondria were then suspended in PBS, transferred into a 96-well clear bottom black