br Apoptosis induction assay br Apoptosis
2.15. Apoptosis induction assay
Apoptosis was measured using Annexin V-Cy5 (Biovision Inc., CA, USA) and 7-aminoactinomycin D (7-AAD) (AAT Bioquest, CA USA) [24,25]. 20,000 cells were seeded into 48-well plates and allowed to adhere for 24 h at 37 °C in a 5% CO2 and 95% air humidified incubator. The cells were treated the next day with peptides at 5, 10, and 20 μM for 2 h in full media. A similar DMSO concentration was used as a control. Following treatment, floating cells were collected, adhered cells were harvested, washed with PBS, and then incubated with An-nexin V-Cy5 (1 μL/mL) and 7-AAD (15 μg/mL) for 20 min at 25 °C as per the manufacturer's protocol. Cells were then placed on ice before measuring fluorescence in a flow cytometer within an hour. Cell debris and doublets were gated out using FSC-A vs FSC-H, and at least 10,000 events were collected. Using the untreated control group, gates were set in the FL-3 (ex/em 488/ > 670 nm) and FL-4 channel (ex/em 640/ 675(25) nm) for 7-AAD and Annexin-V-Cy5 respectively, and identical gates were used for the other treatment groups. Proportion of apoptotic or necrotic cells were averaged from 3 biological repeats.
Cell cycle analysis was performed using 7-AAD (7-aminoactino-mycin D) (AAT Bioquest, CA USA). 20,000 cells were seeded into 48-well plates and allowed to adhere for 24 h at 37 °C in a 5% CO2 and 95% air humidified incubator. The cells were treated the next day for 24 h in full media. Control treatments and peptides were incubated at 15 μM. A similar DMSO concentration was used as a negative control. Following treatment, the cells were harvested, washed with PBS, then incubated with 25 μg/mL of 7-AAD in PBS containing 1% bovine serum albumin (BSA) and 0.15% Triton X for 25 min. Cells were placed on ice before measuring fluorescence in a flow cytometer. Cell debris and doublets were gated out using the FSC-A vs FSC-H, and 10,000 events were collected. The FL-3 channel (ex/em 640/ > 670 nm) was set in a linear range, and BODIPY 493/503 analysis was completed using the FlowJo software
(Flowjo, OR, USA) using the Watson Pragmatic Mod Fit algorithms. The results were expressed as proportion of cells in the G1, S, and G2 phases relative to DMSO control, and averaged from three biological repeats.
2.17. Statistical analysis
All statistical analyses were performed using Graph Pad Prism ver-sion 6.00 for Windows (Graph Pad Software, San Diego, California, www.graphpad.com). Differences among groups were assessed by one-way ANOVA with Tukey's multiple comparison test. Alpha levels were set at 0.05 and a p-value of < 0.05 was set as the criteria for statistical significance. Graphs are annotated with p-values as *p < .05, **p < .01, or ***p < .001. All data are presented as mean ± standard deviation.
3. Results and discussion
3.1. H8R8-based cationic lipids have selective anti-cancer activity
To investigate the effect of the lipid modification of H8R8 on its anti-cancer properties, the half maximum inhibitory concentration (IC50) was evaluated with two breast cancer cells, parental breast cancer cells, EMT6/P, and the permeation glycoprotein (Pgp) overexpressing, MDR variant, EMT6/AR-1. We used the resazurin-based Presto Blue meta-bolic assay as a proxy to evaluate cell survival. Both Str-H8R8 and VES-H8R8 exhibited an IC50 in the low micromolar range while the un-modified peptide control, H8R8, exhibited an IC50 above 300 μM in both cancer cell lines (Fig. 2A, Supplementary Table S1). Similarly to un-modified H8R8, poly(ethylene glycol) (PEG)-modified H8R8 showed no cytotoxicity, confirming the necessity of providing a lipophilic char-acter to H8R8 cationic peptides for anti-cancer activity (Fig. S3A). While not attributed to the cationic amphiphilic structure by the authors, a similar strategy was employed with the cationic Tat peptide, which, when conjugated to paclitaxel, resulted in enhanced anti-cancer activity with increased paclitaxel uptake in MDR cancer cells relative to pacli-taxel alone . Here, the unmodified Tat peptide was non-toxic to the cancer cells while the Tat modified paclitaxel exhibited potent anti-cancer activity in both parental and MDR cancer cells. In our study, both Str-H8R8 and VES-H8R8 had similar activities on each cell line, with an IC50 on EMT6/P of 4.2 ± 0.1 μM and 4.4 ± 0.1 μM, respec-tively; and an IC50 on EMT6/AR-1 of 6.8 ± 0.3 μM and 7.3 ± 0.3 μM, respectively. While the pKa of histidine is 6.0 and intratumoral pH can range between 6.5 and 6.9, we do not anticipate that the more acidic tumor environment would enhance the anti-cancer activity of both Str-H8R8 and VES-H8R8 as the histidine would remain deprotonated [27–29]. Interestingly, due to the amphiphilic nature of the modified peptides, Str-H8R8 and VES-H8R8 formed nanoparticles in PBS ex-hibiting diameters of 11.1 nm ± 0.3 nm and 10.9 nm ± 0.2 nm, re-spectively, and nanoparticle diameters did not change in acidic buffer with pH 5.3 (Fig. S1A, B and Table S2). Importantly, both Str-H8R8 and VES-H8R8 nanoparticles exhibited high critical micelle concentra-tions > 257 μM, indicating that both peptides exist as non-aggregated