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  • br Flowjo OR USA using the Watson Pragmatic

    2022-08-18


    (Flowjo, OR, USA) using the Watson Pragmatic Mod Fit algorithms. The results were expressed as proportion of GSK1838705A 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 [26]. 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
    Str-
    VES-
    unimers in the low 0–20 μM range used (Fig. S1C,D and Table S2). For both lipid-modified cationic peptides, there was a significant increase in IC50 (p < .001) on MDR cancer cells compared to the parental cell line, suggesting that the activity of the H8R8-based amphiphiles was partially dependent on Pgp efflux activity. Pgp is able to efflux both hydrophobic and hydrophilic drugs, thereby reducing the effective intracellular drug concentration. For example, Pgp has been shown to efflux docetaxel and doxorubicin, requiring 100-fold more drug to kill MDR cancer cells than non-resistant cancer cells, whereas lipid-modified cationic pep-tides bypass the efflux capabilities [30,31]. Consistent with the litera-ture, VES alone exhibited some anti-cancer activity, with IC50 values of 23.0 ± 2.0 μM and 36.0 ± 4.6 μM on EMT6/P and EMT6/AR-1, re-spectively [32,33]. Covalent modification of VES to H8R8 led to a 5-fold decrease in IC50 on both EMT6/P and EMT6/AR-1 relative to VES alone.