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  • br Lipid modified cationic peptides induce ROS


    3.4. Lipid-modified cationic peptides induce ROS
    mPTP induction and mitochondrial depolarization typically involve increased cellular levels of ROS, triggering complex signaling cascades that lead to cell death [1]. To test whether this was happening during the treatment of EMT6/AR-1 cells with H8R8-based amphiphiles, ROS levels were evaluated after treatment with Str-H8R8 and VES-H8R8, and compared to controls using 5-carboxy-2,7-dichlorodihydrofluorescein diacetate (CDCFDA) (Fig. 5). After 5 h of incubation with Str-H8R8 or VES-H8R8, ROS levels significantly increased to 200.9 ± 43.5% and 179.2 ± 26.3%, respectively (p < .01), which is similar to ROS levels with exposure to the positive control, hydrogen peroxide, of 266.7 ± 28.6%. Controls including VES, stearic acid, PEG-H8R8 and H8R8, did not increase ROS levels relative to DMSO controls (p > .05). Similar results were observed with the EMT6/P cells (Fig. S7A). Str-H8R8 significantly induced ROS after 2 h of incubation relative to controls whereas, surprisingly, VES-H8R8 did not (p < .001, Fig. S7B). Vitamin E has well-known antioxidant properties whereby the phenolic alcohol acts as a radical scavenger [66,67]. We therefore hypothesized that the delayed induction of ROS by VES-H8R8 was attributed to the cleavable ester present within vitamin E succinate, and subsequent radical scavenging. To test this hypothesis, we synthesized a vitamin E-modified H8R8 with a more stable butyl ether linker (VEB- H8R8) (Fig. S7C). Unexpectedly, however, treating EMT6/AR-1 cells for 2 h with the ether-linked VEB-H8R8 induced ROS levels comparable to those of the ester-linked VES-H8R8 (Fig. S7B). The ether linkage may be oxidized by intracellular reactive oxygen intermediates, cleaving vitamin E and thereby enabling ROS scavenging [68,69].  Journal of Controlled Release 305 (2019) 210–219
    Fig. 5. Reactive oxygen species (ROS) levels are significantly increased upon treatment with H8R8-based amphiphiles. EMT6/AR-1 cells were incubated with treatment groups for 5 h followed by incubation with the CDFDA probe. Flow cytometry was used to determine relative ROS levels to DMSO controls. Data are presented as a mean ± SD (n = 3) and statistical analyses were performed using one-way ANOVA and Tukey's multiple comparison test (N.S. p > .05, **p < .01).
    3.5. Lipid-modified cationic peptides induce apoptosis, necrosis, and Ethylmalonyl Coenzyme A arrest
    Mitochondria depolarization and increased ROS levels often lead to apoptosis [65]; however, synthetic cationic polymers, such as PEI, have been reported to damage cell membranes and cause necrotic cell death [12,22]. To investigate the mechanism of cell death upon treatment with lipid-modified cationic peptides, annexin-V-Cy5 and 7-AAD were used to monitor the levels of apoptosis and necrosis, respectively [70]. Both Str-H8R8 and VES-H8R8 induced death of EMT6/AR-1 cells by apoptosis and necrosis significantly more than DMSO controls (Fig. 6A-
    C) and in a concentration-dependent manner (Fig. 6D-E). Consistent with their IC50 values, 5 μM of each of the lipid-modified cationic peptides had minimal effect compared to the DMSO control in terms of necrosis and apoptosis (p > .05). Interestingly, at 20 μM, VES-H8R8 treatment led predominately to apoptosis (46.9 ± 2.8%), which is si-milar to that observed with VES alone [71], whereas Str-H8R8 resulted in greater necrosis (44.2 ± 2.0%), which may be due to plasma membrane damage as was observed for other amphiphilic peptides [72,73].
    H8R8-based amphiphile treatments were expected to arrest cells in a specific phase of the cell cycle as mitochondria depolarization and in-creased ROS levels should inhibit progression past cell cycle check-points [74]. We studied cell cycle distributions of EMT6/AR-1 cells treated with the H8R8-based amphiphiles by flow cytometry, using 7-AAD as a nucleic dye. Cells treated with Str-H8R8 and VES-H8R8 were arrested in the G1 cell cycle phase relative to DMSO controls (Figs. 6F and S8AeC, p < .01), which is consistent with the reported mechan-isms of VES, pegylated-VES and melittin [71,75–77]. Consequently, there were fewer cells in the S (proliferation) and G2 cell cycle phases when treated with Str-H8R8 and VES-H8R8 vs. DMSO (p < .001). Si-milar results were observed in the EMT6/P cells (Fig. S8D). None of the controls, including VES, stearic acid, PEG-H8R8 and H8R8, significantly arrested the EMT6/P and EMT6/AR-1 cells in any phase (Fig. S8E, F). While it's not clear whether H8R8-based amphiphiles interact with any of the cell cycle regulatory or checkpoint proteins, sonic hedgehog signaling may be implicated based on its involvement with other ca-tionic peptides [77].
    Fig. 6. Apoptosis, necrosis, and G1 cell cycle arrest are induced upon treatment of EMT6/AR-1 with H8R8-based amphiphiles. Representative histograms of (A) VES-H8R8, (B) Str-H8R8, and (C) DMSO treated cells showing annexin-V-Cy5 (apoptosis) and 7-aminoactinomycin D (7-AAD) (necrosis) staining. Identical gates were set up to demonstrate the proportion of live, apoptotic (apop) and necrotic (necro) populations. (D) The proportion of cells in the apoptotic state upon treatment with either H8R8-based amphiphiles at 5, 10, and 20 μM or DMSO controls for 2 h. (E) The proportion of cells in a necrotic state upon treatment with either H8R8-based amphiphiles at 5, 10, and 20 μM or DMSO controls for 2 h. (F) The proportion of cells in G1, S or G2 phase when treated with the H8R8-based amphiphiles relative to that when treated with DMSO. Data are presented as a mean ± SD (n = 3) and statistical analyses were performed using one-way ANOVA and Tukey's multiple comparison test (N.S. p > .05, **p < .01, ***p < .001).