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  • br To better understand mitochondrial depolarization we inve

    2020-08-30


    To better understand mitochondrial depolarization, we investigated VES-H8R8 and Str-H8R8 Fer1 on the induction of mitochondrial permeability transition pore (mPTP). mPTP consists of Fer1 nucleotide translo-case (ANT), cyclophilin D (CypD), and a voltage-dependent anion channel (VDAC), which together form a pore through the outer and inner mitochondrial membrane allowing for solutes < 1500 Da to leak out into the cytosol [57]. The presence of mitochondrial proteins in the cytoplasm induces intrinsic apoptosis [58]. We investigated the in-volvement of mPTP by inhibiting CypD binding with cyclosporine A (CsA), as previously reported [12]; however, since CsA is also a potent inhibitor of the Pgp efflux pump in the EMT6/AR-1 cells (and would result in JC-1 accumulation), we used EMT6/P cells to investigate the role of mPTP (Fig. 4B) [59]. While both Str-H8R8 and VES-H8R8 de-polarized the mitochondria, in the presence of CsA, mitochondria po-larization was maintained at levels similar to those of DMSO and CsA controls (p > .05), demonstrating the involvement of mPTP induction in the mechanism of action of H8R8-based amphiphiles. The CsA-
    Fig. 3. Mitochondrial uptake and time-dependent
    uptake and retention of the H8R8-based amphi-
    philes. (A) Concentration of the dye retained in
    mitochondria isolated from EMT6/AR-1 cells upon a
    3 h treatment of intact cells with either free dye,
    fluorescein, or fluorescein-labeled H8R8-based pep-
    tides. (B) Concentration of the dye retained in mi-
    tochondria isolated from EMT6/AR-1 cells upon a
    1 h treatment of isolated mitochondria with either
    free dye or fluorescein-labeled H8R8-based peptides.
    Free dye was used as a negative control, whereas
    fluorescein modified H8R8 was used as a peptide
    control. (C) Time-dependent uptake and retention of
    fluorescein-labeled H8R8-based amphiphiles in
    Peptides were removed after 24 h, and the cells were
    incubated with fresh medium for another 24 h. Data
    tical analysis performed using one-way ANOVA and
    sensitive mitochondrial depolarization that we observed is consistent with that of guanidine-containing streptomycin and other cationic amphiphilic peptides, such as melittin and mastoparan [60,61].
    Interestingly, decreased mitochondria membrane fluidity can induce mPTP, as may be the case with VES-H8R8 and Str-H8R8 treatment. This mechanism was observed with mastoparan, which interacts with the
    Fig. 4. H8R8-based amphiphiles depolarize mi-tochondria via a mitochondrial permeability transi-tion pore (mPTP) dependent pathway and alter mi-tochondria bioenergetics. (A) The mitochondria membrane polarization of EMT6/AR-1 cells was measured after a 5 h treatment with Str-H8R8, VES-H8R8, or their controls, using the JC-1 probe and flow cytometry. (B) Using EMT6/P cells, mPTP-de-pendent depolarization was validated by co-in-cubating H8R8-based amphiphiles with cyclosporine A (CsA), an mPTP inhibitor, for 2 h. (C) Mitochondrial bioenergetic states of EMT6/AR-1 cells measured after incubation with H8R8-based amphiphiles for 2 h. Mitochondrial membrane po-tential and oxygen consumption rates are normal-ized to DMSO treated cells. Data are presented as mean ± SD (n = 3) and statistical analyses were performed using one-way ANOVA and Tukey's multiple comparison test (N.S. p > .05, *p < .05, **p < .01, ***p < .001).
    lipid phase of the mitochondrial membrane [62,63]. VES alone can also induce mPTP, further supporting this mechanism for the VES-H8R8 treatment. Interestingly, with cationic polymers of higher molecular weight, such as PEI (> 25 kDa), CsA treatment did not maintain mi-tochondria polarization, possibly because PEI itself may form pores in the mitochondria membranes, allowing solutes to leak out [12,64].
    Mitochondria bioenergetics were studied by measuring mitochon-drial respiratory states in intact EMT6/AR-1 cells treated with H8R8-based amphiphiles using real-time measurements of oxygen consump-tion rates (OCR), as previously reported (Fig. 4C and Fig. S6B) [65]. Oligomycin was used to inhibit the mitochondrial F0/F1-ATP synthase, representing mitochondrial proton leak across the mitochondria inner membrane as a proxy for ATP production. Carbonylcyanide-p-tri-fluoromethoxyphenylhydrazone was used to measure the highest ca-pacity of the electron transport chain, providing the maximum re-spiratory rate. All mitochondrial bioenergetics states of EMT6/AR-1 cells were significantly inhibited when treated with H8R8-based am-phiphiles (Fig. 4C). Following a 2 h treatment, the basal respiration of EMT6/AR-1 cells were significantly inhibited compared to DMSO con-trols (p < .001). Furthermore, both cationic amphiphiles significantly inhibited proton leak, ATP production, and maximum respiratory rate relative to DMSO controls, which was expected as the proton current generated by basal respiration supports proton leak and ATP production (p < .001) [65]. Similar results were obtained in the parental cell line, EMT6/P (Fig. S6C). A decrease in ATP production directly affects Pgp-mediated drug efflux. For example, curcumin inhibited ATP production in doxorubicin resistant MCF7 cells, which led to efflux inhibition, and increased accumulation and retention of doxorubicin [30]. The changes in mitochondrial respiration states are consistent with other studies using cationic polymers where, for example, H1299 and C2C12 cells treated with linear or branched PEI had decreased basal respiration, proton leak, maximum respiration rate, and ATP production [22].