Induction of\nParaptotic Cell Death in Cancer Cells\nby Triptycene–Peptide Hybrids and the Revised Mechanism of\nParaptosis II

In previous work, we reported on iridium(III) (Ir(III))\ncomplex-peptide\nhybrids as amphiphilic conjugates (IPH-ACs) and triptycene-peptide\nhybrids as amphiphilic conjugates (TPH-ACs) and found that these hybrid\ncompounds containing three cationic KK(K)GG peptide units through\nC<sub>6</sub>–C<sub>8</sub> alkyl linkers induce paraptosis\nII, which is one of the nonapoptotic programmed cell death (PCD) types\nin Jurkat cells and different from previously reported paraptosis.\nThe details of that study revealed that the paraptosis II induced\nby IPH-ACs (and TPH-ACs) proceeds via a membrane fusion or tethering\nof the endoplasmic reticulum (ER) and mitochondria, and Ca<sup>2+</sup> transfer from the ER to mitochondria, which results in a loss of\nmitochondrial membrane potential (<i>ΔΨ</i><sub>m</sub>) in Jurkat cells. However, the detailed mechanistic studies\nof paraptosis II have been conducted only in Jurkat cells. In the\npresent work, we decided to conduct mechanistic studies of paraptosis\nII in HeLa-S3 and A549 cells as well as in Jurkat cells to study the\ngeneral mechanism of paraptosis II. Simultaneously, we designed and\nsynthesized new TPH-ACs functionalized with peptides that contain\ncyclohexylalanine, which had been reported to enhance the localization\nof peptides to mitochondria. We found that TPH-ACs containing cyclohexylalanine\npromote paraptosis II processes in Jurkat, HeLa-S3 and A549 cells.\nThe results of the experiments using fluorescence Ca<sup>2+</sup> probes\nin mitochondria and cytosol, fluorescence staining agents of mitochondria\nand the ER, and inhibitors of paraptosis II suggest that TPH-ACs induce\nCa<sup>2+</sup> increase in mitochondria and the membrane fusion between\nthe ER and mitochondria almost simultaneously, suggesting that our\nprevious hypothesis on the mechanism of paraptosis II should be revised.