Induction of Paraptotic Cell Death in Cancer Cells by Triptycene-Peptide Hybrids and the Revised Mechanism of...

In previous work, we reported on iridium(III) (Ir(III)) complex-peptide hybrids as amphiphilic conjugates (IPH-ACs) and triptycene-peptide hybrids as amphiphilic conjugates (TPH-ACs) and found that these hybrid compounds containing three cationic KK(K)GG peptide units through C₆-C₈ alkyl linkers induce paraptosis II, which is one of the nonapoptotic programmed cell death (PCD) types in Jurkat cells and different from previously reported paraptosis. The details of that study revealed that the paraptosis II induced by IPH-ACs (and TPH-ACs) proceeds via a membrane fusion or tethering of the endoplasmic reticulum (ER) and mitochondria, and Ca²⁺ transfer from the ER to mitochondria, which results in a loss of mitochondrial membrane potential (ΔΨ_m) in Jurkat cells. However, the detailed mechanistic studies of paraptosis II have been conducted only in Jurkat cells. In the present work, we decided to conduct mechanistic studies of paraptosis II in HeLa-S3 and A549 cells as well as in Jurkat cells to study the general mechanism of paraptosis II. Simultaneously, we designed and synthesized new TPH-ACs functionalized with peptides that contain cyclohexylalanine, which had been reported to enhance the localization of peptides to mitochondria. We found that TPH-ACs containing cyclohexylalanine promote paraptosis II processes in Jurkat, HeLa-S3 and A549 cells. The results of the experiments using fluorescence Ca²⁺ probes in mitochondria and cytosol, fluorescence staining agents of mitochondria and the ER, and inhibitors of paraptosis II suggest that TPH-ACs induce Ca²⁺ increase in mitochondria and the membrane fusion between the ER and mitochondria almost simultaneously, suggesting that our previous hypothesis on the mechanism of paraptosis II should be revised.