Induction of Paraptosis by Cyclometalated Iridium Complex-Peptide Hybrids and CGP37157 via a Mitochondrial Ca²⁺Overload Triggered by Membrane Fusion between Mitochondria and the Endoplasmic Reticulum

Abstract

We previously reported that a cyclometalated iridium (Ir) complex-peptide hybrid (IPH) 4 functionalized with a cationic KKKGG peptide unit on the 2-phenylpyridine ligand induces paraptosis, a relatively newly found programmed cell death, in cancer cells (Jurkat cells) via the direct transport of calcium (Ca 2+ ) from the endoplasmic reticulum (ER) to mitochondria. Here, we describe that CGP37157, an inhibitor of a mitochondrial sodium (Na + )/Ca 2+ exchanger, induces paraptosis in Jurkat cells via intracellular pathways similar to those induced by 4 . The findings allow us to suggest that the induction of paraptosis by 4 and CGP37157 is associated with membrane fusion between mitochondria and the ER, subsequent Ca 2+ influx from the ER to mitochondria, and a decrease in the mitochondrial membrane potential ( ΔΨ m ). On the contrary, celastrol, a naturally occurring triterpenoid that had been reported as a paraptosis inducer in cancer cells, negligibly induces mitochondria-ER membrane fusion. Consequently, we conclude that the paraptosis induced by 4 and CGP37157 (termed paraptosis II herein) proceeds via a signaling pathway different from that of the previously known paraptosis induced by celastrol, a process that negligibly involves membrane fusion between mitochondria and the ER (termed paraptosis I herein).

Design, Synthesis, and Anticancer Activity of Triptycene–Peptide Hybrids that Induce Paraptotic Cell Death in Cancer Cells

“…Paraptosis is a type of programmed cell death (PCD) and is characterized by cytoplasmic vacuolation derived from the swelling of intracellular organelles such as mitochondria and the ER. We previously reported that IPHs 1 – 4 and celastrol (Scheme ), a triterpenoid isolated from Tripterygium wilfordii, induce paraptosis via an influx of Ca 2+ from the ER into mitochondria, the damage of organelles such as mitochondria, ER, and lysosomes, cytoplasmic vacuolization, ,,, as evidenced by the transmission electron microscopic (TEM) analysis and staining experiments with methylene blue . Thus, Jurkat cells were treated with syn- 6 or anti- 6 (5 μM) for 1 h at 37 °C under 5% CO 2 and fixed with glutaraldehyde and osmium tetroxide (OsO 4 ).…”

“…The aforementioned results strongly suggest that TPHs ( syn- 6 and anti- 6 ), like 4 , induce paraptotic cell death. Because it has recently been reported that the transport of Ca 2+ from the ER to mitochondria is crucial in paraptosis induced by IPHs, , we examined the concentrations of Ca 2+ in mitochondria and the cytosol by flow cytometric analysis (Figure a,b) . Jurkat cells were preincubated with Rhod-2/AM (5 μM), an indicator of mitochondrial Ca 2+ , or Rhod-4/AM (5 μM), an indicator of cytosolic Ca 2+ , for 30 min and then treated with syn- 6 or anti- 6 (5 μM) for given incubation times (0, 15, 30, 45, 60 min).…”

“…It is known that programmed cell death is related to a malfunction of intracellular organelles such as mitochondria, the endoplasmic reticulum (ER), and lysosomes. − Our previous reports indicated that 4 induces the degradation of ER and lysosomes . Therefore, we carried out staining experiments of Jurkat cells with MitoTracker Green (a mitochondrial probe), ERTracker Red (an ER probe), and LysoTracker Red (a lysosomal probe) to examine the effect of TPHs on these intracellular organelles.…”

“…The flow cytometry results shown in Figure suggest that Ca 2+ influx into mitochondria is related to the cell death that is induced by syn- 6 and anti- 6 . We next measured mitochondrial membrane potential (MMP, ΔΨ m ) using 1,1′,3,3,3′,3′-hexamethylindodicarbocyanine iodide (DilC1(5)) , (the structure of DilC1(5) is shown in Chart S3), which is accumulated in mitochondria. After incubating Jurkat cells with syn- 6 and anti- 6 (5 μM) for 1 h, the emission of DilC1(5) was decreased compared to the control (Figure e,f,h,i vs Figure b,c), implying that the ΔΨ m is decreased in TPH-induced paraptosis.…”

“…The EC 50 values for 1 , 2 , and 3 that contain three, two, and one KKKGG peptide sequences, respectively, against Jurkat cells were determined to be 1.5, 4.1, and 16.3 μM, respectively, suggesting the existence of a positive relationship between the number of peptides (and positive charges) and anticancer activity. It was also found that IPH 4 containing three KKKGG units at the 4′-position of ppy (2-phenylpyridine) ligands through a C 8 alkyl linker also induces cell death in Jurkat cells . A detailed study of the mechanism for the cell death induced by 1 – 4 suggested that these IPHs induce paraptotic cell death, one of the non-caspase-dependent PCDs, in Jurkat cells.…”