A Helical Polypeptide‐Based Potassium Ionophore Induces Endoplasmic Reticulum Stress‐Mediated Apoptosis by Perturbing Ion Homeostasis

Abstract Perturbation of potassium homeostasis can affect various cell functions and lead to the onset of programmed cell death. Although ionophores have been intensively used as an ion homeostasis disturber, the mechanisms of cell death are unclear and the bioapplicability is limited. In this study, helical polypeptide‐based potassium ionophores are developed to induce endoplasmic reticulum (ER) stress‐mediated apoptosis. The polypeptide‐based potassium ionophores disturb ion homeostasis and then induce prolonged ER stress in the cells. The ER stress results in oxidative environments that accelerate the activation of mitochondria‐dependent apoptosis. Moreover, ER stress‐mediated apoptosis is triggered in a tumor‐bearing mouse model that suppresses tumor proliferation. This study provides the first evidence showing that helical polypeptide‐based potassium ionophores trigger ER stress‐mediated apoptosis by perturbation of potassium homeostasis.


Poly(N ε -Cbz-L-lysine-random-4-hydroxybenzoyl-L-lysine)
4-acetoxybenzoyl chloride was prepared with a slight modification of a previously reported procedure. Poly(N ε -Cbz-L-lysine-random-L-lysine) (1.00 g) was dissolved in anhydrous DMF (10 mL) with trimethylamine (6 equivalents of lysine residues) and 4-acetoxybenzoyl chloride (5 equivalents of lysine residues). The reaction mixture was stirred at RT overnight. To remove the unreacted reagents and isolate the intermediate product, the mixture was precipitated with excessive saturated sodium carbonate solution. A brownish solid was isolated by vacuum filtration and washed with deionized water three times. The brownish solid was suspended in 1 N NaOH solution to deprotect the ester group. The transesterification was done at RT overnight. The cloudy solution was slowly treated with 35 wt% HCl solution until a precipitate was observed. A brownish powder was isolated by filtration and washed with deionized water three times. The brownish powder was lyophilized to obtain poly(N ε -Cbz-L-lysine-random-4-hydroxybenzoyl-L-lysine) (0.93 g). The degree of modification was more than 90%, as evaluated by NMR spectroscopy. ( Figure S4)

Amidation
Prior to preparing the amidating reagent, carboimmide chemistry was used to convert the carboxylic acid group to the NHS ester group. Briefly, acetic acid, butyric acid, hexanoic acid, or octanoic acid solubilized in DMF (5 mL) was converted to the N-hydroxysuccinimide (NHS) ester form using 1-(3-dimethylaminopropyl)-3ethylcarbodiimide (EDC) HCl (3 equivalents of carboxylic acid groups) and NHS (2 equivalents of carboxylic acid groups). The corresponding R-NHS ester (3 equivalents of the lysine residues) was solubilized with TEA which was slowly poured into the AIP solution (2 mL, 1.2 mg/mL in deionized water). The amidation reaction was carried out at RT for 1 day. The corresponding product was isolated by centrifugation and then washed with deionized water three times. The polypeptide was treated with 0.1 N HCl (100 μL) to protonate the product and then dialyzed against deionized water to remove the excessive HCl before lyophilization.

FITC-labelling
AIP (0.2 g) was solubilized in PBS (5 mL) with FITC (3 mL, 0.7 mg/mL in DMSO, Tokyo Chemical Industry, Co., LTD., Japan), and then, the reaction mixture was stirred at RT for 1 day. The unreacted FITC and DMSO were removed by a dialysis method (Molecular weight cutoff: 3500 g/mol, SPECTRUMLABS, USA) before the solution was freeze-dried to obtain FITC-labelled AIP. The FITC-labelled AIP series were synthesized with the method described above.

CD characterization CD spectrometry with different potassium levels
All the AIP samples were solubilized in various concentrations of KCl solutions (0.15, 1.5, 15 and 150 mM). The final concentration was adjusted to 1 mg/mL. CD spectrometry was performed by the same procedure described in the manuscript.

LUV preparation
In a 100 mL dry round bottom flask, EYPC (20 mg/mL in chloroform, Sigma Aldrich, USA) was dried by a rotary evaporator at RT to form a thin phospholipid film. 1 To completely remove the chloroform, the thin film was evaporated under reduced pressure overnight before the buffer (1 mL in 10 mM HEPES and 100 mM KCl) was added while a magnetic bar was stirred at 1000 rpm for 1 h. The lipid solution was rapidly frozen in liquid nitrogen and then melted at RT. The freeze-thaw cycle was repeated 12 times. Extrusion was carried out 19 times by a mini extruder with a polycarbonate membrane (pore size 200 nm, Avanti).

AIP-Lipid CD spectrometry
Prior to the CD measurement, LUVs were prepared in HEPES-buffered solution (10 mM HEPES, 50 mM NaCl, 50 mM KCl). The AIPs solubilized in HEPES-buffered solution were mixed with the LUVs at different molar ratios (100;1, 50:1, 25:1 and 10:1). CD spectrometry was performed by the same procedure described in the manuscript.

HPTS-loaded LUV preparation
A thin phospholipid film was hydrated with HEPES-buffered solution (1 mL in 1 mM HPTS (Sigma Aldrich, USA), 100 mM MCl, and 10 mM HEPES). 1 The free-thaw cycles were repeated 10 times before lipid vesicles were extruded using a mini extruder ( Saturated fluorescence intensity after the addition of 0.1% triton-X100).

Cation selectivity
HPTS-loaded LUV solution (100 μL in 10 mM HEPES, 100 mM MCl, M; Li, Na, K, and Cs) was added to a well (96 well plate). The corresponding treatment of 0.5 N MOH solution (2 μL) was done to form a pH-gradient between the outside and inside of the lipid vesicles. Time-dependent fluorescence intensity was monitored every second until 200 sec. prior to the lysis of the lipid vesicle using a 0.1% triton-X100 solution. Each IF was obtained by the procedure described above.

Anion selectivity
HPTS-loaded LUV solution (100 μL in 10 mM HEPES, 100 mM KX, X; F, Cl, Br, and I) was added to a well (96 well plate). 0.5 N KOH solution (2 μL) was added to each well. Time-dependent fluorescence intensity was measured every second until 200 sec. before the lysis of the LUVs using a 0.1% triton-X100 solution. Each IF was obtained by the procedure described above.

Single channel analysis
A planar lipid bilayer membrane was prepared as in our previous work 2, 3 . In this experiment, 3% DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids, Alabaster, AL, USA) dissolved in n-decane (MP Biomedicals, Irvine, CA, USA) was used to form a lipid bilayer on an 50-80 μm aperture in a 10-μm-thick PTFE film (Goodfellow, Huntingdon, UK). The aperture was fabricated using a spark generator (DAEDALON, Salem, MA, USA). The lipid solution was spread around the aperture of the PTFE film and was then dried for 30 min.
The PTFE film was placed between two chambers, and both chambers were filled with electrolyte solution (

Demonstration of AIPs binding to cell membranes
NCI-H460 cells (15000 cells/well in a 12 well plate) were treated with FITC-labelled AIPs (25 nM  Alexa fluor 488-tagged secondary antibody solution (1% BSA, 0.3% Triton X-100 in PBS, goat anti-rabbit; 1000:1) was added to the wells and incubated for 2 h. In the last step, all the nuclei were stained with DAPI (300 nM in PBS) for 10 min.

Visualization of apoptotic nuclear fragmentation
NCI-H460 cells (80000 cells/well in a 24 well plate) seeded onto a coverslip for a 24 well plate were incubated with the AIP series (0.25 μM) for 12 h. The cells were fixed with 4% para-formaldehyde solution for 10 min. after washing with PBS three times. Thereafter, the cells were stained with DAPI (300 nM) for 10 min. Apoptotic nuclear fragmentation was visualized by CLSM.

VI.
In vivo experiments

Biodistribution study
The formation of tumor xenografts were done as described above (n = 3). When the tumor volume reached approximately 150 mm 3 , IR800CW-labelled AIPs (2 mg/kg) were intravenously injected into the mice. All the NIRF signals were detected at predetermined time points (1, 3, 6, 12, 24, and 48 h). After 2 days, all the organs in each group (tumor, heart, lung, liver, spleen and kidney) were excised, and the NIRF signals were analyzed by a Pearl® Impulse Small Animal Imaging System (LI-COR, Lincoln, NE).

Blood compatibility
Blood (1 mL) obtained from a mouse was centrifuged at 1200 g for 10 min. before the removal of the supernatant.

Assessment of in vivo toxicity
To assess the liver and kidney toxicity, serum samples were collected at 2 days post-injection, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine were analyzed by Seegene (Seoul, Korea).

Immunoblotting of angiogenesis-related proteins in vitro
Electrophoresis was conducted by loading 50 μg of proteins into each well of SDS-PAGE gel before the proteins were transferred to a PVDF membrane (0.2 μm pore size PVDF membrane, Roche, Swiss). The PVDF membrane was treated with the primary antibody solutions (MMP-2; 1000:1, anti-rabbit polyclonal, Cell Signaling Technology, USA, MMP-9; 1000:1, Cell Signaling Technology, Abcam, USA, VEGF; 1000:1, anti-rabbit polyclonal, SantaCruz Biotech, USA). Thereafter, the PVDF membranes were treated with the secondary antibody solutions. The blot signals were visualized by an ECL reagent. Figure S1. Synthetic scheme of AIP series.                             Figure S33. Immunohistochemical assays of the harvested tumor sections for CD31 used as a blood vessel marker. All images were taken by an optical microscope (magnification: 400 X). Figure S34. Demonstration of angiogenesis-related proteins in vitro (VEGF: vascular endothelial growth factor, MMP-2: matrix metalloproteinase-2, MMP-9: matrix metalloproteinase-9) via immunoblotting. GADPH was used as a loading control.