Elsevier

Biomaterials

Volume 197, March 2019, Pages 51-59
Biomaterials

Development of apoptosis-inducing polypeptide via simultaneous mitochondrial membrane disruption and Ca2+ delivery

https://doi.org/10.1016/j.biomaterials.2019.01.006Get rights and content

Abstract

Mitochondria are the primary organelle of regulating apoptosis, and intracellular calcium ions are a key component of pro-apoptosis induction. Herein, we report an artificial apoptosis-inducing polypeptide that destabilizes the mitochondrial membrane and transports calcium ions into the cytosol, thereby synergistically creating severe oxidative conditions. The oxidative stress highly activates an apoptotic signaling cascade, and also inhibits cell migration and invasion in vitro and in vivo. The suggested strategy for simultaneous mitochondrial disruption and perturbed calcium homeostasis demonstrates the applicability of polypeptide-based therapeutics as potent apoptosis-inducers in cancer therapy.

Introduction

Mitochondria not only play a critical role in cell metabolism by producing biological energy but also act as headquarters of apoptosis regulation by modulating several apoptotic signaling pathways. The dysfunction of mitochondria initiates apoptotic biochemical cascades by stimulation of oxidative conditions and the release of apoptosis-mediating proteins such as cytochrome C and apoptosis-inducing factor (AIF) [[1], [2], [3]]. For these reasons, targeting and disruption of mitochondria have been employed as a promising strategy for inducing apoptosis, especially in cancer therapy [[4], [5], [6], [7], [8], [9], [10], [11]]. In order to implement this strategy, both cationic and lipophilic properties are simultaneously endowed in therapeutic molecules [9]. In previous studies, cationic amphipathic helical polypeptides (CAHPs) were used as an apoptosis-inducing agent, targeting and destabilizing the outer mitochondrial membrane (OMM) [[12], [13], [14]]. CAHPs effectively targeted mitochondria due to their lipocationic characteristics and then disturbed OMM, thereby initiating the onset of pro-apoptosis.

The inundation of intracellular Ca2+ ions is another pathway to permeabilize the OMM, accelerating the malfunction of mitochondria [[15], [16], [17], [18], [19]]. The Ca2+ electrochemical potential is maintained by various ion channels, pumps, and exchangers to modulate various cell signaling pathways [20]. Ca2+ signaling is closely relevant to the production of reactive oxygen species (ROS) regulating cell proliferation and apoptosis [21]. The perturbed Ca2+ homeostasis not only exerts severe oxidative stress but also imposes Ca2+ stress on mitochondria, which act as Ca2+ stores, thereby having a detrimental effect on mitochondria and their membranes. Therefore, both OMM destabilization and perturbation of Ca2+ homeostasis lead to severe dysfunction of mitochondria, resulting in remarkable apoptosis.

In this study, we developed a novel ROS-cleavable apoptosis-inducing peptide (RAP) with sidechain modification of an artificial cationic α-helical polypeptide. RAP can induce apoptosis by disrupting the mitochondrial outer membrane and transporting Ca2+ into the cell, simultaneously. RAP consists of a mitochondria-destabilizing moiety (MDM), a Ca2+ ion-delivering moiety (CDM), and a ROS-cleavable linker between these two moieties (Fig. 1). A trimethyl ammonium group with high mitochondria-localizing capability was attached to the outskirt of the MDM to elicit the mitochondria-targeting ability [7]. The side chains of MDM were elongated with alkyl chains and benzene rings to increase hydrophobic interactions to counteract electrostatic repulsion between trimethyl ammonium groups. Reduced electrostatic repulsion facilitates the formation of a stable α-helical structure of MDM, which is required to impart membrane destabilizing ability to RAP. Diethylenetriaminepentaacetic acid (DTPA), a Ca2+ chelator, was conjugated to the side chains of the CDM [22]. DTPA can chelate the extracellular Ca2+ with five carboxylates and three amines and convey Ca2+ into the cell to elevate intracellular Ca2+ concentration. Finally, we designed a ROS-cleavable linker vulnerable to the intracellular ROS-rich environment to connect the two moieties until cell internalization. After RAP was internalized in the cell, the linker was degraded and subsequently the two moieties were separated [23,24]. This ROS-triggered separation enhanced mobility and activity of MDM.

We hypothesized that RAP strongly wreaks damage to mitochondria via simultaneous mitochondrial disruption and intracellular Ca2+ overload. RAP is internalized into cells using membrane destabilizing ability of MDM and degraded into MDM and CDM by cleavage of the linker. MDM targets mitochondria and destabilizes OMM owing to trimethyl ammonium group and α-helical structure, respectively. CDM induces calcium stress of the cells by delivering Ca2+ into the cytosol. Destabilized mitochondria and perturbed Ca2+ homeostasis prompt overproduction of ROS and release of pro-apoptotic proteins. Consequently, synergistic apoptosis is elicited as a result of apoptotic signaling cascades (Fig. 1). In addition, overproduced ROS inhibits the expression of caveolin-1 (cav-1) [25], which has a critical effect on tumor cell migration and invasion by regulating focal adhesion dynamics [26]. Subsequently, the expression of matrix metalloproteinase-2/9 (MMP-2/9) is suppressed and migration as well as invasion of cancer cells is inhibited.

A novel apoptosis-inducing polypeptide RAP was designed to simultaneously destabilize OMM and increase cytosolic Ca2+ stress. To evaluate the functionality of each moiety of RAP, we additionally synthesized three moiety-deficient peptides as control groups: mitochondria-destabilizing peptide (MDP), Ca2+ ion-delivering peptide (CDP), and non-cleavable apoptosis-inducing peptide (NAP) (Fig. S1). The framework of each apoptosis-inducing polypeptide (AIP) was synthesized by N-carboxyanhydride polymerization and the desired moieties were attached to the peripheral side chains by several stepwise reactions (Fig. S1). Detailed synthesis procedures and characterization of AIPs are presented in the supporting information (Figs. S1–4, Table S1).

Section snippets

Synthesis of AIPs

Synthesis methods of AIPs are described in supporting information in detail (Fig. S1).

Determination of secondary structure

Each AIP was dissolved in water at 1 mg/mL and analyzed by a circular dichroism (CD) spectrometer (J-815 spectropolarimeter 150-L type, JASCO, Japan). CD spectra were measured with a quartz cell with 0.01 mm path length in the range of 200 nm–260 nm at RT. Calcium-chelatable polypeptides (CDP, NAP, and RAP) were treated in the same manner as described in the verification of calcium-chelating ability, and

Characterizations

Protein secondary structures of AIPs with variations in Ca2+ and ROS level were evaluated using CD spectrometry. MDM was designed to retain a stable α-helical conformation of polypeptides by maintaining a balance between charge repulsion and hydrophobic interaction. MDP, NAP, and RAP, containing MDM, were confirmed to possess an α-helical structure; however, CDP, lacking MDM, could not form an α-helical structure but rather a random-coil structure (Fig. 2a). To demonstrate the Ca2+-dependency

Discussion

Disrupting mitochondria as a cancer therapy has recently drawn intensive attention from a number of research groups [8,10,11]. Mitochondria-targeting and -destabilizing agents are able to induce apoptosis of cancer cells by releasing apoptosis-mediating proteins from the mitochondrial intermembrane space [10,11]. In addition, disturbing calcium ion homeostasis can provide an outstanding strategy to reinforce the apoptosis-inducing ability of the former cancer therapy strategy [17,18]. This is

Conclusions

In conclusion, we developed apoptosis-inducing polypeptide triggering simultaneous disruption of OMM and perturbed Ca2+ homeostasis. Destabilized OMM and increased cytosolic Ca2+ stress synergistically generated ROS and severely created an oxidative environment. An apoptosis-inducing cascade was initiated by multiple pro-apoptotic signals: overproduced ROS, Ca2+ stress, and release of pro-apoptotic peptides from disrupted OMM. To maximize the functionality of each moiety, the ROS-cleavable

Acknowledgement

This work was financially supported by the Ministry of Science and ICT of Korea (Project No. NRF-2014M3A9E4064580, NRF-2016R1A2B4009619, NRF-2018M3A9E2024583 to Y.C. Kim; NRF-2016M3A9B594235 to C.O. Yun) and the Animal and Plant Quarantine Agency, Gimcheon, Gyenongsangbuk-do, Republic of Korea (N04130146).

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