Characterization of antilytic peptide antibody: application for the detection of lytic‐based hybrid peptide in serum samples

We previously reported that a novel targeted drug termed hybrid epidermal growth factor receptor (EGFR)‐lytic peptide, made by chemical conjugation of targeted binding peptide and cell‐killing, lytic‐peptide components, has selective cytotoxic activity that allows it to discriminate between normal and cancer cells. In addition, in vivo analysis revealed that this hybrid peptide displays significant antitumor activity in a xenograft model of human breast and pancreatic cancer in mice. Here, we characterized antilytic peptide antibody, which was raised from rabbit serum using the antigen of lytic peptide conjugated with keyhole limpet hemocyanin. It was found that antilytic peptide antibody is specific to the lytic peptide as assessed by both ELISA and surface plasmon resonance analysis and can also bind to EGFR‐lytic peptide. Epitope mapping analysis using Biacore showed that two successive lysine regions in the lytic‐peptide sequence are significant for recognition by this antibody. In addition, it was shown that this antibody can detect lytic‐based hybrid peptide in serum samples from mouse blood and also in cultured breast cancer MDA‐MB‐231 cell samples by immunocytochemical staining experiments. It was found that the maximum concentrations of this peptide in serum were reached within 15–30 min of i.v. administration of EGFR‐lytic peptide to mice. These results indicate that this antibody will be a useful tool for the detection of lytic‐based peptides to investigate their in vivo stability and pharmacokinetics. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Semaphorin 3A lytic hybrid peptide binding to neuropilin-1 as a novel anti-cancer agent in pancreatic cancer

We previously reported that novel targeted “hybrid peptide” in which epidermal growth factor receptor (EGFR) binding peptide was conjugated with lytic-type peptide had selective cytotoxic activity to EGFR expressing cancer cells. In this study, we have generated a novel type hybrid peptide, semaphorin 3A lytic (Sema3A-lytic), which is composed of two functional amino acid domains: a sequence derived from Sema3A that binds to neuropilin-1 (NRP1) and a cytotoxic lytic peptide. We found that this hybrid peptide had cytotoxic activity against NRP1-positive pancreatic cancer cell lines such as BxPC-3 and Panc-1, whereas the peptide did not affect the viability of normal cells in vitro. It was also found by affinity analysis that Sema3A peptide binds to NRP1, and two arginines (372R and 377R) in Sema3A peptide are involved in the interaction with NRP1 protein. In addition, confocal microscopy analysis revealed that Sema3A-lytic peptide could not penetrate normal cells regardless of the presence of NRP1 mRNA, suggesting that the ability of Sema3A-lytic peptide to concentrate adjacent to the cell membrane by binding to NRP1 with the target-binding moiety contributes to its selective cytotoxic activity. These results indicate that Sema3A-lytic hybrid peptide would be a possible anti-cancer agent for treatment of human pancreatic cancer.     

Peptide ligand‐mediated endocytosis of nanoparticles to cancer cells: Cell receptor‐binding‐ versus cell membrane‐penetrating peptides

The endocytosis‐mediating performances of two types of peptide ligands, cell receptor binding peptide (CRBP) and cell membrane penetrating peptide (CMPP), were analyzed and compared using a common carrier of peptide ligands‐human ferritin heavy chain (hFTH) nanoparticle. Twenty‐four copies of a CMPP(human immunodeficiency virus‐derived TAT peptide) and/or a CRBP (peptide ligand with strong and specific affinity for either human integrin(α_vβ₃) or epidermal growth factor receptor I (EGFR) that is overexpressed on various cancer cells) were genetically presented on the surface of each hFTH nanopariticle. The quantitative level of endocytosis and intracellular localization of fluorescence dye‐labeled CRBP‐ and CMPP‐presenting nanoparticles were estimated in the in vitro cultures of integrin‐ and EGFR‐overexpressing cancer and human dermal fibroblast cells(control). From the cancer cell cultures treated with the CMPP‐ and CRBP‐presenting nanoparticles, it was notable that CRBPs resulted in quantitatively higher level of endocytosis than CMPP (TAT) and successfully transported the nanoparticles to the cytosol of cancer cells depending on concentration and treatment period of time, whereas TAT‐mediated endocytosis localized most of the nanoparticles within endosomal vesicles under the same conditions. These novel findings provide highly useful informations to many researchers both in academia and in industry who are interested in developing anticancer drug delivery systems/carriers.     

Entry of a cationic lytic-type peptide into the cytoplasm via endocytosis-dependent and -independent pathways in human glioma U251 cells

Cationic lytic-type peptides have been studied for clinical application in various infections and cancers. This study aimed to determine the functions of our specially designed lytic peptide. To investigate the functional mechanism at the cell membrane level, we used giant unilayer vesicles (GUVs) mimicking cell membranes. In GUVs treated with FITC-labeled lytic peptide (lytic-FITC), fluorescence increased in a time-dependent manner. However, no inner fluorescence was detected in GUVs treated with lytic peptide and calcein. Next, distribution of lytic-FITC peptide on the cell membrane and in the cytoplasm was examined in a living human glioma U251 cell line. In the immunocytochemical study, some lytic peptide stains colocalized with early endosome antigen protein 1 (EEA-1). In cells treated with lytic peptide, the immunofluorescence intensity of lytic peptide increased in a concentration and treatment time-dependent manner. Cytotoxic activity of lytic peptide decreased after pretreatment with the endocytosis inhibitors cytochalasin D, chlorpromazine and amiloride. These findings suggest that lytic peptide exerts cytotoxic activity after cellular uptake via an endocytosis pathway. In conclusion, the influx mechanism of lytic peptide was shown to include not only disintegration and pore formation at the cell membrane, but also cell entry via endocytosis dependent and independent pathways.     

Mitochondrially localized EGFR is independent of its endocytosis and associates with cell viability

The molecular mechanism underlying epidermal growth factor receptor (EGFR) localization in mitochondria remains largely unknown. Using immune electron microscopy, we validated that EGFR could be localized on either the outer or the inner membrane of mitochondria. Mutant receptor lacked amino acids 646-660 was flawed in migration onto the organelles, whereas the mutated receptor with a defective endocytosis showed a greater capability of moving onto mitochondria upon stimulation of epidermal growth factor (EGF). Gefitinib, an inhibitor of EGFR kinase, inhibited the receptor endocytosis after short time of treatment, yet, only reduced cell viability as well as the amount of mitochondrial EGFR after longer time of exposure. Moreover, the content of mitochondrial EGFR transfer was decreased when the cells were exposed to the apoptotic inducer etoposide. EGF-induced programmed cell death usually coincided with a decline in mitochondrial EGFR. These data indicated that the mitochondrial-localized EGFR is independent of its internalization and may be correlated with cell survival and participate in the ligand-induced programmed cell death.     

Bovine lactoferricin causes apoptosis in Jurkat T-leukemia cells by sequential permeabilization of the cell membrane and targeting of mitochondria

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that kills Jurkat T-leukemia cells by the mitochondrial pathway of apoptosis. However, the process by which LfcinB triggers mitochondria-dependent apoptosis is not well understood. Here, we show that LfcinB-induced apoptosis in Jurkat T-leukemia cells was preceded by LfcinB binding to, and progressive permeabilization of the cell membrane. Colloidal gold electron microscopy revealed that LfcinB entered the cytoplasm of Jurkat T-leukemia cells prior to the onset of mitochondrial depolarization. LfcinB was not internalized by endocytosis because endocytosis inhibitors did not prevent LfcinB-induced cytotoxicity. Furthermore, intracellular delivery of LfcinB via fusogenic liposomes caused the death of Jurkat T-leukemia cells, as well as normal human fibroblasts. Collectively, these findings suggest that LfcinB caused damage to the cell membrane that allowed LfcinB to enter the cytoplasm of Jurkat T-leukemia cells and mediate cytotoxicity. In addition, confocal microscopy showed that intracellular LfcinB co-localized with mitochondria in Jurkat T-leukemia cells, while flow cytometry and colloidal gold electron microscopy showed that LfcinB rapidly associated with purified mitochondria. Furthermore, purified mitochondria treated with LfcinB rapidly lost transmembrane potential and released cytochrome c. We conclude that LfcinB-induced apoptosis in Jurkat T-leukemia cells resulted from cell membrane damage and the subsequent disruption of mitochondrial membranes by internalized LfcinB.     

Mitochondrially localized EGFR is subjected to autophagic regulation and implicated in cell survival

Although generally acknowledged as a plasma membrane protein, the epidermal growth factor (EGF) receptor has been found in the nucleus and subcellular organelles. Recently, the mitochondrial localization of the EGF receptor (EGFR) was reported; nevertheless, the molecular mechanism underlying EGFR localization in mitochondria is largely unknown. Using immunofluorescence and immunoelectron microscopy, we observed that EGFR did localize within mitochondria. Moreover, EGFR mitochondrial translocation can be increased by rapamycin treatment in A431 cells and greatly reduced by the presence of 3-methyladenine (3-MA), an inhibitor of autophagy. The reduction of mitochondrial EGFR via autophagy inhibition is further confirmed by small interference RNA (siRNA), through which the essential protein Beclin 1 was depleted. Knocking down Beclin 1 markedly decreased the mitochondrial translocation of EGFR that was induced by rapamycin. We also noticed that the content of mitochondrial EGFR transfer is decreased when the cells are exposed to the apoptotic inducer etoposide. Additionally, either EGF treatment or EGFR knockdown by siRNA results in a greater decline of cell viability in cells possessing more mitochondrial EGFRs. Taken together, we conclude that EGFR mitochondrial localization is regulated by either autophagy or programmed cell death and is correlated with cell survival.     

Mitochondrially localized EGFR is subjected to autophagic regulation and implicated in cell survival

Although generally acknowledged as a plasma membrane protein, the epidermal growth factor (EGF) receptor has been found in the nucleus and subcellular organelles. Recently, the mitochondrial localization of the EGF receptor (EGFR) was reported; nevertheless, the molecular mechanism underlying EGFR localization in mitochondria is largely unknown. Using immunofluorescence and immunoelectron microscopy, we observed that EGFR did localize within mitochondria. Moreover, EGFR mitochondrial translocation can be increased by rapamycin treatment in A431 cells and greatly reduced by the presence of 3-methyladenine (3-MA), an inhibitor of autophagy. The reduction of mitochondrial EGFR via autophagy inhibition is further confirmed by small interference RNA (siRNA), through which the essential protein Beclin 1 was depleted. Knocking down Beclin 1 markedly decreased the mitochondrial translocation of EGFR that was induced by rapamycin. We also noticed that the content of mitochondrial EGFR transfer is decreased when the cells are exposed to the apoptotic inducer etoposide. Additionally, either EGF treatment or EGFR knockdown by siRNA results in a greater decline of cell viability in cells possessing more mitochondrial EGFRs. Taken together, we conclude that EGFR mitochondrial localization is regulated by either autophagy or programmed cell death and is correlated with cell survival.     

10H‐3,6‐Diazaphenothiazines triggered the mitochondrial‐dependent and cell death receptor‐dependent apoptosis pathways and further increased the chemosensitivity of MCF‐7 breast cancer cells via inhibition of AKT1 pathways

Breast cancer is one of the leading causes of death in cancer categories, followed by lung, colorectal, and ovarian among the female gender across the world. 10H‐3,6‐diazaphenothiazine (PTZ) is a thiazine derivative compound that exhibits many pharmacological activities. Herein, we proceed to investigate the pharmacological activities of PTZ toward breast cancer MCF‐7 cells as a representative in vitro breast cancer cell model. The PTZ exhibited a proliferation inhibition (IC₅₀ = 0.895 µM) toward MCF‐7 cells. Further, cell cycle analysis illustrated that the S‐phase checkpoint was activated to achieve proliferation inhibition. In vitro cytotoxicity test on three normal cell lines (HEK293 normal kidney cells, MCF‐10A normal breast cells, and H9C2 normal heart cells) demonstrated that PTZ was more potent toward cancer cells. Increase in the levels of reactive oxygen species results in polarization of mitochondrial membrane potential (ΔΨm), together with suppression of mitochondrial thioredoxin reductase enzymatic activity suggested that PTZ induced oxidative damages toward mitochondria and contributed to improved drug efficacy toward treatment. The RT² PCR Profiler Array (human apoptosis pathways) proved that PTZ induced cell death via mitochondria‐dependent and cell death receptor‐dependent pathways, through a series of modulation of caspases, and the respective morphology of apoptosis was observed. Mechanistic studies of apoptosis suggested that PTZ inhibited AKT1 pathways resulting in enhanced drug efficacy despite it preventing invasion of cancer cells. These results showed the effectiveness of PTZ in initiation of apoptosis, programmed cell death, toward highly chemoresistant MCF‐7 cells, thus suggesting its potential as a chemotherapeutic drug.     

Improved mitochondrial targeting effect of HPMA copolymer by SS20 peptide mediation and nonendocytosis pathway

Mitochondrion plays an important role in executing cell programmed death pathway. Therefore, drugs designed to target mitochondria are supposed to make superior contributions to cancer therapy. However, the problem that drugs or drug delivery systems being sequestrated in endosomes/lysosomes needs to be solved for effective drug delivery. Here, mitochondrial targeting and nonendocytic cell entry peptide SS20 modified HPMA copolymer (P‐FITC‐SS20) was synthesized. With SS20 peptide modification, the uptake behavior of HPMA copolymers changed remarkably compared with unmodified ones. The internalization of P‐FITC‐SS20 was not influenced by endocytic inhibitors and temperature. Further, the internalized copolymers were not trapped in endosomes/lysosomes. Although cellular uptake of HPMA copolymer was decreased after SS20 peptide modification, SS20 peptide significantly improved mitochondrial accumulation of HPMA copolymers due to its outstanding mitochondrial targeting ability. Moreover, owing to lower susceptibility to macrophagocyte in blood, P‐SS20‐Cy5 showed longer blood circulation time and enhanced tumor accumulation. The current study validated that SS20 peptide modification is a promising strategy for mitochondrial targeting drug delivery systems and can be further applied to mitochondria associated diseases to improve therapeutic efficacy.     

A Salmonella protein causes macrophage cell death by inducing autophagy

Salmonella enterica, the causative agent of food poisoning and typhoid fever, induces programmed cell death in macrophages, a process found to be dependent on a type III protein secretion system, and SipB, a protein with membrane fusion activity that is delivered into host cells by this system. When expressed in cultured cells, SipB caused the formation of and localized to unusual multimembrane structures. These structures resembled autophagosomes and contained both mitochondrial and endoplasmic reticulum markers. A mutant form of SipB devoid of membrane fusion activity localized to mitochondria, but did not induce the formation of membrane structures. Upon Salmonella infection of macrophages, SipB was found in mitochondria, which appeared swollen and devoid of christae. Salmonella-infected macrophages exhibited marked accumulation of autophagic vesicles. We propose that Salmonella, through the action of SipB, kills macrophages by disrupting mitochondria, thereby inducing autophagy and cell death.     

p35 interacts with α‐tubulin and organelle proteins: Nuclear translocation of p35 in dying cells

We identified heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2, hnRNP A1, the translocase of the transporter outer membrane 40 (TOM40), and α‐tubulin as new interaction partners of anti‐apoptotic protein p35 using MS‐based functional proteomics with GST‐p35 fusion protein as a bait, and using a pull‐down assay with p35‐6His followed by Western blot analysis. p35 was localized in the cytoplasm and in distinct organelles such as the nucleus and mitochondria. p35 was more abundant in the cytoplasm than it was in the nucleus. It co‐localized with α‐tubulin in the cytoplasm in the absence of a death stimulus. However, while cells were undergoing death induced by actinomycin D, cytoplasmic p35 was translocated into the nucleus; this process was inhibited by deletions of the N‐ and C‐terminal domains containing leucine‐rich motifs. Gene delivery of p35 using recombinant adenoviruses inhibited cytoplasmic compartmentalization of hnRNP C1/C2 and hnRNP A1 in dying cells. This study demonstrated translocation of p35 into the nuclei, as well as protection of the hnRNPs from redistribution in cells undergoing death. We propose an active role for p35 in maintaining the integrity of nuclear proteins during cell death.     

killerFLIP: a novel lytic peptide specifically inducing cancer cell death

One of the objectives in the development of effective cancer therapy is induction of tumor-selective cell death. Toward this end, we have identified a small peptide that, when introduced into cells via a TAT cell-delivery system, shows a remarkably potent cytoxicity in a variety of cancer cell lines and inhibits tumor growth in vivo, whereas sparing normal cells and tissues. This fusion peptide was named killerFLIP as its sequence was derived from the C-terminal domain of c-FLIP, an anti-apoptotic protein. Using structure activity analysis, we determined the minimal bioactive core of killerFLIP, namely killerFLIP-E. Structural analysis of cells using electron microscopy demonstrated that killerFLIP-E triggers cell death accompanied by rapid (within minutes) plasma membrane permeabilization. Studies of the structure of the active core of killerFLIP (-E) indicated that it possesses amphiphilic properties and self-assembles into micellar structures in aqueous solution. The biochemical properties of killerFLIP are comparable to those of cationic lytic peptides, which participate in defense against pathogens and have also demonstrated anticancer properties. We show that the pro-cell death effects of killerFLIP are independent of its sequence similarity with c-FLIP_L as killerFLIP-induced cell death was largely apoptosis and necroptosis independent. A killerFLIP-E variant containing a scrambled c-FLIP_L motif indeed induced similar cell death, suggesting the importance of the c-FLIP_L residues but not of their sequence. Thus, we report the discovery of a promising synthetic peptide with novel anticancer activity in vitro and in vivo.     

(20S)‐Protopanaxadiol Ginsenosides Induced Cytotoxicity via Blockade of Autophagic Flux in HGC‐27 Cells

(20S)‐Protopanaxadiol ginsenosides Rg3, Rh2 and PPD have been demonstrated for their anticancer activity. However, the underlying mechanism of their antitumor activity remains unclear. In the present study, we investigated the role of these three ginsenosides on cell proliferation and death of human gastric cancer cells (HGC‐27 cells). The sulforhodamine B (SRB) assay, Western blot analysis, fluorescence microscopy, confocal microscopy, high performance liquid chromatography (HPLC) analysis, flow cytometry, and transmission electron microscopy (TEM) were used to evaluate cell proliferation, apoptosis, and autophagy. The results showed that both Rh2 and PPD were more effective than Rg3 in inhibiting HGC‐27 cell proliferation and inducing cytoplasmic vacuolation, while no significant changes in apoptosis were observed. Interestingly, cytoplasmic vacuolation and blockade of autophagy flux were observed after treatment with Rh2 and PPD. Rh2 obviously up‐regulated the expression of the LC3II and p62. Furthermore, the increase in lysosomal pH and membrane rupture was observed in Rh2‐treated and PPD‐treated cells. When HGC‐27 cells were pretreated with bafilomycin A1, a specific inhibitor of endosomal acidification, cellular vacuolization was increased, and the cell viability was significantly decreased, which indicated that Rh2‐induced lysosome‐damage accelerated cell death. Furthermore, data derived from mitochondrial analysis showed that excessive mitochondrial reactive oxygen species (ROS) and dysregulation of mitochondrial energy metabolism were caused by Rh2 and PPD treatment in HGC‐27 cells. Taken together, these phenomena indicated that Rh2 and PPD inhibited HCG‐27 cells proliferation by inducing mitochondria damage, dysfunction of lysosomes, and blockade of autophagy flux. The number of glycosyl groups at C‐3 position could have an important effect on the cytotoxicity of Rg3, Rh2 and PPD.     

Cell‐assembled nanoclusters of MSC‐targeting Gd‐DOTA‐peptide as a T2 contrast agent for MRI cell tracking

A cyclic peptide CC9 that targets cell membrane of mesenchymal stem cells (MSCs) is coupled with Gd‐DOTA to yield a Gd‐DOTA‐CC9 complex as MRI contrast agent. It is used to label human MSCs (hMSCs) via electroporation. Electroporation‐labeling of hMSCs with Gd‐DOTA‐CC9 induces cell‐assembly of Gd‐DOTA‐CC9 nanoclusters in the cytoplasm, significantly promotes cell‐labeling efficacy and intracellular retention time of the agent. In vitro MRI of labeled hMSCs exhibits significant signal reduction under T₂‐weighted MRI, which can allow long‐term tracking of labeled cell transplants in in vivo migration. The labeling strategy is safe in cytotoxicity and differentiation potential.     

Membrane damage as first and DNA as the secondary target for anti‐candidal activity of antimicrobial peptide P7 derived from cell‐penetrating peptide ppTG20 against Candida albicans

P7, a peptide analogue derived from cell‐penetrating peptide ppTG20, possesses antibacterial and antitumor activities without significant hemolytic activity. In this study, we investigated the antifungal effect of P7 and its anti‐Candida acting mode in Candida albicans. P7 displayed antifungal activity against the reference C. albicans (MIC = 4 μM), Aspergilla niger (MIC = 32 μM), Aspergillus flavus (MIC = 8 μM), and Trichopyton rubrum (MIC = 16 μM). The effect of P7 on the C. albicans cell membrane was examined by investigating the calcein leakage from fungal membrane models made of egg yolk l ‐phosphatidylcholine/ergosterol (10 : 1, w/w) liposomes. P7 showed potent leakage effects against fungal liposomes similar to Melittin‐treated cells. C. albicans protoplast regeneration assay demonstrated that P7 interacted with the C. albicans plasma membrane. Flow cytometry of the plasma membrane potential and integrity of C. albicans showed that P7 caused 60.9 ± 1.8% depolarization of the membrane potential of intact C. albicans cells and caused 58.1 ± 3.2% C. albicans cell membrane damage. Confocal laser scanning microscopy demonstrated that part of FITC‐P7 accumulated in the cytoplasm. DNA retardation analysis was also performed, which showed that P7 interacted with C. albicans genomic DNA after penetrating the cell membrane, completely inhibiting the migration of genomic DNA above the weight ratio (peptide : DNA) of 6. Our results indicated that the plasma membrane was the primary target, and DNA was the secondary intracellular target of the mode of action of P7 against C. albicans. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Ultrastructural changes in Neurospora cells undergoing senescence induced by kalilo plasmids

In N. crassa and N. intermedia, the kalilo plasmid triggers senescence by insertion into mitochondrial DNA. To investigate the cell death pathway induced by this plasmid, juvenile and senescent subcultures of several senescent strains were examined by light and transmission electron microscopy, and at the DNA level. There were no signs of apoptotic events, such as shrinkage of the cytoplasm away from the cell wall, apoptotic bodies, internucleosomal DNA fragmentation or condensation of the cytoplasm while retaining mitochondria and endomembrane structure. Instead, swollen mitochondria lacking cristae and containing amorphous inclusions, and disruption of nuclear and mitochondrial membranes indicated a necrotic mode of cell death.     

Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores

Bax is a key regulator of apoptosis that, under cell stress, accumulates at mitochondria, where it oligomerizes to mediate the permeabilization of the mitochondrial outer membrane leading to cytochrome c release and cell death. However, the underlying mechanism behind Bax function remains poorly understood. Here, we studied the spatial organization of Bax in apoptotic cells using dual‐color single‐molecule localization‐based super‐resolution microscopy. We show that active Bax clustered into a broad distribution of distinct architectures, including full rings, as well as linear and arc‐shaped oligomeric assemblies that localized in discrete foci along mitochondria. Remarkably, both rings and arcs assemblies of Bax perforated the membrane, as revealed by atomic force microscopy in lipid bilayers. Our data identify the supramolecular organization of Bax during apoptosis and support a molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the mitochondrial outer membrane.     

N‐(3‐Oxo‐acyl)‐homoserine lactone induces apoptosis primarily through a mitochondrial pathway in fibroblasts

N‐(3‐Oxododecanoyl)‐l ‐homoserine lactone (C12) is produced by Pseudomonas aeruginosa to function as a quorum‐sensing molecule for bacteria–bacteria communication. C12 is also known to influence many aspects of human host cell physiology, including induction of cell death. However, the signalling pathway(s) leading to C12‐triggered cell death is (are) still not completely known. To clarify cell death signalling induced by C12, we examined mouse embryonic fibroblasts deficient in “initiator” caspases or “effector” caspases. Our data indicate that C12 selectively induces the mitochondria‐dependent intrinsic apoptotic pathway by quickly triggering mitochondrial outer membrane permeabilisation. Importantly, the activities of C12 to permeabilise mitochondria are independent of activation of both “initiator” and “effector” caspases. Furthermore, C12 directly induces mitochondrial outer membrane permeabilisation in vitro. Overall, our study suggests a mitochondrial apoptotic signalling pathway triggered by C12, in which C12 or its metabolite(s) acts on mitochondria to permeabilise mitochondria, leading to activation of apoptosis.