Recombinant HALT-1 induces mitochondrial-associated apoptotic mechanism in HeLa cells

Recombinant HALT-1 induces mitochondrial-associated apoptotic mechanism in HeLa cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Recombinant HALT-1 induces mitochondrial-associated apoptotic mechanism in HeLa cells Lok Wenn Loo, Jung Shan Hwang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7721623/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract The study explored the apoptotic mechanism of Hydra actinoporin-like toxin-1 (HALT-1), an α-pore-forming toxin (α-PFT) produced by Hydra magnipapillata . α-PFT has been known to induce membrane pores in human cells upon contact, leading to the cell death. While previous research has covered HALT-1’s structural, membrane binding, cytolytic, and haemolytic aspects, the detailed information on apoptotic mechanism and cell signalling pathways remain unknown. Our study confirmed previous findings of rHALT-1's dose-dependent cytotoxicity, with a CC 50 of 15.4 µg/mL observed after 24 hours of treatment in our case. Hence, an rHALT-1 concentration below 15.4 µg/mL was selected to examine its apoptotic activity. Real-time Annexin V and DNA dye assays revealed dose- and time-dependent apoptotic patterns, with 12 µg/mL rHALT-1 inducing maximum apoptosis at 7 hours and minimal necrosis. Subsequently, flow cytometric analysis showed mitochondrial membrane potential depolarization without active caspase-3 throughout 6, 12, and 24-hour treatments. Western blot analysis indicated upregulation of apoptotic-inducing proteins (Bad, Bax, cytochrome c, caspase-9) and downregulation of antiapoptotic proteins (Bcl-2, Bcl-xL) at 12 µg/mL of rHALT-1. The absence of active caspases 3, 6, and 8 expressions suggests alternative cell death pathways. In conclusion, the study proposes, for the first time, that rHALT-1 induces apoptosis in HeLa cells by mediating the mitochondrial pathway, although active caspase-3 does not appear to be involved in the execution process. These findings provide a foundation for elucidating the mechanistic basis of rHALT-1 activity and highlight its potential utility in toxin-related research and biotechnological applications. Biological sciences/Biochemistry Biological sciences/Cell biology Biological sciences/Drug discovery Biological sciences/Molecular biology Pore-forming toxin Hydra actinoporin-like toxin-1 Apoptotic pathway mechanism Mitochondrial depolarization Caspases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Actinoporins are a class of α-pore-forming toxins (α-PFTs) produced mostly by sea anemones for the purposes of capturing prey and protecting against predators [ 1 ]. They are small proteins with 18–20 kDa that are typically positively charged with an isoelectric point (PI) greater than 9 [ 2 ]. Secondary structure of actinoporins consists of approximately 50% β-sheets, flanked by N-terminal and C-terminal amphipathic α-helices [ 3 ]. Within the fold, there are conserved tryptophan or tyrosine residues with aromatic rings that play a crucial role in the hydrophobic and electrostatic interactions with the target cell membrane [ 4 – 6 ]. Actinoporin, thus, utilizes this aromatic-rich region and an adjacent phosphocholine (POC) binding site to recognize and bind sphingomyelin (SM) on the cell membrane [ 7 ]. Additionally, actinoporins consist of disulfide linkages to enhance their structural stability and protect them from enzymatic degradation [ 8 , 9 ]. When attacking mammalian cells, oligomeric actinoporins create pores in the cell membrane, and the lumen size is about 1–2 nm in radius which allow monovalent or divalent cations such as K + and Ca 2+ to enter intracellularly [ 10 , 11 ]. Within the subgroup of actinoporins are the Hydra actinoporin-like toxins (HALTs). There are a total of seven HALTs and they are found in the freshwater cnidarian, Hydra magnipapillata [ 12 , 13 ]. These proteins, including HALT-1 to HALT-7, share structural similarities with actinoporins and among them, HALT-1 is the most well-studied [ 12 , 13 ]. HALT-1 shares some similarities with actinoporins but retains its unique characteristics. Like actinoporins, HALT-1 exerts hemolytic and cytolytic effects on human cells through a pore-forming mechanism [ 1 , 14 , 15 ]. The crystal structure of recombinant HALT-1 (rHALT-1) has been resolved at a high resolution of 1.43 Å [ 16 ]. It structurally resembles actinoporins, featuring a β-sandwich fold composed of twelve β-strands, two α-helices, and two 3₁₀ helices. Notably, lysine 76 in the monomeric protein is acetylated, forming N6-acetyl-l-lysine, which interacts with a water molecule through its carbonyl group [ 16 ]. Despite sharing 60% sequence similarity with actinoporins, HALT-1 exhibits some unique structural features that enable it to selectively bind to sulfatide, a sphingolipid with a sulfate head group, rather than sphingomyelin, which is the primary binding site for most actinoporins [ 13 , 16 ]. HALT-1 exerts apoptotic behaviour in human cells when it is used at the IC50, as reported by Ng et al., 2019 [ 17 ]. However, its precise mechanism of triggering cell death remains unknown, particularly its role in the early and late stages of apoptosis after cell binding. There are two primary pathways that govern cell apoptosis, the extrinsic pathway and the intrinsic pathway. In brief, the extrinsic pathway can be initiated by death ligands binding to death receptors on the cell surface. The death receptors are members of the tumor necrosis factor (TNF) receptor gene superfamily. Subsequently, the death receptor recruits adaptor proteins which further activate intracellular caspases, including caspase-8, -10, and − 3, resulting in the execution phase of apoptosis [ 18 ]. On the other hand, the intrinsic pathway is triggered by internal stress signals such as DNA damage or growth factor shortage [ 19 ]. Bcl-2 family proteins, including Bad, regulate the intrinsic pathway, influencing mitochondrial outer membrane permeabilization (MOMP) by interacting with Bcl-xl, Bcl-2, Bax, and Bak [ 20 , 21 ]. MOMP leads to cytochrome c release, binding to Apaf-1, forming the apoptosome, and activating caspase-9 [ 22 ]. The subsequent activation of caspases, like caspase-3 and caspase-6, induces cell shrinkage, membrane blebbing, and nuclear fragmentation, ultimately leading to cell death [ 23 – 25 ]. Studying how other actinoporins interact with and influence these processes may provide further insights into cell death induced by HALT-1. Venoms extracted from sea anemones, Stichodactyla haddoni and Heteractis magnifica , have been proven to induce apoptosis in human cell lines via the death receptor-mediated pathway or/and the mitochondria-mediated pathways [ 26 , 27 ]. However, the particular toxins that cause apoptosis in these sea anemones have yet to be identified. In another study, Kvetkina et al. [ 28 ] have demonstrated that Heteractis crispa actinoporin, rHct-S3, caused apoptosis by cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP). By using Western blot, the authors also showed both the upregulated Bax and the downregulated Bcl-2 [ 28 ]. Furthermore, a study on Sticholysin II (StnII) from Stichodactyla helianthus revealed a unique mechanism of inducing cell death in Raji cells. In the context of programmed cell death, StnII could release calcium from the endoplasmic reticulum, thereby activating the MAPK-ERK pathway and causing mitochondrial depolarization. However, StnII did not activate caspase-3. Instead, it activated the expression of receptor-interacting protein kinase 1 (RIP1), suggesting a distinct form of cell death associated with mitochondrial dysfunction rather than caspase activation [ 29 ]. In summary, these findings contribute to our understanding of diverse apoptotic mechanisms associated with different cytotoxic agents. The cytotoxic dose of HALT-1 is crucial in determining the switch between apoptosis and necrosis. Low doses of pore-forming toxins create small pores that allow selective ion passage, leading to programmed cell death [ 30 , 31 ], while higher doses form larger pores, resulting in ATP depletion and subsequent necrotic cell death [ 32 , 33 ]. The concentration of rHALT-1 may influence the size and properties of the pores formed in the cell membrane [ 12 ]. A recent study by Ng et al. [ 17 ] demonstrated that rHALT-1 induced apoptosis in HeLa, HepG2, and MCF-7 when it was applied at 50% inhibitory concentrations (ranging from 0.30 to 1.62 µM). However, the precise mechanism of action and the involved signaling pathways were not fully elucidated. Our study hypothesized that low doses of rHALT-1 could trigger apoptotic pathways within a short period. To test this, we first determined the sub-lytic concentrations of rHALT-1 for HeLa cells, and then examined phosphatidylserine exposure on the cell membrane and mitochondrial outer membrane polarization. Besides, we also assessed the expression levels of various apoptosis-related proteins using Western blot. Understanding how human cells respond to low doses of rHALT-1 could pave the way for its potential use in future cancer therapies such as immunotoxins (ITs). ITs facilitate the programmed cell death of cancerous cells by coupling a toxic domain with a specific antibody that targets tumour antigens on cancer cell surface [ 34 – 36 ]. Methods Production of recombinant HALT-1 (rHALT-1) protein HALT-1-inserted pET28a plasmid was previously constructed [ 14 ] and the purification procedure was performed as described by Yap et al. [ 37 ]. Briefly, rHALT-1 was overexpressed in IPTG-induced E. coli BL21(DE3). Cells were then lysed under sonication to obtain rHALT-1 in the soluble fraction in binding buffer (20 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0). rHALT-1 was purified under native conditions using Ni-NTA (Nickel-Nitrilotriacetic Acid) resin at a flow rate of 1 mL/min. First, cell lysate containing rHALT-1 was mixed with the resin, and incubated at 4°C for 15 minutes to bind the Ni-NTA resin (ThermoFisher Scientific). After incubation, non-His-tagged proteins were removed with binding and subsequently wash buffers (20 mM sodium phosphate, 300 mM NaCl, 30 mM imidazole, pH 8.0). Finally, 6 mL of elution buffer (20 mM sodium phosphate; 300 mM NaCl, 250 mM imidazole, pH 8.0) was used to release rHALT-1 in 500 µL fractions, which were then collected for SDS-PAGE analysis. The purified rHALT-1 protein obtained from binding affinity purification underwent further purification using SP sepharose ion exchange chromatography. Ten millilitre of rHALT-1 protein was added to the column. The column was then rinsed with 80 mM phosphate buffer (pH 7.4) with increasing NaCl concentrations (60, 120, 150, and 200 mM), culminating in a 200 mM NaCl solution to elute the rHALT-1 protein. Following elution, the ion exchange buffer was replaced with 1x PBS for protein pooling and concentration analysis. Finally, the concentrated rHALT-1 protein in 1x PBS was assessed using SDS-PAGE to confirm the absence of protein degradation or contamination. Cell culture HeLa cells (ATCC® CCL-2™) were cultured in Dulbecco’s Modified Eagle Minimal (DMEM) (Nacalai Tesque, Japan), supplemented with 10% fetal bovine serum (FBS) and 0.5% of Pen-Strep containing 10,000 units/mL of penicillin and 10,000 µg/mL of streptomycin. The cells were incubated at 37°C in a humidified chamber with 95% O 2 and 5% CO 2 . Serum-free medium was used during the treatment period. Cytotoxicity assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed to assess the sublytic toxicity of rHALT-1 in HeLa cells. Cells (1 × 10 4 cells) in a 96-well plate were exposed to respective treatments including 100 µL of various rHALT-1 concentrations (0, 6, 12, 18, 24, and 30 µg/mL), 100 µL of PBS-DMEM (vehicle control) and 100 µL of 100 ng/mL hTRAIL (positive control). To assess the background contributed by DMEM, three wells were added with DMEM only. Cells were then incubated for 24 hours at 37°C. After the treatment, 25 µL of 5 mg/mL MTT solution was loaded into each well, and the plate was incubated for another 3 hours, followed by the removal of supernatant and the addition of 200 µL DMSO to dissolve the insoluble purple formazan crystals. Absorbance was measured at 570 nm (reference at 630 nm) using an Infinite M Plex microplate reader (Tecan, Germany). Cell viability percentages obtained from three experiments (n = 3) were calculated using this equation, % cell viability = {(A t -A b )/(A c -A b )}x100 where A t = cells incubated with rHALT-1 or hTRAIL; A b = blank; A c = negative control and then visualized in mean percentages. CC 50 (µg/mL) and R 2 were determined via Microsoft Excel's exponential trendline, where CC 50 indicates a 50% reduction in cell viability compared to the untreated control. Kinetic evaluation of cell apoptosis and necrosis The RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay Kit (Promega, USA) was employed to track apoptosis and secondary necrosis dynamics by continuously capturing luminescence and fluorescence signals from the same cell samples. In a black 96-well plate with a clear bottom (Corning Incorporated, USA), 1 x 10 4 cells per well were seeded. Cells in the wells were treated with 100 µL of distinct solutions as described in the MTT assay: background control, vehicle control, positive control (100 ng/mL hTRAIL peptide), and various rHALT-1 concentrations (0, 6, 12, 18, 24, and 30 µg/mL), each was supplemented with 1 x detection reagent and incubated at 37°C. The 1 x detection reagent, comprising luminogenic Annexin V stain and a fluorescent dye, was prepared following the manufacturer's instructions. The assay plate was shaken at 500 rpm with linear amplitude for approximately 30 seconds before measurement to ensure thorough mixing. Luminescence (with automatic attenuation) and fluorescence (excitation: 485 nm; emission: 525 nm) signals were recorded every 30 minutes for four hours, shifting to hourly intervals thereafter until the readings stabilised. The initial measurement point was designated as the zero point. Net relative luminescence or fluorescence unit (RLU or RFU) was calculated by subtracting the average luminescence or fluorescence value of background signal from all sample luminescence or fluorescence values Each data point represented the average values from three independent experiments (n = 3). Mitochondrial membrane potential assay Mitochondrial membrane potential (MMP; Δψ) changes were assessed to evaluate apoptotic response induced by rHALT-1 using a MitoScreen JC-1 kit (Becton Dickinson, USA). JC-1 (1st J-aggregate-forming cationic) is a dye that is sensitive to membrane potential and exists as green fluorescence monomers (indicative of low MMP) or red-orange fluorescence aggregates (indicative of high MMP). This study combined JC-1 and BD Pharmingen™ 7-AAD (Becton Dickinson, USA) dyes to distinguish apoptotic and necrotic cells. Viable and early apoptotic cells would remain unstained (7-AAD − ), while necrotic cells would exhibit 7-AAD + staining due to loss of plasma membrane integrity. Following this, viable and apoptotic cells would be differentiated by JC-1 staining into two cell populations with polarized and depolarized MMP. Cell cultures containing 1x 10 6 cells in 5 mL were left untreated and treated with rHALT-1 (12 µg/mL) and hTRAIL (100 ng/mL) for 6, 12, and 24 hours in a 100 mm culture dish. An additional control of unstained and untreated cells was included to establish background signals. Post-treatment, cells were harvested, centrifuged at 400 x g for 5 minutes at room temperature, and washed twice with 1 mL of 1x PBS. Subsequently, cells were incubated with 20 µL of 7-AAD in 0.5 mL of 1x PBS for 15 minutes at room temperature. After incubation and centrifugation, cell pellets were gently resuspended in 0.5 mL of freshly prepared JC-1 working solution and incubated for 10–15 minutes at 37°C. Following this, cells were washed twice with 1x Assay Buffer and then gently resuspended in 0.5 mL of 1x Assay Buffer. The cell suspension was filtered through a 40 µm cell strainer and transferred to polystyrene round-bottom tubes for subsequent analysis using a BD FACSCalibur™ flow cytometer (Becton Dickinson, USA). Data acquisition and analysis were conducted utilizing the BD CellQuest Pro Software (Becton Dickinson, USA). 7-ADD negative cells were further evaluated for the MMP test. The fluorescent signal generated by JC-1 reflects changes in Δψ, indicating whether the MMP is polarized or depolarized. The percentages of 7-AAD⁻ depolarized (JC-1 monomer) and polarized (JC-1 aggregate) cell populations were calculated by dividing the number of depolarized or polarized cells by the total number of 7-AAD⁻ cells. Fold change relative to untreated cells was determined by dividing the percentage of mitochondrial membrane depolarization in treated samples by that of the untreated control (UTC), which served as the baseline with a fold change value of 1.00. Caspase-3 enzymatic activation assay The FITC Active Caspase-3 Apoptosis Kit (Becton Dickinson, USA) combined with BD Pharmingen™ 7-AAD (Becton Dickinson, USA) dye, were used to detect active caspase-3 in rHALT-1-treated cells using flow cytometry. This double staining differentiated cells into live, early apoptotic, late apoptotic, and dead categories. Cells in the early and late stages of apoptosis were identified through caspase-3 staining, while 7-AAD distinguished between live and dead cells. Similar to the MMP assay, cells were treated, harvested, washed with 1x PBS, and stained with 7-AAD dye. However, in this case, cells treated with 12 µg/mL of rHALT-1, untreated control, and positive control (100 ng/mL hTRAIL-treated cells) were stained with anti-caspase-3 antibody. The subsequent sample preparation followed the manufacturer's guidelines by fixing and permeabilizing the cells using 0.5 mL of Cytofix/Cytoperm™ solution for 20 minutes on ice. The cells were pelleted and washed twice with 0.5 mL of 1x BD Perm/Wash™ buffer at room temperature to eliminate other cellular components. After that, the cells were incubated with 120 µL of 1x BD Perm/Wash™ buffer containing FITC rabbit anti-active caspase-3 for 30 minutes at room temperature. Following staining, unbound antibodies were removed by washing with 1.0 mL of 1x BD Perm/Wash™ buffer. The cells were then resuspended in 0.5 mL of 1x BD Perm/Wash™ buffer. Subsequently, the cell suspension was filtered through a 40 µm cell strainer and transferred to polystyrene round-bottom tubes for further analysis using BD FACSCalibur™ flow cytometer (Becton Dickinson, USA) to differentiate cell populations based on caspase-3 and 7-AAD staining patterns. The percentage of the apoptotic cell population exhibiting active caspase-3 was determined based on the fluorescence emitted by the bound anti-active caspase-3 antibodies. The percentage of caspase-3⁺ cells was calculated by dividing the number of cells expressing active caspase-3 by the total number of gated cells. Fold change was determined by dividing this percentage by the percentage of caspase-3⁺ cells in the untreated control (UTC), which served as the baseline with a fold change value of 1.00. Western blot detection of Bcl-2 families, caspases, and cytochrome c Western blot analysis was performed to track the expression levels of apoptotic markers (Bad, Bax, Bcl-2, Bcl-xL, caspase-3, caspase-6, caspase-8, caspase-9, and cytochrome c). Cell pellets (1x 10 6 cells) of untreated, 12 µg/mL rHALT-1, and 100 ng/mL hTRAIL-treated cells at 6, 12, and 24 hours were lysed using 1x RIPA buffer containing 1 mM PMSF for 30 minutes with agitation every 5-minute interval. Whole cell lysate was quantified using the BCA method with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA) and normalized. Ten micrograms of total protein underwent SDS-PAGE on 12% polyacrylamide gels. Proteins were then transferred onto PVDF membranes with 1x transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3, 20% methanol) using a Trans-Blot Turbo Transfer System (Bio-Rad, USA), set at 25 V and 0.8 A for 40 minutes. Membranes were blocked with 5% BSA prepared in 1x triethanolamine-buffered saline solution containing Tween-20 (TBST) for one hour and incubated with primary antibodies overnight at 4ºC or 2 hours at room temperature. The dilution ratios of primary antibodies: β-actin (1:1000), Bax (1:1000), Bad (3:1000), Bcl-2 (1:1000), Bcl-xL (1:1000), caspase-3 (3:1000), caspase-6 (1:1000), caspase-8 (1:1000), and caspase-9 (3:1000), and cytochrome c (1:1000). Following five washes with 1x TBST, each wash lasting 5 minutes with agitation, membranes were exposed to HRP-linked secondary antibodies (1:1000), anti-rabbit IgG or anti-mouse IgG (Cell Signaling Technology, USA) for one hour at room temperature and washed five times again. A chemiluminescent substrate (Promega, USA) was applied, and signals were captured using the LAS 500 ImageQuant™ Chemiluminescent imager (GE Healthcare, USA). Densitometry analysis was performed using ImageJ software (National Institutes of Health, USA), utilizing β-actin as a loading control. Fold change in protein expression was calculated by normalizing to the untreated control. This robust procedure enabled a quantitative comparison of target protein levels across different experimental conditions and time points. Statistical analysis Statistical data were presented as means of three independent experiments (n = 3), with standard deviations displayed as error bars. The level of statistical significance was determined using an unpaired t-test and denoted as two-tailed P values as follows: ns (not significant) = P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0005 vs untreated control. Results Sub-lytic concentrations of rHALT-1 for HeLa cells rHALT-1 induced dose-dependent cytotoxic effects in HeLa cells, as shown in Fig. 1 . A concentration of 6 µg/mL of rHALT-1 exhibited marginal toxicity, while higher concentrations led to decreased cell viability, suggesting increased cytotoxicity. Using the exponential trendline equation from the scatter plot, the CC 50 of rHALT-1 after 24 hours of exposure to cells was estimated to be approximately 15.4 µg/mL, which is consistent with the observed range of 12–18 µg/mL in the bar chart. This result aligns with the findings of Liew et al. [ 14 ], which reported that a concentration as low as 15 µg/mL reduced cell viability by 50% in HeLa cells. In this study, we defined 15 µg/mL or below as the sub-lytic concentrations of rHALT-1 for HeLa cells. Apoptotic and necrotic activities of rHALT-1 Both apoptotic and necrotic activities were observed following treatment with rHALT-1 at various concentrations ranging from 6 to 30 µg/mL. Apoptotic cells, identified through Annexin V luminescent signals, are illustrated in Fig. 2 A, while necrotic cells, labelled using fluorescent probes, are shown in Fig. 2 B. These cellular kinetic activities were monitored in real-time. The initial onset of apoptotic activity was evident within an hour across all rHALT-1 concentrations used to treat HeLa cells (Fig. 2 A). This apoptotic induction is attributed to the externalization of phosphatidylserine from the inner leaflet of the cell membrane [ 38 ]. A small proportion of untreated HeLa cells underwent cell death, with apoptotic activity reaching approximately 30–35% at 12 hours (Fig. 2 A). However, a significant increase in apoptotic cells was observed following treatment with rHALT-1, with the time required to achieve maximal cell death depending on the rHALT-1 concentration. A more than 2-fold increase in the luminescent signal was observed when cells were treated with 6 µg/mL of rHALT-1 for up to 12 hours (Fig. 2 A), with the signal appearing to continue rising beyond the 12-hour mark. At higher concentrations of 12 or 18 µg/mL, apoptotic cells required only 6–7 hours to reach nearly 100% cell death, compared to the longer time needed for cells treated with 6 µg/mL of rHALT-1. When 24 or 30 µg/mL of rHALT-1 was applied, a peak in luminescent signal was observed within 3–5 hours, indicating that the apoptotic activity had reached its maximum stage (Fig. 2 A). Alongside the assessment of apoptotic activity described above, necrotic activity, detected using fluorescent probes, was also observed in HeLa cells following dose-dependent treatment with rHALT-1 (Fig. 2 B). At high concentrations of rHALT-1, necrotic activity increased rapidly, becoming evident at the 3rd hour for 30 µg/mL and at the 5th hour for 24 µg/mL in treated HeLa cells. This coincided with the stage where no further increase in the number of apoptotic cells was observed (Fig. 2 A, Fig. S1 C and S1D). Similar patterns were observed at rHALT-1 concentrations of 12 and 18 µg/mL, where a steep increase in apoptotic activity was evident during the first 7 hours following treatment with rHALT-1, after which the activity slowed down, while necrotic activity gradually increased over the 12-hour period (Fig. 2 , Fig. S1 A and S1B). In contrast, hTRAIL, used as a control, exhibited higher luminescence signals compared to rHALT-1-treated cells after 8 hours, with minimal necrosis over 12 hours, consistent with its role as a typical apoptotic inducer (Fig. 2 A and B). In summary, treatment with rHALT-1 induced both apoptotic and necrotic activities in HeLa cells in a dose- and time-dependent manner. Apoptosis was evident within one hour across all tested rHALT-1 concentrations (6–30 µg/mL). A dose-dependent acceleration of apoptotic activity was observed, with higher rHALT-1 concentrations (≥ 12 µg/mL) leading to rapid cell death within 3–7 hours, while lower concentrations required longer periods (up to 12 hours). Necrotic activity became prominent at higher rHALT-1 concentrations (≥ 24 µg/mL) after apoptotic activity reached its peak, typically after 3–5 hours. A similar trend was observed at 12–18 µg/mL, where necrotic activity gradually increased as the rise in apoptotic activity stabilized. rHALT-1 induced mitochondrial membrane depolarization (MMP) Changes in mitochondrial membrane depolarization (MMP) during apoptosis and cell membrane damage during necrosis can be analysed using flow cytometry analysis with JC-1 and 7-AAD double staining, respectively. To do these, HeLa cells were treated with rHALT-1 at 12 µg/mL. This concentration was chosen due to its notable induction of intracellular apoptotic events in the first 7th hour of rHALT-1 incubation, while exhibiting minimal necrosis compared to other higher concentrations, as mentioned above. Cells with polarized mitochondria were grouped in R4 (Fig. 3 A), while cells with reduced aggregate fluorescence or depolarized mitochondria were grouped in R5 (Fig. 3 A). Apparently, rHALT-1 caused a significant increase in the number of cells with reduced aggregate fluorescence at 6, 12, and 24 hours, indicating a substantial loss of MMP, a characteristic sign of early apoptosis. The percentage of depolarized cells increased significantly to 31.63% ± 2.46 (3.35-fold change) at 6 hours, 37.26% ± 1.06 (2.82-fold change) at 12 hours, and 44.75% ± 2.54 (3.06-fold change) at 24 hours (Fig. 3 A and B). The most pronounced effect occurred at 6 hours, where the Δψ decreased 3.35 times compared to the untreated control. Both rHALT-1 and hTRAIL peptide treatments yielded highly significant results (P ≤ 0.0005 or ≤ 0.001), which were consistent across three replicates. These findings suggest that rHALT-1 induced a time-dependent reduction in MMP, triggering the apoptotic cascade in HeLa cells. No Traces of Caspase-3 Activation Figure 4 shows the percentages of HeLa cells exhibiting active caspase-3 after treatment with 12 µg/mL rHALT-1 and 100 ng/mL hTRAIL at 6, 12, and 24 hours. In untreated cells, the proportions of active caspase-3 in early and late apoptosis remained consistently low, at approximately 1% or less across all three time points. Cells treated with rHALT-1 also exhibited minimal changes in active caspase-3 level (early and late), with overall percentages of 0.41% at 6 hours, 1.04% at 12 hours, and 0.69% at 24 hours. The fold changes relative to the untreated control ranged from 0.38 to 1.09, suggesting no significant induction of caspase 3-mediated apoptosis. On the other hand, hTRAIL-treated cells demonstrated markedly higher level of active caspase 3 in both early and late apoptosis: 69.03% at 6 hours, 73.17% at 12 hours, and 81.01% at 24 hours (Fig. 4 A). Fold changes ranged from 56.70 to 73.40 compared to the untreated control, reflecting substantial apoptosis induction (Fig. 4 B). Therefore, only the positive control indicated a significant increase in apoptosis through caspase-3 activation at all three time points (Fig. 4 A and B), whereas rHALT-1 treated and untreated cells did not possess caspase-3 activation. Statistical analysis confirmed that the results for hTRAIL-treated cells were highly significant (P-value: 0.001 ≤ P ≤ 0.05). rHALT-1 regulated intrinsic apoptotic protein expressions The western blot analysis tracked the expression levels of intrinsic and extrinsic apoptotic markers (Bad, Bax, Bcl-2, Bcl-xL, caspase-3, caspase-6, caspase-8, caspase-9, and cytochrome c) upon treatment with rHALT-1 or hTRAIL across 6, 12, and 24 hours (Fig. 5 A). Bad, a pro-apoptotic protein, gradually increased over 24 hours in UTC, possibly due to a cellular compensatory response to stress or an effort to maintain homeostasis. In contrast, Bad expression peaked at 12 hours in the rHALT-1 and hTRAIL treatment groups indicates that these treatments might be influencing cellular functions differently, potentially altering signaling pathways or gene expression patterns associated with Bad regulation. Bax, another pro-apoptotic protein, rose over time in both rHALT-1 and hTRAIL treatments, peaking at 24 hours for rHALT-1 and at 12 hours for hTRAIL. Anti-apoptotic proteins, such as Bcl-2 and Bcl-xL, were significantly expressed in untreated cells, but their levels decreased following treatments with rHALT-1 or hTRAIL, particularly at the 6-hour mark. Cytochrome c showed consistent, significant upregulation across all treatments and time points. Initiator caspase-9 showed the most prominent expression of its precursor form (47 kDa) at 12 hours following rHALT-1 treatment. Active forms (37 kDa and 35 kDa) were detected at all time points in all samples, with or without treatment. Notably, the expression levels of active caspase-9 were more than one-fold higher in HeLa treated with rHALT-1 or hTRAIL compared to untreated controls (Fig. 5 B). Caspase-8, another initiator caspase, was highly expressed in its precursor form (57 kDa) in untreated and rHALT-1-treated cells, while the active form (43 kDa) was detected only in hTRAIL-treated cells at 6 and 12 hours, with very low expression levels (Fig. 5 B). The procaspase-3 exhibited high expression in rHALT-1-treated cells at 24 hours, with lower but still notable expression at 6 and 12 hours. No active forms of caspase-3 were detected in any rHALT-1 treated cells, whereas active caspase-3 was observed only in hTRAIL-treated cells (Fig. S2 ). It is likely that neither procaspase-6 nor its active form were expressed in both treated and untreated HeLa cells, as no detectable bands corresponding to 34 kDa (inactive form), 20 kDa, or 11 kDa appeared on the Western blot. Although one band appears to have a molecular weight between 25–35 kDa (Fig. 5 and Fig. S4), the presence of multiple higher molecular weight bands suggests that this may be a non-specific signal. Both active and inactive forms of caspase-6 were not detected in hTRAIL-treated cells, likely because hTRAIL primarily activates the extrinsic pathway, involving caspase-8 and caspase-3. Caspase-6 is typically activated downstream of these executioner caspases or through amplification involving mitochondrial pathways, such as cytochrome c release, which are less prominently triggered by hTRAIL in this context. The quantification of protein blots using ImageJ software highlighted significant changes in apoptotic protein expression regulated by 12 µg/mL rHALT-1 (Fig. 5 B). At 6 hours, compared to the untreated control, Bad and Bax were upregulated with fold changes of 1.164 and 2.103, respectively. Bcl-2 and Bcl-xL showed slight downregulation with rHALT-1 treatment with fold changes of 0.827 and 0.638, respectively. Cytochrome c and active caspases-9 (37 and 35 kDa) were consistently upregulated with fold changes of 2.262, 2.352, and 2.033, respectively, indicating increased apoptotic activation. At 12 hours, rHALT-1 treatment resulted in increased expression of Bad, Bax, cytochrome c, and active caspases-9 with fold changes of 1.842, 1.915, 1.567, 1.376 (37 kDa), and 2.131 (35 kDa), respectively, except Bcl-xL showed slight downregulation with a fold change of 0.798, while Bcl-2 displayed nearly no difference with a fold change of 1.033. At 24 hours, rHALT-1 treatment led to further downregulation of Bad, Bcl-2, and Bcl-xL with fold changes of 0.751, 0.805, and 0.551, respectively. Bax, cytochrome c, and two active caspases-9 were upregulated with a fold change of 1.593, 2.385, 1.813 (37 kDa), and 1.297 (35 kDa). The hTRAIL treatment showed similar trends in Bad and Bax upregulation at 6 and 12 h, while Bcl-2 and Bcl-xL were downregulated across the three time points. Cytochrome c and active caspases-9 exhibited decreased fold change of upregulation over time with hTRAIL treatment. Although hTRAIL binds to death receptor 4 (DR4) and death receptor 5 (DR5) on the cell membrane, triggering the extrinsic apoptotic pathway, it can also secondarily activate the intrinsic pathway by engaging mitochondria and promoting cytochrome C release in HeLa cells [ 39 , 40 ]. Discussion Similar to other actinoporins, HALT-1 has been shown to induce apoptosis, adhering to pathways observed in actinoporins such as StnII of Stichodactyla helianthus . However, while the overall apoptotic mechanism remains comparable, the specific intermediate proteins in the HALT-1-mediated signaling cascade may differ, indicating variations in transduction pathways among different actinoporins. The concentration HALT-1 required to trigger apoptosis in HeLa cells is critical. We determined that IC 50 (15.4 µg/mL) or lower could induce apoptosis, whereas higher concentrations lead to necrosis, a pattern consistent with bacterial and sea anemone pore-forming toxins (PFTs) [ 41 , 42 ]. To account for potential dilution errors in all experiments, a concentration of rHALT-1 lower than IC 50 , 12 µg/mL, was used in all subsequent experiments. In addition to HALT-1 dosage, incubation time is another critical factor in apoptosis induction; therefore, treatment durations of 6, 12, and 24 hours were applied across all experiments to demonstrate the apoptotic activity. Bad and Bax might be expressed as early as upon the addition of rHALT-1 to the cells, reaching peak levels between 6 and 12 hours, followed by a decline from 12 to 24 hours. In contrast, Bcl-2 and Bcl-xL expression decreased following HALT-1 treatment, with no significant differences observed across the time points. Similarly, Abdzadeh et al. [ 26 ] also analysed the expression levels of Bcl-2 family proteins in A549 lung cancer cells treated with cytotoxic compounds derived from the mucus of Stichodactyla haddoni . These compounds were found to upregulate Bak and Bax expression while downregulating Bcl-2 in HT-29 cells, leading to morphological changes characteristic of apoptosis [ 26 ]. The authors further indicated that the apoptotic events are dose-dependent, whereas their time-dependency has not been investigated in the same study. Another actinoporin from Heteractis crispa , Hct-S3, could also suppress the migratory activity of colorectal carcinoma HT-29 cells by down-regulating Bcl-2 and up-regulating Bax, and promote apoptosis through the activation of caspase-3, as determined by Western Blotting with specific antibodies [ 43 ]. In fact, Bcl-2 family proteins play a pivotal role in determining the susceptibility of cells to apoptosis [ 44 , 45 ]. Upregulation of pro-apoptotic proteins stimulates the permeabilization of the outer mitochondrial membrane causing the release of apoptogenic factors such as cytochrome c, AIF, Endo G, and Smac/DIABLO to the cytoplasm [ 46 – 49 ]. When HeLa cells were treated with this low concentration of rHALT-1 (12 µg/mL), the Bcl-2 family regulated the cell transition to an apoptotic state, marked by the reduction in mitochondrial potential (ΔΨm). This hallmark arises from the collapse of negative charge within the matrix, leading to a decrease in cationic JC-1 dye uptake and consequently causing JC-1 to exist in its monomeric form within the cytoplasm [ 50 ]. Notably, at the 24-hour time point, mitochondrial membrane depolarization was measured at 44.75%, closely aligning with MTT assay data showing that 38.59% of cells failed to reduce MTT to formazan crystals due to mitochondrial dehydrogenase dysfunction. These findings are the first evidence showing that rHALT-1-induced early apoptosis is associated with mitochondrial dysfunction, as reflected by the loss of ΔΨm in HeLa cells. A study involving crude extracts from sea anemone Heteractis magnifica demonstrated a similar induction of apoptosis in A549 lung carcinoma cells. The treated cells showed a reduction in MMP and the presence of caspase-3 cleavage when exposed to a concentration of 10 µg/mL for 24 hours [ 51 ]. Interestingly, another study highlighted that StnII, as an actinoporin, triggers calcium release mainly from the endoplasmic reticulum, activates the mitogen-activated protein kinase ERK, and disrupts mitochondrial membrane potential [ 29 ]. The study also demonstrated that the StII-induced cell death process does not involve caspase activation or hallmark characteristics of apoptosis and pyroptosis [ 29 ]. Upon disruption of the outer mitochondrial membrane, the release of cytochrome c from mitochondria is a recognized part of the apoptotic effect. According to the western blot results, rHALT-1 induced a time-dependent increase in cytochrome c levels as early as 6 hours after treatment, and this elevation correlates with changes in MMP. The depolarization of MMP and the release of cytochrome c are reliable indicators for detecting early changes in the intrinsic pathway [ 49 , 52 ]. These findings underscore the crucial role of cytochrome c in the rHALT-1-induced apoptotic pathway, suggesting its contribution to the activation of downstream apoptotic effectors. Based on the protein expression, only initiator caspase-9 appeared in its active form following rHALT-1 treatment. Other caspases, including the initiator caspase-8 and the executioner caspases-3, remained in their precursor forms, while the expression of caspase-6 appeared to be not evident. The absence of active caspase-3 was also proven in a flow cytometric assay which differed from the response triggered by hTRAIL, a known apoptosis inducer. This discovery resembles the response to the reaction seen in Raji cells when subjected to sublytic levels of StnII, where, as detailed by Soto et al [ 29 ], the presence of executioner caspase-3 expression is lacking. There is additional evidence suggesting that rHALT-1 might induce cell death through alternative and unique pathways that do not involve the activation of caspase-3, caspase-6, and caspase-8. This discrepancy arises because caspase-3 and caspase-8 possess RGD (Arg-Gly-Asp) binding site, which can be the target of RGD-containing actinoporins such as StnII and FraC [ 53 , 54 ]. The presence of this binding motif in these caspases could potentially allow actinoporins such as FraC to bypass upstream steps, involving the integrin binding to apoptotic initiator activation, instead, they might directly initiate downstream apoptotic executioner activation [ 54 , 55 ]. Instead of RGD, HALT-1 contains a RAG (Arg-Ala-Gly) motif [ 16 ]. If HALT-1 possessed the RGD motif, it could potentially bind directly to the RGD binding site and activate caspase-3 and caspase-8. Moreover, in the conventional apoptotic pathway, intrinsic caspase-9, either alone or in conjunction with extrinsic caspase-8, converges to cleave caspase-3. However, this was not the case for HALT-1, despite its ability to activate caspase-9 production. Therefore, this study suggests the existence of alternative mitochondrial pathways that may facilitate apoptotic execution independently of caspases-3 and − 6. Here we propose a rHALT-1-mediated intrinsic apoptotic pathway (Fig. 6 ). The intrinsic mitochondrial pathway, as illustrated in the schematic, is initiated by rHALT-1 and involves the modulation of key apoptotic proteins. This pathway is primarily regulated by the balance between pro-apoptotic and anti-apoptotic proteins. Upon activation, rHALT-1 upregulates pro-apoptotic proteins such as Bad and Bax while downregulating anti-apoptotic proteins like Bcl-2 and Bcl-xL. These changes lead to mitochondrial membrane depolarization, resulting in the release of cytochrome c (CytC) into the cytoplasm. Once released, cytochrome c interacts with Apaf-1, forming the apoptosome, which subsequently activates caspase-9 (Caps9). However, Fig. 6 indicates that uncertainties regarding the activation of downstream caspases, such as caspase-3 (Caps3) and caspase-6 (Caps6), appear to be either inactive or not involved in rHALT-1-induced apoptosis. Therefore, we speculated that an alternative mitochondrial pathway involving rHALT-1 may be at play. Cytochrome c is not the only molecule released into the cytosol when the mitochondrial outer membrane becomes permeabilized. Other mitochondrial proteins, such as endonuclease G (Endo G) and apoptosis-inducing factor (AIF), are also released from the mitochondrial intermembrane space [ 56 ]. Numerous studies have shown that these proteins can induce cell death in a caspase-independent manner [ 57 , 58 ]. To trigger cell death, Endo G and AIF translocate to the nucleus, where they degrade DNA into fragments. We attempted to detect the expression of Endo G and AIF in HeLa cells following rHALT-1 treatment; however, neither Endo G nor AIF was visible as distinct bands on the Western blot or detectable in qPCR (Fig. S3). Another mitochondrial proteins, Smac and DIABLO, are released into the cytosol, where they antagonize inhibitors of apoptosis proteins (IAPs), thereby facilitating the activation of caspase-3 [ 59 ]. Since our results indicate that HALT-1 induces cell death via a caspase-3-independent pathway, we may exclude the possible involvement of Smac and DIABLO in post-cytochrome c release events. Addressing the limitations in understanding HALT-1-induced apoptosis requires strategic approaches. First, an in-depth analysis of other genes and proteins involved in caspase-3 independent pathway using qPCR and western blot techniques could provide valuable insights. Second, accurately capturing the apoptotic window period by employing sophisticated methods like cell sorting, combined with assays such as TUNEL and MMP, would offer precise endpoints before necrosis onset. Third, enhancing data reliability by expanding the apoptotic inducer panel to include both extrinsic and intrinsic apoptotic inducers would strengthen pathway references. Lastly, utilizing apoptotic inhibitors to block caspases and target anti-apoptotic proteins can dissect intrinsic and extrinsic apoptotic pathways. These strategic methodologies hold significant promise in advancing apoptosis research and broadening the potential therapeutic applications of HALT-1. Conclusion This study provides the first evidence that rHALT-1 induces apoptosis in HeLa cancer cells by modulating multiple proteins associated with the mitochondrial pathway. The findings reveal that a low concentration of rHALT-1 (12 µg/mL) triggers apoptosis, as indicated by phosphatidylserine exposure and mitochondrial dysfunction. Alterations in the expression of key apoptotic proteins, including Bad, Bax, Bcl-2, Bcl-xL, cytochrome c, and caspase-9, suggest the involvement of the intrinsic apoptotic pathway. However, the absence of caspase-3, caspase-6, and caspase-8 activation at this concentration suggests that alternative cell death mechanisms, which are caspase-9-dependent but independent of executioner caspases-3 and − 6, and initiator caspase-8, may be involved. While the precise mechanism of rHALT-1-induced cell death remains unclear, these findings lay a strong foundation for future research. Notably, rHALT-1’s potential as a toxin component presents promising therapeutic applications, particularly in the development of immunotoxins. By selectively inducing apoptosis while preserving the integrity of healthy cells, this approach could provide novel treatment strategies for diseases characterized by dysregulated cell death. Abbreviations α-PFT α-pore-forming toxin AIF apoptosis-inducing factor Caps caspase CC₅₀ 50% cytotoxic concentration DMEM Dulbecco’s Modified Eagle Minimal EndoG endonuclease G FBS fetal bovine serum HALT-1 Hydra actinoporin-like toxin-1 IAPs inhibitors of apoptosis proteins IC50 50% inhibitory concentration ITs immunotoxins JC-1 1st J-aggregate-forming cationic MMP orΔψ mitochondrial membrane potential MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MOMP mitochondrial outer membrane permeabilization Ni-NTA Nickel-Nitrilotriacetic Acid PI isoelectric point POC phosphocholine RIP1 receptor-interacting protein kinase 1 RFU relative fluorescence unit RLU relative luminescence unit SM sphingomyelin PARP poly (ADP-ribose) polymerase StnII Sticholysin ITNF,tumor necrosis factor UTC untreated control. 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01:56:38","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4234781,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials4Looetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7721623/v1/8c732b5c28bb49ceba3e04bf.pdf"},{"id":93639077,"identity":"30076482-89ee-4788-a45a-c7cd31a5398f","added_by":"auto","created_at":"2025-10-16 02:04:38","extension":"xml","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":140534,"visible":true,"origin":"","legend":"","description":"","filename":"b666b9b2641640a1b38a503cdb49145d1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7721623/v1/070acda134d0af04a277e39d.xml"},{"id":93638006,"identity":"c9eb66b3-e91d-44cf-a70f-21abc6470ae0","added_by":"auto","created_at":"2025-10-16 01:56:38","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4236444,"visible":true,"origin":"","legend":"","description":"","filename":"Figures16Looetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7721623/v1/c60bc737ba1e0b7f76c7e479.pdf"},{"id":93638005,"identity":"1e6eed2f-59ab-4a3f-8145-796a795ba254","added_by":"auto","created_at":"2025-10-16 01:56:38","extension":"xml","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":140789,"visible":true,"origin":"","legend":"","description":"","filename":"b666b9b2641640a1b38a503cdb49145d1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7721623/v1/a313a3cf1ab1176852b13496.xml"},{"id":93639732,"identity":"cbefa3ab-3130-4c96-a3e2-44a8cff03d4d","added_by":"auto","created_at":"2025-10-16 02:12:38","extension":"html","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":153187,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7721623/v1/9f7b206f03ceb9f61160e575.html"},{"id":93639730,"identity":"067d420a-cb81-49c8-a600-3a6eb0aea69e","added_by":"auto","created_at":"2025-10-16 02:12:38","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":162082,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytotoxicity of rHALT-1-treated HeLa cells assessed using MTT assay.\u003c/strong\u003e HeLa cells were seeded in a 96-well plate at a density of 1 x 10\u003csup\u003e4\u003c/sup\u003e cells/well and incubated for 16 h. The percentages of cell viability after a 24-h treatment with various concentrations of rHALT-1 (0-30 µg/mL) and positive control (100 ng/mL hTRAIL peptide) relative to the untreated control (set as 100%) were represented in the bar graph. The half-maximal cytotoxic concentration (CC\u003csub\u003e50\u003c/sub\u003e, μg/mL)\u0026nbsp;and R\u003csup\u003e2\u003c/sup\u003e value were determined by using a Microsoft Excel scatter plot with an exponential trendline (y = 107.92e\u003csup\u003e-0.05x\u003c/sup\u003e). The data were collected from three independent experiments of triplicate readings (n = 3) and presented as the mean ± standard deviation (SD) of percentages of cell viability relative to the untreated control. The statistical significances are denoted as follows: ns (not significant): P \u0026gt; 0.05 and **** P ≤ 0.0005 vs untreated control.\u003c/p\u003e","description":"","filename":"Figures16Looetal1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7721623/v1/46ca5db8308978f823781c32.jpg"},{"id":93639073,"identity":"1d1e57c9-2448-4b9d-ac5c-878c145401aa","added_by":"auto","created_at":"2025-10-16 02:04:38","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":356593,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMode of apoptosis and necrosis of rHALT-1-treated HeLa cells assessed using Annexin V and DNA dye.\u003c/strong\u003e HeLa cells were cultured in a 96-well plate at a density of 1 x 10\u003csup\u003e4\u003c/sup\u003e cells/well and incubated overnight for 16 hours. The line graphs represent the trends of \u003cstrong\u003e(A)\u003c/strong\u003e apoptosis and \u003cstrong\u003e(B)\u003c/strong\u003e necrosis observed over 12 hours, with various concentrations of rHALT-1 (0-30 µg/mL) and positive control (100 ng/mL hTRAIL peptide). The data were collected from three independent experiments with triplicate readings (n = 3) and presented as the mean ± SD of relative luminescence or fluorescence units (RLU/ RFU).\u003c/p\u003e","description":"","filename":"Figures16Looetal2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7721623/v1/485e3c17e70897bc7174f1e5.jpg"},{"id":93637994,"identity":"f47ab953-54ac-455c-a4ed-4444ec2de581","added_by":"auto","created_at":"2025-10-16 01:56:38","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":555324,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial membrane potential of HeLa cells treated with 12 µg/mL rHALT-1. \u003c/strong\u003eHeLa cells were seeded in a 10 cm cell culture dish at a density of 1 x 10\u003csup\u003e6\u003c/sup\u003e cells and incubated overnight for 16 hours. Cell apoptosis was detected by flow cytometry with JC-1/ 7-AAD dual staining after 6, 12, and 24 hours of treatment with 12 µg/mL rHALT-1 and 100 ng/mL hTRAIL peptide. \u003cstrong\u003e(A)\u003c/strong\u003e Dot plots of JC-1 aggregate versus JC-1 monomer grouped the healthy/apoptotic population (7-AAD\u003csup\u003e-\u003c/sup\u003e)\u003csup\u003e \u003c/sup\u003einto polarized and depolarized mitochondria. R4 upper gate represents the 7-AAD\u003csup\u003e-\u003c/sup\u003e/ polarized mitochondrial population;\u0026nbsp; R5, lower gate represents the 7-AAD\u003csup\u003e-\u003c/sup\u003e/ depolarized mitochondrial population. Percentage of cell population in gate R4 and R5 is indicated as mean ± SD (standard deviation). \u003cstrong\u003e(B)\u003c/strong\u003e Fold change of depolarization of the mitochondrial membrane.\u003cstrong\u003e \u003c/strong\u003eData were obtained from three independent experiments (n = 3) and presented as the mean cell population in percentages or fold changes ± SD relative to the untreated control. The statistical significances are denoted as follows: ns (not significant) P \u0026gt; 0.05; * ≤ 0.05; ** ≤ 0.01; *** ≤ 0.001; **** P ≤ 0.0005 vs untreated control.\u003c/p\u003e","description":"","filename":"Figures16Looetal3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7721623/v1/906f084dfe87bdceb3f7abd3.jpg"},{"id":93639076,"identity":"97e1390e-0089-44cf-9706-86acefaf9f20","added_by":"auto","created_at":"2025-10-16 02:04:38","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":561850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow cytometric analysis of the effects of rHALT-1 on caspase-3 activity. \u003c/strong\u003eHeLa cells were seeded in a 10 cm cell culture dish at a density of 1 x 10\u003csup\u003e6\u003c/sup\u003e cells and cultured overnight for 16 h. The cells were then left untreated or incubated with 12 µg/mL rHALT-1 or 100 ng/mL hTRAIL peptide for 6, 12, and 24 hours. \u003cstrong\u003e(A)\u003c/strong\u003e Dot plots of active caspase-3 versus 7-AAD grouped the cells into Q1 Active caspase-3\u003csup\u003e+\u003c/sup\u003e/ 7-AAD\u003csup\u003e-\u0026nbsp; \u003c/sup\u003e(live cells with cleaved caspase-3), Q2 Active caspase-3\u003csup\u003e+\u003c/sup\u003e/ 7-AAD\u003csup\u003e+\u0026nbsp; \u003c/sup\u003e(dead cells with cleaved caspase-3), Q3 Active caspase-3\u003csup\u003e-\u003c/sup\u003e/ 7-AAD\u003csup\u003e-\u0026nbsp; \u003c/sup\u003e(live cells without cleaved caspase-3), and Q4 Active caspase-3\u003csup\u003e-\u003c/sup\u003e/ 7-AAD\u003csup\u003e+ \u003c/sup\u003e(dead cells without cleaved caspase-3). \u003cstrong\u003e(B)\u003c/strong\u003e Fold change of active caspase-3 in early and late apoptotic cells. Data were obtained from three independent experiments (n = 3) and presented as the mean cell population in percentages or fold changes ± SD relative to the untreated control. The statistical significances are denoted as follows: ns (not significant): P \u0026gt; 0.05; * ≤ 0.05; ** ≤ 0.01; *** ≤ 0.001; **** P ≤ 0.0005 vs untreated control.\u003c/p\u003e","description":"","filename":"Figures16Looetal4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7721623/v1/61db2dcbf1636a744eca5d21.jpg"},{"id":93639075,"identity":"e4e2b704-84b3-4721-9cf4-25430428aa0c","added_by":"auto","created_at":"2025-10-16 02:04:38","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":368825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWestern blot analysis of apoptotic effects of rHALT-1 on Bcl-2 families, caspases, and cytochrome c proteins expressions. (A) \u003c/strong\u003eWestern blot images. HeLa cells were left untreated (1), and treated with 12 µg/mL rHALT-1 (2) and 100 ng/mL hTRAIL (3) for 6, 12, and 24 hours. Whole-cell lysates (10 µg) were fractionated by 12 % SDS-PAGE and subjected to western blotting analysis. Specific monoclonal antibodies were used to detect the expressions of Bad, Bax, Bcl-xL, Bcl-2, caspase-3, caspase-6, caspase-8, caspase-9, and cytochrome c. β-actin served as an internal control. Data showed results from one of three independent experiments (n = 3). The arrowhead indicates the expressed protein and its corresponding molecular weight in kilodaltons. \u003cstrong\u003e(B) \u003c/strong\u003eFold change of apoptotic protein expression levels. Band intensities were quantified using ImageJ analysis software (NIH). The integrated optical density provided a quantitative measure of the band intensity of protein expression. Each band was normalized to the bands corresponding to β-actin followed by untreated control bands, and the results were presented in bar graphs. Data were collected from three independent experiments (n = 3) and presented as the mean fold change ± SD relative to the untreated control (1.00-fold change, not shown).\u003c/p\u003e","description":"","filename":"Figures16Looetal5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7721623/v1/c39a54f377ed511fcdf030da.jpg"},{"id":93638003,"identity":"f04652b7-ef2c-4c1a-8442-a240be3a9a05","added_by":"auto","created_at":"2025-10-16 01:56:38","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":357000,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of the apoptotic molecular mechanism initiated by rHALT-1.\u003c/strong\u003e This diagram depicts the modulation of factors inducing apoptosis and highlights various proteins associated with the mitochondrial pathway. Orange indicates changes in protein levels within cells, either upregulated (upward arrow) or downregulated (downward arrow). Grey signifies no change in protein levels, and colourless proteins were not included in the study. Green circles are cytochrome c releasing from the mitochondria. Solid arrow (⟶) shows known interaction or activation step, whereas dashed arrow (⤑) with question mark indicates potential interactions or activation steps that were not observed under the current experimental conditions. PS, phosphatidylserine; mPTP, mitochondrial permeability transition pore; Bcl-xL, B-cell lymphoma-extra large; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; Bad, Bcl-2 associated agonist of cell death; Cyt C, cytochrome C; AIF, apoptosis-inducing factor; Endo G, endonuclease G; Apaf, apoptotic protease activating factor-1; Smac/DIABLO, second mitochondria-derived activator of caspases; Caps, caspase.\u003c/p\u003e","description":"","filename":"Figures16Looetal6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7721623/v1/0852968cd0c7278dd4c926aa.jpg"},{"id":98243739,"identity":"e39abb1d-8b8d-4b84-971f-d54268ee780c","added_by":"auto","created_at":"2025-12-15 16:10:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3440437,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7721623/v1/b58afcbf-e359-4908-9317-95cb532ace05.pdf"},{"id":93637997,"identity":"64e13803-252e-4af5-8b7c-c76b894676d7","added_by":"auto","created_at":"2025-10-16 01:56:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":588216,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials13Looetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7721623/v1/beebf51e5a4586183ce06539.pdf"},{"id":93639731,"identity":"3b28c25b-0398-432e-adea-ca2be23f2a73","added_by":"auto","created_at":"2025-10-16 02:12:38","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4234781,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials4Looetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7721623/v1/ec2a2c1386499649d6896064.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Recombinant HALT-1 induces mitochondrial-associated apoptotic mechanism in HeLa cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eActinoporins are a class of α-pore-forming toxins (α-PFTs) produced mostly by sea anemones for the purposes of capturing prey and protecting against predators [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. They are small proteins with 18\u0026ndash;20 kDa that are typically positively charged with an isoelectric point (PI) greater than 9 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Secondary structure of actinoporins consists of approximately 50% β-sheets, flanked by N-terminal and C-terminal amphipathic α-helices [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Within the fold, there are conserved tryptophan or tyrosine residues with aromatic rings that play a crucial role in the hydrophobic and electrostatic interactions with the target cell membrane [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Actinoporin, thus, utilizes this aromatic-rich region and an adjacent phosphocholine (POC) binding site to recognize and bind sphingomyelin (SM) on the cell membrane [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, actinoporins consist of disulfide linkages to enhance their structural stability and protect them from enzymatic degradation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. When attacking mammalian cells, oligomeric actinoporins create pores in the cell membrane, and the lumen size is about 1\u0026ndash;2 nm in radius which allow monovalent or divalent cations such as K\u003csup\u003e+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e to enter intracellularly [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWithin the subgroup of actinoporins are the \u003cem\u003eHydra\u003c/em\u003e actinoporin-like toxins (HALTs). There are a total of seven HALTs and they are found in the freshwater cnidarian, \u003cem\u003eHydra magnipapillata\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These proteins, including HALT-1 to HALT-7, share structural similarities with actinoporins and among them, HALT-1 is the most well-studied [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. HALT-1 shares some similarities with actinoporins but retains its unique characteristics. Like actinoporins, HALT-1 exerts hemolytic and cytolytic effects on human cells through a pore-forming mechanism [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The crystal structure of recombinant HALT-1 (rHALT-1) has been resolved at a high resolution of 1.43 \u0026Aring; [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. It structurally resembles actinoporins, featuring a β-sandwich fold composed of twelve β-strands, two α-helices, and two 3₁₀ helices. Notably, lysine 76 in the monomeric protein is acetylated, forming N6-acetyl-l-lysine, which interacts with a water molecule through its carbonyl group [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Despite sharing 60% sequence similarity with actinoporins, HALT-1 exhibits some unique structural features that enable it to selectively bind to sulfatide, a sphingolipid with a sulfate head group, rather than sphingomyelin, which is the primary binding site for most actinoporins [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHALT-1 exerts apoptotic behaviour in human cells when it is used at the IC50, as reported by Ng et al., 2019 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, its precise mechanism of triggering cell death remains unknown, particularly its role in the early and late stages of apoptosis after cell binding. There are two primary pathways that govern cell apoptosis, the extrinsic pathway and the intrinsic pathway. In brief, the extrinsic pathway can be initiated by death ligands binding to death receptors on the cell surface. The death receptors are members of the tumor necrosis factor (TNF) receptor gene superfamily. Subsequently, the death receptor recruits adaptor proteins which further activate intracellular caspases, including caspase-8, -10, and \u0026minus;\u0026thinsp;3, resulting in the execution phase of apoptosis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. On the other hand, the intrinsic pathway is triggered by internal stress signals such as DNA damage or growth factor shortage [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Bcl-2 family proteins, including Bad, regulate the intrinsic pathway, influencing mitochondrial outer membrane permeabilization (MOMP) by interacting with Bcl-xl, Bcl-2, Bax, and Bak [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. MOMP leads to cytochrome c release, binding to Apaf-1, forming the apoptosome, and activating caspase-9 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The subsequent activation of caspases, like caspase-3 and caspase-6, induces cell shrinkage, membrane blebbing, and nuclear fragmentation, ultimately leading to cell death [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eStudying how other actinoporins interact with and influence these processes may provide further insights into cell death induced by HALT-1. Venoms extracted from sea anemones, \u003cem\u003eStichodactyla haddoni\u003c/em\u003e and \u003cem\u003eHeteractis magnifica\u003c/em\u003e, have been proven to induce apoptosis in human cell lines via the death receptor-mediated pathway or/and the mitochondria-mediated pathways [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, the particular toxins that cause apoptosis in these sea anemones have yet to be identified. In another study, Kvetkina et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] have demonstrated that \u003cem\u003eHeteractis crispa\u003c/em\u003e actinoporin, rHct-S3, caused apoptosis by cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP). By using Western blot, the authors also showed both the upregulated Bax and the downregulated Bcl-2 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Furthermore, a study on Sticholysin II (StnII) from \u003cem\u003eStichodactyla helianthus\u003c/em\u003e revealed a unique mechanism of inducing cell death in Raji cells. In the context of programmed cell death, StnII could release calcium from the endoplasmic reticulum, thereby activating the MAPK-ERK pathway and causing mitochondrial depolarization. However, StnII did not activate caspase-3. Instead, it activated the expression of receptor-interacting protein kinase 1 (RIP1), suggesting a distinct form of cell death associated with mitochondrial dysfunction rather than caspase activation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In summary, these findings contribute to our understanding of diverse apoptotic mechanisms associated with different cytotoxic agents.\u003c/p\u003e\u003cp\u003eThe cytotoxic dose of HALT-1 is crucial in determining the switch between apoptosis and necrosis. Low doses of pore-forming toxins create small pores that allow selective ion passage, leading to programmed cell death [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], while higher doses form larger pores, resulting in ATP depletion and subsequent necrotic cell death [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The concentration of rHALT-1 may influence the size and properties of the pores formed in the cell membrane [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. A recent study by Ng et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] demonstrated that rHALT-1 induced apoptosis in HeLa, HepG2, and MCF-7 when it was applied at 50% inhibitory concentrations (ranging from 0.30 to 1.62 \u0026micro;M). However, the precise mechanism of action and the involved signaling pathways were not fully elucidated. Our study hypothesized that low doses of rHALT-1 could trigger apoptotic pathways within a short period. To test this, we first determined the sub-lytic concentrations of rHALT-1 for HeLa cells, and then examined phosphatidylserine exposure on the cell membrane and mitochondrial outer membrane polarization. Besides, we also assessed the expression levels of various apoptosis-related proteins using Western blot. Understanding how human cells respond to low doses of rHALT-1 could pave the way for its potential use in future cancer therapies such as immunotoxins (ITs). ITs facilitate the programmed cell death of cancerous cells by coupling a toxic domain with a specific antibody that targets tumour antigens on cancer cell surface [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eProduction of recombinant HALT-1 (rHALT-1) protein\u003c/h2\u003e\u003cp\u003eHALT-1-inserted pET28a plasmid was previously constructed [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and the purification procedure was performed as described by Yap et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Briefly, rHALT-1 was overexpressed in IPTG-induced \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3). Cells were then lysed under sonication to obtain rHALT-1 in the soluble fraction in binding buffer (20 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0). rHALT-1 was purified under native conditions using Ni-NTA (Nickel-Nitrilotriacetic Acid) resin at a flow rate of 1 mL/min. First, cell lysate containing rHALT-1 was mixed with the resin, and incubated at 4\u0026deg;C for 15 minutes to bind the Ni-NTA resin (ThermoFisher Scientific). After incubation, non-His-tagged proteins were removed with binding and subsequently wash buffers (20 mM sodium phosphate, 300 mM NaCl, 30 mM imidazole, pH 8.0). Finally, 6 mL of elution buffer (20 mM sodium phosphate; 300 mM NaCl, 250 mM imidazole, pH 8.0) was used to release rHALT-1 in 500 \u0026micro;L fractions, which were then collected for SDS-PAGE analysis.\u003c/p\u003e\u003cp\u003eThe purified rHALT-1 protein obtained from binding affinity purification underwent further purification using SP sepharose ion exchange chromatography. Ten millilitre of rHALT-1 protein was added to the column. The column was then rinsed with 80 mM phosphate buffer (pH 7.4) with increasing NaCl concentrations (60, 120, 150, and 200 mM), culminating in a 200 mM NaCl solution to elute the rHALT-1 protein. Following elution, the ion exchange buffer was replaced with 1x PBS for protein pooling and concentration analysis. Finally, the concentrated rHALT-1 protein in 1x PBS was assessed using SDS-PAGE to confirm the absence of protein degradation or contamination.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eHeLa cells (ATCC\u0026reg; CCL-2\u0026trade;) were cultured in Dulbecco\u0026rsquo;s Modified Eagle Minimal (DMEM) (Nacalai Tesque, Japan), supplemented with 10% fetal bovine serum (FBS) and 0.5% of Pen-Strep containing 10,000 units/mL of penicillin and 10,000 \u0026micro;g/mL of streptomycin. The cells were incubated at 37\u0026deg;C in a humidified chamber with 95% O\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e. Serum-free medium was used during the treatment period.\u003c/p\u003e\n\u003ch3\u003eCytotoxicity assay\u003c/h3\u003e\n\u003cp\u003eMTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed to assess the sublytic toxicity of rHALT-1 in HeLa cells. Cells (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells) in a 96-well plate were exposed to respective treatments including 100 \u0026micro;L of various rHALT-1 concentrations (0, 6, 12, 18, 24, and 30 \u0026micro;g/mL), 100 \u0026micro;L of PBS-DMEM (vehicle control) and 100 \u0026micro;L of 100 ng/mL hTRAIL (positive control). To assess the background contributed by DMEM, three wells were added with DMEM only. Cells were then incubated for 24 hours at 37\u0026deg;C. After the treatment, 25 \u0026micro;L of 5 mg/mL MTT solution was loaded into each well, and the plate was incubated for another 3 hours, followed by the removal of supernatant and the addition of 200 \u0026micro;L DMSO to dissolve the insoluble purple formazan crystals. Absorbance was measured at 570 nm (reference at 630 nm) using an Infinite M Plex microplate reader (Tecan, Germany). Cell viability percentages obtained from three experiments (n\u0026thinsp;=\u0026thinsp;3) were calculated using this equation,\u003c/p\u003e\u003cp\u003e% cell viability = {(A\u003csub\u003et\u003c/sub\u003e-A\u003csub\u003eb\u003c/sub\u003e)/(A\u003csub\u003ec\u003c/sub\u003e-A\u003csub\u003eb\u003c/sub\u003e)}x100 where A\u003csub\u003et\u003c/sub\u003e = cells incubated with rHALT-1 or hTRAIL; A\u003csub\u003eb\u003c/sub\u003e= blank; A\u003csub\u003ec\u003c/sub\u003e= negative control and then visualized in mean percentages. CC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;g/mL) and R\u003csup\u003e2\u003c/sup\u003e were determined via Microsoft Excel's exponential trendline, where CC\u003csub\u003e50\u003c/sub\u003e indicates a 50% reduction in cell viability compared to the untreated control.\u003c/p\u003e\n\u003ch3\u003eKinetic evaluation of cell apoptosis and necrosis\u003c/h3\u003e\n\u003cp\u003eThe RealTime-Glo\u0026trade; Annexin V Apoptosis and Necrosis Assay Kit (Promega, USA) was employed to track apoptosis and secondary necrosis dynamics by continuously capturing luminescence and fluorescence signals from the same cell samples. In a black 96-well plate with a clear bottom (Corning Incorporated, USA), 1 x 10\u003csup\u003e4\u003c/sup\u003e cells per well were seeded. Cells in the wells were treated with 100 \u0026micro;L of distinct solutions as described in the MTT assay: background control, vehicle control, positive control (100 ng/mL hTRAIL peptide), and various rHALT-1 concentrations (0, 6, 12, 18, 24, and 30 \u0026micro;g/mL), each was supplemented with 1 x detection reagent and incubated at 37\u0026deg;C. The 1 x detection reagent, comprising luminogenic Annexin V stain and a fluorescent dye, was prepared following the manufacturer's instructions. The assay plate was shaken at 500 rpm with linear amplitude for approximately 30 seconds before measurement to ensure thorough mixing. Luminescence (with automatic attenuation) and fluorescence (excitation: 485 nm; emission: 525 nm) signals were recorded every 30 minutes for four hours, shifting to hourly intervals thereafter until the readings stabilised. The initial measurement point was designated as the zero point. Net relative luminescence or fluorescence unit (RLU or RFU) was calculated by subtracting the average luminescence or fluorescence value of background signal from all sample luminescence or fluorescence values Each data point represented the average values from three independent experiments (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\n\u003ch3\u003eMitochondrial membrane potential assay\u003c/h3\u003e\n\u003cp\u003eMitochondrial membrane potential (MMP; Δψ) changes were assessed to evaluate apoptotic response induced by rHALT-1 using a MitoScreen JC-1 kit (Becton Dickinson, USA). JC-1 (1st J-aggregate-forming cationic) is a dye that is sensitive to membrane potential and exists as green fluorescence monomers (indicative of low MMP) or red-orange fluorescence aggregates (indicative of high MMP). This study combined JC-1 and BD Pharmingen\u0026trade; 7-AAD (Becton Dickinson, USA) dyes to distinguish apoptotic and necrotic cells. Viable and early apoptotic cells would remain unstained (7-AAD\u003csup\u003e\u0026minus;\u003c/sup\u003e), while necrotic cells would exhibit 7-AAD\u003csup\u003e+\u003c/sup\u003e staining due to loss of plasma membrane integrity. Following this, viable and apoptotic cells would be differentiated by JC-1 staining into two cell populations with polarized and depolarized MMP. Cell cultures containing 1x 10\u003csup\u003e6\u003c/sup\u003e cells in 5 mL were left untreated and treated with rHALT-1 (12 \u0026micro;g/mL) and hTRAIL (100 ng/mL) for 6, 12, and 24 hours in a 100 mm culture dish. An additional control of unstained and untreated cells was included to establish background signals. Post-treatment, cells were harvested, centrifuged at 400 x g for 5 minutes at room temperature, and washed twice with 1 mL of 1x PBS. Subsequently, cells were incubated with 20 \u0026micro;L of 7-AAD in 0.5 mL of 1x PBS for 15 minutes at room temperature. After incubation and centrifugation, cell pellets were gently resuspended in 0.5 mL of freshly prepared JC-1 working solution and incubated for 10\u0026ndash;15 minutes at 37\u0026deg;C. Following this, cells were washed twice with 1x Assay Buffer and then gently resuspended in 0.5 mL of 1x Assay Buffer. The cell suspension was filtered through a 40 \u0026micro;m cell strainer and transferred to polystyrene round-bottom tubes for subsequent analysis using a BD FACSCalibur\u0026trade; flow cytometer (Becton Dickinson, USA). Data acquisition and analysis were conducted utilizing the BD CellQuest Pro Software (Becton Dickinson, USA). 7-ADD negative cells were further evaluated for the MMP test. The fluorescent signal generated by JC-1 reflects changes in Δψ, indicating whether the MMP is polarized or depolarized. The percentages of 7-AAD⁻ depolarized (JC-1 monomer) and polarized (JC-1 aggregate) cell populations were calculated by dividing the number of depolarized or polarized cells by the total number of 7-AAD⁻ cells. Fold change relative to untreated cells was determined by dividing the percentage of mitochondrial membrane depolarization in treated samples by that of the untreated control (UTC), which served as the baseline with a fold change value of 1.00.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCaspase-3 enzymatic activation assay\u003c/h2\u003e\u003cp\u003eThe FITC Active Caspase-3 Apoptosis Kit (Becton Dickinson, USA) combined with BD Pharmingen\u0026trade; 7-AAD (Becton Dickinson, USA) dye, were used to detect active caspase-3 in rHALT-1-treated cells using flow cytometry. This double staining differentiated cells into live, early apoptotic, late apoptotic, and dead categories. Cells in the early and late stages of apoptosis were identified through caspase-3 staining, while 7-AAD distinguished between live and dead cells. Similar to the MMP assay, cells were treated, harvested, washed with 1x PBS, and stained with 7-AAD dye. However, in this case, cells treated with 12 \u0026micro;g/mL of rHALT-1, untreated control, and positive control (100 ng/mL hTRAIL-treated cells) were stained with anti-caspase-3 antibody. The subsequent sample preparation followed the manufacturer's guidelines by fixing and permeabilizing the cells using 0.5 mL of Cytofix/Cytoperm\u0026trade; solution for 20 minutes on ice. The cells were pelleted and washed twice with 0.5 mL of 1x BD Perm/Wash\u0026trade; buffer at room temperature to eliminate other cellular components. After that, the cells were incubated with 120 \u0026micro;L of 1x BD Perm/Wash\u0026trade; buffer containing FITC rabbit anti-active caspase-3 for 30 minutes at room temperature. Following staining, unbound antibodies were removed by washing with 1.0 mL of 1x BD Perm/Wash\u0026trade; buffer. The cells were then resuspended in 0.5 mL of 1x BD Perm/Wash\u0026trade; buffer. Subsequently, the cell suspension was filtered through a 40 \u0026micro;m cell strainer and transferred to polystyrene round-bottom tubes for further analysis using BD FACSCalibur\u0026trade; flow cytometer (Becton Dickinson, USA) to differentiate cell populations based on caspase-3 and 7-AAD staining patterns. The percentage of the apoptotic cell population exhibiting active caspase-3 was determined based on the fluorescence emitted by the bound anti-active caspase-3 antibodies. The percentage of caspase-3⁺ cells was calculated by dividing the number of cells expressing active caspase-3 by the total number of gated cells. Fold change was determined by dividing this percentage by the percentage of caspase-3⁺ cells in the untreated control (UTC), which served as the baseline with a fold change value of 1.00.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eWestern blot detection of Bcl-2 families, caspases, and cytochrome c\u003c/h3\u003e\n\u003cp\u003eWestern blot analysis was performed to track the expression levels of apoptotic markers (Bad, Bax, Bcl-2, Bcl-xL, caspase-3, caspase-6, caspase-8, caspase-9, and cytochrome c). Cell pellets (1x 10\u003csup\u003e6\u003c/sup\u003e cells) of untreated, 12 \u0026micro;g/mL rHALT-1, and 100 ng/mL hTRAIL-treated cells at 6, 12, and 24 hours were lysed using 1x RIPA buffer containing 1 mM PMSF for 30 minutes with agitation every 5-minute interval. Whole cell lysate was quantified using the BCA method with Pierce\u0026trade; BCA Protein Assay Kit (Thermo Fisher Scientific, USA) and normalized. Ten micrograms of total protein underwent SDS-PAGE on 12% polyacrylamide gels. Proteins were then transferred onto PVDF membranes with 1x transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3, 20% methanol) using a Trans-Blot Turbo Transfer System (Bio-Rad, USA), set at 25 V and 0.8 A for 40 minutes. Membranes were blocked with 5% BSA prepared in 1x triethanolamine-buffered saline solution containing Tween-20 (TBST) for one hour and incubated with primary antibodies overnight at 4\u0026ordm;C or 2 hours at room temperature. The dilution ratios of primary antibodies: β-actin (1:1000), Bax (1:1000), Bad (3:1000), Bcl-2 (1:1000), Bcl-xL (1:1000), caspase-3 (3:1000), caspase-6 (1:1000), caspase-8 (1:1000), and caspase-9 (3:1000), and cytochrome c (1:1000). Following five washes with 1x TBST, each wash lasting 5 minutes with agitation, membranes were exposed to HRP-linked secondary antibodies (1:1000), anti-rabbit IgG or anti-mouse IgG (Cell Signaling Technology, USA) for one hour at room temperature and washed five times again. A chemiluminescent substrate (Promega, USA) was applied, and signals were captured using the LAS 500 ImageQuant\u0026trade; Chemiluminescent imager (GE Healthcare, USA). Densitometry analysis was performed using ImageJ software (National Institutes of Health, USA), utilizing β-actin as a loading control. Fold change in protein expression was calculated by normalizing to the untreated control. This robust procedure enabled a quantitative comparison of target protein levels across different experimental conditions and time points.\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical data were presented as means of three independent experiments (n\u0026thinsp;=\u0026thinsp;3), with standard deviations displayed as error bars. The level of statistical significance was determined using an unpaired t-test and denoted as two-tailed P values as follows: ns (not significant)\u0026thinsp;=\u0026thinsp;P\u0026thinsp;\u0026gt;\u0026thinsp;0.05; * P\u0026thinsp;\u0026le;\u0026thinsp;0.05; ** P\u0026thinsp;\u0026le;\u0026thinsp;0.01; *** P\u0026thinsp;\u0026le;\u0026thinsp;0.001; **** P\u0026thinsp;\u0026le;\u0026thinsp;0.0005 vs untreated control.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eSub-lytic concentrations of rHALT-1 for HeLa cells\u003c/h2\u003e\u003cp\u003erHALT-1 induced dose-dependent cytotoxic effects in HeLa cells, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. A concentration of 6 \u0026micro;g/mL of rHALT-1 exhibited marginal toxicity, while higher concentrations led to decreased cell viability, suggesting increased cytotoxicity. Using the exponential trendline equation from the scatter plot, the CC\u003csub\u003e50\u003c/sub\u003e of rHALT-1 after 24 hours of exposure to cells was estimated to be approximately 15.4 \u0026micro;g/mL, which is consistent with the observed range of 12\u0026ndash;18 \u0026micro;g/mL in the bar chart. This result aligns with the findings of Liew et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], which reported that a concentration as low as 15 \u0026micro;g/mL reduced cell viability by 50% in HeLa cells. In this study, we defined 15 \u0026micro;g/mL or below as the sub-lytic concentrations of rHALT-1 for HeLa cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eApoptotic and necrotic activities of rHALT-1\u003c/h2\u003e\u003cp\u003eBoth apoptotic and necrotic activities were observed following treatment with rHALT-1 at various concentrations ranging from 6 to 30 \u0026micro;g/mL. Apoptotic cells, identified through Annexin V luminescent signals, are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, while necrotic cells, labelled using fluorescent probes, are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. These cellular kinetic activities were monitored in real-time. The initial onset of apoptotic activity was evident within an hour across all rHALT-1 concentrations used to treat HeLa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This apoptotic induction is attributed to the externalization of phosphatidylserine from the inner leaflet of the cell membrane [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. A small proportion of untreated HeLa cells underwent cell death, with apoptotic activity reaching approximately 30\u0026ndash;35% at 12 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, a significant increase in apoptotic cells was observed following treatment with rHALT-1, with the time required to achieve maximal cell death depending on the rHALT-1 concentration. A more than 2-fold increase in the luminescent signal was observed when cells were treated with 6 \u0026micro;g/mL of rHALT-1 for up to 12 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), with the signal appearing to continue rising beyond the 12-hour mark. At higher concentrations of 12 or 18 \u0026micro;g/mL, apoptotic cells required only 6\u0026ndash;7 hours to reach nearly 100% cell death, compared to the longer time needed for cells treated with 6 \u0026micro;g/mL of rHALT-1. When 24 or 30 \u0026micro;g/mL of rHALT-1 was applied, a peak in luminescent signal was observed within 3\u0026ndash;5 hours, indicating that the apoptotic activity had reached its maximum stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAlongside the assessment of apoptotic activity described above, necrotic activity, detected using fluorescent probes, was also observed in HeLa cells following dose-dependent treatment with rHALT-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). At high concentrations of rHALT-1, necrotic activity increased rapidly, becoming evident at the 3rd hour for 30 \u0026micro;g/mL and at the 5th hour for 24 \u0026micro;g/mL in treated HeLa cells. This coincided with the stage where no further increase in the number of apoptotic cells was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC and S1D). Similar patterns were observed at rHALT-1 concentrations of 12 and 18 \u0026micro;g/mL, where a steep increase in apoptotic activity was evident during the first 7 hours following treatment with rHALT-1, after which the activity slowed down, while necrotic activity gradually increased over the 12-hour period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and S1B). In contrast, hTRAIL, used as a control, exhibited higher luminescence signals compared to rHALT-1-treated cells after 8 hours, with minimal necrosis over 12 hours, consistent with its role as a typical apoptotic inducer (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B).\u003c/p\u003e\u003cp\u003eIn summary, treatment with rHALT-1 induced both apoptotic and necrotic activities in HeLa cells in a dose- and time-dependent manner. Apoptosis was evident within one hour across all tested rHALT-1 concentrations (6\u0026ndash;30 \u0026micro;g/mL). A dose-dependent acceleration of apoptotic activity was observed, with higher rHALT-1 concentrations (\u0026ge;\u0026thinsp;12 \u0026micro;g/mL) leading to rapid cell death within 3\u0026ndash;7 hours, while lower concentrations required longer periods (up to 12 hours). Necrotic activity became prominent at higher rHALT-1 concentrations (\u0026ge;\u0026thinsp;24 \u0026micro;g/mL) after apoptotic activity reached its peak, typically after 3\u0026ndash;5 hours. A similar trend was observed at 12\u0026ndash;18 \u0026micro;g/mL, where necrotic activity gradually increased as the rise in apoptotic activity stabilized.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003erHALT-1 induced mitochondrial membrane depolarization (MMP)\u003c/h2\u003e\u003cp\u003eChanges in mitochondrial membrane depolarization (MMP) during apoptosis and cell membrane damage during necrosis can be analysed using flow cytometry analysis with JC-1 and 7-AAD double staining, respectively. To do these, HeLa cells were treated with rHALT-1 at 12 \u0026micro;g/mL. This concentration was chosen due to its notable induction of intracellular apoptotic events in the first 7th hour of rHALT-1 incubation, while exhibiting minimal necrosis compared to other higher concentrations, as mentioned above.\u003c/p\u003e\u003cp\u003eCells with polarized mitochondria were grouped in R4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), while cells with reduced aggregate fluorescence or depolarized mitochondria were grouped in R5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Apparently, rHALT-1 caused a significant increase in the number of cells with reduced aggregate fluorescence at 6, 12, and 24 hours, indicating a substantial loss of MMP, a characteristic sign of early apoptosis. The percentage of depolarized cells increased significantly to 31.63% \u0026plusmn; 2.46 (3.35-fold change) at 6 hours, 37.26% \u0026plusmn; 1.06 (2.82-fold change) at 12 hours, and 44.75% \u0026plusmn; 2.54 (3.06-fold change) at 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). The most pronounced effect occurred at 6 hours, where the Δψ decreased 3.35 times compared to the untreated control. Both rHALT-1 and hTRAIL peptide treatments yielded highly significant results (P\u0026thinsp;\u0026le;\u0026thinsp;0.0005 or \u0026le;\u0026thinsp;0.001), which were consistent across three replicates. These findings suggest that rHALT-1 induced a time-dependent reduction in MMP, triggering the apoptotic cascade in HeLa cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eNo Traces of Caspase-3 Activation\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the percentages of HeLa cells exhibiting active caspase-3 after treatment with 12 \u0026micro;g/mL rHALT-1 and 100 ng/mL hTRAIL at 6, 12, and 24 hours. In untreated cells, the proportions of active caspase-3 in early and late apoptosis remained consistently low, at approximately 1% or less across all three time points. Cells treated with rHALT-1 also exhibited minimal changes in active caspase-3 level (early and late), with overall percentages of 0.41% at 6 hours, 1.04% at 12 hours, and 0.69% at 24 hours. The fold changes relative to the untreated control ranged from 0.38 to 1.09, suggesting no significant induction of caspase 3-mediated apoptosis. On the other hand, hTRAIL-treated cells demonstrated markedly higher level of active caspase 3 in both early and late apoptosis: 69.03% at 6 hours, 73.17% at 12 hours, and 81.01% at 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Fold changes ranged from 56.70 to 73.40 compared to the untreated control, reflecting substantial apoptosis induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Therefore, only the positive control indicated a significant increase in apoptosis through caspase-3 activation at all three time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B), whereas rHALT-1 treated and untreated cells did not possess caspase-3 activation. Statistical analysis confirmed that the results for hTRAIL-treated cells were highly significant (P-value: 0.001\u0026thinsp;\u0026le;\u0026thinsp;P\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003erHALT-1 regulated intrinsic apoptotic protein expressions\u003c/h2\u003e\u003cp\u003eThe western blot analysis tracked the expression levels of intrinsic and extrinsic apoptotic markers (Bad, Bax, Bcl-2, Bcl-xL, caspase-3, caspase-6, caspase-8, caspase-9, and cytochrome c) upon treatment with rHALT-1 or hTRAIL across 6, 12, and 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Bad, a pro-apoptotic protein, gradually increased over 24 hours in UTC, possibly due to a cellular compensatory response to stress or an effort to maintain homeostasis. In contrast, Bad expression peaked at 12 hours in the rHALT-1 and hTRAIL treatment groups indicates that these treatments might be influencing cellular functions differently, potentially altering signaling pathways or gene expression patterns associated with Bad regulation. Bax, another pro-apoptotic protein, rose over time in both rHALT-1 and hTRAIL treatments, peaking at 24 hours for rHALT-1 and at 12 hours for hTRAIL. Anti-apoptotic proteins, such as Bcl-2 and Bcl-xL, were significantly expressed in untreated cells, but their levels decreased following treatments with rHALT-1 or hTRAIL, particularly at the 6-hour mark. Cytochrome c showed consistent, significant upregulation across all treatments and time points. Initiator caspase-9 showed the most prominent expression of its precursor form (47 kDa) at 12 hours following rHALT-1 treatment. Active forms (37 kDa and 35 kDa) were detected at all time points in all samples, with or without treatment. Notably, the expression levels of active caspase-9 were more than one-fold higher in HeLa treated with rHALT-1 or hTRAIL compared to untreated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Caspase-8, another initiator caspase, was highly expressed in its precursor form (57 kDa) in untreated and rHALT-1-treated cells, while the active form (43 kDa) was detected only in hTRAIL-treated cells at 6 and 12 hours, with very low expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The procaspase-3 exhibited high expression in rHALT-1-treated cells at 24 hours, with lower but still notable expression at 6 and 12 hours. No active forms of caspase-3 were detected in any rHALT-1 treated cells, whereas active caspase-3 was observed only in hTRAIL-treated cells (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). It is likely that neither procaspase-6 nor its active form were expressed in both treated and untreated HeLa cells, as no detectable bands corresponding to 34 kDa (inactive form), 20 kDa, or 11 kDa appeared on the Western blot. Although one band appears to have a molecular weight between 25\u0026ndash;35 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig. S4), the presence of multiple higher molecular weight bands suggests that this may be a non-specific signal. Both active and inactive forms of caspase-6 were not detected in hTRAIL-treated cells, likely because hTRAIL primarily activates the extrinsic pathway, involving caspase-8 and caspase-3. Caspase-6 is typically activated downstream of these executioner caspases or through amplification involving mitochondrial pathways, such as cytochrome c release, which are less prominently triggered by hTRAIL in this context.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe quantification of protein blots using ImageJ software highlighted significant changes in apoptotic protein expression regulated by 12 \u0026micro;g/mL rHALT-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). At 6 hours, compared to the untreated control, Bad and Bax were upregulated with fold changes of 1.164 and 2.103, respectively. Bcl-2 and Bcl-xL showed slight downregulation with rHALT-1 treatment with fold changes of 0.827 and 0.638, respectively. Cytochrome c and active caspases-9 (37 and 35 kDa) were consistently upregulated with fold changes of 2.262, 2.352, and 2.033, respectively, indicating increased apoptotic activation. At 12 hours, rHALT-1 treatment resulted in increased expression of Bad, Bax, cytochrome c, and active caspases-9 with fold changes of 1.842, 1.915, 1.567, 1.376 (37 kDa), and 2.131 (35 kDa), respectively, except Bcl-xL showed slight downregulation with a fold change of 0.798, while Bcl-2 displayed nearly no difference with a fold change of 1.033. At 24 hours, rHALT-1 treatment led to further downregulation of Bad, Bcl-2, and Bcl-xL with fold changes of 0.751, 0.805, and 0.551, respectively. Bax, cytochrome c, and two active caspases-9 were upregulated with a fold change of 1.593, 2.385, 1.813 (37 kDa), and 1.297 (35 kDa). The hTRAIL treatment showed similar trends in Bad and Bax upregulation at 6 and 12 h, while Bcl-2 and Bcl-xL were downregulated across the three time points. Cytochrome c and active caspases-9 exhibited decreased fold change of upregulation over time with hTRAIL treatment. Although hTRAIL binds to death receptor 4 (DR4) and death receptor 5 (DR5) on the cell membrane, triggering the extrinsic apoptotic pathway, it can also secondarily activate the intrinsic pathway by engaging mitochondria and promoting cytochrome C release in HeLa cells [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSimilar to other actinoporins, HALT-1 has been shown to induce apoptosis, adhering to pathways observed in actinoporins such as StnII of \u003cem\u003eStichodactyla helianthus\u003c/em\u003e. However, while the overall apoptotic mechanism remains comparable, the specific intermediate proteins in the HALT-1-mediated signaling cascade may differ, indicating variations in transduction pathways among different actinoporins. The concentration HALT-1 required to trigger apoptosis in HeLa cells is critical. We determined that IC\u003csub\u003e50\u003c/sub\u003e (15.4 \u0026micro;g/mL) or lower could induce apoptosis, whereas higher concentrations lead to necrosis, a pattern consistent with bacterial and sea anemone pore-forming toxins (PFTs) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. To account for potential dilution errors in all experiments, a concentration of rHALT-1 lower than IC\u003csub\u003e50\u003c/sub\u003e, 12 \u0026micro;g/mL, was used in all subsequent experiments. In addition to HALT-1 dosage, incubation time is another critical factor in apoptosis induction; therefore, treatment durations of 6, 12, and 24 hours were applied across all experiments to demonstrate the apoptotic activity. Bad and Bax might be expressed as early as upon the addition of rHALT-1 to the cells, reaching peak levels between 6 and 12 hours, followed by a decline from 12 to 24 hours. In contrast, Bcl-2 and Bcl-xL expression decreased following HALT-1 treatment, with no significant differences observed across the time points. Similarly, Abdzadeh et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] also analysed the expression levels of Bcl-2 family proteins in A549 lung cancer cells treated with cytotoxic compounds derived from the mucus of \u003cem\u003eStichodactyla haddoni\u003c/em\u003e. These compounds were found to upregulate Bak and Bax expression while downregulating Bcl-2 in HT-29 cells, leading to morphological changes characteristic of apoptosis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The authors further indicated that the apoptotic events are dose-dependent, whereas their time-dependency has not been investigated in the same study. Another actinoporin from \u003cem\u003eHeteractis crispa\u003c/em\u003e, Hct-S3, could also suppress the migratory activity of colorectal carcinoma HT-29 cells by down-regulating Bcl-2 and up-regulating Bax, and promote apoptosis through the activation of caspase-3, as determined by Western Blotting with specific antibodies [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn fact, Bcl-2 family proteins play a pivotal role in determining the susceptibility of cells to apoptosis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Upregulation of pro-apoptotic proteins stimulates the permeabilization of the outer mitochondrial membrane causing the release of apoptogenic factors such as cytochrome c, AIF, Endo G, and Smac/DIABLO to the cytoplasm [\u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. When HeLa cells were treated with this low concentration of rHALT-1 (12 \u0026micro;g/mL), the Bcl-2 family regulated the cell transition to an apoptotic state, marked by the reduction in mitochondrial potential (ΔΨm). This hallmark arises from the collapse of negative charge within the matrix, leading to a decrease in cationic JC-1 dye uptake and consequently causing JC-1 to exist in its monomeric form within the cytoplasm [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Notably, at the 24-hour time point, mitochondrial membrane depolarization was measured at 44.75%, closely aligning with MTT assay data showing that 38.59% of cells failed to reduce MTT to formazan crystals due to mitochondrial dehydrogenase dysfunction. These findings are the first evidence showing that rHALT-1-induced early apoptosis is associated with mitochondrial dysfunction, as reflected by the loss of ΔΨm in HeLa cells. A study involving crude extracts from sea anemone \u003cem\u003eHeteractis magnifica\u003c/em\u003e demonstrated a similar induction of apoptosis in A549 lung carcinoma cells. The treated cells showed a reduction in MMP and the presence of caspase-3 cleavage when exposed to a concentration of 10 \u0026micro;g/mL for 24 hours [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Interestingly, another study highlighted that StnII, as an actinoporin, triggers calcium release mainly from the endoplasmic reticulum, activates the mitogen-activated protein kinase ERK, and disrupts mitochondrial membrane potential [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The study also demonstrated that the StII-induced cell death process does not involve caspase activation or hallmark characteristics of apoptosis and pyroptosis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eUpon disruption of the outer mitochondrial membrane, the release of cytochrome c from mitochondria is a recognized part of the apoptotic effect. According to the western blot results, rHALT-1 induced a time-dependent increase in cytochrome c levels as early as 6 hours after treatment, and this elevation correlates with changes in MMP. The depolarization of MMP and the release of cytochrome c are reliable indicators for detecting early changes in the intrinsic pathway [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. These findings underscore the crucial role of cytochrome c in the rHALT-1-induced apoptotic pathway, suggesting its contribution to the activation of downstream apoptotic effectors. Based on the protein expression, only initiator caspase-9 appeared in its active form following rHALT-1 treatment. Other caspases, including the initiator caspase-8 and the executioner caspases-3, remained in their precursor forms, while the expression of caspase-6 appeared to be not evident. The absence of active caspase-3 was also proven in a flow cytometric assay which differed from the response triggered by hTRAIL, a known apoptosis inducer. This discovery resembles the response to the reaction seen in Raji cells when subjected to sublytic levels of StnII, where, as detailed by Soto et al [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], the presence of executioner caspase-3 expression is lacking.\u003c/p\u003e\u003cp\u003eThere is additional evidence suggesting that rHALT-1 might induce cell death through alternative and unique pathways that do not involve the activation of caspase-3, caspase-6, and caspase-8. This discrepancy arises because caspase-3 and caspase-8 possess RGD (Arg-Gly-Asp) binding site, which can be the target of RGD-containing actinoporins such as StnII and FraC [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The presence of this binding motif in these caspases could potentially allow actinoporins such as FraC to bypass upstream steps, involving the integrin binding to apoptotic initiator activation, instead, they might directly initiate downstream apoptotic executioner activation [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Instead of RGD, HALT-1 contains a RAG (Arg-Ala-Gly) motif [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. If HALT-1 possessed the RGD motif, it could potentially bind directly to the RGD binding site and activate caspase-3 and caspase-8. Moreover, in the conventional apoptotic pathway, intrinsic caspase-9, either alone or in conjunction with extrinsic caspase-8, converges to cleave caspase-3. However, this was not the case for HALT-1, despite its ability to activate caspase-9 production. Therefore, this study suggests the existence of alternative mitochondrial pathways that may facilitate apoptotic execution independently of caspases-3 and \u0026minus;\u0026thinsp;6.\u003c/p\u003e\u003cp\u003eHere we propose a rHALT-1-mediated intrinsic apoptotic pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The intrinsic mitochondrial pathway, as illustrated in the schematic, is initiated by rHALT-1 and involves the modulation of key apoptotic proteins. This pathway is primarily regulated by the balance between pro-apoptotic and anti-apoptotic proteins. Upon activation, rHALT-1 upregulates pro-apoptotic proteins such as Bad and Bax while downregulating anti-apoptotic proteins like Bcl-2 and Bcl-xL. These changes lead to mitochondrial membrane depolarization, resulting in the release of cytochrome c (CytC) into the cytoplasm.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOnce released, cytochrome c interacts with Apaf-1, forming the apoptosome, which subsequently activates caspase-9 (Caps9). However, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e indicates that uncertainties regarding the activation of downstream caspases, such as caspase-3 (Caps3) and caspase-6 (Caps6), appear to be either inactive or not involved in rHALT-1-induced apoptosis. Therefore, we speculated that an alternative mitochondrial pathway involving rHALT-1 may be at play. Cytochrome c is not the only molecule released into the cytosol when the mitochondrial outer membrane becomes permeabilized. Other mitochondrial proteins, such as endonuclease G (Endo G) and apoptosis-inducing factor (AIF), are also released from the mitochondrial intermembrane space [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Numerous studies have shown that these proteins can induce cell death in a caspase-independent manner [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. To trigger cell death, Endo G and AIF translocate to the nucleus, where they degrade DNA into fragments. We attempted to detect the expression of Endo G and AIF in HeLa cells following rHALT-1 treatment; however, neither Endo G nor AIF was visible as distinct bands on the Western blot or detectable in qPCR (Fig. S3). Another mitochondrial proteins, Smac and DIABLO, are released into the cytosol, where they antagonize inhibitors of apoptosis proteins (IAPs), thereby facilitating the activation of caspase-3 [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Since our results indicate that HALT-1 induces cell death via a caspase-3-independent pathway, we may exclude the possible involvement of Smac and DIABLO in post-cytochrome c release events.\u003c/p\u003e\u003cp\u003eAddressing the limitations in understanding HALT-1-induced apoptosis requires strategic approaches. First, an in-depth analysis of other genes and proteins involved in caspase-3 independent pathway using qPCR and western blot techniques could provide valuable insights. Second, accurately capturing the apoptotic window period by employing sophisticated methods like cell sorting, combined with assays such as TUNEL and MMP, would offer precise endpoints before necrosis onset. Third, enhancing data reliability by expanding the apoptotic inducer panel to include both extrinsic and intrinsic apoptotic inducers would strengthen pathway references. Lastly, utilizing apoptotic inhibitors to block caspases and target anti-apoptotic proteins can dissect intrinsic and extrinsic apoptotic pathways. These strategic methodologies hold significant promise in advancing apoptosis research and broadening the potential therapeutic applications of HALT-1.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides the first evidence that rHALT-1 induces apoptosis in HeLa cancer cells by modulating multiple proteins associated with the mitochondrial pathway. The findings reveal that a low concentration of rHALT-1 (12 \u0026micro;g/mL) triggers apoptosis, as indicated by phosphatidylserine exposure and mitochondrial dysfunction. Alterations in the expression of key apoptotic proteins, including Bad, Bax, Bcl-2, Bcl-xL, cytochrome c, and caspase-9, suggest the involvement of the intrinsic apoptotic pathway. However, the absence of caspase-3, caspase-6, and caspase-8 activation at this concentration suggests that alternative cell death mechanisms, which are caspase-9-dependent but independent of executioner caspases-3 and \u0026minus;\u0026thinsp;6, and initiator caspase-8, may be involved.\u003c/p\u003e\u003cp\u003eWhile the precise mechanism of rHALT-1-induced cell death remains unclear, these findings lay a strong foundation for future research. Notably, rHALT-1\u0026rsquo;s potential as a toxin component presents promising therapeutic applications, particularly in the development of immunotoxins. By selectively inducing apoptosis while preserving the integrity of healthy cells, this approach could provide novel treatment strategies for diseases characterized by dysregulated cell death.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eα-PFT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eα-pore-forming toxin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAIF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eapoptosis-inducing factor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCaps\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecaspase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCC₅₀\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e50% cytotoxic concentration\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDulbecco\u0026rsquo;s Modified Eagle Minimal\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEndoG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eendonuclease G\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003efetal bovine serum\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHALT-1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eHydra\u003c/em\u003e actinoporin-like toxin-1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIAPs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003einhibitors of apoptosis proteins\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIC50\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e50% inhibitory concentration\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eITs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eimmunotoxins\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eJC-1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e1st J-aggregate-forming cationic\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMMP orΔψ\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emitochondrial membrane potential\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMTT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMOMP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emitochondrial outer membrane permeabilization\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNi-NTA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNickel-Nitrilotriacetic Acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eisoelectric point\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePOC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ephosphocholine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRIP1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ereceptor-interacting protein kinase 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRFU\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003erelative fluorescence unit\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRLU\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003erelative luminescence unit\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003esphingomyelin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePARP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003epoly (ADP-ribose) polymerase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eStnII\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSticholysin ITNF,tumor necrosis factor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eUTC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003euntreated control.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eL.W.L. performed all experiments, developed the methodology, and wrote the original draft; J.S.H. conceptualized the study, provided resources, prepared funding acquisition, and wrote and edited the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets during and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRam\u0026iacute;rez-Carreto, S., Miranda-Zaragoza, B. \u0026amp; Rodr\u0026iacute;guez-Almaz\u0026aacute;n, C. 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Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2. \u003cem\u003eEMBO J.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e (23), 6627\u0026ndash;6636 (2001).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pore-forming toxin, Hydra actinoporin-like toxin-1, Apoptotic pathway mechanism, Mitochondrial depolarization, Caspases","lastPublishedDoi":"10.21203/rs.3.rs-7721623/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7721623/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe study explored the apoptotic mechanism of \u003cem\u003eHydra\u003c/em\u003e actinoporin-like toxin-1 (HALT-1), an α-pore-forming toxin (α-PFT) produced by \u003cem\u003eHydra magnipapillata\u003c/em\u003e. α-PFT has been known to induce membrane pores in human cells upon contact, leading to the cell death. While previous research has covered HALT-1\u0026rsquo;s structural, membrane binding, cytolytic, and haemolytic aspects, the detailed information on apoptotic mechanism and cell signalling pathways remain unknown. Our study confirmed previous findings of rHALT-1's dose-dependent cytotoxicity, with a CC\u003csub\u003e50\u003c/sub\u003e of 15.4 \u0026micro;g/mL observed after 24 hours of treatment in our case. Hence, an rHALT-1 concentration below 15.4 \u0026micro;g/mL was selected to examine its apoptotic activity. Real-time Annexin V and DNA dye assays revealed dose- and time-dependent apoptotic patterns, with 12 \u0026micro;g/mL rHALT-1 inducing maximum apoptosis at 7 hours and minimal necrosis. Subsequently, flow cytometric analysis showed mitochondrial membrane potential depolarization without active caspase-3 throughout 6, 12, and 24-hour treatments. Western blot analysis indicated upregulation of apoptotic-inducing proteins (Bad, Bax, cytochrome c, caspase-9) and downregulation of antiapoptotic proteins (Bcl-2, Bcl-xL) at 12 \u0026micro;g/mL of rHALT-1. The absence of active caspases 3, 6, and 8 expressions suggests alternative cell death pathways. In conclusion, the study proposes, for the first time, that rHALT-1 induces apoptosis in HeLa cells by mediating the mitochondrial pathway, although active caspase-3 does not appear to be involved in the execution process. These findings provide a foundation for elucidating the mechanistic basis of rHALT-1 activity and highlight its potential utility in toxin-related research and biotechnological applications.\u003c/p\u003e","manuscriptTitle":"Recombinant HALT-1 induces mitochondrial-associated apoptotic mechanism in HeLa cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-16 01:56:33","doi":"10.21203/rs.3.rs-7721623/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-08T05:34:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-06T05:41:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-05T03:24:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"105220890013539058042199006837541389213","date":"2025-10-02T10:55:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"22070783994635982448410562442746054408","date":"2025-10-02T08:04:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-02T02:07:57+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-01T10:54:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-30T08:25:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-30T01:21:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-26T11:53:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3047df40-92cb-4511-a903-8d454a40cc73","owner":[],"postedDate":"October 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55936421,"name":"Biological sciences/Biochemistry"},{"id":55936422,"name":"Biological sciences/Cell biology"},{"id":55936423,"name":"Biological sciences/Drug discovery"},{"id":55936424,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-12-15T16:02:15+00:00","versionOfRecord":{"articleIdentity":"rs-7721623","link":"https://doi.org/10.1038/s41598-025-27491-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-11 15:57:51","publishedOnDateReadable":"December 11th, 2025"},"versionCreatedAt":"2025-10-16 01:56:33","video":"","vorDoi":"10.1038/s41598-025-27491-y","vorDoiUrl":"https://doi.org/10.1038/s41598-025-27491-y","workflowStages":[]},"version":"v1","identity":"rs-7721623","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7721623","identity":"rs-7721623","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}