New Insights into the Bioenergetic and Immunomodulatory Properties of Phospholipases A₂ from Bothrops diporus Venom

Abstract

Phospholipases A₂ (PLA₂s) are key mediators of the cytotoxic and inflammatory activities of snake venoms. While PLA₂ isoforms from Bothrops diporus venom have been characterized and shown to possess antimetastatic and antiangiogenic properties, their impact on mitochondrial bioenergetics and immune modulation has not yet been investigated. In this study, we examined the bioenergetic and immunomodulatory effects of B. diporus PLA₂s using integrated biochemical, metabolic, and multiplex cytokine analyses. In MDA-MB-231 breast cancer cells, pooled PLA₂s induced a dose-dependent decrease in MTT-reducing activity, increased mitochondrial ROS, caused Δψm hyperpolarization, and decreased NADH autofluorescence, collectively indicating sustained mitochondrial stress. Real-time impedance measurements further revealed a marked inhibition of cell proliferation. In human PBMCs, pooled PLA₂s elicited a dynamic cytokine program, inducing early cytotoxic (Granzyme B) and chemotactic (CCL2, CCL3, CCL4) mediators, followed by late pro-inflammatory and regulatory factors such as IL-6, TNF-β, IL-10 and IL-15. Analysis of a single purified PLA₂ isoform (Fraction 6) confirmed activation of the canonical IL-6/TNF-α/IL-1β axis but uniquely induced IL-10 and CCL20, revealing isoform-specific immunomodulatory properties. Altogether, these findings provide the first integrated characterization of mitochondrial and immune perturbations induced by B. diporus PLA₂s, expanding their recognized biological scope and underscoring their potential as molecular templates for novel pharmacological strategies targeting mitochondrial vulnerabilities or modulating the tumor immune microenvironment.

1. Introduction

Phospholipases A₂ (PLA₂s, EC 3.1.1.4) constitute a widely distributed and functionally diverse family of enzymes present in organisms ranging from bacteria to mammals, where they participate in essential biochemical and physiological processes. These enzymes catalyze the hydrolysis of the ester bond located at the sn-2 position of glycerophospholipids, generating free fatty acids and lysophospholipids, which can act either as signaling molecules themselves or as precursors for the biosynthesis of a broad spectrum of lipid mediators, including prostaglandins, leukotrienes and thromboxanes. Through this catalytic activity, PLA₂s play pivotal roles in membrane phospholipid turnover, maintenance of membrane integrity, intracellular and intercellular communication, inflammatory signaling and immune regulation [1,2].

Proteomic studies have demonstrated that PLA₂s are major components of Bothrops venoms and play a central role in the pathophysiology of envenomation, mediating a wide range of biological activities that extend beyond their catalytic properties, including myotoxic, neurotoxic, pro-inflammatory, and hemostatic effects [3]. Despite their well-documented toxic actions, snake venom PLA₂s (svPLA₂s) have also emerged as bioactive molecules with potential pharmacological relevance, particularly in the context of cancer research. These enzymes exert selective cytotoxicity toward tumor cells through mechanisms that involve membrane disruption, mitochondrial stress, oxidative imbalance, apoptosis induction, and metabolic alterations [4,5,6,7].

Mitochondria play a fundamental role in cellular homeostasis and redox regulation, acting both as a major source of reactive oxygen species (ROS) and as the site of energy metabolism controlled by the mitochondrial membrane potential (Δψm) [8]. Under physiological conditions, ROS act as tightly controlled signaling molecules; however, when their production exceeds antioxidant capacity, oxidative stress develops, compromising mitochondrial function and overall cell viability [9]. In cancer biology, oxidative stress has emerged as a decisive factor in tumor development and progression [10], as ROS may either promote or suppress tumor survival through the regulation of proliferation, invasion, metastasis and angiogenesis [11]. Tumor cells often sustain intrinsically elevated ROS levels to support rapid proliferation and adapt by activating alternative metabolic pathways, which provides a survival advantage and contributes to treatment resistance [12]. Accordingly, mitochondrial dysfunction and ROS manipulation have gained increasing attention as therapeutic targets in oncology [13]. Within this framework, tumor cell models offer a relevant platform to evaluate whether bioactive molecules are capable of further modulating mitochondrial redox homeostasis and bioenergetic parameters beyond the intrinsic alterations associated with the malignant phenotype. In this context, svPLA₂s, particularly those derived from Bothrops species, have demonstrated the ability to disrupt mitochondrial function by inducing ROS overproduction and altering Δψm in cancer cells, events that together can lead to mitochondrial destabilization and apoptotic cell death. These findings have positioned svPLA₂s as promising candidates for mechanistically driven anticancer strategies [7,14,15].

In addition to bioenergetic disruption and oxidative stress induction, PLA₂s have also been reported to modulate innate immune responses, particularly through the stimulation of pro-inflammatory cytokine release. Cytokines serve as key mediators of immune communication, and their dysregulation constitutes a critical factor in tumor progression, immune escape and tissue remodeling. Therefore, PLA₂-induced cytokine modulation may influence immune cell recruitment, activation, and effector responses, potentially reshaping the tumor microenvironment and complementing their direct cytotoxic actions [16,17]. By acting at both mitochondrial and immunological levels, PLA₂s may not only exert direct antitumor effects but also contribute to the activation of immune mechanisms involved in tumor recognition and elimination.

We previously isolated and biochemically characterized two basic PLA₂ isoforms from B. diporus venom, an Asp49 PLA₂-I and a Lys49 PLA₂-II–like protein [18]. Later, we demonstrated that both isoforms exert cytotoxic, anti-migratory, anti-invasive and anti-tubulogenic effects on tumor and endothelial cells, and through an integrin-focused in silico approach, we provided the first comparative evidence of their antimetastatic and antiangiogenic potential, with the Lys49 variant being the most active [19]. More recently, using in vivo and ex vivo chorioallantoic membrane (CAM) models, we confirmed the antiangiogenic profile of these venom PLA₂s, showing reduced vascular density and branching, endothelial apoptosis and decreased VEGF expression, reinforcing their relevance as potential angiogenesis-targeting biomolecules [20].

Although these studies have characterized both isoforms and provided evidence of their antimetastatic and antiangiogenic potential, their effects on mitochondrial function, oxidative stress and immune modulation remain largely unexplored. Therefore, this study aims to investigate the impact of PLA₂s isolated from B. diporus venom on mitochondrial dysfunction, ROS generation, cytokine secretion and immune modulation.

2. Results

2.1. Isolation of PLA₂s from Bothrops diporus Venom

The HPLC profile of B. diporus venom from Argentina displayed six major protein peaks eluting at 51–58 min (Figure 1), consistent with the previously reported chromatographic region typically associated with PLA₂s [18]. MALDI-TOF mass spectrometry confirmed that all fractions corresponded to proteins within the expected mass range for venom PLA₂s. While fractions 1–5 showed overlapping patterns that precluded their complete separation, Fraction 6 was identified as a single isoform based on its homogeneous mass signal (Supplementary Data, Figure S1). Homogeneity of the pooled PLA₂s, assessed by SDS-PAGE under reducing and non-reducing conditions, is shown in Supplementary Figure S2.

2.2. Effect of PLA₂s on Redox Balance and mtROS Levels in MDA-MB-231 Cells

2.2.1. MTT-Cellular Redox Capacity

The mitochondrial-dependent redox capacity of MDA-MB-231 cells was evaluated after exposure to the pooled PLA₂ sample at two incubation times (4 and 24 h). The results demonstrated a dose-dependent effect at both time points (Figure 2A,B). Specifically, after 4 h, significant alteration in NADH levels was observed at 25 and 50 μg/mL, resulting in approximately 20% and 23% reductions, respectively, whereas no significant changes were detected at concentrations below 25 μg/mL (Figure 2A). In contrast, after 24 h of incubation, the mitochondrial-dependent redox capacity was significantly reduced at concentrations starting from 5 μg/mL, with more pronounced effects at 25 and 50 μg/mL, leading to approximately 30% and 40% reductions, respectively (Figure 2B).

2.2.2. Mitochondrial ROS (mtROS) Levels

We evaluated the effect of PLA₂s on mitochondrial reactive oxygen species (mtROS) levels in MDA-MB-231 cells. Cells were exposed to PLA₂s at concentrations of 10, 25, and 50 μg/mL for 4 and 24 h. As expected, menadione, a well-established ROS inducer [21], significantly increased mtROS levels (Figure 2E). Similarly, PLA₂ treatment led to a significant elevation in mtROS levels, but only at 25 and 50 μg/mL, with no significant differences detected between the two incubation times (Figure 2C,D).

2.3. Role of PLA₂s in Mitochondrial Function and Proliferation of MDA-MB-231 Cells

2.3.1. Determination of Mitochondrial Membrane Potential (Δψm)

To evaluate whether PLA₂-induced mtROS production was accompanied by changes in mitochondrial membrane potential (Δψm), TMRM fluorescence was measured in MDA-MB-231 cells after 4 and 24 h of treatment. The OXPHOS uncoupler FCCP (2.5 µM) was used as a positive control and induced a marked decrease in TMRM fluorescence, confirming mitochondrial depolarization. In contrast, treatment with PLA₂s at all tested concentrations (25, 40 and 50 μg/mL) resulted in a significant increase in TMRM fluorescence at both incubation times, indicative of mitochondrial hyperpolarization and an increased proton motive force across the inner mitochondrial membrane (Figure 3A,B).

2.3.2. Determination of NADH Levels

To further explore the relationship between mitochondrial stress and bioenergetic imbalance, NADH autofluorescence was measured in MDA-MB-231 cells after exposure to PLA₂s. As shown in Figure 3C, treatment with PLA₂s led to a concentration-dependent decrease in NADH fluorescence, with significant reductions observed from 25 to 50 μg/mL. This decline indicates enhanced NADH oxidation and a reduction in the cellular pool of reduced cofactors, consistent with the hyperpolarized mitochondrial state induced by these enzymes. The loss of NADH suggests an imbalance in mitochondrial redox homeostasis, likely associated with increased electron transport activity and mtROS-driven metabolic stress.

2.3.3. Effect of PLA₂s on MDA-MB-231 Cell Growth Kinetics

The impact of PLA₂s on the proliferative behavior of MDA-MB-231 cells was assessed using real-time impedance monitoring over 96 h. As shown in Figure 3D, control cells followed the expected exponential growth pattern, reaching their maximal normalized cell index (nCI) at approximately 70–72 h. In contrast, PLA₂-treated cells (25 μg/mL) displayed a markedly blunted growth curve. The increase in nCI was modest throughout the experiment, reaching a significantly lower nCI_max and performing so earlier (around 50–55 h). After this peak, the nCI declined and remained stable, indicating a sustained impairment in proliferative capacity. These differences were reinforced by the area-under-the-curve (AUC) analysis (Figure 3E). While early proliferation (0–4 h) was comparable between groups, PLA₂ treatment significantly reduced cumulative growth during the 24–48 h and 48–72 h intervals. Altogether, these findings show that PLA₂s exert a progressive and pronounced inhibitory effect on the proliferation of MDA-MB-231 cells.

2.4. Impact of PLA₂s on Cytokine Production and Immune Mediators

2.4.1. IsoPlexis-IsoCode Analysis

The profile of cytokines and inflammatory mediators demonstrated a time-dependent modulation in response to the pool of PLA₂s. The most overexpressed mediators at 4 h included Granzyme B, IL-13, MCP-1, MIP-1α, MIP-1β, TNF-α, TNF-β and GM-CSF suggesting an early immune activation phase characterized by cytotoxic activity (Granzyme B), Th2-promoting responses (IL-13), and strong pro-inflammatory signaling (TNF-α, TNF-β). High levels of MCP-1, MIP-1α, MIP-1β and GM-CSF indicate the ability to induce an early recruitment of monocytes and macrophages, contributing to the inflammatory response. After 10 h, significant upregulation of markers such as IL-6, MIP-1β, and sCD137 indicate a shift towards a sustained inflammatory response. The increase in IL-6 suggests an amplification of cytokine signaling and immune cell activation, while upregulation of sCD137 points to prolonged T-cell stimulation. Elevated MIP-1β further reinforces the continuous recruitment of immune cells. By 24 h, the overexpressed mediators included Granzyme B, IL-13, IL-6, MCP-1, MIP-1α, sCD137, and TNF-β, indicating a complex immune response involving cytotoxicity, chronic inflammation, and immune regulation. The sustained presence of Granzyme B reflects ongoing cytotoxic activity, while co-expression of IL-6, TNF-β, and sCD137 suggests persistent inflammation and immune cell engagement. Additionally, the maintained elevation of MCP-1 and MIP-1α highlights prolonged recruitment of monocytes/macrophages, potentially leading to tissue remodeling or further immune activation. The strong late induction of IL-10 and IL-15 further suggests the emergence of regulatory and lymphocyte-supporting pathways, consistent with a shift from early inflammation toward a more balanced and sustained immune activation (Figure 4). Overall, these findings suggest that PLA₂s induce a dynamic immune response, with an early activation phase (4 h), a sustained inflammatory response (10 h), and a complex immune modulation at later stages (24 h). The involvement of both pro-inflammatory and regulatory cytokines/chemokines highlights the multifaceted nature of the immune response triggered by PLA₂s, which may contribute to either protective or pathological outcomes depending on the context.

2.4.2. Luminex Assay

Following MALDI-TOF mass spectrometry analysis, Fraction 6 from B. diporus venom was identified to contain a single PLA₂ isoform (item 3.1), making it the fraction selected for further investigation by Luminex. Cytokine profiling of PBMC supernatants stimulated with the purified PLA₂ isoform revealed a predominantly delayed inflammatory and immunomodulatory response. No major changes were detected at 1 h or 4 h. However, by 10 h a significant increase was observed in IL-6, TNF-α, IL-10, MIP-3α (CCL20) and IL-13 (Figure 5). IL-6 and TNF-α represent a robust pro-inflammatory signature, while the concurrent rise in IL-10 indicates the onset of a regulatory feedback mechanism. The induction of MIP-3α suggests activation of chemotactic pathways involved in dendritic cell and lymphocyte recruitment. IL-13 showed a modest but significant increase, consistent with a minor Th2-skewing component. IL-1β exhibited high variability without reaching statistical significance. No substantial changes were detected for IFNγ, IL-4, IL-12p70, IL-15, IL-22 or other markers included in the panel. Overall, the purified PLA₂ isoform triggered a delayed but coordinated inflammatory-regulatory response, distinct from the broader and earlier cytokine activation observed with the PLA₂ pool.

3. Discussion

svPLA₂s are multifunctional proteins capable of altering membrane architecture, triggering oxidative stress, and modulating inflammatory pathways, which together contribute to their broad spectrum of biological activities [2,7,22,23,24]. Although these enzymes are classically associated with the pathophysiology of envenomation, growing evidence indicates that svPLA₂s can profoundly affect mitochondrial dynamics and immune signaling, two central axes that determine cellular responses to stress. In this work, we examined the impact of B. diporus PLA₂s on key parameters of mitochondrial bioenergetics, redox homeostasis, and cytokine secretion using biochemical, metabolic, and immunological approaches. This integrated analysis allowed us to relate mitochondrial alterations observed in tumor cells with immunological responses detected in PBMCs from a complementary biological perspective, providing mechanistic insights into how PLA₂s influence cellular physiology, and revealing additional bioactive properties beyond their traditional toxic actions.

The first analysis performed to explore the cellular impact of B. diporus PLA₂s was the MTT reduction capacity, which provides a sensitive indication of mitochondrial redox activity. Although tumor cells exhibit intrinsic metabolic and mitochondrial alterations, the consistent changes in response to PLA₂ treatment relative to untreated controls indicate that these toxins impose an additional and sustained mitochondrial stress, rather than reflecting basal features of the malignant phenotype. PLA₂ exposure caused a clear, dose-dependent reduction in MTT signal, reflecting an early disturbance in NAD(P)H-dependent oxidoreductase activity within mitochondria [25]. In parallel, PLA₂s induced a significant increase in mtROS levels, a response consistent with membrane perturbation and altered electron flow through the respiratory chain [7]. The combination of reduced MTT activity and elevated mtROS aligns with previous studies showing that svPLA₂s rapidly disrupt oxidative balance and promote oxidative stress [5,14,26]. Supporting this interpretation, Urra et al. [15] emphasized that svPLA₂s can destabilize cellular homeostasis by enhancing mitochondrial ROS generation and impacting metabolic signaling pathways, providing a mechanistic framework consistent with our observations.

To further characterize the mitochondrial disturbances, we examined changes in membrane potential (Δψm) and NADH levels. PLA₂ exposure induced a consistent increase in TMRM fluorescence, indicating Δψm hyperpolarization at both incubation times. This response is consistent with early mitochondrial stress, where elevation of the proton motive force reflects an imbalance between electron transport activity and the efficient utilization of the electrochemical gradient [27]. Alterations in Δψm and mitochondrial integrity have been previously documented for svPLA₂s. For instance, a PLA₂ from Crotalus durissus terrificus induced a decrease in Δψm [28], as does MjTX-I, a PLA₂ isolated from B. moojeni snake venom [29]. In addition to these functional alterations, morphological studies further support mitochondrial involvement in svPLA₂ action. Electron microscopy analyses of tissue injected with BthTX-II, a myotoxic PLA₂ from Bothrops jararacussu, revealed extensive mitochondrial swelling and cristae disorganization, indicating that mitochondrial injury represents a conserved hallmark of svPLA_2-induced cytotoxicity [30]. In parallel, PLA₂ treatment caused a marked decrease in NADH autofluorescence, consistent with enhanced oxidation of reduced cofactors and reflecting a state of bioenergetic stress. It is important to note that mitochondrial hyperpolarization does not necessarily correlate with increased NADH availability, as conditions of mitochondrial stress may promote accelerated NADH oxidation and electron leakage toward ROS generation. Moreover, although NADH autofluorescence reflects both mitochondrial and cytosolic pools, the coordinated alterations observed across multiple mitochondrial parameters strongly support a predominant mitochondrial contribution to the detected redox imbalance. Similar alterations in NADH-linked metabolism have been reported for BaMtx, a Lys49-PLA₂ from Bothrops atrox, which increases ROS production and disrupts mitochondrial redox balance [25].

In agreement with these mitochondrial alterations, the real-time impedance assays revealed that B. diporus PLA₂s exert a sustained inhibitory effect on MDA-MB-231 cell proliferation. Rather than inducing an immediate cytolytic effect, the toxin-treated cells displayed a long-lasting inability to progress through normal proliferative trajectories, a behavior that aligns with the profound disturbances observed in redox balance and mitochondrial function that promotes cell cycle arrest and cytokine secretion [25]. Our results strongly suggest that svPLA₂ from B. diporus generate a persistent bioenergetic constraint that cells are unable to compensate, ultimately compromising their ability to sustain proliferation, possibly due to limitations in the mitochondrial function-dependent anabolic pathways. Collectively, this evidence highlights an underexplored action of svPLA₂ on metabolism that may offer novel approaches for the design of anticancer redox-modulator drugs [15].

Beyond their well-recognized roles as myotoxins and hemostatic disruptors, several Bothrops PLA₂s are now established as potent modulators of leukocytes, stimulating the production of pro-inflammatory cytokines and chemokines. In human PBMCs, the acidic Bl- PLA₂ from B. leucurus induces robust increases in IL-12p40, TNF-α, IL-1β and IL-6, without affecting IL-8 or IL-10, defining a predominantly pro-inflammatory signature [31]. In a different model, the acidic BJ-PLA₂-I from B. jararaca promotes leukocyte recruitment in vivo and selectively elevates IL-6 and IL-1β in peritoneal exudates, together with eicosanoid production [17]. Zuliani et al. [32], further showed that both Asp49 (catalytically active) and Lys49 (catalytically inactive) PLA₂s from B. asper induce marked increases in IL-1, IL-6 and TNF-α in mouse peritoneal exudates, with Asp49 variants eliciting a more intense inflammatory response than their Lys49 counterparts. Within this framework, the cytokine and chemokine responses elicited by B. diporus PLA₂s in PBMCs showed the expected upregulation of classical pro-inflammatory mediators such as IL-6, TNF-α and IL-1β. However, because our study employed high-content multiplex platforms (IsoPlexis and Luminex), we were able to detect a broader array of immune mediators than those assessed in previous studies of viperid PLA₂s. This extended panel included multiple chemokines involved in monocyte and lymphocyte recruitment (CCL2, CCL3, CCL4, CCL20), cytotoxic effector molecules (Granzyme B), and T-cell costimulatory markers (sCD137), indicating that PLA₂ stimulation engages additional layers of immune activation beyond the canonical IL-6/TNF-α/IL-1β axis. These mediators point to coordinated leukocyte trafficking, cytotoxic cell involvement and sustained T-cell activation, mechanistic dimensions that were not previously captured in earlier Bothrops models. Notably, the purified PLA₂ isoform (Fraction 6) recapitulated the core IL-6/TNF-α/IL-1β response but differed from the pool by selectively inducing IL-10 and CCL20 highlighting isoform-specific contributions to cytokine modulation. In this context, IsoPlexis analysis using pooled PLA₂s was employed as a high-content screening approach to capture the broader cytokine landscape and potential synergistic effects among isoforms, whereas Luminex assays were intentionally restricted to Fraction 6, identified by MALDI-TOF as a homogeneous single-isoform preparation, to enable isoform-specific immunological profiling. This pattern indicates that the full immune signature of the venom reflects the additive and non-overlapping contributions of multiple PLA₂ isoforms rather than the action of a single enzyme.

It is worth noting that while the integrated mitochondrial and immunological analyses presented here support a coherent framework linking bioenergetic stress and cytokine modulation induced by B. diporus PLA₂s, the interpretation of these findings should be confined to the scope of the experimental models employed. The identification of a formal mechanism of action and upstream molecular targets remains an open question for future studies.

4. Conclusions

This study highlights the biological and pharmacological relevance of B. diporus PLA₂s by showing that these enzymes simultaneously perturb mitochondrial homeostasis in tumor cells while reconfiguring immune signaling networks in human PBMCs. The differential effects observed between a purified PLA₂ isoform and the pooled preparation support the hypothesis that individual PLA₂s contribute distinct yet complementary components to the overall inflammatory signature of the venom, whereas their combined action may result in additive or amplifying effects. This integrated mode of action enriches current models of venom pathology and underscores the potential of PLA₂s as molecular templates for novel therapeutic approaches in cancer, immune regulation, and inflammation-associated diseases. In particular, the dual ability of these enzymes to impose persistent metabolic stress while reshaping cytokine signaling mirrors key mechanisms exploited by emerging anticancer strategies aimed at targeting mitochondrial vulnerabilities or modulating the tumor immune microenvironment.

5. Materials and Methods

5.1. Venom

B. diporus venom was supplied by the Antivenom Serum Production Center (CEPSAN) of Corrientes, W3400 Argentina, obtained from an undetermined number of adult specimens of either sex collected in this province. After extraction, the venom was vacuum dried, pooled, and stored at −20 °C.

5.2. Isolation of PLA₂s from B. diporus Venom

Basic PLA₂s were purified as previously described by RP-HPLC on a C₁₈ analytical column (250 × 4.6 mm, 5 μm particle; Phenomenex, Torrance, CA, USA) using an Agilent 1220 chromatography system monitored at 215 nm [18]. Peaks corresponding to PLA₂s were manually collected (fractions 1 to 6), dried, and stored at −20 °C. Homogeneity of pooled PLA₂s was assessed by SDS-PAGE using 4–16% gradient gels (Bio-Rad Laboratories, Hercules, CA, USA) under reducing and non-reducing conditions. The molecular identity of the isolated PLA₂ isoforms was further confirmed by MALDI-TOF MS mass spectrometry.

5.3. Analysis of PLA₂ Fractions by MALDI-TOF MS

MALDI-TOF MS analysis was performed on PLA₂ fractions (1–6) to determine the exact mass and detect potential protein contaminants. First, the samples were desalted using C₁₈ ZipTips, eluted in an α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution (5 mg/mL in 70% v/v ACN with 0.1% v/v TFA), and directly spotted onto the MALDI target. A MALDI 8030 mass spectrometer (Shimadzu Corporation, Kyoto, Japan) equipped with a 200 Hz solid-state laser (355 nm) was operated in positive linear mode. The laser power applied to the samples was set to 100, with the pulse extractor set to 13,000 Da and an ion gate blanking of 700 Da.

For subsequent experiments, either the highly pure Fraction 6 or a pooled sample was used. The pooled PLA₂ sample was generated by mixing equal total protein amounts from each collected fraction (fractions 1–6), defined by their RP-HPLC retention time windows.

5.4. MDA-MB-231 Cell Line and Culture Conditions

Cell line MDA-MB-231 (triple-negative breast cancer) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), containing 25 mM glucose and 4 mM glutamine, supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 μg/mL). The culture medium contained no exogenous pyruvate supplementation, and cells were maintained in a humidified atmosphere at 37 °C and 5% CO₂.

5.5. MTT Reduction Assay

The MTT assay was used to assess intracellular MTT reduction to formazan as an indirect measure of mitochondrial-dependent redox capacity in MDA-MB-231 cells. Briefly, 1 × 10⁴ cells/well were seeded in 96-well plates and incubated overnight. The cells were then incubated with increasing concentrations of PLA₂s or PBS as a control, for 4 or 24 h. Then, the cells were washed with PBS and incubated with MTT (0.5 mg/mL) for 1 h. Formazan crystals were solubilized using DMSO, and absorbance values (570 nm) were obtained using a Synergy H1 multireader (Agilent Technologies, USA).

5.6. Determination of Mitochondrial ROS (mtROS) Levels

Mitochondrial ROS generation was quantified in MDA-MB-231 cells. Briefly, 1.0 × 10⁵ cells/well were seeded in 24-well microplates with DMEM supplemented with 10% FBS and incubated for 24 h at 37 °C with 5% CO₂. Cells were then treated with different concentrations of PLA₂ isoforms (5–50 μg/mL) or PBS (Control) for 4 or 24 h. After incubation, cells were gently washed with PBS and stained with the fluorescent probe MitoSOX (5 μM) for 30 min at 37 °C. Menadione (5 μM) was used as a positive control. Finally, cells were washed, collected, and fluorescence intensity was quantified using flow cytometry [33].

5.7. Determination of Mitochondrial Membrane Potential (Δψm)

The effect of PLA₂s on Δψm was assessed using the potentiometric probe tetramethylrhodamine methyl ester (TMRM, Molecular Probes™, Thermo Fisher Scientific, Eugene, OR, USA). Briefly, 1 × 10⁵ MDA-MB-231 cells/well were seeded in 24-well plates with DMEM supplemented with 10% FBS and incubated for 24 h. PLA₂s were then added at 10 to 50 μg/mL concentrations and incubated for 4 or 24 h. Subsequently, cells were incubated with 5 nM TMRM for 15 min, protected from light. Trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP, 2.5 μM) was a positive control for mitochondrial depolarization. Changes in fluorescence were measured using flow cytometry with excitation and emission wavelengths of 488/525 nm, respectively [34].

5.8. Determination of NADH Levels

The NADH autofluorescence was measured in MDA-MB-231 cells (1.5 × 10⁵ cells/mL) plated in 96-well plates and allowed to reach confluence. The cells were then treated with PLA₂s (10–50 µg/mL), or PBS (Control). FCCP (2.5 µM) was used as a positive control for mitochondrial NADH oxidation. Autofluorescence was measured using an excitation wavelength of 340 nm and an emission wavelength of 428 nm with a Synergy H1 multimode reader (Agilent Technologies, USA).

5.9. Determination of Tumor Cell Growth by Impedance Changes

The growth curves were measured using the xCelLingence Real-Time Cell Analysis (RTCA) DP equipment (Agilent Technologies, Santa Clara, CA, USA). First, the background was measured in E-Plate 16 well plates with 50 µL of DMEM High Glucose medium, MDA-MB-231 cells were seeded on this volume at a density of 1.5 × 10⁴ cells/well in 150 µL of final volume in culture medium, and the cell impedance was measured every 15 min for 24 h. Subsequently, they were stimulated with PLA₂s at 25 µg/mL, and cell impedance data were collected every 5 min for 96 h. Cell index (CI) and area under the curve (AUC) data were obtained using RTCA software pro version 2.8.1.

5.10. Cytokines and Immune Mediators

5.10.1. Peripheral Blood Mononuclear Cells (PBMCs)

The PBMCs were obtained from the Orien Biorepository at the University of Virginia Cancer Research Center (IRB HSR #13310). PBMCs were evaluated with Trypan Blue staining to assess viability and only samples with over 90% viability were used.

5.10.2. IsoPlexis-IsoCode Analysis

The PBMCs (1 × 10⁶ cells) were stimulated with the PLA₂s-pool sample (25 μg/mL) for 4, 10 and 24 h or PBS (control) for 24 h. After each time point incubation in a 5% CO₂ incubator, the PBMCs were pelleted by centrifugation at 400× g for 10 min at 4 °C. The supernatants were collected, centrifuged 10,000× g for 10 min, and stored at −80 °C. Cytokine concentrations in the PBMCs supernatant were measured using IsoCode Human Adaptive Immune Panel (GM-CSF, Granzyme B, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-13, IL-15, IL-17A, IP-10, MCP-1, MIP-1α, MIP-1β, Perforin, sCD137, TNF-α, TNF-β). The University of Virginia Spatial Biology Core ran this panel on a Bruker IsoPlexis-IsoSpark instrument per 20 h in duplicate. Data analysis was performed using IsoSpeak software version 3.0.1, IsoSpark instrument software version 1.11.0 (Bruker) and GraphPad Prism version 8. The Heatmap analysis was performed on SRplot web server using complete Euclidean distance method (https://www.bioinformatics.com.cn/srplot. (accessed on 15 February 2026)).

5.10.3. Luminex Assay

The PBMCs (1 × 10⁶ cells) from 3 healthy subjects were stimulated with PLA₂-Fraction 6 (25 μg/mL) for 1, 4 and 10 h or PBS (control) for 10 h. After each time point incubation in a 5% CO₂ incubator, the PBMCs were pelleted by centrifugation at 400× g for 10 min at 4 °C. The supernatants were collected, centrifuged 10,000× g for 10 min, and stored at −80 °C. Cytokine concentrations in the PBMCs supernatant were measured using a Luminex Panel Th17 (GM-CSF, IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, IL-17E/IL-25, IL-17F, IL-21, IL-22, IL-23, IL-27, IL-28A, IL-31, IL-33, MIP-3α, TNFα and TNFβ). The University of Virginia flow cytometry core ran this panel on a Luminex MAGPIX in duplicate according to the manufacturer’s instructions. The statistical analysis was performed using the Two-Way ANOVA and FDR Benjamini–Hochberg method p-value ≤ 0.05 (Prisma version 8).

5.11. Statistical Analysis

Data are presented as mean ± standard deviation (SD) of the indicated number of replicates in each assay. The statistical significance of differences was tested by one-way ANOVA followed by the Tukey HSD post hoc test, considering p < 0.05 as significant. Statistical analyses and graphs were conducted using GraphPad Prism v8 (GraphPad Software, San Diego, CA, USA).