NJ025-1003

Therapeutic Efficacy of Luteolin Against Phorbol 12-Myristate 13-Acetate-Induced Cytotoxicity, Oxidative Stress, and Inflammation in HaCaT Cells 

Himanshi Gahlot1 and Sun Chul Kang1,2*

1Department of Biotechnology, Daegu University, Gyeongsan, 38453, Republic of Korea. 2Dr. Kang Bio Co. Ltd., Gyeongsan, 38428, Republic of Korea

Correspondence to: Sun Chul Kang, sckang@daegu.ac.kr

Received: October 13, 2025; Revised: December 4, 2025; Accepted: January 14, 2026; Published: January 24, 2026

NATPRO J. 2026, 3, 1-8 

https://doi.org/10.23177/NJ025.1003 

Copyright ©  The Asian Society of Natural Products

Abstract

Phorbol 12-myristate 13-acetate (PMA), a potent protein kinase C activator, is a well-established agent that triggers cytotoxicity, oxidative stress, and inflammation. This study investigated the cytoprotective potential of the natural flavonoid luteolin against PMA-induced toxicity in human HaCaT keratinocytes. We demonstrate for the first time that pre-treatment with luteolin significantly alleviates PMA-induced cytotoxicity in HaCaT cells, evidenced by increased cell viability, reduced LDH release, and enhanced clonogenic survival. Mechanistically, luteolin attenuated oxidative stress by scavenging ROS/NO generation, reducing lipid peroxidation (MDA), and restoring antioxidants (GSH, TRX). Luteolin further prevented the PMA-induced elevation of inflammatory mediators (NF-κB, COX-2, IL-6, and IL-1β) and associated DNA damage indicators (γ-H2AX, ERCC1). These findings reveal that luteolin possesses significant cytoprotective, antioxidant, and anti-inflammatory properties against PMA-induced damage, underscoring its potential as a therapeutic agent for inflammatory skin disorders. Future validation in more complex physiological models is warranted to bridge these promising in vitro results to potential clinical applications addressing PMA-related pathologies.

Keywords

luteolin, PMA, HaCaT, inflammation, ROS, NF-κB 

Introduction

Luteolin, a naturally occurring flavonoid abundant in plants such as celery, green pepper, and chamomile, has attracted considerable interest for its potential therapeutic properties, including antioxidant, anti-inflammatory, and anti-carcinogenic effects [1, 2]. PMA, a potent tumor promoter and activator of protein kinase C, is widely utilized to induce cytotoxicity, oxidative stress, and inflammatory responses in cellular models, thereby simulating pathological conditions such as cancer and inflammatory skin disorders [3]. The mechanism of PMA-induced damage, driven by reactive oxygen species (ROS) generation and inflammatory pathway activation, underscores the need to investigate protective agents like luteolin.

Recent studies highlight luteolin's ability to modulate key cellular pathways associated with inflammation and oxidative stress, suggesting its potential role in mitigating PMA-induced cytotoxicity [4]. Its proposed protective effects, including free radical scavenging and inhibition of pro-inflammatory cytokines, are often linked to the suppression of the NF-κB pathway, a central regulator of inflammation [5]. Nevertheless, the specific molecular mechanisms by which luteolin protects against PMA-induced pathologies remain inadequately explored. This gap is particularly relevant in human keratinocyte models, such as the HaCaT cell line, which serves as a standard and reliable system for studying skin-specific inflammatory and oxidative stress responses [6-8].

Hence, this study proposes to examine the therapeutic efficacy of luteolin against PMA-induced cytotoxicity, oxidative stress, and inflammatory signalling in HaCaT keratinocytes. We aim to elucidate the underlying molecular mechanisms by assessing parameters, including ROS/NO production, antioxidant status, and the expression of critical inflammatory and DNA damage response markers. The findings are expected to establish a mechanistic foundation for developing luteolin-based strategies to address PMA-induced cellular damage.  

Materials and methods

Chemicals and reagents

Luteolin (CAS: 491-70-3, PubChem CID: 5280445) was procured from Shanghai Aladdin Biochemical Technologies, while PMA, DMEM, MTT, H2DCFDA, DAPI, acridine orange, Griess reagent, and DMSO were sourced from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) and 1% penicillin–streptomycin were obtained from Gibco (Waltham, MA, USA). All solvents used were of high-quality from Sigma. 

Cell culture

The immortalized human skin keratinocyte HaCaT cells were acquired from the Korean Cell Line Bank (KCLB) and were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO2 until 70-80% confluent. All experiments were carried out using HaCaT cells between passages 15 and 20 post-thawing of a new vial to preserve phenotypic stability and ensure consistent results. This passage range complies with the established standard for HaCaT cells as documented in the literature and mitigates the risk of senescence-associated alterations. 

MTT assay

Cell viability was assessed using the MTT assay followed by [9]. HaCaT cells were cultured in 96-well flat-bottom microtiter plates at a density of 1.0 × 104 cells per well in 200 μL of DMEM in triplicate (n=3) and subsequently incubated at 37 °C for 24 h in a CO2 incubator. The cells were supplemented with fresh media once reaching a confluency of 60-70%. The cells were treated with luteolin (1-40 µM for 12 h), PMA (15-150 nM for 12 h), or luteolin pretreatment (40 µM for 12 h) followed by PMA (100 nM for 12 h). Subsequent to the treatment, cells were carefully rinsed with 1xPBS to eliminate non-adherent dead cells, residual culture media, and cellular debris. This was followed by the addition of 10 µl MTT solution (5 mg/mL in PBS) combined with 90 µl DMEM media to each well, and the cells were incubated for an additional 4 h at 37°C. The formazan crystals were solubilized with 150 µl of DMSO, and absorbance was quantified at 570 nm using a microplate reader. 

Clonogenic assay

For the clonogenic assay, HaCaT cells were inoculated in a 12-well cell culture plate (BD Falcon, CA) at a density of 4.0 × 103 cells per well in triplicate (n=3) and allowed to adhere. After 24 h, cells were treated with luteolin pretreatment (40 µM, 12 h) followed by PMA (100 nM, 12 h) at 37 °C in a CO2 incubator. The cells were then rinsed with 1xPBS and fixed with 4% paraformaldehyde for 10 min, followed by staining with 0.1% (w/v) crystal violet for 10 min. The colonies were counted using ImageJ software. 

Estimation of NO generation

We employed Griess reagent to quantify nitric oxide (NO) concentration. Pre-treatment of HaCaT cells (1.0 × 104 cells per well; n=3) with luteolin (40 µM, 12 h), followed by PMA treatment (100 nM, 12 h), in 96-well flat-bottom microtiter plates. For the NO measurement, 100 µL of Griess reagent was mixed with 100 µL of each well supernatant. The mixture was allowed to incubate for 10-15 min at 37 °C in the dark. The plates were analyzed with an ELISA plate reader (540 nm). To determine the quantity of NO, a standard curve was plotted using varying concentrations of sodium nitrite.

Lactate dehydrogenase activity assay

HaCaT cells were cultured in a 96-well plate at a density of 1.0 × 104 cells per well (n=3) for 24 h. Cells were pre-treated with luteolin (40 µM, 12 h) followed by PMA (100 nM, 12 h), resulting in the release of the extracellular cytosolic enzyme lactate dehydrogenase (LDH). The LDH assay kit (Sigma-Aldrich MAK066) was employed to assess cellular LDH release, and the experiment was conducted in accordance with the manufacturer's guidelines. The intensity of the yellow color formation was measured by using an ELISA plate reader at 540 nm.

Glutathione (GSH) assay

To evaluate GSH levels in HaCaT cells, cells were cultured in a 6-well tissue culture plate at a density of 3.0 × 105 cells per well (n=3) and allowed to adhere for 24 h, and pre-treated with luteolin (40 µM, 12 h) followed by PMA (100 nM, 12 h). Further procedure was followed as per the manufacturer's instructions (BioVision-K264-100 kit). A standard curve was generated using a 0.2 µg/µl working standard solution. Samples and standards were analyzed using a fluorescence plate reader set to excitation/emission wavelengths of 340/420 nm.

Malondialdehyde assay for estimation of lipid peroxidation

To estimate lipid peroxidation, we quantified the concentration of malondialdehyde (MDA) in the cell homogenate samples after treating HaCaT cells 1.0 × 104 cells per well (n=3) in a 96-well flat bottom plate with luteolin pretreatment (40 µM, 12 h) followed by PMA (100 nM, 12 h), adhering to the manufacturer's methodology (Sigma-MAK085 kit). Optical density was assessed at 532 nm utilizing an ELISA plate reader.

Quantification of free radical generation utilizing fluorescent probes

The endogenous ROS level was quantified utilizing a H2DCFDA fluorescent probe. HaCaT cells were seeded in a 6-well cell culture plate on sterile coverslips at a density of 3.0 × 105 cells per well (n=3) and allowed to adhere for 24 h. Subsequently, following pre-treatment with luteolin (40 µM, 12 h) followed by PMA (100 nM, 12 h), HaCaT cells were rinsed with 1xPBS and incubated at 37 °C in the dark with 10 μM H2DCFDA for 30 min. Essentially, in the presence of intracellular H2O2, the non-fluorescent membrane-permeable H2DCFDA is transformed into impermeable fluorogenic 20,70-dichlorofluorescein. The H2DCFDA fluorescence was observed under a fluorescence microscope, and the intensity was measured with ImageJ software.

Detection of nuclear DNA condensation

Apoptotic nuclear morphological modifications can be observed in cells using DAPI staining and fluorescence microscopy. HaCaT cells were cultured in a 6-well cell culture plate on sterile coverslips at a density of 3.0 × 105 cells per well (n=3) in DMEM and pre-treated with luteolin (40 µM, 12 h) followed by PMA (100 nM, 12 h). The cells were rinsed twice with 1xPBS before fixation in ice-cold 4% paraformaldehyde. After an additional rinse, the cells were incubated in a DAPI solution (5 µg/ml) for 30 min at 37 °C in the dark. Post-incubation, cells were rinsed with 1xPBS, and fluorescence intensity was assessed utilizing an inverted fluorescent microscope at 10x magnification.

Immunoblot analysis

Western blot analysis was performed as previously described [10] involved lysing HaCaT cells with RIPA buffer, followed by loading 100 µg protein onto SDS-PAGE. The proteins were transferred to PVDF and probed with primary and secondary antibodies, with band intensities normalized to β-actin loading controls using ImageJ software. The primary antibodies included were iNOS: BS90715 (Bioworld), eNOS: ab66127 (Abcam), nNOS (C7D7): 4231S (Cell Signalling Technology), Trx (FL-105): sc-20146 (Santa Cruz Biotechnology, Inc.), Cox-2 (C-20): sc-1745 (Santa Cruz Biotechnology, Inc.), IL-6: BS6419 (Bioworld), IL-1 beta: BS67752 (Bioworld), NFκB-p65 (K303): BS3157 (Bioworld), Histone H2A.X Antibody: 2595S (Cell Signalling Technology), ERCC1 (D10): sc- 17809 (Santa Cruz Biotechnology, Inc.), β-actin: (2A3): sc-517582 (Santa Cruz Biotechnology, Inc.). The secondary antibodies used in this study were Goat Anti-Rabbit HRP conjugated: A120-101P (Bethyl), Rabbit Anti-Mouse HRP conjugated: A90-117P (Bethyl), and Donkey Anti-Goat HRP conjugated: A50-101P (Bethyl).

Statistical analysis

All quantitative results are expressed as mean ± standard deviation (SD) derived from at least three independent experiments. Statistical significance was assessed using one-way analysis of variance (ANOVA) and subsequently analyzed with Dunnett's/Tukey’s tests. “*” represents the comparison of treated groups with control; * represents p <0.05, ** represents p < 0.01, and *** represents p < 0.001. “#” represents the comparison of luteolin + PMA treated group with PMA alone treatment, with # represents p < 0.05, ## represents p < 0.01, ### represents p < 0.001. All statistical analyses were conducted using GraphPad Prism version 8.0.1.

Results

Luteolin pre-treatment attenuates PMA-induced cytotoxicity and oxidative damage

Luteolin demonstrated a direct cytoprotective effect against PMA-induced toxicity in HaCaT cells. Initial dose-response analysis confirmed that luteolin, at concentrations up to 20 µM, was non-toxic to HaCaT cells (Figure 1A) as previously reported [9]. Subsequently, a sub-cytotoxic concentration of 40 µM luteolin was selected for pre-treatment. Exposure to PMA (100 nM) alone induced significant cytotoxicity, reducing cell viability by approximately 50% (Figure 1B) as previously reported [11]. Pre-treatment with luteolin (40 µM) to PMA significantly restored cell viability, indicating a potent protective effect (Figure 1C). This protection was corroborated by a marked reduction in LDH release in the luteolin + PMA group compared to PMA alone (Figure 1D), confirming the preservation of plasma membrane integrity. To investigate the underlying antioxidant mechanism, indicators of oxidative stress were evaluated. PMA exposure led to a significant increase in lipid peroxidation, as indicated by elevated MDA levels, and a concurrent depletion of the key intracellular antioxidant GSH. Luteolin pre-treatment effectively reversed both these effects, significantly lowering MDA (Figure 1E) and restoring GSH content (Figure 1F).

These findings were further supported by cellular and functional assays. Morphological analysis via colony formation assay revealed that luteolin pre-treatment prevented the PMA-induced cellular shrinkage and decreased cell colonies, demonstrating protection against PMA cytotoxicity (Figure 1G).

Luteolin pre-treatment suppresses PMA-induced ROS/RNS generation and modulates key antioxidant enzymes

Having established that luteolin mitigates the downstream markers of oxidative damage (Figure 1), we investigated its effect on the primary oxidative event: the PMA-induced generation of ROS/RNS. Qualitative fluorescence imaging using the H₂DCFDA probe revealed an intense fluorescent signal in cells treated with PMA (100 nM) alone, indicative of robust ROS generation (Figure 2A). This fluorescence was markedly attenuated in cells pre-treated with luteolin (40 µM), providing evidence of its radical-scavenging capacity.

Quantification of this effect confirmed a significant increase in both intracellular ROS and extracellular NO levels upon PMA treatment (Figure 2B). Consistent with the imaging data, luteolin pre-treatment significantly reduced the levels of NO, demonstrating its dual efficacy against distinct but interconnected reactive species. The overproduction of NO is often driven by the upregulation of nitric oxide synthase (NOS) isoforms.

To investigate this regulatory mechanism, we analyzed the protein expression of key NOS enzymes and the antioxidant protein thioredoxin (TRX) by western blot. PMA strongly upregulated the expression of inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) (Figure 2C). This coordinated upregulation represents a cellular stress response to a toxicity-induced PKC activation and oxidative stress [12]. Pre-treatment with luteolin effectively suppressed the PMA-induced expression of all three NOS isoforms. The inhibition of iNOS is particularly significant, as it is the primary source of sustained, high-output NO during inflammation, which can react with superoxide to form the potent oxidant peroxynitrite [13]. Subsequently, acute PMA exposure depleted the cellular levels of the essential antioxidant protein TRX, indicating a collapse of this key antioxidant defense marker. Luteolin pre-treatment effectively counteracted this depletion, restoring TRX expression (Figure 2C). This restoration of a core component of the antioxidant defense system further underscores luteolin's role in reinforcing redox homeostasis against PMA-induced oxidative stress [14, 15]. 

Protective effects of luteolin against PMA-induced DNA damage and inflammatory signaling

The protective effects of luteolin extended to the preservation of nuclear integrity and the suppression of downstream inflammatory and genotoxic signaling. Assessment of nuclear morphology via DAPI staining revealed that PMA exposure induced significant nuclear condensation and fragmentation, hallmarks of apoptosis and genotoxic stress (Figure 3A). Pre-treatment with luteolin markedly reduced these morphological aberrations, indicating a preservation of nuclear morphology and a reduction in PMA-induced apoptotic events. 

To elucidate the molecular pathways underlying luteolin’s protective nature, we analyzed key proteins involved in inflammation and the DNA damage response. Western blot analysis demonstrated that PMA robustly activated the inflammatory cascade, significantly upregulating the expression of Cyclooxygenase-2 (COX-2) and the pro-inflammatory cytokines IL-6 and IL-1β (Figure 3B). This was accompanied by the increased expression of NF-κB p65, confirming the activation of this master inflammatory regulator. Pre-treatment with luteolin (40 µM) effectively mitigated the PMA-induced upregulation of COX-2, IL-6, and IL-1β, and inhibited NF-κB activation [16]. Concurrently, PMA triggered a DNA damage response, evidenced by a pronounced increase in the phosphorylation of histone H2AX (γ-H2AX), a sensitive marker for DNA double-strand breaks [17]. The expression of the DNA excision repair protein ERCC1 was also elevated, suggesting a compensatory cellular effort to repair PMA-induced DNA lesions. Furthermore, luteolin reduced the levels of γ-H2AX and modulated ERCC1 expression. The suppression of γ-H2AX indicates that luteolin alleviates the primary DNA damage [18], while its effect on ERCC1 may reflect a normalization of the repair process as the genotoxic aberration is reduced. 

Discussion

This study provides compelling evidence that the natural flavonoid luteolin confers robust, multifaceted protection against PMA-induced cellular damage in human HaCaT keratinocytes. By integrating assessments of viability, oxidative stress, inflammation, and DNA integrity, we establish a coherent mechanistic framework for luteolin's action, positioning it as a promising cytoprotective agent against inflammatory skin pathologies via modulation of the cellular redox state. The schematic representation of luteolin’s cytoprotective role in PMA-induced toxicity is shown in Figure 4. PMA, a potent ROS activator, instigates a severe oxidative burst, characterized by the overproduction of ROS and RNS, notably NO. Our data confirm that this initial oxidative assault leads to the depletion of vital antioxidants like GSH and TRX, and results in measurable lipid peroxidation. Luteolin pre-treatment effectively countered this cascade by directly scavenging ROS and suppressing the PMA-induced upregulation of key nitric oxide synthase isoforms (iNOS, eNOS, and nNOS). The inhibition of iNOS is particularly critical, as it prevents the sustained production of NO that can react with superoxide to form peroxynitrite, a highly damaging molecule [13]. Furthermore, luteolin restored the levels of GSH and TRX, indicating its role in not only neutralizing free radicals but also in reinforcing the endogenous antioxidant defense network [14, 15]. This dual action of scavenging radicals and enhanced defenses effectively mitigates the primary oxidative stress.

The alleviation of oxidative stress directly translates into the suppression of the pro-inflammatory signaling cascade. Oxidative species are potent activators of the NF-κB pathway, a master regulator of inflammation [19]. Consistent with this, we observed that PMA-induced oxidative stress led to NF-κB activation and the subsequent upregulation of key inflammatory mediators, including COX-2, IL-6, and IL-1β. Luteolin pre-treatment significantly inhibited this sequence, downregulating both the upstream activator (NF-κB) and its downstream targets, which aligns with the established role of flavonoids in mitigating skin inflammation by targeting the NF-κB and MAPK pathways [20]. This anti-inflammatory efficacy is likely a consequence of luteolin's antioxidant activity, disrupting the feedback loop where ROS activate NF-κB, which induces ROS production [11, 16]. By quenching the initial oxidative signal, luteolin prevents the amplification of the inflammatory response. 

A significant and novel finding of this study is luteolin's protective effect on genomic integrity through reversal of nuclear morphological alterations. The PMA-induced oxidative and inflammatory environment created a genotoxic environment, as evidenced by increased DNA double-strand breaks marked by γ-H2AX phosphorylation and the compensatory upregulation of the DNA repair protein ERCC1. Luteolin treatment significantly reduced γ-H2AX, demonstrating its capacity to alleviate DNA damage. This effect is most likely indirect, stemming from the reduction in oxidative and nitrosative stress that would otherwise cause DNA lesions [17]. The modulation of ERCC1 expression further suggests that luteolin helps normalize the cellular repair response as the genotoxic pressure is reduced [18, 21]. This preservation of DNA integrity adds a crucial dimension to luteolin's cytoprotective profile, linking its antioxidant and anti-inflammatory actions to the prevention of long-term mutagenic consequences.

These results establish a robust in vitro foundation for luteolin's potential as a cytoprotective agent against inflammatory skin damage. The HaCaT cell line was selected as a biologically relevant and well-validated model for epidermal keratinocytes [22, 23], which are primary targets for inflammatory and oxidative stress in vivo. Our focused investigation on this model allowed for a detailed elucidation of the key signaling pathways involved in PMA-induced pathology and luteolin's counteractive mechanisms.

While our in vitro findings are promising, the therapeutic translation of luteolin depends on its bioavailability. Pharmacokinetic studies indicate that luteolin is absorbed and metabolized in humans, with its aglycone form exhibiting biological activity [24]. Notably, luteolin and related flavonoids have demonstrated the ability to penetrate the skin barrier in topical formulations, supporting their potential for direct dermatological application [25]. 

Conclusion and Perspective

Despite multiple investigations validating the pharmacological effects of luteolin, the majority are exclusive to particular disorders or processes. Hence, our findings delineate a logical and interconnected protective mechanism for luteolin. It functions by (1) quenching the initial oxidative burst and restoring antioxidant defenses, (2) thereby inhibiting the activation of the NF-κB inflammatory pathway, and (3) ultimately preserving nuclear and genomic integrity. This multi-targeted efficacy against PMA-induced damage strongly supports the further investigation of luteolin as a preventive or therapeutic candidate for dermatological conditions characterized by inflammation and oxidative stress, such as psoriasis or contact dermatitis.

The findings of this study, while mechanistically detailed, are derived from a simplified in vitro model using HaCaT cells monolayers, though an excellent in vitro system, cannot fully replicate the intricate immune cell interactions and systemic feedback of intact human skin. Future research employing more physiologically integrated models, such as 3D skin equivalents or in vivo studies, is crucial to validate these promising findings. Furthermore, while our multi-parametric approach provides a comprehensive analysis (assessing inflammation, oxidative stress, and DNA damage), subsequent transcriptomic or proteomic analyses would be invaluable for uncovering broader network-level alterations and identifying novel targets, together with formulation strategies to optimize luteolin’s delivery and stability to ultimately bridge the gap between in vitro efficacy and tangible clinical application for inflammatory skin disorders. 

Research funding

This research was supported by the National Research Foundation of Korea grant, NRF RS- 2023-00253438.

Competing interests

The authors declare no financial and non-financial competing interests.

Authors’ contributions

Himanshi Gahlot performed experiments, carried out analysis, and prepared the manuscript. Sun Chul Kang verified and approved the study.

Data availability

Data will be available on request. 

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