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Article

Differential Effects of Apigenin on Normal and Squamous Oral Epithelial Cells Reveal Redox–Autophagy Signaling Vulnerabilities in OSCC

by
Bianca Voicu Balasea
1,
Miruna-Silvia Stan
2,
Miruna Dinescu
3,
Marina Imre
3,
Radu Radulescu
4,
Ana Cernega
5,
Monica Musteanu
6,*,
Alexandra Ripszky
1,4,*,† and
Silviu Mirel Pituru
1,5,†
1
The Interdisciplinary Center for Dental Research and Development, “Carol Davila” University of Medicine and Pharmacy, 19-21 Jean Louis Calderon, 030167 Bucharest, Romania
2
Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Bucharest, 91–95 Splaiul Independentei, 050095 Bucharest, Romania
3
Department of Prosthodontics, Faculty of Dentistry, “Carol Davila” University of Medicine and Pharmacy, 37 Dionisie Lupu Street, District 2, 020021 Bucharest, Romania
4
Department of Biochemistry, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy, 17-23 Plevnei Street, 020021 Bucharest, Romania
5
Department of Organization, Professional Legislation and Management of the Dental Office, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy, 17-23 Plevnei Street, 020021 Bucharest, Romania
6
Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Complutense of Madrid, 28040 Madrid, Spain
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2026, 27(5), 2091; https://doi.org/10.3390/ijms27052091
Submission received: 19 December 2025 / Revised: 12 February 2026 / Accepted: 20 February 2026 / Published: 24 February 2026
(This article belongs to the Section Bioactives and Nutraceuticals)
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Abstract

The aim of this study was to compare the responses of normal human gingival epithelial cells (HGEpiC) and oral squamous cell carcinoma cells (OECM-1) to apigenin, a natural flavonoid, focusing on redox balance, autophagy, and apoptosis. This study is among the first to directly compare apigenin-induced responses in normal and cancerous oral epithelial cells. Cells were exposed to apigenin for 24 or 48 h, with untreated cells as controls. Mitochondrial activity, ATP, ROS (H2O2), and GSH were measured. Proliferation and morphology were monitored using HoloMonitor® M4. Autophagy was assessed by fluorescent vacuole labeling, and apoptosis-related proteins (p-AKT, p-BCL-2, p-p53, p-JNK, caspase-8/9) by Luminex assay. Late apoptosis was evaluated by caspase-3/7 activity. Apigenin elicited a differential response: in HGEpiC cells, it was non-cytotoxic and increased metabolic activity, induced a moderate ROS increase, and activated autophagy as a pro-survival mechanism; in contrast, OECM-1 cells exhibited a significant reduction in metabolic activity, a marked ATP decrease at 24 h, and a pronounced ROS increase. These alterations were associated with reduced autophagy and decreased p-JNK signaling. These findings indicate that apigenin exerted no harmful effects on HGEpiC cells, while inducing redox imbalance in OECM-1 cells, highlighting a context-dependent cellular response.

1. Introduction

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer, accounting for over 90% of all cases of head and neck squamous cell carcinoma (HNSCC) [1]. Mutagenic factors in the oral cavity include smoking, alcohol consumption, HPV infection, and genotoxicity of some modern dental materials, including those used in 3D printing technologies [2]. OSCC is among the most aggressive HNSCC types due to metastasis, recurrence, and resistance to apoptosis. The 5-year survival rate after surgery, radiotherapy, and targeted therapies remains approximately 60% [3]. These challenges require an alternative approach that emphasizes molecular signaling pathways focusing on oxidative stress, autophagy, and apoptosis, and developing treatment methods that distinguish between healthy and cancerous cells.
Reactive oxygen species (ROS) are key mediators linking oxidative stress, autophagy, and apoptosis. In normal cells, moderate levels of ROS act as regulatory signals that maintain cellular homeostasis. In malignant cells, ROS are chronically elevated, promoting proliferation and resistance to apoptosis. Tumor cells utilize ROS as signaling molecules [4,5,6]. Although elevated levels of ROS support tumor cell survival through the PI3K-AKT-mTOR axis, excessive oxidative stress can trigger autophagy or apoptosis [5,6,7,8]. Oxidative stress can activate JNK, which phosphorylates BCL-2, facilitating Beclin-1 release and autophagy initiation. If stress persists, intrinsic apoptosis is triggered through activation of caspase-9 and effector caspases 3/7 [9,10,11].
Apigenin (4′,5,7-trihydroxyflavone) is a natural flavonoid found in chamomile, parsley, and celery, with anti-inflammatory, antioxidant, and antitumor properties via inhibition of PI3K/AKT/mTOR, NF-κB, and MAPK cascades [12,13,14]. It induces apoptosis and inhibits proliferation in several epithelial cancers, such as hepatic, pancreatic, colorectal, pulmonary, breast, prostate, and skin cancers [12,13,14]. In oral tumor cells (SCC-25), apigenin reduces viability, causes cell-cycle arrest, and apoptosis [15], while in normal skin keratinocytes (HaCaT), it shows antioxidant/photoprotective effects [16]. However, its impact on redox status and autophagy remains insufficiently explored, presenting a critical knowledge gap.
Previous studies have examined apigenin in normal oral cell types such as periodontal ligament cells (hPDL), demonstrating anti-inflammatory effects via HO-1 induction [17]. Similarly, investigations using SCC-25 tumor cells compared with HaCaT (a non-oral line) provided limited relevance to oral tissue physiology [15]. Although previous studies have targeted the effect of apigenin in oral carcinoma cell lines, such as SCC-25, SCC-9, and CAL-27 [15,18,19], to date, no research has directly compared the effects of apigenin on HGEpiC versus OECM-1 cells, leaving a gap in understanding its differential redox, autophagic, and apoptotic effects in OSCC. HGEpiC cells are isolated from healthy gingival epithelium [20], whereas OECM-1 cells originate from malignant gingival epithelial tissue and represent a well-established in vitro model of OSCC [21]. These two cell lines were selected because they share a common gingival epithelial origin, allowing a direct comparison between normal and malignant phenotypes.
The novelty of our study lies in providing the first integrative analysis of ROS, ATP, autophagy, and apoptotic pathways in HGEpiC versus OECM-1 cells, combining real-time monitoring of cell morphology using HoloMonitor® M4 with biochemical and molecular assays.
Accordingly, this study aims to comparatively evaluate the effects of apigenin on HGEpiC and OECM-1 cells, with a focus on potential differential modulation of the redox–autophagy–apoptosis triad and elucidating molecular vulnerabilities specific to OSCC cells. If behavioral differences between HGEpiC and OECM-1 cells are observed, they could provide a key starting point for understanding the molecular mechanisms underlying chemotherapy resistance in OSCC.

2. Results

2.1. Impact on Proliferation, Metabolic Activity, and Energy Status

Phase-contrast holographic images provided information on the proliferation and morphology of HGEpiC and OECM-1 cells following exposure to apigenin. HGEpiC cells maintained their characteristic honeycomb organization, and the cultures showed a larger density and structure at 48 h, both in the control and after apigenin treatments (50 and 150 μM), indicating sustained growth (Figure 1a). OECM-1 cells showed proliferative activity in the control, characterized by numerous dimensions (in mitotic and protrusive activity) at all monitored time intervals (0, 24, 48 h), indicating rapid division. Proliferation also continued in OECM-1 cells after apigenin treatment (Figure 1b).
The HoloMonitor® M4 plots support the morphological observations. In HGEpiC cells, proliferation occurred in both control and treated groups, with slightly higher cell confluence observed after apigenin treatment compared with the control at 48 h (Figure 1c). One-way ANOVA revealed a significant main effect of concentration on HGEpiC cell confluence at 24 h (F(2,6) = 5.438, p = 0.045), with lower confluence in the 50 µM group compared with the control (p = 0.046). Time-dependent increases in confluence were observed in the 150 µM group (F(2,6) = 6.125, p = 0.036) and in the control group (Welch ANOVA: F(2,2.819) = 71.95, p = 0.004). Post hoc Tukey test showed significant increases at 48 h versus 0 h in the control and 150 µM groups. In OECM-1 cells, proliferation was also observed in both the control and treated groups, with a slightly lower cell confluence following treatment with 150 μM apigenin compared to the control (Figure 1d). Two-way ANOVA revealed a significant main effect of concentration on OECM-1 cell confluence (F(2,18) = 3.924, p = 0.039) and a strong main effect of time (F(2,18) = 40.919, p < 0.001), with no significant concentration × time interaction (F(4,18) = 0.056, p = 0.994), indicating a consistent concentration effect across time points. Post hoc Tukey test showed significantly lower confluence in the 150 µM group compared with control (p = 0.032), while no differences were observed between 50 µM and control. One-way ANOVA confirmed a significant time-dependent increase in confluence within all groups (control: F(2,6) = 7.628, p = 0.022; 50 µM: F(2,6) = 11.98, p = 0.008; 150 µM: F(2,6) = 55.02, p = 0.001). Post hoc Tukey test showed significant increases at 48 h versus 0 h in all conditions.
Following the MTT test (Figure 1e), in HGEpiC cells, mitochondrial metabolic activity significantly increased only at the highest concentration of apigenin, by 20% compared to the control after 24 h of exposure. One-way ANOVA indicated a significant effect of concentration (F(2,6) = 6.489, p = 0.032). Post hoc Tukey test indicated that 150 µM significantly increased metabolic activity compared with the control at 24 h (p = 0.028), while no significant differences were observed between treated groups and control at 48 h. Two-way ANOVA indicated a time-dependent effect on cell metabolic activity (ANOVA F(1,8) = 28.204, p < 0.001), while the effects of concentration (p = 0.978) and the concentration × time interaction (p = 0.322) were not significant. Post hoc Tukey test indicated that only in the control group did metabolic activity increase significantly from 24 h to 48 h (p = 0.009), suggesting that this increase over time reflects a normal physiological response.
In contrast, OECM-1 cells showed significant reductions in mitochondrial metabolic activity at both doses and time points. After 24 h, a decrease of approximately 19% compared to the control was observed at 50 μM. After 48 h, a more pronounced decrease of 23% (50 μM) and 31% (150 μM) was observed. One-way ANOVA indicated a significant effect of concentration at 24 h (F(2,6) = 6.602, p = 0.031). Post hoc Tukey test indicated that 50 µM significantly reduced metabolic activity compared with the control (p = 0.029). Two-way ANOVA indicated a significant effect of concentration (F(2,12) = 21.24, p < 0.001) and time (F(1,12) = 18.27, p = 0.001), as well as a significant concentration x time interaction (F(2,12) = 4.445, p = 0.036). Post hoc Tukey test indicated that at 48 h, both 50 µM (p = 0.007) and 150 µM (p < 0.001) significantly reduced metabolic activity compared with the control. In the control group, metabolic activity increased from 24 h to 48 h (p = 0.009).
Regarding the total ATP level in HGEpiC cells, at 24 h, treatment with 150 μM apigenin led to a significant 20% decrease in ATP compared with the control, while at 48 h, ATP levels increased by 22% relative to the control (Figure 1f). One-way ANOVA indicated a significant effect of concentration at 24 h (F(2,6) = 8.758, p = 0.017). Post hoc Tukey test indicated that 150 µM significantly reduced ATP level compared with the control (p = 0.017), while no significant differences were observed between treated groups and the control at 48 h.
Two-way ANOVA revealed a significant effect of time on ATP levels (F(1,12) = 35.982, p < 0.001), while the effects of concentration (p = 0.757) and the concentration × time interaction (p = 0.343) were not significant. Post hoc Tukey test indicated that ATP decreased significantly from 24 h to 48 h in the control group (p = 0.008) and in the 50 µM group (p = 0.032), but not in the 150 µM group. Thus, absolute ATP levels are higher at 24 h than at 48 h. However, when compared with the control at the same time point, ATP in treated cells is lower than that of the control at 24 h and higher than that of the control at 48 h.
In contrast, the ATP level in OECM-1 cells compared to the control decreased significantly at 24 h, followed by a marked increase at 48 h. Therefore, at 50 and 150 μM apigenin, a decrease of 35% and 33%, respectively, was observed after 24 h, while after 48 h, a 5.8-fold and 7.7-fold increase, respectively, was observed, suggesting metabolic recovery (Figure 1f). One-way ANOVA indicated a significant effect of concentration (F(2,6) = 48.97, p < 0.001). Post hoc Tukey tests indicated that at 24 h, both 50 µM and 150 µM significantly reduced viability compared with the control (p < 0.001). At 48 h, no significant differences were observed between treated groups and the control, nor between time points.
Despite the reduction in mitochondrial dehydrogenase activity (MTT assay results), intracellular ATP levels increased approximately 5–8-fold at 48 h, suggesting bioenergetic compensation through a metabolic shift toward anaerobic glycolysis for ATP production under oxidative stress.

2.2. Oxidative Stress and Antioxidant Defense

Fluorescent GSH labeling showed an increase in fluorescence intensity at 24 and 48 h in HGEpiC compared to the control, consistent with an increase in GSH level and antioxidant defense (Figure 2a,c). Welch ANOVA indicated a significant effect of time on GSH (F(1,2.851) = 47.02, p = 0.007). Post hoc Games–Howell indicated that only in the control group, GSH increased significantly from 24 h to 48 h (p = 0.007).
In contrast, in OECM-1 cells, fluorescence intensity decreased at 24 h compared with the control, indicating a reduction in GSH levels, whereas at 48 h fluorescence increased, indicating partial recovery of antioxidant capacity (Figure 2b,c). One-way ANOVA revealed a significant increase in GSH levels from 24 h to 48 h in the control group (F(1,4) = 82.66, p < 0.001) and in the 50 µM group (F(1,4) = 101.3, p < 0.001). In the 150 µM group, Welch’s ANOVA confirmed a significant increase in GSH over time (F(1,2.08) = 60.95, p = 0.014). Post hoc tests (Tukey or Games–Howell as appropriate) showed that GSH increased significantly from 24 h to 48 h in all groups.
Specifically, when compared to control, ROS (H2O2) levels in HGEpiC increased mainly at 48 h following apigenin exposure, reaching a statistically significant increase of 83% at 50 μM and approximately 40% at 150 μM (Figure 2d). One-way ANOVA revealed a significant effect of treatment on ROS levels at 48 h (F(2,6) = 6.689, p = 0.03). Post hoc Tukey analysis showed a significant increase in ROS levels in the 50 µM group compared with the control (p = 0.025). At 24 h, no treatment concentration differed significantly from the control.
Further one-way ANOVA comparisons showed that ROS levels in the control group were significantly higher at 24 h than at 48 h (F(1,4) = 17.85, p = 0.013). Similarly, ROS levels in the 150 µM group were significantly higher at 24 h compared with 48 h (F(1,4) = 8.491, p = 0.044). The decrease in absolute ROS levels observed at 48 h occurred in both control and treated groups and therefore cannot be attributed to apigenin treatment.
Surprisingly, apigenin induced a time-dependent response in terms of oxidative stress in OECM-1 cells. Although ROS levels increased approximately 17-fold at 24 h and approximately 3.3-fold at 48 h compared to control, they partially recovered over time. At 24 h, H2O2 treatment significantly increased ROS levels, while at 48 h, ROS levels decreased relative to 24 h, suggesting a partial restoration of antioxidant mechanisms in surviving cells (Figure 2d). Two-way ANOVA revealed a significant main effect of concentration on ROS (F(2,12) = 97.34, p < 0.001), whereas the effect of time (24 h vs. 48 h) was not statistically significant (F(1,12) = 4.27, p = 0.061), and the interaction between time and concentration was also not significant (F(2,12) = 0.67, p = 0.531). Post hoc Tukey HSD tests showed that 150 µM significantly increased ROS compared with control at 24 h and 48 h (all p < 0.001). Also, 150 µM significantly increased ROS compared with 50 µM at 24 h and 48 h (all p < 0.001). No significant differences were observed between control and 50 µM at either time point, nor between the same concentrations across 24 h and 48 h (all p > 0.05). Further one-way ANOVA comparisons demonstrated that ROS levels in the control group were significantly lower at 24 h than at 48 h (F(1,4) = 28.52, p = 0.006).

2.3. Autophagy Modulation

HGEpiC and OECM-1 cells can be observed in both controls and apigenin treatments using phase contrast holographic imaging (Figure 3a) after 24 h. HGEpiC cells retain the characteristic honeycomb-like organization in both the control and treatment. On the other hand, in the case of OECM-1 cells, in the control, a much higher number of dividing cells (round and prominent) is observed; in contrast, after apigenin treatment (50 and 150 μM), the number of dividing cells is visibly reduced.
In HGEpiC cells, fluorescence intensity of the autophagic vacuoles significantly increased by 63% at 50 μM and by 54% at 150 μM, compared to the control, supporting the activation of autophagy as a protective mechanism (Figure 3b,c). One-way ANOVA showed a significant effect of concentration on autophagy (F(2,21) = 29.33, p < 0.001). Tukey post hoc tests indicated significant increases at 50 µM (t(21) = 7.106, p < 0.001) and 150 µM (t(21) = 6.027, p < 0.001) compared with the control, with no difference between concentrations (p = 0.537). Pearson correlation revealed a significant positive association between concentration and autophagy (r = 0.563, p = 0.004).
Conversely, OECM-1 cells showed a significant decrease in the fluorescence intensity of autophagic vacuoles by 14% and by 16% at 50 μM and 150 μM, respectively, compared with the control, indicating inhibition of autophagy (Figure 3b,c). One-way ANOVA showed a significant effect of concentration on autophagy (F(2,27) = 9.636, p < 0.001). Tukey post hoc tests indicated significant reductions at 50 µM (t(27) = −3.433, p = 0.005) and 150 µM (t(27) = −4.086, p < 0.001) compared with control, with no difference between concentrations (p = 0.792). Pearson correlation revealed a significant negative association between concentration and autophagy (r = −0.545, p = 0.002).

2.4. Pro-Survival vs. Pro-Apoptotic Signaling

Multiplex analysis of the parameters of early apoptosis in the phosphorylated or active state performed with the MILLIPLEX® Early Apoptosis 7-Plex kit (Figure 4) provides discrepant data between the molecular pathways and mechanisms of apoptosis and survival activated in the case of HGEpiC cells vs. OECM-1 cells after 48 h of exposure to 50 and 150 μM apigenin.
In HGEpiC cells, apigenin treatment decreased p-AKT and p-BCL-2 levels compared to the control, but induced an increment of the p53 level and both extrinsic (caspase-8) and intrinsic (caspase-9) apoptotic responses. One-way ANOVA showed a significant effect of concentration (F(2,6) = 12.32, p = 0.008). Post hoc Tukey test indicated that the AKT decreased at 50 µM compared with control (p = 0.007) and increased at 150 µM compared with 50 µM (p = 0.034), with no significant difference between 150 µM and control. Pearson correlation showed no significant linear association (r = –0.095, p = 0.808).
On the other hand, in OECM-1 cells, apigenin increased the level of p-AKT and p-BCL-2, decreased p-JNK level, and exhibited only partial caspase-8 and caspase-9 activation at 50 μM, and a lack of response at 150 μM, consistent with impaired apoptotic signaling at higher doses. Welch’s ANOVA showed a significant effect of concentration on JNK levels (F(2,3.09) = 16.56, p = 0.022). Post hoc Games–Howell analysis indicated that 150 µM caused a significant decrease in JNK levels compared with the control (p = 0.03), whereas no difference was observed between 50 µM and the control.
Together, these results indicate that apigenin exerts differential effects on HGEpiC compared with OECM-1 cells. HGEpiC cells maintained proliferation and mitochondrial metabolic activity over time, with only moderate changes in ATP levels, while OECM-1 cells continued to proliferate but exhibited reduced metabolic activity and a transient ATP decrease at 24 h, followed by a marked increase at 48 h. Regarding redox status, HGEpiC cells showed moderate ROS increases accompanied by enhanced GSH-mediated antioxidant defense, whereas OECM-1 cells displayed pronounced ROS accumulation and a transient reduction in GSH at 24 h; at 48 h, ROS levels remained elevated compared with control. Autophagy was activated in HGEpiC cells, whereas it was inhibited in OECM-1 cells. Finally, HGEpiC cells showed decreased p-AKT and modest caspase-3/7 activity, whereas OECM-1 cells exhibited limited apoptotic responses, associated with increased anti-apoptotic signaling (elevated p-AKT and p-BCL-2) and reduced p-JNK levels. Overall, these findings highlight the selective vulnerability of OECM-1 cells to apigenin-induced oxidative stress and suggest that HGEpiC cells preserve homeostasis through coordinated redox, autophagy, and apoptotic responses.

2.5. Late Apoptosis

Regarding late apoptosis in Figure 5, the effector caspase-3/7 was fluorescently labeled. Both HGEpiC (Figure 5a) and OECM-1 (Figure 5b) cells showed detectable caspase-3/7 activation after apigenin exposure. In HGEpiC, caspase-3/7 fluorescence increased slightly but significantly at 48 h, indicating late apoptosis (Figure 5c). Two-way ANOVA showed a significant effect of concentration on caspase activity (F(2,12) = 4.022, p = 0.046) and a significant time × concentration interaction (F(2,12) = 7.547, p = 0.008), while time alone had no effect. Tukey post hoc analysis indicated that at 48 h, both 50 µM (p = 0.048) and 150 µM (p = 0.006) significantly increased caspase 3/7 activity compared to control, with no significant differences between the two concentrations. In the control group, caspase activity was significantly higher at 24 h than at 48 h (p = 0.027).
In OECM-1, caspase-3/7 activation was visible at both concentrations, but overall weaker and attenuated by 48 h, suggesting a limited apoptotic execution in surviving tumor cells (Figure 5c). Caspase-3/7 activity was significantly higher at 24 h compared with 48 h in cells treated with 150 µM (F(1,4) = 11.2, p = 0.029). A similar time-dependent decrease was observed in the 50 µM group, with caspase-3/7 activity being significantly higher at 24 h than at 48 h (F(1,4) = 14.05, p = 0.02). At 48 h, one-way ANOVA revealed a significant effect of treatment on caspase-3/7 activity (F(2,6) = 6.276, p = 0.034), with post hoc analysis indicating significantly lower activity in the 50 µM group compared with the control (p = 0.037).

3. Discussion

Our study is the first to compare the effects of apigenin on redox balance, autophagy, and apoptosis in normal (HGEpiC) versus malignant (OECM-1) gingival epithelium. We selected apigenin because it is a flavonoid known for its antioxidant and pro-apoptotic properties. However, its redox-related mechanisms and differential effects in OSCC remain insufficiently investigated [22,23], which justifies its exploration as a potential adjuvant agent in OSCC therapy. Given the involvement of apigenin in the JAK/STAT, PI3K/Akt/mTOR, MAPK/ERK, NF-κB, and Wnt/β-catenin pathways in multiple cancer types [24], the aim of our study was to assess the effects of apigenin on OECM-1 cells by specifically examining redox homeostasis and the AKT/BCL-2/JNK signaling axis in relation to autophagy and apoptosis, in parallel with HGEpiC cells. To our knowledge, few studies have examined apigenin’s effects on signaling pathways and autophagy specifically in OSCC cells, highlighting the novelty of our approach.
To summarize the differential effects observed, a holistic schematic is presented in Figure 6. Apigenin treatment in HGEpiC cells induced a moderate increase in ROS compared to OECM-1 cells, accompanied by activation of autophagy as a pro-survival adaptive mechanism and the induction of partial apoptosis. In contrast, in OECM-1 cells, apigenin exposure resulted in a pronounced elevation of ROS levels, suppression of autophagic activity, and resistance to apoptosis, effects that are likely associated with the downregulation of phosphorylated JNK (p-JNK).
Our results suggest that apigenin exerts distinct effects on HGEpiC versus OECM-1 cells.
MTT assays showed that apigenin did not exert cytotoxic effects on HGEpiC cells over time; in fact, it promoted a gradual increase in cell viability (p < 0.05). In addition, real-time monitoring using the HoloMonitor® M4 revealed that HGEpiC maintained its characteristic epithelial architecture. These findings are supported by existing literature, where apigenin is classified as having a low cytotoxic profile [25,26].
In contrast, post hoc tests confirmed that both concentrations of apigenin significantly reduced the metabolic activity of OECM-1 cells compared with the control, with a more pronounced effect at 48 h (p < 0.001). This was accompanied by visibly altered morphology in HoloMonitor® M4 images and reduced three-dimensional expansion (Figure 1b), consistent with the literature describing that OECM-1 cells must modify their phenotypic characteristics to adapt to the microenvironment, proliferate, and evade cell death in the presence of apigenin [27,28,29].
In HGEpiC cells, the significant increase in MTT at 24 h versus control (by 20%) indicates proliferation, whereas the 20% decrease in ATP is likely explained by the energy consumption required for activating antioxidant and autophagic mechanisms. Conversely, in OECM-1 cells, mitochondrial activity and ATP levels decreased significantly at 24 h compared with control, consistent with apigenin-induced mitochondrial dysfunction and ATP depletion reported in other malignancies [30]. Interestingly, in our results after 48 h, ATP levels in OECM-1 cells increased markedly despite the persistent reduction in mitochondrial dehydrogenase activity. This suggests a metabolic shift from oxidative phosphorylation to anaerobic glycolysis, serving as a bioenergetic compensation mechanism under conditions of mitochondrial stress; indeed, the literature reports that cancer cells produce ATP through anaerobic glycolysis under stress conditions such as hypoxia or oxidative stress, thereby sustaining survival despite mitochondrial dysfunction [31]. Through this adaptive response, OECM-1 cells likely maintain ATP production to support survival, highlighting the plasticity of cancer cell metabolism in response to apigenin-induced stress.
Glutathione (GSH), the main intracellular thiol, plays a central role in detoxifying H2O2 via glutathione peroxidase GPx, with regeneration through glutathione reductase maintaining redox homeostasis [32,33]. In OECM-1 cells, the significant increase in GSH at 48 h compared with 24 h might suggest an adaptive cellular response to elevated ROS. Regarding oxidative stress, ROS–H2O2 levels in HGEpiC cells increased moderately (83% at 48 h), yet still much lower than the increases of 873% and 331% observed in OECM-1 cells exposed to 150 μM apigenin at 24 h and 48 h, respectively. Pearson correlation confirmed a strong dose-dependent association (r = 0.946, p < 0.001) for OECM-1 cells. These results align with data from Lee et al., who also reported apigenin-induced ROS accumulation [30], further supporting a cancer cell-specific oxidative response. Our findings are consistent with the concept that cancer cells can tolerate higher ROS levels by upregulating antioxidant proteins, supporting survival and resistance to apoptosis [34]. While cancer cells generally have higher ROS than normal cells, they counteract potential damage through antioxidant genes and metabolic adaptations, making the GSH synthesis pathway an attractive therapeutic target [35]. Consequently, targeting ROS regulation represents a promising therapeutic strategy, given the dysregulated redox balance characteristic of cancer cells. Overall, these findings support the potential use of apigenin for redox priming strategies in OSCC therapy, whereby controlled oxidative stress sensitizes tumor cells prior to chemotherapy or radiotherapy [6,36].
Another key aspect in the cancer cell’s defense mechanisms is represented by the modulation of autophagy. Autophagy is a catabolic process through which aggregated proteins and damaged organelles are transported to lysosomes for degradation [37,38]. Basally, autophagy maintains cellular homeostasis and normal growth. Under pathological conditions, it is not always clear whether autophagic alterations act protectively or contribute to damage [39,40]. Autophagy has also been classified as a type II programmed cell death mechanism [37,41].
In HGEpiC cells, autophagy increased as a pro-survival mechanism, correlating with MTT results. In contrast, in OECM-1 cells, autophagy decreased along with reduced GSH and increased ROS at 24 h compared with the control. Contrary to our results, Enrico et al. showed in several tumor lines (HeLa, HepG2, H1299, MCF-7) that GSH depletion promotes autophagy activation [32]. In OECM-1 cells, although GSH decreased following apigenin treatment, autophagy was not activated, suggesting a protective mechanism through which tumor cells inhibit autophagy. Thus, our data clearly suggest that OECM-1 cells did not use autophagy as a survival mechanism, consistent with the reduced MTT results. In contrast to our observations, several studies on malignant cell lines have shown that apigenin-induced autophagy can be pro-survival, and its inhibition can enhance apigenin toxicity through apoptosis induction [12,42,43,44].
The paradox of autophagy in cancer remains unresolved. Autophagy can either protect or suppress tumor cells depending on context and stage. In early stages, autophagy inhibition may be beneficial, whereas in advanced stages, its induction may promote oncogenesis or senescence. Therapeutic strategies must therefore be adapted to the tumor’s molecular profile and autophagic status [45].
At the molecular level (Figure 4), apigenin reduced AKT phosphorylation in HGEpiC cells, promoting the formation of autophagic vacuoles. The decrease in AKT at 50 μM confirms enhanced autophagy, consistent with its known anti-autophagic role [12,44]. In OECM-1 cells, p-JNK levels were significantly decreased (post hoc analysis indicated that 150 μM caused a significant decrease compared with both 50 μM and control). Jun-Mo Moon et al. demonstrated in colorectal cancer that JNK is interconnected with both autophagy and apoptosis, with increased p-JNK expression stimulating both processes in vitro and in vivo [46]. Other studies highlight that JNK inhibition represents a resistance and protective mechanism in oxidative-stress conditions in cancer cells [47,48]. Therefore, in OECM-1 cells, the inhibition of autophagy and partial activation of apoptosis following apigenin treatment can be explained by reduced p-JNK, as an adaptive mechanism of tumor cells. Thus, our results confirm the importance and therapeutic potential of JNK in OSCC treatment, consistent with the existing literature.
In HGEpiC cells, caspase-3/7 activity increased slightly compared with the control at 50 and 150 μM after 48 h (p <0.05), whereas in OECM-1 cells, caspase-3/7 also increased slightly compared with the control after 24 h (p >0.05) (Figure 5). These data suggest that the OECM-1 cells respond to apigenin by reducing the level of autophagy, probably to avoid the pro-death functions of autophagy [45], most likely via JNK dephosphorylation.
In summary, our findings suggested that apigenin exerts differential effects on HGEpiC versus OECM-1 cells, modulating redox balance, autophagy, and apoptosis in a cell-type-dependent manner. These results highlight the potential of apigenin as an adjuvant in OSCC therapy and provide a strong rationale for further mechanistic and in vivo investigations. This study was conducted in vitro on two cell lines; further in vivo studies and exploration of additional OSCC models are warranted to validate these findings.
Raw data of all measured assays, showing mean ± SD values for HGEpiC and OECM-1 cells, are presented in Table 1. Values represent results from confluence, MTT, ATP, GSH, ROS, autophagy, AKT, BCL-2, p53, JNK, Caspase-3/7, Caspase-8, and Caspase-9 measurements at the indicated time points (h).

4. Materials and Methods

Figure 7 summarizes the parameters and intracellular events monitored following exposure of the two cell lines to apigenin for 24 and 48 h. The experimental workflow focused on oxidative stress, mitochondrial function, autophagy, apoptosis, and morphology (Figure 1a), quantifying ROS, GSH, ATP, MTT, autophagic vacuoles, apoptosis-related proteins (p-JNK, p-BCL-2, active caspase-8/9), and monitoring live-cell morphology (Figure 1b), respectively. Early apoptosis markers were quantified using multiplex testing with Luminex 200 technology, while live-cell monitoring was performed with HoloMonitor® M4.

4.1. Cell Culture

Two cell lines were used in this study: HGEpiC and OECM-1 cells.
The HGEpiC cell line was purchased from Innoprot (Bizkaia, Spain; Cat. No. P10864). The cells were cultured in Epithelial Cell Medium-Plus (Innoprot, Bizkaia, Spain; Cat. No. P60106-PLUS), completely supplemented with fetal bovine serum, supplement EpiCGS, and penicillin/streptomycin (P/S) solution 100×, FBS Innoprot, in a humidified environment at 37 °C and 5% CO2. Before seeding, the flasks were pre-treated with poly-L-lysine to improve cell adhesion, according to the manufacturer’s instructions. For the experiments, the cells were seeded in 96-well plates pre-treated with poly-L-lysine at a density of 4 × 104 cells/well. Cells were maintained overnight in a humidified incubator at 37 °C and 5% CO2 to promote cell adhesion before the experiments.
The OECM-1 was purchased from Sigma-Aldrich (St. Louis, MO, USA; Cat. No. SCC180). Cells were cultured in RPMI-1640 medium (VWR, Radnor, PA, USA; Cat. No. 392-0426) supplemented with 5% fetal bovine serum and 1% antibiotics, in a humidified environment at 37 °C and 5% CO2, without pre-treatment of the culture vessel surface with poly-L-lysine. For the experiments, OECM-1 cells were seeded at the same density as normal cells, namely 4 × 104 cells/well in 96-well plates, and maintained overnight at 37 °C and 5% CO2 to promote cell adhesion before the experiments.
Although the two cell lines started from an equivalent cell seeding density, considering the faster division cycle of HGEpiC (for example, doubling time 24–30 h reported for primary human gingival keratinocytes, a biologically comparable epithelial cell population [49]), unlike OECM-1 (doubling time 30–38 h, according to the supplier’s data (Merck/Sigma-Aldrich, Cat. SCC180 [21]), we expect that, over time, a difference in the number of HGEpiC vs. OECM-1 cells will appear, including in the control. Moreover, this statement is supported by the pre-treatment with a special substrate (poly-L-lysine) and the addition of extra serum in the case of HGEpiC, conditions that favor proliferation.

4.2. Biocompound

Apigenin obtained from Cayman Chemical (≥98% purity; CAS No. 520-36-5) was solubilized in dimethyl sulfoxide (DMSO). The stock solutions were sterilized by exposure to UV radiation for 1 h. Prior to use, stock solutions were diluted in complete, cell-type-specific culture medium to obtain final concentrations of 150 µM and 50 µM, with a final DMSO content of ≤0.5%. The concentrations of apigenin (50 μM and 150 μM) were selected based on preliminary viability testing and previously published studies [15]. Higher concentrations were not used to avoid non-specific cytotoxic effects on normal gingival epithelial cells (HGEpiC) and to remain within biologically relevant ranges.

4.3. Live-Cell Imaging—HoloMonitor® M4

Morphological changes, cell migration, and proliferation were monitored using the HoloMonitor® M4 digital holographic microscope (Phase Holographic Imaging PHI AB, Lund, Sweden). Cells were seeded in 96-well plates and exposed to apigenin, with unexposed cells used as controls. A HoloLid™ (PHI AB, Göteborrg, Sweden) was placed over the plate to improve image quality and reduce evaporation. Time-lapse images were captured every 30 min for 48 h and analyzed using the HoloMonitor® App Suite version 4.0.1.546 (PHI AB, Göteborg, Sweden) in a label-free approach. The system was maintained in a humidified incubator at 37 °C and 5% CO2.

4.4. Mitochondrial Metabolic Activity

Mitochondrial metabolic activity was assessed using the MTT reagent from Biotium (Fremont, CA, USA; Cat No. 30006), after 24 and 48 h of incubation of normal and tumor cells with apigenin (50 µM and 150 µM). Following incubation, 10 µL of MTT reagent and 100 µL of culture medium were added per well. Solubilization of formazan crystals was performed with isopropanol, and the absorbance was measured at 570 and 630 nm using a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany).

4.5. Determination of ATP Levels

Intracellular ATP content was determined using the CellTiter-Glo® 2.0 Assay (Promega, Madison, WI, USA; Cat. No. G9241), according to the manufacturer’s instructions. After 24 and 48 h of treatment with apigenin, an equal volume of reagent was added to the culture medium and incubated for 10 min at room temperature, protected from light. Luminescence was measured using the MyGlo® Reagent Reader (Promega, Madison, WI, USA), and results were expressed as relative luminescence units (RLU), corresponding to intracellular ATP concentration.

4.6. Reactive Oxygen Species (ROS)

The intracellular level of hydrogen peroxide (H2O2), the main reactive oxygen species, was determined using the ROS-Glo™ H2O2 Assay Kit (Promega, Madison, WI, USA; Cat. No. G8820) after treatment with apigenin in the two oral cell lines: normal and tumoral. After 24 and 48 h of exposure to apigenin, the ROS-Glo™ reagent was added to the culture medium and incubated for 6 h at 37 °C. Luminescence was recorded using the MyGlo® Reagent Reader (Promega), with signal intensity proportional to the amount of H2O2 in the samples.

4.7. Glutathione (GSH)

Reduced glutathione (GSH), the main intracellular antioxidant, was fluorescently labeled using the BioTracker™ 625 Red GSH Dye (Sigma-Aldrich, St. Louis, MO, USA; Cat. No. SCT036), according to the manufacturer’s protocol. After 24 and 48 h of exposure to apigenin, cells were incubated with the fluorescent dye for 30 min at 37 °C in the dark. Fluorescence images were captured using an Optika IM-3LD4D fluorescence microscope (Optika, Ponteranica, Italy), and fluorescence intensity was quantified using the FLUOstar Omega microplate reader (BMG Labtech, NC, USA).

4.8. Autophagy Activation

Fluorescent labeling of autophagic vacuoles (autophagosomes and autophagolysosomes) was performed using the Autophagy Assay Kit (Merck, Darmstadt, Germany; Cat. No. MAK138) after 24 h of exposure of HGEpiC and OECM-1 cells to 50 and 150 μM apigenin. Images were acquired using an IM-3LD4D fluorescence microscope (Optika, Ponteranica, Italy), with the excitation/emission wavelength filter suitable for the kit ~488/530 nm. Fluorescence intensity was quantified using the FLUOstar Omega microplate reader (BMG Labtech, NC, USA) and correlated with the degree of apoptosis activation.

4.9. Multiplex Assay (Early Apoptosis Markers)

Cell lysates were collected after 48 h of exposure of normal and tumor cells to apigenin (50 μM and 150 μM), using Cell Signaling Lysis Buffer supplemented with Protease Inhibitor Cocktail (Promega, Madison, WI, USA; Cat. No. G6521). The filtered lysates were stored at −80 °C in 1.5 mL Eppendorf tubes for subsequent analyses.
Total Protein Kit, Micro (Sigma-Aldrich, St. Louis, MO, USA; Cat. No. TP0100), based on the Bradford method, was used to determine the total protein concentration. Subsequently, the samples were diluted in Milliplex Assay Buffer to a final concentration of 0.7 mg/mL and analyzed with the MILLIPLEX® MAP 7-Plex Early Apoptosis Magnetic Bead kit (Merck Millipore), which quantifies p-AKT, p-BCL-2, p-p53, p-JNK, and active caspase-8 and -9. Mean fluorescence intensity (MFI) was measured using Luminex 200 technology.

4.10. Caspase-3/7 Activity (Fluorescence)

Late apoptosis, mediated by caspases-3 and -7, was assessed using the Caspase-3/7 Green Detection Reagent Kit (Merck, Darmstadt, Germany; Cat. No. SCT100) after 24 h and 48 h of exposure of both cell lines (HGEpiC and OECM-1) to apigenin. Cells were incubated with the caspase-specific reagent at a final concentration of 5 µM for 30 min at room temperature in the dark.
Images were acquired using an IM-3LD4D fluorescence microscope (Optika, Ponteranica, Italy), with the excitation/emission wavelength filter suitable for the kit ~488/530 nm. Fluorescence intensity was quantified using the FLUOstar Omega microplate reader (BMG Labtech, Ponteranica, Italy).

4.11. Statistical Analysis

All statistical analyses were performed on raw data. Data normality and homogeneity of variances were assessed using the Shapiro–Wilk and Levene’s tests, respectively. For normally distributed variables, group comparisons were performed using one-way or two-way ANOVA, or Welch’s ANOVA when variances were unequal, followed by Tukey post hoc test where appropriate. Correlations were analyzed using Pearson’s correlation, with statistical significance set at p < 0.05.
Results are presented as mean ± SD from triplicate measurements (n = 3). Graphs were generated using Microsoft Excel and show data normalized to 100% of the control (untreated cells).

5. Conclusions

In conclusion, the present study suggests that apigenin elicits cell type-specific and context-dependent responses in HGEpiC and OECM-1 cells. Apigenin preserves cell viability, redox homeostasis, and the balance between autophagy and apoptosis in HGEpiC cells. In contrast, in OECM-1 cells, apigenin induces pronounced redox imbalance and reduces autophagic activity, highlighting a selective vulnerability of the malignant phenotype, accompanied by modulation of JNK signaling. These observations support the potential relevance of redox, autophagy, and JNK signaling as therapeutic entry points in OSCC.
Several limitations of the present study should be acknowledged. The experimental design was restricted to in vitro models, and late apoptosis was assessed mainly through executioner caspase-3/7 activity, without additional multiparametric validation. Therefore, further validation using in vivo models and additional late apoptosis markers is warranted. Future studies should also explore the interplay between autophagy and apoptosis through pathways such as AKT/mTOR.

Author Contributions

Conceptualization, B.V.B. and M.M.; methodology, M.-S.S.; software, R.R.; validation, M.M., M.I., A.R. and S.M.P.; formal analysis, R.R., B.V.B.; investigation, B.V.B.; resources, M.D.; data curation, M.D. and A.C.; writing—original draft preparation, B.V.B.; writing—review and editing, M.I., M.-S.S., A.R. and S.M.P.; visualization, A.C. and B.V.B.; supervision, A.R. and S.M.P.; project administration, A.R. and S.M.P.; funding acquisition, A.R. and S.M.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Acknowledgments

This study was completed with the support of The Interdisciplinary Center for Dental Research and Development, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
AKTProtein kinase B
ANOVAAnalysis of variance
ATPAdenosine triphosphate
BCL-2B-cell lymphoma 2
DMSODimethyl sulfoxide
ERKExtracellular signal-regulated kinase
FBSFetal bovine serum
GSHGlutathione (reduced form)
H2O2Hydrogen peroxide
HaCaTHuman adult low calcium high temperature keratinocytes
HGEpiCHuman gingival epithelial cells
HNSCCHead and neck squamous cell carcinoma
HO-1Heme oxygenase-1
HPVHuman papillomavirus
hPDLHuman periodontal ligament cells
JNKc-Jun N-terminal kinase
MAPKMitogen-activated protein kinase
MFIMean fluorescence intensity
mTORMechanistic target of rapamycin
MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
NF-κBNuclear factor kappa B
OECM-1Oral squamous cell carcinoma cell line
OSCCOral squamous cell carcinoma
PBSPhosphate-buffered saline
PI3KPhosphoinositide 3-kinase
p-AKTPhosphorylated AKT
p-BCL-2Phosphorylated BCL-2
p-JNKPhosphorylated JNK
p-p53Phosphorylated p53
RLURelative luminescence units
ROSReactive oxygen species
SCCSquamous cell carcinoma
SDStandard deviation

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Ijms 27 02091 g001aIjms 27 02091 g001b
Figure 1. Comparative analysis of the effect of apigenin treatment (50 μM and 150 μM) on HGEpiC and OECM-1 cells in terms of proliferation, metabolic activity, and ATP production after 24 and 48 h. (a) Representative holographic phase contrast images of HGEpiC cells at 0, 24, and 48 h following exposure to apigenin. Controls represent untreated cells; (b) Representative holographic phase contrast images of OECM-1 cells at 0, 24, and 48 h following exposure to apigenin. Controls represent untreated cells; (c) Representative plot showing confluence of HGEpiC cells at 0, 24, and 48 h following exposure to apigenin. Controls represent untreated cells. Data are presented as mean± SD (n = 3). One-way ANOVA followed by Tukey post hoc test; 50 µM vs. control * p < 0.05 (24 h); 0 h vs. 48 h * p < 0.05 (150 µM); Welch ANOVA followed by Games–Howell post hoc test; 0 h vs. 48 h ** p < 0.01 (control). (d) Representative plot showing confluence of OECM-1 cells at 0, 24, and 48 h following exposure to apigenin. Controls represent untreated cells. Data are presented as mean ± SD (n = 3). Two-way ANOVA followed by Tukey post hoc test; 150 µM vs. control * p < 0.05; One-way ANOVA followed by Tukey post hoc test; 0 h vs. 48 h (* p < 0.05 control; ** p < 0.01 50 µM; *** p < 0.001 150 µM); (e) Quantification of the metabolic mitochondrial activity of HGEpiC vs. OECM-1 cells by MTT assay after 24 and 48 h of exposure to apigenin. Controls, symbolized as 0 on the graph, represent untreated cells. Data are presented as mean ± SD (n = 3). HGEpiC: One-way ANOVA; 150 µM vs. control * p < 0.05 (24 h); Two-way ANOVA followed by Tukey post hoc test 24 h vs. 48 h ** p < 0.01 (control). OECM-1: One-way ANOVA; 50 µM vs. control * p < 0.05 (24 h); Two-way ANOVA followed by Tukey post hoc test; 50 µM vs. control ** p < 0.01 (48 h); 150 µM vs. control *** p < 0.001 (48 h); control 24 h vs. control 48 h ** p < 0.01. (f) Quantification of the total ATP level in HGEpiC vs. OECM-1 cells after 24 and 48 h of exposure to apigenin. Controls, symbolized as 0 on the graph, represent untreated cells. Data are presented as mean ± SD (n = 3). HGEpiC: One-way ANOVA followed by Tukey post hoc test; 150 µM vs. control * p < 0.05 (24 h); Two-way ANOVA followed by Tukey post hoc test; 24 h vs. 48 h (control) ** p < 0.01; 24 h vs. 48 h (50 µM) * p < 0.05. OECM-1: One-way ANOVA followed by Tukey post hoc test; 50 µM vs. control *** p < 0.001 (24 h); 150 µM vs. control *** p < 0.001 (24 h). The Y-axis is plotted on a log10 scale to allow visualization of large increases.
Figure 1. Comparative analysis of the effect of apigenin treatment (50 μM and 150 μM) on HGEpiC and OECM-1 cells in terms of proliferation, metabolic activity, and ATP production after 24 and 48 h. (a) Representative holographic phase contrast images of HGEpiC cells at 0, 24, and 48 h following exposure to apigenin. Controls represent untreated cells; (b) Representative holographic phase contrast images of OECM-1 cells at 0, 24, and 48 h following exposure to apigenin. Controls represent untreated cells; (c) Representative plot showing confluence of HGEpiC cells at 0, 24, and 48 h following exposure to apigenin. Controls represent untreated cells. Data are presented as mean± SD (n = 3). One-way ANOVA followed by Tukey post hoc test; 50 µM vs. control * p < 0.05 (24 h); 0 h vs. 48 h * p < 0.05 (150 µM); Welch ANOVA followed by Games–Howell post hoc test; 0 h vs. 48 h ** p < 0.01 (control). (d) Representative plot showing confluence of OECM-1 cells at 0, 24, and 48 h following exposure to apigenin. Controls represent untreated cells. Data are presented as mean ± SD (n = 3). Two-way ANOVA followed by Tukey post hoc test; 150 µM vs. control * p < 0.05; One-way ANOVA followed by Tukey post hoc test; 0 h vs. 48 h (* p < 0.05 control; ** p < 0.01 50 µM; *** p < 0.001 150 µM); (e) Quantification of the metabolic mitochondrial activity of HGEpiC vs. OECM-1 cells by MTT assay after 24 and 48 h of exposure to apigenin. Controls, symbolized as 0 on the graph, represent untreated cells. Data are presented as mean ± SD (n = 3). HGEpiC: One-way ANOVA; 150 µM vs. control * p < 0.05 (24 h); Two-way ANOVA followed by Tukey post hoc test 24 h vs. 48 h ** p < 0.01 (control). OECM-1: One-way ANOVA; 50 µM vs. control * p < 0.05 (24 h); Two-way ANOVA followed by Tukey post hoc test; 50 µM vs. control ** p < 0.01 (48 h); 150 µM vs. control *** p < 0.001 (48 h); control 24 h vs. control 48 h ** p < 0.01. (f) Quantification of the total ATP level in HGEpiC vs. OECM-1 cells after 24 and 48 h of exposure to apigenin. Controls, symbolized as 0 on the graph, represent untreated cells. Data are presented as mean ± SD (n = 3). HGEpiC: One-way ANOVA followed by Tukey post hoc test; 150 µM vs. control * p < 0.05 (24 h); Two-way ANOVA followed by Tukey post hoc test; 24 h vs. 48 h (control) ** p < 0.01; 24 h vs. 48 h (50 µM) * p < 0.05. OECM-1: One-way ANOVA followed by Tukey post hoc test; 50 µM vs. control *** p < 0.001 (24 h); 150 µM vs. control *** p < 0.001 (24 h). The Y-axis is plotted on a log10 scale to allow visualization of large increases.
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Figure 2. Differential redox responses to apigenin treatment (50 μM and 150 μM) in HGEpiC cells and OECM-1 cells after 24 and 48 h. Fluorescence microscopy images of the glutathione (GSH) activity in: (a) HGEpiC and (b) OECM-1 cells. Controls represent untreated cells. Scale bar: 100 µm (applies to all images); (c) Quantification of relative GSH fluorescence intensity using FLUOstar Omega microplate reader. Controls, symbolized as 0 on the graph, represent untreated cells. Data are presented as mean ± SD (n = 3). HGEpiC: Welch’s ANOVA followed by Games–Howell post hoc test; 24 h vs. 48 h ** p < 0.01 (control). OECM-1: One-way ANOVA followed by Tukey post hoc test; 24 h vs. 48 h *** p < 0.001 (control); 24 h vs. 48 h *** p < 0.001 (50 µM); Welch’s ANOVA followed by Games–Howell post hoc test; 24 h vs. 48 h * p < 0.05 (150 µM) (d) Quantification of ROS (H2O2) relative luminescence intensity. Controls, symbolized as 0 on the graph, represent untreated cells. Data are presented as mean ± SD (n = 3). HGEpiC: One-way ANOVA followed by Tukey post hoc test; 50 µM vs. control * p < 0.05 (at 48 h); 24 h vs. 48 h * p < 0.05 (control); 24 h vs. 48 h * p < 0.05 (150 µM). OECM-1: Two-way ANOVA followed by Tukey post hoc test; 150 µM vs. control *** p < 0.001 (at 24 h and at 48 h); 150 µM vs. 50 µM *** p < 0.001 (at 24 h and at 48 h); One-way ANOVA; 24 h vs. 48 h ** p < 0.01 (control). The Y-axis is plotted on a log10 scale to allow visualization of large increases.
Figure 2. Differential redox responses to apigenin treatment (50 μM and 150 μM) in HGEpiC cells and OECM-1 cells after 24 and 48 h. Fluorescence microscopy images of the glutathione (GSH) activity in: (a) HGEpiC and (b) OECM-1 cells. Controls represent untreated cells. Scale bar: 100 µm (applies to all images); (c) Quantification of relative GSH fluorescence intensity using FLUOstar Omega microplate reader. Controls, symbolized as 0 on the graph, represent untreated cells. Data are presented as mean ± SD (n = 3). HGEpiC: Welch’s ANOVA followed by Games–Howell post hoc test; 24 h vs. 48 h ** p < 0.01 (control). OECM-1: One-way ANOVA followed by Tukey post hoc test; 24 h vs. 48 h *** p < 0.001 (control); 24 h vs. 48 h *** p < 0.001 (50 µM); Welch’s ANOVA followed by Games–Howell post hoc test; 24 h vs. 48 h * p < 0.05 (150 µM) (d) Quantification of ROS (H2O2) relative luminescence intensity. Controls, symbolized as 0 on the graph, represent untreated cells. Data are presented as mean ± SD (n = 3). HGEpiC: One-way ANOVA followed by Tukey post hoc test; 50 µM vs. control * p < 0.05 (at 48 h); 24 h vs. 48 h * p < 0.05 (control); 24 h vs. 48 h * p < 0.05 (150 µM). OECM-1: Two-way ANOVA followed by Tukey post hoc test; 150 µM vs. control *** p < 0.001 (at 24 h and at 48 h); 150 µM vs. 50 µM *** p < 0.001 (at 24 h and at 48 h); One-way ANOVA; 24 h vs. 48 h ** p < 0.01 (control). The Y-axis is plotted on a log10 scale to allow visualization of large increases.
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Figure 3. Differential autophagy responses to apigenin treatment (50 μM and 150 μM) in HGEpiC and OECM-1 cells after 24 h. (a) Representative holographic phase contrast images of HGEpiC and OECM-1 cells. Controls represent untreated cells; (b) Fluorescence microscopy representative images of autophagic vacuoles in HGEpiC and OECM-1 cells. Scale bar: 100 µm (applies to all images); (c) Quantification of relative fluorescence intensity of autophagic vacuoles using ImageJ software (Version 1.54p). Controls represent untreated cells. Data are presented as mean ± SD (n = 10 OECM-1; n = 8 HGEpiC). HGEpiC: one-way ANOVA followed by Tukey post hoc test; 50 µM *** p < 0.001 vs. control; 150 µM *** p < 0.001 vs. control. OECM-1: one-way ANOVA followed by Tukey post hoc test; 50 µM ** p < 0.01 vs. control; 150 µM *** p < 0.001 vs. control.
Figure 3. Differential autophagy responses to apigenin treatment (50 μM and 150 μM) in HGEpiC and OECM-1 cells after 24 h. (a) Representative holographic phase contrast images of HGEpiC and OECM-1 cells. Controls represent untreated cells; (b) Fluorescence microscopy representative images of autophagic vacuoles in HGEpiC and OECM-1 cells. Scale bar: 100 µm (applies to all images); (c) Quantification of relative fluorescence intensity of autophagic vacuoles using ImageJ software (Version 1.54p). Controls represent untreated cells. Data are presented as mean ± SD (n = 10 OECM-1; n = 8 HGEpiC). HGEpiC: one-way ANOVA followed by Tukey post hoc test; 50 µM *** p < 0.001 vs. control; 150 µM *** p < 0.001 vs. control. OECM-1: one-way ANOVA followed by Tukey post hoc test; 50 µM ** p < 0.01 vs. control; 150 µM *** p < 0.001 vs. control.
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Figure 4. Relative level of phosphorylated or active apoptosis-related proteins quantified after 48 h of HGEpiC cells and OECM-1 cells exposure to apigenin treatment (50 μM and 150 μM). (a) Phospho-AKT (Ser473). Apigenin significantly decreased AKT phosphorylation in HGEpiC, whereas in OECM-1 cells, AKT activity showed a slight, non-significant increase (ns), indicating compensatory pro-survival signaling activation. HGEpiC: one-way ANOVA followed by Tukey post hoc test; 50 µM vs. control ** p < 0.01; 150 µM vs. 50 µM * p < 0.05; (b) Phospho-BCL-2 (Ser70). In HGEpiC cells, phosphorylated BCL-2 showed a mean decrease (−39% at 50 µM; −93% at 150 µM), although variability rendered the 150 µM comparison non-significant; this trend is concordant with reduced p-AKT and supports attenuation of anti-apoptotic signaling. In contrast, in OECM-1 cells, anti-apoptotic protein levels increased by 436% > control 50 μM, 271% > control non-significant, representing tumor cell adaptation to apigenin-induced stress; (c) Phospho-p53 (Ser46). In HGEpiC the p-p53 level increased (ns) at 150 µM apigenin. In OECM-1 cells, no change was significant; (d) Phospho-JNK (Thr183/Tyr185). HGEpiC cells exhibited a slight, nonsignificant increase in JNK activation, while JNK phosphorylation was significantly inhibited in OECM-1 cells, suggesting suppression of stress-induced apoptotic pathways. OECM-1: Welch’s ANOVA 150 µM vs. control * p < 0.05; ((e) Active caspase-8. In HGEpiC cells, apigenin treatment increased caspase-8 activity by 59% at 50 μM (ns) and by 31% at 150 μM (ns) compared to the control. In OECM-1 tumor cells, caspase-8 activity increased by ~50% at 50 μM (ns) and by 12% at 150 μM (ns), indicating a mild, dose-dependent activation of the extrinsic apoptotic pathway; (f) Active caspase-9. In HGEpiC, caspase-9 activity increased by 32% at 50 μM (ns) and by 44% at 150 μM (ns), consistent with activation of the intrinsic (mitochondrial) apoptotic pathway. In OECM-1 cells, caspase-9 activity showed a 39% increase at 50 μM (ns), but no significant change at 150 μM, suggesting partial activation of the intrinsic pathway at lower concentrations and resistance at higher doses. Controls represent untreated cells. Data are presented as mean ± SD (n = 3).
Figure 4. Relative level of phosphorylated or active apoptosis-related proteins quantified after 48 h of HGEpiC cells and OECM-1 cells exposure to apigenin treatment (50 μM and 150 μM). (a) Phospho-AKT (Ser473). Apigenin significantly decreased AKT phosphorylation in HGEpiC, whereas in OECM-1 cells, AKT activity showed a slight, non-significant increase (ns), indicating compensatory pro-survival signaling activation. HGEpiC: one-way ANOVA followed by Tukey post hoc test; 50 µM vs. control ** p < 0.01; 150 µM vs. 50 µM * p < 0.05; (b) Phospho-BCL-2 (Ser70). In HGEpiC cells, phosphorylated BCL-2 showed a mean decrease (−39% at 50 µM; −93% at 150 µM), although variability rendered the 150 µM comparison non-significant; this trend is concordant with reduced p-AKT and supports attenuation of anti-apoptotic signaling. In contrast, in OECM-1 cells, anti-apoptotic protein levels increased by 436% > control 50 μM, 271% > control non-significant, representing tumor cell adaptation to apigenin-induced stress; (c) Phospho-p53 (Ser46). In HGEpiC the p-p53 level increased (ns) at 150 µM apigenin. In OECM-1 cells, no change was significant; (d) Phospho-JNK (Thr183/Tyr185). HGEpiC cells exhibited a slight, nonsignificant increase in JNK activation, while JNK phosphorylation was significantly inhibited in OECM-1 cells, suggesting suppression of stress-induced apoptotic pathways. OECM-1: Welch’s ANOVA 150 µM vs. control * p < 0.05; ((e) Active caspase-8. In HGEpiC cells, apigenin treatment increased caspase-8 activity by 59% at 50 μM (ns) and by 31% at 150 μM (ns) compared to the control. In OECM-1 tumor cells, caspase-8 activity increased by ~50% at 50 μM (ns) and by 12% at 150 μM (ns), indicating a mild, dose-dependent activation of the extrinsic apoptotic pathway; (f) Active caspase-9. In HGEpiC, caspase-9 activity increased by 32% at 50 μM (ns) and by 44% at 150 μM (ns), consistent with activation of the intrinsic (mitochondrial) apoptotic pathway. In OECM-1 cells, caspase-9 activity showed a 39% increase at 50 μM (ns), but no significant change at 150 μM, suggesting partial activation of the intrinsic pathway at lower concentrations and resistance at higher doses. Controls represent untreated cells. Data are presented as mean ± SD (n = 3).
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Figure 5. Late apoptosis in HGEpiC and OECM-1 cells after 24 and 48 h of exposure to apigenin treatment (50 μM and 150 μM). Fluorescence microscopy representative images of caspase- 3/7 activity in: (a) HGEpiC, and (b) OECM-1 cells. Controls represent untreated cells. Scale bar: 100 µm (applies to all images); (c) Quantification of relative fluorescence intensity of caspase- 3/7 activity using FLUOstar Omega microplate reader. Controls, symbolized as 0 on the graph, represent untreated cells. Data are presented as mean ± SD (n = 3). HGEpiC: Two-way ANOVA followed by Tukey post hoc test; 50 µM vs. control * p < 0.05 (48 h); 150 µM vs. control ** p < 0.01 (48 h); 24 h vs. 48 h (Control)* p < 0.05. OECM-1: One-way ANOVA; 24 h vs. 48 h (50 µM) * p < 0.05; 24 h vs. 48 h (150 µM) * p < 0.05; 50 µM vs. control * p < 0.05 (48 h).
Figure 5. Late apoptosis in HGEpiC and OECM-1 cells after 24 and 48 h of exposure to apigenin treatment (50 μM and 150 μM). Fluorescence microscopy representative images of caspase- 3/7 activity in: (a) HGEpiC, and (b) OECM-1 cells. Controls represent untreated cells. Scale bar: 100 µm (applies to all images); (c) Quantification of relative fluorescence intensity of caspase- 3/7 activity using FLUOstar Omega microplate reader. Controls, symbolized as 0 on the graph, represent untreated cells. Data are presented as mean ± SD (n = 3). HGEpiC: Two-way ANOVA followed by Tukey post hoc test; 50 µM vs. control * p < 0.05 (48 h); 150 µM vs. control ** p < 0.01 (48 h); 24 h vs. 48 h (Control)* p < 0.05. OECM-1: One-way ANOVA; 24 h vs. 48 h (50 µM) * p < 0.05; 24 h vs. 48 h (150 µM) * p < 0.05; 50 µM vs. control * p < 0.05 (48 h).
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Figure 6. The holistic model encompasses intracellular aspects of apigenin on HGEpiC versus OECM-1 cells in our study.
Figure 6. The holistic model encompasses intracellular aspects of apigenin on HGEpiC versus OECM-1 cells in our study.
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Figure 7. Experimental workflow and proposed mechanism of apigenin action; (a) Experimental workflow illustrating the comparative analysis of HGEpiC and OECM-1 cells exposed to the biocompound apigenin in terms of oxidative stress, mitochondrial function, autophagy, and apoptosis. Proposed mechanistic model of apigenin action through ROS, autophagy, and apoptosis modulation; (b) Schematic mechanistic model of the intracellular action of apigenin on autophagy and apoptosis via ROS; (c) Timeline of the study, including apigenin treatments and analyses performed.
Figure 7. Experimental workflow and proposed mechanism of apigenin action; (a) Experimental workflow illustrating the comparative analysis of HGEpiC and OECM-1 cells exposed to the biocompound apigenin in terms of oxidative stress, mitochondrial function, autophagy, and apoptosis. Proposed mechanistic model of apigenin action through ROS, autophagy, and apoptosis modulation; (b) Schematic mechanistic model of the intracellular action of apigenin on autophagy and apoptosis via ROS; (c) Timeline of the study, including apigenin treatments and analyses performed.
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Table 1. Raw data presentation, including mean values and standard deviation (SD) for all assays, for the graphs generated.
Table 1. Raw data presentation, including mean values and standard deviation (SD) for all assays, for the graphs generated.
Time (h)Mean valuesSD
Confluence
Control HGEpiC082.52.2
2491.22.2
4898.40.4
A50 HGEpiC067.513.2
2475.09.5
4884.90.5
A150 HGEpiC071.77.0
2478.45.0
4888.15.2
Control OECM-1033.55.3
2451.211.9
4866.312.3
A50 OECM-1038.87.0
2457.513.0
4874.75.0
A150 OECM-1042.33.2
2461.85.6
4879.53.8
MTT
Control HGEpiC240.798.88
481.1310.11
A50 HGEpiC240.853.82
481.0710.6
A150 HGEpiC240.967.42
481.0130.66
Control OECM-1240.555.36
480.707.31
A50 OECM-1240.447.50
480.548.54
A150 OECM-1240.476.52
480.484.03
ATP
Control HGEpiC2450,0838.20
4819,42854.45
A50 HGEpiC2447,638.337.02
4822,72142.33
A150 HGEpiC2439,751.331.68
4823,86175.69
Control OECM-12423,5426.65
484190110.20
A50 OECM-12415,301.334.61
4828,647.671069.22
A150 OECM-12415,964.331.99
4836,456643.68
GSH
Control HGEpiC2489,681.3315.78
48220,801.713.56
A50 HGEpiC24106,644.386.32
48249,292.71.78
A150 HGEpiC24107,971.787.08
48252,4212.08
Control OECM-12471,15429.82
48229,531.39.34
A50 OECM-12458,59944.63
48249,788.33.74
A150 OECM-12466,128.3357.18
48251,3662.51
ROS
Control HGEpiC2479911.47
48424.6628.97
A50 HGEpiC24990.3332.57
4878128.35
A150 HGEpiC24853.3313.02
4859326.96
Control OECM-12475.3364.49
48359.3321.77
A50 OECM-124419.3383.51
4848511.11
A150 OECM-1241411.66385.66
481549.6672.85
Autophagy
Control HGEpiC2447.1419.86
A50 HGEpiC2476.9218.31
A150 HGEpiC2472.3914.77
Control OECM-124118.097.34
A50 OECM-124102.169.42
A150 OECM-12499.129.42
AKT
Control HGEpiC481900.665.72
A50 HGEpiC48120113.33
A150 HGEpic4816917.08
Control OECM-1481922.6612.39
A50 OECM-148264236.23
A150 OECM-1482380.338.36
BCL-2
Control HGEpiC48−5.16128.87
A50 HGEpiC48−3.1614.78
A150 HGEpic48−0.3355.87
Control OECM-1482.33290.93
A50 OECM-1488.66742.30
A150 OECM-14812.5465.71
p53
Control HGEpiC482.66192.43
A50 HGEpiC483361.63
A150 HGEpic489.66471.86
Control OECM-148767.511.91
A50 OECM-1481044.8334.67
A150 OECM-148750.839.16
JNK
Control HGEpiC48276.4117.83
A50 HGEpiC48265.9137.95
A150 HGEpic48322.914.02
Control OECM-148262.5812.86
A50 OECM-148265.7526.5
A150 OECM-148141.755.03
Caspase-8
Control HGEpiC4833.2525.65
A50 HGEpiC4853.0833.83
A150 HGEpic4843.586.26
Control OECM-148137.4124.7
A50 OECM-148206.0859.68
A150 OECM-148155.2519.62
Caspase 9
Control HGEpiC4849.8321.19
A50 HGEpiC486633.39
A150 HGEpic487213.9
Control OECM-14842818.11
A50 OECM-148594.3347.1
A150 OECM-1484532.37
Caspase 3/7
Control HGEpiC24144,472.72.73
48135,789.30.72
A50 HGEpiC24142,8370.4
48143,681.33.23
A150 HGEpic24142,956.71.99
48146,452.31.54
Control OECM-124151,6556.74
48157,517.70.87
A50 OECM-124162,821.32.74
48152,735.31.32
A150 OECM-124161,250.71.04
48156,677.71.11
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Voicu Balasea, B.; Stan, M.-S.; Dinescu, M.; Imre, M.; Radulescu, R.; Cernega, A.; Musteanu, M.; Ripszky, A.; Pituru, S.M. Differential Effects of Apigenin on Normal and Squamous Oral Epithelial Cells Reveal Redox–Autophagy Signaling Vulnerabilities in OSCC. Int. J. Mol. Sci. 2026, 27, 2091. https://doi.org/10.3390/ijms27052091

AMA Style

Voicu Balasea B, Stan M-S, Dinescu M, Imre M, Radulescu R, Cernega A, Musteanu M, Ripszky A, Pituru SM. Differential Effects of Apigenin on Normal and Squamous Oral Epithelial Cells Reveal Redox–Autophagy Signaling Vulnerabilities in OSCC. International Journal of Molecular Sciences. 2026; 27(5):2091. https://doi.org/10.3390/ijms27052091

Chicago/Turabian Style

Voicu Balasea, Bianca, Miruna-Silvia Stan, Miruna Dinescu, Marina Imre, Radu Radulescu, Ana Cernega, Monica Musteanu, Alexandra Ripszky, and Silviu Mirel Pituru. 2026. "Differential Effects of Apigenin on Normal and Squamous Oral Epithelial Cells Reveal Redox–Autophagy Signaling Vulnerabilities in OSCC" International Journal of Molecular Sciences 27, no. 5: 2091. https://doi.org/10.3390/ijms27052091

APA Style

Voicu Balasea, B., Stan, M.-S., Dinescu, M., Imre, M., Radulescu, R., Cernega, A., Musteanu, M., Ripszky, A., & Pituru, S. M. (2026). Differential Effects of Apigenin on Normal and Squamous Oral Epithelial Cells Reveal Redox–Autophagy Signaling Vulnerabilities in OSCC. International Journal of Molecular Sciences, 27(5), 2091. https://doi.org/10.3390/ijms27052091

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