Anticancer Effects of Tangeretin on Apoptosis Induction and Cell Growth Inhibition through Mediating Reactive Oxygen Species in Endometrial Cancer Cells

Abstract

Endometrial cancer, the most common gynecologic malignancy, originates within the epithelial lining of the uterus. Although early diagnosis can lead to effective treatment and improved recovery rates, the worldwide incidence and mortality rates of this cancer continue to rise. The prognosis is particularly poor in cases of metastatic or recurrent disease. Tangeretin, a flavonoid found in citrus fruits, is known for its various biological properties, including antioxidant and anti-inflammatory activities. It has demonstrated anticancer effects against a range of cancers, including bladder, colorectal carcinoma, and breast cancer. However, its effects on endometrial cancer cells have not been previously examined. Here, we investigate the effects of tangeretin on cell viability, proliferation, migration, reactive oxygen species (ROS) generation, and apoptosis in Ishikawa cells, a well-characterized epithelial model used in endometrial research. Our results show that tangeretin treatment significantly inhibits the viability and proliferation of Ishikawa cells. In addition, it suppresses cell migration, as evidenced by wound healing assays. The 2’,7’-dichlorofluorescein diacetate assay revealed that tangeretin enhances ROS generation. Moreover, an annexin V/propidium iodide assay confirmed that tangeretin induces apoptotic death in Ishikawa cells. The expression of B-cell lymphoma 2-associated X protein was analyzed to validate the induction of apoptosis. These findings suggest that tangeretin exhibits anticancer effects on endometrial cancer cells by inhibiting proliferation and migration while promoting apoptosis.

INTRODUCTION

Endometrial cancer, a malignancy originating from the uterus’s inner lining known as the endometrium, is most frequently diagnosed in peri- and post-menopausal women (Passarello et al., 2019; Cao et al., 2023). Endometrial cancers are categorized based on their histopathological features, including endometrioid adenocarcinoma, serous carcinoma, and clear cell carcinoma (Masood and Singh, 2021). Approximately 80% of endometrial cancers fall into the endometrioid adenocarcinoma category (Min et al., 2006; Colombo et al., 2013). Early detection is often facilitated by noticeable symptoms, such as vaginal bleeding (Liu et al., 2025), leading to a higher chance of survival; by contrast, late detection significantly reduces survival rates (AlHilli et al., 2019). The global incidence and mortality rates of endometrial cancer are on the rise, prompting ongoing research aimed at developing effective treatments (Makker et al., 2017; Lortet-Tieulent et al., 2018). Treatment options include surgical resection, radiation therapy, hormone therapy, and chemotherapy (Denschlag et al., 2010), with surgical resection regarded as the most effective method. However, when cancer has metastasized or recurs, the effectiveness of surgical treatment alone is often limited (Ki et al., 2006; Lim et al., 2022).

In recent years, extensive research has focused on the anticancer properties of phytochemicals (Wani et al., 2022; Bhutta and Choi, 2025). Natural bioactive compounds derived from plants, these compounds support health and disease prevention through various physiological properties, including antioxidant, anti-inflammatory, and immune-modulating activities (Muscolo et al., 2024). Phytochemicals are generally considered safe, with fewer side effects and low toxicity (Liu et al., 2021; Cavaco and Faria, 2024; Jeong et al., 2024). One prominent group of phytochemicals is flavonoids, which are abundant in fruits, vegetables, and tea (Panche et al., 2016). Flavonoids are known to exert anticancer effects by inducing cell cycle arrest and promoting apoptosis in cancer cells (Abotaleb et al., 2018; Bhutta et al., 2024).

Tangeretin, a polymethoxyl-flavonoid predominantly found in citrus peels, has a stable structure featuring five methoxy groups attached to a flavone backbone (Fig. 1) (Ha et al., 2007; Chen et al., 2020). This configuration facilitates rapid cellular uptake and enhances resistance to oxidation and degradation, thereby making tangeretin a promising candidate for therapeutic applications (Koirala et al., 2016). It exhibits a range of physiological effects, including anticancer, antioxidant, and anti-inflammatory activities (Raza et al., 2020; Xu et al., 2025). Notably, tangeretin has shown anticancer effectiveness against various malignancies, including bladder, colorectal, and breast cancer, through mechanisms such as apoptosis induction, mitochondrial dysfunction, cell cycle arrest, and inhibition of migration (Lin et al., 2019; Dey et al., 2020; Ko et al., 2020). However, the anticancer effects of tangeretin in endometrial cancer remain unexplored, indicating a need for further investigation.

Fig. 1.

Chemical structure of tangeretin.

This study aimed to investigate the anticancer effects of tangeretin on endometrial cancer using Ishikawa cells, which are derived from an endometrial adenocarcinoma cell line. The diverse effects of tangeretin on cell viability, proliferation, migration, reactive oxygen species (ROS) generation (AlHilli et al., 2019), and apoptosis in Ishikawa cells were confirmed. The results suggest that tangeretin has therapeutic potential in treating endometrial cancer.

MATERIALS AND METHODS

Chemicals

Tangeretin (95% purity) was sourced from Aladdin. The stock solution of tangeretin was dissolved in dimethyl sulfoxide (DMSO), with final DMSO concentrations≤0.1%.

Cell culture

The Ishikawa cell line, derived from human endometrial adenocarcinoma, was obtained from the European Collection of Authenticated Cell Culture. Ishikawa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Welgene), supplemented with 10% fetal bovine serum (R&D Systems), 1% antibiotic-antimycotic solution (Gibco), and 1% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution (Welgene). The cells were maintained in a humidified incubator at 37°C with 5% CO₂.

Cell viability assay (water-soluble tetrazolium, WST assay)

The WST assay was conducted to assess the viability of Ishikawa cells. A Quanti-MAX^TM water-soluble tetrazolium salt-8 cell viability kit (Biomax) was used according to the manufacturer instructions. Ishikawa cells were seeded in 96-well cell culture plates (Sarstedt) at a density of 5×10³ cells per well and maintained in a humidified incubator at 37°C with 5% CO₂. After 24 h, the cells were treated with various concentrations (0－40 µM) of tangeretin for 72 h. Following this, incubation was conducted with the addition of WST solution for 30 min. The optical density was measured at 450 nm using a Neo2 hybrid multimode reader (Agilent).

Colony formation assay

A colony formation assay was performed to evaluate the proliferation of Ishikawa cells. The cells were seeded at a density of 5×10² cells for each well in 6-well cell culture dishes (Sarstedt) and maintained in a humidified incubator at 37°C with 5% CO₂. After 24 h, the Ishikawa cells were treated with tangeretin (0, 10, 20, and 40 µM). After 72 h, the wells were washed with Dulbecco’s phosphate-buffered saline (DPBS, Welgene), and the DMEM medium was replaced with fresh medium. After an additional 10 days of incubation, the Ishikawa cells were washed with DPBS, fixed with 4% paraformaldehyde (GeneAll), and stained with 0.5% crystal violet (Sigma-Aldrich). The stained colonies were photographed, and the colony area was analyzed using ImageJ (National Institutes of Health).

Wound healing assay

To assess the migration of Ishikawa cells, a wound healing assay was conducted. The cells were seeded at a density of 5×10⁵ cells per well (approximately 80%－90% confluency) in 6-well cell culture dishes and maintained in a humidified incubator at 37°C with 5% CO₂. After 24 h, the Ishikawa cells were treated with 0.1% mitomycin C (Sigma-Aldrich). Each well was scratched with a sterilized 200-µL micropipette tip to create wounds of uniform width and length. The wells were then washed with DPBS to remove cell debris, and fresh DMEM medium was added. The Ishikawa cells were further treated with tangeretin (0, 10, 20, and 40 µM) for 72 h. The cells were photographed using a microscope, and the wound area was analyzed using ImageJ.

2’,7’-Dichlorofluorescein diacetate (DCF-DA) assay

Ishikawa cells were seeded at a density of 4×10³ cells per well in 6-well cell culture dishes and maintained in a humidified incubator at 37°C with 5% CO₂. After 24 h, the cells were treated with tangeretin (0, 10, 20, and 40 µM) for 72 h. Following incubation, the media was removed and replaced with 2 µL of DCF-DA (5 µg/mL) solution for 30 min. Fluorescence was then measured and photographed using a microscope. The ROS production was quantified using Cell Sens Dimension Software (Olympus).

Annexin V/propidium iodide (PI) assay

An annexin V/PI assay was conducted to assess the death of Ishikawa cells. The cells were seeded in 6-well cell culture dishes at a density of 5×10³ cells per well and maintained in a humidified incubator at 37°C with 5% CO₂. After 24 h, the cells were treated with tangeretin (0, 10, 20, and 40 µM) for 72 h. Cell death was evaluated using the Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit (Invitrogen). Following washing, the cells were stained with annexin V and PI. Samples were analyzed using fluorescence-activated cell sorting Calibur^TM instruments (BD Biosciences), and the data were processed using the FlowJo program v. 10.8.1 (TreeStar).

Western blot analysis

Western blot analysis was performed to evaluate protein levels in the Ishikawa cells. The cells were treated with tangeretin (0, 20, and 40 µM) for 72 h. Total protein was extracted using the PRO-PREP protein extraction solution (iNtRON Biotechnology), and protein concentration was quantified using bicinchoninic acid reagent (Sigma-Aldrich). A total of 30 µg of protein lysates was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis. The proteins were then transferred to a polyvinylidene difluoride membrane (Bio-Rad). To block the membrane, 5% skim milk was applied for 2 h. The membrane was incubated overnight at 4°C on a shaker with primary antibodies against B-cell lymphoma 2 (Bcl-2)-associated X protein (BAX, 1:1,000; Cell Signaling Technology) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:1,000; Millipore). Following this, the membrane was incubated for 2 h at 4°C with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Bio-Rad, 1:3,000). Target protein bands were detected using LuminoGraph 2 (ATTO) with Supersignal^TM West Femto or Pico (1:4 mix; Thermo Fisher Scientific). Protein expression levels were analyzed with ImageJ software and normalized to GAPDH expression levels.

Statistical analysis

All experiments were conducted independently at least three times, with data presented as mean±standard deviation. Data analysis was performed using a one-way analysis of variance, followed by Dunnett’s post hoc test, using GraphPad Prism 5.01 (GraphPad Software Inc.). P<0.05 was considered statistically significant.

RESULTS

Tangeretin decreased the viability of Ishikawa cells

A WST assay was conducted to confirm the effects of tangeretin on the viability of Ishikawa cells. As Fig. 2 shows, tangeretin (0.16－40 µM) significantly reduced cell viability compared to the control group, which did not receive any treatment. These results indicate that tangeretin treatment resulted in decreased cell viability.

Fig. 2.

Effects of tangeretin on the viability of Ishikawa cells. Ishikawa cells were seeded in 96-well plates at a density of 5×10³ cells per well and treated with tangeretin for 72 h. The water-soluble tetrazolium assay was utilized to examine cell viability. Data are presented as mean±standard deviation from at least three independent experiments. **P<0.01 vs. control group (n=6).

Tangeretin decreased the proliferation of Ishikawa cells

To evaluate the effects of tangeretin on the proliferation of Ishikawa cells, a colony formation assay was conducted. Treatment with tangeretin at concentrations of 10, 20, and 40 µM) significantly reduced the ability of individual Ishikawa cells to proliferate into colonies compared to the control group (0 µM) (Fig. 3A). Specifically, tangeretin markedly decreased the proliferation capacity of Ishikawa cells (Fig. 3B). These results indicate that tangeretin effectively reduces the proliferation of Ishikawa cells.

Fig. 3.

Effects of tangeretin on the proliferation of Ishikawa cells. Ishikawa cells were seeded at a density of 5×10² cells per well in 6-well plates and treated with tangeretin for 72 h. A colony formation assay was employed to assess cell proliferation. (A) Representative image of colony formation. (B) Quantitative analysis of the colony formation of Ishikawa cells. Data are presented as mean±standard deviation from at least three independent experiments. **P<0.01 vs. control group (n=3).

Tangeretin inhibited the migration of Ishikawa cells

The effects of tangeretin on cell migration was assessed using the wound healing assay. As Fig. 4A shows, treatment with tangeretin at 10, 20, and 40 µM led to a reduction in wound closure in Ishikawa cells. The wound closure rate in the experimental group was approximately 20% lower than that in the control group treated with 0 µM tangeretin, particularly at the highest concentration of 40 µM (Fig. 4B). These findings suggest that tangeretin inhibits the migration of Ishikawa cells.

Fig. 4.

Effects of tangeretin on the migration of Ishikawa cells. Ishikawa cells were seeded in 6-well plates using a density of 5×10⁵ cells per well and treated with tangeretin for 72 h. A wound-healing assay was performed to evaluate cell migration. (A) Representative image of wound closure. (B) Quantitative analysis of wound closure in Ishikawa cells. Data are presented as mean±standard deviation from at least three independent experiments. *P<0.05, **P<0.01 vs. control group (n=3). Scale bar=500 µm.

Tangeretin increased the generation of cellular ROS in Ishikawa cells

Tangeretin has been shown to induce oxidative stress in various cancer cell types, resulting in a range of biochemical and physiological responses (Dong et al., 2014; Dey et al., 2020; Mdkhana et al., 2021). To determine whether tangeretin induces oxidative stress in Ishikawa cells, intracellular ROS levels were assessed using DCF-DA staining. The fluorescence intensity of DCF-DA in tangeretin-treated cells (20 and 40 µM) was significantly higher compared to the control group (0 µM) (Fig. 5). These results indicate that tangeretin elevates intracellular ROS levels, suggesting it induces oxidative stress in endometrial cancer cells.

Fig. 5.

Effects of tangeretin on cellular reactive oxygen species (ROS) generation in Ishikawa cells. Ishikawa cells were seeded in 6-well plates using a density of 4×10³ cells per well and treated with tangeretin for 72 h. A 2’,7’-dichlorofluorescein diacetate (DCF-DA) assay was conducted to measure ROS generation. (A) Representative image of DCF-DA staining. (B) Quantitative analysis of the DCF-DA fluorescence ratio in Ishikawa cells. Data are presented as mean±standard deviation from at least three independent experiments. **P<0.01 vs. control group (n=6). Scale bar=200 µm.

Tangeretin induced the apoptosis of Ishikawa cells

To investigate the effects of tangeretin on the apoptosis of Ishikawa cells, an annexin V/PI assay was performed. As Fig. 6 shows, treatment with tangeretin at 40 µM significantly increased the number of apoptotic cells compared to the control group (0 µM). These results suggest that tangeretin induces apoptosis in Ishikawa cells.

Fig. 6.

Effects of tangeretin on the apoptosis of Ishikawa cells. Ishikawa cells were seeded in 6-well plates using a density of 5×10³ cells per well and treated with tangeretin for 72 h. An annexin V/propidium iodide (PI) assay was performed to assess apoptosis. (A) Representative flow cytometry plot of annexin V/PI staining. (B) Percentage of apoptotic cells in Ishikawa cells. Data are presented as mean±standard deviation from at least three independent experiments. **P<0.01 vs. control group (n=3).

Tangeretin upregulated the BAX protein level in Ishikawa cells

To evaluate the effects of tangeretin on BAX protein expression in Ishikawa cells, a western blot assay was conducted. The group treated with tangeretin exhibited increased expression of BAX compared to the control group treated with 0-µM tangeretin (Fig. 7). These findings reveal that tangeretin induces the expression of the tumor suppressor protein BAX in Ishikawa cells.

Fig. 7.

Effects of tangeretin on the expression of B-cell lymphoma 2-associated X protein (BAX) protein in Ishikawa cells. BAX protein expression was assessed using a Western blot assay. (A) Representative image of BAX protein expression. (B) Quantitative evaluation of BAX protein expression in Ishikawa cells. Data are presented as mean±standard deviation from at least three independent experiments. *P<0.05 vs. control group (n=3). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

DISCUSSION

Tangeretin, a flavonoid phytochemical derived from citrus fruits, is well-known for its numerous biological properties, including antioxidant and anti-inflammatory activities, along with its low toxicity (Lee et al., 2016; Ashrafizadeh et al., 2020). Its polymethoxylated structure endows tangeretin with high lipophilicity, which facilitates efficient cellular absorption and enhances metabolic stability. These characteristics contribute to its bioavailability and prolonged biological activity (Koirala et al., 2016), making tangeretin suitable for various therapeutic applications. Numerous studies have explored its potential anticancer effects (Koirala et al., 2016). Tangeretin exerts its biological effects through several mechanisms, including the induction of apoptosis, cell cycle arrest, and suppression of cell proliferation (Meiyanto et al., 2011; Dong et al., 2014; Ko et al., 2020). The present study aimed to evaluate the anticancer effects of tangeretin on Ishikawa cells.

Cell viability is a key indicator for assessing the anticancer potential of a compound, providing insights into its ability to induce cytotoxicity or inhibit cell growth (Kim et al., 2012; Ghasemi et al., 2023). This study demonstrated that tangeretin treatment significantly reduces the viability of Ishikawa cells, indicating its potential to decrease the survival of endometrial cancer cells. Inhibiting cell proliferation is a crucial strategy in cancer prevention and treatment development (Loftus et al., 2022). The observed delays in cell proliferation further support the potential of tangeretin as an indicator of anticancer effects. In addition, tangeretin treatment significantly impaired the colony-forming ability of Ishikawa cells, suggesting that reduced proliferation contributes to diminished cell viability.

The inhibition of cell viability and proliferation implies that cell motility may also be suppressed by limiting cell growth and survival, as cancer cell migration is hindered (Son et al., 2020). The migratory capabilities of cancer cells are critical for metastasis and invasion (Raza et al., 2020). These processes are associated with poor prognosis; therefore, their prevention is vital in cancer research (Yoshida et al., 2000; Kim et al., 2011). This study found that tangeretin significantly reduced the wound healing ability of Ishikawa cells, suggesting that tangeretin may inhibit cell migration, which is a key mechanism in cancer metastasis.

ROS are molecules that indicate oxidative stress within cells. They are known to induce epithelial-to-mesenchymal transition and modify the extracellular matrix, which affects cancer progression and metastasis (Barrera et al., 2021; Chugh and Koul, 2022). Elevated levels of ROS are characteristic of cancer and play a significant role in tumor formation and progression (Li et al., 2024). An excessive increase in ROS can lead to apoptotic cell death by damaging the mitochondrial membrane and inducing endoplasmic reticulum stress (Bhat et al., 2017; Shah and Rogoff, 2021; Muscolo et al., 2024). In the current study, the tangeretin treatment increased intracellular ROS levels in Ishikawa cells, indicating that it enhances oxidative stress in endometrial cancer cells. These findings suggest that the ROS induction by tangeretin in Ishikawa cells may partially contribute to cell death.

Cellular apoptosis is critical factor affecting the effectiveness of chemotherapeutic agents against cancer cells (Hannun, 1997). In this study, we observed an increase in the proportion of both early and late apoptotic cells following tangeretin treatment, highlighting its role in inducing apoptosis in endometrial cancer cells. Apoptosis-related proteins, particularly those in the Bcl-2 family, are vital for regulating the balance between cell survival and death (Ola et al., 2011). The Bcl-2 family members are essential components of the intrinsic apoptotic pathway, activated by various cytotoxic stimuli, including oncogenic stress and chemotherapeutic agents (Cory et al., 2003). BAX, a pro-apoptotic protein, plays a key role in triggering cell death. Its activation leads to mitochondrial membrane permeabilization, resulting in the release of cytochrome c, a critical molecule in the apoptosis process, consequently leading to cell death (Liu et al., 2016). In the present study, we found that tangeretin treatment increased intracellular ROS levels and upregulated the expression of pro-apoptotic BAX, suggesting that oxidative stress may contribute to the induction of apoptosis. However, the precise signaling pathway that connects ROS accumulation to BAX activation remains to be determined. Although BAX upregulation may be a downstream effect of ROS generation, other apoptosis-related pathways, such as caspase activation, may also be involved. These findings indicate that tangeretin induces apoptosis at least in part by promoting ROS accumulation, as supported by the observed increases in intracellular ROS levels and BAX upregulation. Nevertheless, the exact molecular mechanism linking ROS to BAX activation remains unclear. Note that we did not investigate whether caspase activation or other apoptotic regulators play a role in this process. Additional studies, including the use of ROS scavengers (e.g., N-acetyl cysteine) and caspase inhibitors, are necessary to determine whether BAX upregulation results directly from ROS stress or is mediated through a caspase-dependent signaling pathway. The observed increase in ROS levels and upregulation of BAX suggest that tangeretin may activate the intrinsic apoptotic pathway, which is crucial for its anticancer effects.

Although tangeretin may exert its anticancer effects through multiple molecular pathways, the specific contributions of each mechanism to its overall efficacy remain to be clarified (Rizeq et al., 2020; Sharifi-Rad et al., 2023). Although free radicals are recognized as essential signaling molecules that promote metabolic health, antioxidants can have adverse effects if they interfere with free radical signaling (Kim and Lee, 2021). Thus, further investigation is necessary to delineate the specific molecular mechanisms that contribute to tangeretin’s anticancer activity. However, this study did not fully elucidate the mechanisms underlying the anticancer effects of tangeretin in endometrial cancer cells. Furthermore, the lack of in vivo studies examining efficacy, pharmacokinetics, and toxicity limits the translational relevance of these findings. Accordingly, future research is required to explore the therapeutic potential of tangeretin and to support its development as a candidate agent for the management of endometrial cancer.

In conclusion, the current study provides evidence that tangeretin may exert its anticancer effects in Ishikawa cells by reducing cell viability, inhibiting cell proliferation and migration, increasing the generation of cellular ROS, and inducing apoptosis through the upregulation of BAX protein levels. These findings suggest that tangeretin could serve as a supplementary strategy to complement existing treatments for endometrial cancer (Barrera et al., 2021).