Open Access

Esculetin has therapeutic potential via the proapoptotic signaling pathway in A253 human submandibular salivary gland tumor cells

  • Authors:
    • Su-Bin Park
    • Woo Kwon Jung
    • Hyung Rae Kim
    • Hwa-Young Yu
    • Yong Hwan Kim
    • Junghyun Kim
  • View Affiliations

  • Published online on: June 22, 2022
  • Article Number: 533
  • Copyright: © Park et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Esculetin is a natural lactone that is commonly derived from coumarins. According to previous experiments using human cancer cells, esculetin has potent antitumor activity; it also inhibits proliferation and induces the apoptosis of cancer cells. In the present study, the anti‑proliferative effect of esculetin on the submandibular salivary gland tumor cell line, A253, was evaluated via in vitro and in vivo analyses. Furthermore, the anti‑cancer effects of esculetin in A253 cells and a xenograft model of salivary gland tumors were determined using 3‑(4,5‑dimethylthiazol)‑2,5‑diphenyltetrazolium bromide and TUNEL assay, apoptosis protein array, quantitative polymerase chain reaction and western blot analysis. Esculetin (50‑150 µM) was demonstrated to have an anti‑proliferative effect in the A253 cell line in vitro; this observed effect was dependent on the dose and duration of treatment. Esculetin also increased the levels of Bax, cleaved caspase‑3, cleaved‑9 and cleaved poly (ADP‑ribose) polymerase apoptosis‑related proteins, and decreased the expression levels of the Bcl‑2 anti‑apoptotic protein. With respect to apoptosis regulation, esculetin significantly decreased the proliferation of tumor cells in a xenograft model (100 mg/kg/day) for 18 days. Overall, esculetin could be a potential oral anticancer drug against salivary gland cancer.


Head and neck squamous cell carcinoma (HNSCC) is the sixth most common non-skin-related cancer worldwide. Each year, 600,000 patients are diagnosed with this cancer, and 50% succumb (1). Salivary gland carcinoma is a relatively rare malignant tumor, accounting for <5% of HNSCC cases (2). Patients with high-grade salivary carcinoma have a 5-year survival rate of ~40%, whereas those with low-grade salivary carcinoma have a 5-year survival rate of 85-90% (3,4). The standard treatments for major and minor salivary gland tumors include surgical excision, radiotherapy and chemotherapy. Cisplatin, 5-fluorouracil and methotrexate are common drugs used to treat squamous cell carcinoma of the salivary glands (5). However, doxorubicin hydrochloride (Adriamycin), cisplatin, cyclophosphamide or cisplatin with 5-fluorouracil can be used for the primary treatment of patients with recurrent, metastatic or unresectable salivary gland tumors (6,7). Only a few effective treatment options are available for patients with recurrent or unresectable tumors. Despite the effective treatment features of cisplatin, various studies have revealed only a 10-month survival period when this drug is used alone or combined with other medicines. Chemotherapy causes speech/swallowing defects, chronic pain, muscle atrophy and other side effects (8). Therefore, novel chemotherapy and treatment regimens are required for salivary carcinoma (9).

Esculetin, also known as 6,7-dihydroxy coumarin, is derived from coumarin and can be obtained from various plants, such as Citrus limonia, Artemisia capillaries and Euphorbia lathyris (10,11). Pleiotropic biological activity is a well-known characteristic of esculetin. Moreover, esculetin has various advantages, including antioxidant and xanthine oxidase inhibitory activities, platelet aggregation and anticancer behavior (12-16). Esculetin also induces the apoptosis of oral squamous cell carcinoma (OSCC), resulting in the suppression of cancer cell proliferation (13,17). To date, no study has evaluated the effect of esculetin on human salivary gland tumors. Thus, in the present study, in vitro and in vivo experiments were performed to assess the induction of apoptosis and the antiproliferative effect of esculetin on the human submandibular carcinoma A253 cell line.

Materials and methods


Esculetin with a purity >98% was obtained from Tokyo Chemical Industry. DMSO, cisplatin and 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich; Merck KGaA. The apoptosis detection kit for TUNEL-FITC and the human apoptosis proteome profiler kit were supplied by Promega Corporation and R&D Systems, Inc., respectively. All the required antibodies, including Bax (cat. no. ab53154), Bcl-2 (cat. no. ab196495), poly (ADP-ribose) polymerase (PARP; cat. no. ab32139), caspase-3 (cat. no. ab184786), caspase-9 (cat. no. ab184786), and β-actin (cat. no. ab8227), were purchased from Abcam. The A253 cells were obtained from the American Type Culture Collection.

Cell culture

A253 cells were cultured in modified RPMI 1640 medium (Welgene, Inc.) supplemented with 10% fetal bovine serum (Welgene, Inc.), streptomycin (100 µg/ml), and penicillin (100 U/ml), and maintained in an incubator containing 5% CO2 at 37˚C.

Cell viability and apoptosis assay

The MTT assay was used to estimate the effect of esculetin on cell viability. The A253 cells were seeded in a 96-well cell culture plate at a density of 5x104 cells per well. Thereafter, the cultured cells were treated with different doses of esculetin (0, 50, 100 and 150 µM in 0.1% DMSO) for 24 and 48 h. MTT (5 mg/ml; 10 µl) was then added to the cells, which were further incubated at 37˚C for 4 h. After MTT removal, the violet formazan crystals were dissolved in 1 ml DMSO. A microplate reader (540 nm; Tecan Group, Ltd.) was used to obtain the MTT assay results. The cells were cultured on autoclaved coverslips to determine esculetin-treated apoptotic cells. TUNEL staining was conducted to detect apoptotic cells, after 24 and 48 h of pre-incubation with esculetin. Cells were then washed with PBS, and fixed with 4% paraformaldehyde in PBS at room temperature for 20 min. Fixed cells were washed with PBS and treated with the rTdT reaction mix at 37˚C for 1 h; reactions were terminated by immersing in 2X SSC solution for 15 min at room temperature. Cells were stained with 0.2 mg/ml of DAPI in PBS at room temperature for 15 mi and mounted onto glass slides with the mounting medium (Vectashield, Vector Laboratories, Inc.) and analyzed under a fluorescent microscope (Olympus Corporation). Images of at least five random fluorescent fields were captured, and TUNEL-positive cells were counted. Data were expressed as the percentage of apoptotic cells per field.

Western blot analysis

A protein extraction RIPA lysis buffer (Elpis Biotech) containing a Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Inc.) was used to lyse the treated cells. Thereafter, Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Inc.) was used to quantify the extracted protein. Equal amounts of proteins (50 µg/lane) were separated by 12.5% SDS-PAGE gel and transferred to a PVDF membrane (MilliporeSigma). The membranes were then blocked with 5% skimmed milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) at room temperature for 2 h. The primary antibodies used were anti-Bax (1:500), anti-Bcl-2 (1:500), anti-PARP (1:500), anti-caspase-3 (1:500), anti-caspase-9 (1:500), and β-actin (1:1,000). The membranes were incubated with primary antibodies diluted in TBST overnight at 4˚C, washed three times with TBST. Finally, a western blotting detection kit (ECL Western blotting substrate kit; Abcam) was used to observe the protein bands after treatment with a horseradish peroxidase-linked secondary antibody (1:1,000, Advansta) at room temperature for 1 h.

Proteome profiling of human apoptosis array

Different doses of esculetin (0, 50, 100, and 150 µM) were used to treat A253 cells for 48 h. A proteome profiler human apoptosis antibody array kit (cat. no. ARY009, R&D systems) was used to analyze the lysed cells according to the manufacturer's instructions. ImageJ software version 1.52a (National Institutes of Health) was used to desensitize the obtained signals, where the average pixel density was expressed by normalizing the pixel density to untreated samples.

Quantitative polymerase chain reaction (qPCR)

TRIzol® reagent (Thermo Fisher Scientific, Inc.) was used to extract total RNA from the collected cells based on the manufacturer's instructions. Total RNA (1 µg) was reverse transcribed in a final volume of 30 µl using the ImProm-II reverse transcriptase system kit (Promega Corporation) according to the manufacturer's instructions. cDNAs were used for PCR. The primer sequences are summarized in Table I. The AniQ5 Continuous Fluorescence Detector System (Bio-Rad Laboratories, Inc.) and a 2X SYBR® Green PCR Master Mix (cat. no. RR420A, Takara Bio, Inc.) were used to perform the qPCR at 95˚C for 30 sec, followed by 40 cycles of 95˚C for 3 sec and 60˚C for 30 sec and a single cycle of 95˚C for 15 sec, 60˚C for 60 sec, and 95˚C for 15 sec to generate dissociation curves. All PCR reactions were performed in triplicate, and the specificity of the reaction was determined by melting curve analysis. Comparative quantification of each target gene was performed based on cycle threshold (Ct) normalized to β-actin using the 2-ΔΔCq method (18).

Table I

List of primer sequences used in this study.

Table I

List of primer sequences used in this study.

GeneForward primerReverse primer
Tumor xenograft model

A total of 18 male BALB/c nude mice (age, 6 weeks old; weight, 15-20 g) were obtained from Central Lab Animal Inc. Mice were housed under a 12-h light/dark cycle at a temperature of 23±1˚C and 55±5% humidity and were provided with standard rodent pellets and filtered water ad libitum. The protocols approved by the Institutional Animal Care and Use Committee of the Jeonbuk National University Hospital (Jeonju, South Korea; approval no. JBNUH 2021-019) were used for the assessment. Briefly, A253 cells were suspended in RPMI-1640 culture medium containing 10% FBS, and maintained in a humidified atmosphere containing 5% CO2 at a controlled temperature of 37.6˚C. Before tumor inoculation, all mice were anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg). To obtain a xenograft model, mouse shoulders were inoculated with A253 cells at a total of 2x106 cells in 50 µl Dulbecco's modified Eagle's medium (Welgene, Inc.) mixed with 50 µl Matrigel (Becton, Dickinson and Company) for a total volume of 100 µl per injection site. After 7 days, mice were randomly divided into three groups, each containing six mice. Esculetin and cisplatin were dissolved in 0.5% carboxymethylcellulose. Thereafter, esculetin (100 mg/kg per day) or cisplatin (7.5 mg/kg per day), as a positive control, was orally administered for 18 days (19,20). Mice in the negative control group were orally administered the vehicle (0.5% carboxymethylcellulose). The in vivo antitumor activity of esculetin was assessed by measuring tumor size at 3-day intervals. Esculetin was administered for 18 days until the tumor size in the negative control group reached 200 mm3. Tumor volume was calculated using the following equation: π/6 x (a)2 x (b), where ‘a’ and ‘b’, correspond to the shortest and longest tumor diameters, respectively (21). No adverse reactions or compound-related side effects were observed in the mice. Body weight was measured and the mice were sacrificed with ketamine (500 mg/kg) and xylazine (50 mg/kg) to excise the tumor xenografts at necropsy.

H&E and TUNEL staining

For evaluation of the tissue structures, the tumor tissues were fixed in formaldehyde solution (4%) at room temperature for 24 h, dehydrated using gradient ethanol, embedded in paraffin, incised into smaller sections (4 µm) and then stained with H&E. The TUNEL assay was also performed using the one-step TUNEL kit, according to the defined guidelines. After paraffin removal, the sections were incubated in 100 µl Proteinase K (20 µg/ml) for 10 min at room temperature, transferred into a TUNEL reaction mixture with a volume of 50 µl, and placed in a dark incubator for 1 h at 37˚C. A solution of Vectashield Mounting Medium with 4',6-diamidino-2-phenylindole (Vector Laboratories, Inc.) was used to stain the prepared sections. Subsequently, a fluorescence microscope was used to capture images of the prepared slices. Images of at ≥5 random fluorescent fields were captured. In this evaluation, the number of TUNEL-positive apoptotic cells per 100 cells was determined using green fluorescence exerted by the cells.

Statistical analysis

The results are presented as the mean ± standard deviation. One-way analysis of variance was used to analyze the data, followed by Tukey's multiple comparison test. The assessments were conducted using GraphPad Prism 6.0 (GraphPad Prism Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.


Effect of esculetin on the proliferation and apoptosis of A253 cells

Several concentrations of esculetin were used to treat A253 cells for 24 and 48 h to explore the therapeutic potential of esculetin. Further, MTT assays were performed to determine the viability of the cells. The proliferation of A253 cells was inhibited by esculetin after both 24 and 47 h of treatment in a concentration-dependent manner (Fig. 1A). The 50% maximal inhibitory concentration (IC50) values of esculetin at 24 and 48 h were 157.4±30.0 and 78.5±5.3 µM, respectively. TUNEL staining was performed to determine the effect of esculetin on apoptosis. As shown in Fig. 1B and C, the apoptotic cell ratio was enhanced in the treated cancer cells compared with that in the control cells in a concentration-dependent manner. Based on the observed trend, apoptosis was the main cause of the decrease in cell viability of cancer cells treated with esculetin.

Role of esculetin in apoptosis induction via the regulation of apoptosis regulatory factors in A253 cells

The role of esculetin in apoptosis induction through the regulation of apoptosis regulatory proteins in tumor cells has been widely reported (22-24). To determine the expression level of apoptosis-related proteins, A253 cells were treated with various concentrations of esculetin (0, 50, 100 and 150 µM) for 24 or 48 h. The levels of Bax, cleaved caspase-3, cleaved caspase-9 and cleaved-PARP apoptosis-related proteins increased by esculetin after both 24 and 47 h of treatment. However, the level of the anti-apoptotic protein, Bcl-2, decreased in an esculetin dose-dependent manner (Fig. 2). These results indicated that esculetin induced apoptosis in the A253 cell line. To further elucidate the effect of esculetin on the differential expression of pro- and anti-apoptotic proteins, 56 proteins were identified using a human apoptosis proteome profiler array kit (Fig. 3A). As predicted, cleaved caspase-3 was significantly upregulated in cells treated with esculetin compared with untreated control cells. Further, HSP60 expression was found to be raised, whereas the expression levels of the anti-apoptotic proteins, including Bcl-2 and Bcl-x, was decreased in esculetin-treated cancer cells (Fig. 3A and B). Therefore, esculetin may promote A253 cell death through pro-apoptotic protein induction and anti-apoptotic protein reduction. To confirm the aforementioned results, the mRNA expression levels of pro- and anti-apoptotic genes in esculetin-treated A253 cells were evaluated. Caspase-3 and Bax mRNA expression levels were significantly increased, while those of Bcl-2 were decreased in a dose-dependent manner (Fig. 4).

Effect of esculetin on the proliferation and apoptosis of A253 cells

An in vivo examination was performed using a xenograft nude mouse model of A253 cells to determine the role of esculetin in the inhibition of tumor growth. The data revealed the inhibitory effect of esculetin on the growth of tumors (Fig. 5). Esculetin was found to reduce tumor weight and size. In fact, at necropsy, tumor sizes in the vehicle, esculetin and cisplatin-treated groups were 197.9±66.0, 49.8±29.8, and 140±72.0 mm3, respectively. Thus, esculetin suppressed xenograft tumor development in mice by 74% relative to that of vehicle-treated mice. Nevertheless, the body weight of xenograft nude mice was not significantly affected by esculetin. Cisplatin did not significantly suppress A253 tumor growth (P>0.05). Thus, esculetin has more potent antitumor activity than cisplatin. These findings suggest that the growth of human submandibular salivary gland tumors can be suppressed by the application of esculetin.

Effect of esculetin on apoptosis induction in the A253 cell xenograft model

Based on H&E staining, the xenograft tumor tissues of the control group showed vigorous growth, irregular morphology, nuclear hypertrophy, numerous abnormal mitoses, overt dysplasia, giant tumor cells and increased tumor angiogenesis (Fig. 6A). However, the esculetin group rarely showed necrotic lesions, with small nuclei, decreased dysplasia and reduced tumor angiogenesis. Notably, these tumor-related histopathological changes were not suppressed in the cisplatin-treated group. To detect apoptotic cells in tumor tissues, TUNEL analysis was performed, as shown in Fig. 6B and C. The treated group exhibited more TUNEL-positive cells than the control group, which aligns with the in vitro results. Hence, induction of apoptotic cells and inhibition of cell proliferation could occur in an A253 cell xenograft model using esculetin. Overall, both in vitro and in vivo analyses suggested that esculetin induces apoptosis in submandibular salivary gland tumors.


The proliferation, migration and survival of carcinoma cells can occur through several pathways, such as the RAS/RAF/ERK, PI3K and JAK/STAT pathways (25). Some chemotherapy drugs, including cisplatin, 5-fluorouracil, docetaxel, methotrexate, bleomycin and cetuximab, are considered targeted agents. In addition, various signals, such as those of VEGFR, EGFR, tyrosine protein kinase Met and insulin-like growth factor 1 (IGF-1) receptor, have been considered as therapeutic molecular targets (26). Cetuximab is the only EGFR-targeting agent approved by the Food and Drug Administration in the United States, but has insignificant effects due to intrinsic or acquired resistance, despite ubiquitous EGFR expression in HNSCC tumors (26).

In the present study, the antiproliferative and pro-apoptotic activities of esculetin against an HNSCC tumor cell line were investigated using in vitro and in vivo examinations. Based on recent studies, esculetin exerts antitumor activity in several cancer types. For example, esculetin (20-100 µM) inhibits the proliferation and expression of cyclin D1, cyclin-dependent kinase 4 and matrix metalloproteinase-2, and prevents the production of transforming and vascular endothelial growth factors in osteosarcoma LM8 cells (27). Esculetin also induces gastric cancer MGC-803 cell apoptosis by triggering the activation of the mitochondrial apoptotic pathway; this is linked to the mitochondrial membrane potential reduction, Bax/Bcl-2 ratio increase, agitation of the activities of caspase-3 and caspase-9, and enhancement of released cytochrome c from the mitochondria (28). Esculetin prevents cancer cell proliferation and induces the apoptosis of gastric cancer cells, which is mediated by the mitochondrial apoptosis pathway involving IGF-1/PI3K/Akt (28). Similarly, esculetin enhances the activities of caspase-3 and caspase-9, and promotes Bax expression, Bcl-2 expression reduction, mitochondrial membrane potential collapse, cytochrome c release and IGF-1/PI3K/Akt inactivation in SMMC-7721 cells (29). Lee et al (30) reported that the Wnt pathway could be a versatile therapeutic and suppressive target for various human cancer types. The β-catenin-T-cell complex factor, a key canonical Wnt signaling mediator, has been implicated in the development of human colon cancer. Esculetin, with its small molecular structure, is considered an effective Wnt/β-catenin pathway inhibitor. In another study, esculetin was reported to act as an effective lead chemotherapy agent for human metastatic colorectal cancer management; this can be linked to the ability of esculetin to prevent E-cadherin-mediated Wnt signaling via Axin2 inhibitors (31).

In several cancer cell lines, esculetin is known to inhibit cell proliferation and induce apoptosis or cell cycle arrest. Several studies have shown that esculetin inhibits lung cancer cell growth by adversely affecting c-Myc, cyclin D1 and NF-κB (32). Esculetin also causes Akt phosphorylation suppression and enhances protein expression of tumor suppressor phosphatase and tensin homologs by arresting the G1 phase of the cell cycle (33). Esculetin markedly inhibits STAT3 phosphorylation, blocks translocation of STAT3 to the nucleus and restricts the G1/S phase of the cell cycle in laryngeal cancer (34). A Bax/Bcl-2 ratio increase, caspase-3 and -9 activation, and mitochondria-mediated apoptosis pathway induction have been observed in hepatocellular carcinoma cells following the application of this therapy (35). Furthermore, 2-aminoethoxydiphenyl borate-sensitive store-operated Ca2+ entry, Ca2+ release from the endoplasmic reticulum and activation of the mitochondrial apoptosis pathway result in Ca2+ influx via esculetin, ultimately leading to cell cycle arrest in ZR-75-1 human breast cancer (36).

Previously, esculetin was revealed to play an anti-proliferative role in head and neck cancer (13). The induction of apoptosis was caused by the inhibition of the specific protein 1 transcription factor (Sp1) (22), as well as modulation regulation of the p27, p21 and cyclin D1 target genes in OSCC, HSC-4 and HN22, and in human malignant melanoma cell lines (37). Sp1 is an essential transcription factor for a number of genes required for the regulation of multiple aspects of tumor cell survival, growth and angiogenesis (38). Previous studies have demonstrated that Sp1 is a drug target, and several antineoplastic agents have been shown to inhibit Sp1 expression (39). Sp1 is involved in the regulation of apoptosis. In fact, the promoters of a number of anti-apoptotic genes (bcl-2, bcl-x and survivin) and pro-apoptotic genes (bax, trail, fas, fas-ligand, caspase-9 and caspase-3) contain Sp1-binding sites (40). The inhibition of Sp1 expression regulates these genes. Indeed, inhibition of Sp1-DNA binding was shown to induce caspase 9-dependent apoptosis in bone marrow stromal cells (41). The inhibition of Sp1 by esculetin may lead to an increase in levels of Bax, cleaved caspase-3, cleaved-9 and cleaved PARP, and a decrease in Bcl-2 anti-apoptotic protein levels in A253 cells. Although the present study did not provide concrete scientific evidence of the effect of esculetin on the tumorigenesis of A263 cells, the observed results were consistent with its reported effects on various tumors.

The antitumor activity of esculetin was compared with that of cisplatin, which is a well-known anticancer drug. As previously reported, cisplatin is a positive control drug that reduces tumor size (42). In the present study, esculetin displayed more potent antitumor activity than cisplatin. In fact, cisplatin did not suppress A253 tumor growth (P>0.05). Similarly, in previous studies, the tumor growth of Cal-27 human head and neck squamous cell carcinoma cells did not show any growth delay when treated with cisplatin (P>0.05), whereas that of FaDu human head and neck squamous cell carcinoma cells showed a slight but significant growth delay when treated with cisplatin (P<0.01) (43). These results suggest that the antitumor activity of cisplatin varies depending on the head and neck carcinoma cell line used. Notably, the clinical use of cisplatin is often limited by undesirable side effects, such as severe nephrotoxicity and hepatotoxicity (44). Although the precise mechanism for this cisplatin-induced toxicity is not well understood, cisplatin is preferentially taken up and accumulates in the human liver and kidney cells, resulting in enhanced production of reactive oxygen species and a decrease in levels of antioxidant enzymes (45,46).

In the in vitro system of the present study, the effective dose of esculetin to induce apoptosis in A253 cells was as low as 50 µM, whereas the maximum dose was 150 µM. In several previous reports, esculetin (50 and 100 mg/kg/day) dose-dependently inhibited xenograft tumor growth in laryngeal cancer cells (33). Zhu et al (32) reported that esculetin (100 mg/kg/day) suppressed tumor growth in a mouse model of lung cancer. Based on these reports, the oral dosage of esculetin (100 mg/kg/day) in mice was determined. In the in vivo investigation, 150 mg/kg/day of esculetin was identified as the most influential dose. In the present study, there was no difference in body weight between the esculetin-treated and vehicle-treated mice. Injecting 700 mg/kg esculetin per day was suggested to have no adverse effects on the body weight of the mice (34). Esculetin had a median lethal dose (LD50) of 1,450 mg/kg via intraperitoneal injection and >2,000 mg/kg by oral gavage (47). Based on the guidelines of the Organization for Economic Cooperation and Development, an LD50 >2,000 mg/kg indicates the safety of the applied compound (48). Collectively, these results suggest that esculetin has relatively low toxicity in mice, highlighting the safety of esculetin for medical applications.

In the present study, the antiproliferative and pro-apoptotic activities of esculetin against human submandibular salivary gland tumor cells were evaluated. However, the antiproliferative activity of cisplatin was not evaluated in vitro, thereby serving as a study limitation. Previously, Lee et al (49) reported that the IC50 value of cisplatin on the proliferation of A253 cells was ~6.7 µM. In the present study, multiple oral doses of esculetin were required to determine the effective dose range in the animal experiment, which served as another limitation of the study. Therefore, the detailed beneficial role of esculetin in submandibular salivary gland tumor cells requires further study.

Overall, the in vitro and in vivo experiments of the present study confirmed the apoptosis and cell proliferation inhibitory effects of esculetin on human submandibular salivary gland tumors. These findings clearly indicate that esculetin may be a promising treatment option for HSNCC.

Supplementary Material

Original blot images for Fig. 2A.


Not applicable.


Funding: This study was supported by a National Research Foundation of Korea and funded by the Korean government (MEST; grant no. NRF-2019R1A2C1008773).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

SBP, WKJ, HRK, HYY and YHK performed the experiments, collected data and wrote the manuscript. SBP and JK analyzed the data and wrote the manuscript. SBP and JK confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

The animal experiments were approved by the Institutional Animal Care and Use Committee of the Jeonbuk National University Hospital, Jeonju, South Korea (approval no. JBNUH 2021-019).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.



Ferlay J, Shin HR, Bray F, Forman D, Mathers C and Parkin DM: Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer. 127:2893–2917. 2010.PubMed/NCBI View Article : Google Scholar


Laurie SA and Licitra L: Systemic therapy in the palliative management of advanced salivary gland cancers. J Clin Oncol. 24:2673–2678. 2006.PubMed/NCBI View Article : Google Scholar


Bell RB, Dierks EJ, Homer L and Potter BE: Management and outcome of patients with malignant salivary gland tumors. J Oral Maxillofac Surg. 63:917–928. 2005.PubMed/NCBI View Article : Google Scholar


Lima RA, Tavares MR, Dias FL, Kligerman J, Nascimento MF, Barbosa MM, Cernea CR, Soares JR, Santos IC and Salviano S: Clinical prognostic factors in malignant parotid gland tumors. Otolaryngol Head Neck Surg. 133:702–708. 2005.PubMed/NCBI View Article : Google Scholar


Guzzo M, Locati LD, Prott FJ, Gatta G, McGurk M and Licitra L: Major and minor salivary gland tumors. Crit Rev Oncol Hematol. 74:134–148. 2010.PubMed/NCBI View Article : Google Scholar


Kaplan MJ, Johns ME and Cantrell RW: Chemotherapy for salivary gland cancer. Otolaryngol Head Neck Surg. 95:165–170. 1986.PubMed/NCBI View Article : Google Scholar


Lagha A, Chraiet N, Ayadi M, Krimi S, Allani B, Rifi H, Raies H and Mezlini A: Systemic therapy in the management of metastatic or advanced salivary gland cancers. Head Neck Oncol. 4(19)2012.PubMed/NCBI View Article : Google Scholar


Larson DL, Lindberg RD, Lane E and Goepfert H: Major complications of radiotherapy in cancer of the oral cavity and oropharynx. A 10 year retrospective study. Am J Surg. 146:531–536. 1983.PubMed/NCBI View Article : Google Scholar


Pendleton KP and Grandis JR: Cisplatin-based chemotherapy options for recurrent and/or metastatic squamous cell cancer of the head and neck. Clin Med Insights Ther. 2013(10.4137/CMT.S10409)2013.PubMed/NCBI View Article : Google Scholar


Chang WS, Lin CC, Chuang SC and Chiang HC: Superoxide anion scavenging effect of coumarins. Am J Chin Med. 24:11–17. 1996.PubMed/NCBI View Article : Google Scholar


Masamoto Y, Ando H, Murata Y, Shimoishi Y, Tada M and Takahata K: Mushroom tyrosinase inhibitory activity of esculetin isolated from seeds of Euphorbia lathyris L. Biosci Biotechnol Biochem. 67:631–634. 2003.PubMed/NCBI View Article : Google Scholar


Egan D, O'Kennedy R, Moran E, Cox D, Prosser E and Thornes RD: The pharmacology, metabolism, analysis, and applications of coumarin and coumarin-related compounds. Drug Metab Rev. 22:503–529. 1990.PubMed/NCBI View Article : Google Scholar


Kok SH, Yeh CC, Chen ML and Kuo MY: Esculetin enhances TRAIL-induced apoptosis through DR5 upregulation in human oral cancer SAS cells. Oral Oncol. 45:1067–1072. 2009.PubMed/NCBI View Article : Google Scholar


Lin WL, Wang CJ, Tsai YY, Liu CL, Hwang JM and Tseng TH: Inhibitory effect of esculetin on oxidative damage induced by t-butyl hydroperoxide in rat liver. Arch Toxicol. 74:467–472. 2000.PubMed/NCBI View Article : Google Scholar


Okada Y, Miyauchi N, Suzuki K, Kobayashi T, Tsutsui C, Mayuzumi K, Nishibe S and Okuyama T: Search for naturally occurring substances to prevent the complications of diabetes. II. Inhibitory effect of coumarin and flavonoid derivatives on bovine lens aldose reductase and rabbit platelet aggregation. Chem Pharm Bull (Tokyo). 43:1385–1387. 1995.PubMed/NCBI View Article : Google Scholar


Wang CJ, Hsieh YJ, Chu CY, Lin YL and Tseng TH: Inhibition of cell cycle progression in human leukemia HL-60 cells by esculetin. Cancer Lett. 183:163–168. 2002.PubMed/NCBI View Article : Google Scholar


Noguchi M, Kitagawa H, Miyazaki I and Mizukami Y: Influence of esculetin on incidence, proliferation, and cell kinetics of mammary carcinomas induced by 7,12-dimethylbenz[a]anthracene in rats on high- and low-fat diets. Jpn J Cancer Res. 84:1010–1014. 1993.PubMed/NCBI View Article : Google Scholar


Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001.PubMed/NCBI View Article : Google Scholar


Florea AM and Büsselberg D: Cisplatin as an anti-tumor drug: Cellular mechanisms of activity, drug resistance and induced side effects. Cancers (Basel). 3:1351–1371. 2011.PubMed/NCBI View Article : Google Scholar


Osman AM, Telity SA, Telity SA, Damanhouri ZA, Al-Harthy SE, Al-Kreathy HM, Ramadan WS, Elshal MF, Khan LM and Kamel F: Chemosensitizing and nephroprotective effect of resveratrol in cisplatin-treated animals. Cancer Cell Int. 15(6)2015.PubMed/NCBI View Article : Google Scholar


Tikoo K, Kumar P and Gupta J: Rosiglitazone synergizes anticancer activity of cisplatin and reduces its nephrotoxicity in 7, 12-dimethyl benz{a}anthracene (DMBA) induced breast cancer rats. BMC Cancer. 9(107)2009.PubMed/NCBI View Article : Google Scholar


Cho JH, Shin JC, Cho JJ, Choi YH, Shim JH and Chae JI: Esculetin (6,7-dihydroxycoumarin): A potential cancer chemopreventive agent through suppression of Sp1 in oral squamous cancer cells. Int J Oncol. 46:265–271. 2015.PubMed/NCBI View Article : Google Scholar


Park C, Jin CY, Kim GY, Choi IW, Kwon TK, Choi BT, Lee SJ, Lee WH and Choi YH: Induction of apoptosis by esculetin in human leukemia U937 cells through activation of JNK and ERK. Toxicol Appl Pharmacol. 227:219–228. 2008.PubMed/NCBI View Article : Google Scholar


Sakagami H: Apoptosis-inducing activity and tumor-specificity of antitumor agents against oral squamous cell carcinoma. Jpn Dent Sci Rev. 46:173–187. 2010.


Alsahafi E, Begg K, Amelio I, Raulf N, Lucarelli P, Sauter T and Tavassoli M: Clinical update on head and neck cancer: Molecular biology and ongoing challenges. Cell Death Dis. 10(540)2019.PubMed/NCBI View Article : Google Scholar


Wen Y and Grandis JR: Emerging drugs for head and neck cancer. Expert Opin Emerg Drugs. 20:313–329. 2015.PubMed/NCBI View Article : Google Scholar


Kimura Y and Sumiyoshi M: Antitumor and antimetastatic actions of dihydroxycoumarins (esculetin or fraxetin) through the inhibition of M2 macrophage differentiation in tumor-associated macrophages and/or G1 arrest in tumor cells. Eur J Pharmacol. 746:115–125. 2015.PubMed/NCBI View Article : Google Scholar


Wang G, Lu M, Yao Y, Wang J and Li J: Esculetin exerts antitumor effect on human gastric cancer cells through IGF-1/PI3K/Akt signaling pathway. Eur J Pharmacol. 814:207–215. 2017.PubMed/NCBI View Article : Google Scholar


Li J, Li S, Wang X and Wang H: Esculetin induces apoptosis of SMMC-7721 cells through IGF-1/PI3K/Akt-mediated mitochondrial pathways. Can J Physiol Pharmacol. 95:787–794. 2017.PubMed/NCBI View Article : Google Scholar


Lee SY, Lim TG, Chen H, Jung SK, Lee HJ, Lee MH, Kim DJ, Shin A, Lee KW, Bode AM, et al: Esculetin suppresses proliferation of human colon cancer cells by directly targeting β-catenin. Cancer Prev Res (Phila). 6:1356–1364. 2013.PubMed/NCBI View Article : Google Scholar


Kim WK, Byun WS, Chung HJ, Oh J, Park HJ, Choi JS and Lee SK: Esculetin suppresses tumor growth and metastasis by targeting Axin2/E-cadherin axis in colorectal cancer. Biochem Pharmacol. 152:71–83. 2018.PubMed/NCBI View Article : Google Scholar


Zhu X, Gu J and Qian H: Esculetin attenuates the growth of lung cancer by downregulating Wnt targeted genes and suppressing NF-κB. Arch Bronconeumol (Engl Ed). 54:128–133. 2018.PubMed/NCBI View Article : Google Scholar : (In English, Spanish).


Turkekul K, Colpan RD, Baykul T, Ozdemir MD and Erdogan S: Esculetin inhibits the survival of human prostate cancer cells by inducing apoptosis and arresting the cell cycle. J Cancer Prev. 23:10–17. 2018.PubMed/NCBI View Article : Google Scholar


Zhang G, Xu Y and Zhou HF: Esculetin inhibits proliferation, invasion, and migration of laryngeal cancer in vitro and in vivo by inhibiting janus kinas (JAK)-signal transducer and activator of transcription-3 (STAT3) activation. Med Sci Monit. 25:7853–7863. 2019.PubMed/NCBI View Article : Google Scholar


Wang J, Lu ML, Dai HL, Zhang SP, Wang HX and Wei N: Esculetin, a coumarin derivative, exerts in vitro and in vivo antiproliferative activity against hepatocellular carcinoma by initiating a mitochondrial-dependent apoptosis pathway. Braz J Med Biol Res. 48:245–253. 2015.PubMed/NCBI View Article : Google Scholar


Chang HT, Chou CT, Lin YS, Shieh P, Kuo DH, Jan CR and Liang WZ: Esculetin, a natural coumarin compound, evokes Ca(2+) movement and activation of Ca(2+)-associated mitochondrial apoptotic pathways that involved cell cycle arrest in ZR-75-1 human breast cancer cells. Tumour Biol. 37:4665–4678. 2016.PubMed/NCBI View Article : Google Scholar


Liang C, Ju W, Pei S, Tang Y and Xiao Y: Pharmacological activities and synthesis of esculetin and its derivatives: A mini-review. Molecules. 22(387)2017.PubMed/NCBI View Article : Google Scholar


Chu S and Ferro TJ: Sp1: Regulation of gene expression by phosphorylation. Gene. 348:1–11. 2005.PubMed/NCBI View Article : Google Scholar


Safe S, Imanirad P, Sreevalsan S, Nair V and Jutooru I: Transcription factor Sp1, also known as specificity protein 1 as a therapeutic target. Expert Opin Ther Targets. 18:759–769. 2014.PubMed/NCBI View Article : Google Scholar


Black AR, Black JD and Azizkhan-Clifford J: Sp1 and krüppel-like factor family of transcription factors in cell growth regulation and cancer. J Cell Physiol. 188:143–160. 2001.PubMed/NCBI View Article : Google Scholar


Louie JS, Shukla R, Richards-Kortum R and Anandasabapathy S: High-resolution microendoscopy in differentiating neoplastic from non-neoplastic colorectal polyps. Best Pract Res Clin Gastroenterol. 29:663–673. 2015.PubMed/NCBI View Article : Google Scholar


Shirmanova MV, Druzhkova IN, Lukina MM, Dudenkova VV, Ignatova NI, Snopova LB, Shcheslavskiy VI, Belousov VV and Zagaynova EV: Chemotherapy with cisplatin: Insights into intracellular pH and metabolic landscape of cancer cells in vitro and in vivo. Sci Rep. 7(8911)2017.PubMed/NCBI View Article : Google Scholar


Simons AL, Fath MA, Mattson DM, Smith BJ, Walsh SA, Graham MM, Hichwa RD, Buatti JM, Dornfeld K and Spitz DR: Enhanced response of human head and neck cancer xenograft tumors to cisplatin combined with 2-deoxy-D-glucose correlates with increased 18F-FDG uptake as determined by PET imaging. Int J Radiat Oncol Biol Phys. 69:1222–1230. 2007.PubMed/NCBI View Article : Google Scholar


Chirino YI, Hernández-Pando R and Pedraza-Chaverri J: Peroxynitrite decomposition catalyst ameliorates renal damage and protein nitration in cisplatin-induced nephrotoxicity in rats. BMC Pharmacol. 4(20)2004.PubMed/NCBI View Article : Google Scholar


Stewart DJ, Benjamin RS, Luna M, Feun L, Caprioli R, Seifert W and Loo TL: Human tissue distribution of platinum after cis-diamminedichloroplatinum. Cancer Chemother Pharmacol. 10:51–54. 1982.PubMed/NCBI View Article : Google Scholar


Mora Lde O, Antunes LM, Francescato HD and Bianchi Mde L: The effects of oral glutamine on cisplatin-induced nephrotoxicity in rats. Pharmacol Res. 47:517–522. 2003.PubMed/NCBI View Article : Google Scholar


Tubaro A, Del Negro P, Ragazzi E, Zampiron S and Della Loggia R: Anti-inflammatory and peripheral analgesic activity of esculetin in vivo. Pharmacol Res Commun. 20 (Suppl 5):S83–S85. 1988.PubMed/NCBI View Article : Google Scholar


OECD: Test No. 423: Acute Oral toxicity-Acute Toxic Class. Method. In: OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, Paris, 2002.


Lee JH, Hwang EH and Lee SR: A study on the radiosensitivity and chemosensitivity of A-253 cell line in vitro. J Korean Acad Oral Maxillofac Radiol. 27:91–104. 1997.

Related Articles

Journal Cover

Volume 24 Issue 2

Print ISSN: 1792-0981
Online ISSN:1792-1015

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
Spandidos Publications style
Park S, Jung WK, Kim HR, Yu H, Kim YH and Kim J: Esculetin has therapeutic potential via the proapoptotic signaling pathway in A253 human submandibular salivary gland tumor cells. Exp Ther Med 24: 533, 2022
Park, S., Jung, W.K., Kim, H.R., Yu, H., Kim, Y.H., & Kim, J. (2022). Esculetin has therapeutic potential via the proapoptotic signaling pathway in A253 human submandibular salivary gland tumor cells. Experimental and Therapeutic Medicine, 24, 533.
Park, S., Jung, W. K., Kim, H. R., Yu, H., Kim, Y. H., Kim, J."Esculetin has therapeutic potential via the proapoptotic signaling pathway in A253 human submandibular salivary gland tumor cells". Experimental and Therapeutic Medicine 24.2 (2022): 533.
Park, S., Jung, W. K., Kim, H. R., Yu, H., Kim, Y. H., Kim, J."Esculetin has therapeutic potential via the proapoptotic signaling pathway in A253 human submandibular salivary gland tumor cells". Experimental and Therapeutic Medicine 24, no. 2 (2022): 533.