Hinokitiol Exhibits Antitumor Properties through Induction of ROS-Mediated Apoptosis and p53-Driven Cell-Cycle Arrest in Endometrial Cancer Cell Lines (Ishikawa, HEC-1A, KLE)

Hinokitiol is a natural tropolone derivative that is present in the heartwood of cupressaceous plants, and has been extensively investigated for its anti-inflammatory, antioxidant, and antitumor properties in the context of various diseases. To date, the effects of hinokitiol on endometrial cancer (EC) has not been explored. The purpose of our study was to investigate the anti-proliferative effects of hinokitiol on EC cells. Cell viability was determined with an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the quantification of apoptosis and reactive oxygen species (ROSs) was performed by using flow cytometry, while protein expression was measured with the Western blotting technique. Hinokitiol significantly suppressed cell proliferation through the inhibition of the expression of cell-cycle mediators, such as cyclin D1 and cyclin-dependent kinase 4 (CDK4), as well as the induction of the tumor suppressor protein p53. In addition, hinokitiol increased the number of apoptotic cells and increased the protein expression of cleaved-poly-ADP-ribose polymerase (PARP) and active cleaved-caspase-3, as well as the ratio of Bcl-2-associated X protein (Bax) to B-cell lymphoma 2 (Bcl-2). Interestingly, except for KLE cells, hinokitiol induced autophagy by promoting the accumulation of the microtubule-associated protein light chain 3B (LC3B) and reducing the sequestosome-1 (p62/SQSTM1) protein level. Furthermore, hinokitiol triggered ROS production and upregulated the phosphorylation of extracellular-signal-regulated kinase (p-ERK1/2) in EC cells. These results demonstrate that hinokitiol has potential anti-proliferative and pro-apoptotic benefits in the treatment of endometrial cancer cell lines (Ishikawa, HEC-1A, and KLE).


Introduction
The prevalence of endometrial cancer (EC) has rapidly risen worldwide in recent years, including in the United States [1] and in Taiwan [2]. It is estimated that 417,367 new cases of and 97,370 deaths from corpus uteri (also known as endometrial cancer) happened worldwide in 2020 [3]. In general, EC has been histologically classified as either type 1 EC (well-differentiated) or type 2 EC (poorly differentiated), depending on the presence of estrogen. In addition, according to its different clinical features, EC can be subdivided into (i) endometrioid endometrial cancer (EEC, or type 1), (ii) serous endometrial cancer (SEC, or type 2), (iii) clear-cell endometrial cancer (CCEC, or type 2), and (iv) mixed endometrial cancer and uterine carcinoma (USC) [4,5]. To strengthen observers' consensus on the histological classification of tumors and to improve the prognosis of EC patients, many studies have been dedicated to incorporating molecular classification with The Cancer Genome Atlas (TCGA) analysis, which includes TP53 mutations, into routine clinical histological diagnostic evaluations [6]. Mutations in p53 lead to the loss of tumor suppressor activity while promoting the survival and proliferation of malignant tumors [7].
Considering the side effects caused by chemotherapy and radiotherapy, it is critical to seek alternative treatments for malignant cancers, including endometrial cancer. Naturally occurring compounds have a variety of effective phytochemicals and usually have lower toxicity than chemotherapy, so they are widely used in the clinical treatment of various cancers [8,9]. Hinokitiol, also known as β-Thujaplicin, is a natural tropolone derivative with a seven-membered carbon ring and an isopropyl side chain ( Figure 1A). It is widely used in cosmetics and oral-care products due to its strong antifungal and antimicrobial activities.
A large amount of the literature in the public domain has demonstrated the various biological properties of hinokitiol, including its neuroprotective activity [10], as well as its antibacterial [11] and anti-inflammatory [12] effects. In addition, hinokitiol has been shown to have antitumor potential against a variety of cancers; this potential involves reducing metastasis in 4T1 breast cancer cells [13], promoting apoptosis in A549 lung cancer cells [14], inducing senescence and autophagy in HeLa cervical cancer cells [15], disrupting androgen receptor signaling in LNCaP prostate carcinoma cells [16], and alleviating the migration ability [13] and melanogenesis in B16F10 melanoma cells [17]. It is worth mentioning that hinokitiol has the ability to inhibit tumor growth without causing damage to normal tissue; for example, hinokitiol simultaneously inhibits the growth of oral pathogens and oral squamous cell carcinoma cells without inducing toxicity in normal human oral keratinocytes [18].
Although many studies have been conducted on the beneficial effects of hinokitiol on cancer, its antitumor activity and the exact pathophysiological mechanism with respect to EC have not been investigated. Therefore, in this study, we focus on the inhibitory effects of hinokitiol on EC through its effects on cell proliferation, apoptosis, and autophagy.

Hinokitiol Promotes Cell-Cycle Arrest at the G0/G1 Phase in Endometrial Cancer Cells
To assess whether hinokitiol has an effect on the progression of the EC cell cycle, we examined the expression of regulatory proteins of the cell cycle after hinokitiol treatment. Our data revealed that hinokitiol doses of greater than 25 μM significantly upregulated the cell-cycle inhibitor p53 in the Ishikawa (Figure 2A Figure 2I,L) cells. Consequently, we propose that hinokitiol induced an increase in the p53 level and a decrease in cyclin D1 and CDK4 expression, which implies that hinokitiol affected the growth of EC cells through the induction of the arrest of the G0/G1 phase.  (IC 50 ) values of hinokitiol in endometrial cancer cells. Data are expressed as the mean ± SD of three independent experiments; * p < 0.05 and ** p < 0.001 compared to the control group.

Hinokitiol Induces an Anti-Proliferative Effect on Endometrial Cancer Cells
To evaluate the cytotoxicity of hinokitiol, we treated human endometrial cancer cell lines (Ishikawa, HEC-1A, and KLE) with different concentrations of hinokitiol (1, 5, 10, 25, and 50 µM) or dimethyl sulfoxide (DMSO) for 24 and 48 h. Then, 3-(4,5-dimethylthiazol-2yl) -2,5-diphenyltetrazolium bromide (MTT) assays were used to analyze the cell viability. As shown in Figure 1B-D, starting from the dose of 5 µM, hinokitiol induced a sharp decline in the cell viability and significantly inhibited the growth of the Ishikawa, HEC-1A, and KLE cells compared with the DMSO control, especially after 48 h (p < 0.001). The IC 50 after treatment for 48 h was estimated to be 13.33 µM in the Ishikawa cells, 49.51 µM in the HEC-1A cells, and 4.69 µM in the KLE cells ( Figure 1E-G).

Hinokitiol Promotes Cell-Cycle Arrest at the G0/G1 Phase in Endometrial Cancer Cells
To assess whether hinokitiol has an effect on the progression of the EC cell cycle, we examined the expression of regulatory proteins of the cell cycle after hinokitiol treatment. Our data revealed that hinokitiol doses of greater than 25 µM significantly upregulated the cell-cycle inhibitor p53 in the Ishikawa (Figure 2A (G-I), and cyclin D1 (J-L) was assayed through Western blotting analysis. The differences in target proteins were analyzed with Sigma Plot, and data are expressed as the mean ± SD of three independent experiments; * p < 0.05 and ** p < 0.001 compared to the control group.

Hinokitiol Induces Cell Apoptosis via the Caspase Apoptotic Pathway in Endometrial Cancer Cells
Accumulating evidence has suggested that the activation of the p53/p21 pathway promotes apoptosis, and this pathway is part of programmed cell death [19]. To explore whether hinokitiol triggers cell apoptosis, the percentages of apoptotic cells after the treatment were examined by using Annexin-V/PI staining. As the results showed, the numbers of cells in the early apoptosis (annexin V+/PI−) and late apoptosis (annexin V+/PI+) phases increased after the hinokitiol treatment. Interestingly, the treatment had different effects depending on the type of cell line. In detail, in the HEC-1A cells, only the dose of 50 μM significantly increased the number of apoptotic cells, while lower doses showed statisti- (G-I), and cyclin D1 (J-L) was assayed through Western blotting analysis. The differences in target proteins were analyzed with Sigma Plot, and data are expressed as the mean ± SD of three independent experiments; * p < 0.05 and ** p < 0.001 compared to the control group.

Hinokitiol Induces Cell Apoptosis via the Caspase Apoptotic Pathway in Endometrial Cancer Cells
Accumulating evidence has suggested that the activation of the p53/p21 pathway promotes apoptosis, and this pathway is part of programmed cell death [19]. To explore whether hinokitiol triggers cell apoptosis, the percentages of apoptotic cells after the treatment were examined by using Annexin-V/PI staining. As the results showed, the numbers of cells in the early apoptosis (annexin V+/PI−) and late apoptosis (annexin V+/PI+) phases increased after the hinokitiol treatment. Interestingly, the treatment had different effects depending on the type of cell line. In detail, in the HEC-1A cells, only the dose of 50 µM significantly increased the number of apoptotic cells, while lower doses showed statistically insignificant increases ( Figure 3B,E); However, in the Ishikawa cells, doses of hinokitiol greater than 5 µM significantly increased the number of apoptotic cells ( Figure 3A,D). Finally, in the KLE cells, significant induction of apoptosis appeared starting from the dose of 1 µM hinokitiol; the dose of 5 µM achieved the greatest pro-apoptotic effect ( Figure 3C,F). Finally, in the KLE cells, significant induction of apoptosis appeared starting from the dose of 1 μM hinokitiol; the dose of 5 μM achieved the greatest pro-apoptotic effect ( Figure 3C,F). To investigate the molecular mechanisms of hinokitiol-mediated apoptosis in endometrial cancer cells, the expression of the activated PARP, Bax, Bcl-2, and caspase-3 proteins was analyzed with a Western blotting assay. As shown in Figure 4A-C, regardless of the cell line, hinokitiol significantly increased the expression of cleaved PARP ( Figure  4D-F), and the ratio of the pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 was also significantly increased after the hinokitiol treatment ( Figure 4G-I). In addition, the expression of cleaved caspase-3 after hinokitiol treatment has different increasing trends in different endometrial cell lines. Although it did not increase with increasing doses, the expression of cleaved caspase-3 in Ishikawa ( Figure 4J,M), HEC-1A ( Figure 4K,N), and KLE ( Figure 4L,O) cells increased significantly at specific doses. Collectively, these data suggest that hinokitiol might induce EC cell death by altering the activation of caspase. To investigate the molecular mechanisms of hinokitiol-mediated apoptosis in endometrial cancer cells, the expression of the activated PARP, Bax, Bcl-2, and caspase-3 proteins was analyzed with a Western blotting assay. As shown in Figure 4A-C, regardless of the cell line, hinokitiol significantly increased the expression of cleaved PARP ( Figure 4D-F), and the ratio of the pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 was also significantly increased after the hinokitiol treatment ( Figure 4G-I). In addition, the expression of cleaved caspase-3 after hinokitiol treatment has different increasing trends in different endometrial cell lines. Although it did not increase with increasing doses, the expression of cleaved caspase-3 in Ishikawa ( Figure

Hinokitiol Induces Cell Autophagy in Endometrial Cancer Cells
Programmed cell death also occurs through autophagy, which is a complex catabolic process that degrades damaged organelles and proteins through lysosome-mediated degradation and promotes nutrient circulation [20]. We next evaluated whether hinokitiol induces autophagy in EC cells. We assessed the levels of two autophagy regulators, p62/SQSTM1 and LC3, after the treatment with hinokitiol. As shown in Figure 5, after the intervention of hinokitiol, LC3-I was converted into LC3-II, causing the accumulation of LC3-II expression in the Ishikawa ( Figure

Hinokitiol Induces Cell Autophagy in Endometrial Cancer Cells
Programmed cell death also occurs through autophagy, which is a complex catabolic process that degrades damaged organelles and proteins through lysosome-mediated degradation and promotes nutrient circulation [20]. We next evaluated whether hinokitiol induces autophagy in EC cells. We assessed the levels of two autophagy regulators, p62/SQSTM1 and LC3, after the treatment with hinokitiol. As shown in Figure 5, after the intervention of hinokitiol, LC3-I was converted into LC3-II, causing the accumulation of LC3-II expression in the Ishikawa ( Figure 5A,G), HEC-1A ( Figure 5B,H) cells. However, no increasing trend was seen in KLE cells ( Figure 5C,I). As the degradation substrate of autophagic membranes, the expression of p62/SQSTM1 was decreased after the treatment with hinokitiol in the Ishikawa ( Figure 5A,D) and KLE ( Figure 5C,F) cells. However, only a decreasing trend can be observed in HEC-1A cells ( Figure 5B,E), but there is no statistical difference. Collectively, the accumulation of LC3-II and the downregulation of the p62 expression level after the hinokitiol treatment might be interpreted as the induction of autophagy.

Hinokitiol Induces Intracellular Reactive Oxygen Species (ROSs) in Endometrial Cancer Cells
To verify that natural products can activate reactive oxygen species (ROSs) and subsequently induce apoptosis in cancer cells, staining of the oxidative stress indicator H2DCFDA was used. H2O2 was used as the positive control group to confirm the induction of intracellular ROSs. Figure 6 shows representative profiles of ROS generation in the Ishikawa ( Figure 6A), HEC-1A ( Figure 6C), and KLE cells ( Figure 6E). As in the positive control group, hinokitiol had the ability to enhance the fluorescence intensity in the three

Hinokitiol Induces Intracellular Reactive Oxygen Species (ROSs) in Endometrial Cancer Cells
To verify that natural products can activate reactive oxygen species (ROSs) and subsequently induce apoptosis in cancer cells, staining of the oxidative stress indicator H 2 DCFDA was used. H 2 O 2 was used as the positive control group to confirm the induction of intracellular ROSs. Figure 6 shows representative profiles of ROS generation in the Ishikawa ( Figure 6A), HEC-1A ( Figure 6C), and KLE cells ( Figure 6E). As in the positive control group, hinokitiol had the ability to enhance the fluorescence intensity in the three types of cells. Starting from 5 µM, hinokitiol significantly increased the production of ROSs in the Ishikawa cells ( Figure 6B) and KLE cells ( Figure 6F); starting from 10 µM, it significantly increased ROS production in the HEC-1A cells ( Figure 6D). types of cells. Starting from 5 μM, hinokitiol significantly increased the production of ROSs in the Ishikawa cells ( Figure 6B) and KLE cells ( Figure 6F); starting from 10 μM, it significantly increased ROS production in the HEC-1A cells ( Figure 6D).

Hinokitiol Regulates the ERK Signaling Pathway
To determine whether the MAPK pathway is involved in the induction of apoptosis as one of hinokitiol's effects, we measured the changes in ERK1/2 in response to treatment in endometrial cancer cells. As shown in Figure 7

Hinokitiol Regulates the ERK Signaling Pathway
To determine whether the MAPK pathway is involved in the induction of apoptosis as one of hinokitiol's effects, we measured the changes in ERK1/2 in response to treatment in endometrial cancer cells. As shown in Figure 7

Discussion
Accumulating evidence has suggested that the selective induction of cancer cell apoptosis is probably one of the most powerful cancer prevention strategies [9]. Our study revealed that hinokitiol possesses anti-cancer effects with respect to endometrial cancer cells, including Ishikawa, HEC-1A, and KLE cells, via the induction of tumor suppressor protein p53, apoptotic markers (Bax, PARP, and caspase-3), autophagic markers (LC3BII), and ROS levels, while it downregulates cell-cycle-related proteins (cyclin D1 and CDK4 and anti-apoptotic protein Bcl-2 and possibly regulates the ERK signaling pathway. Cell proliferation is achieved through repeated cellular divisions and entry into the cell cycle, where each G0/G, S, and G2/M checkpoint is strictly controlled by cyclin and cyclin-dependent kinases (CDKs) [21]. If the cell cycle of cancer cells loses control of the checkpoints, their ability to proliferate will be affected, thus inhibiting cancer cell proliferation [22]. Wang et al. demonstrated that hinokitiol induced cell-cycle arrest at the G1 phase and significantly downregulated the protein levels of cyclin D1 and cyclin E in a dose-dependent manner in cervical carcinoma HeLa cells [15]. In addition, Chen et al. revealed that β-thujaplicin induced G0/G1 cell-cycle arrest and significantly inhibited cyclin D1, cyclin E, and CDK4 expression in a dose-dependent manner in MCF10DCIS.com breast cancer cells [23]. Our study showed similar results, where hinokitiol treatment for 48 h significantly reduced the expression of cyclin D1 and CDK4 in endometrial cancer cells.
The tumor suppressor p53 is a redox-active transcription factor that maintains genome stability and integrity by regulating diverse cellular functions, including cell-cycle arrest, DNA repair, senescence, and apoptosis [24]. The increase in the p53 level not only inhibits the expression of cyclin during the transition from the G1 to the S phase, but also causes an interaction with anti-apoptotic proteins (Bcl-2 homologous antagonist/killer, Bak; Bcl-2-associated X protein, Bax) that are located in the mitochondria to promote cell apoptosis [24]. We explored the expression of the p53 protein in response to treatment by using Western blotting, and our study showed that hinokitiol prominently induced the expression of p53 phosphorylation in Ishikawa, HEC-1A, and KLE cells. The results of

Discussion
Accumulating evidence has suggested that the selective induction of cancer cell apoptosis is probably one of the most powerful cancer prevention strategies [9]. Our study revealed that hinokitiol possesses anti-cancer effects with respect to endometrial cancer cells, including Ishikawa, HEC-1A, and KLE cells, via the induction of tumor suppressor protein p53, apoptotic markers (Bax, PARP, and caspase-3), autophagic markers (LC3BII), and ROS levels, while it downregulates cell-cycle-related proteins (cyclin D1 and CDK4 and anti-apoptotic protein Bcl-2 and possibly regulates the ERK signaling pathway. Cell proliferation is achieved through repeated cellular divisions and entry into the cell cycle, where each G0/G, S, and G2/M checkpoint is strictly controlled by cyclin and cyclin-dependent kinases (CDKs) [21]. If the cell cycle of cancer cells loses control of the checkpoints, their ability to proliferate will be affected, thus inhibiting cancer cell proliferation [22]. Wang et al. demonstrated that hinokitiol induced cell-cycle arrest at the G1 phase and significantly downregulated the protein levels of cyclin D1 and cyclin E in a dose-dependent manner in cervical carcinoma HeLa cells [15]. In addition, Chen et al. revealed that β-thujaplicin induced G0/G1 cell-cycle arrest and significantly inhibited cyclin D1, cyclin E, and CDK4 expression in a dose-dependent manner in MCF10DCIS.com breast cancer cells [23]. Our study showed similar results, where hinokitiol treatment for 48 h significantly reduced the expression of cyclin D1 and CDK4 in endometrial cancer cells.
The tumor suppressor p53 is a redox-active transcription factor that maintains genome stability and integrity by regulating diverse cellular functions, including cell-cycle arrest, DNA repair, senescence, and apoptosis [24]. The increase in the p53 level not only inhibits the expression of cyclin during the transition from the G1 to the S phase, but also causes an interaction with anti-apoptotic proteins (Bcl-2 homologous antagonist/killer, Bak; Bcl-2associated X protein, Bax) that are located in the mitochondria to promote cell apoptosis [24]. We explored the expression of the p53 protein in response to treatment by using Western blotting, and our study showed that hinokitiol prominently induced the expression of p53 phosphorylation in Ishikawa, HEC-1A, and KLE cells. The results of previous reports are consistent with this observation, as hinokitiol increased the p53 protein level in A549 human lung cancer cells [14]. In addition, an increase in both the p21 and p53 levels was observed in hinokitiol-treated cervical carcinoma HeLa cells [15].
Apoptosis is characterized by cell death in the body through both external and internal pathways; the latter is also known as the mitochondria-dependent pathway [25]. Briefly, overproduction of ROSs downregulates B-cell lymphoma 2 (Bcl-2), while upregulation of Bcl-2-associated X protein (Bax) leads to increased mitochondrial outer membrane permeabilization (MOMP; loss of ∆Ψm), triggers the release of mitochondrial cytochrome C into the cytoplasm, and activates a series of caspases, which, in turn, promote apoptosis [25]. Zhang et al. showed that β-Thujaplicin triggered HepG2 apoptosis and increased cleaved PARP1, cleaved caspase-3, and the Bax/Bcl-2 ratio, which indicated that β-Thujaplicininduced apoptosis is mediated by the mitochondria-dependent pathway [26]. Similarly, our study found that the total number of apoptotic cells largely increased upon the administration of hinokitiol, and simultaneously, the expression of cleaved-PARP, cleavedcaspase-3, and the Bax/Bcl-2 ratio also increased in Ishikawa, HEC-1A, and KLE cells at 48 h, suggesting that the apoptosis induced by hinokitiol was possibly mediated by the mitochondrion-dependent pathway. It's worth mentioning that over-degraded protein may be the reason why the expression of caspase-3 cannot be enhanced with increasing dose, or it may be a non-dominant caspase protein, which requires more experimental verification.
Autophagy is a pivotal step in tissue damage that is regulated in a manner of homeostatic balance between the degradation of damaged organelles and protein aggregates, and the recycling of nutrients [20]. The key biomarkers for autophagic flux are the microtubuleassociated protein light chain 3 (LC3) and p62 (sequestosome-1, SQSTM1). During autophagy, LC3-II, which is converted from LC3-I, is recruited and incorporated into the autophagosome membrane. p62/SQSTM1 acts as a degradation substrate with respect to the autophagic membrane, usually interacts with LC3-II and polyubiquitin, and drives the occurrence of autophagy [27]. Hinokitiol has been shown to promote autophagy in different tissues. A previous study reported that β-Thujaplicin promoted the expression of LC3B-II and decreased the p62 protein level in HepG2 cells, indicating that autophagic events were increased upon β-Thujaplicin treatment [26]. Similarly, our study found that hinokitiol reduced the expression of p62/SQSTM1 and increased the LC3BII/I ratio in Ishikawa, HEC-1A, and KLE cells, indicating that hinokitiol possesses the ability to promote autophagy in endometrial cancer cells. It's worth mentioning that the high expression of LC3B-II protein in the control group suggests that the KLE cells may have been in an autophagy state and cannot be induced anymore.
Excessive ROS can cause damage to cells through mechanisms that involve cell apoptosis, autophagy, and cell arrest [28]. In normal cells, hinokitiol was able to inhibit the H 2 O 2 -induced generation of ROSs, as shown in a study on human corneal epithelial cells, where 100 µmol of hinokitiol significantly suppressed ROS production in human corneal epithelium cells [29]. However, there are distinct results in cancer cells. Recent studies reported that ROS generation is increased in HepG2 cells after exposure to β-Thujaplicin [26]. Several studies have focused on the ROS-mediated apoptosis pathway and explored the effects of natural compounds on the growth of endometrial cancer [30,31]. In the current study, we used H 2 O 2 as a positive control and found that hinokitiol could promote the production of ROSs in the three endometrial cancer cell lines used, and the effect was even greater than that of the positive control group. Thus, we speculate that hinokitiol may inhibit the growth of endometrial cancer through ROS-mediated apoptosis.
The mechanism of action of hinokitiol involves a variety of signaling pathwaysfor example, hinokitiol activates the ERK/MKP-3/proteosome pathway to inhibit the growth of B16F10 melanoma [17]. In addition, hinokitiol activates the AKT/GSK-3β/βcatenin signaling cascade to inhibit the growth of ER-negative MCF10DCIS.com human breast cancer cells [23]. As originally known, the ERK signaling pathway plays a pivotal role in cell survival. A review revealed that DNA damage can promote ERK activation and leads to p53-mediated cellular responses [32], which can even lead to an intrinsic apoptotic pathway, resulting in cell death. However, it is still controversial whether ERK expression is positively or negatively regulated after treatment with natural compounds. Interestingly, in the current study, we observed a phenomenon in which hinokitiol enhanced the expression of phosphorylated ERK1/2 in Ishikawa and HEC-1A cells while reducing it in KLE cells. The results of previous reports are consistent with the observation of increased ERK1/2 phosphorylation in β-Thujaplicin-treated HepG2 cells [26], which suggests that β-Thujaplicin can induce apoptosis via the ERK1/2 pathway.
The advantage of this experiment is to use different types of endometrial cancer cell lines to verify the effect of hinokitiol. In terms of histological classification, Ishikawa belongs to type 1 well-differentiated (G1) EC cells. In addition, HEC-1A and KLE are both type 2 ECs, which are originally established from moderately differentiated (G2) and poorly differentiated (G3) endometrial adenocarcinomas, respectively [5]. On the other hand, we sorted out the following experimental limitations: First, in order to clarify whether hinokitiol can promote apoptosis by promoting ROSs, N-acetylcysteine (a ROS scavenger) should be used to further examine the results of apoptosis. Similarly, U0126 or PD98059 (inhibitors of ERK) should be administered to explore their effects on the expression of apoptosis-related proteins induced by hinokitiol. Finally, we also regret the lack of animal experiments, but due to the limited funds, we could only perform preliminary cancer cell suppression studies and provide a clinical basis for in-depth exploration of hinokitiol in endometrial cancer in the future.

Cell Viability Assays
The influence of hinokitiol on the cell viability of the HEC-1A, Ishikawa, and KLE cell lines was analyzed with an (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA). Briefly, the cells were seeded in 96-well plates (3 × 10 3 per well) and treated with various dosages (1,5,10,25, and 50 µM) of hinokitiol or DMSO (0.05 %) alone as a vehicle control for 48 h. At the indicated time points, the medium was replaced with 100 µL of fresh medium containing 1% serum with 0.5 mg/mL MTT. Following a three-hour incubation at 37 • C, formazan crystals were solubilized with 100 µL of DMSO. The absorbance levels for each sample at 570 nm with a reference wavelength of 630 nm were measured by using a microplate reader (Bio-Tek, Winooski, VT, USA). The data were duplicated at least three times. The average inhibitory concentration (IC50) of hinokitiol was determined by using GraphPad Prism version 9.0 (GraphPad, San Diego, CA, USA).

Western Blotting Analysis
HEC-1A, Ishikawa, and KLE cells were seeded into 10 cm 2 dishes and treated with a gradient of concentrations (1,5,10,25, and 50 µM) of hinokitiol or DMSO (0.05%) alone as a vehicle control for 48 h. After being digested with 0.25% trypsin (w/v), the cells were harvested by centrifugation and washed once with cold phosphate-buffered saline (PBS). Cell lysates were lysed with 100 µL of lysis buffer (mixed with a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland)). Samples containing 20 µg of proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to a polyvinylidene difluoride (PVDF, 0.45 or 0.22 µm, MilliporeSigma, Burlington, MA, USA) membrane (for 100 min at 100 V). The membranes were blocked with 5% bovine serum albumin (BSA, BioShop, Burlington, ON, Canada) for 1 h and were then probed with primary antibodies at 4 • C overnight. Membranes were incubated with the corresponding goat anti-rabbit/mouse antibody IgG (1:10000, Abcam, Cambridge, UK) for 1 h. After reacting with electrochemiluminescence (ECL; Thermo Fisher Scientific) in the dark, chemiluminescence imaging was performed with an eBlot Touch Imager TM (eBlot Photoelectric Technology, Shanghai, China). Densitometric estimations were quantified with the Image J software (National Institutes of Health, NIH, Bethesda, MD, USA). Each experiment was carried out in triplicate, and the results are expressed as means ± SD for each treatment group. All of the raw data from the use of Western blotting are provided in Figure S1 (Supplementary Materials). The primary antibodies, including anti-PARP, anti-Caspase-3, anti-Bax, anti-LC3B, anti-p62/SQSTM1, anti-p-P53, anti-pERK1/2, and anti-ERK1/2, were purchased from Cell Signal Technology (Beverley, MA, USA); Bcl-2 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA); CDK4 and cyclin D1 were purchased from Abcam (Cambridge, UK); anti-horseradish peroxidase (HRP)-conjugated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was purchased from Proteintech (Rosemont, IL, USA). The dilution concentrations of all antibodies used in the Western blotting are provided in Table S1.

Flow Cytometry Analysis
An FITC Annexin V Apoptosis Detection Kit I was purchased from BD Biosciences (Franklin Lakes, NJ, USA). HEC-1A, Ishikawa, and KLE cells were seeded into 6-well plates and treated with a gradient of concentrations (1, 5, 10, 25, and 50 µM) of hinokitiol or DMSO (0.05%) alone as a vehicle control for 48 h. After being digested with 0.25% (w/v) trypsin, all of the cells were harvested by centrifugation and washed twice with cold PBS. The pellets were resuspended in 100 µL of 1× binding buffer and incubated with 2 µL of Annexin V-FITC and 2 µL of PI for 15 min at room temperature in the dark according to the manufacturer's instructions. After the reaction was completed, the 1× binding buffer was replenished to 500 µL in each sample tube. Cell apoptosis was detected within 1 h by using an Attune™ NxT Flow Cytometer (Thermo Fisher Scientific, Waltham, MA, USA). Each experiment was carried out in triplicate, and the results are expressed as means ± SD for each treatment group.

Measurement of Intracellular Reactive Oxygen Species (ROSs)
The general oxidative stress indicator 6-carboxy-2 ,7 -dichlorodihydrofluorescein diacetate (carboxy-H 2 DCFDA) was purchased from Invitrogen™ (Thermo Fisher Scientific, Waltham, MA, USA). To quantitatively assess intracellular ROSs, the cells were treated with different concentrations (1, 5, 10, 25, and 50 µM) of hinokitiol or DMSO (0.05 %) alone as a vehicle control in 6-well plates for 48 h. After being digested with 0.25% (w/v) trypsin, the cells were harvested by centrifugation, washed twice with PBS, and then incubated with 20 µM carboxy-H 2 DCFDA (6-carboxy-2 ,7 -dichlorodihydrofluorescein diacetate) in culture medium without serum at 37 • C in the dark for 45 min. After the reaction was completed, the stained cells were washed once with PBS to remove the residual dye. Except for the positive control group (H 2 O 2 ), for ROSs to be effectively induced, the rest of the samples were kept on ice for later use. For the H 2 O 2 positive control treatment, the medium was refreshed with reduced serum (1%) containing 1 mM H 2 O 2 and incubated for 30 min. ROS levels were assessed with an Attune™ NxT Flow Cytometer (Thermo Fisher Scientific, Waltham, MA, USA) using the FL1 channel with an Ex/Em of~492-495/517-527 nm. Each experiment was carried out in triplicate, and the results are expressed as means ± SD for each treatment group.

Statistical Analysis
The data were analyzed by using SigmaPlot version 12.5 (SoftHome, Taipei, Taiwan, China) or GraphPad Prism version 9.0 (GraphPad, San Diego, CA, USA), and they are expressed as the mean ± standard deviation (SD). Statistical significance was evaluated by using two-tailed Student's t-tests (two groups). A difference between two means was considered statistically significant when p < 0.05 or highly significant when p < 0.001.

Conclusions
In this study, we investigated the antitumor properties of hinokitiol with respect to human endometrial cancer cells and found that hinokitiol regulates the protein expression of p53 and CDK4/cyclin D1, increases the ROS level, and activates apoptosis and autophagy through the ERK1/2 pathway in Ishikawa, HEC-1A, and KLE cells ( Figure 8). Overall, this study might provide fundamental knowledge for understanding the antitumor activity of hinokitiol in endometrial cancer cells, and it is hoped that hinokitiol can potentially be used as an alternative drug for endometrial cancer in the future, pending further studies.
as a vehicle control in 6-well plates for 48 h. After being digested with 0.25% (w/v) trypsin, the cells were harvested by centrifugation, washed twice with PBS, and then incubated with 20 μM carboxy-H2DCFDA (6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate) in culture medium without serum at 37 °C in the dark for 45 min. After the reaction was completed, the stained cells were washed once with PBS to remove the residual dye. Except for the positive control group (H2O2), for ROSs to be effectively induced, the rest of the samples were kept on ice for later use. For the H2O2 positive control treatment, the medium was refreshed with reduced serum (1%) containing 1 mM H2O2 and incubated for 30 min. ROS levels were assessed with an Attune™ NxT Flow Cytometer (Thermo Fisher Scientific, Waltham, MA, USA) using the FL1 channel with an Ex/Em of ~492-495/517-527 nm. Each experiment was carried out in triplicate, and the results are expressed as means ± SD for each treatment group.

Statistical Analysis
The data were analyzed by using SigmaPlot version 12.5 (SoftHome, Taipei, Taiwan, China) or GraphPad Prism version 9.0 (GraphPad, San Diego, CA, USA), and they are expressed as the mean ± standard deviation (SD). Statistical significance was evaluated by using two-tailed Student's t-tests (two groups). A difference between two means was considered statistically significant when p < 0.05 or highly significant when p < 0.001.

Conclusions
In this study, we investigated the antitumor properties of hinokitiol with respect to human endometrial cancer cells and found that hinokitiol regulates the protein expression of p53 and CDK4/cyclin D1, increases the ROS level, and activates apoptosis and autophagy through the ERK1/2 pathway in Ishikawa, HEC-1A, and KLE cells ( Figure 8). Overall, this study might provide fundamental knowledge for understanding the antitumor activity of hinokitiol in endometrial cancer cells, and it is hoped that hinokitiol can potentially be used as an alternative drug for endometrial cancer in the future, pending further studies. Figure 8. A schematic representation of the hypothetical mechanisms of the role of hinokitiol in suppressing endometrial cancer. The upward arrow represents "enhancement"; the downward arrow represents "inhibition"; the dashed arrow represents "the possible pathway induced by hinokitiol"; The unfilled arrow represents the "direction of the mechanism". The upward arrow represents "enhancement"; the downward arrow represents "inhibition"; the dashed arrow represents "the possible pathway induced by hinokitiol"; The unfilled arrow represents the "direction of the mechanism".
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/ijms22158268/s1, Table S1: The dilution concentrations of all antibodies used in Western blotting. Figure S1: The raw data from Western blotting.