Chrysophanol Regulates Cell Death, Metastasis, and Reactive Oxygen Species Production in Oral Cancer Cell Lines

Background Oral cancer belongs to the class of head and neck cancers and can be life threatening if not diagnosed and treated early. Activation of cell death via apoptosis or reactive oxygen species (ROS) accumulation and inhibition of cell cycle progression, migration, and epithelial-to-mesenchymal transition (EMT) may be a good strategy to arrest the development of oral cancer. In this study, we analyzed the possible action of chrysophanol isolated from the rhizomes of Rheum palmatum on the oral cancer cell lines FaDu (human pharynx squamous cell carcinoma) and SAS (human tongue squamous carcinoma) by investigating whether chrysophanol could influence cell death. Method Cell viability was measured by using the MTT assay. For the detection of apoptosis, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and subG1 population analysis were used. We also examined cell cycle progression and ROS levels by flow cytometry. Additionally, the expression of p53, p21, procaspase 3, cyclin D1, CDK4, cdc2, CDK2, E-cadherin, vimentin, and PCNA was evaluated by western blotting. Conclusion Chrysophanol has an anticancer effect on FaDu and SAS cell lines. There is an increase in subG1 accumulation, ROS production, and cell cycle G1 arrest after treatment with chrysophanol. On the other hand, chrysophanol inhibited cell migration/metastasis and EMT. We proposed that chrysophanol may be a good candidate compound on oral cancer treatment in the further.


Introduction
Recently, global cancer statistics sourced from GLOBOCAN 2018 estimating the incidence and mortality of 36 cancers in 185 countries revealed that the incidence of Head and Neck Squamous Cell Carcinoma (HNSCC) was 354.9 thousand new cases with a mortality rate of approximately 50% [1]. In comparison, statistics gathered in 2015 estimated the incidence to be 300 thousands new cases with 48% mortality [2], indicating that both incidence and mortality rate are still rising. HNSCC is derived from a mutation or abnormality in the squamous cell lining of the oral cavity, oropharynx, larynx, or hypopharynx [3], and factors including alcoholic consumption [4], smoking [5], and human papillomavirus (HPV) infection in nonsmokers [6] are known to put people at risk. Tumor recurrence and metastasis of HNSCC may lead to a poor prognosis [7]. e development and progression of HNSCC correlates with cells having several characteristic including limitless replicative potential, genetic instability, metabolic change, self-sufficiency in growth signals, insensitivity to antigrowth signals, ability to avoid cell death, angiogenesis initiation, and ability to invade and metastasize [4,8,9]. However, HNSCC are diagnosed on the basis of TNM staging systems, and clinical treatment modalities, including surgery, chemotherapy, radiotherapy, and developing immunotherapy, are then applied [10]. Since homogeneous treatments based on the TNM staging for different HNSCC tumors in clinical practice, the high mortality rate of the patients is still a limitation [11]. At present, improved technologies and refined algorithms are available to analyze databases of HNSCC profiles, predict cells that may undergo metastasis, and detect tumors in a single cell, revealing factors that correlate with tumor progression and metastasis. On the other hand, it is critical to discover candidate compounds to alleviate carcinogenesis.
is study investigated the pharmacological effects and mechanisms of chrysophanol in HNSCC cell lines, including SAS and FaDu. e results showed that chrysophanol prevents carcinogenesis by decreasing cell proliferation and metastasis, as well as increasing cell apoptosis. We propose that chrysophanol may be a good candidate compound for HNSCC treatment in the future. Anti-cyclin A2, E-cadherin, cyclin E1, cyclin D1, CDK4, cdc2, and CDK2 antibodies were purchased from Cell Signaling (MA, USA). e anti-EpCAM antibody and Apoptosis/Necrosis Assay kit were purchased from Abcam (UK). Anti-vimentin and PCNA were provided from BioLegend (CA, USA) and ABclonal (MA, USA), respectively. Anti-β actin antibody was purchased from Santa Cruz Biotechnology (TX, USA).

Cell
Culture. FaDu (human pharynx squamous cell carcinoma) and SAS (human tongue squamous carcinoma) cell lines were obtained from ATCC and National Defense Medical Center, respectively. Cells were analyzed for mycoplasma and tested negative. Cell lines were cultured in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum and 1% penicillin/streptomycin and incubated in a 5% CO 2 atmosphere at 37°C.

Cell Viability and Cytotoxicity Assay and IC50 Value
Determination. Cell viability and cytotoxicity were assessed by using the MTT assay. Cells (1 × 10 6 ) were seeded in 96well plates with the indicated concentrations of chrysophanol and then treated for 24 hours. To the cells was added 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, ermoFisher Scientific, MA, USA) and incubated at 37°C for 2 hours. Finally, formazan was solubilized with DMSO. e concentration was determined from the optical density at 570 nm. Absorbance was measured with a TECAN infinite M200 PRO (Switzerland). According to a previous study [19], measurements were performed, and the concentration required for 50% inhibition of viability (IC50) was determined graphically from a standard curve drawn by plotting the log of the drug concentration on the X axis and % cell growth inhibition or % cytotoxicity on the Y axis. Additionally, the IC50 values were estimated as the drug concentration at the 50% position on the Y axis. e relationship should be sigmoidal, with the log of the drug concentration on the X axis and "response/ measurement" on the Y axis. e Prism website has some good guidelines for this. We used GraphPad Prism8 software to determine the IC50 value. Moreover, IC50 values were calculated using the nonlinear regression program origin. e average of two measurements (in a duplicate manner) was taken in determination. e IC50 value was derived using curve fitting methods with GraphPad Prism8 statistical software.

Protein Extraction and Western
Blotting. Protein was extracted from cells and subjected to western blotting according to our previous study [19]. e intensities of the reactive bands were analyzed by using Image J.

Apoptosis Staining.
Apoptosis-associated DNA fragmentation was visualized by using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) apoptosis detection kit (Roche), according to the manufacturer's instructions. e cells were finally counterstained with DAPI and analyzed by the Leica DM6000 Microscope (Germany).

Wound Healing Assay.
Cells were plated at an initial density of 1 × 10 5 cells/ml to form a monolayer. During the last 4 h, cytosineβ-D-arabinofuranoside (Sigma) was added at a final concentration of 4 μm to prevent cell proliferation. Cells were washed twice with PBS and incubated with medium containing serum at various concentrations (0, 30, and 70 μm) of chrysophanol for 0, 4, 8, and 12 h. en, cells were wounded by scraping with a pipette tip to make an approximately 400-μm gap in the cell monolayer. Cell migration was observed at different time intervals and photographed at 3 marked locations on each dish using a phasecontrast microscope. e number of migrated cells was counted and averaged. All experiments were carried out in triplicate and repeated at least 3 times.

Statistical
Analysis. Data were expressed as the mean-± standard error of the mean (SEM), and statistical comparisons were calculated by one-way or two-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test. A value of p < 0.05 was considered to indicate the statistical significance.

Chrysophanol Causes Cell Death in HNSCC.
To investigate the cytotoxic effect of chrysophanol on HNSCC, the cell viability of FaDu and SAS was analyzed after treatment with a concentration series of chrysophanol (0, 15, 30, 70, and 100 μm) for 24 hours (Figure 1). e IC50 value at 24 h was 9.64 ± 1.33 μm and 12.60 ± 2.13 μm in FaDu and SAS, respectively. Results showed that chrysophanol (15-100 μm) inhibited cell viability in a concentration-dependent manner (Figures 1(a) and 1(b)). To characterize chrysophanol-induced cell death, the changes in cellular morphology were determined. Phase-contrast microscopy showed that cells treated with chrysophanol (30, 60, and 100 μm) for 24 h underwent marked apoptotic changes, including the formation of membrane blebs and apoptotic bodies (Figure 1(c)). Interestingly, cell viability was decreased dramatically after treatment with 30 μm chrysophanol and reached the threshold at 100 μm ( Figure 1). To further determine the apoptotic effect of chrysophanol on FaDu and SAS, the subG1 population and TUNEL were analyzed ( Figure 2).

Chrysophanol Causes Apoptosis in HNSCC.
It is well known that the process of programmed cell death, or apoptosis, is generally characterized by distinct methods, including morphological changes, subG1 population analysis, caspase-3 expression analysis, and TUNEL staining [20]. Results showed that chrysophanol (30 and 70 μm) caused an increase in the subG1 phase in FaDu and SAS (Figures 2(a) and 2(b)), which is an indicator of apoptosis [21]. Additionally, a decrease in expression of procaspase 3 was detected in chrysophanol (30 and 70 μm) treated FaDu and SAS (Figure 2(c) and supplementary Figure 1), similar to the previous findings in breast and hepatic cancer cell lines [22,23]. DNA fragmentation was also observed in the presence of chrysophanol (30 and 70 μm) treatment ( Figure 2(d)). Taken together, these findings suggest that chrysophanol triggers FaDu and SAS to undergo apoptosis in a dose-dependent manner.

Influence of Chrysophanol on ROS Production and Cell
Cycle Regulation. Kim et al. demonstrated that p53-p21regulated cancer cell invasion and apoptosis are involved in reactive oxygen species (ROS) production [24]. In this study, to further determine the pharmacological mechanism of chrysophanol acting on FaDu and SAS, the level of ROS and the expression of p53 and p21 were measured (Figures 3(a) and 3(b)). e results showed that chrysophanol (30 and 70 μm) caused an increase in ROS production in FaDu and SAS (Figure 3(a)); however, the expression of p53 or p21 was increased in FaDu but decreased in SAS (Figure 3(b) and supplementary Figure 2). We suggest that the conflicting results are based on the different genetic backgrounds. e state of p53 in FaDu was a mutation of CGG to CTG that occurred at codon 248 [25,26], and SAS was shown to express the wild-type p53 genotype [27,28]. e downstream gene p21 was upregulated following p53 activation and mediated cell cycle progression [29]. Moreover, the expression of cyclinD1, CDK4, cdc2, and CDK2 was downregulated under chrysophanol (30 and 70 μm) treatment in FaDu and SAS (Figure 3(c) and supplementary Figure 3). Chrysophanol also arrested the cell cycle at G1 (Figure 3(d)). To further confirm whether chrysophanol caused cell death via ROS production, the results demonstrated that NAC alleviated cell death in 70 μm chrysophanol-treated FaDu and SAS (Figure 3(e)). erefore, the data showed that chrysophanol triggered cell to death via ROS production and G1 phase arrest.

Chrysophanol Inhibited Cell Migration/Metastasis via an EMT-Dependent but ROS-Independent
Pathway. Accumulating evidence demonstrates that EMT plays a critical role in oral cancer invasion, migration, and metastasis [30,31]. To study the effect of chrysophanol on cell   Evidence-Based Complementary and Alternative Medicine migration in FaDu and SAS, the wound healing assay was used. Results showed that chrysophanol strongly inhibited cell migration; however, the inhibitory effect was not reversed after NAC treatment (Figures 4(a) and 4(b)). e data demonstrated that chrysophanol has an antimigration effect via a ROS-independent pathway. Furthermore, loss of E-cadherin is a positive signal for metastasis [32], and vimentin upregulation has been reported in correlation with metastasis and may be a poor prognosis indicator in oral cancer [33]. Results showed that E-cadherin increased but vimentin decreased after 70 μm chrysophanol treatment (Figure 4(c) and supplementary Figures 4(a) and 4(b)). Additionally, proliferating cell nuclear antigen (PCNA) can be identified as a proliferation marker in tumor formation [34]. Conflicting results showed that chrysophanol decreased the PCNA expression in SAS (Figure 4 reported an interesting finding that FaDu has increased intracellular stiffness and an epithelial-type; on the other hand, SAS has no significant difference in intracellular stiffness and a mesenchymal-type [35]. Moreover, some studies reported that the inverse correlation between intracellular stiffness, and invasiveness was revealed in oral cancer cell lines [36,37]. To further make a conclusion that chrysophanol is a novel therapeutic agent for treating oral cancer. We used cisplatin [38] as a positive control to  Figure 5(c)). On the other hand, to elicit substantial inhibition of cell viability (Figure 1), migration is considered a more sensitive function; therefore, usually lower concentrations, subtoxic or subantiproliferative doses,   erefore, we suggest that chrysophanol and cisplatin may alleviate cell migration/ metastasis via an EMT-dependent and -independent pathway, respectively.
In conclusion, chrysophanol significantly induced cell death via apoptosis by inducing ROS accumulation. On the other hand, chrysophanol caused cell cycle arrest and inhibited migration/metastasis via an EMT-dependent but ROS-independent pathway ( Figure 5). Our pharmacological findings support further development of chrysophanol as a novel therapeutic agent for treating oral cancer.

Data Availability
e original data used to support the findings of this study are included within the article.    Taipei City  (TCRD-TPE-107-RT-1).

Supplementary Materials
Supplementary Figure 1: quantification of western blots represented in Figure 2