Chrysophanol-Induced Autophagy Disrupts Apoptosis via the PI3K/Akt/mTOR Pathway in Oral Squamous Cell Carcinoma Cells

Background and Objectives: Natural products are necessary sources for drug discovery and have contributed to cancer chemotherapy over the past few decades. Furthermore, substances derived from plants have fewer side effects. Chrysophanol is an anthraquinone derivative that is isolated from rhubarb. Although the anticancer effect of chrysophanol on several cancer cells has been reported, studies on the antitumor effect of chrysophanol on oral squamous-cell carcinoma (OSCC) cells have yet to be elucidated. Therefore, in this study, we investigated the anticancer effect of chrysophanol on OSCC cells (CAL-27 and Ca9-22) via apoptosis and autophagy, among the cell death pathways. Results: It was found that chrysophanol inhibited the growth and viability of CAL-27 and Ca9-22 and induced apoptosis through the intrinsic pathway. It was also found that chrysophanol activates autophagy-related factors (ATG5, beclin-1, and P62/SQSTM1) and LC3B conversion. That is, chrysophanol activated both apoptosis and autophagy. Here, we focused on the roles of chrysophanol-induced apoptosis and the autophagy pathway. When the autophagy inhibitor 3-MA and PI3K/Akt inhibitor were used to inhibit the autophagy induced by chrysophanol, it was confirmed that the rate of apoptosis significantly increased. Therefore, we confirmed that chrysophanol induces apoptosis and autophagy at the same time, and the induced autophagy plays a role in interfering with apoptosis processes. Conclusions: Therefore, the potential of chrysophanol as an excellent anticancer agent in OSCC was confirmed via this study. Furthermore, the combined treatment of drugs that can inhibit chrysophanol-induced autophagy is expected to have a tremendous synergistic effect in overcoming oral cancer.


Introduction
Oral cancer comprises malignant neoplasms that accrue from the tongue, lip, alveolar, oral cavity, oropharynx, and nasopharynx [1]. Globally, the number of newly diagnosed oral cancer patients is around 360,000, and deaths numbered 180,000 in 2018 [2]. Oral cancer is commonly defined as an OSCC, and more than 90% are squamous-cell carcinomas present in the mucous membranes [3]. Chemotherapy, radiation therapy, and surgery are the generally used methods for cancer management. Surgery is primarily performed for oral cancer treatment, but in an event of recurrence of cancer after surgery, the prognosis is poor [4]. Postoperative adjuvant chemotherapy might be better for preventing relapses in patients than performing surgeries alone with respect to esophageal cancer [5]. Similarly, USA). LC3B and p62 were purchased from Sigma Aldrich (St. Louis, MO, USA); and Akt, p-Akt, Bax, Bcl-2, and β-actin were purchased in Santa Cruz (CA, USA). Secondary antibodies of mouse anti-rabbit IgG and rabbit anti-mouse IgG antibodies were obtained from Enzo Biochem (Farmingdale, NY, USA). The TOPscrip tTM cDNA synthesis kit and TO-Preal TM qPCR 2× PreMIX (SYBR Green with Low ROX) were procured from Enzynomics (Dajeon, Republic of Korea). PCR primers (ATG5, Beclin-1, LC3B, and GAPDH) were obtained from macrogen (Seoul, Republic of Korea).

Cell Culture
CAL-27 and Ca9-22 cells are frequently used cell lines in the field of human oral squamous-cell carcinoma (OSCC) and were obtained from ATCC (Rockveile, MD, USA). CAL-27 and Ca9-22 were cultivated in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) (GE-Healthcare, Chicago, IL, USA) and 1% penicillin-streptomycin at 37 °C in 5% humidified CO₂. The cells were grown in culture media at 70% confluence in the culture dishes.

Cell Proliferation Assay
The MTT assay was conducted to determine the cytotoxicity effects of chrysophanol. In a 96-well plate, both cell lines were grown at 1 × 10⁴ cells/well and treated with various doses of chrysophanol (0-300 μM) for 24 and 48 h. After the treatment with chrysophanol, the media were removed, and 0.05 mg/mL of an MTT solution was added to the media and incubated until the formation of formazan crystals at 37 °C. The formed formazan crystals were liquefied with DMSO, and absorbance was measured at 570 nm using the SpectraMax iD3 (BioTek, Winooski, VT, USA) and calculated as a percentage. Several studies have found that chrysophanol increases apoptosis in various cancer cells, but the mechanisms of apoptosis and autophagy induced by chrysophanol in oral cancer cells and their interrelationships have not yet been studied. In this study, we investigated the effects of chrysophanol on apoptosis and autophagy mechanisms, and furthermore, whether autophagy plays a role in protecting cells in the process of chrysophanol-induced apoptosis or autophagy as the primary response of apoptosis relative to stress stimuli induced by chrysophanol.

Cell Culture
CAL-27 and Ca9-22 cells are frequently used cell lines in the field of human oral squamous-cell carcinoma (OSCC) and were obtained from ATCC (Rockveile, MD, USA). CAL-27 and Ca9-22 were cultivated in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) (GE-Healthcare, Chicago, IL, USA) and 1% penicillinstreptomycin at 37 • C in 5% humidified CO 2 . The cells were grown in culture media at 70% confluence in the culture dishes.

Cell Proliferation Assay
The MTT assay was conducted to determine the cytotoxicity effects of chrysophanol. In a 96-well plate, both cell lines were grown at 1 × 10 4 cells/well and treated with various doses of chrysophanol (0-300 µM) for 24 and 48 h. After the treatment with chrysophanol, the media were removed, and 0.05 mg/mL of an MTT solution was added to the media and incubated until the formation of formazan crystals at 37 • C. The formed formazan crystals were liquefied with DMSO, and absorbance was measured at 570 nm using the SpectraMax iD3 (BioTek, Winooski, VT, USA) and calculated as a percentage.

Colony-Formation Assay
On each well of a 6-well plate, cells were seeded and treated with different doses of chrysophanol for seven days. Methanol at 100% was used to fix cell colonies, and they were dyed with a 1% crystal violet solution for 10 min. Next, the colonies were washed with distilled water three times and dried. The number of colonies was counted using an optical microscope and calculated as a percentage.

Fluorescence Images
To obtain fluorescence images using Hoechst 33342, JC-1, MDC, and AO, cells (1 × 10 4 ) were seeded in a Greiner bio-one 96-well plate (Greiner, Kremsmünster, Austria) and cultured for 24 h until the cells adhered to the well and stabilized. Then, chrysophanol was added to the medium according to the determined concentration, applied to the cells, and cultured for 24 h. Hoechst 33342 was used to observe nuclear morphological changes. Cells were fixed with 4% paraformaldehyde 10 min, and 1 µg/mL of a Hoechst 33342 solution stained the nuclei of the cells at 37 • C. JC-1 was used for verifying the change of mitochondria membrane potential (∆Ψm). The JC-1 dye (2 µg/mL) stained for 30 min at 37 • C. To identify acidic vacuoles, an acridine orange (AO) agent (1 µg/mL) was used to stain the acidic vesicular organelles for 5 min. For autophagic fluorescence observation, the final concentration of 50 µM of the monodansylcadaverine (MDC) solution was stained with autophagic vacuoles for 30 min. After staining, cells were washed 3 times with PBS and mounted using glycerol. Lionheart FX Automated Microscope (BioTek, Winooski, VT, USA) was used to observe changes in cellular fluorescence images. Cells were observed and photographed at ×200 magnification.

Gene Expression Analysis by Real-Time PCR
Cells were seeded on a six-well plate at a density of 2 × 10 5 cells per well and incubated for 24 h to allow the cells to adhere and stabilize in the culture dish. Then, chrysophanol at a defined concentration was applied to the cells, cultured for 24 h, and then harvested. The total RNA was prepared using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA). The total RNA concentration was determined with a microplate spectrophotometer using the SpectraMax iD3 micro reader (BioTek, Winousk, VT, USA). cDNA was synthesized using a total RNA (2 µg) with a TOPscript TM cDNA synthesis kit (Enzynomics, Dajeeon, Republic of Korea) by following the manual. Subsequently, quantitative real-time PCR was executed using the TOPreal TM qPCR 2× PreMIX (SYBR Green with Low ROX) (Enzynomics, Dajeeon, Republic of Korea) with QuantStudio 1 (Applied Biosystems, Foster City, CA, USA). The relative mRNA levels were normalized using GAPDH as a housekeeping gene. The PCR primer sequences are shown in Table 1.

Western Blot Analysis
Cells (1 × 10 6 ) were seeded on a 100 mm culture dish and incubated for 24 h to allow the cells to adhere and stabilize in the culture dish. Then, chrysophanol at a defined concentration was applied to the cells, cultured for 24 h, and then harvested. Cells were lysed in a RIPA (radioimmunoprecipitation assay) buffer (Cell signaling, Danvers, MA, USA), which was mixed with 2 mM PMSF and a 10 µL/mL protein inhibitor cocktail for 2 h. The samples were centrifuged at 13,200 rpm for 30 min. The Bradford protein assay was used to quantify the collected proteins, and 20 µg of protein was used to make loading samples. Samples were loaded based on molecular weights by gel-electrophoresis (SDS-PAGE) with 10% and 12.5% gels at 80 V for 2 h, and then the loaded gels were transferred onto a polyvinylidene difluoride (PVDF) membrane at 20 V for 16 h. The PVDF membrane was incubated with the appropriate primary antibodies blended at 1:1000 in a 5% non-fat dry milk solution overnight at 4 • C. Sequentially, the membrane was washed 5 times for 1 h with a Tris-NaCl-EDT (TNE) buffer and incubated with secondary antibodies at room temperature. Next, the membrane was washed 5 times for 1 h with a TNE buffer again, and the detection of protein was performed using a super signal West Femto (Pierce, Illinois, USA). The protein expression was detected with ImageQuant LAS 500 chemiluminescence (GE Healthcare, Chicago, IL, USA).

Immunofluorescence
The cells (2 × 10 4 ) were placed in an 8-well Lab-Tek II Chambered Slide (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 24 h to allow the cells to adhere and stabilize in the chambered slide. After chrysophanol treatments, the cells were washed with PBS and stained with 100 nM Mitotracker Deep Red at 37 • C for 1 h. Then, cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100 solved in PBS for 10 min. The cells were blocked with 1% BSA-PBS for 1 h and incubated with cytochrome c antibody (1:100) in 1% BSA-PBS with 0.1% Tween 20 at 37 • C for 2 h. Next, the cells were washed with PBS five times for 10 min and then incubated with secondary antibodies conjugated to Alexa Fluor (Alexa 488) (1:100) in 1% BSA-PBS for 2 h. Nuclear staining used the Prolong TM Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA, USA). Finally, fluorescent images were observed and analyzed using a Zeiss LSM 700 laser-scanning confocal microscope (Cal Zeiss, Göettingen, Germany).

Statistical Analysis
GraphPad Prism version 5.0 (San Diego, CA, USA) was used for the statistical analysis. The one-way and two-way ANOVA were used to calculate significance. Significance was assumed to be reached at *** p < 0.001, ** p < 0.01, and * p < 0.05. The graph bar express mean ± SD.

Chrysophanol Reduced Cell Viability and Proliferation in OSCC Cells
CAL-27 and Ca9-22 cells were treated with various concentrations of chrysophanol (0-300 µM) for 24 and 48 h, and then an MTT assay was conducted for both cells to confirm the cytotoxic effect of chrysophanol. After treatment with chrysophanol, the cell's viability was reduced in a dose-dependent manner (Figure 2A , and then the viability of cells was measured using the MTT assay. (C-F)) A colony-formation assay was performed to examine cell proliferation. Cells were treated with chrysophanol for seven days and stained with a 1% crystal violet solution. The number of colonies was converted into a percentage and shown in a histogram. The results are exhibited as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001).

Chrysophanol-Induced Apoptosis via the Caspase Activation in OSCC Cells
During apoptosis, cell morphology was altered by cell shrinkage, chromatin condensation, and apoptotic bodies [40]. Hoechst staining was conducted to observe the morphology changes due to chrysophanol in CAL-27 and Ca9-22 cells. Chrysophanol-treated cells were confirmed to have blue fluorescence, which indicates fragmented and condensed nuclei. Thus, the chrysophanol-treated cells had more morphological changes compared to the control ( Figure 3A,B). The JC-1 dye was used as a practical tool to , and then the viability of cells was measured using the MTT assay. (C-F)) A colony-formation assay was performed to examine cell proliferation. Cells were treated with chrysophanol for seven days and stained with a 1% crystal violet solution. The number of colonies was converted into a percentage and shown in a histogram. The results are exhibited as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001).

Chrysophanol-Induced Apoptosis via the Caspase Activation in OSCC Cells
During apoptosis, cell morphology was altered by cell shrinkage, chromatin condensation, and apoptotic bodies [40]. Hoechst staining was conducted to observe the morphology changes due to chrysophanol in CAL-27 and Ca9-22 cells. Chrysophanol-treated cells were confirmed to have blue fluorescence, which indicates fragmented and condensed nuclei. Thus, the chrysophanol-treated cells had more morphological changes compared to the control ( Figure 3A,B). The JC-1 dye was used as a practical tool to measure the mitochon-drial membrane potential (∆ψM). Green fluorescence is an indicator of monomers, and red fluorescence is an indicator of J-aggregates [41]. As a result of JC-1 staining, chrysophanoltreated cells had higher expression in terms of red fluorescence compared to the control ( Figure 3C,D). To analyze the signaling molecules closely related to apoptosis, Western blot analysis was performed for CAL-27 and Ca9-22 cells. Both cell types were treated with chrysophanol and incubated for 24 h. Protein expression levels that can indicate apoptosis, such as Bax, Bcl-2, caspase-3, caspace-7, and poly (ADP-ribose) polymerase (PARP), were then measured by a Western blot analysis in both cell types. Chrysophanol-treated cells showed caspase-3 and -7 activation, cleaved caspase 3, and PARP upregulation; and antiapoptotic proteins and Bcl-2 were downregulated ( Figure 3E). The relative protein ratios of PARP/cleaved PARP and Bax/Bcl-2 increased in a dose-dependent manner ( Figure 3F,G). These results indicate that chrysophanol treatments significantly increase apoptosis via the caspase activation in OSCC cells.

Chrysophanol-Induced Autophagy in OSCC Cells
Acidic vesicular organelle (AVO) formations have autophagic characteristics [42]. For the detection of AVOs, vital staining with acridine orange (AO) was examined in CAL-27 and Ca9-22 cells by fluorescence microscopy. The control cells presented mostly green fluorescence, indicating the absence of AVOs. Chrysophanol-treated cells had more red fluorescence and minimal green fluorescence. Mature autophagic vacuoles, such as autolysosomes, stained with monodansylcadaverine (MDC), were observed as distinct blue dots within the cytoplasm or perinuclear regions [11]. To detect the chrysophanol effects on the formation of mature autophagic vacuoles, cells were stained with MDC and then observed using a fluorescence microscope. In chrysophanol-treated cells, autophagic vacuole formation increased in quantity and size compared with the control (Figure 4A,B). Next, the expression levels of proteins that indicate autophagy, such as ATG5, beclin-1, and LC3B, were measured by a Western blot analysis. Cells were treated with chrysophanol for 24 h. The expression levels of beclin-1 and ATG5 were upregulated and converted LC3B-I to LC3B-II, which increased dose-dependently in CAL-27 and Ca9-22 cells ( Figure 4C,D). The expression level of mRNA (ATG5, p62/SQSTMI, and MAP1LC3B) was examined using real-time PCR. ATG5, p62/SQSTMI, and MAP1LC3B mRNA levels were dose-dependently upregulated in both cell types. These results revealed that autophagy-related molecules are regulated by chrysophanol in both cells ( Figure 4E-G). Therefore, these experiments provided evidence that chrysophanol treatments cause autophagy in OSCC cells.

3-Methyladenine (3-MA)
, an autophagy inhibitor [43], was used to investigate the effects of autophagy and apoptosis in CAL-27 and Ca9-22 cells. Both cells were treated with 2 mM 3-MA in advance and chrysophanol in the presence or absence of 3-MA. Cells with a single treatment of chrysophanol reflected a higher rate of cell viability than a combination of chrysophanol and 3-MA ( Figure 5A,B). Western blot analysis was performed to determine the protein expression levels. The autophagy-related protein beclin-1 expression and the LC3B conversion level were reduced in the 3-MA combination group ( Figure 5C). Chrysophanol treatments with 3-MA induced the expression of apoptosis-related protein caspase-3 activation and cleaved PARP, and the ratios of cleaved caspase3/caspase 3 and cleaved PARP/PARP improved dose dependently ( Figure 5D-G). When the cells were undergoing apoptosis, the release of cytochrome c activated the caspase cascade [44]. Cytochrome c was shown by immunofluorescence, and it was observed more in co-treatments than in a single treatment. Inhibited autophagy induced the eruption of cytochrome c. (Figure 5H,I). The autophagic process was mediated by anti-apoptotic signals. The results showed that chrysophanol-induced autophagy interrupted apoptosis in OSCC cells.    The expression levels of autophagy-related protein ATG5, beclin-1, LC3B-Ⅰ/LC3B-Ⅱ, and p62 were examined using a Western blot analysis, and the density of protein LC3B-Ⅱ/LC3B-Ⅰratios is shown in the graph. β-actin was used to present a loading control. (E-G) Autophagy-related mRNA expression levels, such as those of ATG5, beclin-1, p62/SQSTM1, and MAP1LC3B, were identified using a realtime PCR. GAPDH was used to present a loading control. Data are expressed as mean ± SD (* p < 0.05).

3-Methyladenine (3-MA)
, an autophagy inhibitor [43], was used to investigate the effects of autophagy and apoptosis in CAL-27 and Ca9-22 cells. Both cells were treated with 2 mM 3-MA in advance and chrysophanol in the presence or absence of 3-MA. Cells with a single treatment of chrysophanol reflected a higher rate of cell viability than a combination of chrysophanol and 3-MA ( Figure 5A,B). Western blot analysis was performed The expression levels of autophagy-related protein ATG5, beclin-1, LC3B-I/LC3B-II, and p62 were examined using a Western blot analysis, and the density of protein LC3B-II/LC3B-I ratios is shown in the graph. β-actin was used to present a loading control. (E-G) Autophagy-related mRNA expression levels, such as those of ATG5, beclin-1, p62/SQSTM1, and MAP1LC3B, were identified using a realtime PCR. GAPDH was used to present a loading control. Data are expressed as mean ± SD (* p < 0.05).  The protein expression levels of autophagy-associated proteins (ATG5, beclin-1, and LC3B) and apoptosis-associated proteins (Bax, Bcl-2, Caspase -3, and PARP) were detected by using a Western blot analysis. β-actin was used to present a loading control. (E-G) Bax/Bcl-2, cleaved caspase -3/caspase -3, and cleaved PARP/PARP ratios were calculated by using the Western blot band density. (H,I) Chrysophanol (0, 150 µM) was treated with both cells in the presence or absence 3-MA, and then the revelation of cytochrome c was visualized by immunofluorescence analyses. The results are exhibited as mean ± SD (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Chrysophanol Produced the Akt/mTOR Signaling Pathway in OSCC Cells
Chrysophanol dose-dependently prompted the phosphorylation of Akt in both cells. The protein expression levels of p-Akt and p-mTOR dose-dependently increased in both cells ( Figure 6A,B). The PI3K inhibitor, LY294002, was used as a tool to measure PI3K/Akt/ mTOR signaling pathways. The cells were pre-treated in the presence or absence of 20 µM of LY294002 for 2 h and then treated with 150 µM of chrysophanol for 24 h. LY294002 treatment cells reflected the downregulation of p-Akt and p-mTOR protein expression ( Figure 7A-C). LY294002 reduced the protein expression levels of beclin-1 and decreased the conversion of LC3B-I to LC3B-II. In contrast, inhibiting the PI3K/Akt pathway induced a caspase cascade, improving Bax and activating caspase-3 and PARP while diminishing Bcl-2. Thus, the cells that were treated with LY294002, and chrysophanol expressed more proteins that were associated with activating apoptosis and autophagy than a single treatment of chrysophanol ( Figure 7D-G). As a result of staining with the JC-1 dye, the cells that were treated with LY294002 and chrysophanol had a lower ∆ψM than single-treatment cells ( Figure 7H,I).

Chrysophanol Produced the Akt/mTOR Signaling Pathway in OSCC Cells
Chrysophanol dose-dependently prompted the phosphorylation of Akt in both cells. The protein expression levels of p-Akt and p-mTOR dose-dependently increased in both cells ( Figure 6A,B). The PI3K inhibitor, LY294002, was used as a tool to measure PI3K/Akt/mTOR signaling pathways. The cells were pre-treated in the presence or absence of 20 μM of LY294002 for 2 h and then treated with 150 μM of chrysophanol for 24 h. LY294002 treatment cells reflected the downregulation of p-Akt and p-mTOR protein expression ( Figure 7A-C). LY294002 reduced the protein expression levels of beclin-1 and decreased the conversion of LC3B-Ⅰto LC3B-Ⅱ. In contrast, inhibiting the PI3K/Akt pathway induced a caspase cascade, improving Bax and activating caspase-3 and PARP while diminishing Bcl-2. Thus, the cells that were treated with LY294002, and chrysophanol expressed more proteins that were associated with activating apoptosis and autophagy than a single treatment of chrysophanol ( Figure 7D-G). As a result of staining with the JC-1 dye, the cells that were treated with LY294002 and chrysophanol had a lower ∆ψM than single-treatment cells (Figure 7H,I).    . (A,B) The expression levels of Akt, p-Akt, mTOR, and p-mTOR were detected using Western blot analyses, and the relative protein p-Akt/Akt ratio was calculated by a Western blot band. (C,E-G) The protein expression levels of autophagy-related proteins (beclin-1, p62, and LC3B) and apoptosis-related proteins (Bax, Bcl-2, caspase -3, and PARP) were evaluated  . (A,B) The expression levels of Akt, p-Akt, mTOR, and p-mTOR were detected using Western blot analyses, and the relative protein p-Akt/Akt ratio was calculated by a Western blot band. (C-G) The protein expression levels of autophagy-related proteins (beclin-1, p62, and LC3B) and apoptosis-related proteins (Bax, Bcl-2, caspase -3, and PARP) were evaluated by Western blot analyses, and then the ratios of apoptosis-regulated proteins are indicated in the histogram. (H,I) Making observations for JC-1, cells were treated with 150 µM chrysophanol in the presence or absence of LY294002. Data are expressed as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001).

Discussion
Natural chemicals extracted from plants are considered good therapeutic agents for cancer therapy because they have fewer side effects and can be used for oral administration [45,46]. Previous studies indicated that chrysophanol induces cell death via the production of ROS and damaged ATP synthesis in liver and ovarian cancer [37,38,47]. Furthermore, chrysophanol has shown anti-cancer effects by anti-proliferation and proapoptotic activities in several cancers, such as choriocarcinoma, breast, and colon cancers [34,36,39]. However, studies on the autophagy effect of chrysophanol on cancer cells are still insignificant. In this study, as a first step toward demonstrating whether chrysophanol affects apoptosis and autophagy activities, our researchers identified an interaction between chrysophanol-induced apoptosis and autophagy in OSCC cells.
The results indicated that chrysophanol reduced cell viability and proliferation in CAL-27 and Ca9-22 cells (Figure 2). Chrysophanol inhibits cell proliferation by the inhibition of the NF-κB and EGFR/mTOR pathways in colon and breast cancers [34,39]. It has been confirmed that the treatment of chrysophanol caused apoptosis via increased morphological changes in OSCC cells. The mitochondrial signaling pathway of apoptosis plays a key role in regulating cell death in response to various stimuli [48]. During apoptosis, mitochondrial outer membrane permeability metastasis is induced by a specific Bcl-2 family [49]. We detected that chrysophanol induced changes in the mitochondrial membrane potential (∆ψM) and indicated that chrysophanol decreased ∆ψM.
If ∆ψM is lower, it could be an apoptotic indicator of the mean of emission apoptogenic factors [50]. Inhibited Bcl-2 protein releases cytochrome c into the cytoplasm. The released cytochrome c binds to the cytochrome c/Apaf-1 complex and oligomerizes with carspace-9 to produce apoptosomes, then activates the carspace cascade containing carspace 3 [51]. Activated caspase-3 cleaves polyADP-ribose polymerase (PARP), causing DNA segmentation, which eventually causes cells to undergo apoptosis [52]. Chrysophanol triggers mitochondrial-mediated apoptosis via the upregulation of the Bax/Bcl-2 ratio and is accompanied by cleavage of caspase-3 and PARP activation in optic nerve meningioma, choriocarcinoma, and breast cancer [33,36,39]. In this study, chrysophanol caused the diminution of Bcl-2 expression, whereas the activations of caspase-3, -7, and -9 and the PARP cleavage were significantly induced in OSCC cells after treatments with chrysophanol. It is suggested that chrysophanol causes apoptosis via caspase activations in OSCC cells (Figure 3).
In the present study, it was confirmed that chrysophanol induces the formation of AVOs and autophagic vacuoles within the cytoplasm or perinuclear regions in OSCC cells. Autophagic biochemical marker expression, such as that of ATG5, beclin-1, LC3, and p62/SQSTM1, has also been verified. The autophagy process is encoded by autophagyrelated genes (ATGs), and it leads to the formation of autophagosome [53]. To initiate the autophagosome membrane elongation process, the ATG5-ATG12 complex's conjugation is necessary [54]. Early studies suggested that beclin-1 (BECN1, also known as ATG6) plays a role in one step of the autophagy process. Beclin-1-Vps 34 complex I promotes the production of autophagosomes [55,56]. The soluble forms of LC3B-I and lipid phosphatidylethanolamine (PE) converted to the autophagic vesicle-associated form, LC3B-II (also known as MAP1LC3B) [57]. For a mature autophagic flux, p62/SQSTM1 (sequestosome-1) targets autophagosomes and links ubiquitinated proteins, and then cellular contents are targeted by sequestosome-1 and degraded in the lysosomes [58]. The upregulated expression level of protein and mRNA, which is associated with autophagy, suggests induced autophagy in OSCC cells when treated with chrysophanol. Chrysophanoltreated cells induced the protein expression levels of beclin-1 and ATG5, and LC3-I was converted to LC3-II. The mRNA expression levels of ATG5, p62/SQSTM1, and MAP1LC3 were considerably induced (Figure 4). This result indicates that chrysophanol significantly induces autophagy in OSCC cells.
The autophagy cell death mechanism remains highly debatable; however, massive autophagy can kill cells. Therefore, autophagy can be considered as a type of cell death [59].
3-MA was used to further investigate the role of autophagy in chrysophanol-induced apoptosis. 3-MA is popularly used as an autophagy inhibitor. It has been reported to degrade the formation of the pre-autophagosome, autophagosome, and autophagic vacuole by blocking phosphoinositide 3-kinase (class III PI3K) activities [60]. In this study, combined chrysophanol and 3-MA treatments showed increased activation of caspase-3 and PARP cleavage and greater release of cytochrome c; on the contrary, the expression levels of beclin-1 and LC3B-II reduced in comparison to a single treatment of chrysophanol. Therefore, autophagy inhibitors increased chrysophanol-induced apoptosis ( Figure 6). The factors that regulate apoptosis and autophagy have been revealed. Beclin-1 is a component of the PI3K III complex demanded for autophagosome formation, and it also binds to Bcl-2 or Bcl-XL via the BH3 domain and then disrupts autophagic activity. Beclin-1 is a wellknown protein that includes autophagic inducers and autophagic inhibitors [61,62]. In another report, the knockdown of beclin-1 was found to expedite apoptosis [63]. In this study, chrysophanol induced apoptosis and autophagy in oral cancer cells. Among them, autophagy induced by chrysophanol is a cytoprotective mechanism of oral cancer cells and plays a role in partially interfering with the apoptosis pathway. The PI3K/Akt/mTOR pathway plays crucial roles in the regulation of autophagic activity. In the regulation of autophagy, the PI3K/Akt/mTOR pathway has been known as a significant player in catabolic processes. [64,65]. Therefore, in this study, we investigated the phosphorylation of the Akt/mTOR pathway in chrysophanol-treated OSCC cells ( Figure 6).
Chrysophanol promotes the phosphorylation of Akt in JEG-3 cells [36], and chrysophanol inhibits the phosphorylation of PI3K/Akt in SNU-C5 cells activated by EGF (epidermal growth factor) [34]. In this study, pan-PI3K inhibitor LY294002, which has been known to inhibit autophagy, was used for verifying chrysophanol-regulated PI3K/Akt pathway activations. PI3K is demanded for autophagic sequestration. Thus, the inhibition of PI3K with LY294002 can hinder autophagic processes [64]. In this study, it was confirmed that the co-treatment of chrysophanol and LY294002 significantly increased pro-apoptotic proteins Bax, cleaved caspase-3, and cleaved PARP while decreasing the conversion of autophagic-related proteins beclin-1 and LC3B-II. That is, the combination of LY294002 with chrysophanol showed the downregulation of autophagy and the upregulation of apoptosis compared with a single treatment of chrysophanol in OSCC (Figure 7).
Taken together, chrysophanol reduced cell viability and induces apoptosis, demonstrating its effectiveness as an anticancer therapeutic, but unfortunately, chrysophanol-induced autophagy was found to interfere with OSCC cell apoptosis ( Figure 8). To overcome this, autophagy induced by chrysophanol has been shown to be synergistic by increasing apoptosis when inhibited via the regulation of the Akt/mTOR pathway. Chrysophanol activates autophagy and in parallel inhibits apoptosis activity. 3-MA, an autophagy inhibitor, inhibits autophagy and cooperates with apoptosis, which is occasioned by chrysophanol. Inhibition of the PI3K/Akt/mTOR signaling pathway with the PI3K inhibitor LY294002 enhances chrysophanol-induced apoptosis and reduces autophagy. Therefore, chrysophanol-induced autophagy acts as a cell protection mechanism and interferes with the apoptosis pathway of OSCC cells.

Conclusions
The purpose of the present study was to investigate the mechanism and role of the autophagy induced by a natural substance, chrysophanol, in oral squamous-cell carcinoma cells. Our study demonstrated that chrysophanol induces autophagy and interferes with apoptosis via the PI3k/Akt/mTOR pathway in oral cancer cells. Therefore, in order to increase the value of chrysophanol as an oral cancer treatment, inhibition of autophagy by chrysophanol is essential, which is expected to have a tremendous synergistic effect on overcoming oral cancer. However, further molecular biological mechanisms and in vivo studies are needed to establish a relationship between autophagy and apoptosis by chrysophanol and provide more solid evidence.

Informed Consent Statement: Not applicable
Data Availability Statement: The data presented in this study are available upon request from the corresponding author.

Conflicts of Interest:
The authors declare no conflicts of interest. Chrysophanol activates autophagy and in parallel inhibits apoptosis activity. 3-MA, an autophagy inhibitor, inhibits autophagy and cooperates with apoptosis, which is occasioned by chrysophanol. Inhibition of the PI3K/Akt/mTOR signaling pathway with the PI3K inhibitor LY294002 enhances chrysophanol-induced apoptosis and reduces autophagy. Therefore, chrysophanol-induced autophagy acts as a cell protection mechanism and interferes with the apoptosis pathway of OSCC cells.

Conclusions
The purpose of the present study was to investigate the mechanism and role of the autophagy induced by a natural substance, chrysophanol, in oral squamous-cell carcinoma cells. Our study demonstrated that chrysophanol induces autophagy and interferes with apoptosis via the PI3k/Akt/mTOR pathway in oral cancer cells. Therefore, in order to increase the value of chrysophanol as an oral cancer treatment, inhibition of autophagy by chrysophanol is essential, which is expected to have a tremendous synergistic effect on overcoming oral cancer. However, further molecular biological mechanisms and in vivo studies are needed to establish a relationship between autophagy and apoptosis by chrysophanol and provide more solid evidence.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.