Ailanthone Inhibits Cell Proliferation in Tongue Squamous Cell Carcinoma via PI3K/AKT Pathway

Tongue squamous cell carcinoma (TSCC) is the most widespread and invasive subtype of oral cancer with high recurrence rates. Ailanthone (AIL) is an active ingredient in the plant extracts of Ailanthus altissima (Mill.) Swingle. Here, we showed that AIL inhibited the proliferation of human TSCC, the cell viability of Cal-27 and Tca8113 was significantly decreased after AIL treatment for 24 h. Hoechst 33258 staining demonstrated apoptotic characteristics (such as chromatin aggregation) after AIL treatment. The ratio of early- and late-apoptotic cells in AIL-treated Cal-27 and TCA8113 cells increased remarkably when compared with the control group. Bcl-2/Bax ratio and the levels of PARP1, caspase-9, and caspase-3 decreased after AIL treatment, accompanied by significant increase of cleaved PARP1, cleaved caspase-9, and caspase-3 in Cal-27 and TCA8113 cells. Meanwhile, AIL led to Cal-27 cell cycle arrest at G2/M phase. Western blot implied decreased levels of CDK1 and cyclin B1 after AIL treatment. The level of phospho-PI3K p55 subunit and p-Akt were significantly downregulated by AIL in both Cal-27 and TCA8113 cells. These findings implied the potential applications of AIL in the treatment of human TSCC.


Introduction
TSCC is the most widespread and highly invasive oral squamous carcinoma [1]. TSCC can easily lead to disorders of speech, swallowing, and chewing, thereby seriously restricting the patients' quality of life [2]. It also has a poor prognosis and a high local recurrence rate, which lead to reduced overall survival rate. In recent years, death rate caused by TSCC has signifcantly increased, and the incidence of TSCC has shifted to a younger age [3]. However, the surgical therapy frequently led to maxillofacial deformity, speech disorder, eating difcult, and other functional disorders, with a high rate of relapse and lymphatic metastasis or distant tissue metastasis [4]. Terefore, it is urgent to explore novel drugs or targets to treat TSCC.
Recent research has focus on the antitumor efcacies of traditional Chinese medicines (TCM) based on their verifed pharmacological efects and fewer adverse reactions. TCM exert their therapeutic efect in a comprehensive way to the whole-body system by maintaining a normal balance. Moreover, the specialists of traditional Chinese medicines tend to elevate the body's endogenous resistance to disease and individualize treatment. Naturopathic therapy with TCM is chosen by a high number of patients, considering that these substances are considered as multicomponent, multitarget, and multistage agents. TCM approach is being accepted by more and more people all over the world. Ailanthone (AIL) is an active ingredient in the plant extracts of traditional Chinese medicine Ailanthus altissima (Mill.) Swingle [5], which has been employed to treat ascariasis, diarrhea, gastrointestinal diseases, bleeding, and infammation [6,7]. Moreover, AIL has been reported to exert growth inhibition in several cancer in vitro and in vivo [8]. Ni et al. found that AIL inhibited cell proliferation in nonsmall cell lung cancer (NSCLC) and suppressed tumor growth in xenografted and orthotopic lung cancer models, which led to prolonged survival of the two types of tumorbearing mice [9]. AIL also induced cell cycle arrest at G0/G1 phase, as indicated by up-regulated levels of p21 and p27 and down-regulated levels of cyclins and CDKs in hepatocellular carcinoma (HCC) [10].
To date, there have been no studies about the efect and mechanism of AIL on human TSCC. Terefore, this study used tongue squamous carcinoma Cal-27 and Tca8113 cells to evaluate the antitumor efect of AIL and tried to clarify its potential mechanism.

MTT Assay.
Cal-27 and Tca8113 cells at a density of 5 × 10 3 were seeded into 96-well plates and kept at 37°C and 5% CO 2 . Te cells were incubated with diferent concentrations of AIL (0.25, 0.5, 1, 2, 4, 8, 16, and 32 μM) for 24 h. Ten, 10 μL MTT (Sigma-Aldrich, Germany) PBS solution was added and subsequently incubated for 4 h. After incubation, the formazan crystals were dissolved with 200 μL DMSO. Te optical density (OD) of each well was measured at 490 nm using a microplate reader (Tecan Group, Switzerland). All OD values were normalized to the control group cells.

Hoechst 33258 Staining.
Morphological changes in cell nuclei were measured using Hoechst 33258 probe, as described previously [11]. Cal-27 and Tca8113 cells at a density of 5 × 10 4 cells/mL were seeded onto 24-well plates and cultured for 24 h. Te cells were incubated with diferent concentrations (0.25, 1, and 4 μM) of AIL for 24 h. After the incubation, the cells were fxed with 4% paraformaldehyde at room temperature for 10 min. After rinse with PBS, the cells were incubated with Hoechst 33258 staining solution in the dark for 15 min. After the cells were washed thrice with PBS, the pictures of the stained nuclei were observed and obtained by a fuorescence microscope (DMI3000B, Leica, Germany).

Evaluation of Apoptosis.
Briefy, 2 × 10 5 Cal-27 and Tca8113 cells were seeded onto 6-well plates and kept at 37°C and 5% CO 2 . After 24 h treatment with AIL (0.25, 1, and 4 μM), the cells were digested and resuspended in 400 μL of Annexin V binding bufer. Ten, 5 μL FITC-conjugated Annexin V solution and 5 μL PI solution were added and incubated for 15 min in the dark at room temperature. Finally, the cells were counted and analyzed by FACScan fow cytometer and BD FACSuite ™ software.

Analysis of Cell Cycle.
Logarithmic growth phase of cells (2 × 10 5 ) i.e., Cal-27 and TCA8113 cells were inoculated on 6-well plates. Te second day, when the cell density reached about 70%, diferent concentrations of AIL were added, followed by overnight incubation. Te 6-well plates were taken out and the cells were collected. Te precooled 75% alcohol was separately added and placed at 4°C for 24 hrs. On the next day, the cells were cleaned with cold PBS, and centrifuged, followed by addition of 100 μL RNase A (Cat. No. CA1050, Solarbio, China), and then incubation at 37°C for 30 min, and addition of 400 μL PI staining solution. Next, the cells were incubated in dark for 0.5 h (at 4°C). Finally, the cell cycle distribution was analyzed by fow cytometry.
2.1.6. Western Blotting. Logarithmic growth phase of 2 × 10 6 cells i.e., Cal-27 and TCA8113 were inoculated on Petri dishes (100-mm). Next, either vehicle or AIL treated the cells. Ten, the cells washing (twice) were carried out with cold PBS, followed by solubilizing in lysis bufer with phosphatase and protease inhibitors. Te BCA protein detection kit was used for protein concentration. Te protein separation using a total of 40 μg cell lysate were used by 10% SDS-PAGE, followed by protein transferring to PVDF membranes (EMD Millipore). In 2 hours, PVDF membranes were blocked with 5% nonfat milk under the condition of room temperature. Ten, the PVDF membrane diluted in 5% nonfat milk in TBST with Tween 20 (0.1%) was incubated with special antibodies or anti-β-actin as housekeeping protein. Subsequently, horseradish peroxidase-conjugated IgG served as secondary antibody were used for the incubation. Te detection of secondary antibodies on the PVDF membrane were used by the enhanced chemiluminescence (ECL) detection reagents (Pierce, Termo Fisher). Densitometry analysis (including integrated density of bands) was carried out via Image J (NIH), followed by normalizing the documented values to beta-actin.

Statistical Analysis.
Each experiment was carried out at least three times and the data were supplied as mean-± Standard Error of Mean (SEM). An unpaired Student's ttests or one-way ANOVA followed by Tukey's post hoc test was performed using SPSS 25.0 statistical software. P < 0.05 was considered statistically signifcant.

AIL Induces Mitochondrial-Mediated Apoptosis in TSCC
Cells. Previous data demonstrated that AIL treatment of TCA8113 and Cal-27 cells subsequently resulted in an increment in the count of apoptotic cells in the early and late stages. To further confrm that AIL induces Cal-27 and TCA8113 cell apoptosis, we employed western blot for detecting the expression of proteins associated with apoptosis. We found that Bcl-2/Bax ratio was signifcantly decreased after 1 μM AIL and 4 μM AIL in both TCA8113 and Cal-27 cells in comparison with the control group (Figures 2(a), 2(b), 2(e), and 2(f)). We further found that following AIL exposures, the expression of PARP1, caspase-9 and -3 underwent a decrease, simultaneously accompanied by cleaved PARP1, cleaved caspase-9 and -3 accumulating in signifcant quantities within TCA8113 and Cal-27 cells (Figures 2(c), 2(d), 2(g), and 2(h)). Te results implies that the inherent apoptotic pathway was primarily responsible for apoptosis induced by AIL in TCA8113 and Cal-27 cells.

AIL Triggers the Cell Cycle Arrest at G2/M Phase in Cal-27 Cells but Does Not Infuence TCA8113 Cells.
To ascertain if the cell cycle progression is afected by AIL, we subjected Cal-27 and TCA8113 cells to incubation with various concentrations of AIL for a duration of 24 h, followed by measurement and analysis of cell cycle distribution. It was evident that only Cal-27 cells treated with 4 μM AIL manifested a considerable decrease in the ratio of cells within the G0/G1 state, whereas the number of AIL-treated Cal-27 cells at the G2/M phase exhibited a signifcant increment upon treating with 1 μM and 4 μM AIL compared with control (Figures 3(a) and 3(b)). Tereafter, we made a thorough assessment of the molecular mechanism regulating cell cycle arrest in AIL-treated Cal-27 cells. Western blotting was employed to estimate the G2/M-related proteins. As evident from Figures 3(c) and 3(d), AIL decreased the level of CDK1 and cyclin B1, which implied that AIL induced the arrest of G2/M in Cal-27 cells. We also analyzed the cell cycle change in TCA8113 cells. Te percentage of cells at diferent cell cycle phases did not difer among the AIL treatments in TCA8113 cells (Figures 3(e) and 3(f )). Terefore, we did not further detect the transition in the levels of proteins taking part in the cell cycle.

AIL Causes Blocking of the PI3K/AKT Pathway in TSCC
Cells. To probe into the mechanistic pathway controlling apoptosis and G2/M phase arrest mediated by AIL, an analysis of the levels of PI3K/AKT pathway-associated proteins was carried out. As illustrated in Figures 4(a) and 4(b), the level of phospho-PI3K p55 subunit was signifcantly downregulated by AIL in Cal-27 cells. As evident in Figure 4(b), the extent of expression of PI3K was not found to signifcantly alter in Cal-27 cells. From Figures 4(a) and 4(b), the phosphorylation of AKT at the Ser473 site also decreased in Cal-27 cells after AIL treatment. In Cal-27 cells, only after the treatment with 4 μM AIL, the expression of total AKT was signifcantly decreased (Figure 4(b)). In TCA8113 cells, there was an obvious decrease in the level of phospho-PI3K p55 subunit and phosphorylation of AKT at the Ser473 site by AIL (Figures 4(c) and 4(d)). Te level of PI3K was signifcantly increased after 0.25, 1, and 4 μM AIL in TCA8113 cells (Figure 4(d)), while the level of total AKT was signifcantly decreased after the treatment with 0.25, 1, and 4 μM AIL (Figure 4(d)).

Discussion
TSCC is an aggressive form of cancer of the oral cavity and has the highest rate of prevalence and recurrence among the various kinds of oral cancers [1]. In 2015, within China, a total of 48,100 new cases and 22,100 deaths associated with TSCC were reported [12]. Although the clinical outcomes have been improved after the advances in surgical therapy, chemotherapy, and radiotherapy, the prognosis, in general, and the overall survival rates of TSCC patients have not elevated much in the past decade. Hence, it is necessary to explore and fnd improved drugs for developing novel therapeutic approaches for TSCC patients.      Evidence-Based Complementary and Alternative Medicine TCM has an extensive background of successful utilization in the treatment and prevention of disorders and diseases. Many studies and clinical observations have revealed that traditional herbal Chinese medicines as well as their extracts have powerful inhibitory characteristics against tumors [13]. AIL, acquired from the Ailanthus altissima, has anticancer potential toward a multitude of cancer cells. In this study, we found that the cell viabilities of TCA8113 and Cal-27 cells were signifcantly decreased following AIL treatment. Further study showed that exposure to AIL led to chromatin aggregation, and the count of apoptotic cells in the early and late stages of apoptosis among Cal-27 and TCA8113 cells was obviously increased after the treatment with AIL.
It has been reported that the Bcl-2 family proteins play a vital role for cancer cell apoptosis. Tere are two major types of Bcl-2 family proteins: pro-survival members (e.g., Bcl-2) and pro-apoptotic members (e.g., Bax) [14]. Bcl-2 can be induced by various factors, including growth factor deprivation, glucose deprivation, lipid peroxidation, and many antitumor drugs [15]. Te increased level of Bcl-2 has been confrmed to protect against apoptosis [16]. Several clinical studies demonstrated that the higher level of Bcl-2 is related with a poor prognosis in many tumors' types, including pancreatic carcinoma and oral tongue squamous cell carcinoma [17,18]. Bax, a pro-apoptotic protein, can facilitate mitochondrial membrane permeabilization and activate caspase-3/9, resulting in cell apoptosis [19]. Multiple studies have suggested that decreased Bcl-2/Bax ratio in tumor cells triggers activation of caspase-9/-3 and subsequently promotes the mitochondrial-mediated apoptosis [20,21]. Consistent with the above results, our data showed that decreased Bcl-2/Bax ratio and increased cleaved caspase-9 and -3 in AIL-treated Cal-27 and TCA8113 cells.
One important characteristic of tumor is uncontrolled cell proliferation, and several clinical drugs exert antitumor efects by suppressing cell proliferation through arresting tumor cell cycle [22]. AIL has been reported that it inhibits Huh7 cell proliferation by blocking cell cycle [10]. In our study, we found that AIL induced cell cycle arrest at G2/M phase in Cal-27 cells, while no signifcant change of cell cycle distribution in TCA8113 cells after AIL treatment. We supposed that this may be due to the diferent patientsderived TSCC cells with diferent cellular metabolic patterns [23]. Cal-27 cells are epithelial cells isolated from a White male with a lesion in the middle of the tongue, and TCA8113 cells are isolated from a biopsy section of tongue carcinoma. Likewise, many studies have showed that the same drug/ compound has an obvious diferent efect in one type of tumor cells from diferent patients. For example, one study reported that curcumin sensitized TRAIL-induced cytotoxicity in malignant glioma U87MG cells but not in malignant glioma U251MG cells [24]. Several recent studies showed that cyclin d kinase 1 (CDK1) and cyclin B1 are closely related to G2/M cell cycle [25,26]. Next, we examined the levels of CDK1 and cyclin B1 in AIL-treated Cal- 27 cells, and found that the levels of CDK1 and cyclin B1 were remarkably decreased after treatment with AIL. Tese fndings implied that AIL could induce Cal-27 cells G2/M phase arrest through regulation of CDK1 and cyclin B1.
In human cancers, PI3K-AKT pathway is the most recurrently triggered pathway regulating survival, diferentiation, cell growth, and cell apoptosis of tumor cells in response to a wide array of signals, and PI3K/AKT inhibitors demonstrate a more efective and also synergize with a variety of chemotherapeutics [27][28][29][30]. Phosphorylated AKT promotes the apoptosis-related protein Bcl-2 and cell cycle-related protein cyclin D [31]. Te decline in the expression of AKT induces cell apoptosis while inhibiting Bcl-2 expression [32]. It was reported by Zhang et al. that in colorectal cancer, the inhibited phosphorylation of PI3K and AKT reduced Bcl-2 expression subsequently promoting the mitochondrial apoptosis induced by oxaliplatin [33]. In this work, we found that the decreased Bcl-2 expression was accompanied by downregulation of p-PI3K and p-AKT, eventually leading to TCA8113 and Cal-27 cells apoptosis. In addition, according to several reports, the action of the CDK1 and cyclin B1 can be promoted by PI3K/AKT pathway [34,35]. Our data  To the best of our awareness, the current work is a pioneer report demonstrating the antitumor efect of AIL on tongue squamous cell carcinoma. For a better understanding of the therapeutic capability of AIL, additional in vivo experiments are needed to verify the antitumor ability of AIL. Our observations conclusively demonstrated that AIL instigated cell cycle arrest at G2/M and suppressed the PI3K/ AKT pathway to cause apoptosis of tongue squamous cell carcinoma cells. Terefore, this study suggested that AIL showed prospects of being developed into an efective novel therapeutic agent focusing to target the PI3K/AKT pathway in TSCC.

Data Availability
Te data used and/or analyzed during this study are available from the corresponding author upon request.

Ethical Approval
Tis article does not contain any studies with human participants or animals performed by any of the authors.

Conflicts of Interest
Te authors declare that they have no conficts of interest. , p-AKT, and AKT in TCA8113 cells treated with or without AIL. * P < 0.05, * * P < 0.01, and * * * P < 0.001 versus the control.