XIAP inhibitor embelin induces autophagic and apoptotic cell death in human oral squamous cell carcinoma cells

Abstract Embelin is an active ingredient of traditional herbal remedies for cancer and other diseases. Recently, it has been suggested that autophagy may play an important role in cancer therapy. However, little data are available regarding the role of autophagy in oral cancers. Therefore, we conducted this study to examine whether Embelin modulates autophagy in Ca9‐22. Our results showed that Embelin had anticancer activity against the Ca9‐22 human tongue squamous cell, and we observed that autophagic vacuoles were formed by MDC and AO. We also analyzed Embelin‐treated Ca9‐22 cells for the presence of biochemical markers and found that it directly affected the conversion of LC3‐II, the degradation of p62/SQSTM1, full‐length cleavage formation of ATG5‐ATG12 complex and Beline‐1, and caspase activation. Rescue experiments using an autophagy inhibitor showed Embelin‐induced cell death in Ca9‐22, confirming that autophagy acts as a pro‐death signal. Furthermore, Embelin exhibited anticancer activity against Ca9‐22 via both autophagy and apoptosis. These findings suggest that Embelin may potentially contribute to oral cancer treatment and provide useful information for the development of a new therapeutic agent.


| I N TR ODU C TI ON
Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone) is an inhibitor of X-linked inhibitor of apoptosis protein (XIAP); it is derived from the fruit of Embelia ribes and has been demonstrated to possess therapeutic properties, such as anticancer, antioxidant, anti-inflammation, antidiabetes, and antihelminthic qualities. 1,2 XIAP is the most potent member of the inhibitors of apoptosis proteins (IAP) gene family. XIAP binds and inhibits caspase and therefore inhibits cell migration and invasion and induces apoptosis. 3 Previous studies have demonstrated the potential of Embelin as an antitumor agent to induce cell growth inhibition and apoptosis in different human cancers. [4][5][6] Autophagy is an evolutional phenomenon by which long-lived proteins and damaged organelles within cells are digested in lysosomes. 7,8 Autophagy also promotes cancer cell survival under conditions of stress and functions as a defense mechanism in response to various anticancer drugs. 9,10 Therefore, the induction of autophagic cell death by anticancer reagents has been recognized as an important component of cancer therapy. [11][12][13] Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer and is responsible for a substantial portion of cancerrelated deaths, affecting nearly 500 000 patients annually worldwide. 14 OSCC is one of the most persistent malignancies and remains incurable despite aggressive therapies. 15 Patients with OSCC are currently treated with classical treatment modalities consisting of surgery, radiotherapy, and/or chemotherapy, but OSCC still shows significant mortality rates. [16][17][18]  were also used. The p62/SQSTM1, caspase-9, ATG5-ATG12 complex, GAPDH, mouse antiactin antibody, mouse antirabbit IgG antibody, and rabbit antimouse IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California). All other chemicals and reagents were purchased from Sigma unless otherwise specified.

| MTT assay
Cells were placed in a 96-well plate and were incubated for 24 h. Then they were treated with various doses of Embelin (2.5-300 lM) for 24 h.
After cells were treated with 500 lg/mL of thiazolyl blue tetrazolium bromide (MTT solution), they were incubated at 378C with 5% CO 2 for 4 h. The medium was aspirated and formed formazan crystals were dissolved in DMSO. Cell viability was measured by an ELISA reader (Tecan, Männedorf, Switzerland) at 570 nm excitatory emission wavelength.

| Hoechst staining
After embelin treatment, cells were harvested and cytocentrifuged onto a clean, fat-free glass slide with a cytocentrifuge. Cells were stained in 4 lg/mL Hoechst 33342 for 10 min at 378C in the dark and washed twice in PBS. The slides were mounted with glycerol. The samples were observed and photographed under an epifluorescence microscope (Carl Zeiss, G€ oettingen, Germany). The number of cells that showed condensed or fragmented nuclei was determined by a blinded observer from a random sampling of 3 3 10 2 cells per experiment.
Three independent experiments were conducted.        Figure 5A,B). A combined treatment with both 3MA and Embelin 5 lM showed that the conversion rate of LC3-I to LC3-II was decreased compared to a single treatment of Embelin on Ca9-22 cells ( Figure   5C). We also intended to determine whether 3-MA could promote Embelin-induced apoptosis by assessing cell viability. The 1-mM 3-MA pretreatment and the Embelin-treated group showed a greater decrease in cell viability than the group that received a single Embelin treatment ( Figure 6A). Next, to find out whether 3MA could promote apoptosis, our results were verified by MMP and apoptosis-related proteins. The group that was given a combination of 3-MA and Embelin exhibited down-regulation of the procaspase 9, 3, PARP, and MMP, and PARP cleaved form was shown ( Figure 6B

| D I SCUSSION
Autophagy is widely known as an important process in cell physiology for both cell survival and death. 19 Autophagy begins with the elimination of cytoplasmic organelles in a double-membrane vacuole, an autophagosome, which delivers them to a degradative organelle, the vacuole/lysosome, for breakdown and eventual recycling of the resulting macromolecules. Because numerous recent studies have shown that increased autophagic activity is associated with cell death, 12,20 autophagy is now considered to be a type of cell death. Embelin is an active ingredient in traditional herbal medicine used to treat inflammation and cancer 21 ; many studies have shown that Embelin has a cytotoxic effect and inhibits cell proliferation in various cancer cell types, 6,[22][23][24] presumably by activating the cell's apoptosis machinery. 5,23,25 However, no reports to date have examined the relevance of apoptosis and autophagy for human oral cancer treatment.
Previous studies in our laboratory have also revealed that Embelin We also analyzed Embelin-treated Ca9-22 cells for the presence of biochemical markers including p62/SQSTM1, LC3, ATG5-ATG12 complex, and Beclin-1, which are associated with autophagy. Embelin treatment directly affected the conversion of LC3-II, the degradation of p62/SQSTM1 and full-length Beclin-1, and cleavage formation of ATG5-ATG12 complex and Beline-1 ( Figure 4C). Several previous studies have reported that a relationship may exist between LC3 and p62/ SQSTM1, and p62/SQSTM1 has been shown to selectively decrease in cells undergoing autophagy. [26][27][28][29] The process of autophagy mediates a nonspecific bulk degradation pathway that is responsible for the destruction of the majority of long-lived proteins and some organelles.
ATG12-ATG5 conjugation systems are necessary for the formation of the autophagosome. 30 Beclin-1 (Bcl-2-interacting protein-1) is a key protein in autophagy signaling, and it works in conjunction with Vps34, UVRAG, AMBRA-1, and Barkor to assemble the PI3KC3 complex during the initiation of autophagosome formation. [31][32][33] Several recent FIG URE 5 Embelin-induced autophagy was inhibited by 3-MA in Ca9-22 cells. Cells were pretreated with 1 mM 3-MA for 1 h, and then exposed to 5 lM Embelin for 24 h. A and B, Vital staining was then performed using acridine orange, it is observed with the ratio red fluorescence quantified by flow cytometry. C, The expression levels of autophagy-related protein LC3. As an autophagy control, cells were cultured in EBSS for 6 h. Data were expressed as the mean 6 SD (n 5 3) and analyzed by one-way ANOVA using Dunnett's multiplecomparison test (*P < .05, **P < .01 for the difference between the control and treatment groups). [Color figure can be viewed at wileyonlinelibrary.com] studies using different cell types and stimuli also reported that the caspase-mediated cleavage of Beclin-1 and ATG proteins enhances apoptosis. [34][35][36][37] Our results showed that Embelin led to the degradation of caspase-9 and caspase-3, and it assumes a decisive role on Beclin-1 ( Figure 3). To further clarify the role of autophagy in Ca9-22, our study demonstrated that Embelin-induced cell death was increased by 3-MA, an inhibitor of autophagy. Combination treatment with 3MA and Embelin showed various evidences of cell death as down-regulation of cell survival rate, mitochondrial membrane potential, and cell death related proteins. This result suggests that Embelin-induced autophagy is a prosurvival signal, it could act as an obstructive factor in oral cancer prevention. (Figure 6). This is the first report of Embelin-induced apoptosis and autophagy in Ca9-22 cells. As Embelin clearly can induce autophagy and is involved in the survival of Ca9-22 cells, the combination of Embelin and an effective autophagy inhibitor could be a potentially useful therapy for oral cancer treatment. The identification of the molecular mechanism by which Embelin acts will provide useful information for its development as a novel therapeutic agent for the management of Ca9-22.