Thymoquinone inhibits the metastasis of renal cell cancer cells by inducing autophagy via AMPK/mTOR signaling pathway

Abstract Thymoquinone (TQ, 2‐methyl‐5‐isopropyl‐1,4‐benzoquinone), a bioactive constituent extracted from the seeds of Nigella sativa, has been proved to exert anti‐tumor efficiency in various cancers. Autophagy is a self‐digestion phenomenon, and its role in tumor formation and progression remains controversial. In the present study, we investigated the effects of TQ on renal cell cancer (RCC) cell lines (786‐O and ACHN) using wound healing assay, transwell assay and western blot analysis. We found that TQ effectively inhibited the metastatic capacity of RCC cells in vitro, which was also verified in a xenograft model. Meanwhile, we observed LC3 puncta and detected the expression of LC3 in TQ‐treated RCC cells, and then found that autophagy was induced by TQ in 786‐O and ACHN cell lines. In addition, TQ inhibited the migration and invasion as well as the EMT in RCC cells in an autophagy‐dependent manner. To further explore the underlying mechanism, we detected the AMPK/mTOR signaling pathway. The results indicated that TQ inhibited the metastasis of RCC cells by inducing autophagy via AMPK/mTOR signaling pathway. In conclusion, our findings provide a novel therapeutic strategy that aims at TQ‐induced autophagy in RCC treatment.

has been proved to exert wide-ranging pharmacological activities, with anti-tumor, anti-inflammatory, anti-oxidant, anti-microbial, anti-convulsant and anti-diabetic properties. [6][7][8][9] Therefore, TQ has been regarded as a promising agent for numerous diseases. Previous studies have revealed that TQ may exhibit high anti-tumor efficiency against various cancers, such as breast cancer, gastric cancer, cervical cancer, colon cancer, central nervous system cancer, ovarian cancer, prostate cancer, bladder cancer and renal cell cancer. [10][11][12][13][14][15][16][17][18] It is well known that invasion and metastasis play vital roles in the progression of tumors. Recent research has shown that TQ can inhibit metastasis and EMT in prostate cancer via the transforming growth factor-beta/Smad2/3 signaling pathway. 19 Jun Li and his team also indicated that TQ inhibited the migration and invasion abilities in cervical cancer cells, which might be regulated via the expression of Twist1, Zeb1 and E-cadherin directly. 12 However, there are few studies that consider the effect of TQ on metastasis in renal cell cancer.
Autophagy is a self-digestion phenomenon, first identified by Christian de Duve in 1963, which is evolutionarily conserved from yeast to mammals. During the autophagy process, a doublemembrane vesicle, named autophagosome or autophagic vacuole, sequesters cytoplasmic proteins and organelles and then delivers them to lysosome, where they are degraded. 20 The role of autophagy in tumors constantly changes with the progression of tumors. In the early stages of tumor formation, autophagy suppresses cancer initiation through eliminating inflammation, genome instability and tissue damage. Alternatively, as a tumor grows, autophagy promotes tumor growth through providing substrates for metabolism. 21,22 Emerging observations indicate that there is an intricate relationship between autophagy and metastasis in cancer. On the one hand, autophagy helps to support viability for the potentially metastatic cancer cells. On the other hand, autophagy tends to inhibit early metastasis and to reverse of epithelial-mesenchymal transition (EMT) in cancer cells. 23,24 Hence, in the present study, we aim to explore the effect of TQ on metastasis in RCC and the role of autophagy in this process. We also elucidate the underlying molecular mechanisms. at 37°C in a humidified incubator with 5% CO 2. Invitrogen (Carlsbad, CA, USA). Protease inhibitor and phosphatase inhibitor were obtained from Roche (Basel, Switzerland).

| Cell viability assay
786-O and ACHN cells were plated into 96-well culture dishes (Corning, NY, USA) at a density of 5 × 10 3 cells/well with complete culture medium. After being incubated overnight, the medium was removed and replaced with fresh medium containing different concentrations of TQ (0, 10, 20, 40, 60, 80 and 100 μmol/L) for 24 or 48 hours, respectively.
Then the medium was removed and 20 μL CCK8 was added into each well and incubated for 4 hours at 37°C. The optical density (OD) of each well was measured at 490 nm by microplate reader.

| Transwell migration and invasion assay
We used 24-well plates with chambers of 8μm pore size (Millipore, Germany) in the transwell analysis. In the migration assay, 1 × 10 4 RCC cells were seeded in the upper chamber. In the invasion assay, 2 × 10 4 cells were seeded in the upper chamber pretreated with Matrigel. Cells in the upper chamber were resuspended in 200 μL serum-free medium with different concentrations of TQ. The lower chamber was filled with 800 μL complete medium containing the same concentration of TQ as in the upper chamber. After incubation for 24 hours, the non-migrated or non-invaded cells in the upper chamber were removed by cotton swab, and the migrated or invaded cells were fixed with 4% paraformaldehyde for 30 minutes and then stained with .1% crystal violet for another 30 minutes. Then, the cells were counted under a microscope and 4 random fields for each chamber were selected for statistical analysis.

| Wound healing assay
Renal cell cancer cells were seeded into 6-well plates with complete medium. We used 200-μL pipet tips to scratch wounds when the cells had grown to 90%. The plates were washed 3 times to remove the floated cells and then filled with serum-free medium containing different concentrations of TQ for 24 hours. The cells were observed at different time points and the wound closure was determined by inverted microscope.

| Tandem fluorescence microscopy
Renal cell cancer cells were plated into 6-well dishes with complete medium. When cells grew to 70%, the medium was discarded and replaced with serum-free medium. Then, the cells were transfected with tandem fluorescent mRFP-EGFP-tagged LC3 expressing plasmids using Lipofectamine 2000. After 6 hours, the medium containing Lipofectamine 2000 was abandoned and new complete medium was added to the dishes to maintain cells for 18 hours. Next, cells were treated with TQ for 24 hours and then fixed with 4% paraformaldehyde. Then, the expression of enhanced green fluorescent protein (EGFP) and mRFP was observed by fluorescence microscopy (Olympus, Tokyo, Japan).

| Western blot
Renal cell cancer cells were lysed by RIPA buffer containing protease inhibitor or phosphatase inhibitor and PMSF for 10 minutes on ice. Then, the cell debris was collected and centrifuged at 12 400 g for 15 minutes. The supernatants were transferred into new tubes, and protein concentrations were detected by BCA Protein Assay Kit (Heart, Xi'an, China). The supernatants were mixed with 1/5 volume of 6X buffer and then boiled for 5 minutes. The whole proteins were subjected to 10%-15% SDS-PAGE and then transferred to PVDF membranes. The membranes were first sealed with 10% skim milk for 2 hours at room temperature and then incubated with primary antibodies overnight at 4°C. Subsequently, the membranes were washed with TBST 3 times and incubated with secondary antibodies for 1 hour. The membranes were visualized and measured using an ECL system (Bio-Rad, CA, USA).

| In vivo experiments
All animal experiments were performed in accordance with standard ethical guidelines and approved by the Ethics Committee of Xi'an Jiaotong University. Ten male nude mice were included in the study.
All mice were 5 weeks old and each weighted 19-22 g. Harvested 786-O cells (1 × 10 6 cells) were suspended in 200 μL serum-free medium containing 100 μL Matrigel, and subcutaneously injected into the right flank of every mouse. When the tumors had grown to approximately 100-150 mm 3 in size, the mice were randomly divided into 2 groups (5 in each group) and intraperitoneal injected with DMSO (control group) or TQ 20 mg/kg, respectively, every 3 days.
During the treatment, the tumor volumes were calculated and the mice were weighted with the same frequency. After 30 days, tumors were harvested, weighted and analyzed. The volume was calculated using the following formula: tumor volume = (length × width 2 ) ×.5.
To establish the metastatic tumor model, luciferase-tagged 786-O cells were injected into mice via tail veins. Then, the mice were divided into 2 groups and received the same treatment as above. After 30 days, the mice were intraperitoneal injected with D-luciferin (150 mg/kg). Ten minutes later, mice were anesthetized with 10% chloral hydrate (.004 mL/g) and imaged using the IVIS Lumina II with Living Image Software.
The lung metastatic tumors were then harvested and stained with H&E.

| Immunohistochemical assay
Renal tumors were separated from xenograft mice and fixed with 10% formaldehyde for 24 hours. Then, they were embedded in paraffin and cut into 5μm-thick sections. After that, the tissue sections were subjected to deparaffinization, rehydration, endogenous peroxidase blocking and antigen retrieval. Next, the sections were blocked with 1% BSA for 10 minutes. Subsequently, they were incubated with primary antibodies overnight and appropriate secondary antibodies for 1 hour. The sections were then visualized using a DBA kit following the manufacturer's instructions.

| Statistical analysis
All data were presented as mean ± SD of 3 independent experiments and analyzed using GraphPad Prism 5.2 software. In all cases, differences were considered statistically significant when P-value <.05.

| Thymoquinone suppresses migration, invasion and epithelial-mesenchymal transition in renal cell cancer cells
To choose proper concentrations of TQ in the present study, first we  (Table S1). As shown in Figure 1A Figure 1D). In addition, we observed EMT-related markers (E-cadherin and vimentin) in TQ-treated ACHN by fluorescent immunostaining and found that the results were consistent with those for western blot analysis ( Figure S2). These results suggest that TQ effectively inhibits migration, invasion and the EMT process in RCC cells in vitro.  Figure 2D). Collectively, these data indicate that TQ can induce autophagy in RCC cells.

| Inhibition of autophagy attenuates thymoquinone-induced anti-tumor effects in renal cell cancer cells
Accumulated evidence has suggested that there is an association between autophagy and metastasis. We hypothesized that autophagy induced by TQ may affect the migration and invasion in RCC cells.

| AMPK/mTOR pathway is involved in anti-tumor effects of autophagy induction by thymoquinone in renal cell cancer cells
Numerous studies have indicated that autophagy can be mediated by various pathways, including PI3K/AKT/mTOR, AMPK/mTOR, MAPK/ERK1/2, P53/DRAM and HGF/MET. 25 Among these pathways, the AMPK/mTOR signaling pathway plays a vital role in the process of autophagy. It positively regulates autophagy through increasing AMPK phosphorylation and decreasing mTOR phosphorylation. To confirm whether the AMPK/mTOR signaling pathway is involved in the induction of autophagy by TQ in RCC cells, the protein levels of AMPK, p-AMPK (Thr172), mTOR, p-mTOR (Ser2448) and p-S6K (Thr389) were detected after treatment with different concentrations of TQ. As expected, the expression of p-AMPK was elevated by TQ, while expressions of p-mTOR and p-S6K were reduced, which indicated the activation of the AMPK/mTOR pathway ( Figure 4A). Then, we pretreated the RCC cells with AICAR (an activator of AMPK) or compound C (an inhibitor of AMPK) and detected the expression of p-AMPK, p-mTOR and LC3. As shown in Figure 4B, the effects of TQ on LC3 could be receded when the AMPK/mTOR signaling pathway was inhibited by compound C, which suggested the clinical performance of mTOR inhibitors in relation to avoiding drug resistance is still less than satisfactory at present. Therefore, drugs that target multiple components of pathways, such as autophagy/AMPK/mTOR, are urgently needed.

| Anti-tumor effects of thymoquinone on renal cell cancer cells in vivo
We further investigated the effects of TQ on RCC cell growth in a xenograft tumor model. As shown in Figure 5A, tumors in the group treated with TQ grew more slowly than those in the control group.  Figure S4). These data indicate that TQ inhibits RCC cell growth and metastasis in vivo.  EMT can transform the epithelial phenotype into mesenchymal phenotype, leading to an increase in cell motility and invasiveness. 29 Accumulating research has suggested that EMT has a close correlation with the migratory and invasive abilities of cancer cells.

| D ISCUSS I ON
Then, we detected EMT-related proteins in our study. The results showed that TQ could upregulate epithelial markers and downregulate mesenchymal markers, suggesting that TQ could inhibit EMT in RCC cells. To observe the effects of TQ on tumor growth in vivo, we calculated the volume of the tumors and found that tumors in the group treated with TQ grew more slowly when compared to those in the control group. Moreover, the tumors in the TQ-treated group showed higher expression of E-cadherin and lower expression of N-cadherin, which concurred with our findings in vitro. We further established a metastatic tumor model and the results indicated that TQ significantly inhibited metastasis compared with the control group, which was consistent with our expectations.
Autophagy is a catabolic cellular process in which the dysfunctional cytoplasmic constituents are removed to maintain metabolic balance. It occurs not only in normal cells but also in cancer cells. 30 It is widely accepted that autophagy plays a complex role in oncogenesis: it represses tumor growth in the early stages on the one hand but is required for progression to advanced or invasive tumors on the other hand. 24 During the past 5 years, autophagy induced by plantderived drugs has been widely studied in various tumors. In the present study, we first demonstrated that TQ-induced autophagy in a concentration-dependent manner in RCC cells, which was verified by inverted microscope, LC3 turnover assay and western blot.
There is an intricate connection between autophagy and metastasis. Autophagy is usually induced during the progression of various cancers to metastasis and it can promote tumor metastasis through numerous mechanisms, including promoting a stem cell phenotype, production of secreted factors and modulating the Rho signaling pathway. 24 In contrast, autophagic cells can act as a barrier against tumor progression through inhibiting the tumor transfer from primary sites to secondary sites. 31 A study has shown halofuginoneinduced autophagy suppresses breast cancer cell migration and invasion. 32 Meanwhile, in osteosarcoma cells, the activation of autophagy is associated with the suppression of EMT. 33 In the study, we used 3-MA to inhibit autophagy and observed the effects of TQ on migration, invasion and EMT in RCC cell lines 786-O and ACHN.
We, therefore, found that inhibition of autophagy could attenuate TQ-induced anti-metastasis effects in RCC cells.
The mTOR signaling pathway has been reported to be activated in nearly 100% of RCC, and inhibitors targeting the mTOR signaling seem to offer a promising therapy against RCC. In our supplementary experiments, we compared TQ with everolimus.
The results indicated that everolimus showed more anti-tumor effects as compared to TQ. However, the emergency of drug resistance has limited the application of mTOR inhibitors in clinical treatment and agents targeting multiple pathways have been widely studied. Because the mTOR signaling pathway plays a vital role in the process of autophagy, it is important to study the relationship between the mTOR pathway and autophagy. The mTOR includes 2 functionally and biochemically distinct complexes, mTORC1 and mTORC2. 34 Evidence has shown that mTORC1, but not mTORC2, plays a vital role in the regulation of autophagy.
The mTORC1 inhibition increases autophagy, while activation of mTORC1 represses this process. 34 AMPK is a cellular energy sensor that regulates homeostatic balance. It positively regulates autophagy by inhibiting the activity of mTORC1 through direct phosphorylation of its receptor, a regulatory-associated protein of mTOR. 25 To verify whether TQ induced autophagy through the AMPK/mTOR signaling pathway, we detected the main molecules in this pathway and found that the expression of p-AMPK was elevated by TQ, while expressions of p-mTOR and p-S6K were reduced, accompanied by an increase in LC3. Moreover, we observed an upregulation of LC3 in TQ-treated RCC cells when activating the AMPK/mTOR pathway, while a downregulation when inhibiting this pathway; taken together, we can conclude that TQ could induce autophagy via the AMPK/mTOR pathway. Then, the migration and invasion experiments showed that the AMPK inhibitor attenuated the anti-metastatic effects of autophagy induced by TQ, which suggested that TQ could inhibit the metastasis of RCC cells by inducing autophagy through the AMPK/mTOR pathway.
In conclusion, our study demonstrated that TQ exerted antimetastatic properties in RCC cell lines 786-O and ACHN. In addition, these characteristics, including inhibiting migration, invasion and EMT, were mainly dependent on TQ-induced autophagy.
Furthermore, we revealed that the mechanism by which TQ-induced autophagy inhibited the metastasis of RCC cells was regulated through the AMPK/mTOR pathway. These findings indicated the potential role of TQ-induced autophagy in cancer treatment and suggested that combining TQ with autophagy inhibitors might enhance its cancer suppression effects.

D I SCLOS U R E
The authors have no conflict of interests.