
Renal cell carcinoma (RCC) is the most common type of kidney cancer and accounts for approximately 3% of total malignancies (Cairns, 2010). If RCC is detected at the initial stage, it can be successfully removed by surgery, with a 5-year survival rate of nearly 70-80%. However, the early stage of RCC is usually asymptomatic, which makes it difficult to diagnose early. Thus, by the time it is discovered, RCC patients often present with metastasis to other organs, such as to the lymph, lung, and bone, which significantly lowers the survival rate (Bianchi
Inflammation is a strong driving force for tumor metastasis (Wu and Zhou, 2009). During inflammatory responses, prostaglandin E2 (PGE2) is synthesized from arachidonic acids by cyclooxygenase-2 (COX-2) (Nakanishi and Rosenberg, 2013). PGE2 then binds to its four G protein-coupled receptors, EP1-EP4, stimulating cancer cell growth, invasion, and metastasis (Sugimoto and Narumiya, 2007). In addition to overproduction of PGE2, the aberrant expression of its receptors can also amplify PGE2 signaling, which can facilitate cancer promotion and metastasis. Previously, we found that EP2 and EP4 receptors were responsible for PGE2-dependent migration in a human RCC Caki-1 cell line (Woo
The PGE2-EP2/EP4 axis can recruit matrix metalloproteinases (MMPs), which are pivotal for cancer metastasis (Yen
Thymoquinone (2-isopropyl-5-methyl-1,4-benzoquinone; TQ) is a monoterpene present in the seeds of black cumin,
TQ (purity 99%), gelatin, eosin Y, hematoxylin, and β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). EP2 antibody (#101750), PGE2, butaprost, and were purchased from Cayman Chemical (Ann Arbor, MI, USA). SB203580 was obtained from Calbiochem (San Diego, CA, USA). LY294002, Akt (#9272), p-Akt (#9271), p38 (#9212), and anti-rabbit IgG horseradish peroxidase-conjugated antibodies were obtained from Cell Signaling Technologies (Beverly, MA, USA). MMP-2 (ab92536) and MMP-9 (ab76003) antibodies were purchased from Abcam (Cambridge, UK). The p-p38 antibody (#sc-17852) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA).
Human renal cancer Caki-1 cells were provided by Dr. T. K. Kwon (Keimyung University, Daegu, Korea) and were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin G and 100 µg/mL streptomycin) at 37°C under a humidified 95% air/5% CO2 mixture (v/v).
Caki-1 cells were seeded in 96-well plates at a density of 2×103 cells/well and were treated with 1, 5, and 10 μM TQ for 24, 48, and 72 h. Following treatment, the cells were incubated with fresh medium containing 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; AMRESCO, Solon, OH, USA) and incubated at 37°C for an additional 4 h. The medium was removed, and formazan was dissolved in 100 μL of dimethyl sulfoxide. The plates were then shaken for 5-10 min, and the absorbance at 570 nm was measured using a microplate reader (Tecan Trading, Mannedorf, Switzerland). The relative cell viability (%) was expressed as a percentage relative to the vehicle treated group.
The cells were harvested and lysed with RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) and the resulting protein samples were quantified using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Equal amounts of protein extracts were denatured by boiling at 100°C for 5 min in Laemmli sample buffer (Bio-Rad, Hercules. CA, USA). The proteins were separated by 8-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 buffer (TBS-T) (10 mM Tris, 150 mM NaCl, pH 7.5, and 0.1% Tween 20) for 1 h at room temperature, and incubated with primary antibodies (diluted 1:1,000) overnight at 4°C. The membranes were then washed three times for 10 min each with TBS-T buffer and incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies. The membranes were then washed three times for 10 min each with TBS-T buffer, and incubated with Super-signal pico-chemiluminescent substrate or dura-luminol substrate (Thermo Fisher Scientific) according to the manufacturer’s instructions, and visualized using an ImageQuant LAS 4000 system (Fujifilm Life Science, Tokyo, Japan).
The wound healing assay was conducted to determine the migratory ability of cells. Briefly, the cells were cultured at a concentration of 5×105 cells/mL in 6-well plates, and incubated until the cell density reached 90%. The cell monolayers were wounded by scratching with a 10-μL pipette tip, washed with phosphate-buffered saline (PBS) to remove detached cells, and then incubated in DMEM supplemented with 5% FBS containing appropriate reagents according to the experimental design. After 24 h at 37°C, the medium was replaced with PBS and washed twice. The gap-closure was then examined using a microscope (Olympus, Tokyo, Japan).
The invasion capacity of Caki-1 cells was determined
The gelatin zymography assay was performed to assess the proteolytic activities of MMPs by measuring their capability to degrade gelatin. To assess the enzymatic activity of MMPs, culture supernatants were collected, mixed with non-reducing sample buffer, and subjected to 10% SDS-PAGE containing 0.25% gelatin. The gel was washed three times with wash buffer (2.5% Triton X-100), and then incubated in incubating buffer (1 M Tris-HCl at pH 7.5, 5% NaN3 and 1 M CaCl2) overnight at 37°C. The gel was then stained with Coomassie Blue (Bio-Rad) and the MMP-9 enzymatic activity was quantified using the LAS 4000 imaging system (Fujifilm Life Science, Tokyo, Japan).
All data were obtained from more than three independent experiments. Statistical analysis was conducted using the paired Student’s
TQ is a phytochemical extracted from black cumin seeds and classified as a monoterpene based on its chemical structure as shown in Fig. 1A. To evaluate its effect on RCC migration, we utilized a human metastatic RCC Caki-1 cell line. Caki-1 cells were treated with various concentrations (1, 5, and 10 µM) of TQ for 24, 48, and 72 h. The MTT assay showed that TQ did not induce either cell death or cell proliferation, even after 72 h post-treatment (Fig. 1B). All the subsequent experiments were, therefore, performed at 24 h after treatment.
While TQ did not induce cancer cell death in Caki-1 cells, both wound-healing migration (Fig. 2A, upper panel) and invasion (Fig. 2A, bottom panel) assays showed that TQ significantly inhibited the migratory/invasive activity of Caki-1 cells with a high metastatic potential. Thus, we examined the effect of TQ on the activity and expression of MMP-9, a crucial enzyme for wound healing, cell spreading, and migration. Treatment with TQ reduced the proteolytic activity of MMP-9 in a concentration-dependent manner in Caki-1 cells (Fig. 2B). In addition to its activity, TQ also attenuated MMP-9 expression at the protein level (Fig. 2C). However, TQ was not able to modulate either the protein expression or enzymatic activity of MMP-2 (Fig. 2B, 2C). Taken together, these data suggested that TQ inhibited migration of human metastatic renal carcinoma Caki-1 cells by suppressing MMP-9.
Previously, we reported that the PGE2-EP2/EP4 axis was crucial for the migratory activity in Caki-1 cells (Woo
To verify the effect of TQ on PGE2-EP2 axis-induced migration, Caki-1 cells were co-treated with TQ plus either PGE2 or butaprost, an EP2 selective agonist. As previously reported, we confirmed that both PGE2 and butaprost remarkably accelerated wound closure (Fig. 3A, 3C) and invasion (Fig. 3B, 3D) of Caki-1 cells. In the presence of TQ, however, the PGE2- or butaprost-induced wound healing and invasion were markedly decreased (Fig. 3). These results showed that TQ suppressed the PGE2-EP2 axis-stimulated migration/invasion in Caki-1 cells.
As shown in Fig. 2, TQ inhibited migration and MMP-9 activity, simultaneously suppressing the expression of EP2, phosphorylation of Akt, p38, and the MMP-9 upstream regulator. We thus assumed that Akt and p38 might be responsible for the PGE2/EP2 axis-induced MMP-9 activation and subsequent migration in Caki-1 cells. To test this hypothesis, we examined whether the EP2 selective agonist, butaprost, regulated the Akt and p38 signaling pathways in Caki-1 cells. Butaprost induced phosphorylation of both Akt and p38 in Caki-1 cells (Fig. 4A). Moreover, butaprost increased MMP-9 proteolytic activity (Fig. 4B) as well as protein expression levels (Fig. 4C) in a concentration-dependent manner. These data indicated that the PGE2-EP2 axis may stimulate migration through activation of Akt and p38 in Caki-1 cells.
To verify the involvement of the Akt and p38 signaling pathways, pharmacological inhibitors of Akt and p38, LY294002 and SB203580, respectively, were selected for subsequent studies. First, the effect of the PI3K/Akt inhibitor LY294002 on butaprost-induced Caki-1 cell migration was examined. The wound healing assay showed that treatment with LY294002 significantly reduced closure of open scratches stimulated by butaprost in Caki-1 cells (Fig. 5A, upper panel). In addition, LY294002 attenuated butaprost-induced invasion (Fig. 5A, bottom panel), further supporting the involvement of PI3K/Akt signaling in the PGE2-EP2 axis-promoted migration. Western blot analysis confirmed the inhibitory effect of LY294002 on Akt activation in Caki-1 cells (Fig. 5B). Together, the results showed that inhibition of the PI3K/Akt pathway by LY294002 treatment blocked butaprost-induced MMP-9 expression (Fig. 5B) as well as its enzymatic activation (Fig. 5C).
We also investigated the effects of the p38 inhibitor, SB203580, in the same manner as shown in Fig. 5. SB203580 also had similar inhibitory effects on butaprost-induced Caki-1 cell migration/invasion (Fig. 6A). SB203580 treatment inactivated p38 in a concentration-dependent manner, which led to the attenuation of MMP-9 induction by butaprost (Fig. 6B, 6C).
Although cancer treatment strategies have been evolving rapidly, including patient-derived xenograft and immunotherapies, the treatment of metastatic RCC using cancer therapies has not undergone similar advances. At the time of diagnosis, metastasis is found in more than half of RCC patients, who often cannot be treated using conventional chemotherapy or radiotherapy (Grimm
The metastatic process can be initiated by cancer cell migration, requiring proteolytic degradation of the ECM that acts as a physical barrier against cell migration (Nabeshima
TQ is a phytochemical derived from the seeds of
In response to inflammatory stimuli, COX-2 induces eicosanoid synthesis from arachidonic acids, which can modulate cell proliferation, inflammation, and immune responses (Nakanishi and Rosenberg, 2013). Among the eicosanoids produced by COX-2 activation, PGE2 is the most potent protumorigenic eicosanoid that promotes cell proliferation, proinflammatory cytokine stimulation, and cancer cell migration (Greenhough
Here, we found that activation of PI3K/Akt and p38 was a missing link between the PGE2-EP2 axis and MMP-9 in Caki-1 cells. It has been reported that proinflammatory cytokines, such as IL-1β and TNFα, promote the secretion of MMP-9 via upregulation of PI3K/Akt and mitogen-activated protein kinases (Ruhul Amin
As described in Fig. 7, TQ inhibits the migration of RCC cells via inactivation of Akt and p38, as well as decreased MMP-9 activity, by decreasing PGE2-EP2 expression in RCC cells. However, more investigations are needed to determine whether Akt and p38 occur continuously or independently in regulating the activity of MMP-9 in RCC. Taken together, these findings suggested that TQ is a promising antimetastatic drug to treat human RCCs. The phytochemical TQ has long been used as a food additive, ensuring its nontoxicity, which should benefit patients during long-term therapy. Moreover, it can be utilized as an adjuvant for combination therapy or immunotherapy, which would be helpful, at least in part, in the establishment of the most effective treatment strategy in clinical settings.
This research was supported by the Basic Research Grant of Keimyung University in 2019.
The authors declare that they have no competing interests.
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