Caki, A498 and ACHN cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The culture medium used throughout these experiments was Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 20 mM HEPES buffer and 100 µg/ml gentamycin. The mouse kidney cells, TMCK-1, were a gift from Dr T.J. Lee (Yeungnam University, Korea). z-VAD-fmk was purchased from R&D Systems. Thymoquinone was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Anti-Bcl-2 (sc-783), anti-Bcl-xL (sc-634), anti-Mcl-1 (sc-819) and anti-cIAP2 (sc-7944) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-XIAP (610762) was purchased from BD Biosciences (Bedford, MA, USA). Anti-PARP (#9542) antibody was obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-actin (A5441) antibody was obtained from Sigma (St. Louis, MO, USA). Other reagents were purchased from Sigma Chemical Co.

For flow cytometry, the cells were resuspended in 100 µl of phosphate-buffered saline (PBS), and 200 µl of 95% ethanol was added while the cells were being vortexed. The cells were then incubated at 4°C for 1 h, washed with PBS, resuspended in 250 µl of 1.12% sodium citrate buffer (pH 8.4) with 12.5 µg of RNase and incubated for an additional 30 min at 37°C. The cellular DNA was then stained by adding 250 µl of a propidium iodide solution (50 µg/ml) to the cells for 30 min at room temperature (14). The stained cells were analyzed by fluorescent-activated cell sorting on a FACScan flow cytometer to determine the relative DNA content, which was based on the red fluorescence intensity.

For the western blot experiments, the cells were washed with cold PBS and lysed on ice in modified RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM Na₃VO₄ and 1 mM NaF) containing protease inhibitors (100 µM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin and 2 mM EDTA). The lysates were centrifuged at 10,000 × g for 10 min at 4°C and the supernatant fractions were collected. The proteins were separated by SDS-PAGE electrophoresis and transferred to Immobilon-P membranes. The specific proteins were detected using an enhanced chemiluminescence (ECL) western blotting kit according to the manufacturer's instructions.

DNA fragmentation was performed using the Cell Death Detection ELISAPLUS kit (Boehringer Mannheim, Indianapolis, IN, USA). Briefly, cells were centrifuged for 10 min at 200 × g, the supernatant was removed, and pellet was lysed for 30 min. After centrifuging the plate again at 200 × g for 10 min, and the supernatant that contained the cytoplasmic histone-associated DNA fragments was collected and incubated with an immobilized anti-histone antibody. The reaction products were incubated with a peroxidase substrate for 5 min and measured by spectrophotometry at 405 and 490 nm (reference wavelength) with a microplate reader. The signals in the wells containing the substrate alone were subtracted as background.

To evaluate DEVDase activity, cell lysates were prepared after their respective treatments with thymoquinone. Assays were performed in 96-well microtiter plates by incubating 20 mg of cell lysates in 100 µl of reaction buffer (1% NP-40, 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 10% glycerol) containing a caspase substrate [Asp-Glu-Val-Asp-chromophore-p-nitroanilide (DVAD-pNA)] at 5 mM. Lysates were incubated at 37°C for 2 h. Thereafter, the absorbance at 405 nm was measured with a spectrophotometer.

Total cellular RNA was extracted from cells using TRIzol reagent (Life Technologies, Gaithersburg, MD, USA). Complementary DNA was synthesized from 2 µg of total RNA using M-MLV reverse transcriptase (Promega, Madison, WI, USA). The cDNA for c-FLIP, Bcl-2 and actin were amplified by a PCR using specific primers: c-FLIP (forward) 5′-CGGACTATAGAGTGCTGATGG-3′ and (reverse) 5′-GATTATCAGGCAGATTCCTAG-3′; Bcl-2 (forward) 5′-GTCCTCAGCCCTCGCTCT-3′ and (reverse) 5′-CACCTAATTGGGCTCCATCT-3′; actin (forward) 5′-GGCATCGTCACCAACTGGGAC-3′ and (reverse) 5′-CGATTTCCCGCTCGGCCGTGG-3′. PCR products were analyzed by agarose gel electrophoresis and visualized using ethidium bromide staining.

Transient transfection was performed in 6-well plates. One day before the transfection, Caki cells were plated at ~60–80% confluence. The Bcl-2/-3254 promoter- or NF-κB-luciferase plasmid was transfected into the cells using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA). To assess the promoter-driven expression of the luciferase gene, the cells were collected and disrupted by sonication in lysis buffer (25 mM Tris-phosphate pH 7.8, 2 mM EDTA, 1% Triton X-100 and 10% glycerol), and aliquots of the supernatants were used to analyze the luciferase activity according to the manufacturer's instructions (Promega).

Intracellular accumulation of ROS was determined using the fluorescent probes 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). H2DCFDA is commonly used to measure ROS generation. Caki cells were pretreated with NAC for 30 min, and then added with thymoquinone. Cells were stained with the fluorescent dye H2DCFDA for an additional 10 min. Then, cells were observed using a fluorescence microscope (Axiovert 200M; Carl Zeiss, Oberkochen, Germany).

Rhodamine 123 and DiOC₆ (Molecular Probes, Carlsbad, CA, USA) uptake by mitochondria is directly proportional to its membrane potential. Caki cells 4 h after treatment were incubated with Rhodamine 123 (5 µM) and DiOC₆ for 30 min in the dark at 37°C (15). The cells were harvested and suspended in PBS. The MMP was subsequently analyzed using a flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA).

Caki cells (1.2×10⁶ cells/ml) were harvested, washed once with ice-cold PBS and gently lysed for 2 min in 80 µl ice-cold lysis buffer [250 mM sucrose, 1 mM EDTA, 20 mM Tris-HCl (pH 7.2), 1 mM DTT, 10 mM KCL, 1.5 mM MgCl₂, 5 µg/ml pepstatin A, 10 µg/ml leupeptin and 2 µg/ml aprotinin]. Lysates were centrifuged at 12,000 × g at 4°C for 10 min to obtain the supernatants (cytosolic extracts free of mitochondria) and the pellets (fraction that contains mitochondria). The resulting cytosolic fractions were used for western blot analysis with an anti-cytochrome c antibody.

The data were analyzed using a one-way ANOVA followed by post hoc comparisons (Student-Newman-Keuls) using the Statistical Package for Social Sciences version 22.0 (SPSS, Inc., Chicago, IL, USA).

We first determined the cytotoxic effect of thymoquinone against Caki cells and found that after treatment of thymoquinone (25, 50 and 75 µM) for 24 h apoptotic population (sub-G1) was increased in a dose-dependent manner (Fig. 1A). Furthermore, we used caspase inhibitor zVAD to examine the association of caspases in apoptosis induced by thymoquinone, and we found that after inhibition of caspase apoptotic population was decreased even at a higher concentration of thymoquinone which was further validated by DEVDase activity suggesting that thymoquinone induced caspase-dependent apoptosis (Fig. 1B and C). Moreover, the level of cytoplasmic histone was observed to be increased that is an outcome of DNA fragmentation thus thymoquinone-induced DNA fragmentation which resulted in increased cytoplasmic histone (Fig. 1D). We further determined the expression pattern of apoptotic and anti-apoptotic proteins to understand the molecular mechanism underlying the thymoquinone toxicity, as depicted in Fig. 1E, thymoquinone enhanced the cleavage of PARP protein in a dose-dependent manner. Expression of anti-apoptotic proteins such as c-FLIP and Bcl-2 was downregulated, but expression of Mcl-1, Bcl-xL and cIAP2 did not change in western blot examination (Fig. 1E).

We found that thymoquinone downregulated the expression of c-FLIP in our previous experiment, next we decided to validate our data. We then examined the expression pattern of c-FLIP at the transcriptional level, results of RT-PCR analysis showed that there was no any change in the expression of c-FLIP at the transcriptional level (Fig. 2A). Therefore, we investigated whether thymoquinone modulates the protein stability of c-FLIP in Caki cells. Cells were treated with cycloheximide (CHX), an inhibitor of de novo protein synthesis, in the presence or absence of thymoquinone. CHX gradually decreased c-FLIP, but co-treatment with CHX and thymoquinone reduced more c-FLIP protein expression (Fig. 2B). To investigate the importance of down-regulation of c-FLIP expression on thymoquinone-induced apoptosis, c-FLIP protein was overexpressed in Caki cells. Overexpression of c-FLIP markedly inhibited thymoquinone-induced increase of sub-G1 cell population (Fig. 2C) and PARP cleavage (Fig. 2D). These results indicate that downregulation of c-FLIP may be involved in thymoquinone-mediated apoptosis.

We evaluated the effect of thymoquinone on the expression pattern of Bcl-2, another major regulator of the anti-apoptotic mechanism. Results are depicted in Fig. 3, where we first determined the expression of Bcl-2 at the transcriptional level and found that Bcl-2 mRNA expression was reduced in a dose-dependent manner (Fig. 3A). Furthermore, we validated our data with Bcl-2 promoter assay and found that thymoquinone reduced the Bcl-2 promoter activity in a dose-dependent manner (Fig. 3B). Moreover, we determined the effect of thymoquinone on NF-κB expression as it is associated with expression of Bcl-2 protein. NF-κB luciferase activity was reduced after treatment of thymoquinone. In addition, overexpression of p65, a subunit of NF-κB heightened the Bcl-2 promoter activity in case of thymoquinone treated cells, which was further confirmed by immunoblot analysis (Fig. 3D). We examined the efficacy of thymoquinone to induce apoptosis in Bcl-2 overexpressed Caki cells and observed that overexpression of Bcl-2 reversed the apoptotic potential of thymoquinone as evidenced by flow cytometric analysis (Fig. 3E). Moreover, thymoquinone-induced cleavage of PARP protein was blocked in Bcl-2 overexpressed cells (Fig. 3E), suggesting the downregulation of Bcl-2 plays a critical role in thymoquinone-induced apoptosis.

The induction of ROS production plays an important role in apoptosis. Therefore, we carried out H2DCFDA staining to determine the level of intracellular ROS. Thymoquinone (75 µM) potentially induced the generation of intracellular ROS within 10 min, and then slightly reduced after 30 min of treatment (Fig. 4A and B). Furthermore, we determined the effect of thymoquinone-induced ROS on cell death with or without using the ROS scavenger N-acetylcysteine (NAC). Thymoquinone-induced ROS generation caused apoptosis in Caki cells which was reduced after using the ROS scavenger (Fig. 4C). We observed increased intensity of MitoSOX Red dye which detects mitochondrial ROS production (Fig. 4D). Furthermore, we determined the MMP, as the exaggerated production of ROS leads to the mitochondrial damage. Rhodamine 123 and DiOC₆ fluorometry data revealed the loss of MMP in time-dependent manner at 75 µM thymoquinone treatment (Fig. 4E and F). Treatment with thymoquinone caused cytochrome c release into cytoplasm (Fig. 4G). These results suggest that thymoquinone reduces the MMP levels and induces cytochrome c release.

Although, our results demonstrated that thymoquinone potentially induced apoptosis in Caki cells, we further examined the efficacy of thymoquinone against other renal carcinoma cell line for example ACHN and A498 and found that thymoquinone has efficacy to induce apoptosis in all the renal carcinoma cells used in the present study (Fig. 5A and B). However, there was the insignificant cytotoxic effect of thymoquinone on normal TMCK-1 cells neither on both morphology and cell population analysis, even at a higher concentration of 75 µM which induced the apoptosis in Caki cells (Fig. 5C and D) suggesting the safe use of thymoquinone.