Caki cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Dulbecco's modified Eagle's medium, containing 10% fetal bovine serum (FBS), 20 mM HEPES buffer and 100 μg/ml gentamicin was used as the culture medium in these experiments. PCR primers were purchased from Bioneer (Daejeon, Korea). The anti-Bcl-2, anti-Mcl-1 and anti-cIAP-2 were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti cFLIP_L antibody was obtained from ALEXIS Corp. (San Diego, CA, USA). Anti-PARP, and anti-caspase-3 antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-XIAP antibody was supplied by R&D systems (Minneapolis, MN, USA). Gambogic acid and the other chemicals were purchased from Sigma (St. Louis, MO, USA). Recombinant human TRAIL was obtained from KOMA Biotech (Seoul, Korea).

The cell counts were performed using a hemocytometer. Approximately 0.5×10⁶ Caki cells were suspended in 100 μl of PBS, and 200 μl of 95% ethanol was added while vortexing. The cells were incubated at 4°C for 1 h, washed with PBS, and resuspended in 250 μl of 1.12% sodium citrate buffer (pH 8.4) together with 12.5 μg of RNase. Incubation was continued at 37°C for 30 min. The cellular DNA was then stained by applying 250 μl of propidium iodide (50 μg/ml) for 30 min at room temperature. The stained cells were analyzed by fluorescent activated cell sorting (FACS) on a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA) to determine the relative DNA content based on the red fluorescence.

The cellular lysates were prepared by suspending 1.2×10⁶ cells in 100 μl of lysis buffer (137 mM NaCl, 15 mM EGTA, 0.1 mM sodium orthovanadate, 15 mM MgCl₂, 0.1% Triton X-100, 25 mM MOPS, 100 μM phenylmethylsulfonyl fluoride, and 20 μM leupeptin, adjusted to pH 7.2). The cells were disrupted by sonication and extracted at 4°C for 30 min. The proteins were electro-transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA, USA). The detection of specific proteins was carried out using an ECL Western blotting kit according to the manufacturer's instructions.

For transfection, the cells were plated onto 6-well plates at a density of 0.5×10⁶ cells/well and grown overnight. The cells were co-transfected with 2 μg of various plasmid constructs and 1 μg of pCMV-β-galactosidase plasmid for 5 h using the Lipofectamine method. After transfection, the cells were cultured in 10% FBS medium with a vesicle (DMSO) or drug for 24 h.

The expression of cFLIP_L mRNA was determined by RT-PCR. The total cellular RNA was extracted from the cells using an easyBlue reagent (Life Technologies, Seongnam, Korea), and the cDNA was prepared using M-MLV reverse transcriptase (Gibco-BRL, Gaithersburg, MD, USA), according to the manufacturer's instructions. The cellular RNA sample was reverse-transcribed with a random primer and then amplified by PCR, the GAPDH primer set was used as the internal control. The following primers were used to amplify the cFLIP_L and GAPDH. The sequences of the sense and antisense primer for cFLIP_L were 5′-CGG ACT ATA GAG TGC TGA TGG-3′ and 5′-GAT TAT CAG GCA GAT TCC TAG-3′, respectively. The sequences of the sense and antisense primer for GAPDH were 5′-AGG TCG GAG TCA ACG GAT TTG-3′ and 5′-GTG ATG GCA TGG ACT GTG GT-3′, respectively. The PCR products were analyzed by electrophoresis on a 1.5% agarose gel and detected by UV light.

The cells were fixed with 1% paraformaldehyde on a slide glass for 30 min at room temperature. After washing with PBS, 300 nM 4′,6′-diamidino-2-phenylindole (Roche, Mannheim, Germany) was added to the fixed cells for 5 min, and the cells were examined by fluorescence microscopy.

The data were analyzed by a one-way ANOVA followed by post-hoc comparisons (Student-Newman-Keuls) using the Statistical Package for Social Sciences 8.0 (SPSS Inc., Chicago, IL, USA).

To examine the anti-effects of GA in human renal cancer cells, Caki cells were treated with various concentrations of GA. With increasing GA concentration, the GA-treated Caki cells progressively showed the typical features of apoptosis, including cell shrinkage, rounding and detachment of the cell from the plate (Fig. 1A). Cell death was next determined by flow cytometry analysis to detect the hypodiploid cell populations. As shown in Fig. 1B, treatment of Caki cells with GA resulted in a significant increase in the accumulation of sub-G1 phase cells in a dose-dependent manner. In addition, the treatment of Caki cells with GA strongly led to a reduction of the protein levels of 32-kDa precursor together with the concomitant cleavage of PARP, a substrate protein of caspases (Fig. 1C).

This study next examined whether the activation of caspase pathway plays a critical role in GA-induced apoptosis. As shown in Fig. 2A, GA-mediated apoptosis was prevented partly by a pretreatment with a general and potent inhibitor of caspases, z-VAD-fmk. In contrast, treatment with z-VAD-fmk completely prevented these caspase-related events, such as the cleavage of PARP (Fig. 2B). These results suggest that the GA-induced cell death was mediated partly by the caspase-dependent pathway and caspase-independent cell death in the presence of z-VAD-fmk. Because AIF is involved in the induction of apoptotic cell death through the caspase-independent pathway, this study examined whether AIF plays a role in GA-induced apoptotic cell death. The translocation of AIF was analyzed by the observation of its release from the mitochondria and translocation to the nucleus by fluorescence microscopy. As shown in Fig. 2C, fluorescence microscopy showed that AIF was translocated to the nucleus and caused nuclear condensation after the treatment with GA. This suggests that GA-induced apoptotic cell death in Caki cells is mediated by AIF translocation from the mitochondria into the nucleus via a caspase-independent pathway.

Further experiments were conducted to determine if ROS generation induced by GA is associated directly with the induction of apoptosis because numerous studies have documented that GA induces apoptosis through ROS accumulation in human cancer cells (15,16). On the contrary, as shown in Fig. 3A and B, pretreatment with N-acetylcysteine (NAC), a ROS scavenger, did not prevent GA-induced cell death. In addition, pretreatment with NAC failed to prevent the cleavage of PARP (Fig. 3C). These results suggest that ROS generation is not critical for the induction of apoptosis by GA.

This study next examined whether the downregulation of cFLIP_L is critical to stimulate GA-induced apoptosis. The overexpression of cFLIP_L in Caki cells attenuated GA-mediated apoptosis, whereas treatment with GA-induced apoptosis in Caki/vector cells (Fig. 4C). This suggests that cFLIP_L downregulation also contributes to GA-induced apoptosis. The cleavage of PARP and procaspase-3 was also analyzed in both cell lines. As shown in Fig. 4D, the ectopic expression of cFLIP_L attenuated the cleavage of PARP and procaspase-3 induced by GA treatment in Caki cells. The levels of cFLIP_L and cFLIPs expression were checked after a treatment with various concentration of GA to determine if GA specifically downregulates cFLIP_L expression without affecting the levels of cFLI_Ps. The treatment with GA decreased the level of cFLIP_L expression in Caki cells. In contrast, no changes in the cFLIPs mRNA and protein levels were detected in the GA-treated cells (Fig. 4E and F). These results suggest that GA suppressed cFLIP_L expression with little effect on cFLIP expression in the present system. Interestingly, we found that GA treatment induced the cleavage of p65 protein, a subunit of nuclear factor-κB (NF-κB), subsequently reduction of p65 protein level in Caki cells (Fig. 4G). This result suggested the possibility that GA-induced cFLIP_L downregulation might be caused by inhibiting NF-κB pathway in our system.

In an attempt to search for novel strategies to overcome TRAIL resistance in cancer cells, this study examined the effects of a combination treatment of GA and TRAIL in Caki cells. The co-treatment of Caki cells with GA and TRAIL resulted in a marked increase in the accumulation of sub-G1 phase cells compared to Caki cells treated with GA or TRAIL alone (Fig. 5A). In addition, a combined treatment with GA and TRAIL induced chromatin condensation paralleled by nuclear fragmentation (Fig. 5B). Finally, the combinational treatment of Caki cells with GA and TRAIL led to a decrease in the protein levels of procaspase-3 with the concomitant cleavage of PARP protein (Fig. 5C).

This study next examined whether activation of the caspase pathway plays a critical role in GA plus TRAIL-induced apoptosis. As shown in Fig. 5D, GA plus TRAIL-induced apoptosis was prevented by a pretreatment with z-VAD-fmk, as determined by FACS analysis. In addition, z-VAD-fmk prevented these caspase-related events including the cleavage of procaspase-3 and PARP (data not shown). These results suggest that a combined treatment of GA and TRAIL-induced apoptosis was mediated by caspase-dependent apoptosis pathways. Furthermore, NAC partly prevented GA plus TRAIL-mediated apoptosis in Caki cells (Fig. 5E).