The Caki cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The culture medium used throughout these experiments was Dulbecco’s modified Eagle’s medium (DMEM), containing 10% fetal calf serum (FCS), 20 mM HEPES buffer and 100 μg/ml of gentamycin. Anti-Bcl-2, anti-PARP, anti-pro-caspase-3, anti-Mcl-1, and anti-actin antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-c-FLIP antibody was purchased from Alexis Corp. (San Diego, CA, USA). Dioscin was isolated from Polygonatum zanlanscianse PAMP) and were directly added to cell cultures at the indicated concentrations. N-acetyl-L-cysteine (NAC) and pan-caspase inhibitor (Z-VAD-FMK) were purchased from Calbiochem (San Diego, CA, USA).

The root of Dioscorea nipponica Makino was obtained from the Uiseong Medicinal Farm (Uiseong, Korea). Three kilograms of the roots were extracted three times with 5 liters of methanol each time. After filtration, the extract was evaporated in vacuo to give 115 g of dry sample. The following procedures of purification of dioscin based on silica-gel chromatography were the same as previously reported (12). The purified compound was identified as dioscin by analyses of IR spectroscopy (Perkin-Elmer, Shelton, CT, USA) and 1H- and 13C-NMR spectroscopy (Bruker AMX 300, Rheinsten, Germany).

The purity of dioscin was confirmed by HPLC analysis as was previously reported (12). Dioscin and its derivatives, such as prosapogenin A and prosapogenin C were determined by HPLC system comprising an SCL-10A system controller, LC-10AD pump and SPD-10A UV detector (Shimadzu, Japan). The analytical column was a Mightysil RP-C18 GP-250 (Kanto Chemical Co., USA). The mobile phase for HPLC consisted of 75% acetonitrile (v/v) with a flow rate of 0.7 ml/min. The column temperature was maintained at 30˚C. A 10 μl of the sample dissolved in methanol (1 mg/ml) was injected into the HPLC system, and the UV absorption at 215 nm was recorded. The retension time of dioscin was 3.25 min and the purity of dioscin was identified as above 98.5%.

Cellular lysates were prepared by suspending 6×10⁵ 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 phenylmethlsulfonyl fluoride, and 20 μM leupeptin, adjusted to pH 7.2). The cells were disrupted by sonication and extracted at 4˚C for 30 min. Lysates containing proteins were quantified using BCA protein assay kit (Pierce, Rockford, IL, USA). The proteins were electrotransferred to Immobilon-P membranes (Millipore Corp., Bedford, MA, USA). Detection of specific proteins was carried out with an ECL western blotting kit (Millipore) according to the manufacturer’s instructions.

Cell counts were performed using a hemocytometer. Approximately 1×10⁶ Caki cells were suspended in 100 μl of PBS, and 200 μl of 95% ethanol were 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 FACScanto flow cytometer for relative DNA content based on red fluorescence.

Total cellular RNA was extracted from cells using the Easy-blue Total RNA Extraction kit (iNtRon, Sungnam, Korea). A cDNA was synthesized from 5 μg of total RNA using M-MLV reverse transcriptase (Promega, Madison, WI, USA). The cDNAs for c-FLIP, Bcl-2 and actin were amplified by PCR with specific primers. The sequence of the sense primer for c-FLIPL was 5′-CGG ACT ATA GAG TGC TGA TGG-3′ and the antisense primers were 5′-GAT TAT CAG GCA GAT TCC TAG-3′. PCR products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide.

Three or more separate experiments were performed. Statistical analysis was done by paired Student’s t-test or ANOVA. A P-value <0.05 was considered to have pronounced difference between experimental and control groups.

To investigate the effect of dioscin-induced apoptosis, human renal carcinoma Caki cells were treated with various concentrations of dioscin. Two established criteria were subsequently used to assess apoptosis in this study. Apoptosis was determined in Caki cells using flow cytometry analysis demonstrating hypo-diploid DNA. Fig. 1A shows treatment with dioscin in Caki cells resulted in a markedly increased accumulation of sub-G1 phase in a dose-dependent manner of dioscin. Because cells undergoing apoptosis executed the death program by activating caspases and cleavage of PARP, we analyzed expression levels of pro-caspase-3, and cleavage of PARP. As demonstrated in Fig. 1B, exposure to dioscin led to a reduction of the 32-kDa precursor, accompanied by a concomitant revealed cleavage of PARP. Next, we analyzed nuclear condensation, which is another hallmark of apoptosis. Combinatory treatment with dioscin plus TRAIL induced nuclear condensation in Caki cells. In contrast, nuclear condensation in Caki cells treated with TRAIL alone or dioscin alone was barely detected.

In an attempt to search for novel strategies to overcome TRAIL resistance in cancer cells, we investigated the effect of the combined treatment with dioscin and TRAIL in Caki cells. Co-treatment of Caki cells with dioscin and TRAIL resulted in a markedly increased accumulation of sub-G1 phase cells, compared with Caki cells treated with dioscin or TRAIL alone (Fig. 2A). In addition, combinatory treatment of Caki cells with dioscin and TRAIL strongly stimulated reduction of the protein levels of pro-caspases 3, Bcl-2, Mcl-1, and c-FLIP_L (Fig. 2B).

To investigate the underlying mechanisms involved in dioscin enhanced TRAIL-induced apoptosis, we analyzed the changes in the expression levels of various apoptosis-regulating proteins. Bcl-2, Mcl-1 and c-FLIP_L protein expressions were decreased by the indicated concentrations of dioscin-treated Caki cells in a dose-dependent manner. To further elucidate the mechanism responsible for the changes in amounts of proteins level, we determined the levels of Bcl-2, Mcl-1 and c-FLIP_L mRNAs by RT-PCR. c-FLIP_L and Mcl-1 mRNA levels remain constant through the dioscin treatment at different doses in Caki cells. We found that dioscin treatment of Caki cells dose-dependently decreased the mRNA levels of Bcl-2 from RT-PCR analysis, suggesting that dioscin modulates Bcl-2 expression at the transcriptional level and c-FLIP_L and Mcl-1 at the post-transcriptional level (Fig. 3).

We next examined whether activation of caspase pathway plays a critical role in dioscin plus TRAIL-induced apoptosis. As shown in Fig. 4A, dioscin plus TRAIL-induced apoptosis was completely prevented by pre-treatment with a general and potent inhibitor of caspases, z-VAD-fmk, as determined by FACS analysis. These results suggest that the combined treatment with dioscin and TRAIL-induced apoptosis was mediated by caspase-dependent apoptosis pathways. We also found that z-VAD-fmk prevented all these caspase-related events such as cleavage of pro-caspase-3 and PARP (Fig. 4B). Pretreatment with z-VAD–fmk recovered Mcl-1 protein which were downregulated by combination treatment with dioscon plus TRAIL to basal level, but z-VAD-fmk partly blocked dioscin plus TRAIL-induced downregulation of c-FLIP_L protein, indicating that the decreased c-FLIP_L protein level was partly caused by caspase activation. These results suggested the possibilities that the decreased c-FLIP_L protein was partly caused by caspase-independent pathways (Fig. 4B).

To further clarify the underlying mechanisms of the decreased c-FLIP_L protein level in dioscin-treated cells, we performed c-FLIP_L protein stability test. Caki cells were treated with cycloheximide (CHX) and dioscin for different doses. We found that the degradation of c-FLIP_L protein was facilitated by dioscin treatment (Fig. 4C), implying that dioscin treatment caused reduction of c-FLIP protein stability.

Numerous investigations have documented that ROS may play an important role during apoptosis induction (13,14). It has been reported that dioscin increases ROS production in various cancer cells (5,10). Therefore, we investigated whether ROS generation is directly associated with dioscin plus TRAIL-induced apoptosis. As shown in Fig. 5A, dioscin plus TRAIL-induced apoptosis was completely prevented by pretreatment with NAC, as determined by FACS analysis. As shown in Fig. 5B, pretreatment with NAC decreased the increased expression levels of c-FLIP_L and Bcl-2 by dioscin treatment to basal levels, dioscon-induced downregulation of c-FLIPL protein was partly blocked by NAC treatment.

We examined whether dowregulation of c-FLIP_L by dioscon is critical to stimulate TRAIL-induced apoptosis. Overexpression of c-FLIP_L in Caki cells significantly attenuated dioscin-facilitated TRAIL-induced apoptosis, whereas co-treatment with dioscin plus TRAIL induced significant apoptosis in Caki/vector cells (Fig. 6). This result suggests that c-FLIP_L downregulation also contributes to dioscin-facilitated TRAIL-induced apoptosis.