RCE-4 (Fig. 1A) was isolated from the whole plant of Reineckia carnea and purified at the Hubei Key Laboratory of Natural Products Research and Development (China Three Gorges University); it was dissolved in DMSO at a stock concentration of 10 mM, and diluted to the indicated concentration with RPMI-1640 medium. Antibodies against Bax, Bcl-2, cytochrome c and HRP-labeled secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Annexin V-FITC/PI apoptosis assay kit, Cell Cycle assay kit and JC-1 mitochondrial membrane potential (Δψm) assay kit were obtained from KeyGen Biotech Co., Ltd. (Nanjing, China). The NE-PER nuclear and cytoplasmic extraction kit were obtained from Thermo Scientific Pierce.

The cancer cell lines CaSki, HT-29, CNE-2 and the normal cell lines Marc-145 and MDCK, were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Science, Shanghai, China. All cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum, 25 mM HEPES buffer, 2 mmol/l L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Cultures were incubated in a humidified atmosphere of 5% CO₂ at 37°C.

Cells (1×10⁴/well) were seeded in supplemented culture medium (100 μl/well) in a 96-well plate and incubated for 24 h. Then the medium was replaced with a drug-containing medium, and the cells were further incubated for 48 h. All experiments were run in parallel with controls (0.1% DMSO) and the cell viabilities were evaluated by MTT assays. The absorbance of formazan formed was measured at 570 nm by a microplate reader. Each experiment was repeated at least 3 times.

CaSki cells treated with DMSO or RCE-4 were collected by trypsinization, washed twice with PBS, and then fixed with 0.5 ml of ice-cold glutaraldehyde (2.5% in 0.1 cacodylate buffer, pH 7.4) at 4°C overnight. The subsequent steps were performed according to standard procedures, including fixing, incubation, rinsing, gradient dehydration, embedding and ultrathin sections. Ultrathin sections were doubly stained with uranyl acetate and lead citrate and analyzed by transmission electron microscopy (Hitachi H-7500).

Early apoptosis was assessed by detecting surface exposure of phosphatidylserine (PS) in cells using an Annexin V-FITC/PI kit. Briefly, CaSki cells (2×10⁵) were seeded into a 100-ml culture flask and incubated for 12 h. Then, cells were treated with RCE-4 of 2.5, 5 and 10 μM for 24 h. The cells (both adherent and floating cells) were collected and treated according to the manufacturer’s instructions. Finally, the cells were analyzed with FITC/PI double-staining using a flow cytometer (Beckman Coulter, USA) with the single beam at 488 nm excitation.

To investigate the effect of RCE-4 on the cell cycle distribution, CaSki cells (2×10⁶) were subcultured into culture flasks and treated with 2.5, 5 and 10 μM of RCE-4 for 24 h. The cells were resuspended in 2 ml of 70% ice-cold ethanol solution and fixed at 4°C overnight. After washing with PBS, the pellets were resuspended in 100 mg/ml PI solution containing 100 mg/ml RNase, and then incubated at 37°C for at least 30 min. The cellular DNA content was then detected by flow cytometer.

The change in mitochondrial membrane potential (Δψm) was measured using a JC-1 fluorescent probe assay kit, according to the kit’s instructions. Briefly, following RCE-4 treatment of 2.5, 5 and 10 μM for 24 h, the cells were washed with PBS and incubated for 30 min with JC-1 at 37°C. After washing in PBS twice, the cells were subjected to two-color analysis by a flow cytometer.

To examine the role of Bcl-2 family members in RCE-4-induced apoptosis, we measured the gene expression of Bax and Bcl-2 using qPCR. CaSki cells were treated with 10 μM of RCE-4 for 0, 6, 12 and 24 h in 6-well plates. Total cellular RNA was isolated using the TRIzol Reagent. The qPCR reaction was carried out on the LightCycler 2.0 instrument (software v4.0; Roche Applied Science) using the double-stranded DNA dye SYBR-Green I strategy. The oligonucleotide sequences of the PCR primers used herein were designed based on the human mRNA encoding the respective genes. Quantification was based on threshold cycle (Ct) difference performed according to the ΔΔCt method, using the following equation: expression ratio=2^-ΔΔCt, where ΔΔCt = (Ct target - Ct reference) time x - (Ct target - Ct reference) time 0. The expression level of each target gene was normalized to that of glyceraldehyde-3 phosphate dehydrogenase (GAPDH), which fulfils the requirements for validation of reference genes.

CaSki cells (6×10⁶) were treated with 10 μM RCE-4 for 0, 6, 12 and 24 h. The cells were harvested and washed with cold PBS twice. Cell pellets were lysed in 40 μl lysis buffer (20 mM HEPES/NaOH, pH 7.5, 250 mM sucrose, 10 mM KCl, 2 mM MgCl₂, 1 mM EDTA, 1 mM DTT, protease inhibitor cocktail) for 20 min on ice. The lysis solution was centrifuged at 25,000 × g for 10 min at 4°C and protein contents in the supernatant were measured using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA, USA). The protein expression of Bax, Bcl-2, and cytochrome c were detected by western blot analysis. Briefly, equal amounts of protein were electrophoresed on 12% SDS acrylamide. Following electrophoresis, the proteins were transferred from the gel to a PVDF membrane. Non-specific binding was blocked with 5% skim milk in TBST buffer (5 mM Tris-HCl, pH 7.6, 136 mM NaCl, 0.05% Tween-20) at 4°C overnight. Blots were incubated at 37°C for 4 h each with primary and secondary antibody conjugated with peroxidase (HRP)-labeled anti-rabbit/mouse IgG. Blots were developed with ECL western blotting detection reagents (Multi Sciences). Experimental values were normalized to β-actin reactivity.

Statistical analyses were performed using Prism software (GraphPad, San Diego, CA, USA). Data are presented as the means ± SEM. Data were analyzed by one-way ANOVA for multiple comparisons. P<0.05 was considered to indicate statistically significant differences.

The structure of RCE-4 was identified as (1β,3β,5β,25S)-spirostan-1,3-diol1-[α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranoside] (Fig. 1A).

MTT assays were performed to investigate the effects of RCE-4 on the proliferation of tumor and normal cells. After treatment for 48 h with different concentrations, RCE-4 exhibited the greatest growth inhibitory effect against CaSki cells, followed by HT-29, CNE2 cells and much less cytotoxicity to Marc 145 and MDCK normal cells (~6-fold higher IC₅₀) (Fig. 1B). The IC₅₀ values were 3.37, 4.31, 4.81, 18.73 and 19.75 μM for CaSki, HT-29, CNE2, Marc-145 and MDCK, respectively. These results indicated that RCE-4 had a good growth-inhibitory effect on CaSki cells in a dose-dependent manner and relatively high selectivity.

To evaluate cellular ultrastructure for indications of the mode of death, we compared RCE-4-treated and untreated CaSki cells using transmission electron microscopy. Inspection of the ultrastructural details revealed the presence of RCE-4-induced apoptosis. CaSki control cells (Fig. 2A) showed a clear nucleus, nuclear membrane and nucleoli, irregular cell surface with cells very densely packed together. Following treatment with different concentrations of RCE-4 (2.5, 5 and 10 μM), cells exhibited pronounced morphological changes and typical apoptosis features (Fig. 2B-D), including cell shrinkage, nuclear fragmentation, and chromatin condensation. The nucleus appeared to have broken down and their volume decreased. The nucleoli were also absent and no subcellular organelles were observed. Furthermore, irregular cell surface disappeared and a large number of vacuoles in the cytoplasm was present.

To further differentiate between apoptosis or necrosis, the cytotoxic effects of RCE-4 on CaSki cells were evaluated using the early marker of apoptosis Annexin V, and the dead cell marker PI. Numerous studies have reported that advanced nuclear fragmentation is preceded by alteration in the plasma membrane, such as PS externalization (17). Hence, cells treated with RCE-4 of 2.5, 5 and 10 μM for 24 h were double stained with Annexin V-FITC/PI and analyzed by flow cytometry. Apoptotic cells were determined by counting the percentage of early apoptosis in the upper left quadrant (Annexin V⁺/PI^-), and late apoptosis in the upper right quadrant (Annexin V⁺/PI⁺). Treatment with different doses of RCE-4 (2.5, 5 and 10 μM) for 24 h resulted in separately 9.5, 23.3 and 26.3% early apoptosis compared with the control (0.8%) (Fig. 3A). These results suggest that RCE-4 can effectively induce apoptosis, and, particularly, early apoptosis. The necrotic cell population (Annexin V^-/PI⁺) did not change apparently following exposure to different concentrations of RCE-4, indicating that apoptosis is the preferential cell death induced by RCE-4 in CaSki cells.

An experiment was performed to evaluate the effect on the cell cycle phase distribution of CaSki cells after treatment with 2.5, 5 and 10 μM of RCE-4. Results shown in Fig. 3B indicate that there was a significant increase in the Sub-G1 DNA fraction (4.9, 7.5, 25.5 and 27.1%) at a concentration of 0-10 μM in a dose-depended manner, which responded to apoptotic cells. Results of this experiment demonstrated that RCE-4 arrested the cell cycle progression of CaSki cells at S phase. Compared with the control (5.8%), RCE-4 (2.5, 5 and 10 μM) led to an S phase increase of 8.4, 11.3 and 12.5%, respectively.

Apart form PS externalization, dissipation of Δψm has also been reported to be an early apoptosis event in several different systems. It is commonly used to detect mitochondrial depolarization that occurs in early apoptosis (18). To signal the loss of Δψm, JC-1 probe was applied to test the occurrence of apoptosis. As shown in Fig. 4, the majority of the untreated cells were identified in the upper right quadrant (FITC⁺/PI⁺). This corresponded to mitochondria with a polarized Δψm. However, following treatment with different concentrations of RCE-4 for 24 h, cells moved towards the upper/left region (FITC⁺/PI^-) and Δψm began to decrease, suggesting disruption of mitochondrial function. As can be seen in Fig. 4, RCE-4 treatment significantly decreased the Δψm in CaSki cells, compared with the control. These results were dose-dependent. These findings demonstrated that the RCE-4-induced apoptosis in CaSki cells involved mitochondria dysfunction associated with dissipation of the Δψm.

The Bcl-2 family plays an important regulatory role in apoptosis, either as an activator (Bax) or inhibitor (Bcl-2). Since RCE-4 showed the ability to interfere with the Δψm, we hypothesized that Bax and Bcl-2 are involved in the RCE-4-induced apoptosis. Therefore, we investigated the gene expression level of Bax and Bcl-2 using qPCR. As depicted in Fig. 5A, Bax gene expression increased following treatment with 10 μM RCE-4 and reached its peak at 6 h (~4.5-fold higher compared to the untreated). In addition, Bcl-2 gene expression was clearly suppressed as it was found to decrease 2-fold at 6 h and remained lower during the treatment hours (Fig. 5B). The increase in Bax and the decrease in Bcl-2 expression significantly elevated the Bax/Bcl-2 expression ratio (Fig. 5C).

In order to further prove that the anti-proliferative effect of RCE-4 was due to mitochondria-mediated apoptosis, CaSki cells were treated with RCE-4 at 10 μM for 6, 12 and 24 h and proteins implicated in apoptosis were evaluated using western blot analysis. The release of cytochrome c from mitochondria into cytosol induces the mitochondrial-dependent apoptotic pathway. Cytochrome c gradually increased in cytosol with a time-dependent increase as exposed to RCE-4 (Fig. 5D). We further investigated the involvement of Bcl-2 and Bax, the key regulatory factor, when cytochrome c was released into the cytosol during apoptosis induction. RCE-4 treatment resulted in the upregulation of Bax and downregulation of Bcl-2, leading to an increase in the Bax/Bcl-2 ratio (Fig. 5D). These results strongly indicate that apoptosis induced by RCE-4 in CaSki cells occurs via the mitochondria-dependent signal pathway.