Human laryngeal cancer HEp-2 cells were obtained from the American Type Cell Culture Collection and were cultured as previously described (20). Oridonin was isolated from Rabdosia rubescens and was identified by comparing its physical and spectroscopic (¹H NMR, ¹³C NMR) data with those reported in the literature (21). The purity was measured by HPLC (column: 4.6×150 mm, Inertsil ODS-SP, 5 μm; solvent phase: methanol/H₂O, 55:45) and determined to be 99.6%. Oridonin was dissolved in DMSO to obtain a stock solution. The DMSO concentration was kept below 0.05% in all the cell cultures so that it had no detectable effect on cell growth or cell death.

HEp-2 cells were incubated in 96-well tissue culture plates. After a 24-h incubation, the cells were treated with, or without, pan-caspase inhibitor (z-VAD-fmk), caspase-9, -8, -3, -1 inhibitors [Ac-LEHD-cmk (C9i), z-IETD-fmk, z-DEVD-fmk or Ac-YVAD-cmk], 3-MA or NAC (Sigma) at the given concentrations for 1 h and subsequently treated with oridonin for 24 h. The cytotoxic effect was measured by MTT assay as described elsewhere (20).

HEp-2 cells were seeded into 6-well culture plates. After a 24-h incubation, the cells were treated with, or without, C9i or NAC at the given concentrations for 1 h and subsequently incubated with oridonin for 24 h. The cellular morphology was observed using a phase contrast microscope (Leica, Nussloch, Germany).

HEp-2 cells were treated with 36 μM oridonin for the indicated time periods. The collected cells were fixed with PBS containing 3% glutaraldehyde, and postfixed with PBS containing 1% OsO₄. The samples were dehydrated in graded alcohol solutions, then embedded and sectioned. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined using a JEM-1200 transmission electron microscope (Jeol, Tokyo, Japan) (22).

After chemical treatment, 1×10⁶ cells were harvested. The collected cells were fixed in 70% ethanol, stained for DNA content with propidium iodide (PI), and measured by flow cytometry (Becton-Dickinson, Franklin Lakes, NJ, USA) as previously described (20). The sub-G1 DNA content was used as an indicator of apoptosis (23).

After treatment with 36 μM oridonin for the indicated time periods, the cells were incubated with 10 mM DCF-DA (Sigma) at 37°C for 30 min. The intracellular ROS caused oxidation of DCF-DA to the fluorescent compound DCF. Then, the cells were harvested and the pellets were suspended in 1 ml PBS. Samples were analyzed at an excitation wavelength of 480 nm and an emission wavelength of 525 nm by FACScan flow cytometry (18).

The mitochondrial membrane potential was measured using the fluorescent dye rhodamine-123 (Sigma) by flow cytometry as previously described (20).

After incubation with 36 μM oridonin for the fixed times, cells were cultured with 0.05 mM monodansylcadaverine (MDC) at 37°C for 60 min. The cellular fluorescent changes were observed under a fluorescence microscope (Olympus). The fluorescence intensity of cells was analyzed by FACScan flow cytometry (24).

A caspase-9 fluorimetric assay kit from Chemicon International (Temecula, CA, USA) was used according to the manufacturer's instructions. Briefly, cell lysates were incubated with peptide substrate, LEHD-pNA (Ac-Leu-Glu-His-Asp-pNA), in assay buffer [100 mM NaCl, 50 mM HEPES, 10 mM DTT, 1 mM EDTA, 10% glycerol, 0.1% CHAPS (pH 7.4)] for 2 h at 37°C. The release of p-nitroaniline was monitored at 405 nm. Caspase-9 activity in cell lysates was expressed as the x-fold increase from baseline controls.

HEp-2 cells were transfected with caspase-9 small interfering RNA (siRNA) and control siRNA (Shanghai GenePharma, China) by Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The sequences of the sense strands of the RNAs used in this study were as follows. Control siRNA: UUC UCC GAA CGU GUC ACG. Caspase-9 siRNA-1: CGG UGA AAG GGA UUU AUA ATT. Caspase-9 siRNA-2: CCA AAG UUG UCG AAG CCA ATT CGU UGA. Caspase-9 siRNA-3: GUG ACA UCU UUG UGU CCU ATT.

HEp-2 cells at 70% confluence were transfected with control pcDNA3.1-3FLAG and pcDNA3.1-hCASP9-3FLAG plasmid (Shanghai GeneChem, China). Transfection of cells was performed by using Lipofectamine 2000 reagent according to the manufacturer's protocol. The effectiveness of transfection was detected by western blot analysis.

HEp-2 cells were collected and then washed twice with ice-cold PBS. The cell pellets were resuspended in ice-cold homogenizing buffer (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethanesulfonyl fluoride, 10 μg/ml pepstatin and 10 μg/ml leupeptin). The cells were then homogenized and centrifuged at 14,000 g at 4°C for 60 min. The supernatant was used as the cytosol fraction and the pellet was resuspended in lysis buffer as the membrane fraction (25).

Western blot analysis was performed as previously described (20) using corresponding antibodies against Beclin 1, LC3, Bax, Bcl-2, AIF, cytochrome c, ICAD, FADD, caspase-9, -8, -3, Hsp90, β-actin (Santa Cruz). Representative blots from three independent experiments are shown.

All the presented data and results were confirmed in at least three independent experiments. The data are expressed as means ± SD. Statistical comparisons were made by Student's t-test and P<0.05 was considered statistically significant.

Some of our preliminary studies have indicated that oridonin is able to inhibit the proliferation of human laryngeal squamous carcinoma HEp-2 cells, and the cell death is shown to be typical of apoptosis (17,20). Since it is well known that caspase family members play a pivotal role in the cell apoptosis process, the effect of caspase inhibitors on oridonin-induced HEp-2 cell death was examined. Unexpectedly, the C9i enhanced cell death due to oridonin stimuli, while z-DEVD-fmk, z-IETD-fmk and Z-VAD-fmk blocked the cytotoxity of the response to oridonin (Fig. 2A).

Further, we investigated whether inhibition of caspase-9 affected drug sensitivity. As shown in Fig. 2B, pre-treatment of HEp-2 cells with 5–20 μM C9i markedly enhanced the inhibitory ratio in a dose-dependent manner after treatment with oridonin. In addition, we examined cellular morphological changes by phase contrast microscopy. At 24 h, some of oridonin-treated HEp-2 cells became round, with shrunken nuclei and membrane blebbing, while untreated cells did not show these apoptotic characteristics (Fig. 2C). Whereas, the C9i-pre-treated group showed more marked apoptotic changes in a dose-dependent manner (Fig. 2C). Quantification of apoptotic cells by flow cytometric analysis also showed that combined treatment with oridonin and C9i caused more significant apoptosis, and the apoptotic ratio was observed to be significantly increased from 15.08% in oridonin alone-treated cells to 23.59, 31.54 and 37.26% in 5, 10 and 20 μM C9i pre-treated cells, respectively (Fig. 2D).

Dysregulation of cellular redox status can be a potent mechanism of cell death (26). Therefore, we tested the possibility that oridonin, with or without C9i, induces apoptosis through ROS accumulation. Compared with the control group, ROS generation was significantly increased after exposure to oridonin for 24 h. C9i effectively increased oridonin-induced intracellular ROS generation (Fig. 3A and B). As expected, the use of NAC, a well known ROS scavenger (27), almost completely blocked ROS generation induced by oridonin alone or in combination with C9i (Fig. 3A and B). Whereas, NAC not only completely prevented HEp-2 cell apoptosis induced by oridonin alone, but also reversed the cell apoptosis induced by combined treatment with oridonin and C9i (Fig. 2C and D). Collectively, these results demonstrated that caspase-9 inhibition amplified oridonin-induced oxidative stress, which leads to enhanced apoptosis.

It has been reported that ROS generation can lead to mitochondrial damage and membrane depolarization (28). Therefore, we investigated whether ROS targeted the mitochondria and thereby decreased the ΔΨm in our model. As shown in Fig. 3C and D, exposure of HEp-2 cells to oridonin for 24 h caused a marked loss of ΔΨm compared with the control group and C9i pre-treatment produced a progressive loss of ΔΨm. Notably, pre-treatment with NAC reversed the disruption in ΔΨm induced by oridonin alone or in combination with C9i (Fig. 3C and D). These results suggested that the oridonin, with or without C9i, was capable of inducing mitochondrial dysfunction and that the loss of ΔΨm might be affected directly by ROS generation.

To determine the possible causes and consequences of the ΔΨm decrease, we studied the presence of two proteins, pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2, which are involved in the regulation of mitochondrial permeability and two other proteins, cytochrome c and AIF, which are usually released from mitochondria during apoptosis. We found that oridonin treatment significantly increased and decreased levels of Bax and Bcl-2 proteins, respectively, in a time-dependent manner (Fig. 3E). The expression of cytochrome c was markedly elevated. Simultaneously, a visible increase in AIF levels was also detectable after 12- and 24-h incubation (Fig. 3E). However, NAC and catalase (CAT, hydrogen peroxide decomposer) pre-treatment completely reversed the changes in Bax, Bcl-2, cytochrome c and AIF (Fig. 3E).

To assess whether the Fas-mediated pathway was activated in oridonin-treated cells, the expressions of Fas-associated protein with death domain (FADD), caspase-8 and -3 were determined by western blot analysis. The expression of FADD was markedly elevated and there was clear cleavage of procaspase-8 after oridonin administration. A time-dependent cleavage of procaspase-3 was also observed in oridonin-treated cells (Fig. 3F). ICAD, an inhibitor of caspase-activated DNase (CAD), is a substrate for caspases such as caspase-3. When caspase-3 is activated by apoptotic stimuli, ICAD is cleaved, resulting in the release of CAD which appears to cause DNA fragmentation in the nuclei (29). As shown in Fig. 3F, oridonin induced degradation of ICAD in HEp-2 cells in a time-dependent manner. Addition of NAC and CAT markedly inhibited the changes in FADD, caspase-8, -3 and ICAD (Fig. 3F).

We first investigated the effect of oridonin on cell autophagy. The ultrastructural details displayed that the oridonin-treated cells possessed typical characteristics of autophagy. These characteristic changes included extensive cytoplasmic vacuolization, and some autophagic vacuoles contained degraded organelles (Fig. 4A). The formation of autophagic vacuoles was further assessed by MDC, a fluorescent dye specifically staining autophagosomes. As shown in Fig. 4A, control cells presented diffused staining, and oridonin treatment resulted in an extensive punctuate MDC staining pattern. Next, the levels of Beclin 1 and LC3, two important proteins involved in autophagy (10,30), were examined by western blot analysis. As shown in Fig. 4D, the level of Beclin 1 and conversion from LC3-I to LC3-II both increased with time after oridonin administration. These findings apparently reveal that autophagy is induced as a response of HEp-2 cells to oridonin treatment.

To explore the involvement of caspase-9 in the modulation of autophagy, the autophagic ratio was measured by flow cytometry. Compared with the oridonin treatment group, the autophagic ratio was significantly reduced by the combined use of oridonin and C9i (Fig. 4C). Accordingly, the treatment of C9i markedly suppressed Beclin 1 upregulation and the conversion from LC3-I to LC3-II (Fig. 4D), suggesting the autophagy-promoting effects of caspase-9.

To investigate the role of autophagy in oridonin-induced apoptosis in HEp-2 cells, 3-MA, a specific inhibitor of autophagy, was introduced. Treatment with 3-MA prior to the addition of oridonin induced a significant reduction in the number of MDC-labeled fluorescent particles in the cells (Fig. 4A). Flow cytometric analysis also indicated that pre-treatment with 3-MA markedly reduced the autophagic ratio compared with the group treated with oridonin alone (Fig. 4B). Simultaneously, oridonin-induced Beclin 1 activation as well as the modification of LC3-I to LC3-II were markedly blocked when the cells were pre-treated with 3-MA (Fig. 4D). However, pre-treatment of HEp-2 cells with 0.4–1.6 mM of 3-MA dramatically increased the inhibitory rate of cell growth in a dose-dependent manner after oridonin treatment (Fig. 4E). Further, inhibition of autophagy increased the oridonin-induced sub-G1 cell proportion (an index of apoptosis) in HEp-2 cells (Fig. 4F). Collectively, these results suggested that 3-MA inhibited oridonin-mediated autophagy and it also sensitized the cells to the cytotoxic actions of oridonin with induction of apoptosis. In short, oridonin-induced autophagy inhibits apoptosis in our model.

In the cytosol, cytochrome c binds to Apaf-1, allowing recruitment of caspase-9 and formation of apoptosome, resulting in caspase-9 activation and execution of cell death (31,32). Here, the expression of critical apoptosome complex-related proteins was examined by means of western blot analysis. As shown in Fig. 5A, the level of cytochrome c in mitochondria began to decrease at 6 h, which was consistent with the increase in cytochrome c in the cytosol. However, no apparent change was observed in the protein level of pro-caspase-9, and cleaved-caspase-9 could not be detected (Fig. 5A). Recently, several investigators have described negative regulation of the apoptosome complex by Hsp90 (33,34). Therefore, the effect of oridonin on expression of Hsp90 was examined. The result shows that oridonin treatment increased levels of Hsp90 in a time-dependent manner in HEp-2 cells (Fig. 5A).

Next, we directly examined the effect of drug treatments on caspase-9 activation using functional assays. The result shows that caspase-9 activity was activated in a time-dependent manner, and the maximal activity was seen after a 24-h incubation (Fig. 5B). As expected, marked suppression of the high levels of caspase-9 was noted in cells subjected to combination-treatment with C9i (Fig. 5B).

To determine whether the results obtained with C9i could be corroborated by another method, we turned to RNA interference using siRNA. Here, the role of caspase-9 in oridonin-mediated apoptosis was studied by knock-down of caspase-9 using 3 different specific siRNAs. The expression of capase-9 was markedly suppressed in HEp-2 cells when transfected with caspase-9 siRNA-1 while only slight downregulation was found with the other two siRNAs (Fig. 6A). Therefore, caspase-9 siRNA-1 was selected as a valid candidate for the subsequent investigation. As shown in Fig. 6A, caspase-9 siRNA-1 effectively and specifically suppressed HEp-2 caspase-9 protein in a dose-dependent manner. In contrast, an identical amount of control siRNA had no effect on caspase-9. We then tested whether caspase-9-deficient HEp-2 cells would undergo a heightened response to apoptotic stimuli. As shown in Fig. 6B, caspase-9-deficient HEp-2 cells were significantly more sensitive to oridonin than control siRNA-treated cells. Quantification of apoptotic cells by flow cytometric analysis showed that the incidence of apoptosis after oridonin treatment was increased by >10% in caspase-9 siRNA-1-transfected cells when compared with control siRNA (Fig. 6C). Next, the degree of ROS generation was also determined by flow cytometric analysis. The percentage of DCF-positive cells was highest in caspase-9 siRNA-1-transfected cells treated with oridonin (Fig. 6D). However, caspase-9-deficient HEp-2 cells showed a much reduced autophagic ratio after oridonin treatment when compared with those transfected with control siRNA (Fig. 6E). Accordingly, oridonin treatment, with or without control siRNA, enhanced both the expression level of Beclin 1 and the conversion from LC3-I to LC3-II, while these enhancements were markedly suppressed in HEp-2 cells transfected with caspase-9 siRNA-1 (Fig. 6F).

To test the ability of caspase-9 to inhibit oridonin-induced apoptosis, we transfected HEp-2 cells with a CAS9-expressing plasmid (pcDNA3.1-hCASP9-3FLAG) or control plasmid (pcDNA3.1-3FLAG). After 24, 48 or 72 h of transfection, the effectiveness of transfection was detected by western blotting. As shown in Fig. 7A, caspase-9 protein level was significantly increased in a time-dependent manner in the HEp-2 cells transiently transfected with the CAS9-expressing plasmid. In contrast, the control plasmid had no effect on caspase-9 expression. We then tested whether increased caspase-9 expression would promote survival and diminish apoptosis in oridonin-treated HEp-2 cells. As shown in Fig. 7B, the inhibitory ratio of control plasmid-transfected cells is 42.80%, while, the inhibitory ratio of CAS9-expressing plasmid-transfected cells is 29.64%, thus demonstrating caspase-9-mediated increase in survival. Moreover, quantification of apoptotic cells by flow cytometric analysis showed that the incidence of apoptosis after oridonin treatment was decreased by nearly 10% in CAS9-expressing plasmid-transfected cells when compared with control plasmid (Fig. 7C). Next, the degree of ROS generation was also determined by flow cytometric analysis. The percentage of DCF-positive cells was significantly decreased in CAS9-expressing plasmid-transfected cells compared with control plasmid (Fig. 7D). However, CAS9-expressing plasmid-transfected HEp-2 cells showed a much increased autophagic ratio after oridonin treatment when compared with those transfected with control plasmid (Fig. 7E). Accordingly, oridonin treatment, with or without control plasmid, enhanced both the expression level of Beclin 1 and the conversion from LC3-I to LC3-II, while these enhancements were markedly augmented in HEp-2 cells transfected with CAS9-expressing plasmid (Fig. 7F).