MC-3 and YD-15 cell lines were obtained from the Fourth Military Medical University and Yonsei University, respectively. Both cell lines were maintained in either DMEM/F12 or RPMI-1640 medium (Welgene, Inc.) supplemented with 10% fetal bovine serum (Welgene, Inc.) and 100 U/ml each penicillin (Welgene, Inc.) in a humidified atmosphere containing 5% CO₂ at 37°C. During the culture process, the cells were routinely investigated under a microscope (CKX53, Olympus Corp.) for fungal and mycoplasma contamination and the mycoplasma removal agent (MP Biomedicals, LLC) was used to treat the mycoplasma-free cultures if deemed necessary. All experiments were performed with cells cultured at 50‑60% confluency. Oridonin (chemical structure shown in Fig. 1A) was purchased from Abcam and cycloheximide (CHX) were obtained from Sigma-Aldrich; Merck KGaA. Each chemical was dissolved in dimethyl sulfoxide (DMSO), aliquoted and stored at ‑20°C. Stock solutions were diluted with culture medium to the indicated concentrations (final DMSO concentration, 0.1%).

The effects of oridonin on cell viability were investigated using Trypan blue exclusion assay. Cells were incubated with the vehicle control (0.1% DMSO) or oridonin for 48 h at 37°C, stained with 0.4% trypan blue solution (Gibco; Thermo Fisher Scientific, Inc.) for 2 min at room temperature, and viable cells were counted using a hemocytometer (Marienfeld).

The Live/Dead and Viability/Cytotoxicity assay kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used to determine the cytotoxicity of oridonin in the MEC cell lines. Calcein-AM is retained in living cells, producing intense green fluorescence through intracellular esterase activity. Ethidium homodimer-1 enters dead cells with damaged membranes and binds to nucleic acids, producing bright red fluorescence. Briefly, cells were stained with 2 µM calcein‑AM and 4 µM ethidium homodimer-1 and incubated for 30 min at room temperature. Cells were analyzed under a fluorescence microscope (Leica DM5000B; Leica Microsystems GmbH) with a suitable excitation and emission filter.

Whole‑cell lysates were extracted with RIPA lysis buffer (EMD Millipore) containing phosphatase inhibitors (Thermo Fisher Scientific) and protease inhibitor cocktails (Roche Diagnostics GmbH). Protein concentrations were measured using a DC protein assay kit (Bio‑Rad Laboratories, Inc.). Proteins at the same concentration (20 µg) were separated by SDS-polyacrylamide gel electrophoresis in 12% acrylamide gel (Bio‑Rad Laboratories, Inc.) and electrotransferred to a polyvinylidene fluoride membrane (Pall Corporation). The membranes were blocked with 5% skim milk dissolved in Tris‑buffered saline‑Tween‑20 buffer (T‑BST) for 1 h at room temperature. The membranes were then washed with T‑BST and incubated with primary antibodies overnight at 4°C. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. Rabbit anti-human polyclonal antibodies against cleaved caspase-3 (cat. no. 9664; 1:1,000), cleaved poly (ADP-ribose) polymerase (PARP; cat. no. 9541; 1:1,000), Bcl‑xL (cat. no. 2764; 1:1,000), Bak (cat. no. 3814; 1:1,000), Bax (cat. no. 2772; 1:1,000), Bim (cat. no. 2819; 1:1,000) and MCL-1 (cat. no. 4572; 1:1,000) were obtained from Cell Signaling Technology, Inc. Mouse anti-human monoclonal antibodies against active‑Bak (cat. no. AM04; 1:1,000) was purchased from EMD Millipore. Mouse anti-human monoclonal antibodies against active‑Bax (cat. no. 556467; 1:1,000) was obtained from BD Biosciences. Goat anti‑human polyclonal antibodies against t‑Bid (cat. no. 34325; 1:1,000) and mouse anti-human monoclonal antibodies against β-actin (cat. no. sc47778; 1:3,000) were purchased from Santa Cruz Biotechnology, Inc. Antibody‑bound proteins were detected using enhanced chemiluminescence Western blotting Luminol reagent (Santa Cruz Biotechnology, Inc.) and visualized using a LAS-500 imaging system (GE Healthcare Life Sciences). The densitometric analysis of the western blots was performed using ImageJ software (version 1.51k, NIH).

Flow cytometry was performed to analyze the cell cycle and apoptosis of the MEC cell lines. Cells were harvested after being treated with various concentrations of oridonin (0-30 µM) for 48 h, washed twice with PBS, and fixed with 70% ethanol at ‑20°C for overnight. Cells were then re‑suspended in PBS containing 20 µg/ml RNase A and propidium iodide (P4170, Sigma-Aldrich; Merck KGaA) for 15 min at 37°C. DNA contents were detected using a fluorescence-activated cell sorter (FACS) Calibur (BD Biosciences) and relative DNA content was calculated with Cell Quest software (BD Biosciences).

DAPI solution (Sigma-Aldrich; Merck KGaA) was used to investigate the nuclear morphological changes of apoptotic cells. Cells seeded on 60 mm² plates were treated with various concentrations of oridonin (0-30 µM) for 48 h. Following treatment, the cells were harvested, washed twice with PBS, and fixed with 100% methanol at room temperature for 10 min. The cells were washed again with PBS, plated on coated glass slides, and stained with 2 µg/ml of DAPI solution for 1 min at room temperature. The morphological changes of the cells were observed under a fluorescence microscope (Leica DM5000B, Leica Microsystems GmbH).

Apoptosis was measured using a FITC Annexin V apoptosis detection kit (BD Pharmingen). Harvested cells were washed twice with PBS and stained with Annexin V-FITC and PI dye at room temperature for 15 min. The stained cells were then analyzed using a FACSCalibur flow cytometer and calculated using Cell Quest software (BD Biosciences).

Total RNA was isolated using the Easy‑BLUE total RNA extraction kit (iNtRON Biotechnology) and cDNA was synthesized using a cDNA synthesis kit (Enzo Life Sciences, Inc.). The resulting target cDNA was subjected to PCR using HiPi PCR PreMix (ELPISBIOTECH, Inc.) and amplified using the following primers: MCL-1 sense, 5′-GAG GAG GAG GAC GAG TTG TA-3′ and antisense 5′-CCT TAC GAG AAC GTC TGT TGT GAT AC-3′; and GAPDH sense, 5′-CGG AGT CAA CGG ATT TGG TCG TAT-3′ and antisense, 5′-AGC CTT CTC CAT GGT GGT GAA GAC‑3′. The amplification of MCL‑1 and GAPDH was performed for 28 cycles (30 sec at 95°C, 35 sec at 60°C and 45 sec at 72°C). The amplified PCR products were detected using 2% agarose gel electrophoresis and visualized by ethidium bromide staining. The densitometric analysis of the amplified PCR products was performed using ImageJ software (version 1.51k, NIH).

The resulting target cDNA was subjected to PCR using AMPIGENE qPCR Green Mix Hi‑Rox (Enzo Life Sciences, Inc). qPCR was performed by a StepOne Plus Real-Time PCR System (Applied Biosystems) and the resulting target cDNA was amplified using the following primers: MCL-1 sense, 5′-GTA TCA CAG ACG TTC TCG TAA GG-3′ and antisense, 5′-CCA CCT TCT AGG TCC TCT ACA T-3′; and GAPDH sense, 5′-GTG GTC TCC TCT GAC TTC AAC-3′ and antisense, 5′-CCT GTT GCT GTA GCC AAA TTC‑3′. The amplification of MCL‑1 and GAPDH was performed for 40 cycles (2 min at 95°C 10 sec at 95°C and 30 sec at 60°C). Each PCR product was run in triplicate. The relative MCL-1 mRNA expression was calculated using the 2^−ΔΔCq method (25).

Cycloheximide (Sigma-Aldrich; Merck KGaA) chase assay was carried out to examine whether oridonin affected the half-life of MCL-1 protein. In brief, the cells were treated with 0.05 µg/ml of CHX 1 h prior to oridonin treatment, and were subsequently treated with or without oridonin for the indicated periods of time (0‑12 h). Western blot analysis was then performed.

Changes in ΔΨm were determined using a MitoScreen kit (BD Pharmingen). Harvested cells were washed twice with PBS and incubated with JC‑1 solution at 37°C for 30 min. The cells washed twice using 1X Assay Buffer and JC‑1 fluorescence was analyzed by flow cytometry (FACSCalibur, BD Biosciences).

The open reading frame of the human MCL-1 (NM_021960) gene was amplified from cDNA using the following primers: MCL-1 sense, 5′-GAA TTC ATG TTT GGC CTC AAA AGA-3′, with an included EcoRI site; and MCL-1 antisense, 5′-GAA TTC CTA TCT TAT TAG ATA TGC-3′, with an included EcoRI site and cloned into a pGEM^®-T Easy Vector System (Promega Corporation). The genes were cloned into the multiple cloning site of pcDNA3.1(+) vector (Invitrogen; Thermo Fisher Scientific, Inc.). The MC‑3 and YD-15 cells were transfected with the empty pcDNA3.1 or the pcDNA3.1‑ MCL‑1 vector (0.5 µg) construct using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

Statistical analysis was performed using SPSS 22 software (SPSS, Inc.). A one-way analysis of variance (ANOVA) was applied with Tukey's post hoc test and all data are presented as the means ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

To investigate the growth-inhibitory effects of oridonin, the MC-3 and YD-15 cells were incubated with various concentrations of oridonin (0‑30 µM) for 48 h. The results revealed that statistical significance was observed at the 15 and 30 µM concentrations of oridonin for the MC‑3 cells and at the 6.25, 12.5 and 25 µM concentrations for the YD‑15 cells (Fig. 1B). To determine the cytotoxic effects of oridonin, a Live/Dead assay was performed under the same conditions as a Trypan blue exclusion assay in the MEC cell lines. As illustrated in Fig. 1C, treatment with oridonin led to an increase in the ratio of dead cells (red fluorescence) in a concentration-dependent manner. These results indicate that oridonin inhibits the viability and induces the death of MEC cell lines.

To determine the type of cell death induced by oridonin, the levels of PARP and caspase-3, as markers of apoptosis, were detected by western blot analysis. In response to oridonin treatment, the cleavage of caspase-3 and PARP was markedly increased compared with the vehicle control (Fig. 2A). To examine the effects of oridonin on the sub G₁ cell population, flow cytometry was performed. The cell population in the sub G₁ phase increased from 8.96- to 13.31-fold in the MC-3 and YD‑15 cell lines treated with oridonin (Fig. 2B). When visualizing cell apoptosis by DAPI staining, oridonin was found to significantly increase the number of apoptotic nuclei with condensation or fragmentation (white arrows) in the MEC cell lines (Fig. 2C). To further verify the apoptotic effects of oridonin, Annexin V/PI double staining was performed. The results revealed that the number of Annexin-positive cells was significantly increased in a concentration‑dependent manner (Fig. 2D). Taken together, these results suggest that oridonin enhances the apoptotic death of human MEC cell lines.

To elucidate the fundamental mechanisms of oridonin-induced apoptosis, the expression of Bcl‑2 family was identified as a key mediator of apoptosis. The results of western blot analysis revealed that treatment with oridonin significantly decreased the expression level of MCL‑1 protein and increased PARP cleavage in a concentration- and time-dependent manner (Fig. 3A and D); however, oridonin did not affect the expression of other anti-apoptotic proteins (e.g., Bcl‑xL) in both cell lines (Fig. S1). To determine whether MCL-1 is regulated at the transcriptional level, RT-PCR and qPCR were performed. The effect of oridonin on MCL‑1 mRNA levels was variable in both cell lines (Fig. 3B, C and E). To further determine the effect of oridonin on MCL-1 protein turnover, chasing analysis was performed using cycloheximide (CHX), an inhibitor of protein synthesis, followed by western blot analysis. As a result, the MCL-1 protein levels were markedly decreased by simultaneous treatment with oridonin and CHX compared to CHX treatment only (Fig. S2, top panel). The half-life of MCL-1 protein in the cells treated with both oridonin and CHX was approximately 11.5 and 10.8 h compared with 23.3 and 44.0 h in the CHX-treated group in the MC-3 and YD-15 cells, respectively (Fig. S2 bottom panel). These results suggest that the mode of action of oridonin is through the inhibition of the MCL-1 protein level.

To verify whether the effects of oridonin-mediated apoptosis are dependent on MCL-1, the two cell lines were transfected with an empty vector or a MCL-1 expression vector. MCL-1 overexpression significantly restored cell viability and PARP cleavage, which were previously affected by oridonin in both cell lines (Fig. 4A and B). This observation was further confirmed by the result that the ratio of apoptotic nuclei in MCL-1 overexpressing cells was reduced compared with the control (Fig. 4C). These results suggest that the principal mechanism of oridonin-mediated apoptosis may involve the targeting of MCL-1 protein in human MEC cell lines.

The majority of apoptotic stimuli require mitochondrial outer membrane permeabilization (MOMP) by pro‑apoptotic proteins, such as Bax, Bak, Bim and t‑Bid (26). In the present study, JC‑1 staining was performed to determine whether oridonin induces apoptosis through mitochondrial dysfunction. Oridonin exerted a significant decrease in red fluorescence compared to the vehicle control, confirming that the loss of ΔΨm was induced in both cell lines (Fig. 5A). Subsequently, to further examine the mitochondrial‑dependent pathway involved in oridonin-mediated apoptosis, the expression levels of pro-apoptotic proteins were analyzed. As shown in Fig. 5B, the expression of t‑Bid was markedly increased by oridonin in a concentration-dependent manner, whereas the levels of other pro‑apoptotic proteins, such as Bak, Bax and Bim were not commonly affected in both cell lines (Fig. S1). To ascertain the involvement of MCL‑1 in t‑Bid expression during oridonin-induced apoptosis, an MCL-1 expression vector was used. As was expected, the ectopic expression of MCL‑1 protein significantly decreased the expression of t‑Bid in the oridonin-treated cells (Fig. 5C). These results suggest that t‑Bid may be an essential downstream molecule of MCL‑1 during the oridonin-induced apoptosis of MEC cell lines.