Wogonin was from National Institute of the Control Pharmaceutical and Biological Products (Beijing, China). Cisplatin, butylated hydroxyanisole (BHA) and N-acetyl-L-cysteine (NAC) were purchased from Sigma (St. Louis, MO). Z-VAD-FMK was from Calbiochem (La Jolla, CA). ROS-sensitive fluorescent dye 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H₂DCFDA) and dihydroethidium (DHE) were purchased from Molecular Probes (Eugene, OR). Antibodies against active caspase-3, poly (ADP-ribose) polymerase (PARP) were from BD Bioscience (San Diego, CA). Anti-β-actin antibody was from Protein Tech (Chicago, IL).

A549 (a non-small cell lung cancer cell line) and HeLa (a cervical cancer cell line) were from American Type Culture Collection (ATCC, Manassas, VA) and grown in RPMI-1640 supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 U/ml penicillin and 100 μg/ml streptomycin. The cultured cells were kept in a 37°C humidified incubator with 5% CO₂. For cell death assay, cells were seeded in 96-well plate and after overnight culture were then treated as indicated in each figure legend. Cell death was assessed based on the release of lactate dehydrogenase (LDH) using a cytotoxicity detection kit from Promega (Madison, WI) as described previously (14). All the experiments were repeated 3–5 times and the average is shown in each figure.

Cells were treated as indicated in figure legend and cell lysates were prepared by lysing cells with M2 buffer [20 mmol/l Tris-HCl (pH 7.6), 0.5% NP40, 250 mmol/l NaCl, 3 mmol/l EDTA, 3 mmol/l EGTA, 2 mmol/l DTT, 0.5 mmol/l phenylmethylsulfonyl fluoride, 20 mmol/l β-glycerophosphate, 1 mmol/l sodium vanadate and 1 μg/ml leupeptin]. Cell lysates were then subjected to SDS-PAGE and analyzed by western blotting using specific antibodies. The proteins were seen by enhanced chemiluminescence (Millipore, Billerica, MA) using Bio-Rad Image station (Hercules, CA). Each experiment was repeated at least 3 times and representative results are shown.

Apoptosis was detected by flow cytometric analysis using an Annexin V-FITC Apoptosis Detection kit (Nanjing KeyGen Biotech, Nanjing, China). Cells were treated as indicated in the figure legend, and then were double stained with Annexin V-FITC and propidium iodide (PI) following manufacturer’s instruction. The stained cells were analyzed by flow cytometry (Beckman Coulter, Inc., Brea, CA). Cells that are in early apoptosis are Annexin V-FITC positive and PI negative; and cells that are in late apoptosis or already dead are both FITC Annexin V and PI positive.

Cells were seeded in 12-well plates and after overnight culture were then treated as indicated in each figure legend. Thirty minutes before collecting cells, H₂O₂-sensitive fluorescent dye CM-H₂DCFDA (5 μM) or superoxide-sensitive dye DHE (5 μM) was added. ROS were detected by flow cytometry (Beckman Coulter, Inc.) as reported previously (15).

All numerical data are expressed as mean ± standard deviation (SD). Statistical significance was examined by Student’s paired-sample t-test using the SPSS statistics software package (IBM SPSS, Chicago, IL) and P<0.05 was used for significance.

Aiming to overcome chemoresistance to cisplatin in cancer cells, we first investigated whether wogonin is able to enhance the anticancer activity of cisplatin. We treated the A549 cells with 10 μM of wogonin, 10 μM of cisplatin or both for 60 h and cell death was observed microscopically. As shown in the representative images (Fig. 1A), while cisplatin or wogonin caused limited cell death, co-treatment of these agents resulted in significantly enhanced cytotoxicity. To more quantitatively measure cell death, A549 cells were treated with increasing concentrations of wogonin (5–20 μM) and a fixed concentration of cisplatin (7.5 μM) and cell death was detected by LDH release assay. The results showed that while cisplatin alone caused about 25% cell death in A549 cells, wogonin synergistically sensitized A549 cells to cisplatin-induced cell death in a dose-dependent manner (Fig. 1B). The synergism that killed about 80% of cells was detected at the highest dose of wogonin (20 μM), a concentration of wogonin alone that only caused moderate cell death (~12%). Conversely, a similar dose-dependent potentiation of cytotoxicity was detected when increasing concentrations of cisplatin with a fixed wogonin dose was used (Fig. 1C). The sensitization of cisplatin’s anticancer activity was further validated in the cervical cancer cell line HeLa. A similar dose-dependent synergism either with fixed concentration of wogonin or with fixed concentration of cisplatin was observed (Fig. 1D and E), suggesting wogonin is able to sensitize cancer cells to cisplatin-induced cytotoxicity.

We next investigated whether wogonin potentiates cisplatin-induced cell death through enhancing apoptosis. HeLa cells were treated with wogonin, cisplatin alone or both. The cells were stained with Annexin V-FITC and PI, apoptosis was analyzed by flow cytometry. As shown in Fig. 2A, both early apoptotic (Annexin V-FITC positive and PI negative, 3.5%) and late apoptotic (both Annexin V-FITC and PI positive, 10.3%) cell population were significantly increased in wogonin and cisplatin co-treated cells compared to the samples individually treated with cisplatin (3.1% and 4.7%) or wogonin (1.9% and 1.5%), indicating that the enhanced cell death is mainly through apoptosis. The activation of caspases was also detected by western blotting. Whereas the activation of caspase cascade was barely detected in wogonin or cisplatin alone treated cells, the activation of caspase-3 and cleavage of the caspase-3 substrate PARP were significantly enhanced in cisplatin and wogonin co-treated A549 cells and HeLa cells (Fig. 2B). The pan-caspase inhibitor Z-VAD-FMK effectively suppressed the synergistic cytotoxicity induced by wogonin and cisplatin co-treatment (Fig. 3), further confirming that the sensitization to cisplatin by wogonin is through enhancement of apoptosis in cancer cells.

It has been well established that ROS, a group of reactive oxygen-containing species including superoxide, hydrogen peroxide (H₂O₂) and hydroxyl radical, are important signaling mediators for cell death pathways. Our previous work has demonstrated that wogonin induces intracellular accumulation of H₂O₂ in cancer cells, which contributes substantially to the synergistic cytotoxicity induced by wogonin plus TNF (13). The role of ROS in wogonin plus cisplatin-induced synergistic cytotoxicity was thus investigated. Cells were treated with wogonin, cisplatin or both, stained with two ROS-specific dyes, CM-H₂DCFDA that is specific for hydrogen peroxide (H₂O₂) or DHE that is specific for superoxide, and then analyzed by flow cytometry. As expected, wogonin induced strong intracellular H₂O₂ accumulation in both A549 cells and HeLa cells, as indicated by significant rightward shift of the peaks of wogonin treated cells compared with that of the control cells (Fig. 4A). The treatment with wogonin plus cisplatin showed similar trend and even more striking extent of H₂O₂ induction as treated by the wogonin alone. On the contrary, wogonin and cisplatin had marginal effect on cellular superoxide level in A549 and HeLa cells (Fig. 4B), which is consistent with our previous results (13). Then, we treated cells with ROS scavengers BHA or NAC to remove H₂O₂. As shown in Fig. 5A, these two scavengers effectively suppressed the synergistic cytotoxicity induced by wogonin and cisplatin co-treatment in both HeLa and A549 cells, which is well-correlated to significant reduction of H₂O₂ levels in the cells (Fig. 5B). Taken together, these results strongly suggest that wogonin-induced H₂O₂ accumulation substantially contributes to the potentiated cytotoxicity caused by cisplatin and wogonin co-treatment.