Gomisin J (PubChem CID: 3001686) (Fig. 1) was isolated from the fruits of Schisandra chinensis following the previously published method (28). It was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg dry weight/ml and stored at −20°C.

Each cell line was cultured in the appropriate media. Nine breast cancer cell lines (MCF7, MDA-MB-231, BT20, BT549, T47D, SKBR3, MDA-MB-453, HS578T and MDA-MB-468), two colon cancer cell lines (HCT116 and HT-29), and two cervical cancer cell lines (HeLa and SiHa) were cultured in Dulbecco's modified Eagle's medium (DMEM; Welgene, Inc., Daejeon, Korea) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1 % antibiotic-antimycotic solution (cat. no. 15240-062; Gibco-BRL; Thermo Fisher Scientific, Inc.). Normal human MCF10A mammary epithelial cell line was grown in DMEM/F12 medium (cat. no. 11330-032; Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 20 µg/ml epidermal growth factor (EGF) (cat. no. E9644), 100 µg/ml cholera toxin (cat. no. C-8052), 10 µg/ml insulin (cat. no. I-9278) and 0.5 mg/ml hydrocortisone (cat. no. H-0888; all from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 5% horse serum (cat. no. 16050-122; Invitrogen; Thermo Fisher Scientific Korea Ltd., Seoul, Korea) and 1% antibiotic-antimycotic solution. All cells were cultured at 37°C in a humidified atmosphere composed of 95% air and 5% CO₂. The SiHa cell line was obtained from the Korean Cell Line Bank (KCLB; Seoul, Korea; cat. no. 30035) and the other cell lines were purchased from the American Type Culture Collection (ATCC; Mannassas, VA, USA).

Each PCD was mainly analyzed through western blotting with corresponding marker or regulatory proteins. In brief, MCF7 and MDA-MB-231 cells (2×10⁶/well) were seeded in a 100-mm diameter culture dish containing DMEM and treated with 30 µg/ml gomisin J for 72 h. Cells were centrifuged (micro centrifuge; Smart R17 refrigerated; Hanil Science Co., Ltd., Daejeon Korea) at 3,000 × g at 4°C for 3 min, washed in ice-cold phosphate-buffered saline (PBS) buffer, then lysed in radio immunoprecipitation assay (RIPA) lysis buffer. The amount of protein was quantified using a protein assay kit (Bio-Rad Laboratories Korea, Seoul, Korea) and 10 or 30 µg of proteins were loaded in each lane. Subsequently, they were separated on a 10 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a an Immobilon-P^® PVDF transfer membrane (cat. no. IPVH00010; EMD Millipore, Billerica, MA, USA). Membranes were then blocked using 5% skimmed milk in PBS for 30–60 min at 20–25°C and incubated overnight at 4°C with the primary antibodies. After washing with Tris-buffered saline containing 1% Tween-20 (product code T1027; CAS#9005-64-5; Biosesang, Seongnam, Korea), membranes were incubated with the corresponding secondary antibodies. Immunodetection was performed using the PowerOpti-ECL Western Blotting Detection reagent (Bio-Rad Laboratories). The antibodies used in this study were as follows: A mouse monoclonal antibody against BAX (dilution 1:5,000; cat. no. sc-20067) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). A rabbit polyclonal antibody against PARP (dilution 1:3,000; cat. no. 9542S) was obtained from Cell Signaling Technology (Danvers, MA, USA). A rabbit polyclonal antibody against cyclophilin A (CypA) (dilution 1:3,000; cat. no. BML-SA296) was supplied by Enzo Life Sciences, Inc. (Farmingdale, NY, USA). A goat polyclonal antibody against γ-tubulin (dilution 1:5,000 dilution; cat. no. sc-7396) was purchased from Santa Cruz Biotechnology, Inc. (CA, USA). BAX and PARP were used to detect for apoptosis and cyclophilin A (CypA) was used for necroptosis. Additionally, γ-tubulin was used as a loading control. Necroptosis was checked by assessing the levels of extracellular CypA biomarker protein that were released from necroptotic cells to an extracellular location (29). The result of the western blot analysis was quantified using ImageJ software program (version 1.51u) (Image Processing and Analysis in Java; http://imagej.nih.gov/ij/) supplied by the National Institutes of Health (Bethesda, MD, USA) and relative intensity vs. β-actin is represented in the bar graphs.

Cell viability was analyzed using a Cell Viability, Proliferation and Cytotoxicity assay kit (EZ-CYTOX; cat. no. EZ-3000; Seoul, Korea) according to the manufacturer's instructions in triplicate. Briefly, cells (2×10⁴/well) were seeded in 96-well cell culture plates containing DMEM medium and exposed to 1, 5, 10 or 30 µg/ml gomisin J. Cell viability was evaluated by measuring absorbance at 450 nm in a Gemini XPA microplate reader (Molecular Devices, San Jose, CA, USA). Cell viability was also checked by Trypan blue staining under same conditions, in which the number of live or dead cells were counted by using an EVE automatic cell counter (NanoEnTek Inc., Pleasanton, CA, USA).

Statistical analysis was performed by using Student's t-test to compare two different groups and one-way ANOVA for multiple groups, followed by Bonferroni's multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.

First, we tested the anticancerous effect of gomisin J on the proliferation and viability of two human breast cancer cell lines, MCF7 and MDA-MB-231. In particular, MCF7 was considered to be a good model system for testing our hypothesis since it has been reported to be highly resistant to many pro-apoptotic anticancer drugs, most likely owing to the absence of the key proteins necessary for apoptotic cell death (e.g., caspase-3). In brief, MCF7 and MDA-MB-231 cell lines were plated onto 24-mm culture dishes and allowed to form a confluent monolayer for 24 h. These cells were screened with various concentrations of gomisin J (0, 25, 50 and 100 µg/ml) for 5 days, and their cell density as well as morphological changes were observed under an optical microscope with IS capture software (KI-400F; Korea LabTech Corp., Seongnam, Korea). Notably, gomisin J exhibited a strong cytotoxic effect on MCF7 and MDA-MB-231 cells at higher concentrations of 25 µg/ml (data not shown). Accordingly, the cytotoxicity of gomisin J was quantified in detail by evaluating its effects on cell growth rate (Fig. 2) and morphological changes (Fig. 3) at four different concentrations (0, 1, 5, 10 and 30 µg/ml) for 0, 24, 48 and 72 h. In both MCF7 and MDA-MB-231 cell lines, the cell growth rate was highly delayed in comparison with those of the DMSO-only treated control cells (Fig. 2A-D). In addition, the number of cells decreased at relatively high concentrations (30 µg/ml) after treatment for 72 h, which implied the induction of cell death (Fig. 2A-D). These data indicated that gomisin J greatly suppressed the proliferation and viability of cancer cells in a time- and concentration-dependent manner. Notably, our data revealed that MCF10A cells appeared to be much less sensitive to gomisin J treatment, to a lesser degree than that observed in the breast cancer cells, which may suggest reduced adverse or no side effects in normal cells (Fig. 2E and F). In a further experiment, examination under an optical microscope with IS capture software (KI-400F; Korea LabTech Corp., Seongnam, Korea) also revealed similar results as the cell growth data (Fig. 3).

Collectively, our data indicated that gomisin J functioned as an anticancer drug by inhibiting cell proliferation and by inducing cell death in MCF7 and MDA-MB 231 cancer cells.

Our main goal for cancer treatment is to understand and overcome the resistance of malignant cancer cells to cell death, mainly apoptosis. Our data revealed that gomisin J treatment at concentrations above 30 µg/ml significantly induced cell death in MCF7 and MDA-MB-231 cancer cells. In order to clarify whether gomisin J can induce necroptosis as well as apoptosis, both cancer cell lines were treated with either DMSO (as a control) or 30 µg/ml of gomisin J for 72 h, and then the two types of PCD were assessed by employing western blot analysis. At first, we evaluated necroptosis by assessing the levels of extracellular CypA biomarker protein that were released from necroptotic cells. In this case, the export of CypA into the extracellular space was greatly increased in MCF7 cells but only slightly in MDA-MB-231 cells after 72 h, which indicated a much higher level of induction of necroptosis in MCF7 cells compared with MDA-MB-231 cells (Fig. 4). Apoptosis was also investigated through analysis of typical apoptotic markers, BAX and PARP. BAX protein levels were significantly increased in MDA-MB-231 cells (Fig. 4A and B). In contrast, they were only slightly increased in MCF7 cells in comparison with that in DMSO-treated control cells (Fig. 4A and B). PARP was also cleaved at a much higher level in MDA-MB-231 cells than in MCF-7 cells (Fig. 4A and B). These data again indicated the high resistance of MCF7 cells to apoptosis.

Collectively, this result strongly indicated that gomisin J can render apoptotic-resistant MCF7 cells more sensitive to necroptosis than apoptosis.

Cytotoxic effect of gomisin J on the proliferation and viability of various cancer cell lines. The cytotoxicity of gomisin J to MCF7 and MDA-MB-231 cells indicated the possibility of similar effects on other cancer cell lines. Therefore, this investigation was extended to different types of breast cancer (BT20, BT549, T47D, SKBR3, MDA-MB-453, HS578T and MDA-MB-468), colon cancer (HCT116 and HT-29) and cervical cancer (HeLa and SiHa) cell lines. The cytotoxic effect of gomisin J on each cell line was assessed as mentioned in Materials and methods after treatment of the cells with either DMSO (control) or 10 or 30 µg/ml gomisin J for 24 or 48 h. The cell viability markedly decreased in all tested cancer cell lines treated with gomisin J in comparison with that in untreated control cells (Fig. 5A and B).

Collectively, our results indicated that gomisin J has a strong inhibitory activity against the proliferation of various cancer cells, indicating that the antitumor activity of gomisin J may be used as an effective anticancer drug in various types of cancer cells.