(−)-Asarinin from the Roots of Asarum sieboldii Induces Apoptotic Cell Death via Caspase Activation in Human Ovarian Cancer Cells

Two tetrahydrofurofurano lignans (1 and 2), four phenylpropanoids (3–6), and two alkamides (7 and 8) were isolated from the EtOAc-soluble fraction of the roots of Asarum sieboldii. The chemical structures of the isolates were identified by analysis of spectroscopic data measurements, and by a comparison of their data with published values. The isolates (1, 2, 4–8) were evaluated for their cytotoxicity against human ovarian cancer cells (A2780 and SKOV3) and immortalized ovarian surface epithelial cells (IOSE80PC) using a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay. Of the isolates, (−)-asarinin (1) exhibited the most potent cytotoxicity to both A2780 and SKOV3 cells. A propidium iodide/annexin V-fluorescein isothiocyanate (V-FITC) double staining assay showed that (−)-asarinin (1) induces apoptotic cell death in ovarian cancer cells. In addition, (−)-asarinin (1) increased the activation of caspase-3, caspase-8, and caspase-9 in ovarian cancer cells. Pretreatment with caspase inhibitors attenuated the cell death induced by (−)-asarinin (1). In conclusion, our findings show that (−)-asarinin (1) from the roots of A. sieboldii may induce caspase-dependent apoptotic cell death in human cancer cells.


Cytotoxicity of the Extract and Solvent Fractions against Human Ovarian Cancer Cells
The 70% EtOH extract of the roots of A. sieboldii and two solvent partitions (EtOAc-and water-soluble fractions) from the 70% EtOH extract were investigated for their cytotoxicity against human ovarian cancer cells (A2780 and SKOV3) using MTT assays ( Table 1). The 70% EtOH extract showed a significant cytotoxicity against A2780, with an observed IC 50 value of 31.5 ± 16.83 µg/mL. Of the solvent partitions, the EtOAc-soluble fraction exhibited more potent cytotoxicity than the water-soluble fraction, against both ovarian cancer cells (IC 50 values were 19.89 and 118.47 µg/mL in A2780 and SKOV3, respectively). Thus, we attempted to identify the cytotoxic constituents in the EtOAc-soluble fraction. Our data on the EtOAc-soluble fraction were consistent with previous results [11] on the cytotoxic activity of the EtOAc-soluble fraction of Asiasari radix against several human cancer cell lines including SKOV3 cells.

(−)-Asarinin (1)-Induced Apoptotic Cell Death in Human Ovarian Cancer Cells
We found that (−)-asarinin (1) increased the fractionation of the nuclei accumulated at Sub G1 in A2780 and SKOV3 cells, but it failed to induce cell cycle arrest, which is one mechanism of growth inhibition (Figure 2A,B). Therefore, we further explored the molecular mechanism of the cytotoxic activity of (−)-asarinin (1) in human ovarian cancer cells. Dysfunction of apoptosis signaling pathways is associated with cancer development [32]. Therefore, compounds that promote apoptosis in cancer cells are considered as good candidates for anti-cancer chemotherapeutics. To examine whether (−)-asarinin (1)-induced cell death is mediated by the induction of apoptosis, two ovarian cancer cells A2780 and SKOV3 cells were treated with (−)-asarinin (1) and subsequently co-stained with PI and Annexin V-FITC. Treatment with (−)-asarinin (1) increased the percentage of Annexin V-FITC positive/PI negative cells by up to 43% in A2780 cells and 48% in SKOV3 cells ( Figure 2C,D). These results suggest that the (−)-asarinin (1)-induced cell death is associated with the induction of apoptosis in human ovarian cancer cells.

(−)-Asarinin (1)-Induced Apoptotic Cell Death in Human Ovarian Cancer Cells
We found that (−)-asarinin (1) increased the fractionation of the nuclei accumulated at Sub G1 in A2780 and SKOV3 cells, but it failed to induce cell cycle arrest, which is one mechanism of growth inhibition (Figure 2A,B). Therefore, we further explored the molecular mechanism of the cytotoxic activity of (−)-asarinin (1) in human ovarian cancer cells. Dysfunction of apoptosis signaling pathways is associated with cancer development [32]. Therefore, compounds that promote apoptosis in cancer cells are considered as good candidates for anti-cancer chemotherapeutics. To examine whether (−)-asarinin (1)-induced cell death is mediated by the induction of apoptosis, two ovarian cancer cells A2780 and SKOV3 cells were treated with (−)-asarinin (1) and subsequently co-stained with PI and Annexin V-FITC. Treatment with (−)-asarinin (1) increased the percentage of Annexin V-FITC positive/PI negative cells by up to 43% in A2780 cells and 48% in SKOV3 cells ( Figure 2C,D). These results suggest that the (−)-asarinin (1)-induced cell death is associated with the induction of apoptosis in human ovarian cancer cells.

(−)-Asarinin (1) Induced Caspase-Dependent Cell Death in Human Ovarian Cancer Cells
The activation mechanisms that trigger apoptosis are often referred to as the extrinsic and intrinsic pathways [33,34]. The intrinsic pathway (also called the mitochondria-mediated pathway) is activated directly by a variety of intracellular death stimuli. On the other hand, the extrinsic pathway (also called the receptor-mediated pathway) is characterized by the activations of cellsurface death receptors by the binding of extracellular ligands. Both extrinsic and intrinsic apoptotic pathways are highly dependent on the activation of caspases, which play a critical role in the proteolysis of specific targets [35]. Caspase-8 and caspase-9 are the initiator caspases for the extrinsic and intrinsic apoptotic pathways, respectively, while the effector caspase caspase-3 can be stimulated by activation of the initiator caspases in both pathways. To identify the mechanisms involved in (−)asarinin (1)-induced apoptotic cell death, we investigated the activation of an effector caspase, caspase-3 and initiator caspases caspase-8 and caspase-9. (−)-Asarinin (1) markedly stimulated the activation of caspase-3, caspase-8, and caspase-9 in both A2780 and SKOV3 cells (Figure 3). To further confirm the involvement of caspases in (−)-asarinin (1)-induced apoptosis, the effect of caspase inhibitors on (−)-asarinin (1)-induced cell death was investigated. z-DEVD-fmk (a specific caspase-3 inhibitor), z-IETD-fmk (a specific caspase-8 inhibitor), and z-LEHD-fmk (a specific caspase-9 inhibitor) considerably attenuated (−)-asarinin (1)-induced cell death in both A2780 and SKOV3 cells (Figure 4). These results show that (−)-asarinin (1)-induced apoptosis is mediated by the caspasedependent pathway in human ovarian cancer cells.

Plant Meterial
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Cell Culture
Human ovarian cancer cell lines (A2780 and SKOV3 cells) were obtained from the American Type Culture Collection (ATCC), and immortalized ovarian surface epithelial cell lines (IOSE80PC) were provided by Dr. N. Auersperg (University of British Columbia, Vancouver, British Columbia, Canada) and Dr. A. Godwin (Fox Chase Cancer Center, Philadelphia, PA, USA). Cells were cultured in the Roswell Park Memorial Institute (RPMI) 1640, supplemented with penicillin (100 U/mL), streptomycin sulfate (100 µg/mL), and 5% fetal bovine serum (FBS) (Life Technologies, Inc., Grand Island, NY, USA) in a humidified atmosphere of 5% CO 2 -95% air at 37 • C.

MTT Assay
A MTT assay was performed to evaluate the cell viability. The MTT was obtained from Molecular Probes Inc. (Eugene, OR, USA). Briefly, the cells (1.0 × 10 5 /well) were seeded in a 96-well plate and incubated for 24 h. The cells were treated with extracts (0.125-200 µg/mL) and compounds (0.125-200 µM) and incubated for 48 h. MTT solution was added into each well (final concentration; 0.5 mg/mL) and the plates were incubated for an additional 4 h. The medium was removed and 50 µL of dimethyl sulfoxide (DMSO) was added. The optical density was measured at 540 nm using a microplate spectrophotometer (SpectraMax; Molecular Devices, Sunnyvale, CA, USA). To investigate the involvement of caspases in asarinin (1)-induced cell death, a MTT assay was also performed using caspase inhibitors. The cells were pretreated with caspase inhibitors (50 µM) for 30 min, and treated with (−)-asarinin (1) (A2780, 40 µM; and SKOV3, 60 µM) for 48 h. Caspase-3 inhibitor z-DEVD-fmk, caspase-8 inhibitor z-IETD-fmk, and caspase-9 inhibitor z-LEHD-fmk were purchased from Calbiochem (Bad Soden, Germany).

Cell Cycle Analysis
The cells were treated with (−)-asarinin (1) and incubated for 48 h. At the time of collection, the cells were harvested and washed twice with ice-cold phosphate buffered saline (PBS). The cells were fixed and permeabilized with 70% ice-cold ethanol at −20 • C for 4 h. The cells were washed once with PBS and resuspended in a staining solution containing propidium iodide (50 µg/mL) and RNase A (5 mg/mL). The cell suspensions were incubated for 30 min at room temperature in a dark place. After 30 min the suspensions were analyzed by fluorescence-activated cell sorting (FACS) cater-plus flow cytometry (guava easy cyte TM , Merk Millipore, Darmstadt, Germany) using 5000 cells per group.

Annexin V-FITC/PI Double Staining
Annexin V-FITC was obtained from BD Biosciences (San Jose, CA, USA). The cells were treated with (−)-asarinin (1) and incubated for 48 h. The cells were rinsed twice with ice-cold PBS and suspended with 100 µL of binding buffer (10 mM HEPES/NaOH, 140 mM Nacl, 2.5 mM CaCl 2 , PH 7.4). A total of 5 µL of FITC-conjugated annexin V and 5 µL of PI (50 mg/mL) were added into the cell suspension, and the mixture was incubated in a dark place at room temperature for 15 min. The cells were analyzed using FACS cater-plus flow cytometry (guava easy cyte TM ); at least 10,000 cells per each group were counted.

Western Blot Assay
The cells were treated with (−)-asarinin (1) and incubated for 48 h. The cells were rinsed twice with ice-cold PBS and lysed with protein lysis buffer (Intron Biotechnology, Seoul, Korea) containing protease inhibitors (0.5 mM PMSF and 5 µg/mL aprotinin). The lysates were mixed with 5X sodium dodecyl sulfate (SDS) sample buffer and boiled for 5 min for denaturation. Total protein was run on 10-12% SDS-PAGE gels and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was immunoblotted using specific primary antibodies overnight at 4 • C following blocking with 5% non-fat dry milk for 30 min-1 h. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:1000-2000) at room temperature for 1−2 h. After washing, immunepositive bands were visualized using an ECL chemiluminescent system and analyzed by Image Quant LAS-4000 (Fujifilm Life science, Tokyo, Japan). Anti-caspase-3 and b-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Caspase-9 antibody was purchased from Cell Signaling (Beverly, MA, USA). Caspase-8 antibody was obtained from BD Biosciences (San Jose, CA, USA).

Statistical Analysis
One-way ANOVA and Student's t-test were performed to determine statistically significant differences. p-values of less than 0.05 were regarded as statistically significant.