br similar chemical structure to kusunokinin except at
similar chemical structure to kusunokinin, except at the α-butyr-olactone ring (Fig. 1). Moreover, bursehernin also has a same molecular formula (C21H22O6) and molecular weight (370.401 g/mol) with ku-sunokinin. Bursehernin has shown cytotoxic activity against many cancer cell line such as nasopharyngeal, prostate, breast and colon cancer . Moreover, 7,7´-dihydroxybursehernin, a derivative of bursehernin isolated from G. thunbergii, inhibits the expression and activity of COX and NOS enzymes in macrophages leading to a physical blockage of NF-κB protein .
Kusunokinin and bursehernin have main chemical structure like etoposide and teniposide, which are chemotherapeutic drugs from lignan group. Both etoposide and teniposide inhibit topoisomerase II [22,23]. In previous study, we found that (-)-kusunokinin inhibited topoisomerase II and bcl-2, and increased p53, p21, cytochrome C, caspase -8, -7 and -3 . However, yield of (-)-kusunokinin was low and required many extraction techniques. Here, we attempted to che-mically synthesize kusunokinin so as to overcome the limitations of low natural yield and time required. Moreover, bursehernin, kusunokinin derivative, also was synthesized. Various cancers and normal cell lines were used to test cytotoxicity using MTT assay. The levels of proteins associated with cell proliferation were verified using Western blotting. Finally, flow cytometry was used to determined Cycloheximide arrest, multi-caspase activity and apoptosis. r> 2. Materials and methods
( ± )-Kusunokinin and its derivative, ( ± )-bursehernin, were syn-thesized following the procedure reported by Ganeshpure and Stevenson . Based on HPLC data, the compound purity was higher
than 99% (Fig. 2). Both compounds were dissolved in DMSO to prepare the stock solution. These racemic kusunokinin and bursehernin were used throughout the study.
2.2. Cell lines and culture conditions
468) were obtained from American Type Culture Collection (Manassas, VA, USA). A mouse fibrosarcoma (L-929), a colorectal adenocarcinoma (HT-29), three cholangiocarcinoma (KKU-K100, KKU-M055, and KKU-M213) and immortal cholangiocyte (MMNK-1) cell lines were kindly donated by Associate Professor Dr. Jasadee Kaewsrichan (Drug Delivery System Excellence Center, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Songkhla, Thailand), Associate Professor Dr. Ruedeekorn Wiwattanapatapee (Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Songkhla, Thailand), and Dr. Mutita Junking (Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand). All cell lines were cultured in growth media (Dulbecco’s modified Eagle medium (DMEM)) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1% penicillin and 1% streptomycin (Gibco BRL, NY, USA). The cells were maintained at incubation con-dition of 37 °C in 5% CO2.
2.3. Cytotoxicity assay
Cytotoxicity was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described. All of cell lines were seeded into 96 well plate and treated with various concentrations of individual ( ± )-kusunokinin or ( ± )-bursehernin for
Fig. 4. Eﬀect of ( ± )-kusunokinin and ( ± )-bursehernin on level of cell cycle-related proteins. (A, C) MCF-7 and (B, D) KKU-M213 cells were treated with IC50 concentration of ( ± )-kusunokinin or ( ± )-bursehernin for 24, 48 and 72 h. Then, cells were harvested and protein levels which related to cell cycle (topoisomerase II, STAT 3, cyclin D1, and p21) were detected using Western blotting. Fold change of each protein was measured by densitometry quantitation using ImageJ software and normalized with GAPDH, loading control. The representative data are mean ± SD of two independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 were considered to indicate a statistically significant diﬀerences compared to control group at 0 h.
72 h. The half of maximal inhibitory concentration (IC50) values were determined and calculated as previously described .
According to the US NCI plant screening program, a pure compound is generally considered to have in vitro cytotoxicity activity with an IC50 value of not more than 4 μg/ml equivalent to 10.8 μM of kusunokinin .
MCF-7 and KKU-M213 cell lines were seeded into 12-well plates at a density of 2 × 105 and 1 × 105 cells per well, respectively. The cells were grown overnight followed by incubation with ( ± )-kusunokinin or ( ± )-bursehernin at an IC50 concentration for 72 h. After treatment, the cells were harvested by trypsinization and fixed with 1 ml of ice cold 70% ethanol for 3 h. Then, the cells were stained with propidium iodide (PI) and incubated in the dark at room temperature for 30 min. The percentage of the cells in G0/G1, S, and G2/M phase of cell cycle was analyzed using MUSE® Cell Analyzer (Merck Millipore, Germany) with a Millipore's Muse® Cell Cycle Kit (Catalog No. MCH100106, Merck Millipore, Germany). These experiments were performed in tri-plicate.