br Kusunokinin and bursehernin enhanced multi caspase activi
3.5. ( ± )-Kusunokinin and ( ± )-bursehernin enhanced multi-caspase activity
Fig. 7. Eﬀect of ( ± )-bursehernin on multi-caspase in KKU-M213 cells. (A, C) KKU-M213 3X FLAG Peptide were treated with ( ± )-bursehernin at IC50 concentration for 24, 48, 72, and 96 h. (B, D) KKU-M213 cells were also treated with various concentrations of ( ± )-bursehernin at 1.85, 3.70, and 7.40 μM for 48 h. Multi-caspases were determined using the Muse® MultiCaspase Kit. (A, B) The plot is depicted the eﬀect of ( ± )-bursehernin treatment on KKU-M213 cells. (C, D) Each plot is a representative figure of the three replicates of each determination. The graph is depicted the percentage of cell with multi-caspase activity. Values are mean ± SD (n = 3). The statistical analysis of the data was carried out by Student t-test. *p < 0.05 and **p < 0.01 were considered to indicate a statistically significant diﬀerences compared to control group at 0 h.
Taken together, the multi-caspase activity results suggest that ( ± )-kusunokinin and ( ± )-bursehernin can induce caspase-dependent apoptotic cell death in breast cancer and cholangiocarcinoma cell lines, respectively.
In our previous study, we reported that (-)-kusunokinin, the active compounds from P. nigrum, showed anticancer activity against breast
( ± )-kusunokinin, racemic compounds, showed strong cytotoxicity against breast cancer cells including MCF-7 and MDA-MB-468 cells with IC50 value of 4.30 ± 0.65 and 5.90 ± 0.44 μM, respectively. Surprisingly, a synthetic ( ± )-kusunokinin showed a stronger cyto-toxicity with IC50 value of 7.57 ± 0.92 μM on MDA-MB-231 more than the extracted (-)-kusunokinin, which may be due to a purity of synthetic kusunokinin. In addition, macelignan, a diarylbutane-type of lignans from Myristica fragrans has shown antiproliferative eﬀects on MDA-MB-231 cells with IC50 value of 7.67 ± 0.27 μg/ml (23.35 ± 0.82 μM)  which is less eﬀective than ( ± )-kusunokinin. (-)-Hinokinin and other lignan compounds which are extracted from A. malmeana, have similar structure to (-)-kusunokinin, however, they have less eﬀect against A. gemmatalis than (-)-kusunokinin .
Fig. 8. Proposed pathway of ( ± )-kusunokinin and ( ± )-bursehernin on cell proliferation and apoptosis.
The blunt end and arrow lines represent the inhibition of action resulting in the decreasing of protein, and the induction of action leading to the increasing of protein and activity, respectively.
( ± )-kusunokinin demonstrated strong cytotoxicity against HT-29 cells with IC50 value of 5.51 ± 0.99 μM which was higher than (-)-cubebin and ( ± )-bursehernin. Previously, (-)-kusunokinin has shown antic-ancer activity on colon cancer SW-620 cells with IC50 value of 2.60 ± 0.34 μg/ml (5.75 ± 0.43 μM) . HT-29 cells are non-me-tastatic and moderately diﬀerentiated whereas SW620 cells are meta-static and poorly diﬀerentiated . To understand the action of ku-sunokinin, the molecular specific of (-)-kusunokinin on colon cancer will be addressed in further study. The diﬀerent action of extracted (-)-kusunokinin, ( ± )-synthetic kusunokinin and ( ± )-bursehernin could be also due to structure, purity and characterization of cell lines. MCF-7, MDA-MB-468, and MDA-MB-231 cells are non-invasive (ER+, PR+/- and HER2+), poorly metastatic (ER-, PR- and HER2-), and highly metastatic (ER-, PR- and HER2-), respectively [30–32]. Our previous study using computational analysis showed that (-)-burse-hernin, a configurational isomer of ( ± )-kusunokinin, bind to FMS ki-nase receptor, Hsp90, adenylate cyclase 10 (ADCY10), mitogen-acti-vated protein kinase kinase (MEK1), human epidermal growth factor receptor 2 (HER2) and α-tubulin. HER2 is a member of the epidermal growth factor receptor family having tyrosine kinase activity . This receptor protein activates variety of signaling pathways (MAPK, PI3K and PKC), causing in cell proliferation survival, diﬀerentiation, angio-genesis and invasion . This protein could be one of target protein of ( ± )-kusunokinin on MCF-7 cells. Moreover, FMS protein is found to be higher in cancer tissue more than normal tissue . HT29 and SW620 exhibit FMS protein  that could be the target for our interested compounds, kusunokinin and bursehernin.
Surprisingly, ( ± )-bursehernin revealed cytotoxicity to well diﬀer-entiation (KKU-M213) cells more than poorly diﬀerentiation cells
(KKU-K100 and KKU-M055). Curcumin, a flavonoid compound from the rhizome of Curcuma longa, also has a potential anticancer activity against KKU-K100, KKU-M156 and KKU-M213 cells. Curcumin inhibits NF-kB and STAT3 which are involved in tumor cell survival and pro-liferation and activates PPAR-ɣ and death receptor4. Down-regulation of NF-kB and STAT3 by curcumin leads to decreasing of bcl-2, bcl-xL, cIAP-1, cIAP-2, c-FLIP, surviving, XIAP, cyclin D1 and c-Myc in cho-langiocarcinoma cells . Our previous result also showed (-)-kusu-nokinin down-regulated bcl-2 on MCF-7 and MDA-MB-468 cells . In this study, we now report that synthetic ( ± )-kusunokinin and ( ± )-bursehernin inhibited topoisomerase II, STAT3, and cyclin D1, and induced p21 protein. Curcumin induces G2/M arrest and apoptosis and inhibits cell proliferation via interference in microtubule assembly on MCF-7 cells . Similarly, ( ± )-bursehernin induced cell cycle arrest at G2/M phase which was related to down-regulation of STAT3 and cyclin D1. However, ( ± )-kusunokinin tended to induce cell cycle arrest at both S and G2/M phases, giving a hint that this phenomenal could be due to down-regulation of cyclin D1 and cyclin B1, respec-tively (Fig. 8).