• Journal of Microbiology and Biotechnology
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• v.25 no.3
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• pp.413-417
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• 2015

Recently, we isolated HY253, a novel decahydrofluorene analog with a molecular structure of 7,8a-divinyl-2,4a,4b,5,6,7,8,8a,9,9a-decahydro-1H-fluorene-2,4a,4b,9a-tetraol from the roots of Aralia continentalis, which is known as Dokwhal (獨活), a traditional medicinal herb. Moreover, we previously reported its cytotoxic activity on cancer cell proliferation in human lung cancer A549 and cervical cancer HeLa cells. The current study aimed to evaluate its detailed molecular mechanisms in cell cycle arrest and apoptotic induction in human hepatocellular carcinoma HepG2 cells. Flow cytometric analysis of HepG2 cells treated with $60{\mu}M$ HY253 revealed appreciable cell cycle arrest at the G1 phase via inhibition of Rb phosphorylation and down-regulation of cyclin D1. Furthermore, using western blots, we found that up-regulation of cyclin-dependent kinase inhibitors, such as p21^CIP1 and p27^KIP1, was associated with this G₁ phase arrest. Moreover, TUNEL assay and immunoblottings revealed apoptotic induction in HepG2 cells treated with $60{\mu}M$ HY253 for 24 h, which is associated with cytochrome c release from mitochondria, via down-regulation of anti-apoptotic Bcl-2 protein, which in turn resulted in activation of caspase-9 and -3, and proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). Accordingly, we suggest that HY253 may be a potent chemotherapeutic hit compound for treating human liver cancer cells via up-regulation and activation of the p53 gene.

• Archives of Pharmacal Research
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• v.24 no.1
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• pp.55-63
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• 2001

Aucubin, an irdoid g1ucoside, was investigated to determine whether it has a stimulating effect on $\alpha$-amanitin excretion in $\alpha$-amanitin intoxicated rats, and whether there is binding activity to calf thymus DNA. High-performance liquid chromatography (HPLC) analysis of $\alpha$-amanitin in rat urine allowed quantitative measurement of the $\alpha$-amanitin concentration with a detection limit of 50${mu}g/ml$. In this system, a group treated with both $\alpha$-amanitin and aucubin showed that o(-amanitin was excreted about 1.4 times faster than in the $\alpha$-amanitin only treated group. Our previous results showed that the toxicity of $\alpha$-amanitin is due to specific inhibition of RNA polymerase activity and the resultant blockage of the synthesis of certain RNA species in the nucleus. However, no significant activity change on RNA polymerase from Hep G2 cells was observed when aucubin was treated with $\alpha$-amanitin at any concentration tested. Nevertheless, aucubigenin inhibited both DNA polymerase (IC50, 80.5${mu}g/ml$) and RNA polymerase (IC50, 135.0${mu}g/ml$) from the Hep G2 cells. The potential of both $\alpha$-amanitin and aucubin to interact with DNA were examined by spectrophotometric analysis. $\alpha$-Amanitin showed no significant binding capacity to calf thymus DNA, but aucubin was found to interact with DNA, and the apparent binding constant ($K_{app}$) and apparent number of binding sites per D7A phosphate ($B_{app}$) were 0.45$0.45{\times}$$10^4$ $M^{-1}$ and 1.25, respectively.