Anti-inflammatory and Anti-proliferative Diterpenes from Croton stellatopilosus Ohba

Abstract

One acyclic diterpene (plaunotol) and three cyclic diterpenes (plaunol A, plaunol E and plaunol F) were isolated from leaves and stems of Croton stellatopilosus Ohba (Euphorbiaceae). Their physical properties and structures were determined by means of UV, IR, MS, 'H-NMR and "C-NMR spectroscopies. Anti-inflammatory potential using cell-based assay in lipopolysaccharide-induced murine macrophage RAW264.7 cells of plaunol A was evaluated. It exhibited inhibitory activity on nitric oxide (NO) production with an ICs of 11.69 M. The MTT assay indicated the cytotoxic effect at concentration > 30 PM of plaunol A. Transcription profile analysis of inducible nitric oxide synthase (iOS) and cyclooxygenase-2 (COX-2) genes in the RAW264.7 cells using qRT-PCR technique revealed that plaunol A inhibited the NO production by suppressing the iOS and Cox 2 mRNAs. Anti-proliferative activity of plaunotol, plaunol A, plaunol E and plaunol F against the four human cancer cells including HeLa, HT-29, MCF-7 and KB cells was investigated using the MTT assay, Planotol, plaunol A and plaunol E exhibited the cytotoxicity in types of cancer cells with the IC,, ranging from 60-80 M while plaunol F did not. No cytotoxic effect in human gingival fibroblast (HGF) cells (normal cell) was observed at concentration > 100 M. Determination the effect of diterpenes on cell cycle was performed using MuseTM cell cycle reagent. At concentration of plaunotol 75 M induced the cell cycle arrest at G0/G1 phase of Hela, Sphase of MCF-7 and G2/M phase of HT-29 and KB cells. At concentration of 75 M plaunol A induced the cell cycle arrest at G2/M phase in Hela, HT-29 and KB cells and S phase in MCF-7, while plaunol E did at G2/M phase in all types of cells. These results suggested that plaunotol, plaunol A and plaunol E performed moderate cytotoxic effect by causing cell cycle arrest during cell division. Potential on apoptosis of diterpenes using double staining of Muse annexin-V/7-AAD following flow cytometry was investigated. At concentrations of 75 M and 150 M, plaunotol, plaunol A and plaunol E caused apoptosis in all types of cancer cells. The results suggested that plaunol E has higher potency on apoptosis than plaunotol and plaunol A, respectively. Effect of plaunotol and plaunol E on apoptotic-associated genes such as TNF-a, BCL-2, BAK and BAX using GRT-PCR was determined. Plaunotol at concentrations of 50 uM and 75 M suppressed TNF-a and BCL-2, but not BAK and BAX genes in HeLa, HT-29 and MCF-7 cells. Plaunotol inhibited all genes mRNA in KB cells. The ratio of BCL-2/BAX that indicted the involvement of those genes on apoptosis, revealed the mechanism of plaunotol. The reduction of ratio observed in all types of cells. This result indicated plaunotol performed apoptosis via death signaling and mitochondrial dependent pathway. Plaunol E has no effect on TNF-a, BCL-2, BAK and BAX mRNA levels in Hela, HT-29 and KB cells. In contrast, it suppressed the genes expressions in MCF-7. Effect of plaunol E on caspase-3, -8 and -9 in MCF-7 was investigated. It enhanced caspase-3, -8 and -9 with 2.67,6.33 and 533 fold, respectively. Thus, plaunol E caused apoptosis by activation of caspases. In conclusion, plaunol A performed anti-inflammatory activity by suppressed iNOS and COX-2 genes in RAW264.7 cells in the similar manner of plaunotol and plaunol F. The present study illustrated the potential of plaunotol, plaunol A and plaunol E on anti-proliferative activity in four types of human cancer cell lines. The results suggested that plaunotol and plaunol E have affected on apoptosis induction and they exhibited a potential for further anti-cancer development