Apoptosis-inducing activity of compounds screened and characterized from cinobufacini by bioassay-guided isolation

Abstract

Abstract. cinobufacini (huachansu), an aqueous extract from the skin of the toad Bufo bufo gargarizans cantor, is a traditional chinese medicinal preparation widely used in clinical cancer therapy in China. Here, we screened and identified active compounds of cinobufacini and investigated their apoptosisinducing effect on HepG 2 cells. Screening was performed using bioassay-guided isolation. The effects of different fractions on the proliferation of HepG 2 cells were detected by the MTT assay. The extraction and isolation of active fractions were performed by chloroform extraction, silica column chromatography, preparative thin-layer chromatography and high-performance liquid chromatography. Nuclear magnetic resonance (NMR) imaging and electron ionization-mass spectrometry (EI-MS) were used to identify the structure of the active compounds. The extent of cell apoptosis was detected by Hoechst 33258 staining and flow cytometric analysis. Western blot analysis was used to detect the expression of the apoptosis-related proteins Bax and Bcl-2. Through bioassay-guided isolation, two compounds were isolated from cinobufacini. nMr and ei-MS data revealed these compounds to be resibufogenin and cinobufagin. Cinobufagin was determined to be the more efficient of the two in inhibiting the proliferation of HepG 2 cells. Hoechst 33258 staining and flow cytometric analysis indicated that cinobufagin induced marked changes in apoptotic morphology and significantly increased the proportion of apoptotic cells in HepG 2 cells. Western blot analysis showed that cinobufagin up-regulated Bax expression and down-regulated Bcl-2 expression. In conclusion, we screened and identified two anti-proliferation compounds of cinibufacini, resibufogenin and cinobufagin. The most effective compound, cinobufagin, inhibited cell proliferation by inducing the apoptosis of HepG 2 cells. This was potentially triggered by regulation of the Bax/ Bcl-2 ratio.

Efficient Killing Effect of Osteosarcoma Cells by Cinobufacini and Cisplatin in Combination

“…However, little is known about its killing effect on osteosarcoma. Experimental pharmacodynamics and clinical study both indicate that cinobufacini has obvious killing effect on many kinds of tumor cells, and the mechanism may be related with the apoptosis-inducing effect on tumor cells (Qi et al, 2008;Qi et al, 2010b;Wang et al, 2010). Our research found that cinobufacini may inhibit the proliferation of OS-732 cells in a dose-dependent manner which was consistent with the above research results.…”

Toad skin extract cinobufatini inhibits migration of human breast carcinoma MDA-MB-231 cells into a model stromal tissue

“…Some reports have suggested that apoptosis of cancer cell lines was induced by bufosteroids, unique steroid compounds contained in toad skin extract and toad venom, such as bufalin and cinobufagin (15)(16)(17)(18)(19). Decrease in MDA-MB-231 cell numbers observed in our experiments may in inhibiting metalloproteinase activity and result in suppressing cancer cell migration in the collagen gels.…”

Resibufogenin suppresses transforming growth factor‐β‐activated kinase 1‐mediated nuclear factor‐κB activity through protein kinase C‐dependent inhibition of glycogen synthase kinase 3

“…22 Resibufogenin shows strong cytotoxic activities against human cancer cells through induction of apoptosis or G 1 -phase arrest. 23,24 Moreover, the growth inhibition effect of RB against human cancer cells was comparable to or stronger than paclitaxel. 19 However, the effect and precise mechanism of RB on pancreatic cancer has not been elucidated.…”

“…Resibufogenin is one of the major active components in Huachansu, and it is regarded as a representative compound for the quality control of Huachansu . Resibufogenin shows strong cytotoxic activities against human cancer cells through induction of apoptosis or G 1 ‐phase arrest . Moreover, the growth inhibition effect of RB against human cancer cells was comparable to or stronger than paclitaxel .…”