In vitro anti-cancer activity of chamaejasmenin B and neochamaejasmin C isolated from the root of Stellera chamaejasme L

Abstract

Aim: To examine the anti-cancer effects of chamaejasmenin B and neochamaejasmin C, two biflavonones isolated from the root of Stellera chamaejasme L (known as the traditional Chinese herb Rui Xiang Lang Du) in vitro.

Methods: Human liver carcinoma cell lines (HepG2 and SMMC-7721), a human non-small cell lung cancer cell line (A549), human osteosarcoma cell lines (MG63, U2OS, and KHOS), a human colon cancer cell line (HCT-116) and a human cervical cancer cell line (HeLa) were used. The anti-proliferative effects of the compounds were measured using SRB cytotoxicity assay. DNA damage was detected by immunofluorescence and Western blotting. Apoptosis and cell cycle distribution were assessed using flow cytometry analysis. The expression of the related proteins was examined with Western blotting analysis.

Results: Both chamaejasmenin B and neochamaejasmin C exerted potent anti-proliferative effects in the 8 human solid tumor cell lines. Chamaejasmenin B (the IC(50) values ranged from 1.08 to 10.8 μmol/L) was slightly more potent than neochamaejasmin C (the IC(50) values ranged from 3.07 to 15.97 μmol/L). In the most sensitive A549 and KHOS cells, the mechanisms underlying the anti-proliferative effects were characterized. The two compounds induced prominent expression of the DNA damage marker γ-H2AX as well as apoptosis. Furthermore, treatment of the cells with the two compounds caused prominent G(0)/G(1) phase arrest.

Conclusion: Chamaejasmenin B and neochamaejasmin C are potential anti-proliferative agents in 8 human solid tumor cell lines in vitro via inducing cell cycle arrest, apoptosis and DNA damage.

Figure 1

Chemical structure of neochamaejasmin C and chamaejasmenin B.

Figure 2

Chamaejasmenin B and neochamaejasmin C induced DNA damage. (A) A549 cells were exposed to the compounds for 48 h, and protein extracts were immunoblotted for γ-H2AX detection. (B) KHOS cells were exposed to the compounds for 48 h, and protein extracts were immunoblotted for γ-H2AX detection. (C) A549 cells were treated with chamaejasmenin B (2 μmol/L) for 48 h and imaged by immunofluorescence for γ-H2AX (green) and nuclei (blue). γ-H2AX expression is indicated by a punctate appearance in the nucleus. Scale bar=40 μm.

Figure 3

Chamaejasmenin B and neochamaejasmin C induced apoptosis. (A) Cells were treated with the compounds for 48 h and analyzed for apoptosis. The experiments were repeated three times, and error bars represent the standard deviation. ^bP<0.05, ^cP<0.01 (two-sided Student's t-test). (B) A549 cells were exposed to the compounds for 48 h, and protein extracts were immunoblotted with specific antibodies against p53, XIAP, Mcl-1, caspase-3, cleaved-caspase-3, and PARP. (C) A549 cells were pretreated with the pan-caspase inhibitor Boc-D-fmk (10 μmol/L) for 1 h and then treated with 4 μmol/L chamaejasmenin B for 48 h. The cells were analyzed for apoptosis by flow cytometry.

Figure 4

Chamaejasmenin B and neochamaejasmin C induced G₀/G₁ arrest. (A) and (B) Cells treated with the compounds for 48 h were collected and processed for cell cycle distribution analysis.

Figure 5

Effect of chamaejasmenin B and neochamaejasmin C on the expression of cell cycle regulators. A549 cells were treated with the compounds for 48 h, and the expression of p21^CIP1, CDK2, cyclin E, and Rb was evaluated using Western blotting.