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. 2013 Jan 29;11(2):316-31.
doi: 10.3390/md11020316.

Asperolide A, a marine-derived tetranorditerpenoid, induces G2/M arrest in human NCI-H460 lung carcinoma cells, is mediated by p53-p21 stabilization and modulated by Ras/Raf/MEK/ERK signaling pathway

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Asperolide A, a marine-derived tetranorditerpenoid, induces G2/M arrest in human NCI-H460 lung carcinoma cells, is mediated by p53-p21 stabilization and modulated by Ras/Raf/MEK/ERK signaling pathway

Cuiting Lv et al. Mar Drugs. .

Abstract

Here we first demonstrate that asperolide A, a very recently reported marine-derived tetranorditerpenoid, leads to the inhibition of NCI-H460 lung carcinoma cell proliferation by G2/M arrest with the activation of the Ras/Raf/MEK/ERK signaling and p53-dependent p21 pathway. Treatment with 35 μM asperolide A (2 × IC(50)) resulted in a significant increase in the proportion of G2/M phase cells, about a 2.9-fold increase during 48 h. Immunoblot assays demonstrated time-dependent inhibition of G2/M regulatory proteins. Moreover, asperolide A significantly activated MAP kinases (ERK1/2, JNK and p38 MAP kinase) by phosphorylation, and only the inhibition of ERK activation by PD98059 reversed downregulation of G2/M regulatory proteins CDC2, and suppressed upregulation of p21 and p-p53 levels. Transfection of cells with dominant-negative Ras (RasN17) mutant genes up-regulated asperolide A-induced the decrease of cyclin B1 and CDC2, suppressed Raf, ERK activity and p53-p21 expression, and at last, abolished G2/M arrest. This study indicates that asperolide A-induced G2/M arrest in human NCI-H460 lung carcinoma cells relys on the participation of the Ras/Raf/MEK/ERK signaling pathway in p53-p21 stabilization. An in vivo study with asperolide A illustrated a marked inhibition of tumor growth, and little toxcity compared to Cisplatin therapy. Overall, these findings provide potential effectiveness and a theoretical basis for the therapeutic use of asperolide A in the treatment of malignancies.

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Figures

Figure 1
Figure 1
The chemical structure and cell proliferation inhibition effect of asperolide A. (A) Chemical structure of asperolide A; (B) NCI-H460 cells were treated with 3.5, 7, 14, 28 or 56 μM of asperolide A for 48h. Then, cell proliferation inhibition was analyzed by MTT assay. Values are means ±SD from three independent experiments.
Figure 2
Figure 2
Asperolide A-induced cell cycle arrest and apoptotic cell death in NCI-H460 cells. (A) Asperolide A caused cell cycle arrest at the G2/M phase. Cells were treated with vehicle and 35 μM asperolide A for 12, 24, and 48 h, and cell cycle distribution was assessed by flow cytometry; (B) Annexin V-FITC/PI staining for apoptosis in NCI-H460 cells was assessed after 12, 24 or 48 h of treatment with 35 μM asperolide A by flow cytometry analysis; (C) The percentage of cells in different phases of the cell cycle was represented by a bar diagram; (D) The diagram showed the apoptosis rate of (C). (E) Present the change of G2/M arrest and apoptosis compared to control and 48 h treated groups after 72 h. Values were expressed as means ±SD of three independent experiments. ** p< 0.01 compared with control.
Figure 3
Figure 3
The responsibility of ERK activation for asperolide A-induced p53-p21 regulated cell cycle arrest. (A) NCI-H460 cells were treated with or without 35 μM asperolide A for indicated time, and then cells were harvested and lysed. cyclin B1, CDC2, p-CDC2 (Tyr15), cdc25C, p-cdc25C (Ser216), p-p53 (Ser15), p21 (Waf1/Cip1) were analyzed by Western blotting assay. GAPDH was used as an equal loading control; (B) Total and phosphorylated MAPK members (JNK, p38, ERK) after treatment with 35μM asperolide A for indicated time; (C) NCI-H460 cells were pre-treated with 20μM MEK inhibitor (PD98059), 10μM JNK inhibitor (SP600125), or 20μM P38 inhibitor (SB203580) for 2h, followed by treatment with or without 35 μM asperolide A for 48 h and the total or activation forms of MEK, JNK and p38 were evaluated by western blotting; (D) Cells were pre-incubated in absence or presence of MAPK inhibitors, then treated with 35 μM asperolide A, followed by immunoblotting assay performed with antibodies specific for CDC2, p-p53 and p21. Results are representative of three separate experiments. GPDH is shown as protein loading control.
Figure 4
Figure 4
The effects of a dominant negative RasN17 mutant gene on asperolide A-induced G2/M arrest. (A) Ras, Ras-GTP, p-c-Raf (Ser338) were detected after asperolide A-treatment for 48h by western blotting analysis; (B and C) Cells transfected with a dominant-negative RasN17 mutant gene or not were treated with or without 35 μM asperolide A for 48 h, then harvested for cell cycle analysis or (D) western blotting assay against p-c-Raf, p-ERK, p-p53, p21, cyclin B1 and CDC2. GAPDH was used to ensure equal protein loading. Values were means ±SD of three independent experiments. Differences were considered statistically significant at * p < 0.01 when compared with asperolide A treatment in none transfected NCI-H460 cells.
Figure 5
Figure 5
Effect of asperolide A on tumor growth in a xenograft model. (A) BALB/c male athymic mice were injected 5 × 106 NCI-H460 cells s.c. for the development of subcutaneous tumors. The mice were randomized into 3 groups (N = 9) and treated with Vehicle (1% DMSO), 2.5 mg/kg Cisplatin or 5 mg/kg asperolide A i.v. according to the protocol in panel (A); (B) Tumor image from various treatment groups; (C) Average tumor mass at sacrifice. ## p < 0.001; (D) Tumor volume measurements; (E) Body weight of mice from control, Cisplatin and asperolide A treated groups.
Figure 6
Figure 6
(A) The probable effect of asperolide A on the Ras/Raf/MEK/ERK signaling pathway; (B) Mechanisms of asperolide A-induced G2/M cell cycle arrest.

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