Antitumor Activity of Asperphenin A, a Lipopeptidyl Benzophenone from Marine-Derived Aspergillus sp. Fungus, by Inhibiting Tubulin Polymerization in Colon Cancer Cells

Abstract

Marine-derived microorganisms are a valuable source of novel bioactive natural products. Asperphenin A is a lipopeptidyl benzophenone metabolite isolated from large-scale cultivation of marine-derived Aspergillus sp. fungus. The compound has shown potent antiproliferative activity against various cancer cells. However, the underlying mechanism of action remained to be elucidated. In this study, we demonstrated the antitumor activity and molecular mechanism of asperphenin A in human colon cancer cells for the first time. Asperphenin A inhibited the growth of colon cancer cells through G₂/M cell cycle arrest followed by apoptosis. We further discovered that asperphenin A can trigger microtubule disassembly. In addition to its effect on cell cycle, asperphenin A-induced reactive oxygen species. The compound suppressed the growth of tumors in a colon cancer xenograft model without any overt toxicity and exhibited a combination effect with irinotecan, a topoisomerase I inhibitor. Moreover, we identified the aryl ketone as a key component in the molecular structure responsible for the biological activity of asperphenin A using its synthetic derivatives. Collectively, this study has revealed the antiproliferative and antitumor mechanism of asperphenin A and suggested its possibility as a chemotherapeutic agent and lead compound with a novel structure.

Keywords: Aspergillus sp. fungus; antimitotic agent; asperphenins; colon cancer; marine microorganism metabolite; tubulin polymerization.

Figure 1

Structures of asperphenins and synthetic derivatives: Asperphenin A (1); asperphenin B (2); cycloasperphenin A (3); cycloasperphenin B (4); 7-hydroxyasperphenin A (5); 7-epi-hydroxyasperphenin A (6); 7-hydroxyasperphenin B (7); 7-epi-hydroxyasperphenin B (8); 7, 15(S)-dihydroxyasperphenin A (9); 7,15(S)-dihydroxyasperphenin B (10); 7,15(R)-dihydroxyasperphenin B (11).

Figure 2

Effect of AspA on cell cycle progression and tubulin polymerization in RKO cells. (A,B) Cells were treated with the indicated concentrations of AspA for 48 h (A) or 5 μM of AspA for indicated times (B) and then analyzed by flow cytometry. (C,D) The levels of G₂/M regulatory proteins in cells were determined after treatment with various concentrations of AspA for 48 h (C) or 5 μM of AspA over time (D) by western blot analysis. β-Actin was used as a loading control. (E) Inhibitory effect of AspA on tubulin polymerization. The tubulin assembly in the presence of vehicle or indicated compounds was analyzed by measuring the fluorescence emitted at 450 nm using 360 nm as an excitation wavelength for 60 min at 37 °C. Paclitaxel (TXL) and vinblastine (VBL) were used as reference compounds. The data are presented as means ± SD.

Figure 3

Effect of AspA on the induction of apoptosis and intracellular ROS in RKO cells. (A) Cells were treated with the indicated concentrations of AspA for 48 h. Collected cells were stained with Annexin V-FITC and PI, and analyzed by flow cytometry. (B,C) The expression of apoptosis-associated proteins was observed in cells treated with the indicated concentrations of AspA for 48 h (B) or 5 μM of AspA for the indicated times (C) by western blot analysis. β-Actin was used as a loading control. tBid, truncated Bid. (D) Cells were treated with AspA alone or with 5 mM of NAC for 24 h. The cells were stained with DCFH-DA and then analyzed by flow cytometry.

Figure 4

Effect of combined treatment with AspA and irinotecan or paclitaxel (TXL) on cell proliferation in RKO cells. (A,B) Cells were treated with the indicated concentrations of either irinotecan (A) or TXL (B) with or without AspA for 48 h. The cell proliferation was measured by SRB assay and the combination effect was determined by calculating combination index (CI) values. The data are presented as means ± SD.

Figure 5

Antitumor activity of AspA in a nude mouse xenograft model. (A) RKO xenograft-bearing nude mice were treated intraperitoneally with the indicated drugs three times per week for 21 days (n = 5 per group). For combination, 4 mg/kg of AspA and 4 mg/kg of irinotecan were administered. Tumor volumes were measured every 3–4 days. (B) Ki67 protein expression in RKO cell xenograft tumors was determined by immunohistochemical analysis. Scale bar, 100 μm. (C) The bodyweight of mice bearing RKO xenografts was measured every 3–4 days during the treatment with the indicated drugs (n = 5 per group). The error bars represent the means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.005 by t-test.