Two Novel Compounds Isolated from the Marine Fungal Symbiont of Aspergillus unguis Induce Apoptosis and Cell Cycle Arrest in Breast Cancer Cells: In vitro Study

Abstract

Purpose: A promising feature of marine sponges is the potential anticancer efficacy of their secondary metabolites. The objective of this study was to explore the anticancer activities of compounds from the fungal symbiont of Aaptos suberitoides on breast cancer cells.

Methods: In the present research, Aspergillus unguis, an endophytic fungal strain derived from the marine sponge A. suberitoides was successfully isolated and characterized. Subsequently, ethyl acetate extraction and isolation of chemical constituents produced was performed. The structures of the isolated compounds were identified using several spectroscopic methods, ie, UV, NMR, and mass spectrometry. Thereafter, MDA-MB-231, MCF-7 breast cancer cells and HaCat cells were treated with the isolated compounds. Not only viability, apoptosis, and cell cycle analyses were conducted, but also the mRNA expression of MCL1, BCL2L1, AKT1 and CDK2 were evaluated.

Results: The extract showed cytotoxic activity in breast cancer cells. Two novel compounds were successfully isolated and identified, ie, Unguisol A (15.1 mg) and Unguisol B (97.9 mg). Both compounds share the same basic skeleton and comprise an aromatic ring which is attached to a sulphur-containing, seven-membered ring via an oxygen atom. This marked the first-time isolation of Unguisol A and Unguisol B from A. unguis, highlighting their novelty. Both compounds induced early apoptosis (p < 0.01) and cell cycle arrest at the S phase (p < 0.05) in MDA-MB-231 cells, but not in HaCat cells. Both compounds suppressed BCL2L1 and AKT1 mRNA expression (p < 0.01).

Conclusion: Two novel compounds were isolated from A. unguis. Unguisol A and Unguisol B induced apoptosis in MDA-MB-231 breast cancer cells via BCL2L1 mRNA downregulation, while both compounds induced cell cycle arrest at the S phase through AKT1 mRNA downregulation.

Keywords: Aspergillus unguis; Porifera; drug screening assays; triple negative breast neoplasms.

Graphical abstract

Figure 1

Cytotoxicity of A. unguis extract on TNBC and normal cells. MDA-MB-231 and HaCaT cells were exposed to A. unguis extract for 72 hours before the MTT assay was conducted. Data represented the average of triplicate measurements from three independent experiments.

Figure 2

Compound S01 (Unguisol A) Structure and 2D NMR Correlations.

Figure 3

Compound S02 (Unguisol B) Structure and 2D NMR Correlations.

Figure 4

Compound S02 triggered death in MDA-MB-231 cells more potently and selectively than compound S01. MDA-MB-231 and HCC-1954 cells were exposed to compounds S01 (A) and S02 (B) for 72 hours before the MTT assay was conducted. Data represented the average of triplicate measurements from three independent experiments.

Figure 5

MDA-MB-231 cells were induced apoptosis and suppressed mRNA expression of BCL2L1 but not MCL1. MDA-MB-231 cells and HaCaT cells were exposed to DMSO as control, compound S01, and S02 or cisplatin 30nM for 48 hours before Annexin V/PI staining (A and C) as well as mRNA isolation were conducted. Data (B and D) represented the average of duplicate measurements from two independent experiments. MCL1 (E) and BCL2L1 (F) mRNA expression represented the fold average of triplicate measurements from three independent experiments. *, p < 0.05; **, p < 0.01.

Figure 6

Both compounds S01 and S02 induced cell cycle arrest at the S phase and suppressed mRNA expression of AKT1 but not CDK2 in MDA-MB-231 cells. MDA-MB-231 cells and HaCaT cell were exposed to DMSO as control, compound S01, and S02 for 48 h before conducting cell cycle analysis (A) and mRNA isolation (C and D) followed by RT qPCR. Paclitaxel 1nM were used as positive control. Data (B) represented the average of duplicate measurements from two independent experiments. AKT1 (C) and CDK2 (D) mRNA expression represented the fold average of triplicate measurements from three independent experiments. *, p < 0.05; **, p < 0.01, ***, p < 0.001.