Development and synthesis of diffractaic acid analogs as potent inhibitors of colorectal cancer stem cell traits

Abstract

In recent years, evidence for the anti-cancer activity of lichen secondary metabolites has been rapidly increasing. In this study, we synthesised analogues of diffractaic acid, a lichen secondary metabolite, and evaluated their ability to suppress colorectal cancer stem potential. Among the 10 compounds after H/CH₃/benzylation of the diffractaic acid structure or modifications in an aromatic hydrophobic domain, TU3 has a more inhibition effect on the stem potential of colorectal cancer compared to other compounds. The compound TU3 targets ALDH1 and suppresses key signalling pathways such as WNT, STAT3, NF-κB, Hedgehog, and AP-1. Inhibition of these signalling pathways by TU3 contribute to attenuate the survival mechanisms of colorectal cancer stem cell and thus inhibit cancer progression.

Keywords: ALDH1; Colorectal cancer stem cells; Diffractaic acid analogs; Lichen secondary metabolites; Structure-based modifications.

Fig. 1

Structures of naturally occurring bioactive depsides.

Fig. 2

Evaluation of Spheroid Formation in CSC Cell Lines Treated with Diffractaic Acid and Its Analogs. Representative images of spheroid formation by CSC221 (A) and CaCo2 (B) cells treated with diffractaic acid and its analogs at 10 μM concentration for 10–14 days, and quantitative analysis of the number of spheroids formed following each treatment. Data represent the mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 3

Compound TU3 suppress spheroid formation in CSC221 and CaCo2 cell lines in a dose-dependent manner. Representative images of spheroid formation by CSC221 (A) and CaCo2 (B) cells treated with TU3 at 10 μM concentration for 10–14 days, and quantitative analysis of the number of spheroids formed following each treatment. Flow cytometric analysis of cell cycle distribution. CSC221 (C) and CaCo2 (D) cells were treated with indicated concentrations of TU3 for 48 h incubation. Data represent the mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 4

TU3 targets colorectal cancer stem cells by blocking cellular signaling. Western blot analysis of ALDH1, Gli1, STAT3, NF-κB, β-catenin, c-Myc and Cyclin-D1 protein levels in CSC221 (A) and CaCo2 (B) cells treated with indicated concentrations of TU3 and incubated 48 h. (C–D) Quantitative analysis of protein expression. Data represent the mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001; difference compared with DMSO-treated.

Fig. 5

Schematic showing how the TU3 supresses the colorectal cancer stem cell. TU3 strongly suppresses the ALDH1 expression by regulated the STAT, NF-κB, AP-1, WNT, and HH signaling pathways.

Fig. 6

Synthetic procedure of monomer for diffractaic analogs. (i) Na, BnOH, refluxed, 24 h; (ii) K₂CO₃, Alkyl halide, DMF, 50 °C, 10 h; (iii) KOH, MeOH, rt, 12 h; (iv) SO₂Cl₂, Diethyl ether, 0 °C, 15 min and 37 °C, 30 min.

Fig. 7

Synthetic procedure of depside ester, TU-01, and TU-02.

Fig. 8

Synthetic procedure of TU-03, and TU-04.

Fig. 9

Synthetic procedure of TU-05.

Fig. 10

Synthetic procedure of TU-06.

Fig. 11

Synthetic procedure of TU-07.

Fig. 12

Synthetic procedure of TU-08.

Fig. 13

Synthetic procedure of TU-09.

Fig. 14

Synthetic procedure of TU-10.