Antimetastatic effect of halichondramide, a trisoxazole macrolide from the marine sponge Chondrosia corticata, on human prostate cancer cells via modulation of epithelial-to-mesenchymal transition

Abstract

Halichondramide (HCA), a trisoxazole-containing macrolide isolated from the marine sponge Chondrosia corticata has been shown to exhibit cytotoxicity and antifungal activities. In our previous study, HCA was also found to exhibit antiproliferative activity against a variety of cancer cells. However, the precise mechanism of action of HCA in the antitumor activity remains to be elucidated. In the present study, we identified the antimetastatic activity of HCA in the highly metastatic PC3 human prostate cancer cells. HCA showed potent growth inhibitory activity of the PC3 cells with an IC50 value of 0.81 µM. Further analysis revealed that HCA suppressed the expression of a potential metastatic biomarker, phosphatase of regenerating liver-3 (PRL-3), in PC3 cells. The suppression of PRL-3 by HCA sequentially down-regulates the expression of phosphoinositide 3-kinase (PI3K) subunits p85 and p110. The antimetastatic effect of HCA was also correlated with the down-regulation of matrix metalloproteases (MMPs) and the modulation of cadherin switches N-cadherin and E-cadherin. In addition, HCA also effectively suppressed the migration and invasion of PC3 cells. These findings suggest that halichondramide might serve as a potential inhibitor of tumor cell metastasis with the modulation of PRL-3.

Figure 1

The growth inhibitory activity of halichondramide (HCA) in PC3 prostate cancer cells. (A) PC3 cells (1 × 10⁵ cells/mL) were treated with HCA for 72 h; subsequently, antiproliferative activity was determined by the SRB protein dye method as described in the Experimental Section. The data are represented as the mean percentages ± SD for each HCA-treated group relative to the DMSO-treated control group.Each experiment was performed in triplicate (n = 3). * p < 0.05, ** p < 0.01 compared with the control group; (B) The morphological changes of PC3 cells treated with HCA for 72 h were observed under a phase-contrast microscope and photographed (at 40×magnification).

Figure 2

The effects of HCA on the mRNA expression of PRL-3 and its down-stream genes and metastatic biomarkers. PC-3 cells (4 × 10⁵ cells/mL) were treated with HCA for 24 h, and the mRNA expression levels of PRL-3 and metastasis biomarkers were determined by real-time RT-PCR analysis. The data represent the mean values ± SD of nine experiments. * p < 0.05, ** p < 0.01 compared with the control group.

Figure 3

The effects of HCA on the expression of PRL-3 and its associated proteins. PC-3 cells (2 × 10⁵ cells/mL) were treated with HCA for 24 h; subsequently, the protein expression levels of PRL-3 and its associated proteins were analyzed by Western blotting as described in the Experimental Section.

Figure 4

The inhibitory effects of HCA on cell migration and invasion. (A) The effect of HCA on cell migration was analyzed by the wound healing assay. PC3 cells (1×10⁵ cells/mL) were seeded in six-well plates and incubated for 48 h.An artificial wound was made that consisted of a scratch with a micropipette tip. The wound healing process was monitored during 72 h of incubation with various concentrations of HCA. Images of the wounds were photographed at 0 and 72 h under an inverted microscop; (B)The cell invasion assay was performed in a Matrigel-coated chamber system as described in the Experimental Section. PC3 cells (1 × 10⁵ cells/chamber) were plated in the upper chambers of Matrigel-coated Transwell insert. The lower chambercontained 0.1 mg/mL BSA as a chemoattractant in the medium. The inserts were incubated for 24 h; subsequently, the cells that had invaded the outer surface of the membrane were fixed, stained, and photographed.

Figure 5

The inhibitory effects of HCA on gelatin degradation. The effect of HCA on the MMPs expression were also analyzed by the gelatin zymographic method. HT-1080 cells (5 × 10⁴ cells/mL) were treated with HCA for 72 h; subsequently, as described in the Experimental Section, the proteolytic activities of the MMPswere assessed by the degradation of gelatin.

Figure 6

Chemical structure of halichondramide.