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. 2020 Nov;20(5):166.
doi: 10.3892/ol.2020.12029. Epub 2020 Aug 27.

Monascin accelerates anoikis in circulating tumor cells and prevents breast cancer metastasis

Kung-Yen Chen  1 , Jui-An Lin  1   2 , Han-Yun Yao  1 , An-Chih Hsu  1 , Yu-Ting Tai  1   2 , Bing-Ying Ho  3
Affiliations

Monascin accelerates anoikis in circulating tumor cells and prevents breast cancer metastasis

Kung-Yen Chen et al. Oncol Lett. 2020 Nov.

Abstract

Anoikis resistance has been observed in various types of cancers in which anchorage-independent growth is a crucial step for cancer metastasis. Therefore, agents interfering with this specific cancer cell behavior may be integrated into novel antimetastatic strategies. Monascin (MS), a secondary metabolite found in Monascus species, is a known potent chemopreventive compound used for treating metabolic complications; however, the effect of MS on anoikis resistance has not been investigated. In this study, 4T1 breast cells were treated with MS under either suspension or adhesion conditions. The higher cytotoxicity of MS was more potent against suspended cells than against adherent cells. This selective cytotoxicity was due to the induction of anoikis, which was evidenced by changes in cell aggregation, caspase activity, and Annexin V/propidium iodide binding as well as the results of systemic metastasis in an animal model. Furthermore, MS inhibited E-cadherin and β-catenin expression in the cells; the treated cells formed spherical aggregates, which suggested that anchorage-independent growth was prevented by MS. These results provide new insights into the mechanisms underlying the growth-preventing effect of MS on cancer cells and indicate the potential ability of MS to suppress metastasis.

Keywords: anoikis; circulating tumor cell; metastasis; monascin.

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Figures

Figure 1.
Figure 1.
Inhibitory effects of MS on the viability of breast cells. (A) 4T1 breast cancer cells and NMuMG normal breast cells were treated with MS for 48 h. A growth inhibition of 50% was observed at 23.8 and 160.0 µM in 4T1 and NMuMG cells, respectively. (B) Cell survival of 4T1 cells cultured on the polyHEMA-coated plates treated with MS for 24, 48, and 72 h was compared with that of the cells under normal adhesion conditions at the same time points. (C) Trypan blue dye exclusion was used to measure the percentage of dead cells in different culture types treated with and without 20 µM MS for 48 h. The relative percentage of dead cells under the suspension condition was compared with that under the attachment condition. Points indicate the mean ± standard deviation of at least three independent experiments that were each performed in triplicate. ***P<0.001 vs. control. MS, monascin; Ctrl, control.
Figure 2.
Figure 2.
Induction of apoptosis by MS in 4T1 breast cancer cells. (A) Fluorescence-activated cell sorting analysis of Annexin V/PI staining. The results indicated early apoptosis, defined as Annexin V-positive and PI-negative cells (Q1-UL), and late apoptosis, defined as Annexin V-positive and PI-positive cells (Q1-UR). 4T1 cells were cultured on a general culture plate (att) or a polyHEMA-coated plate (un_att), treated with 20 µM (MS) or without MS for 48 h, and then labeled with Annexin V-FITC and PI. The results are expressed as means ± SD of three independent experiments. ***P<0.001 vs. control.(B) Western blot analysis for determining the effect of MS treatment for 48 h on PARP and cleavage caspase-3 expression under attachment and suspension conditions. MS, monascin; Ctrl, control; att, attached; un_att, unattached; PI, propidium iodide.
Figure 3.
Figure 3.
MS inhibits 4T1 breast cancer cell aggregation under the suspension condition. (A) Phase-contrast images of 4T1 cells on the polyHEMA-coated plate treated with or without 20 µM MS for 48 h. Quantitative counts of clustered 4T1 cells reveal a significant decrease in MS treatment. (B) Western blot analysis of E-cadherin and β-catenin expression to determine the effect of MS treatment for 48 h on the floating cells. *P<0.05 vs. control. MS, monascin; Ctrl, control.
Figure 4.
Figure 4.
MS treatment reduces tumor growth in a tail vein-injected mouse model. (A) Before injection of 4T1 cells expressing the firefly luciferase gene (4T1-Luc), female BALB/c mice were treated (oral gavage) with 100 or 500 mg/kg MS (or vehicle PBS control) for 1 week. Then, the mice were intravenously injected with 4T1-Luc cells (1×105) through the tail vein, and the treatment (MS or vehicle PBS) continued for 4 weeks. (B) For 1 week after tumor injection, bioluminescence signals were not detected in any of the living mice. Pulmonary metastasis susceptibility was observed via detection of bioluminescence signals in the vehicle PBS-treated mice after sacrifice; however, no signal was detected in the MS-treated groups. (C) Bioluminescence signals [mean photon flux (photons/s)] were assessed in the areas over the lungs on days 14, 21 and 28. n=5 mice per group. MS, monascin.
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