Korean J Physiol Pharmacol.  2024 Jan;28(1):21-30. 10.4196/kjpp.2024.28.1.21.

Inhibition of Wnt/ββ-catenin signaling by monensin in cervical cancer

• Fu B
• Fang L
• Wang R
• Zhang X

• KMID: 2550276
• DOI: http://doi.org/10.4196/kjpp.2024.28.1.21

Abstract

The challenging clinical outcomes associated with advanced cervical cancer underscore the need for a novel therapeutic approach. Monensin, a polyether antibiotic, has recently emerged as a promising candidate with anti-cancer properties. In line with these ongoing efforts, our study presents compelling evidence of monensin's potent efficacy in cervical cancer. Monensin exerts a pronounced inhibitory impact on proliferation and anchorage-independent growth. Additionally, monensin significantly inhibited cervical cancer growth in vivo without causing any discernible toxicity in mice. Mechanism studies show that monensin's anti-cervical cancer activity can be attributed to its capacity to inhibit the Wnt/β-catenin pathway, rather than inducing oxidative stress. Monensin effectively reduces both the levels and activity of β-catenin, and we identify Akt, rather than CK1, as the key player involved in monensin-mediated Wnt/β-catenin inhibition. Rescue studies using Wnt activator and β-catenin-overexpressing cells confirmed that β-catenin inhibition is the mechanism of monensin’s action. As expected, cervical cancer cells exhibiting heightened Wnt/β-catenin activity display increased sensitivity to monensin treatment. In conclusion, our findings provide pre-clinical evidence that supports further exploration of monensin's potential for repurposing in cervical cancer therapy, particularly for patients exhibiting aberrant Wnt/β-catenin activation.

Keyword

Monensin; Oxidative stress; Uterine cervical neoplasms; Wnt beta-catenin signaling pathway

Figure

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  Fig. 1 Monensin inhibits cervical cancer in vitro. (A) Proliferation of cervical cancer cells incubated with monensin for 72 h was assessed by BrdU proliferation assay. (B) Apoptosis of cervical cancer cells incubated with monensin for 72 h was assessed by flow cytometry of Annexin V percentage. (C) Representative images of anchorage-independent growth of CaSki cells in the presence of monensin. (D) Anchorage-independent growth of cervical cancer cells in the presence of monensin was measured by anchorage-independent colony formation assay and quantitative analysis by Image J software. Monensin at 2.5, 5 and 10 µM was used. N = 3. BrdU, 5-bromo-2’-deoxyuridine; DMSO, dimethyl sulfoxide. *p < 0.05 represents significant difference compared with cells without monensin treatment.

• 

  Fig. 2 Monensin inhibits cervical cancer in a ROS-independent manner. (A) Intracellular ROS level of cervical cancer cells incubated with monensin for 24 h. (B) Proliferation of cervical cancer cells incubated with monensin in the absence or presence of NAC. Representative images (C) and quantification (D) of anchorage-independent growth of cervical cancer cells incubated with monensin (10 µM) in the absence or presence of NAC. The cells were given a pre-treatment of NAC at a concentration of 10 mM before being exposed to monensin. N = 3. ROS, reactive oxygen species; NAC, N-acetylcysteine; DMSO, dimethyl sulfoxide. *p < 0.05 represents significant difference compared with cells without monensin treatment; n.s represents no significant differences between cells with and without NAC.

• 

  Fig. 3 Monensin inhibits Wnt/β-catenin signaling in cervical cancer cells. (A, B) Western blotting showing the levels of β-catenin, p-β-catenin, Akt and p-Akt in CaSki and HeLa cells treated with monensin. Transcriptional activity level of β-catenin (C) and mRNA level of Wnt-targeted genes (D) in CaSki and HeLa cells treated with monensin. Monensin at 10 µM was used. Results were relative to control (set up as 1). N = 3. DMSO, dimethyl sulfoxide. *p < 0.05 represent significant difference compared with cells without monensin treatment.

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  Fig. 4 Monensin inhibits cervical cancer in a β-catenin-dependent manner. (A) Western blotting showing the levels of β-catenin in CaSki and HeLa cells after transfecting β-catenin overexpression plasmid. Transcriptional activity level of β-catenin (B), proliferation (C) and anchorage-independent growth (D) in CaSki and HeLa cells transfected with β-catenin overexpression plasmid and incubated with monensin. Monensin at 10 µM was used. N = 3. *p < 0.05 represent significant difference between cells transfected with vector and cells transfected with β-catenin overexpression plasmid in the presence of monensin.

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  Fig. 5 Lithium chloride (LiCl) reverses the inhibitory effects of monensin in cervical cancer cells. (A) Western blotting showing the levels of β-catenin in CaSki and HeLa cells incubated with LiCl and monensin. Proliferation (B) and anchorage-independent growth (C) in CaSki and HeLa cells incubated with LiCl and monensin. The cells were given a pre-treatment of LiCl at a concentration of 20 mM before being exposed to monensin. (D) Western blotting showing the levels of β-catenin and (E) transcription activity level of β-catenin in CaSki, SiHa and HeLa cells. N = 3. DMSO, dimethyl sulfoxide. *p < 0.05 represent significant differences between cells with and without LiCl.

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  Fig. 6 Monensin inhibits cervical cancer growth in mice. (A) Mice body weight in control and monensin-treated groups on day 0 and day 30. (B) Representative images of tumors in mice treated with monensin and control. Mice were euthanized at day 30 and tumors were dissected. N = 3. (C) CaSki tumor size in mice treated with monensin at different times. (D) Immunoblotting of phosphor- and total β-catenin and Akt of tumor lysates pooled from control and monensin groups. N = 6. *p < 0.05 represent significant difference compared with mice without monensin treatment.

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