Cervical cancer continues to pose a significant challenge in the realm of gynecological health, despite the rapidly evolving knowledge regarding its prevention and treatment. While advancements in radiotherapy technology and the introduction of the anti-vascular endothelial growth factor agent bevacizumab have yielded improvements in clinical outcome, the overall prognosis for women afflicted with recurrent or metastatic cervical cancer remains disheartening [1,2]. A growing body of evidence has drawn a compelling connection between the unfavorable prognosis of cervical cancer and the abnormal activation of the Wnt/β-catenin pathway. This pathway plays a pivotal role in governing various cellular processes, including differentiation, proliferation, metastasis, epithelial-to-mesenchymal transition, recurrence, chemoresistance, and survival, across a spectrum of cancers [3,4]. Thus, the components of the Wnt/β-catenin pathway have emerged as key targets in the development of anti-cancer therapeutics over the past two decades.

Monensin, an ionophore antibiotic produced by Streptomyces cinnamonensis, has been traditionally employed to combat bacterial, fungal, and parasitic infections [5]. Notably, polyether ionophore antibiotics, including monensin, salinomycin, and narasin, have garnered attention for their capacity to induce apoptosis and arrest the cell cycle in various cancer cell types [6-11]. Moreover, these antibiotics have demonstrated effectiveness in surmounting multidrug-resistant cancer cells and eradicating cancer stem cells [7,12-16]. Monensin has exhibited inhibitory effects on anaplastic thyroid cancer, ovarian cancer, leukemia, glioblastoma, non-small cell lung cancer, melanoma, and breast cancer [9-12,17-20]. However, its impact on cervical cancer had not been explored until now. The anticancer attributes of monensin are ascribed to its capability to induce oxidative stress and inhibit signaling pathways involving mTOR, MYB, and growth factor receptors. Several studies have reported that monensin exerts inhibitory effects on canonical Wnt/β-catenin signaling [21-23].

Our hypothesis centered on the potential high efficacy of monensin in combating cervical cancer. To investigate this, we conducted a comprehensive examination of monensin's anticancer properties employing both a cervical cancer cell culture system and a xenograft mouse model. Our research delved into understanding the mechanisms underlying monensin's actions, with particular emphasis on its impact on oxidative stress and the Wnt/β-catenin signaling pathway.

Cell lines, culture, antibodies and chemicals

The human cervical cancer cell lines HeLa, CasKi and SiHa (Guangzhou Jennio Biotech Co. Ltd) were subjected to authentication through short tandem repeat profiling conducted by Precision Genomics Biotechnology. These cell lines were cultured in RMPI1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C, 5% CO₂. Antibodies specific to phospho-β-catenin, phospho-Akt and their corresponding total were from Cell Signaling Technology. β-actin antibody was from Santa Cruz Biotechnology. Monensin, N-acetylcysteine (NAC) and lithium chloride (LiCl) were purchased from Selleckchem and were reconstituted as per manufacturer’s instructions.

Proliferation assay

Cell proliferation was assessed using the 5-bromo-2'-deoxyuridine (BrdU) cell proliferation assay kit (Millipore), and the assay was conducted following the manufacturer's guidelines. Cells were seeded at a density of 8 × 10³ cells per well in a 96-well plate. Following a 72-h incubation period with varying concentrations of monensin in RPMI 1640 containing 10% FBS, the supernatants were aspirated, and BrdU reagent was introduced into each well, followed by a 2-h incubation period. The absorbance of each well was subsequently measured at 450 nm using a Tecan Microplate reader.

Anchorage-independent colony formation

Cell colony formation was assessed using the anchorage-independent colony formation assay, a method consistent with our previous study [24]. Soft agar plates were prepared by layering 400 µl of 0.7% Bacto agar containing RPMI 1640 and 10% FBS. Upon solidification, an additional layer of 400 µl of 0.3% Bacto agar containing RPMI 1640 and 10% FBS was added as the top agar. Cells, seeded at a density of 1,000 cells per well, were cultured in soft agar medium supplemented with monensin. The medium was refreshed every other day. After 7–10 days of incubation, the colonies were stained using crystal violet (Sigma) and subsequently analyzed using Image J version 1.8.0.

Cell apoptosis assay

Cell apoptosis was evaluated using flow cytometry based on Annexin V staining following the manufacturer's protocol. In brief, cervical cancer cells were seeded at a density of 5 × 10⁶ cells per well in a 6-well plate. After 72 h of incubation with varying concentrations of monensin in RPMI 1640 supplemented with 10% FBS, the cells were detached using trypsin and subsequently stained with FITC Annexin V Apoptosis Detection kit along with 7-AAD (BioLegend). This staining was performed at 4°C in the dark for 30 min, followed by flow cytometry analysis using a MACSQuant X instrument (Miltenyi Biotec).

Cellular reactive oxygen species (ROS) assay

Intracellular ROS levels were measured using a cellular ROS assay kit (Abcam). Cells were plated in a 96-well plate at a density of 2 × 10⁴ cells per well. Following a 24-h incubation with varying concentrations of monensin in RPMI 1640 supplemented with 10% FBS, the culture supernatant was removed, and the cells were exposed to the ROS red dye working solution. This was followed by a 30-min incubation period, after which fluorescence intensity was quantified at excitation/emission wavelengths of 520/605 nm using a Fluorescence Microplate Reader from Molecular Devices.

Quantitative real-time polymerase chain reaction (qPCR)

After a 24-h exposure to monensin, total RNA was extracted utilizing TRIzol reagent from Invitrogen. Subsequently, quantitative PCR (qPCR) was conducted in triplicate to assess the expression levels of AXIN2, C-MYC, LEF1, and GAPDH, employing the SYBR Green PCR Master Mix kit from Takara. The primer sequences used were consistent with those previously described [25], and they are listed in Supplementary Table 1. qPCR was executed using a CFX96 Real-Time System from Bio-Rad, and data analysis was carried out using MyiQ software from Bio-Rad. GAPDH served as the loading control for gene expression normalization.

β-catenin overexpression

To achieve β-catenin overexpression, cells were subjected to transient transfection with either 5 µg of pcDNA or pcDNA-β-catenin (Addgene plasmid #16828) in 6-well plates, with a seeding density of 1 million cells per well. Subsequently, after 48 h of transfection, cells were detached and collected for various cellular activity assays.

TOPFlash/FOPFlash luciferase reporter assay

To evaluate the transcriptional activity of β-catenin, we employed the dual TOPFlash/FOPFlash luciferase reporter assay kit from Promega. Initially, cells were transiently transfected with 1 µg of Renilla luciferase and 10 µg of TopFlash using Dharmafect Transfection Reagent. This transfection was carried out in 96-well plates with a seeding density of 2.5 × 10⁴ cells per well. Subsequently, transfected cells were subjected to drug treatments for 24 h. The assessment of both firefly and Renilla luciferase activity was conducted following the manufacturer's instructions. Firefly luciferase activity was normalized against Renilla luciferase activity, and the fold increase in TOPFlash activity compared to FOPFlash was reported.

Cervical cancer growth in mice

The animal study was conducted in accordance with the guidelines set forth by the Institutional Animal Care Committee of Hubei University of Arts and Science, under Approval No. 2019-16R, and adhered to the Guide for the Care and Use of Laboratory Animals. CaSki cells were subcutaneously injected into mice at a concentration of 6 × 10⁵ cells per mouse. One week post tumor inoculation, mice were randomly assigned to treatment groups, and the treatments were initiated on the same day. The treatment groups were as follows: The Control group and the Monensin group, where mice received 50 µl of 20% dimethyl sulfoxide (DMSO) in normal saline, and 1 mg/kg of monensin in 20% DMSO, respectively. These treatments were administered intraperitoneally every 24 h. Tumor dimensions, including length and width, were measured using a capillar. The mice were euthanized after 30 days.

Statistical analyses

Data are represented by means ± standard deviation of at least three independent experiments. p < 0.05 was considered to be statistically significant. The “n” in the figure legends indicates the number of mice or independent cell culture preparations.

Monensin is more effective in inhibiting proliferation and anchorage independent growth than inducing apoptosis in multiple cervical cancer cell lines

We conducted experiments to evaluate the impact of monensin at various concentrations on the growth and survival of cervical cancer cells. Utilizing three different cervical cancer cell lines, each representing a distinct in vitro model for cervical cancer with diverse cellular and genetic characteristics [26], we observed that monensin exhibited a dose-dependent reduction in the proliferation of all tested cell lines, as evidenced by the quantification of BrdU incorporation (Fig. 1A). Specifically, at a concentration of 10 µM, monensin led to approximately 100%, 60%, and 30% growth inhibition in CaSki, SiHa, and HeLa cells, respectively. We further assessed the induction of apoptosis by monensin using flow cytometry with Annexin V staining. The results indicated that monensin did not significantly induce apoptosis in SiHa and HeLa cells. It slightly promoted apoptosis, with an increase of up to approximately 20%, in CaSki cells (Fig. 1B and Supplementary Fig. 1).

Figure 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.

Subpopulations with highly proliferative, invasive and stem cell-like properties uniquely display anchorage independent growth in soft agar [27]. Using anchorage-independent colony formation assay, we showed that monensin, at concentrations of 2.5 µM, 5 µM, and 10 µM, potently decreased anchorage independent growth in three cervical cancer cell lines (Fig. 1C, D and Supplementary Fig. 2). Consistent with its inhibitory effect on proliferation, monensin at 10 µM resulted in approximately 100%, 80%, and 50% growth inhibition in CaSki, SiHa, and HeLa cells, respectively.

Taken together, our results indicate the following: 1) CaSki cells are more sensitive to monensin treatment compared to SiHa and HeLa cells, and 2) monensin is more effective at inhibiting proliferation and anchorage-independent growth than inducing apoptosis in multiple cervical cancer cell lines.

Monensin inhibits Wnt/β-catenin signaling in cervical cancer cells

Our subsequent investigations focused on HeLa and CaSki cell lines, representing the least sensitive and most sensitive responses to monensin, respectively. Previous research has suggested that inducing oxidative stress is the underlying mechanism behind monensin's anti-cancer activity [9,28,29]. To investigate whether oxidative stress contributes to the inhibitory effects of monensin in cervical cancer, we measured ROS level in cervical cancer cells after monensin treatment. To determine whether oxidative stress plays a role in the inhibitory effects of monensin in cervical cancer, we assessed the levels of intracellular ROS in cervical cancer cells following monensin treatment. While monensin did lead to a significant increase in intracellular ROS levels in both CaSki and HeLa cells (Fig. 2A), the antioxidant NAC did not reverse the effect of monensin in terms of decreasing cell proliferation and anchorage-independent growth (Fig. 2B–D). These findings indicate that ROS is not the primary mechanism responsible for the observed biological effects of monensin in cervical cancer cells, and other molecular mechanisms are likely involved.

Figure 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.

Our investigation revealed a significant reduction in β-catenin levels in both CaSki and HeLa cells following monensin treatment (Fig. 3A, B). We then delved into the phosphorylation status of β-catenin at key sites, including S33/37/T41, which are associated with GSK-3β phosphorylation and subsequent degradation; Ser 675, indicating nuclear accumulation and increased transcriptional activity of β-catenin, and S45, which serves as the phosphorylation site for casein kinase 1 and subsequent phosphorylation by GSK-3β [30]. Interestingly, we observed a decrease in the phosphorylation of β-catenin at Ser 33/37/T41 in both CaSki and HeLa cells (Fig. 3A, B). Furthermore, phosphorylation at Ser 675, indicative of enhanced transcriptional activity, was reduced in these cells upon monensin treatment. In contrast, phosphorylation at S45 remained unaffected by monensin. It is well established that β-catenin undergoes ubiquitination and subsequent degradation in the 26S proteasome through phosphorylation. However, the concurrent reduction in both total β-catenin levels and its phosphorylation levels at S33/37/T41 implies that the decrease in β-catenin in monensin-treated cervical cancer cells is not attributable to β-catenin degradation at this site. This suggests the involvement of additional mechanisms contributing to the observed decrease in β-catenin levels. Akt is known to phosphorylate β-catenin at Ser552, leading to an increase in its transcriptional activity. In line with the reduction in phosphorylated β-catenin at Ser552, we observed a decrease in phosphorylated Akt at Ser473 following monensin treatment (Fig. 3A, B). Additionally, we noted a significant decrease in Wnt/β-catenin transcriptional activity induced by monensin (Fig. 3C), as assessed by TOPFlash/FOPFlash luciferase reporter assay. Moreover, the mRNA levels of Wnt-targeted genes, including AXIN2, C-MYC, and LEF1, exhibited a substantial decrease ranging from 50% to 90% in cells treated with monensin (Fig. 3D). These findings collectively underscore the inhibitory effect of monensin on Wnt/β-catenin signaling.

Figure 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.

Monensin inhibits in cervical cancer cells in a Wnt/β-catenin-dependent manner

To validate that the inhibition of Wnt/β-catenin signaling is indeed the molecular mechanism of monensin's action in cervical cancer, we conducted rescue experiments employing two distinct approaches. The first approach involved the use of cells overexpressing β-catenin, while the second approach utilized LiCl, a compound known to inhibit β-catenin degradation and activate Wnt signaling [31]. The underlying hypothesis was that if monensin exerts its inhibitory effect on cervical cancer cells by suppressing Wnt/β-catenin signaling, then monensin's effectiveness should be reduced or nullified in β-catenin-overexpressing cells and LiCl-treated cells.

We transiently transfected CaSki and HeLa cells using β- catenin overexpression plasmid and validated that these cells displayed much higher level of β-catenin compared to control cells (Fig. 4A). As expected, the overexpression of β-catenin notably reversed the inhibitory effect of monensin on the transcriptional activity of Wnt/β-catenin in CaSki and HeLa cells (Fig. 4B). Of note, monensin demonstrated significantly reduced effectiveness in inhibiting proliferation and anchorage-independent growth in β-catenin-overexpressing cells (Fig. 4C, D). Additionally, we observed nearly complete restoration of monensin-induced β-catenin reduction in cervical cancer cells when LiCl was added (Fig. 5A). The anti-proliferative and anti-anchorage-independent growth activities of monensin were also reversed by the presence of LiCl (Fig. 5B, C). These findings provide clear evidence that monensin's mechanism of action involves the targeting of Wnt/β-catenin in cervical cancer cells.

Figure 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.

Figure 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.

Interestingly, we further found that β-catenin level and transcriptional activity of Wnt/β-catenin were significantly higher in CaSki cells compared to HeLa cells (Fig. 5D, E). This discrepancy in β-catenin activity may explain the differential sensitivity of CaSki and HeLa cells to monensin treatment.

Monensin inhibits cervical cancer growth in vivo

We conducted a comprehensive assessment of the in vivo efficacy and toxicity of monensin using a cervical cancer xenograft model established by implanting CaSki cells beneath the skin of SCID mice. Monensin treatment was initiated once tumors reached a size of approximately 150 mm³ and continued for a duration of 30 days. Throughout the study, we closely monitored the mice by measuring their body weight to assess general toxicity and tracking tumor size every 5 days to evaluate the anti-cancer efficacy. Our results showed that monensin administered at a dose of 1 mg/kg for 30 days had no significant impact on the body weight of the mice (Fig. 6A). Conversely, we observed a substantial reduction in tumor size starting from day 10 of treatment with monensin (Fig. 6B, C). Consistent with in vitro findings, immunoblotting analysis revealed a reduction in both total and phosphorylated β-catenin, as well as p-Akt in tumors derived from mice subjected to monensin treatment (Fig. 6D). These findings suggest that monensin, when administered at a non-toxic dose, effectively inhibits the growth of cervical cancer in vivo, primarily through the suppression of β-catenin.

Figure 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.

Drug repurposing has garnered attention in recent years in the anti-cancer drug discovery program because it has significant advantages over conventional drug development to identify targeted therapies. Monensin, a prime example of natural polyether ionophore antibiotics, is known for its ability to form pseudomacrocyclic complexes with metal cations, which contributes to its antimicrobial properties [32]. In this study, we provide the first evidence that the inhibition of the Wnt/β-catenin pathway by monensin has demonstrated significant activity in cervical cancer. This discovery underscores the potential of repurposing monensin as a novel therapeutic strategy in the treatment of cervical cancer.

The cell lines, we selected for demonstration of the biological effects of monensin model in vitro cervical cancer with diverse cellular and genetic origins. Our results demonstrated that while all tested cell lines were inhibited by monensin, they exhibited varying degrees of sensitivity. It's noteworthy that monensin did not induce apoptosis in two out of the three cell lines, which is in contrast to some previous studies reporting its pro-apoptotic effects in cancer cells [20,28,33]. This discrepancy is likely attributed to differences in monensin concentrations and the specific disease models employed. In contrast, our study consistently showed that monensin, at low micromolar concentrations, exerted significant inhibitory effects on the proliferation of cervical cancer cells. This aligns with the majority of research indicating that monensin possesses potent inhibitory effects on cancer cell growth, even in cases of chemoresistant cells [11,16]. The remarkable ability of monensin to inhibit anchorage-independent growth in cervical cancer cells suggests its potential in targeting cervical cancer stem cells, a hypothesis supported by previous studies demonstrating monensin's capacity to inhibit stemness maintenance in melanoma stem cells and its toxicity to cancer stem-like cells [12,34]. Furthermore, while the majority of studies on monensin's anti-cancer activities have focused on in vitro cell culturing systems, our research extends this understanding by demonstrating its in vivo efficacy using a cervical cancer xenograft mouse model. Importantly, we utilized a dose of monensin that proved to be both effective and non-toxic, highlighting a promising therapeutic window for monensin in cancer treatment. Previous studies have also indicated that monensin exhibits greater toxicity towards tumors than normal cells, making it a potentially favorable candidate for selective cancer therapy [9,35]. Notably, monensin has been shown to suppress colorectal tumor growth without adverse effects on nonmalignant intestinal mucosa [21].

While numerous studies have previously suggested that the anti-cancer properties of monensin are linked to its ability to induce oxidative stress [9,28,29], our research has demonstrated that this mechanism does not apply to monensin's actions in cervical cancer. Instead, we have provided evidence that monensin attenuates the Wnt signaling pathway, resulting in reduced nuclear and cytoplasmic β-catenin levels and a significant decrease in the transcription of Wnt target genes. This observation aligns with our hypothesis that monensin may target cervical cancer stem cells, as the Wnt/β-catenin pathway plays a critical role in these cells' biology. Several previous studies have also highlighted monensin's capacity to target cancer stem cells. For instance, monensin has shown selective toxicity to gastric cancer stem-like cells [34] and has acted as a potent inhibitor of melanoma stem cells [12]. Additionally, related polyether ionophore antibiotics like salinomycin have been found to inhibit metastatic colorectal cancer growth and interfere with Wnt/β-catenin signaling in colorectal cancer stem cells [36]. Salinomycin has also suppressed the tumorigenicity of hepatocellular carcinoma stem cells and Wnt/β-catenin signaling [37]. Our work, along with others in the field, suggests that targeting cancer stem cells via the Wnt/β-catenin pathway might be a common feature of polyether ionophore antibiotics. Aberrant activation of the Wnt/β-catenin pathway has been observed in cervical cancer, and targeting this pathway has shown therapeutic promise [3,4]. A significant finding of our work is that cervical cancer cells with higher level of Wnt/β-catenin signaling are more sensitive to monensin. Further exploration of the relationship between Wnt/β-catenin activity levels and the efficacy of Wnt inhibitors in cervical cancer in vivo would be an intriguing avenue for future research.

While various studies have laid the groundwork for monensin's anticancer effects using in vitro cell culturing models, our work focuses on the application of monensin in cervical cancer and demonstrates the significant inhibitory effects of monensin on cervical cancer growth in vivo without causing toxicity in mice. This aspect underscores the potential translational significance of our findings. Furthermore, our work dispels the notion that oxidative stress, commonly attributed to monensin's anticancer activity in various cancer types, governs its action in cervical cancer. Instead, our research reveals that monensin acts by suppressing the Wnt/β-catenin pathway, with the involvement of Akt in this process.

In summary, our study provides promising results that underscore the potential of monensin as a candidate for further investigation as a therapeutic option for cervical cancer, either as a standalone treatment or in combination with chemotherapy. While research on the repurposing of monensin as an anti-cancer drug is still in its early stages, our findings confirm its anti-cancer activity and are expected to expedite the initiation of clinical trials to evaluate monensin as a repurposed medication for cancer treatment.

None.

This work was supported by a central grant from Xiangyang Central Hospital Research Center Central Grant (CG2019-XJCD65).

The authors declare no conflicts of interest.

Supplementary data including one table and two figures can be found with this article online at https://doi.org/10.4196/kjpp.2024.28.1.21