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.

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.

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 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).

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.