SW480 and SW1116 cells were acquired from ATCC and grown in DMEM medium (Biological Industries, Israel) supplemented with 10% FBS (Biological Industries, Israel), 1% penicillin/streptomycin (Coolaber, Beijing, China), and 2.5% HEPES buffer (Procell, Wuhan, China) in an incubator with a humidified air atmosphere of 5% CO₂ at 37°C.

Cells were seeded at a density of 1 × 10⁴ cells/well in a 96-well plate. After being cultured overnight, cells were treated with ivermectin (Figure 1) (MCE Chemicals, Shanghai, China) at the indicated concentrations for 12, 24, or 36 h or cells were pretreated with N-Acetyl-l-cysteine (NAC, 5 mM) (Aladdin, Shanghai, China) for 1 h and then were cultured in ivermectin (20 μM) for 6 h. Then, 10 μL of CCK-8 solution was added to each well and incubated at 37°C for 1 h. The absorbance was detected at 450 nm by a microplate reader (SpectraMax i3x, Molecular Devices, United States). The cell viability was calculated as follows: (absorbance of drug-treated sample/absorbance of control sample) × 100.

Colorectal cancer cells were plated at 1 × 10⁵ cells/well in twelve-well plates. After being cultured overnight, the cells were treated with ivermectin at the indicated dose for 24 h. Cell morphology was evaluated using an optical microscope.

Apoptosis was determined using the Annexin V-FITC/7-AAD Apoptosis Kit. Briefly, after colorectal cancer cells were exposed to ivermectin (0, 5, 10, and 20 μM) for 6 h, cells were centrifuged at 1,500 rpm for 5 min, washed, and suspended in PBS. Then, cells were stained with Annexin V-FITC and 7-AAD for 15 min. In addition, cells (1 × 10⁶ cells/well) were cultured onto 6-well plates overnight and treated with indicated concentrations (0, 2.5, 5, and 10 μM) of ivermectin. Then, the cells were harvested and resuspended in PBS and fixed with 70% ethanol, and left at −20°C overnight. After 12 h of fixation, cells were centrifuged, washed, and resuspended in cold PBS. Then, the cells were added with 100 μL of RNase and incubated at 37°C for 30 min. The PI staining solution was then added and incubated at 4°C for 30 min. Cells were acquired by flow cytometry (FACSCanto Plus), and data were analyzed using Flowjo 10.0 software. The percentage of Q2 (early apoptosis, Annexin V⁺7-AAD^-) plus Q3 (late apoptosis, Annexin V⁺7-AAD⁺) region was counted as the percentage of apoptosis cells.

Caspase 3/7 assay was performed using the Caspase 3/7 Activity Apoptosis Assay Kit (Sangon Biotech, Shanghai, China). Briefly, after colorectal cancer cells (SW480 or SW1116) were treated with different concentrations of ivermectin (0, 5, 10, and 20 μM) for 6 h, we added 100 μL of Caspase 3/7 reagent into each well and mixed using a plate shaker. The Caspase 3/7 activity was then determined using a microplate reader (SpectraMax i3x). The Caspase 3/7 activity was expressed as a fold of the untreated control (Con) treatment.

After colorectal cancer cells (SW480 and SW1116) were treated with 0, 5, and 10 μM ivermectin for 6 h, they were collected, washed with PBS, and lysed with RIPA buffer. Protein quantification was determined using BCA Protein Assay Kit (EpiZyme Biotechnology, Shanghai, China). Equal amounts of protein were loaded onto an SDS-PAGE gel for electrophoresis and transferred to nitrocellulose. After blocking with 5% nonfat milk for 1 h, the membranes were incubated with the primary antibody [Bcl-2 (1:2000), Bax (1:2000), PARP (1:20,000) (all from Proteintech, Rosemont, IL, United States), and β-actin (1:5000) (Sigma-Aldrich)] on a 4°C shaker overnight. The membranes were then incubated with a secondary antibody for another 1 h at room temperature. A chemiluminescent gel imaging system detected the change in target protein expression (Universal Hood II, Bio-Rad, Hercules, CA, United States).

For total ROS measurement, colorectal cancer cells (SW1116) were seeded (1 × 10⁵ cells/well) in a 12-well plate and incubated overnight. Cells were treated with different concentrations of ivermectin for another 6 h, and then they were co-cultured with DCFH-DA (Invitrogen, Carlsbad, CA, United States) and DAPI (Biolegend, San Diego, CA, United States) for 20 min at 37°C in the dark. The cell fluorescence was photographed by fluorescence microscopy (OLYMPUS, Tokyo, Japan).

For the mitochondrial ROS measurement, colorectal cancer cells (SW1116) were seeded in a 12-well plate and incubated overnight. After that, cells were treated with 0, 2.5, 5, 10, and 20 μM ivermectin for another 6 h, and then they were tinted with oxidation of MitoSOX Red (Invitrogen, Carlsbad, CA) and DAPI, which is oxidized by superoxide in the mitochondria, emitting red fluorescence. Cultures were incubated for 10 min at 37°C and washed twice with warm HBSS. Production of mitochondrial ROS was analyzed using MitoSOX Red. The cell fluorescence was photographed by fluorescence microscopy.

All experiments were repeated at least three times, and data were presented as mean ± S.E.M. The statistics were analyzed using a one-way or two-way ANOVA analysis (ANOVA) followed by Tukey’s test using Prism 9.0 software (Graphpad Software). p values are *, p < 0.05, ^#, p < 0.05;

To explore the effect of ivermectin on the proliferation of colorectal cancer cells, we used different concentrations of ivermectin (0, 2.5, 5, 10, 15, 20, and 30 μM) to culture colorectal cancer cells SW480 and SW1116. CCK-8 assay was performed to measure SW480 and SW1116 cancer cell proliferation after cells were incubated for 12, 24, and 36 h. As shown in Figure 2, the cell viability of SW480 and SW1116 cells decreased dose-dependently by ivermectin treatment (Dose, D: p < 0.01). Furthermore, ivermectin inhibited SW480 and SW1116 cell viability in a time-dependent manner (Time, T: p < 0.01). Finally, Table 1 showed that the IC₅₀ of SW480 cells treated with ivermectin for 12, 24, and 36 h was 16.17 ± 0.76 μM, 15.34 ± 0.81 μM, and 12.11 ± 0.97 μM, respectively; and the IC₅₀ of SW1116 cells treated with ivermectin for 12, 24, and 36 h were 7.60 ± 0.62 μM, 6.27 ± 0.70 μM and 5.76 ± 0.81 μM, respectively. The present data indicate that ivermectin might have more sensitive to SW1116 cells than that of SW480 cells.

To study the impact of ivermectin on the cell morphology of colorectal cancer cells, we treated colorectal cancer cells SW480 and SW1116 with different concentrations of ivermectin and then observed the alteration of cell morphology under an optical microscope. After 24 h culture, the cell morphology changed significantly. For the SW480 cells, as the ivermectin concentration increased, the cells became more and more sparse. Especially at 20 μM, the cells lost their original shape, became rounded, and shrunk or floated in the medium (Figure 3). Consistent with IC₅₀ of ivermectin, ivermectin had more sensitivity to SW1116 cells since ivermectin at 5 μM resulted in the cells shrunk; when the concentration of ivermectin was 10 μM, the cells became round, shrunk, and floated in the medium, and the concentration of ivermectin increased to 20 μM, most of the cells shed and floated in the culture medium (Figure 3). These results suggest that ivermectin could promote the death of colorectal cancer cells in a dose-dependent manner.

To determine whether ivermectin decreased the cell viability and the cell morphology of colorectal cancer cells via inducing cell apoptosis, we cultured colorectal cancer cells SW480 and SW1116 cells with indicated concentrations of ivermectin for 6 h, and apoptosis was evaluated by flow cytometry using Annexin V-FITC/7-AAD co-staining. As shown in Figure 4, ivermectin increased the proportion of apoptosis cells of SW1116 cells from 9.48% in the control group to 10.5%, 19.87%, and 30.5% in 5, 10, and 20 μM, respectively. Like this, ivermectin increased the proportion of apoptosis SW480 cells from 4.65% in the control cells to 8.51, 12.27, and 12.66% in 5, 10, and 20 μM, respectively. The results indicated that ivermectin had a dose-dependent effect on the induction of colorectal cancer cell apoptosis.

Caspase-3 plays a vital role in the initiation of cell apoptosis. Caspase-3 typically exists in the cytoplasm in the form of zymogen (32KD). Caspase-3 activated by upstream signaling molecules can cleave the downstream key apoptosis proteins in the early stages of apoptosis and ultimately lead to apoptosis. In this study, the Caspase 3/7 Activity Apoptosis Assay kit was used to determine the effect of ivermectin on cell apoptosis. As shown in Figure 5, ivermectin increased Caspase 3/7 activity of SW480 and SW1116 cells in a dose-dependent manner.

Bax and Bcl-2 are critical molecules in the endogenous apoptotic pathway. PARP (poly ADP-ribose polymerase) is a DNA repair enzyme, cleaved into Cleaved-PARP by the Caspase family protein, and cannot perform the repair function. The Western blot assay was used to determine the changes of apoptosis-related proteins Bax, Bcl-2, PARP, and Cleaved-PARP after treatment of colorectal cancer SW480 and SW1116 cells with ivermectin at the indicated doses. As the concentration of ivermectin increased, the expression of the proapoptotic protein Bax increased significantly, and the expression of the antiapoptotic protein Bcl-2 decreased; that is, the expression of the Bax/Bcl-2 ratio was gradually increasing (Figure 6). Also, the expression of Cleaved-PARP increased following the increase of ivermectin concentration (Figure 6).

To determine total intracellular total ROS and mitochondrial ROS levels, we treated SW1116 cells with ivermectin at the indicated concentrations for 6 h. After that, the cells were stained by DCFH-DA or MitoSOX with DAPI; cells were visualized using a fluorescence microscope. With the increase of the ivermectin concentration, the fluorescence of DCFA-DA (Figure 7) and MitoSOX (Figure 8) intensity gradually increased, indicating that ivermectin dose-dependently could increase the total and mitochondrial ROS content.

To further illuminate the relationship between ROS and mitochondrial signal pathways in ivermectin-induced apoptosis, colorectal cancer cells were pretreated with ROS inhibitor NAC (5 mM) for 1 h and cultured in ivermectin (20 μM) for another 6 h. The cells were stained by MitoSOX and detected by flow cytometry. Compared with the control (Con, 0 µM) group, the peak shifted significantly to the right after treatment with ivermectin (20 µM); the peak shifted to the left after NAC administration (Figures 9A,B). This result illustrated that the NAC inhibited the ivermectin-induced ROS accumulation. Furthermore, this study used the CCK-8 kit to detected cell viability. For colorectal cancer cells SW480 (Figure 9C) and SW1116 (Figure 9D), compared with the control group without ivermectin, the cell viability decreased after ivermectin treatment could be reversed by administration of NAC.

The intercellular phase is divided into stationary phase (G0), early DNA synthesis phase (G1), DNA synthesis phase (S), and late DNA synthesis phase (G2/M). Propidium iodide (PI) single staining combined with flow cytometry could be used to detect the cell cycle changes after treating SW480 and SW1116 cells with different concentrations of ivermectin for 12 h. The fluorescent dye PI can bind to DNA in cells. The higher the DNA content, the more fluorescent dyes it binds, and the stronger the fluorescence intensity detected by flow cytometry. The cell cycle stages are divided, and the number of cells in different cycles is measured. Our results showed that the proportion of SW480 cells in the control group (0 µM) in the S phase is 5.31%. The proportion of S phase in each experimental group after 2.5, 5, and 10 μM ivermectin treatments were 25.2, 31.8, and 32.4%, showing an upward trend, indicating that ivermectin could block SW480 in the S phase, slow down the process of S phase entering G2/M phase, block cell growth, and division, and inhibit tumor cell proliferation (Figures 10A,B). It could also be seen from the same principle that the proportions of the S phase of SW1116 cells treated with 0, 2.5, 5, and 10 μM for 12 h were 15, 23, 29, and 37%, respectively (Figures 10A,C).