Tumor growth suppression of ivermectin in gastric cancer cell lines and primary gastric cancer organoids

Introduction

Ivermectin is an avermectin family drug discovered by scientists Satoshi Omura and William C. Campbell as an effective antiparasitic agent (1,2), causing them to win the 2015 Nobel Prize in Physiology or Medicine. Recently, suggestions have been made regarding its other uses besides its parasitic effect, namely its anticancer and antiviral effects. It was effective in vitro on coronavirus disease 19 (COVID-19) (3) but was ineffective in in vivo human studies (4). Several studies have demonstrated the anticancer effects of ivermectin (5,6) in various types of malignant tumor cell lines, including ovarian cancer, breast cancer, colorectal cancer, and leukemia. Ivermectin has been reported to inhibit signaling pathways such as P21-activated kinase 1 (PAK1) (7), epidermal growth factor receptor (EGFR), extracellular signal-regulated kinases (ERKs), AKT serine/threonine kinase 1 (Akt), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which are involved in tumor progression and drug resistance (8-12).

Furthermore, ivermectin exhibits antiproliferative effects in gastric cancer by inhibiting the expression of Yes-associated protein 1 (YAP1), thereby reducing tumor growth and increasing cell death (13-15). Recent studies have reported that ivermectin promotes apoptosis in melanoma cells by inhibiting transcription factor binding to IGHM enhancer 3 (TFE3)-dependent autophagy and impairing lysosomal function, as evidenced by alterations in lysosomal associated membrane protein 1 (LAMP1) expression (16). These results suggest that ivermectin is a potential therapeutic anticancer agent. However, the anticancer effects of ivermectin have been mostly evaluated using two-dimensional (2D) gastric cancer cell lines, which offer advantages such as simplicity, cost-effectiveness, and ease of handling. However, the flattened culture method lacks the ability to study complex cell-cell interactions, and cell signaling is not as representative as it would be in an in vivo environment. Additionally, compared to 2D cultures, three-dimensional (3D) cultures technologies more closely resemble the in vivo cell environment. Therefore, 3D cultures are expected to provide more accurate predictions for drug discovery (17).

This study investigated the anticancer effects of ivermectin using a 2D gastric cancer cell line and patient-derived gastric cancer organoids. In addition, we evaluated the combined effects of ivermectin and cisplatin to assess their potential synergistic anticancer activity. We present this article in accordance with the MDAR reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-710/rc).

Methods

Cell culture

Human gastric cancer cell lines SNU719 and SNU620 were purchased from the Korean Cell Line Bank (KCLB, Seoul, South Korea) and cultured in the Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS). Cells were then seeded on culture plates and routinely cultured at 37 ℃ in an incubator with a humidified atmosphere containing 5% CO₂. SNU719 and SNU620 were selected because they represent biologically distinct gastric cancer subtypes. SNU719 is an Epstein-Barr virus (EBV)-positive gastric cancer model known to exhibit sensitivity to modulation of apoptosis-related pathways, whereas SNU620 was derived from a poorly differentiated metastatic gastric cancer. Using these two complementary cell lines, we were able to compare ivermectin activity in a heterogeneous 2D gastric cancer environment.

Drug treatment in gastric cancer cells

SNU719 and SNU620 GC cells were seeded at a density of 3×10³ cells/well in a 96-well plate (SPL Life Sciences, Pocheon, South Korea) with 0.2 mL of medium. After 24 h, both cell lines were treated with ivermectin (Sigma-Aldrich, MA, USA) at concentrations of 0, 5, 10, 15, and 20 µM and cultured for 48 and 96 h. The SNU719 cells were seeded at a density of 2×10⁴ cells/well in a 96-well plate containing 0.2 mL of medium. Following 24 h of incubation, the cells were treated with ivermectin (0, 0.5, 1, 2, and 5 µM) and ivermectin-cisplatin (Sigma-Aldrich) combination (1+1, 2+2 µM) and cultured for 48 and 96 h.

Cell Counting Kit (CCK)-8 assay

The viability of gastric cancer cells was evaluated using a CCK-8 assay kit (Promega, WI, USA). After drugs treatment, 2×10⁴ cells/well were seeded in 96-well plates and cultured for 48 h. Cells were washed twice with phosphate-buffered saline (PBS). Finally, 100 µL of CCK-8 reagent was added to each well and shaken vigorously on a shaker for 5 min. The optical density was measured at 450 nm using a microplate reader (Berthold, Bad Wildbad, Germany) after 30 min in the dark. The percentage of each concentration relative to the control was expressed as cell viability.

Western blot and antibodies

SNU719 cells were seeded at a density of 1×10⁶ cells per well in 12-well plates and cultured for 24 h at 37 °C in a humidified incubator with 5% CO₂. After harvest of cells stimulated with indicated conditions, lysates were prepared by solubilizing cell pellets in radioimmunoprecipitation assay (RIPA) buffer for 30 min at 4 °C and centrifuged Eluted samples with sodium dodecyl sulfate (SDS) loading buffer and heat denatured were loaded for separation on 10% SDS-polyacrylamide gel electrophoresis (PAGE) gels, transferred onto nitrocellulose membranes, and blocked in 5% skim milk in tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) for 1 h. After rinse with TBS-T, membranes were incubated with indicated immunoblotted antibodies in 5% skim milk in TBS-T at 4 °C overnight. Excess primary antibodies were removed by washing the membrane four times with TBS-T. Membranes were then incubated with 0.1 µg/mL peroxidase-labeled secondary antibodies (against rabbit or mouse) for 2 h at room temperature (RT). After four washes with TBS-T, bands were visualized with enhanced chemiluminescence (ECL) western blotting detection reagents (Thermo Fisher Scientific, Waltham, MA, USA) with an Invitrogen iBright Imagers system. All detected bands were quantified by ImageJ and were normalized with the intensity of the loading control proteins. The antibodies used included YAP1 (SC-101199), CTGF (ab6992), BAX (Cell signaling, #2772S) cleaved caspase-3 (cell signaling #9661), β-actin (HRP-60008)

Cell apoptosis analysis with annexin V assay

SNU719 cells were treated with various drug concentrations were gently dissociated with trypsin/ethylenediaminetetraacetic acid (EDTA) and stained for annexin V and propidium iodide (PI) using the Annexin V-FITC Apoptosis Detection Kit according to manufacturer’s instructions. The stained cells were immediately analyzed with a BD FACS Calibur system (BD Biosciences, Germany)

Patient-derived organoids

Primary cancer tissues were collected from patients with gastric cancer before treatment at the Kyungpook National University Hospital. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. Written informed consent was obtained from all patients, and the study was approved by the Institutional Review Board of Kyungpook National University Hospital (No. KNUMC 2016-04-013-003). GC7 and GC15 organoids were selected because they were derived from two independent gastric cancer patients exhibiting distinct molecular backgrounds and growth characteristics. These differences enabled the evaluation of ivermectin responses across heterogeneous 3D tumor models. Primary 3D cultures were established following a modified procedure based on the method described by Kondo et al. (18). Briefly, fresh tissue samples were placed on a sterile bench and rinsed with chilled Hank’s balanced salt solution (HBSS; Gibco, NY, USA). The tissues were sectioned into small pieces (approximately 4 mm³) using a feather surgical blade and rinsed twice with chilled HBSS. Tumor pieces were incubated with digestion medium, including Dulbecco’s modified Eagle medium/F-12 (DMEM/F) supplemented with GlutaMaX (Gibco), Liberase Dispase-High (Roche Applied Science, Mannheim, Germany), and antibiotic-antimycotic at 37 ˚C for 1 h. Liberase-treated samples were sequentially passed through a 100 µm cell strainer (Corning, NC, USA) and 40 µm cell strainer (Corning). The fragments retained on the cell strainer were retrieved and subsequently cultured in a non-coated culture dish (SPL, Pocheon, Korea) in stem cell medium (StemPro hESC SFM; Thermo Fisher Scientific, MA, USA) supplemented with basic fibroblast growth factor (Invitrogen, CA, USA), antibiotic-antimycotics, and 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). To minimize bacterial and fungal contamination, the medium was supplemented with penicillin, streptomycin, and antifungal agents (penicillin/streptomycin and amphotericin B).

Primary organoid culture and passage

Organoids were seeded in a Matrigel matrix (Corning) and HBSS at a 7:3 ratio, then incubated for 30 min in a 37 °C, humidified atmosphere containing 5% CO₂. Next, 500 µL of culture medium was added into the four-well plates. The culture medium was replaced periodically, and organoids exceeding 1,000 µm in size were subjected to passaging.

Drug treatment in organoids

Organoids were treated with ivermectin and cisplatin (Sigma-Aldrich) for GC7 and GC15 organoids. After seeding three organoids of 300–500 µm size in Matrigel onto 96-well plates, and incubated for 3 days, organoids were treated with ivermectin alone (0, 25, 50, 100 µM) or co-treated with a combination of ivermectin and cisplatin (25+20, 50+40, 100+60 µM). Following which, the organoids were incubated at 37 ˚C in a humidified incubator with 5% CO₂ for 48 or 96 h.

Measuring live and dead cells

Subsequently, the organoids were washed with HBSS, organoids were treated with DMEM/F and PI at a final concentration of 20 µg/mL and incubated at room temperature for 5 min in the dark. After washing with PBS, organoids were treated with DMEM/F and calcein AM at a final concentration of 10 µM. Then, organoids were stained for 2 h at 37 ℃. After washing with PBS, the stained organoids embedded in Matrigel were measured using a fluorescence microscope Olympus IX81 (Olympus, Tokyo, Japan). Nonfluorescent calcein AM is converted into green fluorescent calcein in live cells, while the nuclear dye PI emits red fluorescence in dead cells. The fluorescence areas of live and dead cells were measured using the polygon selection tool in the ImageJ 1.50i toolbar.

Total RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA from SNU719 cells and GC organoids was extracted using TRIzol reagent (Ambion, CA, USA). DNA contamination was removed using a TURBO DNA-free kit (Invitrogen). Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using SuperScript III reverse transcriptase and Random Hexamers (Invitrogen). Including primers for connective tissue growth factor (CTGF) (forward 5'-ACCAATGACAACGCCTCC-3' and reverse 5'-TTGGAGATTTTGGGAGTACGG-3'), Yes1 associated transcriptional regulator (YAP1) (forward 5'-CCCAGATGACTTCCTGAACAG-3' and reverse 5'-CCATCTCCTTCCAGTGTTCC-3'), TFE3 (forward 5'-ATCACTGTCAGCAACTCCTG-3' and reverse 5'-CTGTCGTTAATGTTGAATCGCC-3'), LAMP1 (forward 5'-GCTCTTCCAGTTCGGGATG-3' and reverse 5'-TAGGAATTGCCGACTGTGG-3') and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control (forward 5'-ACCCAGAAGACTGTGGATGG-3' and reverse 5'-TCTAGACGGCAGGTCAGGTC-3'), Reverse transcription-polymerase chain reaction (RT-PCR) were performed using PowerUp SYBR Green Master Mix (Applied Biosystems, CA, USA) in a ViiA 7 Real-Time PCR system (Applied Biosystems). The tested genes are related to the anticancer mechanism of ivermectin.

Statistical analysis

For continuous variables, data were expressed as mean ± standard deviation, and statistical analyses were performed using Student’s t-test. Statistical tests were two-sided, and P<0.05 was considered statistically significant.

Results

Effect of ivermectin alone and cisplatin combination in SNU719 and SNU620

Cell viability analysis using the CCK-8 assay showed reduced viability only in SNU719 cells after 48 (Figure 1A) and 96 h (Figure 1B). These results suggest that ivermectin suppressed cell viability in SNU719 gastric cancer cells.

Figure 1 Ivermectin suppresses SNU719 cell viability and enhances the effect of cisplatin. Cell viability of SNU719 and SNU620 gastric cancer cell lines was evaluated using a CCK-8. (A) Cell viability of SNU719 and SNU620 cells treated with various ivermectin concentrations (0, 5, 10, 15, and 20 µM) and incubated for 48 h and (B) 96 h. (C) SNU719 cells were treated with increasing concentrations of ivermectin alone (0, 0.5, 1, 2 and 5 µM) and then incubated for 48 h. (D) SNU719 cells were co-treated with various concentrations of ivermectin-cisplatin, and then incubated for 48 h. CCK-8, Cell Counting Kit-8.

Synergistic effect of ivermectin and cisplatin combination on cell viability in SNU719

Treatment with ivermectin alone for 48 h resulted in a dose-dependent decrease in cell viability in SNU719 cells (Figure 1C). Co-treatment with ivermectin and cisplatin for 48 h further decreased cell viability (Figure 1D), suggesting the potential synergistic effect of combination therapy.

Effect of ivermectin and cisplatin combination on messenger RNA (mRNA) expression in SNU719

To investigate the decreases in cell viability induced by ivermectin in SNU719 cells, the mRNA expression levels of TFE3, LAMP1, YAP1, and CTGF were analyzed. SNU719 gastric cancer cells were treated with various ivermectin concentrations and incubated for 48 h. After which, in RT-PCR analysis, no significant reduction was observed in the mRNA expression levels of TFE3 and LAMP1 (Figure 2A,2B), whereas that of CTGF and YAP1 was significantly decreased (Figure 2C,2D). These results suggest that ivermectin induced reduction in cell viability in SNU719 cells may be associated with apoptosis.

Figure 2 Effect of ivermectin on the mRNA expression in SNU719. The mRNA expression levels of TFE3, LAMP1, CTGF, and YAP1 were analyzed in SNU719 cells treated with various concentrations of ivermectin for 48 h. (A-D) Relative mRNA levels of TFE3, LAMP1, CTGF, and YAP1 were measured by RT-qPCR. Expressed as means ± standard deviation, using GAPDH as internal control. CTGF, connective tissue growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LAMP1, lysosomal associated membrane protein 1; mRNA, messenger RNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; TFE3, transcription factor binding to IGHM enhancer 3; YAP1, yes1 associated transcriptional regulator.

Protein expression changes after ivermectin treatment in SNU719

To investigate whether the transcriptional changes observed in RT-PCR were reflected at the protein level, western blot analysis was performed in SNU719 cells following ivermectin treatment. Consistent with the RT-PCR results, the protein levels of YAP1 and CTGF were decreased in ivermectin treated cells compared with the control group (Figure 3A). In contrast, no significant change was observed in the expression of the pro-apoptotic protein BAX. Protein band intensity analysis using ImageJ confirmed the reduction of YAP1 and CTGF protein levels, whereas BAX expression remained relatively unchanged in ivermectin-treated cells (Figure 3B). Additional western blot analyses were performed to examine the protein levels following with ivermectin alone or combination with cisplatin at 48, 72 h. At 48 h, the protein levels of YAP1 and CTGF did not show significant changes compared with the control, while cleaved caspase-3 appeared to decrease following treatment (Figure S1A,S1B). At 72 h, YAP1 and CTGF expression showed a slight increase depending on drug concentration, whereas cleaved caspase-3 expression appeared reduced (Figure S1C,S1D). To assess time dependent changes, western blot analysis was performed at 16, 24, 48, and 72 h after ivermectin alone or combination with cisplatin treatment. YAP1 and CTGF expressions appeared reduced at earlier time points (Figure S2A,S2B) but showed a slight increase at later time points (Figure S2C,S2D). These findings suggest that ivermectin reduces YAP1 and CTGF expression at early time point, whereas later time points show less consistent changes in protein expression.

Figure 3 Effects of ivermectin on apoptosis related protein expression and apoptotic cell death in SNU719. SNU719 Cells were treated with ivermectin (0, 2, or 4 µM) and incubated for an additional 24 h. (A) Western blot analysis of YAP1, CTGF, and BAX protein expression. (B) Quantification of protein band intensities. Relative protein expression levels were measured using ImageJ and normalized to β-actin. (C) Flow cytometry plots showing apoptotic cell populations following ivermectin treatment. (D) The percentage of apoptotic cells is presented as a bar graph. Statistical comparisons among groups were performed using one-way ANOVA followed by Tukey’s post hoc test. Statistical significance was defined as: ns, not significant; *, P<0.05; **, P<0.01; ****, P<0.0001. ANOVA, analysis of variance; CTGF, connective tissue growth factor; PI, propidium iodide; YAP1, yes1 associated transcriptional regulator.

Ivermectin induces apoptosis in SNU719

To further investigate whether ivermectin induces apoptosis in SNU719, Annexin V-FITC/PI double staining followed by flow cytometry analysis was performed after ivermectin treatment. Cells were treated with ivermectin (2, 4 µM) for 24 h and subsequently analyzed. Flow cytometry analysis showed that the proportion of apoptotic cells increased in a dose-dependent manner following ivermectin treatment (Figure 3C). Quantitative analysis further confirmed that the percentage of apoptotic cells (Figure 3D). Additionally flow cytometry analysis was performed after 48 h of treatment with ivermectin alone or in combination with cisplatin. Flow cytometry analysis showed that the proportion of apoptotic cells decreased following treatment (Figure S3). These results suggest that ivermectin induces apoptotic cell primarily at an early time point in SNU719 gastric cancer cells.

Ivermectin-cisplatin combination-induced cell death in patient-derived gastric cancer organoids

We investigated the effects of ivermectin and ivermectin-cisplatin combination on patient-derived gastric cancer organoids, GC7 and GC15. Bright-field microscopic images of GC7 organoids revealed a slight increase in organoid size over 4 days after ivermectin treatment. However, fluorescence microscopy revealed a concentration-dependent decrease in green fluorescence and increase in red fluorescence, indicating that cell death increased with the increasing concentrations of ivermectin (Figure 4A). Fluorescence images were analyzed using the ImageJ software to calculate the percentage area of PI staining, which demonstrated a dose-dependent increase in dead cells in GC7 organoids treated with ivermectin alone. Treatment with 100 µM ivermectin resulted in a 48.25% cell death, as indicated by the red fluorescence area (Figure 4B). Compared to those treated with ivermectin alone. GC7 organoids co-treated with cisplatin, showed minor volume increase (Figure 4C). Additionally, the ivermectin-cisplatin co-treatment (100+60 µM) showed an 82.48% increase in red fluorescence area, suggesting a synergistic effect between ivermectin and cisplatin that instigates cell death in patient-derived gastric cancer organoids (Figure 4D).

Figure 4 Ivermectin-induced cell death in patient-derived gastric cancer organoids (GC7 and GC15) and enhances the effect of cisplatin. Patient-derived gastric cancer organoids (GC7 and GC15) were treated with ivermectin alone or combination with cisplatin for 4 days. (A) GC7 organoids were treated with various concentrations of ivermectin alone. (B) Quantification of live (green) and dead (red) fluorescence areas in GC7 organoids with ivermectin alone. (C) GC7 organoids were co-treated with various concentrations of ivermectin-cisplatin. (D) Quantification of live and dead fluorescence areas in GC7 organoids following ivermectin-cisplatin combination treatment. (E) GC15 organoids were treated with various concentration ivermectin alone. (F) Quantification of fluorescence areas in GC15 organoids with ivermectin alone. (G) GC15 organoids were co-treated with various concentrations of ivermectin-cisplatin. (H) Quantification of fluorescence areas in GC15 organoids following combination treatment. Fluorescence areas were quantified using ImageJ software. Magnification 40×. scale bars: 300 µm. GFP, green fluorescent protein (Calcein AM stain); RFP, red fluorescent protein (PI stain). AM, acetoxymethyl ester; PI, propidium iodide.

In GC15 organoids, treatment with ivermectin alone resulted in a dose-dependent increase in cell death, except for the maximum concentration (100 µM), which reduced the effect instead. Fluorescence microscopy also confirmed a dose-dependent increase in cell death following ivermectin treatment (Figure 4E). Treatment with 50 µM ivermectin resulted in a 43.63% increase in red fluorescent area (Figure 4F). Co-treatment with 100 µM ivermectin and 60 µM cisplatin further increased cell death, with a 65.77% increase in red fluorescent area, demonstrating the combination efficacy of ivermectin and cisplatin in GC15 organoids (Figure 4G,4H).

Effect of ivermectin-cisplatin combination on mRNA expression in patient-derived gastric cancer organoids

To assess TFE3, LAMP1, CTGF, and YAP1 mRNA expression in patient-derived gastric cancer organoids, GC7 and GC15 organoids were treated with 50 µM ivermectin alone or combined with 40 µM cisplatin for 48 h, with untreated organoids as controls. In GC7 cells, compared to combination treatment (3.1 and 1.3 times), ivermectin alone exhibited higher TFE3 and LAMP1 (6.5 and 1.6 times; Figure 5A,5B). CTGF remained unchanged, and YAP1 increased by 1.7 times with ivermectin alone and 1.5 times with combination treatment (Figure 5C,5D). In GC15 cells, ivermectin alone increased TFE3 and LAMP1 by 4.0 and 2.4 times, whereas the combination elevated their levels further (6.0 and 2.7 times; Figure 5E,5F). CTGF and YAP1 mRNA levels also increased with combination treatment (Figure 5G,5H). The synergistic effect of ivermectin and cisplatin was more pronounced in the GC15 organoids than in the GC7 organoids.

Figure 5 Effect of ivermectin and ivermectin-cisplatin combination on mRNA expression in patient-derived gastric cancer organoids. Patient derived cancer organoids (GC7 and GC15) were treated with ivermectin alone or combination with cisplatin for 48 h. (A-D) The mRNA levels of TFE3, LAMP1, CTGF, and YAP1 were measured in GC7 organoids treated with ivermectin alone or co-treated with ivermectin-cisplatin. (E-H) The mRNA levels of TFE3, LAMP1, CTGF, and YAP1 were measured in GC15 organoids treated with ivermectin alone or co-treated with ivermectin-cisplatin. Data expressed as means ± standard deviation, using GAPDH as internal control. CTGF, connective tissue growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LAMP1, lysosomal associated membrane protein 1; mRNA, messenger RNA; TFE3, transcription factor binding to IGHM enhancer 3; YAP1, yes1 associated transcriptional regulator.

Discussion

This study showed that ivermectin reduced the viability of SNU719 gastric cancer cells in a dose-dependent manner, whereas no such effect was observed in SNU620 cells. SNU719 cells treated with the ivermectin-cisplatin combination exhibited a greater decrease in cell viability than those treated with ivermectin alone. Ivermectin also modulated the expression of apoptosis-related genes (CTGF and YAP1) and autophagy-related marker (LAMP1). Western blot analysis showed that the protein expression levels of YAP1 and CTGF were reduced in SNU719 cells at 24 h following ivermectin treatment, whereas the expression of the BAX did not show a significant change. Additional time-course western blot analysis revealed that the suppression of YAP1 and CTGF protein expression was most prominent at 24 h, whereas the effect was attenuated at 48 h and showed a rebound tendency at 72 h. Interestingly, the expression of cleaved caspase-3 showed a dose-dependent decrease at 48 h and 72 h. Flow cytometry analysis using Annexin V-FITC/PI double staining demonstrated that ivermectin increased the proportion of apoptotic cells in a dose-dependent manner at 24 h. However, at 48 h of treatment ivermectin alone or in combination with cisplatin, the proportion of apoptotic cells decreased compared with the earlier time point. In patient-derived organoids (GC7 and GC15), the ivermectin-cisplatin combination was more effective in inducing cell death. However, ivermectin increased the mRNA expression of apoptosis-related markers in patient-derived organoids.

Ivermectin has been commonly used as a drug for parasitic infections (1,2). Recently, researchers have focused on ivermectin’s ability to inhibit tumor growth in various cancers (5-16). TFE3 and TFEB are master transcription factors of autophagy (19), and the inhibition of reactive oxygen species-TFE3-dependent autophagy in human melanoma cells increases apoptosis. The relative mRNA levels of TFE3 and LAMP1 increase with the increasing concentration of ivermectin (16). In a previous study, ivermectin demonstrated anticancer effects in the gastric cancer cell line MK1; this was attributed to the reduced expression of the apoptosis-related genes CTGF and YAP1 (13). However, its mechanism of action in cancer remains unclear. Cisplatin is widely used as a chemotherapeutic agent for advanced cancers, including gastric cancer, and combination drug. In the flattened culture method, cell-to-cell interactions and signals are not represented as they are in an in vivo environment; therefore, 3D technologies more closely resemble the in vivo cell environment than the 2D cell cultures (17,20). Subsequently, we compared the effects of ivermectin and cisplatin on 2D and 3D cell cultures.

In the present study, our data revealed that ivermectin reduced the survival of SNU719 gastric cancer cells in a dose-dependent manner. When combined with cisplatin, ivermectin exhibited a stronger effect, suggesting that it enhanced the vulnerability of cancer cells to cisplatin by promoting cell death. In addition, ivermectin treatment in SNU719 cells resulted in decreased expression of the apoptosis-related genes CTGF and YAP1 at early time points, whereas their expression became attenuated or showed a rebound tendency at later time points. These findings are consistent with previous studies reporting that ivermectin exerts anticancer effects by modulating signaling pathways related to autophagy and apoptosis (13). Ivermectin reduced the protein expression of YAP1 and its downstream transcriptional target CTGF, whereas the expression of the pro-apoptotic protein BAX remained largely unchanged. This pattern suggests that the antiproliferative effect of ivermectin may be associated with suppression of the Hippo-YAP1 signaling pathway rather than direct activation of the classical mitochondria-mediated apoptotic pathway (14,21). The downregulation of CTGF, a well-known YAP1-driven pro-survival mediator, further supports the possibility that ivermectin interferes with the transcriptional activity of YAP1 in gastric cancer cells.

Similarly, flow cytometry analysis showed that the proportion of apoptotic cells increased at 24 h following ivermectin treatment. This result is consistent with previous reports demonstrating that ivermectin induces apoptosis and promotes cancer cell death (16,21). However, at 48 h, the proportion of apoptotic cells decreased compared with the earlier time point. This attenuation of apoptosis may reflect temporal changes in cellular responses to ivermectin. One possible explanation is that the initial wave of apoptosis selectively eliminates the most drug-sensitive cells, leaving behind a relatively resistant cell population at later time points. Alternatively, prolonged exposure to ivermectin may activate adaptive survival responses, including autophagy or compensatory reactivation of pro-survival signaling pathways. It is also possible that cells undergo alternative forms of cell death that are not detected by Annexin V-FITC/PI staining, resulting in an apparent reduction in the apoptotic fraction at later time points. These temporal dynamics suggest that the maximal apoptotic response to ivermectin in SNU719 cells may occur during the early phase of treatment. We also observed that the response to ivermectin differed between SNU719 and SNU620 cells. These results highlight the need for further studies to elucidate the molecular mechanisms underlying differential sensitivity of cancer cells to ivermectin.

In patient-derived organoids GC7 and GC15, ivermectin-cisplatin combination was more effective at inducing cell death than ivermectin alone, with differential responses noted between the organoid types. In particular, GC7 cells exhibited a stronger induction of cell death in response to combination therapy. Therefore, the therapeutic efficacy of ivermectin may depend on tumor type or patient-specific conditions. In contrast to the suppression of anti-apoptotic genes observed in the 2D cell lines, ivermectin upregulated these genes in gastric cancer organoids, indicating that ivermectin may induce cell death in 3D organoid models via a mechanism distinct from that observed in 2D cultures, highlighting the need for further research. The observed discrepancies in mRNA expression between the 2D and 3D cultures were likely due to the inherent limitations of 2D models in replicating in vivo microenvironments. In contrast, 3D cultures had better reflect the complex cellular interactions and signaling pathways found in living tissues, resulting in gene expression profiles that closely resemble those observed in vivo.

Conclusions

In conclusion, the differential effects observed between SNU719 cells and patient-derived organoids emphasize the importance of individualized cancer treatment approaches. Ivermectin exhibits morphological anticancer properties in both 2D cell cultures and organoids. In addition, ivermectin treatment reduced the protein expression of YAP1 and CTGF and induced apoptotic cell death at early time point in SNU719 gastric cancer cells. However, the effects on anti-apoptotic gene expression differed between 2D and 3D cultures. These findings show that despite its potential as a cancer therapy, the anticancer effects observed in 2D cell line experiments may not fully reflect clinical efficacy, which could limit its clinical application. Further research is required to evaluate the therapeutic efficacy and limitations of ivermectin treatment.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-710/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-710/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-710/prf

Funding: This work was supported by the National Research Foundation, Republic of Korea (Nos. NRF-2019R1F1A1063990 and NRF-2022R1A2C2013044, to S.Y.N.), Biomedical Research Institute grant, Kyungpook National University Hospital (2019, to S.Y.N.), and Korea Health Industry Development Institute (No. RS-2025-25410994) (to S.Y.N.). The funders had no role in the design and conduct of the study, including collection, management, analysis, and interpretation of the data, and preparation, review, or approval of the manuscript.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-710/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. Written informed consent was obtained from all patients, and the study was approved by the Institutional Review Board of Kyungpook National University Hospital (KNUMC 2016-04-013-003).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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