The human colorectal cancer cell line HCT8 (cat. no. 1101HUM-PUMC000104; Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College) and its 5-FU-resistant derivative, HCT8R (cat. no. MXC149; ShangHai MEIXUAN Biological Science and Technology Ltd.), were kindly provided by the research group of Professor Xi (Hubei University of Medicine, Shiyan, China). Both cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin. Cultures were maintained in a humidified incubator at 37°C containing 5% CO₂ to support optimal cell growth. To inhibit autophagic activity, the HCT8R cells were co-incubated with an autophagy inhibitor, 3-methyladenine (3-MA; 5 mM), 5-FU (20 µg/ml) and LOP (20 µM) for 24 h at 37°C.

RPMI-1640 medium was purchased as a powder from Gibco; Thermo Fisher Scientific, Inc., FBS was obtained from Zhejiang Tianhang Biotechnology Co., Ltd., the antibiotics were sourced from Beyotime Institute of Biotechnology, and LOP, 5-FU and 3-MA were purchased from MedChemExpress.

HCT8 and HCT8R cell metabolic activity was assessed using the CCK-8 kit (cat. no. C0038; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Briefly, cells in the logarithmic growth phase were seeded at 1×10³ cells/well in 96-well plates (100 µl/well). After 24 h of adhesion, the medium was replaced with fresh medium containing 5-FU (0, 10, 20, 40 and 80 µg/ml) and LOP (0, 10, 20, 40 and 80 µM). To evaluate the combined effects of 5-FU and LOP on cell viability, HCT8R cells were treated with 5-FU (20 µg/ml), dimethyl sulfoxide (DMSO, 0.2% v/v) or LOP (10 and 20 µM). The cells were then incubated for 24 h, followed by the addition of 10 µl CCK-8 reagent/well and further incubation for 2 h at 37°C. Culture medium without cells served as the blank control. Absorbance at 450 nm was measured using a microplate reader (BioTek Synergy H1; Biotek; Agilent Technologies, Inc.).

Exponentially growing HCT8R cells were counted and seeded into 48-well plates at a density of 10,000 cells/well. After 24 h of adherence to the plates, the cells were treated with 5-FU (20 µg/ml), DMSO (0.2% v/v) or LOP (20 µM) for 48 h at 37°C. To assess cell proliferation, the cells were incubated with EdU diluted in culture medium at a 100:1 ratio (200 µl/well) for 6 h at 37°C. Following EdU labeling, the medium was subsequently removed and the cells were fixed with 4% paraformaldehyde (Biosharp Life Sciences) for 30 min at room temperature. Fixed cells were then incubated with 2 mg/ml glycine for 5 min to neutralize residual paraformaldehyde, followed by permeabilization with 0.5% Triton X-100 for 20 min at room temperature. The EdU reaction solution was prepared according to the manufacturer's protocol (Beyotime Institute of Biotechnology) and applied to the cells (100 µl/well) for 30 min in the dark to ensure precise labeling at room temperature. Subsequently, the cells were counterstained with DAPI (Beyotime Institute of Biotechnology) for 10 min to visualize the nuclei at room temperature. Finally, fluorescent images were acquired using an inverted fluorescence microscope.

Exponentially growing HCT8R cells were seeded into 6-well plates at a density of 1,000 cells/well. Once the cells had formed small colonies, they were treated with 5-FU (20 µg/ml), DMSO (0.2% v/v) or LOP (20 µM) for 48 h at 37°C. After 6–8 days, colony formation was assessed under a microscope. When distinct colonies were visible in the control group, the culture medium was aspirated and the cells were washed three times with PBS. Cells were subsequently fixed with 4% paraformaldehyde for 30 min at room temperature, and colonies were then stained with crystal violet for 30 min at room temperature to enhance visualization. Excess stain was removed with multiple PBS washes and the plates were left to air-dry prior to imaging. Crystal violet-stained colonies (>50 cells) were visualized using an inverted light microscope (IX71; Olympus Corporation). Colony counts per group were manually quantified in triplicate and averaged.

Exponentially growing HCT8R cells were treated with trypsin until they formed a single-cell suspension, and ~5×10⁵ cells/well were seeded into 6-well plates. The cells were subsequently cultured at 37°C in a humidified atmosphere containing 5% CO₂ for 24 h to allow them to reach 100% confluence. A uniform scratch was introduced using a 200-µl pipette tip and the cells were subsequently washed three times with PBS to remove any detached cells. Fresh RPMI-1640 medium supplemented with 2% FBS was then added to each well. Initial images were captured to confirm that the scratch that had been made was uniform, centered and perpendicular to the plane of the plate. Following treatment with 5-FU (20 µg/ml), DMSO (0.2% v/v) or LOP (20 µM) for 24 h at 37°C, additional images were acquired under a light microscope. Cell migration into the scratch area was quantified using ImageJ (version 4.1; National Institutes of Health), and the wound healing rate was calculated as: (0 h scratch width −24 h scratch width)/0 h scratch width ×100 to assess cell motility.

For the Transwell assay experiments in HCT8 and HCT8R cells, 600 µl RPMI-1640 medium supplemented with 20% FBS was added to the lower chamber, with or without 3-MA (5 mM), 5-FU (20 µg/ml) and LOP (10 and 20 µM). Subsequently, 200 µl suspension containing 10⁵ cells/ml in FBS-free medium was added to the upper chamber (pore size, 8 µm; Corning, Inc.), which was placed into a 24-well plate. The cells were cultured for 48 h to allow for migration. After the incubation period, the insert was carefully removed and washed once with PBS. The cells that had migrated to the lower surface of the insert were subsequently fixed with 4% paraformaldehyde for 30 min at room temperature and washed three times with PBS. The interior of the insert was gently swabbed with a soft cotton swab to remove non-adherent cells. Finally, staining was performed using crystal violet for 20 min at room temperature, and excess stain was removed by multiple washes with PBS. Images were captured under a light microscope.

Exponentially growing HCT8R cells were plated in 6-well plates, and cultured for 24 h until they reached 70–80% confluence. Transfection was subsequently performed using Lipo8000^™ reagent (Beyotime Institute of Biotechnology) in combination with 2 µg pEGFP (cat. no. 165830; Addgene, Inc.) or pEGFP-microtubule-associated protein 1 light chain 3 (LC3) plasmid (cat. no. 24920; Addgene, Inc.) and Opti-MEM^™ serum-free medium (Gibco; Thermo Fisher Scientific Inc.). After 36 h of transfection at 37°C, the cells were subsequently transferred to confocal dishes, and allowed to continue growing for an additional 24 h. Once cells had reached ~50% confluence, they were incubated with either 5-FU (20 µg/ml), DMSO (0.2% v/v) or LOP (20 µM) for 24 h at 37°C. Imaging was performed using an Olympus FV3000RS super-resolution laser confocal microscope (Olympus Corporation). All samples were analyzed under identical imaging conditions to ensure consistency and reproducibility.

HCT8R cells were harvested into 1.5-ml Eppendorf tubes containing RIPA lysis buffer (Beyotime Institute of Biotechnology) for protein extraction at 4°C. The protein concentration was subsequently quantified using a bicinchoninic acid protein assay kit, and 20 µg protein samples were separated by SDS-PAGE on 12% gels and transferred to a PVDF membrane (MilliporeSigma). Non-specific binding was blocked using PBS containing 5% nonfat milk for 2 h at room temperature. The membrane was subsequently incubated overnight at 4°C with primary antibodies (diluted with PBS) raised against LC3 (1:1,000; cat. no. 18725-1-AP), Beclin (1:1,000; cat. no. 11306-1-AP) (both from Proteintech Group, Inc.) and β-actin (1:1,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.). Following incubation with the primary antibodies, the membrane was washed and incubated with horseradish peroxidase-conjugated anti-rabbit (diluted with PBS, 1:5,000; cat. no. 31460; Invitrogen; Thermo Fisher Scientific, Inc.) and anti-mouse secondary antibodies (diluted with PBS, 1:5,000; cat. no. 31430; Invitrogen; Thermo Fisher Scientific, Inc.) for 2 h at room temperature. Finally, the blots were visualized using an enhanced chemiluminescence detection system (Bio-Rad Laboratories, Inc.). Images were semi-quantified using ImageJ (version 4.1; National Institutes of Health).

For apoptosis detection, the HCT8R cells were seeded in 6-well plates at a density of 4×10⁵ cells/well. Cells were treated with 5-FU (20 µg/ml) and LOP (10 or 20 µM) for 48 h at 37°C. Both adherent and floating cells were then harvested and washed with ice-cold PBS. Cells from different experimental groups were then digested with trypsin (without EDTA), combined with the culture supernatant and centrifuged at 112 × g for 4 min at 4°C to pellet the cells. Subsequently, the pellet was gently resuspended in pre-chilled binding buffer and adjusted to a concentration of 1–5×10⁶ cells/ml. Aliquots of the cell suspension (100 µl) were then mixed with 5 µl FITC-Annexin V and 5 µl PI, gently mixed, and incubated in the dark at room temperature for 8–10 min. Subsequently, 400 µl binding buffer was added, and the mixture was gently mixed on a vortex mixer. Finally, apoptosis was assessed using a flow cytometer (SA3800; Sony Biotechnology Inc.) within 1 h, with an Annexin V-PI reagent kit purchased from Invitrogen; Thermo Fisher Scientific, Inc.

HCT8R cells (5×10⁶ cells/100 µl PBS) were subcutaneously injected into the lateral abdominal region of female nude BALB/c mice (age, 4 weeks; weight, 20–24 g; n=3/group; total n=9) that were housed under the following specific pathogen-free conditions: Temperature, 23±1°C; humidity, 55±5%; 12-h light/dark cycle, and with free access to standard chow and drinking water. The mice were randomly divided into the control (vehicle: 0.9% NaCl + 0.1% DMSO, i.p. daily), 5-FU monotherapy (25 mg/kg i.p., three times per week) and combination (5-FU as above + LOP 2 mg/kg/day i.p., three times per week) groups. All procedures followed protocols approved by the Hubei University of Medicine Animal Research Committee (approval no. 2023-034). Treatment was initiated at day 7 post-inoculation when tumors reached 100±10 mm³ and continued for 21 days. The humane endpoints requiring euthanasia included ≥20% body weight loss within 48 h, tumor diameter >1.5 cm, impaired mobility or signs of distress (labored breathing/vocalization). Animal health and behavior were assessed twice daily (8:00 a.m./6:00 p.m.) using validated scoring sheets, and tumor volume (V=LxW²/2) and weight were measured every 48 h, with the endpoint being day 28 or when tumor volume exceeded 1,500 mm³. Welfare provisions included: Individual ventilated cages with environmental enrichment (paper tunnels, chew blocks), and euthanasia by cervical dislocation under deep anesthesia (5% isoflurane for induction and 2% for maintenance). Death was verified by the cessation of respiration, an absent heartbeat, fixed dilated pupils and bilateral diaphragmatic transection. All 9 mice were sacrificed, with no unexpected deaths recorded and no mice excluded from the analysis.

Statistical analyses were performed using GraphPad Prism 9 (Dotmatics). Comparisons between two groups were made using unpaired Student's t-test, whereas comparisons among multiple groups were performed by one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the malignant potential of HCT8 and HCT8R cells, Transwell assays were performed, which revealed that the migratory capability of the drug-resistant HCT8R cells was significantly higher compared with that of HCT8 cells (Fig. 1A). The preliminary dose-response assessments revealed marked insensitivity of HCT8R cells to 5-FU monotherapy, with conventional therapeutic concentrations (10–80 µg/ml) failing to induce significant inhibition of cell viability (Fig. S1). Subsequently, the inhibitory effects of LOP on cell viability were assessed using the CCK-8 assay. The results demonstrated that LOP inhibited the viability of both HCT8 and HCT8R cells, with IC₅₀ values of ~24.74 and 34.29 µM, respectively. These findings suggested that LOP was able to effectively suppress the viability of both the parental and 5-FU-resistant colorectal cancer cell lines (Fig. 1B and C).

To further examine the impact of LOP on the sensitivity of HCT8R cells to 5-FU, additional CCK-8 assays were performed. The results showed that the HCT8R cells exhibited resistance to 5-FU, as increasing concentrations of the drug did not significantly inhibit cell proliferation; however, treating the cells with 20 µM LOP in addition to 5-FU led to a marked enhancement of the inhibitory effect of 5-FU on cell proliferation (Fig. 1D). Further analysis revealed that treating the cells with 5-FU (20 µg/ml) alone did not substantially alter HCT8R cell proliferation; by contrast, compared with in the 5-FU group, the addition of LOP caused a significant improvement in the sensitivity of these cells to 5-FU in a concentration-dependent manner (Fig. 1E).

The EdU assay results demonstrated that the proliferative activity of the HCT8R cells remained unaffected by the addition of 5-FU or DMSO alone; however, treatment with LOP led to a significant reduction in the number of proliferating HCT8R cells (Fig. 2A). Colony formation assays were subsequently performed, which further demonstrated that treatment with LOP caused a marked decrease in the number of colonies formed by HCT8R cells, whereas treatment with 5-FU alone or DMSO did not lead to any significant inhibitory effects (Fig. 2B).

To confirm the ability of LOP to enhance the inhibitory effect of 5-FU on HCT8R cell proliferation in vivo, a xenograft model was generated using nude mice. HCT8R cells were implanted into nude mice, which were then randomly divided into control, DMSO and LOP treatment groups. With the exception of the control group, the other groups were injected intraperitoneally with 5-FU (25 mg/kg per week, three times per week), whereas the LOP treatment group was injected intraperitoneally with LOP (2 mg/kg per day). The results demonstrated that treatment with LOP led to a significant enhancement of the inhibitory effect of 5-FU on tumor growth (Fig. 2C), as evidenced by a marked reduction in tumor weight and volume in the LOP treatment group (Fig. 2D and E). Compared with in the 5-FU group, the 5-FU and LOP co-treatment group demonstrated a stronger tumor growth inhibitory effect. Collectively, these findings suggested that LOP is able to effectively suppress cell proliferation both in vitro and in vivo.

Tumor cell migration is a critical factor for both tumor invasion into normal tissues and subsequent metastasis. After performing wound healing assays, it was observed that treatment with 5-FU or DMSO alone did not significantly affect the migratory capabilities of drug-resistant HCT8R cells; however, the addition of 20 µM LOP led to a marked inhibition of cell migration compared with in the 5-FU group (Fig. 3A). These findings were further validated by employing Transwell assays, which demonstrated that LOP was able to effectively suppress HCT8R cell migration in a dose-dependent manner (Fig. 3B).

The balance between tumor cell proliferation and apoptosis is a crucial determinant of drug sensitivity. The aforementioned experiments demonstrated that LOP can effectively inhibit the proliferation, invasion and migration of 5-FU-resistant cells; however, its impact on apoptosis has not yet been fully elucidated. To investigate this possibility, Annexin V-PI staining experiments were performed for apoptosis detection. Flow cytometric analysis revealed that treatment with 5-FU alone did not cause any significant induction of apoptosis; by contrast, the addition of LOP markedly increased the proportions of late apoptotic cells compared with in the 5-FU group (Fig. 3C).

To elucidate the molecular mechanism underlying the ability of LOP to reverse 5-FU resistance, the expression levels of the autophagy markers LC3 and Beclin were assessed using western blot analysis. The results demonstrated that LOP treatment led to an upregulation of Beclin expression and an increase in the LC3-II/I ratio compared with in the 5-FU group (Fig. 4A), indicating the activation of autophagy.

To further investigate this phenomenon, HCT8R cells were transfected with EGFP or EGFP-LC3 plasmids, and autophagic vesicles were visualized using confocal microscopy. The results obtained showed that treatment with LOP in combination with 5-FU resulted in dispersed, dense fluorescent spots in the cytoplasm of resistant cells, characteristic of autophagic vesicles; by contrast, cells treated with 5-FU or DMSO alone exhibited diffuse fluorescence without the presence of autophagic vesicles (Fig. 4B).

To confirm that the reversal of HCT8R cell resistance by LOP was associated with autophagy activation, the autophagy inhibitor 3-MA was used to block autophagy. Western blot analysis confirmed a significant downregulation of Beclin following 3-MA treatment (Fig. 4C). Moreover, Transwell assays indicated that treatment with 3-MA led to a reversal of the inhibition of migration caused by LOP (Fig. 4D) compared with in the group not treated with 3-MA, highlighting the importance of autophagy as a target of the action of LOP.

Taken together, these findings suggested that LOP may enhance autophagy to inhibit cell proliferation, invasion and migration, while also promoting apoptosis, thereby reversing 5-FU resistance in HCT8 colorectal cancer cells.