Abstract
Background/Aim: Colorectal cancer (CRC) remains a major cause of cancer mortality. Lenvatinib, a multi-kinase inhibitor, has emerging anticancer potential, but its effects in CRC are not fully defined. The aim of this study was to identify potential treatment mechanism and efficacy of Lenvatinib on CRC in vitro.
Materials and Materials: Human CRC cell lines HT-29 and HCT-116 were treated with lenvatinib. Cell viability (MTT assay), proliferation (colony formation), apoptosis (flow cytometry), migration, and invasion (Transwell assay) were assessed. Key signaling pathways were analyzed by western blot.
Results: Lenvatinib reduced viability in a dose- and time-dependent manner (IC50≈30 μM at 24 h) and suppressed colony formation. Apoptosis occurred via extrinsic (Fas/Fas-L upregulation, cleaved caspase-8) and intrinsic (cleaved caspase-9, mitochondrial membrane potential loss) pathways. ERK phosphorylation and downstream STAT3/NF-κB activation were inhibited. Migration and invasion were markedly reduced.
Conclusion: Lenvatinib inhibits CRC cell growth, migration, and invasion by inducing dual-pathway apoptosis and inactivating the ERK/STAT3/NF-κB signaling axis, supporting its potential as a therapeutic strategy for colorectal cancer.
Introduction
Colorectal cancer (CRC), the third most commonly diagnosed cancer and the second leading cause of cancer-related death worldwide, is projected to double in incidence by 2035 due to genetic, lifestyle, and inflammatory risk factors (1, 2). The standard and conventional treatment modalities for colorectal cancer (CRC) primarily include surgical resection for resectable lesions. For unresectable CRC, radiotherapy, chemotherapy, immunotherapy, and targeted therapy are commonly employed. In addition, various combination treatment strategies are widely applied in clinical practice, such as targeted therapy combined with chemotherapy or immunotherapy-based regimens. Conventional therapies lack specificity and are prone to adverse effects and drug resistance, highlighting the urgent need for novel molecularly targeted strategies to broaden cancer treatment options (3-5).
Lenvatinib, an orally multitarget tyrosine kinase inhibitor (TKI), exerts activity against VEGFR, FGFR, PDGFRα, KIT, and RET. Through simultaneous suppression of VEGF and FGF signaling, lenvatinib effectively disrupts angiogenesis, remodels the tumor microenvironment toward an immune-responsive phenotype, and mitigates FGF-mediated resistance to VEGF blockade. These mechanisms underlie its broad therapeutic potential, reflected in its demonstrated efficacy and acceptable safety profile across multiple malignancies, with current approvals for thyroid carcinoma, hepatocellular carcinoma, renal cell carcinoma, and endometrial carcinoma (6-9).
A previous single-center, single-arm phase II clinical trial evaluated the efficacy and safety of oral lenvatinib (24 mg/day) in patients with unresectable metastatic colorectal adenocarcinoma who had failed multiple lines of standard therapy. The results showed a disease control rate of 70%, a median progression-free survival of 3.6 months, and a median overall survival of 7.4 months. The most common grade ≥3 adverse event was hypertension. The study indicated that lenvatinib demonstrated promising antitumor activity in this patient population, with toxicities remaining within an acceptable range (10). These findings suggest that lenvatinib may provide potential therapeutic benefits for colorectal cancer, and further investigation into its antitumor mechanisms could help clarify its role in CRC treatment. Therefore, the present study aimed to investigate the effects of lenvatinib on apoptosis and survival pathways in CRC cells.
Materials and Methods
Cell culture. The colorectal cancer cell lines HT-29 (RRID:CVCL_0320) and HCT-116 (RRID:CVCL_0291) were obtained from a certified cell bank. Cells were maintained in RPMI-1640 medium (Thermo Fisher Scientific, Fremont, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific), penicillin (100 U/ml), and streptomycin (100 μg/ml) (11, 12). Cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO2. The reagents were listed in Table I.
Reagents used in the study.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The inhibitory effect of Lenvatinib on HT-29 nd HCT-116 cells was evaluated using the MTT assay. Cells were first cultured to 70-80% confluency, and approximately 5×103 cells were seeded into each well of a 96-well plate. Various concentrations of Lenvatinib (0, 10, 15, 20, 25, and 30 μM) were then added. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 24 and 48 h. After incubation, the culture medium was removed, and MTT solution was added to each well, followed by a further 2 h incubation. The MTT solution was then discarded, and dimethyl sulfoxide (DMSO) was added to dissolve the purple formazan crystals. Absorbance was measured at 570 nm using a microplate reader (SpectraMax® iD3s, Molecular Devices, San Jose, CA, USA) (13). Cell viability was calculated, and a concentration-response curve was generated to assess the effects of Lenvatinib.
Transwell assay for migration and invasion. Cell migration and invasion were assessed using Transwell chambers (8 μm pore size; Corning). For the migration assay, uncoated inserts were used, whereas for the invasion assay, inserts were pre-coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). HT-29 and HCT-116 cells were trypsinized, counted, and resuspended in serum-free medium. Approximately 1×105 cells were seeded into the upper chamber, while the lower chamber was filled with medium containing 10% FBS as a chemoattractant. Chambers were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 24-48 h. After incubation, non-migrated or non-invaded cells remaining on the upper surface of the membrane were removed with a cotton swab. Cells that had migrated or invaded to the lower membrane surface were fixed with a methanol-glacial acetic acid solution and stained with 0.1% crystal violet. The membranes were rinsed with PBS, and stained cells were visualized and imaged using a light microscope (EVOS M5000 Imaging System, Thermo Fisher Scientific, Waltham, MA, USA) (14).
Flow cytometry analysis. HT-29 and HCT-116 cells were harvested after treatment and collected into centrifuge tubes. Following centrifugation, the supernatant was discarded, and the cell pellets were washed once with phosphate-buffered saline (PBS). Cells were then incubated with individual fluorescently conjugated antibodies targeting cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Fas/FasL, or DIOC6 (for mitochondria membrane potential), according to the manufacturer’s instructions. Staining was performed in the dark for at least 30 min at room temperature. After staining, cells were washed with PBS and resuspended in an appropriate buffer. Flow cytometric analysis was performed using a NovoExpress® flow cytometer (Agilent, Santa Clara, CA, USA), and data were analyzed with FlowJo software (BD Pharmingen™) to quantify the proportion of positively stained cell populations (15). Antibodies information was listed in Table II.
Primary antibodies used in this study for flow cytometry.
Western blot analysis. Treated cells were collected, and total protein was extracted using NP-40 lysis buffer supplemented with protease and phosphatase inhibitors. Lysates were clarified by centrifugation, and protein concentrations were determined using a BCA Protein Assay Kit. Equal amounts of protein were denatured by heating and separated by SDS-PAGE, followed by transfer onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated with primary antibodies against ERK, STAT3, NF-κB, and β-actin (loading control) overnight at 4°C. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence (ECL) reagents and captured with a UVP ChemiDoc-It™ imaging system. Protein band intensities were quantified using ImageJ software (version 1.50; NIH, USA) (16). Antibodies information was listed in Table III. The experiment was repeated three times.
Primary antibodies used in this study for western blot.
Statistical analyses. Statistical analyses and graph generation were performed using GraphPad Prism software 8.0; GraphPad Software, San Diego, CA, USA). All statistical tests were two-tailed, and p-values <0.05 were considered statistically significant.
Results
Lenvatinib induces cytotoxicity and apoptosis in colorectal cancer cells. First, we evaluated whether lenvatinib treatment could induce cytotoxicity in HT29 and HCT116 cells using the MTT assay (Figure 1A-B). Lenvatinib reduced cell viability in both HT29 and HCT116 cells in a dose- and time-dependent manner. The IC50 values of lenvatinib for HT29 and HCT116 cells at 24 h were approximately 30 μM. We also evaluated whether lenvatinib affects the proliferation of CRC cells. As shown in Figure 1C, the total number of colonies was markedly reduced by lenvatinib in a dose-dependent manner. Next, we investigated whether lenvatinib could also trigger apoptosis signaling in colorectal cancer (CRC) cells. As shown in Figure 1D, cleaved caspase-3 levels were increased by 1.5- to 2-fold compared to untreated controls, indicating activation of the apoptotic pathway. In summary, our results suggest that lenvatinib induces cytotoxicity in CRC cells and is associated with the activation of apoptosis signaling.
Cytotoxic and apoptosis-inducing effects of lenvatinib in CRC lines. (A-B) MTT assay to assess the cytotoxic effects of lenvatinib on HT-29 and HCT-116 colorectal cancer cell lines after treatment at various concentrations and time points. (C) Colony formation following lenvatinib treatment was assessed across a range of concentrations. (D) Flow cytometric analysis of cleaved caspase-3 expression in HT29 and HCT116 cells following lenvatinib treatment, used to assess activation of apoptosis signaling. Data are expressed as mean±SD from three independent experiments. **p<0.01, ***p<0.0005 vs. 0 μM.
Lenvatinib induces extrinsic apoptosis in colorectal cancer cells. To further elucidate the mechanism by which lenvatinib induces apoptosis in colorectal cancer (CRC) cells, we performed flow cytometric analysis using various apoptosis-related markers. First, we examined the expression of the death receptor Fas and its ligand (Fas-L) following lenvatinib treatment. As shown in Figure 2A-B, lenvatinib markedly increased the expression of both Fas and Fas-L in CRC cells. This upregulation occurred in a dose-dependent manner, with expression levels elevated approximately 1.5-2-fold compared to untreated controls. Next, we assessed the downstream signaling events of death receptor activation, focusing on caspase-8. Western blot analysis revealed that lenvatinib treatment led to a pronounced increase in the cleaved (active) form of caspase-8, a key executor of extrinsic apoptosis (Figure 2C). Taken together, these findings indicate that lenvatinib activates the extrinsic apoptotic pathway in CRC cells through upregulation of Fas/Fas-L and subsequent activation of caspase-8.
Extrinsic apoptosis induced by lenvatinib in CRC lines. HT29 and HCT116 cells were treated with varying concentrations of lenvatinib, harvested, and stained with fluorophore-conjugated antibodies against Fas, Fas ligand (Fas-L), or cleaved caspase-8. Flow cytometry was used to assess expression levels of (A) Fas, (B) Fas-L, and (C) cleaved caspase-8. Data are expressed as mean±SD from three independent experiments.
Lenvatinib induces intrinsic apoptosis in colorectal cancer cells. Next, we examined whether the apoptosis induced by lenvatinib in colorectal cancer (CRC) cells was associated with the mitochondrial (intrinsic) apoptotic pathway. To address this, we assessed key markers typically activated during mitochondria-mediated apoptosis, including cleaved caspase-9 and loss of mitochondrial membrane potential (ΔΨm). As shown in Figure 3A, lenvatinib treatment increased the level of cleaved caspase-9 by approximately two- to three-fold compared with the untreated control group. In addition, the proportion of cells exhibiting mitochondrial membrane potential loss was markedly elevated following lenvatinib treatment (Figure 3B). These findings indicate that lenvatinib-induced apoptosis in CRC cells is mediated not only through the extrinsic pathway but also via the intrinsic, mitochondria-dependent pathway, suggesting a multifaceted mechanism of cell death induction.
Intrinsic apoptosis induced by lenvatinib in CRC lines. HT-29 and HCT-116 cells were treated with varying concentrations of lenvatinib, harvested, and stained with fluorophore-conjugated antibodies against cleaved caspase-9. Mitochondrial membrane potential (ΔΨm) was assessed using [DIOC6] staining. Flow cytometry was used to evaluate (A) expression levels of cleaved caspase-9 and (B) the proportion of cells exhibiting mitochondrial membrane potential loss. Data are expressed as mean±SD from three independent experiments.
Lenvatinib inhibits the ERK/STAT3/NF-κB signaling axis and reduces migration and invasion in colorectal cancer cells. To further investigate the molecular mechanisms underlying the anti-CRC effects of lenvatinib, we performed western blot analysis to examine key signaling pathways. As shown in Figure 4A-B, lenvatinib treatment led to a marked, dose-dependent reduction in ERK phosphorylation. Consistently, treatment with a selective ERK inhibitor also suppressed CRC cell growth in a dose- and time-dependent manner (Figure 4C). This inhibitory effect extended to downstream effectors, with phosphorylation of STAT3 and NF-κB significantly reduced in both HT-29 and HCT-116 cells. Given that ERK activation is known to regulate STAT3 and NF-κB signaling, these results suggest that lenvatinib suppresses CRC cell growth and survival, at least in part, through inactivation of the ERK/STAT3/NF-κB axis. Furthermore, both lenvatinib and the ERK inhibitor markedly reduced the migration and invasion capacities of CRC cells (Figure 4D-E), highlighting their potential to impair metastatic behavior.
Lenvatinib inhibits the ERK/STAT3/NF-κB signaling axis and reduces migration and invasion in colorectal cancer cells. (A, B, C) HT-29 and HCT-116 cells were treated with increasing concentrations of lenvatinib or a selective ERK inhibitor for the indicated times. Phosphorylation levels of ERK, STAT3, and NF-κB, as well as total protein levels, were analyzed by western blotting. β-actin served as a loading control. (D-E) Invasion and migration were assessed using Transwell assays. Cells were treated with lenvatinib or the ERK inhibitor, and migrated/invaded cells were fixed, stained, and quantified. Data are presented as mean ± SD from three independent experiments. **p<0.01, ***p<0.0005 vs. 0 μM.
Discussion
Our results demonstrated that lenvatinib significantly inhibited the growth of HT-29 and HCT-116 cells and markedly increased the activity of cleaved caspase-3, a key hallmark of apoptosis (Figure 1). Anticancer drugs are known to induce apoptosis through activation of death receptors or by eliciting intrinsic stress, such as DNA damage and inhibition of cell division, thereby initiating both extrinsic and intrinsic signaling pathways (17, 18). Therefore, we further evaluated the effects of lenvatinib on these pathways. The results revealed that, in the extrinsic pathway, lenvatinib significantly increased the activity of death receptor FAS, FASL, and cleaved caspase-8 (Figure 2); furthermore, in the intrinsic pathway, it effectively reduced mitochondrial membrane potential and enhanced cleaved caspase-9 activity (Figure 3). Collectively, these findings indicate that lenvatinib induces apoptosis in colorectal cancer cells by simultaneously activating both the extrinsic and intrinsic pathways.
Constitutive activation of oncogenic kinases and transcription factors plays a critical role in tumor progression. Aberrant activation of ERK, NF-κB, and STAT3 upregulates downstream effector proteins, thereby promoting cell survival and enhancing invasive potential. ERK functions as a central kinase within the RAF/MEK/ERK signaling cascade, whereas NF-κB and STAT3 serve as key transcription factors that can be activated through multiple upstream pathways (19-22). Persistent activation of these signaling molecules has been strongly associated with poor clinical outcomes in CRC, while inhibition of their activity has been shown to suppress CRC cell survival and invasion (23-26). In this context, our findings demonstrate that treatment with lenvatinib significantly attenuates the activation of ERK, STAT3, and NF-κB in CRC cells (Figure 4A-B). Moreover, both lenvatinib and PD98059 effectively inhibit the migratory and invasive capacities of CRC cells (Figure 4D).
This study demonstrated that lenvatinib exerts potent antitumor effects in colorectal cancer cells by inhibiting proliferation, promoting apoptosis, and reducing migratory and invasive capacities. Mechanistically, lenvatinib triggers apoptosis through concurrent activation of the extrinsic and intrinsic pathways, while suppressing the constitutive activation of ERK, STAT3, and NF-κB. Collectively, these findings indicate that lenvatinib not only promotes apoptosis but also disrupts survival signaling, underscoring its potential as a therapeutic strategy for colorectal cancer.
Conclusion
Lenvatinib exerts potent antitumor effects in colorectal cancer cells by inhibiting proliferation, promoting apoptosis, and reducing migratory and invasive capacities. Mechanistically, lenvatinib triggers apoptosis through concurrent activation of the extrinsic and intrinsic pathways, while suppressing the constitutive activation of ERK, STAT3, and NF-κB. Collectively, these findings indicate that lenvatinib not only promotes apoptosis but also disrupts survival signaling, underscoring its potential as a therapeutic strategy for colorectal cancer.
Footnotes
Authors’ Contributions
CH Wu, FT Hsu, CH Chen, and PE Wang were responsible for conducting experiments, data analysis, and preparation of the manuscript draft. LL Hsieh, HY Huang, and CH Chen contributed to the study conception and design, supervised the research activities, and prepared the final version of the manuscript.
Conflicts of Interest
The Authors report no conflicts of interest.
Funding
This study was supported by the Changhua Show Chwan Memorial Hospital, Changhua, Taiwan (ID: SRD-110004).
Artificial Intelligence (AI) Disclosure
During the preparation of this manuscript, a large language model (ChatGPT-5, OpenAI) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning-based image enhancement tools.
- Received September 26, 2025.
- Revision received October 28, 2025.
- Accepted November 4, 2025.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
