Ⅰ. INTRODUCTION
Mucoepidermoid carcinoma (MEC) represents the most common malignant tumor of the salivary gland, characterized by considerable heterogeneity in histologic grade, cellular differentiation, and clinical outcome (1-4). Despite complete surgical resection remains the cornerstone of treatment, patients with high-grade or advanced MEC frequently develop locoregional recurrence or distant metastasis, resulting in dismal clinical outcomes (5, 6). Moreover, the limited efficacy and substantial systemic toxicity of conventional chemotherapeutic regimens highlight an urgent need for novel therapeutic approaches that achieve greater tumor selectivity and improved safety profiles.
Microtubules are essential components of the cytoskeleton, orchestrating cell shape maintenance, intracellular trafficking, and chromosome segregation during mitosis (7, 8). Pharmacologic disruption of microtubule polymerization has long served as an effective anticancer strategy (8, 9); however, classical microtubule inhibitors such as vinblastine and colchicine cause irreversible cytoskeletal damage and severe adverse effects (10), limiting their therapeutic window. KX-01 (KX2-391or Tirbanibulin) is a next-generation microtubule- targeting compound that reversibly binds to the colchicine- binding site of β-tubulin, inducing transient mitotic arrest and apoptosis while minimizing toxicity in normal cells (11, 12). This reversible interaction mechanistically distinguishes KX-01 from traditional tubulin inhibitors and underlies its favorable tolerability observed in both preclinical and clinical studies.
KX-01 has exhibited robust antiproliferative activity across several epithelial malignancies, including breast and squamous cell carcinoma, primarily through disruption of microtubule dynamics (12, 13). Importantly, KX-01 has been approved by the U.S. Food and Drug Administration for the topical treatment of actinic keratosis(14), underscoring its favorable safety profile and pharmacologic stability. Despite its clinical success, the therapeutic potential and underlying mechanism of KX-01 in salivary gland MEC remain undefined. Because apoptosis constitutes a fundamental mode of programmed cell death and serves as a key determinant of anticancer efficacy – and given our previous investigations delineating apoptotic mechanisms in MEC models (2-4, 6) – we hypothesized that KX-01 may induce apoptosis in MEC cells. The present study was therefore designed to characterize the apoptotic response elicited by KX-01 in human salivary MEC cell lines (MC3 and YD15).
Ⅱ. MATERIALS and METHODS
1. Cell culture and reagents
Two salivary gland–derived MEC cell lines were used in this study. The MC3 cell line was generously provided by Professor Wu Junzheng (Fourth Military Medical University, Xi’an, China), and the YD15 cell line was obtained from the Korean Cell Line Bank (Seoul, Republic of Korea). Cells were cultured in DMEM/F12 or RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin under standard conditions (37 °C, 5% CO₂, humidified atmosphere). All experiments were conducted using cells at approximately 50–60% confluence. KX-01 was purchased from MedChemExpress (Cat. HY-10340). All reagents were dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C until use.
2. Cell viability assay
Cell viability after KX-01 treatment was assessed using the trypan blue exclusion assay. MC3 and YD15 cells were seeded in six-well plates and exposed to 80 nM KX-01 for 24 h. After incubation, cells were stained with 0.4% trypan blue (Gibco; Thermo Fisher Scientific, Waltham, MA, USA), and viable cell numbers were quantified using a Corning® Cell Counter (Corning Inc., Corning, NY, USA).
3. Soft agar assay
A soft agar assay was performed to assess anchorage-independent growth. The bottom agar layer (0.5%) was made by mixing 2× Basal Medium Eagle (BME), L-glutamine, gentamicin, phosphate-buffered saline (PBS), FBS, and 1.25% agar. Each well was precoated with 3 mL of the bottom agar, followed by a top agar layer (0.3%). After solidification at room temperature (RT) for 1 h, 200 μL of culture medium containing DMSO or KX-01 was added every three days. Cells were incubated at 37 °C for 7 days. Colony formation was observed using a light microscope (Olympus Corporation) at ×40 magnification, capturing four random fields per well for quantification. Colony number and size were measured using ImageJ software version 1.53t (National Institutes of Health, Bethesda, MD, USA).
4. Live/Dead assay
Cell viability was assessed using the LIVE/DEAD™ Viability/Cytotoxicity Kit (Cat. no. L3224; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. Cells were treated with the indicated concentration of KX-01 for 24 h at 37 °C, followed by staining with Calcein AM (4 μM) and ethidium homodimer-1 (2 μM) prepared in PBS. After 1h-incubation at RT, cells were visualized under a digital inverted fluorescence microscope (Nikon Corporation), and images were analyzed using ImageJ software.
5. 4′-6-Diamidino-2-phenylindole (DAPI) staining
Nuclear morphological changes indicative of apoptosis was examined using DAPI staining (Sigma-Aldrich). Cells were seeded in 60-mm dishes and treated with KX-01 for 24 h. Following treatment, cells were harvested, washed twice with PBS, and fixed with 100% methanol for 10 min at RT. Fixed cells were washed again with PBS, mounted onto glass slides, and stained with DAPI (2 μg/mL). Nuclear morphology was then visualized and analyzed under a fluorescence microscope.
6. Annexin V/PI double staining
Apoptosis was assessed using the FITC-Annexin V Apoptosis Detection Kit (BD Pharmingen) according to the manufacturer’s instructions. Both adherent and floating cells were collected, washed twice with ice-cold PBS, and centrifuged at 180 × g for 5 min at 4 °C. Cell pellets were resuspended in Annexin V binding buffer and incubated with 5 μL Annexin V-FITC for 15 min at RT in the dark, followed by staining with 2 μL propidium iodide (PI; Cat. no. P4170, MilliporeSigma). Stained samples were analyzed by flow cytometry, and at least 10,000 events were recorded per sample. Cells positive for Annexin V only were classified as early apoptotic, whereas cells positive for both Annexin V and PI were identified as late apoptotic.
7. Cell cycle distribution analysis
Following KX-01 treatment, MC3 and YD15 cells were harvested, washed with PBS, and fixed in 70% ethanol at −20 °C overnight. Fixed cells were incubated with 20 μ g/mL PI and RNase A (20 μg/mL) for 15 min at 37 °C. DNA content was analyzed using an LSRFortessa™ Cell Analyzer (BD Biosciences), and at least 10,000 events were acquired per sample. Data were processed with BD FACSDIVA™ software (version 6.0), and the sub-G1 population was quantified using FlowJo software (versions 9/10; FlowJo LLC).
8. Western blot analysis
Total protein was extracted using RIPA lysis buffer (MilliporeSigma) supplemented with 1% phosphatase inhibitor (Thermo Fisher Scientific, Inc.) and protease inhibitor cocktail (Roche Diagnostics, GmbH). Protein concentrations were determined using a DC Protein Assay Kit (Bio-Rad Laboratories, Inc.). Equal amounts of protein (30– 50 μg) were normalized, mixed with 5× sample buffer, and heated at 95 °C for 5 min, separated on 8–15% SDS-polyacrylamide gels, transferred onto Immuno-Blot PVDF membranes (MilliporeSigma), and blocked with 5% skim milk in TBST for 1 h at RT, incubated with primary antibodies overnight at 4 °C, and then with HRP-conjugated secondary antibodies for 2 h. Protein bands were visualized using a WestGlow™ PICO PLUS chemiluminescent substrate (Biomax, Inc.) and detected with an ImageQuant™ LAS 500 system (GE Healthcare Life Sciences) or x-ray film. Band intensities were quantified by densitometry using ImageJ software version 1.53t.
9. Statistical analysis
Statistical analyses were performed using SPSS version 26.0 (SPSS, Chicago, IL, USA). Differences between control and treatment groups were evaluated using the Student’s t-test or one-way ANOVA. Graph were generated with GraphPad Prism version 10.4.1 (GraphPad Software, San Diego, CA, USA) and all data are presented the means ± standard deviations (SD) of three independent experiments. A p-value of less than 0.05 considered statistically significant.
Ⅲ. RESULTS
1. KX-01 reduces cell viability in MC3 and YD15 cells
To determine the growth-inhibitory effects of MEC cells, MC3 and YD15 cells were treated with 80 nM KX-01 for 24 h and analyzed for morphological and viability changes (Fig. 1A). Phase-contrast microscopy revealed pronounced morphologic alterations, including cell rounding, detachment, and loss of cell–cell adhesion, which are hallmarks of microtubule disruption and apoptosis. Consistently, trypan blue exclusion assays demonstrated a significant reduction in viable cells relative to DMSO-treated controls (Fig. 1B). These results indicate that KX-01 markedly decreases MEC cell viability within 24 h, consistent with its reported ability to induce mitotic arrest and apoptosis in epithelial malignancies.
2. KX-01 suppresses anchorage-independent growth and promotes cell death in MEC cells
The effect of KX-01 on the tumorigenic potential of MEC cells was assessed using a soft agar colony formation assay. Treatment with 80nM KX-01 for 7 days exhibited a pronounced reduction in both the number and size of colonies compared with the DMSO control, demonstrating the inhibition of the anchorage-independent growth (Fig. 2A). To further confirm the cytotoxicity, Live/Dead staining was conducted to distinguish viable (green) and dead (red) cells. Following treatment, a marked increase in red fluorescence was observed in both cell lines (Fig. 2B), indicating enhanced cell death. These findings show that KX-01 not only impairs anchorage-independent growth-a key feature of malignant transformation, but also enhances cell death in MEC cells, supporting its strong antitumor efficacy.
3. KX-01 induces apoptotic nuclear morphology and Annexin V–positive cell populations
To determine whether KX-01 induces apoptosis in MEC cells, DAPI staining, Annexin V/PI double staining, and cell cycle analyses were performed. DAPI staining revealed extensive chromatin condensation and nuclear fragmentation following 24 h of KX-01 treatment, in contrast to the intact, round nuclei observed in control cells (Fig. 3A). Flow cytometric analysis further demonstrated a significant in Annexin V–positive and sub-G1 cell populations in both MC3 and YD15 cells (Fig. 3B, 3C) confirming apoptotic DNA fragmentation and cell membrane asymmetry. Together, these results demonstrate that KX-01 robustly induces apoptotic cell death in MEC cells.
4. KX-01 activates apoptotic signaling pathways by increasing cleaved PARP and cleaved caspase-3 expression
To investigate the molecular mechanism underlying KX-01-induced apoptosis, the expression of cleaved PARP (c-PARP) and cleaved caspase-3 (c-caspase-3), key markers of apoptotic signaling, was analyzed by Western blotting. Treatment with 80 nM KX-01 for 24 h markedly increased c-PARP and c-caspase-3 levels compared with the control (Fig. 4A). Time-course analysis revealed a gradual increase in both proteins following KX-01 exposure, with maximal activation observed at later time points, demonstrating a time-dependent progression of apoptosis (Fig. 4B). These findings confirm that KX-01 induces caspase-mediated apoptotic cell death rather than nonspecific cytotoxicity in salivary gland MEC cells.
Ⅳ. DISCUSSION
Salivary gland carcinoma, including MEC, remains a major therapeutic challenge due to its limited responsiveness to conventional chemotherapy and radiotherapy, as well as the high recurrence rate associated with advanced disease (15-17). The efficacy of existing cytotoxic agents is further constrained by their severe systemic toxicity, emphasizing the need for safer and more selective anticancer compounds (18). In this context, KX-01 represents a promising candidate owing to its unique mechanism of action and favorable clinical safety profile. Previous structural and biochemical analyses demonstrated that KX-01 reversibly binds to the colchicine-binding site of β-tubulin, transiently inhibiting microtubule polymerization and inducing mitotic arrest without causing irreversible cytoskeletal disruption (11). This mechanism is distinct from classical tubulin inhibitors such as colchicine and vinblastine, which permanently destabilize microtubules and cause substantial cytotoxicity in normal tissues (10). The reversible nature of KX-01’s tubulin interaction provides a mechanistic basis for its lower toxicity and excellent tolerability observed in both preclinical and clinical settings (11). Beyond its microtubule-targeting activity, KX-01 has been reported to interfere with multiple oncogenic signaling cascades. In particular, inhibition of the Src–FAK–STAT3 signaling axis has been documented in breast and squamous carcinoma models. (12). These findings collectively suggest that the antitumor efficacy of KX-01 results from a coordinated blockade of both cytoskeletal and signaling networks. Despite the well-documented anticancer efficacy of KX-01 in several other malignancies (12, 13), its therapeutic potential in salivary gland tumors has not yet been investigated. Thus, in the present study, we demonstrated for the first time that KX-01 exerts potent antitumor effects in MEC cells. KX-01 treatment markedly reduced cell viability and anchorage-independent colony formation. It also induced characteristic nuclear changes on DAPI staining and increased the sub-G1 population and Annexin V–positive apoptotic fraction. Furthermore, Western blot analysis revealed elevated levels of cleaved PARP and cleaved caspase- 3. Collectively, these results provide the first experimental evidence supporting the antitumor efficacy of KX-01 in salivary gland carcinoma. The present findings provide mechanistic insight into its apoptotic activity and lay the foundation for future studies aimed at expanding its application to this rare and treatment-resistant cancer. While our results identify apoptosis as a principal outcome of KX-01 treatment, further studies are required to elucidate additional signaling pathways involved in the antitumor effects of KX-01 in MEC.
In conclusion, this study provides the first evidence that KX-01 exerts potent antitumor effects in MEC cells by inducing apoptosis. These findings highlight the therapeutic potential of KX-01 as a low-toxicity, clinically feasible compound for the treatment of salivary gland malignancies. Further studies are warranted to elucidate the underlying signaling mechanisms and to assess its efficacy in vivo for future clinical application.
