
Head and neck squamous cell carcinoma, including hypopharyngeal carcinoma represented by the FaDu cell line, is one of the most aggressive malignancies1,2). Despite advancements in therapeutic interventions, the prognosis for hypopharyngeal carcinoma remains poor due to its high metastatic potential and resistance to conventional treatments such as chemotherapy and radiotherapy3,4). The need for novel therapeutic strategies targeting molecular pathways that induce apoptosis in cancer cells has become increasingly important for improving treatment outcomes5).
Reversine (Fig. 1), a 2,6-disubstituted purine derivative, was initially identified for its ability to induce myoblast dedifferentiation into multipotent progenitor cells6). Recent studies have demonstrated its anticancer properties across various tumor cell lines, including osteosarcoma7,8), gastric cancer9), and oral squamous cell carcinoma10). Reversine exerts its tumor-suppressive effects by inhibiting cell proliferation, inducing cell cycle arrest, and promoting apoptosis through the intrinsic and extrinsic apoptotic pathways11).
We hypothesized that reversine activates key apoptotic pathways leading to the induction of apoptosis in FaDu hypopharyngeal carcinoma cells. Reversine inhibits tumor cell proliferation and promotes apoptosis, demonstrating its potential as an anticancer agent. Specifically, it is expected to exert these effects through the activation of caspase-3, cleavage of poly adenosine diphosphate-ribose polymerase (PARP), and nuclear fragmentation. This study aimed to elucidate the mechanisms through which Reversine inhibits cell proliferation and induces apoptosis in FaDu cells. By focusing on caspase-3 activation, PARP cleavage, and nuclear changes, the study sought to provide critical insights into the molecular mechanisms underlying the pro-apoptotic effects of Reversine.
Using a range of assays, including the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay, DNA fragmentation analysis, immunoblotting, and fluorescence imaging, this study investigated the potential of Reversine as a therapeutic agent for hypopharyngeal carcinoma. These findings suggest that Reversine targets both the intrinsic and extrinsic apoptotic pathways, highlighting its promise as a novel therapeutic strategy for hypopharyngeal carcinoma.
The human-derived FaDu cell line was purchased from the American Type Culture Collection (HTP-43; ATCC, Manassas, VA, USA). FaDu cells were grown in Minimum Essential Medium (WELGENE, Gyeongsan, Korea) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA ) at 5% CO2 and 37°C incubator.
The effects of reversine on FaDu cell viability were also investigated. FaDu cells were seeded in 96-well plates at a density of 2×105 cells/well. They were incubated for overnight in an incubator with a 5% CO2 and 37°C, allowing them to adhere to wells. Reversine was treated at concentrations of Control, 15 μM, and 30 μM. Reversine was purchased from Sigma-Aldrich (St. Louis, MO, USA). After culturing for 24 hours, the MTT reagent (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma-Aldrich) was added and incubated for 4 hours. Spore residue crystals were dissolved in dimethyl sulfoxide (Duksan C&P Co. Ltd., Ansan, Korea) and the absorbance was measured at 570 nm (SpectraMax ABS Plus; Molecular Devices, San Jose, CA, USA). The absorbance of the experimental group treated with reversine was expressed as a percentage (%) compared with that of the Control group.
Changes in the proportion of living and dead FaDu cells treated with reversine were analyzed by fluorescence. FaDu cells were inoculated into 8-well chamber silde (Lab-Tek I Chamber Slide; Thermo Fisher Scientific, Rockford, IL, USA) at a density of 2×104 cells/well. The cells were incubated overnight, and then reacted with Control, 15, and 30 μM/ml concentrations of reversine for 24 hours. After the reaction, the culture medium was removed and washed with 1× phosphate buffered saline (PBS; WELGENE). A live/dead cell imaging kit (488/570; Thermo Fisher Scientific) was used. A 2X stock solution was prepared by mixing green and red vials included in the kit, and 200 μl was added to each well and stained for 15 minutes in an incubator at 37°C. Stained FaDu cells were compared and analyzed between living and dead cells using a fluorescence microscope (Eclipse Ni-U; Nikon Instruments Inc., Melville, NY, USA).
Hematoxylin and eosin (H&E) staining was performed to observe morphological changes in FaDu cells. FaDu cells were seeded in an 8-well chamber at a density of 2×104 cells/well. The cells were incubated overnight and then reacted with Control, 15 μM, and 30 μM concentrations of reversine for 24 hours. The cells were rinsed in PBS, fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 minutes, before the H&E staining kit (Abkam, Cambridge, United Kingdom) was used. This was performed by incubating with hematoxylin for 5 minutes, bluing reagent for 15 seconds, eosin for 3 minutes, and washing with ethanol. Cells were imaged using an inverted retardation microscope (Nikon Instruments Inc.).
FaDu cells were seeded in an 8-well chamber at a density of 2×104 cells/well. Reversine was treated with Control, 15, and 30 μM and then incubated for 24 hours. The cells were then washed with PBS and fixed with 4% paraformaldehyde. The washed cells were stained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; F. Hoffmann-La Roche Ltd., Mannheim, Germany) (1 mg/ml) for 20 minutes. Nuclear condensation in reversine-treated FaDu cells was observed using fluorescence microscopy (Eclipse Ni-U; Nikon Instruments Inc.).
DNA fragmentation assay was conducted to evaluate apoptosis in FaDu hypopharyngeal carcinoma cells following treatment with Control, 15 μM, and 30 μM concentrations of reversine for 24 hours. After treatment, the cells were collected, rinsed in PBS at 4°C, and transferred to a 15 ml conical tube. The cells were centrifuged at 1,200 RPM for 3 minutes and the supernatant was carefully removed. The supernatant was discarded, and DNA fragmentation lysis buffer (approximately 100 μl, depending on pellet size) was added to resuspend the cell pellet. An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1; Sigma-Aldrich) solution was added to the supernatant, mixed gently for 5 minutes, and centrifuged again at 13,000 RPM at 4°C for 30 minutes. The supernatant was then transferred to a new tube and twice the volume of cold 100% ethanol and 1/10 volume of 3 M sodium acetate (WELGENE) were added. The solution was centrifuged again under the same conditions, the supernatant was discarded, and the resulting pellet was air-dried. The pellet was resuspended in 20∼30 μl of nuclease-free water, and 5 μl of RNase A (10 mg/ml) was added. Samples were incubated at 37°C for 1 hour to degrade any residual RNA. A 2% agarose gel was prepared, loaded onto the gel, and electrophoresis was performed.
FaDu cells were seeded in 6-well plates and treated with Control, 15 μM, and 30 μM concentrations of reversine for 24 hours. After treatment, cells were harvested and washed twice with ice-cold PBS. The cells were lysed using RIPA buffer (Thermo Fisher Scientific) containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) and incubated on ice for 30 minutes. The lysates were centrifuged at 13,000 RPM at 4°C for 15 minutes, and the supernatants were collected. Protein concentration was determined using the bicinchoninic acid assay (Thermo Fisher Scientific). Equal amounts of protein (20∼30 μg) from each sample were separated on a 10% SDS-PAGE gel and transferred onto PVDF membranes (Thermo Fisher Scientific) using a semi-dry transfer system. Membranes were blocked with 5% non-fat dry milk (Sigma-Aldrich) in tris-buffered saline containing 0.1% Tween-20 (TBST; Cell Signaling Technology Inc., Danvers, MA, USA) for 1 hour at room temperature to prevent non-specific binding. After blocking, the membranes were incubated overnight at 4°C with primary antibodies targeting caspase-3, cleaved caspase-3, PARP, cleaved PARP, and β-actin (used as a loading control) at appropriate dilutions (Cell Signaling Technology Inc.). The membranes were washed thrice with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature (Santa Cruz Biotechnology Inc., Dallas, TX, USA). After washing thrice with TBST, the protein bands were visualized using an enhanced chemiluminescence detection system (Sigma-Aldrich). Band intensities of cleaved caspase-3 and cleaved PARP were quantified relative to those of total caspase-3 and PARP, respectively. This analysis demonstrated a dose-dependent activation of caspase-3 and PARP cleavage, indicating apoptosis induction in FaDu hypopharyngeal carcinoma cells by reversine treatment.
Data analysis was conducted on experiments performed in triplicate or more. Results are expressed as the mean±SEM. SEM represents the standard error of the mean. Statistical comparisons between two groups were evaluated using the Student’s t-test, while multi-group comparisons were assessed using one-way ANOVA. Statistical than 0.05 was deemed at significant (IBM SPSS Statistics, Version 22.0, IBM Corp., Armonk, NY, USA).
Reversine’s impact on FaDu cell viability was assessed using the MTT assay. After treating FaDu cells with Control, 15 μM, and 30 μM concentrations of reversine for 24 hours, cell viability significantly decreased in a dose-dependent manner (Fig. 2A). The most pronounced reduction was observed in the 30 μM treatment group, demonstrating the potent inhibitory effect of reversine on cell proliferation (p<0.01).
Additionally, live/dead staining was performed to confirm cell viability. In the Control group, most cells exhibited green fluorescence, indicating live cells. However, in the reversine-treated groups (15 μM and 30 μM), the proportion of red fluorescent dead cells increased in a dose-dependent manner (Fig. 2B). These findings indicate that reversine not only inhibits cell proliferation, but also induces cell death in FaDu cells.
H&E staining was used to observe morphological changes in the cells. In the Control group, cells showed intact nuclei and cytoplasm (Fig. 3A). However, reversine treatment induced characteristic morphological changes associated with cell death in a dose-dependent manner. In the 15 μM group, cell shrinkage, cytoplasmic condensation, and nuclear condensation were observed. These effects were more prominent in the 30 μM group, where cell detachment and the formation of cellular debris were evident.
DAPI staining was used to observe nuclear changes induced by reversine treatment (Fig. 3B). While intact nuclei were observed in the Control group, nuclear condensation and fragmentation increased in a dose-dependent manner in reversin-treated groups. In particular, nuclear fragmentation was prominent in the 30 μM group, a hallmark of apoptotic cell death.
A DNA fragmentation analysis was performed to confirm these observations. DNA laddering, a distinct pattern of fragmented DNA, was detected in reversine-treated groups, indicating apoptosis-induced DNA degradation (Fig. 3C). In contrast, DNA fragmentation was not observed in the Control group. These results confirm that reversine induces apoptosis through DNA degradation pathways in FaDu cells.
To investigate the activation of apoptotic pathways, Western blot analysis was performed to evaluate caspase-3 and PARP levels in FaDu cells treated with reversine at concentrations of Control, 15 μM, and 30 μM. The total caspase-3 and PARP levels remained consistent across all groups. However, the activated forms of cleaved caspase-3 and cleaved PARP increased in a dose-dependent manner following reversine treatment (Fig. 4).
The cleaved caspase-3 levels were most elevated in the 30 μM group, indicating robust activation of caspase-3, which promotes apoptosis. Similarly, PARP cleavage was observed, with intact PARP cleaved into cleaved PARP, suggesting the inhibition of DNA repair mechanisms. In the Control group, cleaved caspase-3 and cleaved PARP were minimally detected, confirming that reversine specifically activates apoptotic pathways.
Quantitative analysis of band intensities revealed a significant increase in the levels of cleaved caspase-3 and cleaved PARP compared to those of total caspase-3 and PARP, respectively. These results strongly supported the conclusion that reversine induces apoptosis in FaDu cells by activating caspase-dependent apoptotic pathways.
The present study investigated the effects of reversine on FaDu hypopharyngeal carcinoma cells, focusing on its ability to suppress cell viability and induce apoptosis12). The findings demonstrate that reversine effectively inhibits cell proliferation and induces apoptosis in a dose-dependent manner, highlighting its potential as a therapeutic agent for hypopharyngeal carcinoma13).
First, the MTT assay revealed a significant reduction in cell viability following reversine treatment, confirming its inhibitory effects on FaDu cell growth. Live/dead staining further supported these results, showing an increase in dead cells (red fluorescence) with increasing concentrations of reversine. These results suggest that reversine exerts cytotoxic effects on FaDu cells, likely through apoptosis rather than other forms of cell death, such as necrosis.
Morphological analyses using hematoxylin & eosin staining showed characteristic apoptotic features, such as cell shrinkage, nuclear condensation, and the formation of apoptotic bodies, particularly at higher reversine concentrations. This was further validated by DAPI staining, which revealed nuclear fragmentation in treated cells, a hallmark of apoptosis. DNA fragmentation analysis confirmed these observations, as the appearance of a DNA ladder pattern in treated cells indicated the activation of endonucleases, a critical step in the apoptotic process14).
Mechanistically, the study demonstrated that reversine activates intrinsic apoptotic pathways. Western blot analysis revealed increased cleavage of caspase-3 and PARP, two critical proteins involved in apoptosis. Cleaved caspase-3 serves as an executioner enzyme, orchestrating downstream events in apoptosis, while PARP cleavage is associated with DNA repair inhibition and the progression of apoptotic cell death. The dose-dependent increase in cleaved caspase-3 and cleaved PARP levels strongly supports the hypothesis that reversine induces apoptosis through caspase activation15).
The observed increases in cleaved caspase-3 and PARP cleavage strongly indicate the involvement of the intrinsic (mitochondria-mediated) apoptotic pathway in inducing cell death. Previous studies on reversine have shown its ability to disrupt mitochondrial membrane potential and modulate the activity of apoptotic proteins such as Bcl-2 and Bax, which are critical regulators of the intrinsic pathway16). Additionally, reversine has been reported to inhibit survival pathways, including the Akt/mTOR signaling cascade, further sensitizing cancer cells to apoptosis10). These mechanisms may collectively contribute to the observed effects in FaDu cells.
However, while the apoptotic effects of reversine are evident, its potential toxicity and off-target effects on normal cells remain to be investigated17). reversine, as a small molecule kinase inhibitor, could potentially disrupt critical signaling pathways in healthy cells, leading to unintended side effects18). Furthermore, comprehensive studies are required to assess reversine’s toxicity, including its effects on normal tissues and overall safety17). These studies are crucial to establish its feasibility and safety for therapeutic used. One of the most significant findings of this study is the demonstration of reversine’s ability to inhibit cell proliferation and induce apoptosis in hypopharyngeal carcinoma cells. The ability of reversine to target these cancer cells through apoptosis induction suggests its potential as a novel therapeutic agent19). Its mechanism of action—suppressing survival pathways and activating apoptotic cascades—provides a strong rationale for further exploration of reversine as part of combination therapies to enhance treatment efficacy and overcome resistance to existing therapies20).
However, several limitations should be addressed in future studies. While the current findings confirm the pro-apoptotic effects of reversine in vitro, in vivo studies are required to validate its efficacy and safety in animal models of hypopharyngeal carcinoma. Additionally, the precise molecular targets of reversine in FaDu cells remain to be elucidated. Further studies should focus on identifying key signaling pathways and molecular interactions that mediate its anti-cancer effects21). Moreover, investigating whether reversine enhances the efficacy of existing chemotherapeutic agents could offer additional perspectives on its potential application in combination therapy22).
Reversine effectively inhibits the viability of FaDu cells in a dose-dependent manner, induces characteristic morphological changes in apoptosis, promotes DNA fragmentation, and activates the apoptosis signaling pathway through caspase-3 and PARP cleavage. These findings demonstrate the potential of reversine as a candidate anticancer agent that inhibits cell proliferation and induces apoptosis of FaDu cells. This highlights its potential as a novel therapeutic strategy for hypopharyngeal carcinoma cancers. The originality of this study lies in the focus on unstudied hypopharyngeal cancer with respect to the effects of reversine. Previous studies have primarily explored the effects of reversine on different tumor types, but this study has revealed the cell death-promoting effects and underlying mechanisms of reversine in particular in FaDu cells. This highlights the novel therapeutic objectives of reversine and highlights its diversity and relevance. Furthermore, this study reveals that not only does reversine inhibit cell viability, but it also activates both intrinsic (mitochondria-mediated) and extrinsic (dead receptor-mediated) apoptosis pathways in FaDu cells, providing new insights to the existing knowledge base. This dual-path activation increases understanding of the mechanisms of action of reversine and represents its potential to overcome therapeutic resistance, associated with hypopharyngeal carcinoma cancer. These findings suggest that reversine holds promise as a treatment option, warranting further investigation into its therapeutic potential.
Conflict of interest
No potential conflict of interest relevant to this article was reported.
Ethical approval
This study does not require IRB review because it is an experimental paper using commercially available cells.
Funding
None.
Data availability
The data and materials of this article are included within the article. The data supporting the findings of this study are available from the corresponding author upon reasonable request.
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