ABSTRACT
Objectives
Senescent cells release a senescence-associated secretory phenotype, promoting polyploid giant cancer cells (PGCCs) to emerge, fostering tumor heterogeneity and resistance. Pentagamavunone-1 (PGV-1) emerges as a promising agent inducing senescence and prometaphase arrest, resulting in permanent cytotoxicity. This study was aimed to investigate the effect of PGV-1 in dysregulating mitosis through the modulation of PGCCs and senescence in low MYCN-expressing hepatocellular carcinoma (HCC) cells JHH4.
Materials and Methods
To assess the physiological effects of PGV-1, several in vitro tests were done including the MTT assay, cell cycle assay, May-Grünwald-Giemsa staining, senescence associated-β-galactosidase (SA-β-Gal) assay, and apoptosis assay. The protein levels of the apoptosis regulatory protein were evaluated using western blot analysis. The interaction of PGV-1 toward the protein that plays a role in PGCCs formation was simulated by molecular docking and molecular dynamics (MD).
Results
The cytotoxic assay revealed that PGV-1 inhibited the proliferation of JHH4 liver cancer cells permanently. Inhibition of cell proliferation by PGV-1 was associated with the modulation of G2/M phase, particularly mitotic arrest and formation of PGCCs. The SA-β-Gal verified that PGV-1 induced senescence in cells (p<0.01), inducing PGCCs formation. Apoptotic mechanisms were validated via Annexin V staining, which showed the level of cleaved poly (ADP-ribose) polymerase (p<0.001). Molecular docking and MD simulations suggested that PGV-1 could interfere with the conformation of the chromosomal passenger complex (CPC) protein, particularly disrupting essential interactions within the inner centromere protein, Survivin, and Borealin.
Conclusion
PGV-1 induced strong cytotoxicity in HCC cells by disrupting mitosis leading to PGCC formation, senescence, and subsequent apoptotic cell death.
INTRODUCTION
Polyploid giant cancer cells (PGCCs) and cellular senescence play multifaceted roles in the cell cycle machinery of hepatocellular carcinoma (HCC). PGCCs are characterized by abnormal polyploidization, leading to errors in mitotic spindle assembly and chromosomal segregation during mitosis.1 Aberrant centrosome function in PGCCs contributes to the enlarged cell size and genomic instability, defining the characteristic morphology of PGCCs.2, 3 PGCCs exhibit a senescence-like phenotype, contributing to chemoresistance by evading the cytotoxic effects of chemotherapy drugs.4 Simultaneously, the irreversible growth arrest characteristic of cellular senescence can create a microenvironment rich in senescence-associated secretory phenotype (SASP) factors, influencing the behavior of neighboring cancer cells, including the emergence of PGCCs.5, 6 The dynamic interaction between PGCCs and cellular senescence promotes heterogeneity within the tumor, fostering adaptability and resistance mechanisms.7 Understanding the crosstalk between PGCCs and senescence is crucial for developing chemotherapeutic agents targeting cell cycle machinery in HCC.
The induction of senescence and dysregulation of mitosis represent two primary physiological effects of pentagamavunone-1 (PGV-1) (Figure 1a) in various cancer cells. PGV-1 triggers senescence through transient elevation in reactive oxygen species (ROS) by interacting with several ROS-metabolizing enzymes.8 PGV-1 promotes prometaphase arrest, leading to permanent cytotoxic effects on leukemia.8 In both triple-negative breast cancer (TNBC) and non-TNBC cells, PGV-1 increased polyploid cells as an indication of mitotic catastrophe.9-11 Functional network analysis with molecular docking studies revealed that PGV-1 could target several mitotic checkpoints such as aurora kinase A (AURKA), aurora kinase B (AURKB), polo-like kinase 1 (PLK1), and cyclin-dependent kinase 1 (CDK1).12 AURKB is a member of the chromosomal passenger complex (CPC) localized at the centromere from prophase to the metaphase-anaphase transition. The CPC consists of four proteins: the AURKB, the scaffold protein inner centromere protein (INCENP) and two targeting proteins, Survivin and Borealin. Reducing or disrupting the activity of any component within CPC results in cytokinesis defects, leading to subsequent multinucleation, resembling the phenotype of PGCCs.13-15 The effect of PGV-1 on the CPC complex remains unexplored.
Dysregulation of mitosis and induction of senescence by PGV-1 also persist in MYCN-expressing HCC, including HepG2 and JHH7 cells.15, 16 During mitosis, aberrant MYCN expression contributes to centrosome duplication, spindle formation, and chromosome segregation in HCC.17 MYCN also activates CDK1 to promote mitosis through Aurora A-PLK1 cascades.18 However, PGV-1 does not affect the mRNA level of MYCN, suggesting that PGV-1 targets different proteins in the dysregulation of centrosomes during mitosis in HepG2 and JHH7.16 These distinctive phenotypic changes induced by PGV-1, especially in the cell cycle machinery of HCC, provide attractive attributes for targeting PGCCs.
In this study, we aimed to investigate the effect of PGV-1 in dysregulating mitosis through the modulation of PGCCs and senescence in low MYCN-expressing HCC, JHH4 cells. Similar to other cancer types, PGV-1 is expected to exhibit potent cytotoxic effect in JHH4 cells. We identified changes in the phenotype of JHH4 cells based on the cell cycle profile, mitotic phase profile, the presence of senescent cells, and predicted CPC-related targets through computational analysis.
MATERIALS AND METHODS
Compound
PGV-1 was obtained from our compound collection and synthesized as previously reported.19 For each biological assay, PGV-1 is dissolved in dimethyl sulfoxide to prepare a stock solution. These stock solutions are then diluted for various concentrations for specific experiments.
Cell culture and reagents
JHH4 cells were kindly provided by Professor Yoshitaka Hippo and Dr. Yusuke Suenaga (JRBC® CRL-2359) from Chiba Cancer Center Research Institute, Japan. The cells were maintained under standard conditions (37 °C, 5% CO2) in high glucose Dulbecco’s Modified Eagle Medium (Gibco USA) enriched with HEPES (Sigma), 10% fetal bovine serum (Gibco USA), and 1% penicillin-streptomycin (Gibco USA).
MTT assay
A 96-well plate was seeded with 1 × 104 cells per well, and the cells were cultured until reaching 80% confluency. PGV-1 (0.1-10 µM) was then administered to the cells in repeated doses, followed by incubation for 24 and 48 hours. After the incubation period, MTT (Sigma) reagent was added, and absorbance was measured at 595 nm using a multi-plate reader (BioRad). The absorbance values were converted into percentages of cell viability using Hill’s equation to determine the half-maximal inhibitory concentration (IC50) value.20
Cell cycle analysis
In brief, JHH4 cells were cultured and treated with PGV-1 for 24 hours. The cells were trypsinized and washed with cold 1X phosphate-buffered saline (PBS). Fixation was performed by adding 70% ethanol for 30 minutes, followed by replacement with 1X PBS. The cells were incubated with a staining solution containing 200 µL 1X propidium Iodide and RNase, prepared according to the kit protocols (Abcam, ab139418), for 20-30 min. The distribution of the cell cycle BD fluorescence-activated cell sorting Calibur flow cytometer.
May-Grünwald Giemsa staining
JHH4 cells were seeded in a 6-well plate at a density of 2.5 × 104 cells per well and incubated with PGV-1 for 24 hours. After removing the medium and washing with 1X PBS, the cells were stained with May-Grünwald stain solution for 5 min, followed by two washes with 1X PBS. Additional staining was performed using 1:20 v/v Giemsa solution, followed by air drying. The mitotic phase was evaluated based on the chromosomal formation observed under a phase-contrast microscope.10
Senescence associated-β-galactosidase (SA-β-gal) assay
PGV-1 was applied to JHH4 cells that had been seeded at a density of 3 × 106 cells per well on a 6-well plate and incubated for 24 hours. After two washes with 1X PBS, the cells were fixed for 10 to 20 minutes and then washed again with 1X PBS. Cells were stained using an X-gal solution for 72 hours at 37 °C. The presence of senescence was indicated by the green color under the microscope.21
Apoptosis assay
Apoptosis assays were performed according to the manufacturer’s instructions (Apoptosis Detection Kit I, Cat. 556547). JHH4 cells were treated with PGV-1 for 24 hours. After treatment, cells were harvested using trypsin and washed with cold 1x PBS. The cells were then resuspended in 1x binding buffer, and equal amounts of fluorescein isothiocyanate Annexin V and propidium iodide were added. The cells were gently vortexed and incubated at 25 °C in the dark for 15 minutes. Apoptotic cells were detected using the FACSCalibur flow cytometer.
Western blotting
Briefly, JHH4 cells were seeded at a density of 1 × 106 cells per well in a 6-well plate and treated with or without PGV-1 for 24 hours. After treatment, cell lysates were collected using a radioimmunoprecipitation assay buffer. The samples were separated by Sodium dodecyl sulfate – polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After blocking with 5% bovine serum albumin for 2 hours, the membranes were incubated with primary antibodies: anti-poly(ADP-ribose) polymerase (PARP) antibody (Cell Signaling Technology, PARP (46D11) Antibody #9532) at a 1:1000 dilution, and anti-β-actin antibody (Cell Signaling Technology, #3700) at a 1:5000 dilution, which served as the housekeeping protein. The membranes were probed overnight with primary antibodies at 4 °C with gentle agitation. After washing with PBST, the membranes were incubated with an anti-rabbit immunoglobulin G (IgG) secondary antibody (anti-rabbit IgG, horseradish peroxidase-linked Antibody #7074) for 1 hour at room temperature. Chemiluminescent signals were detected using the ImageQuantTM TL with enhanced chemiluminescence Western Blotting Substrate (Bio-Rad). ImageJ software was then used to semi-quantify and normalize the intensity of each band relative to the housekeeping protein (anti-β-actin antibody).16
Molecular docking
Molecular docking was performed to predict the binding interaction between PGV-1 and CPC protein, which is among the proteins involved in PGCC generation. The crystal structure of CPC was obtained from the PDB (ID: 2QFA). The binding site for the molecular docking simulation was identified based on the core of CPC, which consists of the interacting amino acids among INCENP, Borealin, and Survivin.22 The molecular docking simulation was carried out using the default protocol in molecular operating environment, with slight modifications.23 The conformation of PGV-1 with the lowest docking score was selected for binding interaction analysis.
Molecular dynamics
The stability and conformational changes of PGV-1 in complex with CPC were evaluated by molecular dynamics (MD) simulation. Nanoscale molecular dynamics 2.14 was used for the MD simulation, and the resultant trajectories were visualized with visual molecular dynamics 1.9.4.24, 25 The ligands and protein were parameterized using CGenFF and CHARMM36, accessed through the CHARMM-GUI web server.26 The preparation included adding water molecules in a cubic box with 20-Å padding for solvation and neutralization, followed by the introduction of K+ and Cl– ions. The complex then underwent 70 ps of minimization and a 1000 ps equilibration simulation. To further refine the MD simulation, a 1000 ps production run was performed under the NPT ensemble, maintaining a temperature of 303 K and pressure of 1 atm. The MD results were analyzed based on conformational changes and root mean square deviation (RMSD).
Statistical analysis
The data are expressed as the mean ± standard deviation or standard error (SE) from three independent experiments. Statistical analysis was performed using analysis of variance, followed by the Student’s t-test with a 95% and 99% confidence level (p<0.01; p<0.001). All analyses were conducted using GraphPad Prism software, version 9.0 (GraphPad, San Diego, CA, USA).
RESULTS
PGV-1 permanently inhibits proliferation by inducing G2/M arrest
The establishment of a permanent cytotoxic effect has proven to be a crucial factor in the advancement of chemotherapeutic agents, addressing the challenge of relapse incidence upon the discontinuation of treatment.27 Based on wash-out experiments, PGV-1 (Figure 1a) exerted strong cytotoxic effects, particularly after withdrawal, in chronic myeloid leukemia, breast cancer, colon cancer, and hepatocellular carcinoma (HCC), with an IC50 range of 1-7 µM for 72 h treatment.8, 15, 28 However, in high MYCN HCC JHH7, PGV-1 exhibited strong cytotoxicity only after 96 h without affecting MYCN mRNA levels.16 We are then interested in evaluating the cytotoxicity of PGV-1 toward HCC with low expression of MYCN, the JHH4 cell line. The MTT assay revealed the dose-dependent cytotoxic effect of PGV-1, with IC50 values of 10 µM and 1.5 µM for 24 and 48 hours, respectively (Figure 1b). Upon the withdrawal of PGV-1 after three days, the potent cytotoxic effect of PGV-1 remained until six days of observation, indicating the permanent inhibition of proliferation (Figure 1c). The potent cytotoxicity of PGV-1 in JHH4, as reported in other types of cancer cells, confirmed its potential novel phenotypic cytotoxic effect. The induction of a persistent cytotoxic effect has the potential to disrupt the cell cycle. Cell cycle modulation of PGV-1 is associated with G2/M arrest in various cancer cells.8, 15, 28, 29 We found that PGV-1 consistently induced G2/M arrest in JHH4 and increased the sub-G1 population (Figure 1d). In addition, PGV-1 increased the number of hyperdiploid cells in a dose-dependent manner (Figure 1e). The presence of hyperdiploid cells could indicate an abnormality of genetic material, which could lead to cancer survival or death.2
PGV-1 triggers multiple arrests in mitosis and increases senescence cells
The precise regulation of mitosis plays a pivotal role in the action of chemotherapeutic drugs, as aberrations in cell division are a hallmark of cancer.30 In MYCN-expressing HCC, PGV-1 specifically demonstrated prometaphase and metaphase arrest.15, 16 The May-Grünwald Giemsa staining was used to analyze the mitosis phase affected by PGV-1. At low concentrations, PGV-1 induced chromatin condensation resulting in mitotic catastrophe (Figure 2a). Higher concentrations of PGV-1 triggered metaphase arrest, anaphase abnormality, and PGCCs (Figure 2a). The emergence of PGCCs, characterized by an abnormal number of chromosomes, suggested a unique phenotypical change in PGV-1 in JHH4 cells.
Uncontrolled mitotic activity, which triggers genomic instability, could lead to senescence as a protective mechanism to curb the unbridled proliferation of cancer cells.31 A reduced prevalence of senescent cells has been implicated in the progression of hepatocarcinogenesis within HCC. Conversely, an elevated abundance of senescent cells is associated with the activation of anti-tumoral mechanisms.6 The involvement of PGV-1 in eliciting metaphase arrest associated with the senescence arrest in HCC is reported.16 We found, by SA-β-gal assay, that a low level of cellular senescence was observed in untreated cells and significantly increased in PGV-1-treated cells in a dose-dependent manner (Figure 2b, c). Notably, senescent cells were also identified within the PGCCs, suggesting a connection between genomic rearrangements and the effects of PGV-1 treatment.
The apoptosis mechanism as the final step of the permanent cytotoxic effect by PGV-1
Evidence of senescence in PGCCs facilitated genetic rearrangement in cancer, which led to cancer chemoresistance.3 The failure of PGCCs regulation could lead to abnormality in cell division, resulting in cell death.32 We conducted an annexin-V staining to confirm the apoptosis induction of PGV-1, which triggers the PGCCs and senescence in JHH4. PGV-1 increases the incidence of early and late apoptosis (Figure 3a, b). Apoptosis induction by PGV-1 was also identified by the increased protein level of cleaved PARP (Figure 3c, d). Our results confirmed that the induction of senescence and apoptosis by PGV-1 acted as mechanisms to restrict the proliferation of PGCCs, preventing further genomic instability and tumor progression.
Binding interaction of PGV-1 in the chromosomal passenger complex
AURK-PLK1 cascades involving CEP192 and TPX2 tightly regulate the transition of prometaphase to metaphase in mitosis. During preparation to enter anaphase, PLK1 and AURKB form a complex with CPC, consisting of INCENP, Survivin, and Borealin, to regulate chromosome attachment to the mitotic spindle and ensure proper chromosome segregation (Figure 4a).18 We hypothesize that the persistent metaphase arrest caused by PGV-1, leading to mitotic catastrophe and PGCCs, was associated with the disruption of the CPC complex. Our molecular docking simulation, focusing on the core of CPC complex, showed PGV-1 interacting with INCENP by forming hydrogen bonding with Gln38 and hydrophobic bonding with Asp27. In addition, PGV-1 formed hydrophobic bonding with Thr127 on survivin (Figure 4b). The multiple interactions of PGV-1 on the core structure might affect the conformational stability of the CPC.
MD simulations were conducted to investigate the stability and potential conformational changes of the CPC complex further. At 500 ps, PGV-1 slightly modified the conformation of the CPC complex (Figure 5a). CPC showed a significant conformational change in the presence of PGV-1 after 1000 ps (Figure 5a). The conformational change was located mainly in the helical structure of INCENP, Survivin, and Borealin, suggesting the binding interaction promoted destabilization of the CPC complex (Figure 5a). The destabilization of CPC in the presence of PGV-1 was also indicated by an increase in RMSD value up to 9.2 Å (Figure 5b). Our MD simulations suggest that PGV-1 promotes a conformational change in the CPC complex, potentially contributing to the dysregulation of chromosome localization during the transition from metaphase to anaphase.
DISCUSSION
PGV-1 has previously exhibited potent cytotoxicity in HepG2 and JHH7 cells without affecting the mRNA level of MYCN.15, 16 This study explored the distinctive phenotypic changes induced by PGV-1 in JHH4 cells with low MYCN expression, contributing valuable foundational knowledge for the development of chemotherapeutic agents for HCC. Examining these phenotypic changes is crucial for elucidating potential signaling pathways activated by PGV-1 in HCC. The utilization of cells with low MYCN expression enables us to distinguish the correlation between MYCN/NCYM signaling regulation and the mechanism of action of PGV-1. While the specific target of PGV-1 remains elusive, our study highlights a cluster of essential targets regulated by PGV-1 in HCC.
HCC is classified as a malignant cancer with only a 10% survival rate for patients within five years of diagnosis.33 The high rate of recurrence is a major factor contributing to the poor prognosis of HCC compared to other liver cancers.34 Given the limited treatment options for HCC, there is a critical need for potent and effective cytotoxic chemotherapeutic agents. PGV-1 exhibited potent cytotoxicity in JHH4, greater than in HepG2 and JHH7 after a 48-hour treatment, suggesting that MYCN signaling may not be the primary target of PGV-1. Our results also confirm the persistent cytotoxicity of PGV-1 even after removal from the culture medium, aligning with previous reports.8, 15, 16This observation could indicate the ability of PGV-1 to overcome the recurrence phenomenon in HCC. Revealing the molecular mechanism of the permanent cytotoxic effect of PGV-1 would be essential for further development of chemotherapeutic agents for HCC. The typical cytotoxic effect of PGV-1 in HCC and other cancer types is associated with G2/M arrest, especially in prometaphase or metaphase.15, 16, 28 We consistently found that metaphase arrest in JHH4-treated PGV-1 led to mitotic catastrophe. Results from various cancer cells highlight the shared protein targets of PGV-1 in promoting metaphase arrest. Previous bioinformatic and molecular docking studies propose AURKA, AURKB, PLK1, CDK1, KIF11, and WEE1 as possible protein targets of PGV-1.9, 10, 12 Elucidating the specific role of metaphase arrest is a crucial requirement for uncovering the precise cytotoxicity of PGV-1.
Metaphase arrest in cancer could either lead to cell death mechanisms or facilitate the promotion of PGCCs for cancer survival. Constitutive endoreplication and endomitosis at metaphase arrest resulted in centrosomal dysregulation and genomic rearrangement, generating PGCCs.1, 35 We found that PGV-1 promotes PGCCs alongside metaphase arrest cells, indicating a connection between both processes. Upon committing to mitosis, it was crucial that subsequent events, such as chromosome segregation and cytokinesis, proceeded without errors. We consider CPC as another target of PGV-1. CPC emerged as a pivotal coordinator overseeing chromosome condensation, orchestrating spindle assembly, rectifying aberrant microtubule-kinetochore interactions, impacting chromosome alignment, signaling to the spindle checkpoint, and aiding in the completion of cytokinesis. Furthermore, disrupting the interaction within the CPC emerged as a promising strategy to impede HCC growth. A prior study demonstrated that the absence of either Survivin or Borealin resulted in HCC regression after hepatocyte mitotic arrest, hypertrophy, and genome polyploidy.36, 37 mirroring the effect of PGV-1 on JHH4.
By performing molecular docking and molecular dynamic simulation of PGV-1 on the complex of CPC, we found that PGV-1 interacted with the essential amino acid sequence that contributes to the interaction of INCENP, Borealin, and Survivin protein-protein interaction, resulting in destabilization of the complex formation. New approaches in anticancer strategies, aiming to restrict CPC function by degrading CPC members or destabilizing the CPC complex, show promise.38 Interestingly, a previous investigation emphasized the pivotal role of targeting INCENP or interrupting the interaction between INCENP and Aurora B kinase in efficiently restraining the growth of neuroblastoma cell lines in both MYCN-WT and MYCN-amp cells.39 Our findings align with this report, showcasing a MYCN-independent impact.
PGV-1 also induces senescence, particularly in PGCCs of JHH4. To control abnormal cell division, PGCCs enter senescence to reorganize cell division for the release of daughter cells that are more resistant to chemotherapeutic agents.6, 35 In contrast, PGV-1 triggers apoptosis in JHH4, as evidenced by the increase in cleaved PARP. These findings reveal the mechanism behind the lasting cytotoxic impact of PGV-1 in JHH4. This involves inducing mitotic arrest, forming PGCCs, increasing cellular senescence, and facilitating ultimately hindering the progression of HCC (Figure 6). Notably, no reported studies have demonstrated the promotion of PGCCs associated with irreversible cytotoxic effects.
Our findings highlight PGV-1 as a promising chemotherapeutic agent with multifaceted mechanisms that address key challenges in cancer treatment, including drug resistance, tumor recurrence, and metastasis. Unlike conventional therapies, PGV-1 demonstrates permanent cytotoxicity, even after drug withdrawal. This durable cytotoxic effect is important to improve survival rates among cancer patients, particularly in highly recurrent malignancies like HCC. As research advances toward clinical trials, PGV-1 holds promise not only for extending survival but also for transforming the therapeutic landscape of aggressive and recurrent cancers.
CONCLUSION
In conclusion, the low MYCN level in JHH4 cells established a crucial model for investigating the distinctive cytotoxic effects of PGV-1 in HCC, focusing on the observation of phenotypic changes. Our consistent findings highlighted the robust cytotoxic effect of PGV-1 in HCC cells, manifested by the induction of arrests across various mitotic phases. This suggested a potential interference with the CPC, resulting in multiple defects during mitotic arrest and the formation of PGCCs. The revelation that PGV-1 initiated senescence in PGCCs, ultimately leading to apoptotic cell death, is noteworthy. These novel insighted into the unique attributes of PGV-1 in JHH4 cells contribute to the understanding of potential therapeutic strategies, particularly in the pursuit of inducing permanent PGCCs death in HCC.
