Antiproliferative Activity of N-Acylhydrazone Derivative on Hepatocellular Carcinoma Cells Involves Transcriptional Regulation of Genes Required for G2/M Transition

Liver cancer is the second leading cause of cancer-related death in males. It is estimated that approximately one million deaths will occur by 2030 due to hepatic cancer. Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer subtype and is commonly diagnosed at an advanced stage. The drug arsenal used in systemic therapy for HCC is very limited. Multikinase inhibitors sorafenib (Nexavar®) and lenvatinib (Lenvima®) have been used as first-line drugs with modest therapeutic effects. In this scenario, it is imperative to search for new therapeutic strategies for HCC. Herein, the antiproliferative activity of N-acylhydrazone derivatives was evaluated on HCC cells (HepG2 and Hep3B), which were chemically planned on the ALL-993 scaffold, a potent inhibitor of vascular endothelial growth factor 2 (VEGFR2). The substances efficiently reduced the viability of HCC cells, and the LASSBio-2052 derivative was the most effective. Further, we demonstrated that LASSBio-2052 treatment induced FOXM1 downregulation, which compromises the transcriptional activation of genes required for G2/M transition, such as AURKA and AURKB, PLK1, and CDK1. In addition, LASSBio-2052 significantly reduced CCNB1 and CCND1 expression in HCC cells. Our findings indicate that LASSBio-2052 is a promising prototype for further in vivo studies.


Introduction
Liver cancer is the second and third leading cause of cancer-related deaths, respectively, in the male and female sexes [1].It is estimated that approximately one million deaths will occur by 2030 due to this type of cancer.Hepatocellular carcinoma (HCC) is the most common subtype of primary liver cancer (around 75-85% of all diagnosed cases) [2].
Hepatocarcinogenesis is an intricate process characterized by multiple stages marked by genetic and epigenetic alterations.Key events in the development of HCC include chronic inflammation, heightened cell proliferation, resistance to apoptosis, and the emergence of tumor stem cells [2].
The risk factors for HCC are well-established and include chronic viral infection (Hepatitis B or Hepatitis C), excessive alcohol consumption, and aflatoxin B1 uptake.Importantly, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) have become significant risk factors for HCC in recent years [3,4].
Commonly, HCC is diagnosed at an advanced stage, and the patients are treated with systemic therapy.The multikinase inhibitors sorafenib (Nexavar ® ) and lenvatinib (Lenvima ® ) have been used as first-line treatments for advanced HCC with modest therapeutic responses [2].
In addition to the gene pathways targeted by VEGFR2 inhibitors, advancements in cancer treatment could also involve pathways related to the cell cycle [7].These genes play a pivotal role in regulating cell growth and division, making them a significant focal point.The inhibition of key cell cycle genes, such as aurora kinases and cyclins, holds the potential to halt the uncontrolled proliferation of cancer cells [8][9][10].This approach is noteworthy for its potential to minimize the side effects commonly associated with traditional treatments that often impact healthy cells.By specifically targeting the highly active cell cycle genes prevalent in rapidly proliferating cancer cells, treatments can become more precise and effective [7,11].This strategy represents a promising avenue for combating cancer in a personalized and accurate manner.
Despite the introduction of new drugs for the treatment of HCC, the 5-year survival rate is still very low, corresponding to 10% for local tumors and 3% for metastatic tumors [12,13], which motivates the constant search for new agents capable of improving therapeutic proposals for HCC.
Therefore, it is crucial to search for new drug candidates for HCC.The purpose of this study was to evaluate the effects of N-acylhydrazone derivatives (LASSBio-2027, LASSBio-2029, and LASSBio-2052) on the proliferative behavior of hepatocarcinoma cells.These derivatives were chemically planned on the ALL-993 scaffold, which is a potent inhibitor of vascular endothelial growth factor 2 (VEGFR2) [14].For this purpose, we utilized HepG2 and Hep3B cells, which are widely employed in cancer research due to their distinct characteristics.HepG2 cells typically express TP53, whereas Hep3B cells lack both TP53 alleles.Additionally, HepG2 cells do not harbor the integrated hepatitis B virus (HBV) genome, unlike Hep3B cells, which contain integrated HBV genome segments.These genetic differences between the cell lines can significantly influence their responses to various treatments, providing valuable insights for the development of more effective therapies against hepatocellular carcinoma [15].
The results indicate that all target derivatives, especially LASSBio-2052, effectively reduce HepG2 and Hep3B cell proliferation by modulating key regulators of the G2/M transition.This involves decreased mRNA levels of AURKA, AURKB, PLK1, CDK1, and FOXM1.

Cell Lines and Culture Cell Conditions
Liver cancer cell lines (HepG2 and Hep3B) were used in the present study, which were purchased from the Rio de Janeiro Cell Bank.Primary fibroblasts (PFB), derived from human skin, were used as a non-transformed cell model.They were kindly provided by Prof. Dr. Angel Mauricio de Castro Gamero from the Institute of Nature Sciences at UNIFAL-MG (approved by the ethics committee-Process: 2.082.524).Cell cultures were maintained in DMEM/F12 (Dulbecco's Modified Eagle's Medium plus F12, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Cultilab, Campinas, SP, Brazil).Cells were grown in a humidified atmosphere of 95% air and 5% CO 2 at 37 • C, and subcultures were performed regularly.The stocks were maintained in liquid nitrogen.
For biological assays, the substances were solubilized in DMSO at 20 mM, and then diluted in culture medium immediately before treatment.The final concentration of DMSO in the culture medium was 0.1% (v/v) and did not affect cell viability under tested experimental conditions.

Cell Viability-Sulforhodamine B (SRB) Colorimetric Assay
Cell viability was determined by the SRB assay, which correlates cellular protein content with the viability rate [16].HepG2, Hep3B, and PFB were seeded into 96-well plates (1 × 10 4 cells/well).The cultures were treated for 48 h with the compounds LASSBio-2027, LASSBio-2029, and LASSBio-2052 at different concentrations (0-200 µM) to determine the IC 50 values.Cell monolayers were fixed with 10% (w/v) trichloroacetic acid at 4 • C for 1 h and stained with SRB (0.4% in 1% acetic acid) for 1 h.Next, the samples were washed repeatedly with 1% (v/v) acetic acid to remove unbound SRB.The protein-bound dye was dissolved in a 10 mM Tris-base solution (30 min) for optical density (OD) determination at 540 nm, using 690 nm as a reference value in a microplate reader.IC 50 values were calculated using GraphPad Prism ® 8.0 software (GraphPad Software, Inc., San Diego, CA, USA).
In the next step, we evaluated the proliferation kinetics of HCC cells.For this, the cells were treated with LASSBio-2052 at 10 or 20 µM (HepG2) and 20 or 40 µM (Hep3B), and viability was determined at 0, 24, and 48 h.The data are presented as mean ± standard deviation (SD) from at least three independent experiments.

Clonogenic Assay
To evaluate the long-term proliferation ability of HCC cells, a clonogenic assay was performed [17].The cells were seeded at low density into 35-mm plates (1000 cells/plate).HepG2 cells were treated with LASSBio-2052 at 10 or 20 µM for 48 h and recovered in a drug-free medium for 12 days.Hep3B cells were treated with LASSBio-2052 at 20 or 40 µM for 48 h and recovered in a drug-free medium for 12 days.After the recovery time, the colonies were fixed with methanol for 30 min and stained with crystal violet for 5 min.The quantification of colonies was performed using a stereomicroscope (20× magnification).Only colonies with >50 cells were considered for analysis.The data are presented as the mean ± SD of three independent experiments.

Cell Cycle Analysis
The cell cycle distribution pattern was determined by flow cytometry.Cells were seeded into 35-mm Petri plates at a density of 1 × 10 5 cells/plate.Cells were treated with LASSBio-2052 at 10 or 20 µM (HepG2) and 20 or 40 µM (Hep3B) for 48 h.Afterward, the cells were collected by enzymatic digestion (Trypsin-EDTA solution, Sigma-Aldrich, St. Louis, MO, USA) and fixed with 75% ethanol at 4 • C overnight.Subsequently, the DNA was stained with a propidium iodide (PI) solution (90 µg/mL) containing RNase (2.5 mg/mL) for 1 h at 4 • C. The analysis was performed using a flow cytometer (Guava easyCyte 8HT, Hayward, CA, USA).The data are presented as the mean ± SD of three independent experiments.

Gene Expression Profile Determined by qPCR
Expression of the target genes (CCNB1, CCND1, CDKN1A, CDK1, PLK1, FOXM1, AU-RKA, and AURKB) was evaluated by real-time PCR (RT-qPCR).The sequences of primers are shown in Table S1.Briefly, cells were seeded into 35-mm Petri plates at a density of 2 × 10 5 cells/plate.Cells were treated with LASSBio-2052 at 10 or 20 µM (HepG2) and 20 or 40 µM (Hep3B).After 24 h of treatment, the cells were collected by enzymatic digestion, and total RNA was extracted using the RNeasy ® Mini kit (Qiagen, Mississauga, ON, Canada).Total RNA concentrations were measured by a spectrophotometer using a NanoDrop ® ND 1000 (NanoDrop Technologies, Wilmington, DE, USA).Then, 1 µg of total RNA was incubated with DNase (1 U; ThermoFisher, Waltham, MA, USA) and subjected to reverse transcription (RT) using the High-Capacity cDNA Reverse Transcription Kit ® (Ther-moFisher).Relative quantification of mRNA was performed as previously described [18,19].The data are presented as the mean ± SEM of four independent experiments.The raw cycle threshold values of the samples are in Table S2.

Apoptosis Evaluation
Apoptosis was evaluated by phosphatidylserine externalization detection using the Annexin V-FITC/PI Kit (#V13242, Invitrogen, Waltham, MA, USA).Cells were seeded into 35-mm Petri plates at a density of 1 × 10 5 cells/plate.Cells were treated with LASSBio-2052 at 10 or 20 µM (HepG2) and 20 or 40 µM (Hep3B) for 48 h.The cells were collected by enzymatic digestion (Trypsin/EDTA, Sigma-Aldrich) and centrifuged at 200× g for 5 min at 4 • C. The samples were washed with ice-cold PBS, and 100 µL of a mixture solution containing buffered Annexin V-FITC and PI was added.After 20 min of incubation at room temperature in a dark chamber, the samples were analyzed by flow cytometry using GuavaSoft 2.7 software.The data are presented as the mean ± SD of three independent experiments.

Survival Analysis of HCC Patients
We plotted the overall survival probability of HCC patients using Kaplan-Meier plots by separating the samples into two groups (patients with high and low expression of each gene altered by LASSBio-2052).We used gene expression and survival data from The Cancer Genome Atlas (TCGA).Specifically, we generated plots for AURKA, AURKB, PLK1, CDK1, CCNB1, CCND1, FOXM1, and CDKN1A.We also generate Kaplan-Meier plots composed of the average expression of all the combined genes downregulated by LASSBio-2052 (AURKA, AURKB, PLK1, CDK1, CCNB1, CCND1, and FOXM1) which we named Signature.The mean expression of the Signature was calculated per TCGA sample.For the CDKN1A gene, in addition to evaluating it in the same way as the other genes in all patients, we also performed an analysis separating patients with wild-type TP53 and patients with mutated TP53, resulting in three analyses for this gene.The samples were split by median groups representing low and high expression, and log-rank p-values were displayed.The plots were generated with the 'survminer' version 0.4.9 [20] package in R Statistical Software v4.1.0[21].Molecular profiles and patient metadata were obtained using the package "cBioPortalData" v2.6.1 [22], using studyId = "lihc_tcga" and molecularProfileId = "lihc_tcga_rna_seq_v2_mrna_median_all_sample_Zscores".

Statistical Analysis
The results were tested for significance using one-way analysis of variance (ANOVA), followed by a Dunnett post-test using GraphPad Prism ® 8.0 software.p-values < 0.05 were considered statistically significant.

LASSBio-2052 Has Antiproliferative Activity on Hepatocarcinoma Cells
The chemical structures of the three N-acylhydrazone derivatives developed from the original ALL-993 molecule differ only concerning the substituents on the aryl group (Figure 1A).To test the effect of these different substituents on HCC cells, we evaluated the viability of HepG2 and Hep3B cells treated with different concentrations of the molecules.Following a 48 h treatment, there was a reduction in cell viability for all compounds tested; notably, LASSBio-2052 exhibited the highest efficacy (Figure 1B,D).The determined IC 50 values for LASSBio-2052 were approximately 20 and 40 µM on HepG2 and Hep3B cells, respectively (Figure 1D).Additionally, LASSBio-2052 underwent evaluation in human dermal primary fibroblasts (PFB) (Figure 1C).The IC 50 for LASSBio-2052 in PFB could not be ascertained with precision since the molecule was not cytotoxic enough to PFB (Figure 1D).Nonetheless, an estimation can be made from the graph, suggesting that the IC 50 of LASSBio-2052 in PFB was approximately 200 µM, greater than that observed in cancer cells.maintained in DMEM/F12 (Dulbecco's Modified Eagle's Medium plus F12, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Cultilab, Campinas, SP, Brazil).Cells were grown in a humidified atmosphere of 95% air and 5% CO2 at 37 °C, and subcultures were performed regularly.The stocks were maintained in liquid nitrogen.

Synthesis of the N-Acylhydrazone Derivatives
The substances LASSBio-2027, LASSBio-2029, and LASSBio-2052 (Figure 1A) were synthesized and characterized as previously described by Pauli et al. (2020) [14].We chose concentrations relative to the IC 50 and IC 50 /2 of each cell type to evaluate the growth kinetics of cells at 0, 24, and 48 h after treatment (Figure 2A-D).IC 50 concentrations (20 µM for HepG2; 40 µM for Hep3B) prevented growth and even reduced the number of cells in both cell types after 48 h (Figure 2A,B).Concentrations of IC 50 /2 (10 µM for HepG2; 20 µM for Hep3B) also prevented cell growth, but to a lesser extent (Figure 2A,B).
We chose concentrations relative to the IC50 and IC50/2 of each cell type to evaluate the growth kinetics of cells at 0, 24, and 48 h after treatment (Figure 2A-D).IC50 concentrations (20 µM for HepG2; 40 µM for Hep3B) prevented growth and even reduced the number of cells in both cell types after 48 h (Figure 2A,B).Concentrations of IC50/2 (10 µM for HepG2; 20 µM for Hep3B) also prevented cell growth, but to a lesser extent (Figure 2A,B).Photomicrographs of the cells at the same treatment time confirmed these results, showing that there was a lower confluence of cells after 48 h (Figure 2C,D).Furthermore, signs of cytotoxicity were evident in cultures treated with IC 50 concentrations (20 µM for HepG2; 40 µM for Hep3B) (Figure 2C,D).Specifically, after 48 h of treatment, HepG2 cultures displayed cellular debris (Figure 2C), while Hep3B cultures exhibited rounded cells partially adhered to the substrate (Figure 2D).
The clonogenic assay confirmed these results, showing that LASSBio-2052 was able to inhibit cell growth in the long term, evaluated after 12 days in cells previously treated for 48 h (Figure 2E-H).

LASSBio-2052 Inhibits Cell Cycle Progression in Hepatocarcinoma Cells
We evaluated cell cycle progression to better understand the mechanism associated with the antiproliferative activity of LASSBio-2052 on HepG2 and Hep3B cells (Figure 3A-D).The molecule inhibited cell cycle progression once there was an increase in G2/M population in both HepG2 (≈60%) (Figure 3A,C) and Hep3B (≈40%) cells (Figure 3B,D), treated with LASSbio-2052 for 48 h compared with controls (≈25% for both HepG2 and Hep3B).Additionally, we observed an increased sub-G1 population in response to treatment in both cells, which is indicative of cell death.
Photomicrographs of the cells at the same treatment time confirmed these results, showing that there was a lower confluence of cells after 48 h (Figure 2C,D).Furthermore, signs of cytotoxicity were evident in cultures treated with IC50 concentrations (20 µM for HepG2; 40 µM for Hep3B) (Figure 2C,D).Specifically, after 48 h of treatment, HepG2 cultures displayed cellular debris (Figure 2C), while Hep3B cultures exhibited rounded cells partially adhered to the substrate (Figure 2D).
The clonogenic assay confirmed these results, showing that LASSBio-2052 was able to inhibit cell growth in the long term, evaluated after 12 days in cells previously treated for 48 h (Figure 2E-H).

LASSBio-2052 Inhibits Cell Cycle Progression in Hepatocarcinoma Cells
We evaluated cell cycle progression to better understand the mechanism associated with the antiproliferative activity of LASSBio-2052 on HepG2 and Hep3B cells (Figure 3A-D).The molecule inhibited cell cycle progression once there was an increase in G2/M population in both HepG2 (≈60%) (Figure 3A,C) and Hep3B (≈40%) cells (Figure 3B,D), treated with LASSbio-2052 for 48 h compared with controls (≈25% for both HepG2 and Hep3B).Additionally, we observed an increased sub-G1 population in response to treatment in both cells, which is indicative of cell death.

LASSBio-2052 Modulates Expression Profiles of Regulators of Cell Cycle
Based on cell cycle analysis, we evaluated the expression of genes associated with the regulation of the G2/M transition.Thus, we determined the expression of genes encoding mitotic kinases CDK1, AURKA (Aurora A), AURKB (Aurora B), and PLK1, cyclin-dependent kinase inhibitor CDKN1A (p21), and CCNB1 (cyclin B).In both HepG2 and Hep3B, LASSBio-2052 at 20 µM was able to reduce the mRNA abundance of all positive

LASSBio-2052 Modulates Expression Profiles of Regulators of Cell Cycle
Based on cell cycle analysis, we evaluated the expression of genes associated with the regulation of the G2/M transition.Thus, we determined the expression of genes encoding mitotic kinases CDK1, AURKA (Aurora A), AURKB (Aurora B), and PLK1, cyclindependent kinase inhibitor CDKN1A (p21), and CCNB1 (cyclin B).In both HepG2 and Hep3B, LASSBio-2052 at 20 µM was able to reduce the mRNA abundance of all positive regulators of the cell cycle, such as AURKA, AURKB, PLK1, CDK1, and CCNB1 (Figure 4A,B).On the contrary, the expression level of CDKN1A was significantly increased in both treated cell types (Figure 4A,B).For HepG2, we also evaluated the expression profile of CCND1 (cyclin D1), which was also reduced in cells treated with 20 µM.Intriguingly, the lower concentration increased the expression of the CCNB1 gene in HepG2 cells and the PLK1 gene in Hep3B cells, while the higher concentration decreased the expression of these genes in the same cell type.
the PLK1 gene in Hep3B cells, while the higher concentration decreased the expression of these genes in the same cell type.
We also investigated the expression of FOXM1, a transcriptional activator involved in the M phase that regulates the expression of several cell cycle genes, such as CCNB1 and CCND1 [23].We found that LASSBio-2052 reduced the expression of FOXM1 in a dose-dependent manner in both HepG2 (Figure 4A) and Hep3B cells (Figure 4B).We also investigated the expression of FOXM1, a transcriptional activator involved in the M phase that regulates the expression of several cell cycle genes, such as CCNB1 and CCND1 [23].We found that LASSBio-2052 reduced the expression of FOXM1 in a dose-dependent manner in both HepG2 (Figure 4A) and Hep3B cells (Figure 4B).

Cytotoxic Activity of LASSBio-2052 on HCC Cells Involves Apoptosis Induction
Since cell cycle analysis indicated an increase in sub-G1 population, an indication of cell death in response to treatment (Figure 3), we investigated the pro-apoptotic potential of LASSBio-2052 on HepG2 and Hep3B cells using an annexin V assay (Figure 5A-D).Treating both cell types with the higher concentrations (IC 50 ) increased the annexin-positive cells (Figure 5A-D), suggesting that LASSBio-2052 can induce apoptotic cell death in HCC, especially in Hep3B cells.(Figure 5C,D).

Cytotoxic Activity of LASSBio-2052 on HCC Cells Involves Apoptosis Induction
Since cell cycle analysis indicated an increase in sub-G1 population, an indication of cell death in response to treatment (Figure 3), we investigated the pro-apoptotic potential of LASSBio-2052 on HepG2 and Hep3B cells using an annexin V assay (Figure 5A-D).Treating both cell types with the higher concentrations (IC50) increased the annexin-positive cells (Figure 5A-D

Gene Expression Changes Induced by LASSBio-2052 Are Associated with the Better Overall Survival of HCC Patients
LASSBio-2052 was able to reduce the expression of genes related to the cell cycle.Therefore, we investigated whether the reduction in the expression of these genes has clinical relevance.For this, we utilized gene expression data from TCGA from patients with HCC (Figure 6).The reduction in the expression of the AURKA (Figure 6A), AURKB (Figure 6B), PLK1 (Figure 6C), CDK1 (Figure 6D), CCNB1 (Figure 6E), and FOXM1 (Figure 6G) genes, all downregulated by LASSBio-2052, is associated with better overall patient survival.CCND1 expression was not associated with overall survival (Figure 6F, p > 0.05).When we combined all the genes, creating an average of the expression of all of them (Signature), the association was also significant.This suggests that, in addition to the expression of individual genes, the set of genes that was downregulated by LASSBio-2052 is associated with better overall patient survival.
We also tested the expression of the CDKN1A tumor suppressor, the only one that was upregulated by LASSBio-2052.In this case, there was no statistically significant

Gene Expression Changes Induced by LASSBio-2052 Are Associated with the Better Overall Survival of HCC Patients
LASSBio-2052 was able to reduce the expression of genes related to the cell cycle.Therefore, we investigated whether the reduction in the expression of these genes has clinical relevance.For this, we utilized gene expression data from TCGA from patients with HCC (Figure 6).The reduction in the expression of the AURKA (Figure 6A), AU-RKB (Figure 6B), PLK1 (Figure 6C), CDK1 (Figure 6D), CCNB1 (Figure 6E), and FOXM1 (Figure 6G) genes, all downregulated by LASSBio-2052, is associated with better overall patient survival.CCND1 expression was not associated with overall survival (Figure 6F, p > 0.05).When we combined all the genes, creating an average of the expression of all of them (Signature), the association was also significant.This suggests that, in addition to the expression of individual genes, the set of genes that was downregulated by LASSBio-2052 is associated with better overall patient survival.
We also tested the expression of the CDKN1A tumor suppressor, the only one that was upregulated by LASSBio-2052.In this case, there was no statistically significant difference between gene expression and patient overall survival (Figure 6I).We then separated the patients according to mutations in the TP53 gene.Patients with no mutation in TP53 also did not show changes in overall survival according to CDKN1A expression (Figure 6J).However, patients with TP53 mutations showed better overall survival when CDKN1A was upregulated (Figure 6K).This suggests that treatment with LASSBio-2052 would be an interesting therapeutic approach, particularly for patients with TP53 mutations, present in 30% of the patients analyzed.
difference between gene expression and patient overall survival (Figure 6I).We then separated the patients according to mutations in the TP53 gene.Patients with no mutation in TP53 also did not show changes in overall survival according to CDKN1A expression (Figure 6J).However, patients with TP53 mutations showed better overall survival when CDKN1A was upregulated (Figure 6K).This suggests that treatment with LASSBio-2052 would be an interesting therapeutic approach, particularly for patients with TP53 mutations, present in 30% of the patients analyzed.

Discussion
In the present study, we evaluated the antiproliferative activity of N-acylhydrazone derivatives, namely LASSBio-2027, LASSBio-2029, and LASSBio-2052.These compounds differ due to the presence of distinct groups at the aryl group.LASSBio-2027 has a phenyl group, LASSBio-2029 has a 4-chlorophenyl group, and the LASSBio-2052 has the strongly electron withdrawing group trifluoromethyl (CF3) in the para-position of the phenyl group.Based on IC 50 values, LASSBio-2052 emerged as the most potent prototype when tested against hepatocellular carcinoma cells (HepG2 and Hep3B), in contrast to its previously reported effects on estrogen-positive breast cancer cells, where LASSBio-2052 was not so cytotoxic to MCF-7 cells [18].This discovery suggests that the presence of CF3 in LASSBio-2052 holds significance for its cytotoxic activity and selectivity, specifically towards hepatocellular carcinoma cells.Interestingly, fluorine-containing compounds represent more than 50 percent of the best-selling drug molecules approved by the US Food and Drug Administration (FDA) [24].
LASSBio-2052 effectively inhibited the proliferation of HepG2 and Hep3B cells in both short-and long-term scenarios, even when concentrations as low as the IC 50 were employed.These findings are promising, given that both cell lines exhibit genetic alterations commonly observed in HCC, such as mutations in the CTNNB1 gene and TERT promoter [2,25].Additionally, the Hep3B cell line harbors parts of the hepatitis B virus (HBV) genome integrated [26] and does not express TP53 due to deletion in both alleles [15].Thus, the Hep3B cell line serves as a representative model for aggressive tumors, which are often resistant to treatment and associated with a poor prognosis [26][27][28].Importantly, HBV integration is estimated to occur in 85-90% of HCCs associated with HBV, contributing to increased genetic damage and chromosomal instability in infected neoplastic cells [29].
Cell cycle analysis revealed an increase in G2/M and sub-G1 populations in both HCC cell lines following treatment with LASSBio-2052.These findings suggest that this compound is able to modulate cell cycle regulators and induce cell death.Moreover, we demonstrated that the elevated sub-G1 population was a result of apoptosis induction.Additionally, we assessed the gene expression profiles of positive and negative regulators of the cell cycle [11], considering the observed increase in the G2/M population induced by LASSBio-2052.The activation of mitotic kinases AURKA/B, CDK1, and PLK1 is crucial for G2/M transition and mitosis [30,31].Consistent with previous data, RT-qPCR analysis revealed that LASSBio-2052 led to the downregulation of genes encoding mitotic kinases (AURKA, AURKB, PLK1, and CDK1) in HepG2 and Hep3B cells.Furthermore, the relative expression of mRNA for CCNB1 (cyclin B1) in both HepG2 and Hep3B and CCND1 (cyclin D1) in HepG2 was significantly lower in LASSBio-2052-treated samples compared to control groups.
Overexpression of AURKA/B, CDK1, and PLK1 has been reported in several malignant tumors, including HCC [30,32,33].A recent study has demonstrated that the overexpression of AURKA, CDK1, and PLK1 is positively correlated with tumor grades and stage.Thereby, these genes may be used as prognostic biomarkers for HBV-induced HCC.Furthermore, there is a strong correlation between high expression of AURKA, CDK1, and PLK1 and the infiltration of immune cells [33].The coordinated overexpression of FOXM1 and AURKA has been linked to the worst overall survival in sorafenib-treated patients with HCC [34].
While positive regulators of the cell cycle were downregulated by LASSBio-2052 treatment, the negative regulator of the cell cycle, CDKN1A (encoding p21), was significantly upregulated.In Hep3B, the relative expression of this gene was approximately 8-fold higher in samples treated with LASSBio-2052 at 20 µM, while in HepG2, the relative expression of CDKN1A was around 2-fold.These results are interesting because the transcriptional activation of CDKN1A typically occurs preferentially by TP53, and as mentioned before, Hep3B cells are TP53 deficient.Thus, the pronounced upregulation of CDKN1A in response to LASSBio-2052 treatment in Hep3B cells likely occurred through TP53-independent pathways.It has been reported that the viral oncogenic protein HBx (expressed by Hep3B) inhibits the expression of CDKN1A at the transcriptional level by negatively regulating the SP1 transcriptional factor [35].Hence, it is plausible that LASSBio-2052 might have modulated the HBx/SP1 pathway.Alternatively, the elevated expression of CDKN1A in Hep3B might be a consequence of epigenetic regulation.It has been reported that CDKN1A gene transcription can be repressed by epigenetic mechanisms.Lou et al. (2019) [36] demonstrated that antisense lncRNAs, such as GATA3-AS1, promote cell proliferation and metastasis in HCC by suppressing proto-oncogenes, including CDKN1A.In gastric cancer, it was shown that CDKN1A was epigenetically silenced by the HOXA-AS2-EZH2 complex [37].
We also assessed the expression profile of FOXM1, a proliferation-associated transcription factor belonging to the forkhead box superfamily of proteins [38].It has been reported that FOXM1 is overexpressed in HCC and sorafenib-resistant HCC samples.The overexpression of FOXM1 has been positively associated with a poor prognosis in HCC patients [39,40].The ectopic expression of FOXM1 in HepG2 cells induced proliferation, while the opposite was observed when FOXM1 was silenced in Hep3B [39].FOXM1 knockdown also suppressed cell proliferation and induced G2/M cell cycle arrest in Huh7 cells [40].The molecular mechanism by which FOXM1 is required for G2/M transition and mitosis onset involves the transcription activation of several genes, including mitotic kinases and cyclin B [41,42].In the present study, we demonstrated that FOXM1 and its downstream genes were downregulated by LASSBio-2052 in both cell lines, reinforcing its antitumor potential in HCC.Our findings suggest that LASSBio-2052 might be useful in treating tumors refractory to sorafenib.

Conclusions
In conclusion, our study focused on three N-acylhydrazone derivatives, with LASSBio-2052 demonstrating the highest efficacy, reducing cell viability in both HepG2 and Hep3B HCC cells.Importantly, LASSBio-2052 exhibited minimal cytotoxicity in human dermal primary fibroblasts.LASSBio-2052 inhibited the growth kinetics of both cell types, as confirmed by short-and long-term assays demonstrating its inhibitory effect on HCC cell growth.Cell cycle analysis indicated G2/M arrest and an increased sub-G1 population, indicative of cell death, which was confirmed by increased apoptosis.Molecular analysis showed that LASSBio-2052 downregulated key cell cycle regulators such as AURKA, AU-RKB, PLK1, CDK1, CCNB1, and FOXM1 and upregulated CDKN1A.These changes in gene expression correlated with improved overall survival in patients with HCC.Additionally, upregulation of CDKN1A was positively associated with overall survival in patients with TP53 mutations.In summary, our findings demonstrate that LASSBio-2052 has promising antiproliferative and pro-apoptotic effects in HCC cells.Further in vivo investigations are warranted to validate these findings and explore LASSBio-2052's translational implications in the treatment of HCC.

Figure 1 .
Figure 1.(A) Chemical structure of AAL-993 and N-acylhydrazone derivatives (NAH).(B,C) Doseresponse curves.HepG2, Hep3B, and primary fibroblasts were treated for 48 h with NAH derivatives.(D) IC50 values were determined after 48 h treatment.* The IC50 for LASSBio-2052 in human dermal primary fibroblasts (PFB) was estimated visually from the graph once its cytotoxic effect on normal cells was not sufficient to determine the exact IC50 value.

Figure 1 .
Figure 1.(A) Chemical structure of AAL-993 and N-acylhydrazone derivatives (NAH).(B,C) Doseresponse curves.HepG2, Hep3B, and primary fibroblasts were treated for 48 h with NAH derivatives.(D) IC 50 values were determined after 48 h treatment.* The IC 50 for LASSBio-2052 in human dermal primary fibroblasts (PFB) was estimated visually from the graph once its cytotoxic effect on normal cells was not sufficient to determine the exact IC 50 value.

Figure 2 .
Figure 2. (A,B) Cell viability rate was determined in HepG2 (C) and Hep3B (D) cultures at 0, 24, and 48 h after treatment.(C,D) Representative images obtained by phase microscopy showing morphological aspects of cells.(E,F) Representative images from the clonogenic assay.The cells were treated for 48 h and recovered in fresh medium for 12 days.(G,H) Quantification of the number of colonies relative to control DMSO.*** p < 0.001 according to ANOVA followed by a Dunnett post-test.

Figure 5 .
Figure 5. (A,C) Representative dot plots from the annexin assay.Cells were treated for 48 h with LASSBio-2052.(B,D) Determination of apoptotic cells considering the cell population positive for the Annexin V assay.* p < 0.05, ** p < 0.01, *** p < 0.001 according to ANOVA followed by a Dunnett post-test.

Figure 5 .
Figure 5. (A,C) Representative dot plots from the annexin assay.Cells were treated for 48 h with LASSBio-2052.(B,D) Determination of apoptotic cells considering the cell population positive for the Annexin V assay.* p < 0.05, ** p < 0.01, *** p < 0.001 according to ANOVA followed by a Dunnett post-test.

Figure 6 .
Figure 6.Reduced expression of genes downregulated by LASSBio-2052 is associated with improved overall survival in patients with hepatocellular carcinoma.Overall survival probability analysis using samples from Liver Hepatocellular Carcinoma (TCGA, Firehose Legacy, study ID: "lihc_tcga").(A-G) Single gene analysis.(H) Gene signature made with the average of all genes

Figure 6 .
Figure 6.Reduced expression of genes downregulated by LASSBio-2052 is associated with improved overall survival in patients with hepatocellular carcinoma.Overall survival probability analysis using samples from Liver Hepatocellular Carcinoma (TCGA, Firehose Legacy, study ID: "lihc_tcga").(A-G) Single gene analysis.(H) Gene signature made with the average of all genes downregulated by LASSBio-2052.(I-K) CDKN1A analysis using the whole dataset (I), patients with TP53 not mutated (J), and patients with TP53 mutation (K).Log-rank p-values are presented.