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Research Article
2025
:22;
88
doi:
10.25259/Cytojournal_273_2024

AZ-628 sensitizes donafenib in hepatocellular carcinoma by targeting tyrosine kinase pathway and ferroptosis

, , , , , ,
1Department of General Surgery, Division of Hepatobiliary and Pancreatic Surgery, The Second Hospital of Dalian Medical University, Dalian, China
2Department of Gastroenterology, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, China
3Department of Hematology, The First Affiliated Hospital of China Medical University, Shenyang, China.
Author image

*Corresponding author: Zhenming Gao, Department of General Surgery, Division of Hepatobiliary and Pancreatic Surgery, The Second Hospital of Dalian Medical University, Dalian, China. gaozhenmingdl@163.com

Received: , Accepted: ,
© 2025 The Author(s). Published by Scientific Scholar
Licence
This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

How to cite this article: Yu T, Zhao X, Dong C, Lu H, Wang Y, Luo X, et al. AZ-628 sensitizes donafenib in hepatocellular carcinoma by targeting tyrosine kinase pathway and ferroptosis. CytoJournal. 2025;22:88. doi: 10.25259/Cytojournal_273_2024

Abstract

Objective:

Hepatocellular carcinoma (HCC) represents a primary liver tumor characterized by rapid disease progression and unfavorable clinical outcomes. Most patients with HCC are identified in advanced stage, where targeted therapies are considered an effective treatment method for advanced disease. The tyrosine kinase inhibitor (TKI) donafenib has shown efficacy in managing HCC. However, drug resistance often occurs after treatment with donafenib, which limits its widespread clinical application. Thus, this study aims to identify small-molecule TKIs that can enhance the sensitivity of HCC to donafenib.

Material and Methods:

The HCC cells HepG2 and SNU449 were treated with five drugs, namely, dimethyl sulfoxide, AZ-628, SU-5402, TG-101209, and SPP-86, combined with donafenib to determine half-maximal inhibitory concentration values. RNA sequencing data obtained from The Cancer Genome Atlas (TCGA) were analyzed using Differential Expression analysis for Sequence data 2 (DESeq2)/limma and Gene Set Enrichment Analysis (GSEA). The effects of AZ-628 on proliferation, viability, apoptosis, and migration were assessed. The expression level of early growth response gene 1 (EGR1) was measured through Western blotting/quantitative polymerase chain reaction and silenced by cell transfection. Donafenib-resistant HepG2 cells negative control shRNA (shNC)/shRNA targeting EGR1 (shEGR1) were treated with AZ-628 combined with donafenib. Ferrous ion (Fe2+) and reactive oxygen species levels were measured after Erastin/RSL3 induction. The synergy between AZ-628 and donafenib was analyzed using Combenefit2. In vivo, tumor growth, and Ki67 expression were evaluated in nude mice treated with DMSO, AZ-628, donafenib, or their combination.

Results:

This study showed that AZ-628 reduced donafenib resistance in HCC by targeting the tyrosine kinase (TK) pathway. Cell counting kit-8 and colony formation assay validated that AZ-628 significantly improved the sensitivity of HCC cells to donafenib (P < 0.0001). Rescue experiments showed that AZ-628 regulated HCC cell proliferation and drug resistance through EGR1 (P < 0.001). In addition, AZ-628 was found to affect donafenib resistance in HCC by regulating epithelial–mesenchymal transition, apoptosis, and ferroptosis (P < 0.0001). In vivo experiments demonstrated a combined anti-tumor efficacy of AZ-628 and donafenib in HCC models (P < 0.0001).

Conclusion:

The findings of this study reveal a new combination therapy targeting the TK pathway for the treatment of HCC and provide a theoretical foundation for addressing donafenib resistance.

Keywords

AZ-628
Donafenib
Ferroptosis
Hepatocellular carcinoma
Tyrosine kinase

INTRODUCTION

Hepatocellular carcinoma (HCC) is characterized by high malignancy and poor prognosis, and it is challenging to treat.[1] Recent advancements in treatment strategies have improved the outcomes of HCC.[2] However, patients with HCC are frequently diagnosed during advanced disease stages, and treatment options for advanced disease are particularly complex.[3] Surgical resection and liver transplantation have therapeutic potential, but are limited to select patients due to limitations in tumor extent and eligibility criteria.[4] Targeted therapies show great application potential in the treatment of advanced HCC. For example, drugs such as sorafenib, lenvatinib, and regorafenib selectively inhibit specific molecules that drive tumor growth.[5] These therapies block pathways necessary for cancer cell proliferation and angiogenesis.[6] Although targeted therapies can prolong survival and improve outcomes for some patients, their effectiveness varies, and resistance can develop over time.[7] Therefore, developing novel strategies to enhance the sensitivity of HCC to targeted therapies is necessary.

The tyrosine kinase (TK) pathway plays an important role in regulating cellular growth, proliferation, and differentiation by transferring phosphate groups to tyrosine residues on proteins, thereby initiating intracellular signaling cascades.[8] Early growth response gene 1 (EGR1), a transcription factor belonging to the E26 transformation-specific (ETS) family, is a nucleic acid–binding protein that regulates gene expression and various biological processes, including cell proliferation, apoptosis, and development.[9] In addition, this mechanism plays a role in modulating viral thymidine kinase messenger RNA (mRNA) expression levels through the MEK/ERK/ERG-1 signaling cascade.[10] The EGR1 gene is aberrantly activated in tumors, including HCC. It facilitates neoplastic cell proliferation, survival, and angiogenic processes, which contribute to tumor progression.[11,12] TK pathways, including those involving EGR1, are frequently dysregulated in HCC and, hence, serve as potential therapeutic targets.[13] Inhibitors targeting TK pathways, such as sorafenib and lenvatinib, interfere tumor growth and angiogenesis in HCC.[14] Tyrosine kinase inhibitors (TKIs) are targeted drugs that exert potent anti-tumor effects by inhibiting the activity of TKs, which are crucial enzymes involved in signaling pathways promoting cancer growth.[15] In liver cancer, particularly HCC, TKIs such as sorafenib and lenvatinib are used to selectively inhibit certain receptors, including vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor.[16] TKIs disrupt tumor angiogenesis and cellular proliferation by inhibiting these receptors, effectively delaying tumor growth and progression.[17] Although TKIs have shown application potential in extending lifespan and enhancing therapeutic results for advanced HCC, their efficacy may vary, and resistance can develop after long-term use.[18] However, TKIs remain valuable components of HCC treatment regimens. Donafenib, a deuterated form of sorafenib, has high bioavailability,[19] and it targets multiple TKs and receptors, including v-raf murine sarcoma viral oncogene homolog (BRAF) and VEGFR.[20] It was developed primarily for the treatment of HCC, which is a common type of primary liver cancer.[21] The therapeutic potential of donafenib in HCC and possibly other cancers is associated with its ability to inhibit tumor cell proliferation and angiogenesis, both of which are critical processes in tumor growth and metastasis.[22] However, the application of donafenib is limited due to its potential adverse effects, drug resistance developing over time, pharmacokinetic challenges in patients with liver dysfunction, and potential drug interactions. Combination therapy, in which two or more therapeutic agents are used concurrently, has been used to address the limitations of targeted therapy. It may improve therapeutic efficacy, overcome resistance, and minimize the risk of side effects in some cases.[23] In HCC, the combined use of two or more TKIs may improve treatment outcomes by targeting multiple tumor-related pathways simultaneously.[24] For example, the combination of sorafenib and regorafenib, which are multi-kinase inhibitors, has been used to treat HCC. These two TKIs share some targets, but they also have distinct targets. Therefore, their combined use may enable the broad inhibition of kinases, enhance therapeutic efficacy, and delay resistance.[25] Combination therapy has demonstrated promising therapeutic potential in clinical trials, with evidence of improved response rates and survival outcomes.[26] However, to date, studies on combination therapy have focused more on in vitro models.

AZ-628 is a highly effective inhibitor of receptor-interacting protein kinase 3 (RIP3).[27] Its inhibitory effects on RIP3 have been investigated in various diseases, including cancer.[28] Furthermore, preclinical studies have demonstrated that AZ-628 enhances the susceptibility of malignant cells to undergo necroptosis.[29] However, whether AZ-628 is a specific TKI, remains unclear. Based on previous reports, AZ-628 inhibits several TKs, making it a promising candidate for molecularly targeted cancer therapy. A study investigating checkpoint inhibitors reported that AZ-628 plays a role in predicting the outcome of immunotherapy in patients with HCC.[30] However, to date, no studies have demonstrated the specific effects and mechanisms of action of AZ-628 as a TKI in HCC.

Herein, we found that AZ-628 effectively attenuated donafenib resistance in HCC by modulating the TK pathway. In addition, AZ-628 influenced donafenib resistance by interfering with epithelial–mesenchymal transition (EMT), apoptosis, and ferroptosis. Multi-angle results demonstrated that AZ-628 and donafenib had synergistic anti-tumor effects against HCC. These findings highlight the potential application of AZ-628 as an adjuvant drug for enhancing the therapeutic effectiveness of donafenib in HCC.

MATERIAL AND METHODS

Cell culture

The human HCC cell lines Hep2G (SCSP-510), Huh7 (SCSP-526), and Hep3B (SCSP-5045), as well as the human embryonic kidney cell line HEK-293T (SCSP-502), were purchased from the Institute of Life Sciences, Chinese Academy of Sciences (Shanghai, China) and cultured in a Dulbecco’s modified eagle medium (11966025)/Roswell Park Memorial Institute 1640 (RPMI1640; 12633020) medium (Gibco, Massachusetts, USA) supplemented with 10% fetal bovine serum (10099158, Gibco, Massachusetts, USA). The HCC cell line SNU449 (CRL-2234) was purchased from the USA Type Culture Collection (ATCC, Virginia, USA) and cultured in an RPMI1640 (12633020, Gibco, Massachusetts, USA) medium supplemented with 10% fetal bovine serum (10099158, Gibco, Massachusetts, USA). The files of mycoplasma detection and short tandem repeat authentication have been uploaded in the supplementary materials.

Supplementary File

Cell transfection

A target-specific short hairpin RNA (shRNA) for EGR1 was synthesized and purchased from Ribobio (Guangzhou, China). The sequence of shEGR1 was as follows: 5'-GACTCTTGGGAGGGAGTTA-3'. The shRNA plasmids were constructed on the basis of PLKO.1 (#10878, Addgene, Massachusetts, USA), PMD2.G(#12259, Addgene, Massachusetts, USA) and PsPAX2(#12260, Addgene, Massachusetts, USA). This mature shRNA was introduced into HEK293T cells through Lipofectamine 2000 (11668500, Invitrogen, California, USA) to establish a lentivirus-packed cell line. After 48 h, the cell culture supernatant containing packaged lentiviral particles was harvested, and the particles were introduced into SNU449 and HepG2 cells for infection. After 2 days of incubation, the culture medium was replaced with a fresh medium supplemented with corresponding puromycin (A1113803, Thermo, MA, USA) to facilitate the selection and screening of stably transfected cells.

Protein extraction and Western blotting

After adherent cells were washed with pre-chilled phosphate-buffered saline (PBS; 10010072, Gibco, Massachusetts, USA), they were treated with an appropriate volume of Radio Immunoprecipitation Assay lysis buffer (AR0102S, Boster, Wuhan, China) for subsequent protein extraction. The extracted proteins were quantified, separated through electrophoresis, and transferred to polyvinylidene fluoride membranes (ISEQ00010, Solarbio, Beijing, China). The membranes were blocked using non-fat milk, followed by incubation with target-specific primary and matched secondary antibodies. Finally, protein bands were detected and visualized through enhanced chemiluminescence (A38556, Thermo, MA, USA) in the ChemiDoc XRS+ Imaging System (Bio-Rad, California, USA). The following primary antibodies were purchased: anti-EGR1 (1:1000, #97249, Cell Signaling, MA, USA) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:1000, #2118, Cell Signaling, MA, USA). The following secondary antibodies were purchased: anti-rabbit IgG (1:3000, #7074, Cell Signaling, MA, USA).

RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA was extracted from tissues and cells using an RNA extraction kit (80504, QIAGEN, Dusseldorf, Germany) in accordance with the manufacturer’s protocol. For cDNA synthesis, reverse transcription of isolated RNA was performed using the HiScript reverse transcription kit (R312-01/02, Vazyme Biotech, Nanjing, China). qRTPCR was performed using Bio-Rad CFX96 real-time PCR operator (BIO-RAD, California, USA) and SYBR qPCR Mix (CN830A, Takara, Dalian, China). GAPDH served as the reference gene for normalizing target gene expression. The changes in mRNA were determined using the 2−ΔΔCt method. The primers used for PCR analysis are as follows: EGR1-F, 5'-AGCCCTACGAGCACCTGAC-3'; EGR1-R, 5'-GGTTTGGCTGGGGTAACTG-3'; GAPDH-F, 5'GATTCCACCCATGGCAAATTC-3'; GAPDH-R, 5'-CTGGAAGATGGTGATGGGATT-3'.

Cell counting kit-8 (CCK8) assay

HCC cells were inoculated with a 96-well plate with 3 × 103 cells/well. After the cells adhered to the well surface, they were treated with drugs. Following CCK8 reagent (A311-01, Vazyme, Nanjing) incubation for 2 h, cell viability was determined by measuring absorbance at 450 nm through a microplate reader (Envision, Perkin Elmer, Massachusetts, USA).

Colony formation assay

Cells were plated at 1000 cells/well in six-well plates. After the cells adhered to the well surface, they were exposed to varying concentrations. After treatment, the cellular samples were immobilized using 4% paraformaldehyde (P1110, Solarbio, Beijing, China) and subsequently subjected to staining with a crystal violet solution (G1062, Solarbio, Beijing, China) at optimal concentrations. Subsequently, cell colonies were counted manually under a microscope (BX53, Olympus, Tokyo, Japan).

Transwell migration assay

The migratory potential of cells was evaluated through Transwell chamber experiments to determine the impact of AZ-628 (HY-11004, MCE, New Jersey, USA) on cell motility. Cells were cultured and treated with AZ-628 at different concentrations. The drug-treated cells (5 × 104) were added to the upper chamber, whereas a culture medium supplemented with 10% FBS was added to the lower chamber (3491, Corning, New York, USA). After 48 h of incubation, the cells were fixed, stained, observed, and analyzed. Three random fields were selected for photography and cell counting through a microscope (BX53, Olympus, Tokyo, Japan).

Apoptosis assay

The cells in the ocular growth phase were washed and centrifuged to obtain a single-cell suspension (12,000 g, 10 min). A total of 5 μL of Annexin V (R37176, Invitrogen, CA, USA) and 10 μL of PI (100 μg/mL; ST511, Beyotime, Shanghai) were added to the cell suspension. The mixture was carefully agitated and maintained in the dark at refrigerated temperatures (2–8°C) for 5 min. Subsequently, the cells were analyzed using flow cytometry (C6, BD Biosciences, New Jersey, USA).

Iron assay

The Iron Assay Kit (ab83366, Abcam, Cambridge, UK) was used to detect the concentration of Fe2+. First, 5 × 106 cells were seeded in a 10 cm × 10 cm dish, after 12 h of drug treatment, washed with PBS, and lysed cells using a homogenizer in iron assay buffer on ice. Then centrifuged (13,000 g, 10 min) at 4℃ to remove the magazine. The supernatants were incubated with an iron-reducing agent for 30 min at room temperature (RT). Next, 100 μL of iron probe was added to each sample, and the reaction was incubated at RT for 1 h in the dark. Finally, the absorbance at 593 nm was measured using a microplate reader for comparison.

Reactive oxygen species (ROS) assay

BODIPY581/591C11 (D3861, ThermoFisher Scientific, MA, USA) was used to measure lipid ROS level. A total of 1 × 105 cells were seeded into 12-well plates and cultured in 5% carbon dioxide (CO2) at 37°C. Next, 1.5 μM BODIPY 581/591C11 dye was added to each 12-well plate and incubated for 30 min. Then, the cells were washed 2 times with a Hank’s balanced salt solution (HBSS; 14025076, ThermoFisher Scientific, MA, USA) and resuspended in HBSS for analysis using flow cytometry.

Donafenib-resistant (DR) cell line construction

HepG2 or SNU449 cells were seeded at a density of 1 × 104 cells per well in 96-well culture plates and incubated at 37°C with 5% CO2 for 24 h. The half-maximal inhibitory concentration (IC50) of the drug on the cells was determined using the CCK8 assay (HepG2, IC50 9.53 μM; SUN449, IC50 12.52 μM). Subsequently, the cells in the logarithmic phase with a confluence of 80–90% were selected, and an initial drug concentration of 1/10 to 1/5 of the IC50 value of the parental cells was applied for intervention. When the cell density reached 50%, the drug-containing medium was discarded, and the cells were washed 2 times with PBS before being replaced with a drug-free medium for continued cultivation. After the cells re-proliferated to 80–90% confluence, the aforementioned drug treatment procedure was repeated for 8 cycles. Once the cells exhibited stable growth at a specific drug concentration, the drug concentration was incrementally increased while maintaining the same treatment protocol until a drug-resistant cell line capable of stable proliferation at the final drug concentration was obtained. The IC50 value of the drug-resistant cell line was measured, and the resistance index (RI) was calculated as follows: RI = IC50 of drug-resistant cell line/IC50 of parental cell line. Based on the established criteria, an RI value >5 indicates that the cell line possesses significant drug resistance, thereby meeting the screening standards for drug-resistant cell strains.[31]

Combined drug experiment in mice

Four-week-old male BALB/C nude mice with an average body weight of 15 g were obtained from Liaoning Changsheng Biotechnology Co., Ltd., and under specific pathogen-free conditions in a controlled vivarium environment. All animal-related procedures were performed in accordance with the guidelines proposed by the Institutional Review Board at China Medical University. Subcutaneous xenograft mouse models of HCC, SNU-449 cells were implanted subcutaneously at a density of 1 × 10^6 cells per injection site in each immunocompromised mouse to initiate neoplastic growth. Tumor volume was measured regularly. After it reached 50 mm3, the mice were randomly divided into the following four groups, with 5 mice in each group: Normal control (treated with dimethyl sulfoxide [DMSO], D8371, Solarbio, Beijing, China), donafenib (10 mg/kg, HY-10201S, MCE, New Jersey, USA), AZ-628 (20mg/kg), and donafenib (10mg/kg) plus AZ-628 (20 mg/kg) groups. The respective drugs were orally administered to the mice in each group for 28 days. The mice were euthanized by intraperitoneal injection of sodium pentobarbital (100–200 mg/kg). Tumors were harvested from the mice and weighed for further analysis.

Immunohistochemical analysis

After isolating the tumors in mice, they were fixed in formalin (G2160, Solarbio, Beijing, China), embedded in paraffin (YA0012, Solarbio, Beijing, China), and sectioned at 4 μm thicknesses using a microtome. Following dewaxing and rehydration, tissue sections underwent antigen retrieval to unmask epitopes for immunostaining. Subsequently, the sections were sealed using 3% hydrogen peroxide and incubated with a Ki67 (1:800, #9027, Cell Signaling, MA, USA) antibody overnight at 4℃. Following extensive washing steps, tissue sections were incubated with a horseradish peroxidase–conjugated immunohistochemistry detection reagent (diluted 1:1, catalog no. 8114S, Cell Signaling) at RT for 1 h. Finally, the sections were stained with hematoxylin to visualize specific cell structures.

Data collection and differential gene expression analysis

The RNA sequencing (RNA-seq) data of HCC samples were obtained from TCGA. The data were preprocessed and standardized to account for technical and sample-specific variations. Differential expression analysis was performed using the R packages DESeq or limma. Subsequently, the ggplot2 and pheatmap packages in R were used to visualize differentially expressed genes in TCGA-HCC.

GSEA

The gene expression data of HCC and normal samples were preprocessed and standardized to ensure quality and comparability. Genes were ranked on the basis of their expression levels in the samples, and the ranked gene set was compared with a predefined gene set typically representing specific biological functions or pathways. Subsequently, enrichment scores were calculated to assess the extent to which the gene set was clustered or enriched within the ranked gene list.

Molecular docking

The interactions between the predicted drugs and the target genes were evaluated using AutoDock software, and the results were visualized using PyMol software (3.1.0, Schrödinger, LLC) from https://pymol.org/.

Statistics analysis

All numerical results were expressed as mean ± standard deviation, which were obtained from three or more independent experimental trials. Intergroup differences were assessed using two-tailed Student’s t-tests, with statistical significance defined as P < 0.05. One-way analysis of variance (ANOVA) was used for multiple groups, with appropriate post-hoc tests for multiple comparisons. Differences among multiple groups containing two variables were analyzed using two-way ANOVA followed by Bonferroni’s post hoc analysis. The Statistical Package for the Social Sciences 23.0 and GraphPad Prism 8.0 were used for analysis.

RESULTS

AZ-628 enhanced the sensitivity of HCC to donafenib

Donafenib, a multi-target TKI, is an orally administered drug used in targeted therapy. It suppresses tumor cell growth by inhibiting TKs involved in multiple signaling pathways in tumor cells. It is commonly used as a first-line drug for treating patients with advanced HCC or those with early-stage HCC who are not eligible for surgical resection or liver transplantation. However, long-term use of donafenib may lead to the development of resistance in some patients, resulting in the loss of its effectiveness. Current research focuses on combination therapies targeting multiple pathways, development of novel targeted drugs, and immunotherapy. In this study, novel small molecules that can reduce donafenib resistance in HCC were screened. Four targeted TKIs, namely, AZ-628, SU-5402, TG-101209, and SPP-86, were identified through molecular docking [Supplementary Figure 1a-l]. Subsequently, SNU449 and HepG2 cells were treated with these drugs, and the IC50 value of donafenib combined with different drugs was determined using the CCK8 assay. The findings indicated that the IC50 value of donafenib combined with different drugs decreased remarkably in cells treated with AZ-628, indicating that AZ-628 may enhance the sensitivity of HCC cells to donafenib (P < 0.0001); [Figure 1a-j and Table 1].

Supplementary Figures
AZ-628 could enhance the sensitivity of HCC to donafenib. (a-j) The CCK8 experiment showed the cell viability of five drugs (DMSO, AZ-628, SU-5402, TG-101209, and SPP-86) combined with different concentrations (0, 1, 2, 3, and 4 μM) of donafenib in the HCC cells SNU449 and HepG2. n = 3. Data are expressed as mean ± SD. ✶✶✶✶P < 0.0001. SD: Standard deviation, DMSO: Dimethyl sulfoxide, HCC: Hepatocellular carcinoma, CCK8: Cell counting kit-8.
Figure 1:
AZ-628 could enhance the sensitivity of HCC to donafenib. (a-j) The CCK8 experiment showed the cell viability of five drugs (DMSO, AZ-628, SU-5402, TG-101209, and SPP-86) combined with different concentrations (0, 1, 2, 3, and 4 μM) of donafenib in the HCC cells SNU449 and HepG2. n = 3. Data are expressed as mean ± SD. ✶✶✶✶P < 0.0001. SD: Standard deviation, DMSO: Dimethyl sulfoxide, HCC: Hepatocellular carcinoma, CCK8: Cell counting kit-8.
Table 1: The IC50 value of donafenib combined with different drugs.
HepG2
Drugs Donafeinib IC50
DMSO 2.78 μM
AZ-628 1.15 μM
SU5402 2.93 μM
TG101209 2.83 μM
SPP-86 2.59 μM
SNU449
Drugs Donafeinib IC50
DMSO 2.52 μM
AZ-628 1.43 μM
SU5402 2.85 μM
TG101209 2.81 μM
SPP-86 2.69 μM

DMSO: Dimethyl sulfoxide, IC50: Half-maximal inhibitory concentration

AZ-628 suppressed the proliferation of HCC cells

To validate the role of AZ-628 as a potential anti-tumor drug and its influence on donafenib resistance, normal liver cells (L02) and HCC cells were treated with AZ-628, and the IC50 values were determined using the CCK8 assay. Cytotoxicity assessment showed that the IC50 value of L02 cells treated with AZ-628 was substantially greater than that of HCC cells treated with AZ-628 (P < 0.0001); [Supplementary Figure 1m-r], indicating that AZ-628-targeted cancer cells have no significant toxic effects on normal cells. To elucidate the molecular mechanism underlying AZ-628 in the progression of HCC, transcriptomic profiling was performed using RNA-seq on HepG2 cells treated with either AZ-628 or DMSO, followed by differential expression analysis and GSEA. A total of 1864 significant differentially expressed genes were identified between the AZ-628 and DMSO groups, including 948 upregulated and 916 downregulated genes (Padj < 0.05) [Figure 2a]. The bubble plot demonstrated that these differentially expressed genes were primarily enriched in pathways related to cell proliferation [Figure 2b and Supplementary Figure 2a-d]. To assess the impact of AZ-628 on HCC cell growth, varying doses of the compound were administered to the cancer cells. Using the CCK8 assay, the IC50 values of AZ-628 in HepG2 and SNU449 cells were determined to be approximately 10–20 μM. Therefore, 0, 10, and 20 μM were selected for subsequent experiments (P < 0.001) [Table 1]. The results of colony formation and CCK8 assays indicated that the proliferative ability of HCC cells decreases with the increase of AZ-628 concentration (P < 0.001) [Figure 2c-f]. Collectively, these results demonstrate the inhibitory effect of AZ-628 on HCC cell growth.

AZ-628 could inhibit the proliferation of HCC cells. (a) Heat maps showed the differential genes of HepG2 cells after RNA-seq treatment with AZ-628 and DMSO. Red indicated upregulation, and green indicated downregulation. Padj < 0.05 and |log2FC| > 1. (b) The bubble map showed the enrichment analysis results for GSEA, with the size of the circles representing the number of genes involved in each GO item. (c-f) Colony formation and CCK-8 assays were used to evaluate the cellular proliferative capacity. HepG2 and SNU449 cell lines were exposed to varying concentrations of AZ-628 (10 μM and 20 μM), with DMSO-treated cells serving as the negative control. n = 3. Data are presented as mean ± SD derived from triplicate experiments (✶✶✶P < 0.001, ✶✶✶✶P < 0.0001). SD: Standard deviation, HCC: Hepatocellular carcinoma, RNA-seq: RNA sequencing. CCK8: Cell counting kit-8, DMSO: Dimethyl sulfoxide.
Figure 2:
AZ-628 could inhibit the proliferation of HCC cells. (a) Heat maps showed the differential genes of HepG2 cells after RNA-seq treatment with AZ-628 and DMSO. Red indicated upregulation, and green indicated downregulation. Padj < 0.05 and |log2FC| > 1. (b) The bubble map showed the enrichment analysis results for GSEA, with the size of the circles representing the number of genes involved in each GO item. (c-f) Colony formation and CCK-8 assays were used to evaluate the cellular proliferative capacity. HepG2 and SNU449 cell lines were exposed to varying concentrations of AZ-628 (10 μM and 20 μM), with DMSO-treated cells serving as the negative control. n = 3. Data are presented as mean ± SD derived from triplicate experiments (✶✶✶P < 0.001, ✶✶✶✶P < 0.0001). SD: Standard deviation, HCC: Hepatocellular carcinoma, RNA-seq: RNA sequencing. CCK8: Cell counting kit-8, DMSO: Dimethyl sulfoxide.

AZ-628 inhibited the proliferation and enhanced the sensitivity of HCC cells to donafenib by the TK pathway

To determine specific mechanisms through which AZ-628 exerts anti-cancer effects against HCC, pathway-based functional annotation was conducted on the identified differentially expressed genes that were significantly enriched in the TK pathway (P = 5.604 × 10−6) [Supplementary Figure 3a], which was consistent with our hypothesis. Among the genes involved in the TK pathway, EGR1 was identified as a promising target due to its functional significance. Moreover, the fold change of EGR1 induced by AZ-628 was found to be the most substantial within this pathway. To investigate the mechanistic involvement of EGR1 in the antitumor activity of AZ-628, SNU-449, and HepG2 cells were treated with AZ-628 at different concentrations. The findings indicate that the expression level of EGR1 notably decreased with the increase of AZ-628 concentration (P < 0.001) [Figure 3a-f]. This finding strongly indicated that AZ-628 modulated the expression of EGR1. Furthermore, to verify whether the regulatory effects of AZ-628 relied on EGR1, based on the median expression level of EGR1, HCC samples obtained were stratified into high-expression and low-expression cohorts. Differentially expressed genes, including 167 upregulated and 27 downregulated genes, were identified between the two cohorts (Padj < 0.05) [Figure 3g]. Subsequently, the upregulated genes in the TCGA dataset were compared with the downregulated genes induced by AZ-628, and the downregulated genes in the TCGA dataset were intersected with the upregulated genes induced by AZ-628. The results revealed a total of 30 co-regulated genes [Figure 3h and i and Supplementary Figure 3b-d], indicating that the regulatory effects of AZ-628 rely on EGR1 in HCC. Rescue experiments were conducted to confirm these findings. CCK8 and clone formation demonstrated that AZ-628 effectively suppressed the proliferation of shNC HCC cells. Furthermore, AZ-628 could still inhibit cell proliferation after slightly knocking down EGR1. However, these inhibitory effects were attenuated in HCC cells with EGR1 knockdown (P < 0.001) [Figure 3j-o and Supplementary Figure 3e-g]. To further investigate whether AZ-628 resistance to donafenib in HCC is dependent on EGR1, a DR HepG2 cell line was constructed. DR HepG2 was treated with AZ-628 alone or in combination with donafenib, and its proliferation ability was tested using the CCK8 and colony formation assays. The results showed that the combination of AZ-628 and donafenib significantly reduced the resistance of DR HepG2 to donafenib. However, in DR HepG2 with EGR1 knockdown, the combination of AZ-628 and donafenib did not significantly improve the sensitivity of DR HepG2 to donafenib (P < 0.0001) [Figure 4a-f]. Collectively, these findings indicate that AZ-628 exerts dual anti-tumor effects on HCC cells, inhibiting proliferative capacity while potentiating donafenib responsiveness through modulation of TK-mediated signaling cascades.

AZ-628 could inhibit the proliferation of HCC cells by inhibiting the tyrosine kinase pathway. (a-f) The expression levels of EGR1 in SNU449 and HepG2 cell lines following AZ-628 treatment at varying doses (10 μM and 20 μM)were analyzed through Western blotting and qPCR. n = 3. (g) Volcano plot analysis revealed differentially expressed genes. Padj < 0.05 and |log2FC| > 1. (h and i) Intersecting genes were visualized using Venn diagrams. (j-m) SNU449 and HepG2 cells were treated with AZ-628 and DMSO for 48 h, and the proliferation of EGR1-silenced cells and sh-NC-transfected cells was detected by cell colony formation. n = 3. (n and o) SNU449 and HepG2 cells were treated with AZ-628 and DMSO for 48 h, and the cell viability of EGR1-silenced cells and sh-NC transfected cells was detected by CCK8 formation. n = 3. Data are expressed as mean ± SD, with ✶✶P < 0.01, ✶✶✶P < 0.001, ✶✶✶✶P < 0.0001. ns: No statistical difference, SD: Standard deviation, HCC: Hepatocellular carcinoma, DMSO: Dimethyl sulfoxide, CCK8: Cell counting kit-8, qPCR: Quantitative polymerase chain reaction, EGR1: Early growth response gene 1.
Figure 3:
AZ-628 could inhibit the proliferation of HCC cells by inhibiting the tyrosine kinase pathway. (a-f) The expression levels of EGR1 in SNU449 and HepG2 cell lines following AZ-628 treatment at varying doses (10 μM and 20 μM)were analyzed through Western blotting and qPCR. n = 3. (g) Volcano plot analysis revealed differentially expressed genes. Padj < 0.05 and |log2FC| > 1. (h and i) Intersecting genes were visualized using Venn diagrams. (j-m) SNU449 and HepG2 cells were treated with AZ-628 and DMSO for 48 h, and the proliferation of EGR1-silenced cells and sh-NC-transfected cells was detected by cell colony formation. n = 3. (n and o) SNU449 and HepG2 cells were treated with AZ-628 and DMSO for 48 h, and the cell viability of EGR1-silenced cells and sh-NC transfected cells was detected by CCK8 formation. n = 3. Data are expressed as mean ± SD, with ✶✶P < 0.01, ✶✶✶P < 0.001, ✶✶✶✶P < 0.0001. ns: No statistical difference, SD: Standard deviation, HCC: Hepatocellular carcinoma, DMSO: Dimethyl sulfoxide, CCK8: Cell counting kit-8, qPCR: Quantitative polymerase chain reaction, EGR1: Early growth response gene 1.
AZ-628 could decrease the resistance of HCC cells to donafenib. (a) DR HepG2 cell lines were constructed. The CCK8 assay was used to detect the cell viability after treatment with AZ-628 alone or in combination with donafenib. n = 3. (b) DR sh-EGR1 HepG2 cell lines were constructed. The CCK8 assay was used to detect the cell viability after treatment with AZ-628 alone or in combination with donafenib. n = 3. (c and d) The clonogenic potential of DR HepG2 cells was assessed following treatment with AZ-628, either alone or in combination with donafenib, utilizing colony formation assays. n = 3. (e and f) The clonogenic potential of DR sh-EGR1 HepG2 cells was assessed following treatment with AZ-628, either alone or in conjunction with donafenib, utilizing colony formation assays. n = 3. Data are expressed as mean ± SD, with ✶✶✶✶P < 0.0001. ns: No statistical difference, SD: Standard deviation, HCC: Hepatocellular carcinoma, DR: Donafenib-resistant, CCK8: Cell counting kit-8, sh-EGR1: Short hairpin Early growth response gene 1.
Figure 4:
AZ-628 could decrease the resistance of HCC cells to donafenib. (a) DR HepG2 cell lines were constructed. The CCK8 assay was used to detect the cell viability after treatment with AZ-628 alone or in combination with donafenib. n = 3. (b) DR sh-EGR1 HepG2 cell lines were constructed. The CCK8 assay was used to detect the cell viability after treatment with AZ-628 alone or in combination with donafenib. n = 3. (c and d) The clonogenic potential of DR HepG2 cells was assessed following treatment with AZ-628, either alone or in combination with donafenib, utilizing colony formation assays. n = 3. (e and f) The clonogenic potential of DR sh-EGR1 HepG2 cells was assessed following treatment with AZ-628, either alone or in conjunction with donafenib, utilizing colony formation assays. n = 3. Data are expressed as mean ± SD, with ✶✶✶✶P < 0.0001. ns: No statistical difference, SD: Standard deviation, HCC: Hepatocellular carcinoma, DR: Donafenib-resistant, CCK8: Cell counting kit-8, sh-EGR1: Short hairpin Early growth response gene 1.

AZ-628 modulates the sensitivity of HCC to donafenib modulation through EMT, apoptosis, and ferroptosis

EMT has been associated with increased invasiveness, metastatic potential, and contributes to therapeutic resistance development. In addition, alterations in programmed cell death pathways, such as apoptosis, have been associated with the acquisition of chemoresistance phenotypes in malignant cells. Transcriptomic profiling through RNAseq coupled with differential expression analysis revealed that the genes altered by AZ-628 were significant pathway-enriched related to EMT and apoptosis (Padj < 0.05) [Figure 5a and Supplementary Figure 4a and b]. Therefore, we hypothesized that AZ-628 regulates donafenib resistance in HCC through these pathways. To validate this hypothesis, apoptosis of DR SNU449 and HepG2 cells treated with AZ-628 at different concentrations was assessed. Flow cytometry revealed a marked, concentration-dependent increase in the proportion of apoptotic cells (P < 0.0001) [Figure 5b-e]. The Transwell assay indicated that the migratory ability of HCC cells decreased significantly with the increase of AZ-628 concentration (P < 0.0001) [Figure 5f-i]. To further investigate the effect of AZ-628 on regulated cell death, a ferroptosis-related experiment was performed after using AZ-628. First, HCC cell lines were induced with low concentrations of Erastin (2 μM) or RSL3 (1 μM) alone, and the changes in Fe2+ concentration and lipid ROS level were detected. The results indicated that low concentrations of ferroptosis inducers could slightly influence Fe2+ concentration and lipid ROS level in HCC cells. Next, HCC cells were treated with AZ-628 in combination with low concentrations of Erastin (2 μM) or RSL3 (1 μM), showing significant upregulation of Fe2+ concentrations and lipid ROS levels in HCC cells (P < 0.0001) [Figure 6a-f]. These results indicate that AZ-628 influences HCC resistance to donafenib through EMT, apoptosis, and ferroptosis.

AZ-628 may affect the resistance of HCC to donafenib through the EMT and apoptosis pathways. (a) The bubble map showed the enrichment analysis results of AZ-628-induced differentially expressed genes. (b-e) Flow cytometric analysis was performed to quantify apoptotic cells in HepG2/DR and SNU449/DR cultures following exposure to varying concentrations of AZ-628 (10 and 20 μM). (f-i) To assess the impact of AZ-628 on cell migration, transwell experiments were conducted using HepG2/DE and SNU449/DR cell lines exposed to different doses of the compound (10 and 20 μM). The magnification is ×50 and scale bar is 200 μm. n = 3. Data are presented as mean ± SD derived from triplicate experiments (✶✶✶✶P < 0.0001). SD: Standard deviation, HCC: Hepatocellular carcinoma, DR: Donafenib-resistant, EMT: Epithelial–mesenchymal transition.
Figure 5:
AZ-628 may affect the resistance of HCC to donafenib through the EMT and apoptosis pathways. (a) The bubble map showed the enrichment analysis results of AZ-628-induced differentially expressed genes. (b-e) Flow cytometric analysis was performed to quantify apoptotic cells in HepG2/DR and SNU449/DR cultures following exposure to varying concentrations of AZ-628 (10 and 20 μM). (f-i) To assess the impact of AZ-628 on cell migration, transwell experiments were conducted using HepG2/DE and SNU449/DR cell lines exposed to different doses of the compound (10 and 20 μM). The magnification is ×50 and scale bar is 200 μm. n = 3. Data are presented as mean ± SD derived from triplicate experiments (✶✶✶✶P < 0.0001). SD: Standard deviation, HCC: Hepatocellular carcinoma, DR: Donafenib-resistant, EMT: Epithelial–mesenchymal transition.
AZ-628 may affect the resistance of HCC to donafenib through ferroptosis. (a and b) Following independent induction with low-dose Erastin (2 μM) or RSL3 (1 μM), intracellular Fe2+ levels in HepG2/DR and SNU449/DR cells were quantified after exposure to either DMSO or varying concentrations of AZ-628. n = 3. (c-f) HepG2/DR and SNU449/DR cells were induced with Erastin (2 μM) or RSL3 (1 μM), and ROS levels were detected after treatment with DMSO or AZ-628 at different concentrations. n = 3. Data are presented as mean ± SD derived from triplicate experiments (✶P < 0.05, ✶✶P < 0.01, ✶✶✶✶P < 0.0001). SD: Standard deviation, HCC: Hepatocellular carcinoma, DR: Donafenib-resistant, DMSO: Dimethyl sulfoxide, ROS: Reactive oxygen species, Fe2+: Ferrous ion.
Figure 6:
AZ-628 may affect the resistance of HCC to donafenib through ferroptosis. (a and b) Following independent induction with low-dose Erastin (2 μM) or RSL3 (1 μM), intracellular Fe2+ levels in HepG2/DR and SNU449/DR cells were quantified after exposure to either DMSO or varying concentrations of AZ-628. n = 3. (c-f) HepG2/DR and SNU449/DR cells were induced with Erastin (2 μM) or RSL3 (1 μM), and ROS levels were detected after treatment with DMSO or AZ-628 at different concentrations. n = 3. Data are presented as mean ± SD derived from triplicate experiments (✶P < 0.05, ✶✶P < 0.01, ✶✶✶✶P < 0.0001). SD: Standard deviation, HCC: Hepatocellular carcinoma, DR: Donafenib-resistant, DMSO: Dimethyl sulfoxide, ROS: Reactive oxygen species, Fe2+: Ferrous ion.

AZ-628 and donafenib synergistically inhibited the malignant progression of HCC in vitro

The prolonged use of donafenib leads to resistance, thereby limiting its therapeutic effects against tumors. The findings indicate that AZ-628 suppresses the proliferation of HCC cells through the TK pathway. However, whether the combination of AZ-628 and donafenib yields synergistic therapeutic effects remains unclear. Consequently, SNU449 and HepG2 cells were exposed to a combination of AZ-628 and donafenib. The findings demonstrated that the two drugs had significant synergistic effects on the cells [Figure 7a and b and Supplementary Figure 5a-f]. In addition, clone formation and the CCK8 assay showed that the joint utilization of AZ-628 and donafenib significantly decreased the number of cell colonies and inhibited cell proliferation (P < 0.001) [Figure 7c-h]. These findings indicate that AZ-628 and donafenib work together to suppress HCC progression in vitro.

AZ-628 and donafenib synergistically inhibit the malignant progression of HCC cells in vitro. (a and b) The synergistic effects of AZ-628 and donafenib combination therapy were analyzed using Combenefit2 software. (c-f) HepG2 and SNU449 cells were subjected to treatments with DMSO, AZ-628, donafenib, and a combination of AZ-628 and donafenib, and the proliferative capacity of these cells was evaluated using colony formation assays. n = 3. (g and h) HepG2 and SNU449 cells were treated with DMSO, AZ-628, donafenib, and AZ-628 combined with donafenib, and the cell viability was determined using the CCK8 assay. n = 3. Data are expressed as mean ± SD, with ✶✶P < 0.01, ✶✶✶P<0.001, ✶✶✶✶P < 0.0001. SD: Standard deviation, HCC: Hepatocellular carcinoma, DMSO: Dimethyl sulfoxide, CCK8: Cell counting kit-8.
Figure 7:
AZ-628 and donafenib synergistically inhibit the malignant progression of HCC cells in vitro. (a and b) The synergistic effects of AZ-628 and donafenib combination therapy were analyzed using Combenefit2 software. (c-f) HepG2 and SNU449 cells were subjected to treatments with DMSO, AZ-628, donafenib, and a combination of AZ-628 and donafenib, and the proliferative capacity of these cells was evaluated using colony formation assays. n = 3. (g and h) HepG2 and SNU449 cells were treated with DMSO, AZ-628, donafenib, and AZ-628 combined with donafenib, and the cell viability was determined using the CCK8 assay. n = 3. Data are expressed as mean ± SD, with ✶✶P < 0.01, ✶✶✶P<0.001, ✶✶✶✶P < 0.0001. SD: Standard deviation, HCC: Hepatocellular carcinoma, DMSO: Dimethyl sulfoxide, CCK8: Cell counting kit-8.

AZ-628 and donafenib exerted synergistic therapeutic effects against HCC in vivo

To investigate the potential synergistic effects of AZ-628 and donafenib in vivo, we established xenograft tumor models by injecting SNU449 cells into nude mice (n = 20). The mice were randomly assigned to four groups: DMSO (control), AZ-628, donafenib, and combination treatment groups. The mice were continuously treated with the respective drugs for 28 days, and tumor burden parameters were measured every 4 days. All mice survived throughout the treatment. As anticipated, the mice in the AZ-628 and donafenib groups had lower tumor volume and weight than those in the DMSO group. In addition, tumor growth was inhibited most significantly in the combination treatment group (P < 0.0001) [Figure 8a-c]. After 28 days, tumor tissues were collected from mice for immunodetection of the proliferation marker Ki67. The results indicated that the proliferation index, as measured by Ki-67 immunopositivity, statistically and significantly decreased in the AZ-628-treated and donafenib-treated cohorts relative to the vehicle control (DMSO) group, whereas it was lowest in the combination treatment group (P < 0.0001) [Figure 8d and e]. Collectively, these findings indicated that AZ-628 and donafenib exerted synergistic therapeutic effects against HCC in vivo. The schematic representation illustrating the proposed molecular mechanisms is presented in Supplementary Figure 6.

AZ-628 and donafenib synergistically treat HCC in vivo. (a) The mice were treated with DMSO, AZ-628, donafenib, AZ-628, and donafenib for 28 days. Four groups of mouse tumor photos. n = 5. (b) Change trend of tumor volume in mice treated with DMSO, AZ-628, donafenib, AZ-628, and donafenib. n = 5. (c) Tumor weights across the four groups. n = 5. (d and e) The expression of the tumor species Ki67 in the mice after treatment with the four drugs was detected by immunohistochemistry. The magnification is ×20 and ×200 and scale bar is 200 μm and 20 μm. n = 5. Data are expressed as mean ± SD, with ✶✶✶✶P < 0.0001. SD: Standard deviation, HCC: Hepatocellular carcinoma, DMSO: Dimethyl sulfoxide.
Figure 8:
AZ-628 and donafenib synergistically treat HCC in vivo. (a) The mice were treated with DMSO, AZ-628, donafenib, AZ-628, and donafenib for 28 days. Four groups of mouse tumor photos. n = 5. (b) Change trend of tumor volume in mice treated with DMSO, AZ-628, donafenib, AZ-628, and donafenib. n = 5. (c) Tumor weights across the four groups. n = 5. (d and e) The expression of the tumor species Ki67 in the mice after treatment with the four drugs was detected by immunohistochemistry. The magnification is ×20 and ×200 and scale bar is 200 μm and 20 μm. n = 5. Data are expressed as mean ± SD, with ✶✶✶✶P < 0.0001. SD: Standard deviation, HCC: Hepatocellular carcinoma, DMSO: Dimethyl sulfoxide.

DISCUSSION

HCC is often diagnosed at an advanced stage, and it typically has a poor prognosis. Targeted therapy is an effective treatment option for HCC. The TK pathway is critically involved in the initiation and progression of HCC. TKIs influence the TK pathway primarily by blocking the activity of specific TKs. These small molecules interfere with the phosphorylation of tyrosine residues in key signaling proteins, thereby disrupting the downstream signaling cascades of the TK pathway.[32,33] TKIs impede cancer cell growth and survival by targeting the aberrant activation of TKs, which drive cellular proliferation, differentiation, and survival.[34] This targeted approach is particularly effective in cancers in which the TK pathway is dysregulated due to genetic mutations or overexpression of specific receptors. Therefore, TKIs are an essential class of drugs for the precise treatment of cancer.[35] Donafenib, a TKI, has been recently approved as a first-line treatment for patients with unresectable HCC who have not undergone systemic therapy.[36] As a deuterated form of sorafenib, donafenib has high bioavailability. Considering that drug resistance may develop after long-term use of donafenib, its widespread clinical application is challenging, and its role in combination therapy is under investigation. In this study, AZ-628 could enhance the sensitivity of HCC to donafenib by scanning small-molecular TKIs. However, RNA-seq was performed between the DMSO and AZ-628 groups in HCC cells; thus, there may be limitations for the mechanism investigation. Therefore, DR HCC cells will be studied further in the future to illustrate more the mechanism of drug resistance in HCC. Combination therapy may enhance the efficacy of donafenib by targeting multiple pathways involved in tumor growth and progression.[37] Donafenib has been combined with anti-PD-1 antibodies and trans-arterial chemoembolization as a potential triple therapy for unresectable HCC.[38] The combined use of TKIs can mutually increase drug sensitivity.[25,39] This study demonstrated that AZ-628, a drug with TKI activity, improved the sensitivity of HCC to donafenib through the TK pathway. This strategy may be used to overcome donafenib resistance in HCC.

An unavoidable problem associated with the use of chemotherapy or targeted therapy is drug resistance developed during treatment.[40] Drug resistance in cancer treatment involves a complex interplay of molecular and phenotypic changes that enable malignant cells with therapeutic resistance mechanisms. The two key mechanisms underlying the development of drug resistance include molecular phenotypic changes, such as EMT, and alterations in cell death pathways, particularly apoptosis and ferroptosis.[41] During EMT, cancer cells undergo phenotypic changes characterized by the loss of cell adhesion, increased motility, and enhanced resistance to apoptosis.[42] Such molecular alterations enable cancer cells to invade surrounding tissues, evade immune responses, and resist the cytotoxic effects of drugs.[6] Apoptotic resistance is a hallmark of cancer and a major factor contributing to the development of drug resistance.[43] Cancer cells can acquire mutations or alterations in key regulators of apoptosis, such as Bcl-2 family proteins, to promote cell survival during treatment.[44] These variations allow cancer cells to evade treatment-induced cell death and continue proliferation. An in-depth understanding of the mechanisms underlying drug resistance is crucial for developing effective strategies for overcoming resistance and improving treatment outcomes in cancer. Targeted therapies that address specific molecular changes associated with drug resistance and combination therapies that block multiple resistance mechanisms are actively being investigated in cancer research and clinical practice. In this study, AZ-628 was found to regulate the EMT and apoptosis of HCC cells by increasing their sensitivity to donafenib. RNA-seq analysis between the DMSO and AZ-628 groups in HCC cells revealed that the cell death and EMT pathways were significantly enriched after AZ-628 treatment. This finding indicates that AZ-628 influences cell death and the EMT pathway. Further experiments validated that AZ-628 could promote apoptosis and inhibit EMT of HCC cells, thereby decreasing the resistance of HCC to donafenib. However, the detailed mechanism through which AZ-628 regulates apoptosis and EMT remains unclear. Therefore, we aimed to investigate such a mechanism in the future.

The TK pathway is crucial to cell growth, differentiation, and signaling.[45] In HCC, the dysregulation of the TK pathway contributes to cancer progression. In addition, the aberrant activation of TK receptors and downstream signaling pathways promotes tumor growth and angiogenesis in HCC. Therefore, TKs are promising therapeutic targets for HCC. TKIs exert anti-tumor effects by blocking specific signaling pathways that drive cancer growth. TKIs such as sorafenib and lenvatinib target aberrant signaling, thereby inhibiting tumor angiogenesis and proliferation.[3] Although TKIs cannot cure diseases, they can prolong survival, and they are important therapeutic agents for advanced HCC. EGR1, which is also known as ETS-related gene 1, is a transcription factor involved in the TK pathway.[46] It regulates the expression level of genes related to cell growth, differentiation, and development.[47] In addition, it has been associated with tumor development and progression in various cancer types, including HCC.[48] The overexpression or dysregulation of EGR1 can contribute to the aggressive behavior of HCC.[49] EGR1 can regulate genes involved in cellular proliferation, neovascularization, and metastatic potential, which are key hallmarks of cancer progression. Therefore, it can drive tumor growth and metastasis in HCC. Understanding the role of EGR1 in the TK pathway is essential for developing targeted therapies. Inhibiting EGR1 or its downstream targets in the TK pathway may suppress tumor growth and angiogenesis in HCC. Moreover, elucidating the precise mechanisms of EGR1 and its crosstalk with the TK pathway may guide novel therapeutic strategies for HCC and other cancers. This finding demonstrated that AZ-628 regulated the proliferation of HCC cells through EGR1, thereby increasing their sensitivity to donafenib. This finding indicates that AZ-628 can be used in combination with donafenib to overcome donafenib resistance in HCC. EGR1 is a key node in the TK pathway. Our findings indicate that EGR1 RNA levels changed when HCC cells were treated with AZ-628. Further assays validated that knocking down EGR1 could influence the sensitivity of HCC to donafenib. Rescue experiments showed that AZ-628 depends on EGR1 to exert its function. However, there may be other mechanisms for regulating the sensitivity of HCC to donafenib. Thus, in the future, we will integrate multi-omics to further investigate the mechanism through which AZ-628 regulates the sensitivity of HCC to donafenib in the future.

AZ-628 is a novel ATP-competitive inhibitor of Raf kinases, including B-Raf and B-RafV600E.[50] It has been shown to inhibit ERK more effectively than dabrafenib, a type I RAF inhibitor, in cells expressing several BRAF mutants in lung cancer.[51] AZ-628 suppresses anchorage-dependent and -independent growth, leading to cell cycle arrest and triggering apoptosis in colon and melanoma cells harboring B-RafV600E mutations.[52] Although studies investigating the effects of AZ-628 in HCC are limited, a previous study reported a novel scoring system based on gene expression profiling, in which AZ-628 was identified as a potential therapeutic drug for HCC. AZ-628 prevents the activation of several TK receptors, including VEGFR2.[53] This study shows that AZ-628 and donafenib exert synergistic therapeutic effects against HCC in vitro and in vivo. In the future, the safety, feasibility, and effect of AZ-628 will be further investigated.

SUMMARY

This study reveals that the TK pathway represents a therapeutic target for AZ-628 to reverse donafenib resistance in HCC cells. AZ-628 may influence donafenib resistance by modulating EMT, apoptosis, and ferroptosis. The cooperative interaction of AZ-628 and donafenib exerts synergistic anti-tumor effects against HCC in vitro and in vivo. Collectively, this study proposes a novel combination therapy involving TKIs for addressing donafenib resistance in HCC and provides valuable insights into the further development of therapeutic strategies for HCC.

AVAILABILITY OF DATA AND MATERIALS

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

ABBREVIATIONS

ANOVA: One-way analysis of variance

BRAF: V-raf murine sarcoma viral oncogene homolog

CCK8: Cell counting kit-8

CO2: Carbon dioxide

DESeq2: Differential Expression analysis for Sequence data 2

DMSO: Dimethyl sulfoxide

DR: Donafenib-resistant

EGR1: Early growth response gene 1

EMT: Epithelial–mesenchymal transition

ETS: E26 transformation-specific

Fe2+: Ferrous ion

GSEA: Gene Set Enrichment Analysis

HCC: Hepatocellular carcinoma

IC50: Half-maximal inhibitory concentration

mRNA: Messenger RNA

PBS: Phosphate-buffered saline

qRT-PCR: Quantitative reverse transcription polymerase chain reaction

RIP3: Receptor-interacting protein kinase 3

RNA-seq: RNA sequencing

ROS: Reactive oxygen species

shEGR1: shRNA targeting EGR1

shNC: Negative control shRNA

shRNA: Short hairpin RNA

TCGA: The Cancer Genome Atlas

TK: Tyrosine kinase

TKI: The tyrosine kinase inhibitor

VEGFR: Vascular endothelial growth factor receptor

AUTHOR CONTRIBUTIONS

ZMG: Substantial contributions to the conception and design of the study; XWL: Substantial contributions to the acquisition; TYY: Substantial contributions to analysis, and interpretation of data; CYD: Substantial contributions to the drafting and revision of the manuscript; XYZ: Confirmed the authenticity of all the raw data; HL and YHW: Gave final approval of the version to be published. All authors read and approved of the final manuscript. All authors meet ICMJE authorship requirements.

ACKNOWLEDGMENT

Thanks for the support from all the members of group.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Animal protocol was performed in accordance to the fundamental principles indicated by the International Council for Laboratory Animal Science (ICLAS) and all animal-related procedures were conducted in accordance with the guidelines proposed by the Institutional Review Board at China Medical University (CMU2022607). Informed consent to participate is not required, as this study does not involve human subjects.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

EDITORIAL/PEER REVIEW

To ensure the integrity and highest quality of CytoJournal publications, the review process of this manuscript was conducted under a double-blind model (authors are blinded for reviewers and vice versa) through an automatic online system.

FUNDING: Not applicable.

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