Synergistic antitumor effect of oroxylin A and donafenib in hepatocellular carcinoma through tumor protein p53 signaling pathway activation

Abstract

BACKGROUND

The clinical application of donafenib in advanced hepatocellular carcinoma (HCC) is restricted by its limited therapeutic efficacy and a variety of treatment-associated adverse events. These factors collectively underscore the need for more effective and well-tolerated therapeutic strategies.

AIM

To investigate the effects and underlying mechanisms of oroxylin A in combination with donafenib on HCC through in vivo and in vitro studies.

METHODS

The antitumor efficacy of oroxylin A, donafenib, and their combination was assessed in xenograft mouse models and MHCC-97H/PLC-PRF-5 cell lines. Tumor growth was monitored using fluorescence live imaging. Cell viability, colony formation, and apoptosis were assessed using Cell Counting Kit-8, clonogenic, and flow cytometry assays, respectively. Molecular mechanisms were investigated by assessing the expression of tumor protein p53 (TP53) signaling-related regulators via quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry. Public datasets and Kaplan-Meier analysis were used to analyzed the relationship between their expression and patient survival.

RESULTS

The combination of oroxylin A and donafenib demonstrated superior anti-tumor efficacy in vivo compared to monotherapies, without inducing significant hepatorenal toxicity. The combination therapy demonstrated a stronger anti-proliferative and pro-apoptotic effects than two monotherapies in two HCC cell lines. Mechanistically, the drug combination synergistically activated the TP53 signaling pathway. Oroxylin A primarily targeted the cyclin-dependent kinase 9-murine double minute 2 (MDM2)/MDM4 axis to stabilize TP53, while donafenib suppressed the vascular endothelial growth factor receptor/B-rapidly accelerated fibrosarcoma proto-oncogene serine/threonine kinase axis to activate TP53. Clinical data has verified that the upregulation of key components of this pathway (TP53, MDM2, MDM4, and cyclin-dependent kinase 9) in patients with HCC is associated with a poor overall survival.

CONCLUSION

Oroxylin A and donafenib exert a synergistic anti-tumor effect in HCC by co-activating the TP53 signaling pathway through distinct but complementary molecular axes. This combination strategy presents a promising and viable therapeutic approach to overcome the limitations of donafenib monotherapy in the treatment of HCC.

Key Words: Hepatocellular carcinoma; Oroxylin A; Donafenib; Combination therapy; Xenograft tumor; TP53 signaling pathway

Core Tip: This study demonstrates that the combination of oroxylin A and donafenib exerts a potent synergistic antitumor effect against hepatocellular carcinoma in vitro and in vivo. Mechanistically, this synergy is achieved through the convergent activation of the tumor-suppressive tumor protein p53 signaling pathway. This work provides a novel and effective therapeutic strategy for hepatocellular carcinoma.

INTRODUCTION

Hepatocellular carcinoma (HCC) is a prominent pathological type of primary liver cancer that is primarily caused by cirrhosis, which is the result of established risk factors (hepatitis B virus or hepatitis C virus, alcohol-related liver disease or metabolic dysfunction-associated steatotic liver disease) and less common risk factors (hereditary hemochromatosis, primary sclerosing cholangitis, primary biliary cholangitis, autoimmune hepatitis, and other chronic hepatitis)[1-4]. The five-year survival rate of HCC is only 18%, which is only lower than that of pancreatic cancer[5]. Owing to the insidious onset of HCC and its atypical early symptoms, over 50% of patients with HCC are diagnosed at an advanced stage; hence, systematic treatment is an important treatment option[6].

Donafenib, a deuterium derivative of sorafenib, is a novel molecular targeted drug for unresectable HCC[7]. It demonstrates anti-HCC effects by suppressing the rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinases (ERK) signaling pathway and the expression of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptors[7]. Donafenib displayed improved overall survival (OS), safety, tolerability, quality-adjusted life years and lower cost than sorafenib, serving as a promising alternative for first-line systemic treatment of advanced HCC[8,9]. However, donafenib still has limitations, such as various adverse events, and similar progression-free survival, objective response rate and disease control rate to sorafenib[7,10]. Given the limitations of monotherapy with molecularly targeted drugs, combination strategies, particularly those integrating traditional Chinese medicine (TCM), have emerged as a promising frontier in HCC management[11]. TCM, with its multi-component and multi-target characteristics, has long been used as an adjunctive therapy for cancer in Asia[12,13]. Accumulating evidence suggests that certain Chinese herbal medicines and their active compounds can enhance the efficacy of conventional chemotherapeutic or targeted agents while mitigating their side effects[14-16], offering a holistic approach for advanced HCC treatment. However, there are no relevant studies on the combination of donafenib and TCM for the treatment of HCC.

Oroxylin A is a natural flavonoid compound that is derived from the TCM Scutellaria baicalensis[17]. Oroxylin A exhibits a broad range of pharmacological activities, such as inducing apoptosis in tumor cells, blocking the tumor cell cycle, inhibiting tumor metastasis and invasion, and exerting antioxidant, antiviral, and anti-inflammatory effects[17]. Oroxylin A exerts an anti-HCC effect by inducing apoptosis in HCC cells and reshaping the immune microenvironment of HCC mice models in experimental studies[18]. Notably, oroxylin A has been approved by the National Medical Products Administration and is currently undergoing phase I or II clinical trials for HCC therapy. The present study systematically investigated the therapeutic potential and underlying molecular mechanisms of oroxylin A in combination with donafenib for HCC through in vitro and in vivo experiments. We aim to provide a robust experimental foundation for its future clinical application.

MATERIALS AND METHODS

Chemicals and regents

Oroxylin A (provided by the China Pharmaceutical University). Donafenib (Zelgen, #H20210020, China). Dulbecco’s modified Eagle’s medium (Gibco, #12800-017, MA, United States). Minimum essential medium (MEM) (Gibco, #12561056, MA, United States). RPMI-1640 medium (Gibco, #11875093, MA, United States). Fetal bovine serum (Gibco, #10099-141, MA, United States). MEM (non-essential amino acids; 100 ×; Gibco: #11140050, MA, United States). Penicillin-streptomycin solution (Gibco, #15140122, MA, United States). Dimethyl sulfoxide (DMSO) (Aladdin, #D274279, China). Alanine aminotransferase (ALT) detection kit (Solarbio, #BC1555, China). Aspartate aminotransferase (AST) detection kit (Solarbio, #BC1565, China). Creatinine detection kit (Elabscience, #E-BC-K188-M, China). Urea nitrogen detection kit (Elabscience, #E-BC-K183-M, China). Cell Counting Kit-8 (CCK-8) kit (Beyotime, #C0038, China). Annexin V-FITC/PI apoptosis detection kit (Elabscience, E-CK-A211, China). RNA extraction kit (Qiagen, #74034, Germany). TSA multiplex fluorescence staining kit (Aifang Biological, #AFIHC037, China). RNA reverse transcription kit (Invitrogen, #18080-051, CA, United States). SYBR fluorescence quantitative PCR detection kit (TaKaRa, #RR420A, Japan). 1 × phosphate-buffered saline (PBS) (Solarbio, #P1020-500 mL, China). Crystal violet solution (Solarbio, #G1062, China).

Cell culture and treatment

The MHCC-97H cell line was acquired from Saibaikang Biotechnology Co., Ltd, China. The PLCPRF-5 cell line was acquired from Punuosai Biotechnology Co., Ltd, China. The cell lines were identified in the cell bank of the Chinese Academy of Sciences through STR, and the mycoplasma test was negative. The MHCC-97H cell line was cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 1% dual antibody. The PLCPRF-5 cell line was cultured in MEM containing 1 × nonessential amino acids, 10% fetal bovine serum, and 1% dual antibody. The culture conditions for all cell lines were 5% CO₂, 37 °C temperature, and 80% humidity.

The stock solutions of oroxylin A and donafenib were prepared in DMSO at concentrations of 5 mmol/L and 1.25 mmol/L, respectively. The stock aliquots were maintained at a temperature of -20 °C. The stock solutions were thawed and diluted with the corresponding complete culture medium to achieve the desired final working concentrations prior to cell treatment. All treatment groups, including the vehicle control, were maintained at a final concentration of 0.1% (v/v, equivalent to one part per thousand) of DMSO. The cells were subjected to the following treatments for 48 hours after they reached approximately 70%-80% confluence: DMSO group (culture medium containing 0.1% DMSO); oroxylin A group (culture medium containing 50 μM oroxylin A); donafenib group (culture medium containing 12.5 nM donafenib; oroxylin A + donafenib group (culture medium containing 50 μM oroxylin A and 12.5 nM donafenib). The culture medium was replaced with the corresponding drug-containing or control medium during the treatment period.

HCC transplantation tumor model in mice

The specific pathogen free grade, immunodeficient BALB/c nude mice were purchased from Changsha Yizhixing Biotechnology Co., Ltd. (Hunan Province, China). The mice were maintained in sterile cages and standard environments (22 ± 3 °C, 40%-55% humidity, and 12 hours light/dark cycle). All mice underwent one week of adaptive feeding before the start of experiment. The subcutaneous HCC transplantation model was established by injecting 0.2 mL of MHCC-97H cells (5 × 10⁶ cells/mL) into the armpit of the right forelimb of each mouse approximately 0.3-0.5 cm from the back. The data were recorded as d0 (before drug treatment), and fluorescence live imaging was performed on the nude mice seven days after tumor formation. All mice were randomly assigned to one of four groups (n = 8 per group, four males and four females) using the random number table method: Placebo control, oroxylin A, donafenib, or oroxylin A + donafenib. For 21 days, mice were administered placebo (0.2% DMSO), oroxylin A (30 mg/kg), donafenib (50 mg/kg), or a combination of oroxylin A (30 mg/kg) and donafenib (50 mg/kg) every two days for 21 days. Live imaging was conducted on mice on the 7^th (d7), 14^th (d14), and 21^st (d21) days of the administration period. The mice were euthanized and dissected after live imaging was completed on d21. Tumor volume was measured using a caliper, and the tumor was weighed by two investigators who blind to the experimental groups. The study was approved by the Animal Ethics Committee of International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University School of Medicine (Approval No. 22-12-22).

Drug safety assessment in vivo

Blood samples (100 μL) were obtained from the tail vein of mice on days of d0, d7, d14, and d21. The samples were centrifuged at 3000 rpm for 20 minutes after being placed in Eppendorf (EP) tubes at room temperature for 2 hours. The resulting serum was carefully aspirated and transferred to a clean 1.5 mL EP tube with a pipette for subsequent analysis. The concentration of ALT, AST, Cr, and blood urea nitrogen (BUN) in mice before and after administration were measured using corresponding kit and microplate reader.

Half maximal inhibitory concentration determination

The half maximal inhibitory concentration (IC₅₀) values for oroxylin A and donafenib were determined using a CCK-8 assay. The cells were seeded in 96-well plates and allowed to adhere overnight. Subsequently, the culture medium was replaced with a fresh medium containing serially diluted oroxylin A (0, 2.5, 5, 10, 15, 20, 25, 37.5, 50, 62.5, 80, 105, 150, 200, 300, 500, 750, and 1000 μM) or donafenib (0, 1, 2, 3, 4, 5, 6, 7.5, 10, 12.5, 15, 20, 30, 50, 75, 100, 125, and 150 nM). The final DMSO concentration was ≤ 0.1% in all wells. At 37 °C, the plates were incubated for an additional 2 hours after the addition of 10 μL of CCK-8 reagent to each well after a 24 hours incubation period. The optical density at 450 nm was then measured with a microplate reader. Cell viability was normalized to the untreated control cells, and the IC₅₀ values were calculated by fitting the dose-response data to a four-parameter logistic model with GraphPad Prism software (version 8.0).

Cell proliferation assay

The cell proliferation ability was assessed using the CCK-8 assay. With a final concentration of 0.75 × 10⁴ cells/well, HCC cells (1 × 10⁵/mL) were seeded into 96-well plates at a concentration of 100 μL per well. The culture media containing drugs or DMSO control was added, and the cells were cultured for an additional 48 hours. After 72 hours, 10 μL of CCK-8 solution (Beyotime, #C0038, China) was added to the cells, which were then incubated for 1 hour. The OD value was assessed at 450 nm.

Colony formation assay

HCC cells were inoculated into six-well plates at a density of 200 cells per well and cultured overnight in standard conditions (37 °C, 5% CO₂, and 80% humidity). The drug-containing complete medium was refreshed every two days for a period of two weeks after cell attachment was confirmed the following day. The cells were washed with PBS and stained with 0.1 % crystal violet solution for 15 minutes until a visible cell colony formed. The colonies were imaged, and the number of colonies was quantified under a microscope after several PBS washes. A cluster of more than 50 cells was considered a colony.

Cell apoptosis assay

Cell apoptosis was induced through flow cytometry and detected using an annexin V-FITC/PI apoptosis detection kit (Elabscience, E-CK-A211, China) in accordance with the manufacturer’s instructions. All experiments were repeated three times.

Immunohistochemical examination

The hospital professional technicians used standardized processing procedures to obtain the tumor and adjacent tissue samples, which were subsequently processed into paraffin tissue chip slices by Aifang Technology Biotechnology Company. A TSA multiplex fluorescence staining kit (#AFIHC037) was used in immunohistochemistry experiments after the acquisition of the tissue chip.

The Cancer Genome Atlas

RNA-sequencing expression (level 3) profiles and corresponding clinical information for HCCwere downloaded from The Cancer Genome Atlas (TCGA) dataset (https://portal.gdc.com). The expression distribution of tumor protein p53 (TP53), cyclin-dependent kinase 9 (CDK9), cell division cycle 7 (CDC7), murine double minute 2 (MDM2), MDM4, and B-Raf proto-oncogene serine/threonine kinase (BRAF). The Wilcox test was used to compare the statistical differences between the two groups, and the results are presented as the mean ± SD. The Pearson χ² test or Fisher’s exact test was employed to analyze the relationship between clinical characteristic variables and TP53, CDK9, CDC7, MDM2, and MDM4.

Knockdown of target genes in HCC cell lines

Genepharma, Shanghai, supplied all small interfering RNAs (siRNAs) and negative controls. A density of 1.0 × 10⁵ cells per well was used to seed cells into 12-well plates, and the cells were cultured overnight. Then, 100 μL of Opti-MEM was combined with 7.5 μL of Lipo RNAi MAX and 100 pM siRNA, respectively. Each mixture was incubated at room temperature for 5 minutes. Finally, the two mixtures were gently combined and incubated at room temperature for an additional 20 minutes. Subsequently, the original culture medium in the 12-well plate was aspirated and replaced with 1 mL of fresh serum-free basal medium. The cells were incubated at 37 °C under 5% CO₂ for 6 hours after the siRNA-lipofectamine complexes were added. The medium was replaced with complete medium containing serum after this period, and the cells were cultured for an additional 24 hours to facilitate the knockdown of the target gene. The cells were subsequently incubated for an additional 24 hours after the drugs were directly administered into the culture medium. Cells were harvested and subjected to western blot analysis following treatment to evaluate the expression levels of pertinent proteins. Supplementary Table 1 contains the siRNA sequences employed in this investigation.

Quantitative real-time polymerase chain reaction

The RNA-easy Isolation Reagent (Qiagen, #74034, Germany) was employed to isolate total RNA from HCC cells in accordance with the manufacturer’s protocol. NanoDrop (ThermoFisher Scientific, MA, United States) was employed to quantify the RNA concentration. Subsequently, the Invitrogen cDNA synthesis kit (Invitrogen, #18080-051, CA, United States) was employed to synthesize cDNA from 1 μg of total RNA. Quantitative real-time polymerase chain reaction (RT-qPCR) was conducted in a 20 μL reaction system with a TaKaRa kit (TaKaRa, #RR420A, Japan). The relative gene expression levels were quantified using the 2^-ΔΔCt method and normalized for glyceraldehyde-3-phosphate dehydrogenase. All primer sequences are listed in Supplementary Table 2.

Western blot

Protein was extracted using the radio-immunoprecipitation assay lysis buffer. The protein concentration was determined with the bicinchoninic acid assay method. Subsequently, equal amounts (30 μg) of protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was then blocked with 3% BSA at room temperature for 1 hour, and the primary antibodies were incubated at 4 °C overnight. The membrane was incubated with a corresponding secondary antibody for 1 hour at room temperature and washed again under the same conditions after being washed three times with tris-buffered saline with Tween 20. The protein bands were imaged with an eBlot Touch Imager (eBlot, #XLi, Shanghai) and visualized using an enhanced chemiluminescence solution (Thermo, #34580, MA, United States). All antibodies used are listed in Supplementary Table 3.

Statistics analysis

All data are presented as the mean ± SD of three independent experiments. Statistical analysis was performed using GraphPad Prism (version 8.0). Comparisons of multiple groups for a single independent variable were performed through repeated-measures analysis of variance, and Tukey’s post hoc test was employed for pairwise comparisons. The log-rank test. A P value of < 0.05 was considered statistically significant, and specific levels were denoted as follows: ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001.

RESULTS

Synergistic antitumor effect of oroxylin A and donafenib in HCC in vivo

To evaluate the potential synergistic antitumor effect of oroxylin A and donafenib against HCC in vivo, a 21-day combined administration regimen was implemented in male and female xenograft mice (Figure 1A). The experimental groups did not exhibit any substantial differences in terms of body size, skin condition, or general health (Figure 1B). The combination treatment group exhibited a substantial decrease in tumor weight and volume compared to the oroxylin A or donafenib monotherapy groups, as well as the placebo group, as evidenced by in vivo fluorescence imaging over the 21 days period (Figure 1C and D). The absence of sex-dependent differences in treatment response was suggested by the consistent therapeutic effect of oroxylin A and donafenib in male and female mice (Figure 1E and F). Venous blood was collected from mice during in vivo imaging to evaluate the potential effects on liver and kidney function following the co-administration. The serum levels of ALT, AST, Cr, and BUN were assessed. The results indicated that the combination treatment did not result in any substantial changes in any of these biochemical indices, with all values remaining within the normal physiological range (Figure 1G-J, Table 1). Collectively, these findings indicate that the combination of oroxylin A and donafenib is a viable and promising therapeutic approach for the treatment of HCC.

Figure 1 Synergistic antitumor effect of oroxylin A and donafenib in hepatocellular carcinoma in vivo. A: Schematic illustration of the in vivo experimental procedures in a rodent model; B: Representative photographs showing the general status of mice in each group; C-F: In vivo fluorescence images of tumors in female (C) and male (E) mice at 7 days, 14 days, and 21 days post-treatment, enabling non-invasive, longitudinal, and sex-specific monitoring of tumor progression. Tumor volume and weight were measured in female (D) and male (F) mice after surgical resection at the endpoint (day 21); G-J: Serum levels of alanine aminotransferase (G), aspartate aminotransferase (H), blood urea nitrogen (I) and creatinine (J) were quantified every 7 days following treatment with oroxylin A and/or donafenib. Data are presented as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. PC: Placebo control; OA: Oroxylin A; DF: Donafenib; OA + DF: Oroxylin A + donafenib; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BUN: Blood urea nitrogen; Cr: Creatinine.

Table 1 the concentration of alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen and creatinine in each group after treatment with oroxylin A and/or donafenib.

Index	Treatment	d0	d7	d14	d21
ALT (U/L)	Control	21.02	20.03	25.12	21.75
	Oroxylin A	19.64	19.89	22.84	22.14
	Donafenib	24.14	19.70	21.79	24.58
	Oroxylin A + donafenib	26.87	21.91	21.79	20.00
AST (U/L)	Control	104.83	114.41	107.77	117.92
	Oroxylin A	105.37	115.67	102.81	121.66
	Donafenib	99.10	113.13	101.34	112.74
	Oroxylin A + donafenib	115.21	110.32	111.39	126.61
BUN (μg/mL)	Control	153.75	183.21	142.78	153.50
	Oroxylin A	165.93	182.93	149.81	138.22
	Donafenib	166.48	168.75	148.67	149.67
	Oroxylin A + donafenib	154.05	161.61	154.82	136.39
Cr (μmol/L)	Control	22.90	23.98	28.58	26.99
	Oroxylin A	26.49	21.97	27.01	28.91
	Donafenib	22.00	26.56	28.58	26.72
	Oroxylin A + donafenib	24.40	23.69	29.36	28.64

d0: Before drug treatment; d7: Drug treatment for 7 days; d14: Drug treatment for 14 days; d21: Drug treatment for 21 days; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BUN: Blood urea nitrogen; Cr: Creatinine.

Synergistic anti-proliferative and pro-apoptotic effects of oroxylin A and donafenib on HCC cells

The IC₅₀ of oroxylin A and donafenib in the MHCC-97H and PLC-PRF-5 cell lines was assessed. The IC₅₀ values of oroxylin A against MHCC-97H and PLC-PRF-5 cells were 58.36 ± 2.07 μM and 53.86 ± 2.61 μM, respectively, as indicated by the CCK-8 results (Figure 2A). Donafenib’s IC₅₀ values were 11.36 ± 0.9 nM and 11.67 ± 0.89 nM (Figure 2B). In accordance with the in vivo results, the combined treatment of oroxylin A and donafenib suppressed cell proliferation (Figure 2C) and colony formation ability (Figure 2D and E), while simultaneously increasing cell apoptosis (Figure 2F and G). Notably, the combination therapy demonstrated a stronger anti-proliferative and pro-apoptotic effects than two monotherapies in HCC cells.

Figure 2 Synergistic anti-proliferative and pro-apoptotic effects of oroxylin A and donafenib on hepatocellular carcinoma cells. A and B: The half-maximal inhibitory concentrations (IC₅₀) of oroxylin A (A) and donafenib (B) were determined in MHCC-97H and PLC-PRF-5 cell lines; C-E: MHCC-97H and PLC-PRF-5 cell proliferation were detected using Cell Counting Kit-8 assay (C) and colony formation assay (D and E) after treatment with oroxylin A and/or donafenib; F and G: Cell apoptosis was assessed using flow cytometry after treatment with oroxylin A and/or donafenib in MHCC-97H (F) and PLC-PRF-5 (G) cell lines. Data are presented as mean ± SD. ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. DMSO: Dimethyl sulfoxide; OA: Oroxylin A; DF: Donafenib; OA + DF: Oroxylin A + donafenib.

Synergistic activation of the TP53 signaling pathway by oroxylin A and donafenib in HCC cells

MHCC-97H and PLC-PRF-5 cells were treated with drugs for 24 hours to elucidate the mechanism that underlies the synergistic lethality of the oroxylin A and donafenib combination. The mRNA and protein expression of TP53 signaling-related regulators (MDM2, MDM4, BRAF, CDK9, and VEGFR) were subsequently analyzed using RT-qPCR and western blot. TP53 mRNA expression was significantly elevated by the combination treatment relative to that in the control and monotherapy groups (Figure 3A and B). Furthermore, oroxylin A primarily reduced the mRNA expression levels of MDM2, MDM4, and CDK9, whereas donafenib decreased the mRNA expression of BRAF and VEGFR (Figure 3A and B). CDK9 mRNA levels were decreased by oroxylin A and donafenib (Figure 3A and B). The western blot results indicated that the protein expression of TP53 and CDC7 was increased by the combination of oroxylin A and donafenib, and the protein expression of pMDM2, MDM2, pMDM4, MDM4, BRAF, and VEGFR decreased. Additionally, the mRNA expression of BRAF and VEGFR was decreased by donafenib (Figure 3C). These results collectively suggest that the TP53 signaling pathway is synergistically activated as a result of the combination treatment, which significantly alters the expression of TP53-associated proteins in HCC cells.

Figure 3 Synergistic activation of the TP53 signaling pathway by oroxylin A and donafenib in hepatocellular carcinoma cells. A and B: The mRNA expression of tumor protein p53, murine double minute 2 (MDM2), MDM4, cyclin-dependent kinase 9, B-rapidly accelerated fibrosarcoma proto-oncogene serine/threonine kinase, vascular endothelial growth factor receptor, and cell division cycle 7 in MHCC-97H (A) and PLC-PRF-5 (B) cells was measured using quantitative real-time polymerase chain reaction after treatment with oroxylin A and/or donafenib; C: The protein levels of tumor protein p53, MDM2, MDM4, phosphorylated MDM2, phosphorylated MDM4, cyclin-dependent kinase 9, and B-rapidly accelerated fibrosarcoma proto-oncogene serine/threonine kinase, vascular endothelial growth factor receptor, and cell division cycle 7 were examined using Western blot analysis in both cell lines following treatment with oroxylin A and/or donafenib. Data are presented as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001, ns: P > 0.05. DMSO: Dimethyl Sulfoxide; OA: Oroxylin A; DF: Donafenib; OA + DF: Oroxylin A + donafenib; MDM2: Murine double minute 2; MDM4: Murine double minute 4; CDK9: Cyclin-dependent kinase 9; TP53: Tumor protein p53; CDC7: Cell division cycle 7; BRAF: B-rapidly accelerated fibrosarcoma proto-oncogene serine/threonine kinase; VEGFR: Vascular endothelial growth factor receptor; p-MDM2: Phosphorylated murine double minute 2; p-MDM4: Phosphorylated murine double minute 4.

Upregulation of TP53, MDM2, MDM4, and CDK9 correlates with poor prognosis in patients with HCC

In tumor tissues, immunohistochemical analysis of the tissue microarrays of patients with HCC revealed the positive expression of CDK9, MDM2, pMDM2, MDM4, and pMDM4, whereas adjacent normal tissues exhibited negative or minimal expression for these markers (Figure 4A). In agreement with these results, the mRNA expression profiles from the TCGA and Gene Expression Omnibus (GEO) databases were analyzed, revealing a substantial increase in the expression of TP53, MDM2, and CDK9 in HCC tissues in comparison to normal controls (Figure 4B). Conversely, no discernible difference in MDM4 expression was found between the tumor and normal samples (Figure 4B). Additionally, the OS of HCC patients was significantly diminished by the elevated expression levels of TP53, MDM2, MDM4, and CDK9 (Figure 4C).

Figure 4 Upregulation of tumor protein p53, murine double minute 2, murine double minute 4, and cyclin-dependent kinase 9 correlates with poor prognosis in hepatocellular carcinoma patients. A: The expression of murine double minute 2 (MDM2), phosphorylated MDM2, MDM4, phosphorylated MDM4, cell division cycle 7 (CDC7), and cyclin-dependent kinase 9 (CDK9) in clinical hepatocellular carcinoma (HCC) specimens was assessed using immunohistochemical analysis; B: The mRNA levels of tumor protein p53, MDM2, MDM4, CDC7, CDK9, and B-rapidly accelerated fibrosarcoma proto-oncogene serine/threonine kinase were analyzed in HCC patients using datasets from The Cancer Genome Atlas and Gene Expression Omnibus repositories; C: Kaplan-Meier survival analysis were performed based on the expression of tumor protein p53, MDM2, MDM4, CDC7, CDK9, and B-rapidly accelerated fibrosarcoma proto-oncogene serine/threonine kinase and their regulatory proteins, showing a significant correlation with overall survival in HCC patients. Data are presented as mean ± SD. ^bP < 0.01, ^dP < 0.0001, ns: P > 0.05. CDC7: Cell division cycle 7; CDK9: Cyclin-dependent kinase 9; MDM2: Murine double minute 2; TP53: Tumor protein p53; MDM4: Murine double minute 4; BRAF: B-rapidly accelerated fibrosarcoma proto-oncogene serine/threonine kinase.

Oroxylin A and donafenib activate TP53 signaling via the CDK9-MDM2/MDM4 and VEGFR/BRAF axes, respectively

To investigate the underlying molecule mechanism of oroxylin A and donafenib, MHCC-97H and PLC-PRF-5 HCC cells were treated with increasing concentrations of oroxylin A/donafenib and transfected with siRNAs targeting CDK9, MDM4, and MDM2, respectively. Western blot results showed that transfection of siRNAs effectively downregulated the protein levels of CDK9 (Figure 5A), MDM4 (Figure 6A), and MDM2 (Figure 7A) in two HCC cell lines. CDK9 knockdown augmented oroxylin A-induced decrease in MDM2/MDM4 and pMDM2/MDM4 expression, but did not affect BRAF or VEGFR levels (Figure 5A). Furthermore, CDK9 knockdown rescued the oroxylin A-induced upregulation of TP53 (Figure 5A), but did not alter the donafenib-induced increase in TP53 or decrease in VEGFR/BRAF (Figure 5B). Further investigations have demonstrated that knockdown of either MDM2 or MDM4 had no effect on the oroxylin A-induced upregulation of CDK9 but effectively prevented the oroxylin A-mediated activation of TP53 (Figures 6A and 7A). In contrast, the donafenib-induced suppression of VEGFR/BRAF was unaffected by MDM2 or MDM4 knockdown (Figures 6B and 7B). Collectively, oroxylin A primarily targeted the CDK9-MDM2/MDM4 axis to stabilize TP53, while donafenib suppressed the VEGFR/BRAF axis to activate TP53.

Figure 5 Knockdown of cyclin-dependent kinase 9 affects oroxylin A/donafenib-induced dysregulation of tumor protein p53 signaling-related proteins. A and B: The protein expression of tumor protein p53, cell division cycle 7, cyclin-dependent kinase 9, murine double minute 2 (MDM2), phosphorylated MDM2, MDM4, phosphorylated MDM4, B-rapidly accelerated fibrosarcoma proto-oncogene serine/threonine kinase, and vascular endothelial growth factor receptor was detected using western blot analysis after the genetic silencing of cyclin-dependent kinase 9 and exposure to oroxylin A (A) or donafenib (B) for 24 hours in MHCC-97H and PLC-PRF-5 cell lines. OA: Oroxylin A; DF: Donafenib; CDK9: Cyclin-dependent kinase 9; BRAF: B-rapidly accelerated fibrosarcoma proto-oncogene serine/threonine kinase; CDC7: Cell division cycle 7; MDM4: Murine double minute 4; p-MDM4: Phosphorylated murine double minute 4; MDM2: Murine double minute 2; p-MDM2: Phosphorylated murine double minute 2; TP53: Tumor protein p53; VEGFR: Vascular endothelial growth factor receptor.

Figure 6 Knockdown of murine double minute 4 affects oroxylin A/donafenib-induced dysregulation of tumor protein p53 signaling-related proteins. A and B: The protein expression of tumor protein p53, cell division cycle 7, cyclin-dependent kinase 9, murine double minute 2 (MDM2), phosphorylated MDM2, MDM4, phosphorylated MDM4, B-rapidly accelerated fibrosarcoma proto-oncogene serine/threonine kinase, and vascular endothelial growth factor receptor was detected using western blot analysis after the genetic silencing of cyclin-dependent kinase 9 and exposure to oroxylin A (A) or donafenib (B) for 24 hours in MHCC-97H and PLC-PRF-5 cell lines. OA: Oroxylin A; DF: Donafenib; MDM4: Murine double minute 4; BRAF: B-rapidly accelerated fibrosarcoma proto-oncogene serine/threonine kinase; CDC7: Cell division cycle 7; CDK9: Cyclin-dependent kinase 9; p-MDM4: Phosphorylated murine double minute 4; MDM2: Murine double minute 2; p-MDM2: Phosphorylated murine double minute 2; TP53: Tumor protein p53; VEGFR: Vascular endothelial growth factor receptor.

Figure 7 Knockdown of murine double minute 2 affects oroxylin A/donafenib-induced dysregulation of tumor protein p53 signaling-related proteins. A and B: The protein expression of tumor protein p53, cell division cycle 7, cyclin-dependent kinase 9, murine double minute 2 (MDM2), phosphorylated MDM2, MDM4, phosphorylated MDM4, B-rapidly accelerated fibrosarcoma proto-oncogene serine/threonine kinase, and vascular endothelial growth factor receptor was detected using western blot analysis after the genetic silencing of cyclin-dependent kinase 9 and exposure to oroxylin A (A) or donafenib (B) for 24 hours in MHCC-97H and PLC-PRF-5 cell lines. OA: Oroxylin A; DF: Donafenib; MDM2: Murine double minute 2; BRAF: B-rapidly accelerated fibrosarcoma proto-oncogene serine/threonine kinase; CDC7: Cell division cycle 7; CDK9: Cyclin-dependent kinase 9; MDM4: Murine double minute 4; p-MDM4: Phosphorylated murine double minute 4; p-MDM2: Phosphorylated murine double minute 2; TP53: Tumor protein p53; VEGFR: Vascular endothelial growth factor receptor.

DISCUSSION

The present study is the first to evaluate the effect and underlying mechanism of oroxylin A in conjunction with donafenib in the management of HCC in laboratory settings and living organisms. The investigation began with the development of a mouse HCC transplantation tumor model to facilitate in vivo animal experimentation. Subsequently, in vitro assessments targeting cell proliferation, monoclonal formation, and facilitation of apoptosis were conducted across three distinct HCC cell lines. The mechanism was determined through immunohistochemical evaluation, RT-qPCR, western blot analysis, and the silencing of target genes within the HCC cell lines and by exploring TCGA and GEO databases.

The synergistic anticancer effect of the combined treatment of oroxylin A and donafenib was demonstrated on the HCC mouse xenograft model. This combination effectively amplified the suppression of solid tumor growth, showing no discernible differences between genders and demonstrating a favorable safety profile. The IC₅₀ values of oroxylin A and donafenib were within 46.8-58.36 μM and 11.09-11.67 nM, respectively, in the cell culture experiments. The synergistic effects of oroxylin A and donafenib in suppressing cell proliferation, colony formation, and inducing apoptosis in the HCC cell lines were superior to the effects observed with individual treatments of oroxylin A or donafenib. Furthermore, the TP53 signaling pathway was found as a promising target for anti-HCC therapy through an immunohistochemical assessment of liver cancer and adjacent tissues from HCC patients, in conjunction with analyses from TCGA and GEO. Oroxylin A inhibits HCC progression through the CDK9-MDM2/MDM4-TP53 axis, as demonstrated by subsequent investigations using RT-qPCR, western blotting, and gene knockdown experiments in HCC cell lines. Conversely, donafenib achieves its anticancer effects by targeting the CDK9-BRAF/VEGFR-TP53 axis. Contributing to the anti-angiogenic effects and suppression of tumor cell proliferation collectively.

Cellular mutations, epigenetic changes, and changes in copy numbers are the primary factors contributing to the development of HCC at the molecular level[19]. These reactive biomarkers are essential in the complex signal transduction pathways of targeted therapy and resistance mechanisms in HCC. This study explores the primary targets of the combination of oroxylin A and donafenib in order to uncover the underlying functions and effects within the mechanism of action.

The p53 protein, which contains 393 amino acid residues, is encoded by the TP53 gene, which is located on the short arm of chromosome 17 (17p13.1)[20]. The actual molecular weight of p53 is 43.7 kDa[21], containing an N-terminal transactivation domain (residue 1-61), a proline rich domain (proline rich domain, residue 61-92), a DNA binding domain (residue 94-292) connected to a tetrameric domain (residue 326-353), and a C-terminal regulatory domain (residues 353-390) structurally[22,23]. The connecting bodies between DNA-binding domain and tetrameric domain, as well as transactivation domain and C-terminal regulatory domain, comprise over 40% of the regions in p53 that are essentially disordered[24]. As a modular protein, p53 interacts with a diverse array of chaperone proteins owing to these unordered regions[24]. P53 is located in the nucleus and cytoplasm, where it specifically binds to DNA and regulates numerous genes[25,26]. The level of p53 protein in cells is extremely low under normal conditions as a result of the strict regulation of negative regulatory factors MDM2 and MDM4, which promote p53 degradation through ubiquitination[27,28]. Upon exposure to tumor cells, p53 ubiquitination is inhibited, resulting in a rapid increase in intracellular p53 protein levels[21]. Post-translational modifications (such as phosphorylation, acetylation, and methylation) are employed to activate and stabilize the accumulated p53[21]. The tetramers of the stable p53 are formed in the nucleus, where they bind to target DNA and regulate gene transcription, resulting in modifications to downstream signaling pathways[29-33]. As a response to cellular stress, p53 activates various genes involved in cell apoptosis and cell cycle by transcription, preventing cell differentiation with DNA mutated or impaired, and terminating cellular processes[34,35], thereby inhibiting the growth of tumor cells[29-33,36-40]. Meanwhile, p53 regulates numerous “non-classical” pathways, including metabolic homeostasis, iron removal, stem cell differentiation, autophagy, aging, and the tumor microenvironment[41-46]. The TP53 gene typically undergoes mutations or losses in HCC cells because of the absence of chromosome 17[47,48]. The mutant-type p53 protein (mt-p53) that is produced when TP53 undergoes missense mutations is responsible for the promotion of tumor transformation[49,50]. mt-p53 can induce the loss of wild-type p53 (wt-p53) function, dominant negative inhibition of wt-p53, and the acquisition of oncogenic functions[51]. mt-p53 affects various cellular responses, including genomic instability, metabolic reprogramming, and the tumor microenvironment. It also promotes the proliferation, invasion, metastasis, and drug resistance of cancer cells[52].

CDK9 is a member of the serine/threonine kinase family and is dependent on cyclin. CDK9 has a molecular weight of approximately 42 kDa and is composed of an N-terminus [five β structures (β 1-5) and one α Spiral (αC), residues 16-108] and a C-terminus [seven α-Spiral (α D-J) and four β chain (β 6-9), residues 109-330][53]. The N-terminus contains an ATP binding site and an activated T ring that contains a critical T186 residue[54]. The activity of CDK9 and its interaction with cyclin T are determined by the cis self-phosphorylation of T186 and three C-terminal phosphorylation sites[55-57]. Proper regulation of CDK9 kinase activity is crucial for maintaining transcriptional homeostasis in cells. CDK9 binds to cyclin T and forms a p-TEFb complex, and then CDK9 acts as a catalytic subunit of p-TEFb to phosphorylate RNA polymerase II (RNA Pol II) at the Ser2 site and regulates RNA transcription elongation[54]. CDK9 also plays a crucial role in the repair of damaged DNA[58] and interacts with other cytokines, including mammalian rapamycin target, anti-silencing function of 1B histone chaperone, signal transduction and transcription 3, and bromine containing domain protein 4[59]. In the past decade, the mis-localization and abnormalities in CDK9 expression and activation can promote abnormal transcription and DNA repair programs, leading to tumor progression[60]. The activated CDK9 in the p-TEFb complex can promote tumor cell proliferation, anti-apoptosis, migration, angiogenesis, metabolism, and stemness through the transcriptional regulation of target genes[59]. The current research has demonstrated that CDK9 is highly expressed in HCC and has the ability to phosphorylate MDM2 at the Ser166 site, thereby promoting the degradation of wt-p53 and reducing the stability of the wt-p53 protein[61].

The BRAF protein, which is involved in the MAP kinase/ERK signaling pathway, is encoded by the BRAF gene, which is located on chromosome 7q34[62]. BRAF is a protein with a molecular weight of approximately 69 kDa. It is composed of three conserved domains: Conserved region-1, which is composed of amino acids 150-290 and contains the RAS binding domain (amino acids 155-227); conserved region-2, which is composed of amino acids 360-375 and is a hinge region rich in serine; and conservative region-3, which is also known as the BRAF kinase domain, is composed of amino acids 457-717 and can phosphorylate common sequences on protein substrates[63-66]. BRAF is activated in normal cells by binding to RAS, which is stimulated by a growth factor. This binding sends signals to downstream kinases MEK and ERK through a series of continuous phosphorylation and activation events. Subsequently, the activated ERK phosphorylates a variety of cytoplasmic and nuclear proteins to promote the proliferation, differentiation, and survival of normal cells[67,68]. Approximately 200 BRAF mutation alleles have been identified in human tumors, and nearly 30 distinct BRAF mutations have been functionally characterized to date[44]. BRAF can be classified into three categories based on the activity: In contrast, type 1 BRAF mutants function as active monomers and are not dependent on the stimulation of the upstream regulatory factor RAS GTPase. Type 2 BRAF mutants are also active dimers and are not dependent on RAS GTPase stimulation. However, type 3 BRAF mutants require RAS signaling for optimal activation[68-70]. The abnormally high expression of the BRAF cascade in the pathogenesis of HCC regulates the downstream MEK/ERK pathway, promotes tumor cell proliferation, invasion, and metastasis, as well as cell death through apoptosis[71-73]. Additionally, it facilitates the expression of matrix metalloproteinases, alters the adhesion of tumor cells, degrades the extracellular matrix and basement membrane, and facilitates tumor invasion and metastasis[72,73].

VEGFR is a tyrosine kinase receptor that is typical in its structure. It is composed of three components: An extracellular vascular endothelial growth factor (VEGF) binding region with seven immunoglobulin-like domains, a transmembrane domain of the receptor, and an intracellular signaling domain that includes tyrosine activation structures[74,75]. VEGFRs have three primary types. VEGFR1 and VEGFR2 are mainly expressed in vascular endothelial cells and responsible for angiogenesis and regeneration. By contrast, VEGFR3 is primarily expressed in endothelial lymphocytes, where it is the primary factor in the generation of lymphatic vessels[76,77]. Upon binding to its ligand VEGFs, VEGFR undergoes receptor homodimerization or heterodimerization, which results in the activation of tyrosine kinase and the self-phosphorylation of tyrosine residues in the receptor’s intracellular domain. Subsequently, the phosphorylated tyrosine and surrounding amino acid residues establish binding sites for connecting molecules, thereby initiating a variety of intracellular signaling pathways. These pathways ultimately lead to the growth, proliferation, and maturation of endothelial cells and the formation of new blood vessels[78]. The primary pro-angiogenic signal is produced by ligand-activated VEGFR2 given that VEGFR1 has a high affinity for VEGF that is one order of magnitude higher than that of VEGFR2. However, its tyrosine kinase activity is approximately ten times weaker than that of VEGFR2[79]. The body’s mechanism of angiogenesis is tightly regulated by the equilibrium between the pro-angiogenic and anti-angiogenic factor groups. In pathological conditions, this equilibrium is disrupted, and angiogenesis is not suppressed, resulting in an excessive proliferation of blood vessels. Effective angiogenesis is essential for the regeneration of cells in the liver, which is a highly vascularized organ[80]. In HCC, angiogenesis, which is mediated by VEGF, plays a clear regulatory role in the progression of the tumor[81]. The expression of VEGF and VEGFRs (including VEGF-1, -2, and -3) has been observed to increase in HCC cell lines and tissues and in sera from patients with HCC[80,82-84]. The liver tumors of the majority of patients with HCC exhibited an elevated expression of VEGF mRNA, which is linked to a low survival rate and tumor progression[85]. VEGFR-3 upregulation is also associated with hepatitis B antigen x[86]. Additionally, the high expression of VEGF is typically associated with disease recurrence, vascular invasion, portal vein thrombosis, and a poor prognosis following resection[85].

This research is unique in that it is the first to investigate the impact and operational method of utilizing oroxylin A in conjunction with donafenib for the treatment of HCC in vitro and in vivo settings. Additionally, this investigation identifies the TP53 signaling pathway as a prospective target of donafenib, thereby opening up novel pathways for the development of anti-HCC medications. Furthermore, it reveals the synergistic inhibitory effect of oroxylin A from Scutellaria, a TCM, when combined with donafenib against HCC. Consequently, it introduces new perspectives on the integration of TCM into the clinical management of HCC and proposes novel therapeutic strategies for HCC treatment in clinical practice. Finally, the research offers preliminary insights into the molecular interactions between oroxylin A and donafenib and their impact on the TP53 signaling pathway through in vitro cellular experiments, thereby establishing a new theoretical foundation for future clinical trials. The study under discussion is subject to a number of limitations that require careful consideration. The primary objective of the in vivo experiments conducted in this research was to observe the inhibitory effects and safety of drug treatment on solid tumors in HCC xenograft mice over a 21-day period. However, the potential long-term effects of the drugs administered are not taken into account during this timeframe. Consequently, future research should aim to address this gap in understanding. Second, the current body of literature has demonstrated that BRAF inhibitor-targeted therapy is effective only for types 1 and 2 mutants, and type 3 mutants, which are dependent on RAS signal transduction, do not exhibit comparable results[68]. Regrettably, the mechanism elucidated in this study does not delve into the distinct mutations of BRAF, requiring additional research to examine the effects of the combination of oroxylin A and donafenib on a variety of BRAF mutants. Finally, the investigation of the mechanism that underpins the combined treatment of oroxylin A and donafenib was restricted to in vitro environments. It is essential to validate the efficacy of this combination in an in vivo setting in future research endeavors, as the efficacy of drugs can be influenced by factors such as metabolism in vivo.

CONCLUSION

Our findings demonstrate that oroxylin A and donafenib exert a synergistic anti-tumor effect in HCC by co-activating the TP53 signaling pathway, providing new experimental evidence for drug development for HCC. This combination strategy presents a promising and viable therapeutic approach to overcome the limitations of donafenib monotherapy in the treatment of HCC.