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. 2025 Apr 29;33(1-2):6. doi: 10.1007/s44446-025-00001-x

Enhancing hepatocellular carcinoma treatment: synergistic cytotoxicity and mechanistic insights of Ramucirumab and 5-Azacytidine combination therapy

Hadeel Almasoud 1,✉, Fares A Alzahrani 1, Saud Alarifi 1, Badr Aldahmash 1, Bader Almutairi 1, Abdullah A AlKahtane 1, Khadijah N Yaseen 1, Bashayer Aljuhani 1, Saad Alkahtani 1
PMCID: PMC12102452  PMID: 40397219

Abstract

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality, with limited therapeutic options and poor prognosis for advanced stages. This study investigates the synergistic anticancer effects of Ramucirumab (RAM), a VEGFR-2 inhibitor, and 5-Azacytidine (5-Aza), a hypomethylating agent, on HCC cells, focusing on mechanisms of cytotoxicity, DNA damage, apoptosis, and cell cycle modulation. HuH-7 cells were treated with RAM and 5-Aza, alone and in combination, across varying concentrations. Cell viability was assessed using the Neutral Red Uptake assay, while DNA damage and apoptosis were evaluated through the TUNEL assay and protein array analysis. The expression of cell cycle and inflammatory genes was analyzed using quantitative real-time PCR (qRT-PCR). Result shows combination treatment significantly enhanced cytotoxicity compared to individual dose-dependent therapies. DNA damage was markedly increased in RAM-treated cells, with upregulation of apoptotic proteins CAS3, BID, BAD, p53, and FAS observed. In contrast, apoptotic proteins were markedly decreased in combination-treated cells. Cell cycle arrest was evident through the downregulation of key regulatory genes, including MCM2, MCM3, cyclin B1, and CDK2. Inflammatory cytokines IL-1β and IL-6 were repressed, while COX2 expression was elevated, suggesting oxidative stress as a possible mechanism. In conclusion, the synergistic effects of RAM and 5-Aza in HCC cells are mediated through increased damage to DNA, apoptosis, and arrest of cell cycle, offering potential treatment strategy for advanced HCC. Further experiments conducted in vivo are warranted to validate these findings and optimize treatment regimens.

Keywords: Hepatocellular Carcinoma, Ramucirumab, 5-Azacytidine, Apoptosis, Cell Cycle Arrest, Oxidative stress

Introduction

Liver cancer continue to be a major contributor of cancer related mortality worldwide, with hepatocellular carcinoma (HCC) accounting for approximately 80% of cases (Sayiner et al. 2019). Despite advances in treatment strategies, the prognosis for advanced HCC remains dismal due to its aggressive nature and limited therapeutic options. Standard treatments such as surgical resection, ablation, and systemic therapies, including targeted agents like sorafenib, often yield suboptimal outcomes (Bosi et al. 2023). These treatments target molecular pathways like the RAF/MEK/ERK signaling cascade, which is essential for the growth and survival of tumor (Li et al. 2016). However, their effectiveness is curtailed by compensatory activation of alternative pathways and insufficient induction of apoptosis. Hence, there is an urgent need for innovative treatment approaches that effectively disrupt the molecular drivers of HCC progression.

The hypomethylating drug 5-Azacytidine (5-Aza) has been widely studied for its ability to reverse aberrant DNA methylation, a hallmark of many cancers, including HCC (Seelan et al. 2018). By inhibiting DNA methyltransferases (DNMTs), 5-Aza restores the epigenetically silenced tumor suppressor genes expression including p16INK4a and MLH1, leading to reduced cell proliferation, enhanced apoptosis, and metastasiss suppression (Gailhouste et al. 2018). Furthermore, 5-Aza has been demonstrated to enhance cancer cells immunogenicity through increasing the expression of cancer-testis antigens and stimulating an immune response against cancers (Sulek et al. 2017). Despite its potential, the clinical efficacy of 5-Aza as a monotherapy is limited by issues such as rapid degradation, systemic toxicity, and acquired resistance mechanisms, including the upregulation of compensatory pathways like PI3K/AKT (Acharya and Singh 2022). Combining 5-Aza with complementary agents may enhance its anticancer efficacy and circumvent resistance.

Ramucirumab (RAM), a fully human monoclonal antibody targeted receptor 2 for vascular endothelial growth factor (VEGFR-2), has emerged as an effective anti-angiogenic (agent) for HCC (Calvetti et al. 2015). By blocking VEGFR-2-mediated signaling, RAM inhibits angiogenesis, a critical process for tumor growth and metastasis, thereby starving tumors of oxygen and nutrients. Additionally, RAM has been shown to interfere with cell survival pathways, sensitizing cancer cells to apoptosis by disrupting the interplay between VEGF signaling and pro-survival proteins such as Bcl-2 (Refolo et al. 2020). The antiangiogenic effects of RAM are complemented by its potential to disrupt the progression of cell cycle, possibly by altering downstream targets linked to through modulation of VEGFR-2, including cyclin D1 and CDK4/6. These properties make RAM an encouraging option for combination treatment, particularly with epigenetic drugs like 5-Aza, to amplify antitumor effects through dual targeting intracellular signalling and the tumor microenvironment.

The intent of this research is to inspect the synergistic anticancer effects of combo treatment of 5-Aza with RAM in cells of HCC and provide mechanistic insights into their cytotoxic effects. We assume that this combination will not only enhance the anticancer potential of 5-Aza but also overcome resistance by targeting complementary pathways, including angiogenesis, apoptosis, and cell cycle regulation. By integrating analysis of cellular and molecular mechanism, this work seeks to unravel the intricate interplay between anti-angiogenic therapy and epigenetic alteration, laying the groundwork for novel combination strategies that could significantly provide better results for individuals with advanced HCC.

Methodology

Cell culture and treatment

Human hepatoma (HUH-7) cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). Dulbecco’s Modified Eagle Medium was used to cultivate HuH-7 cells, (DMEM; Gibco, Thermo Fisher Scientific, USA) added to with 10% fetal bovine serum (FBS; Sigma-Aldrich, USA) and 1% penicillin–streptomycin (Gibco, Thermo Fisher Scientific, USA). Cells were kept in humidified environment at 37°C containing 5% CO₂. For experimentation purpose, cells were seeded in six well-plates at density of 1 × 105 cells/well and left overnight to adhere. Treatment of cells were done for 24 h. with varying concentrations of RAM and/or 5-Aza (5-Azacytidine, Sigma-Aldrich, USA). Based on MTT assay three selected concentrations (50 µg/mL, 100 µg/mL, and 150 µg/ mL) of RAM were investigated and one dose of 5-Aza at 1 µM/mL (Almasoud et al. 2024).

Assessment of Neutral Red Uptake (NRU)

The seeded of HUH-7 cells was done at a density of (5 × 105) cells/ mL in ninety-six well- plates and allowed incubation for 24 h at 37°C. After this initial incubation, the treatment of cells with RAM at concentrations of 50, 100, and 150 µg/ mL was carried out, 5-Aza alone, and combinations of 5-Aza with RAM at the specified concentrations for 24 h. Following treatment, NRU dye (200 µL) was supplemented to individual well and allowed to incubate for 3h. (37°C). Subsequently, 100 µL of 0.5 g CaCl2 was used to fix the cells, and then de-staining was done for 15 min on a shaker. A microplate spectrophotometer was used to detect absorbance at 540nm.

TUNEL assay

HUH-7 cells were seeded on coverslips in 6-well plates and allowed to incubate (37°C) for 24 h. After this incubation period, the treatment of cells was carried out with RAM at 50, 100, and 150 µg/mL, 5-Aza alone, and combinations of 5-Aza with RAM at the specified concentrations. After that, the fixing of cells was done with 1% paraformaldehyde and PBS was used for washing. The cell viability was assessed through TUNEL assay. After 15 min of incubation in 70% alcohol, 51 µL of DNA labelling solution was added, and the cells were cultured for an additional hour. Following the addition of Propidium Iodide/RNase staining buffer, the cells were incubated for half an hour. The coverslips were inverted onto microscopic slides for confocal microscopy viewing after being cleaned with PBS and rinse buffer solution.

Apoptotic protein expression analysis by protein array

To investigate the expression of apoptotic pathway proteins, a Human Apoptosis Antibody Array (Catalog No. AAH-APO-1–8, Manufacturer, USA) was employed, which enables the simultaneous detection of multiple proteins of cell death from cell lysates. The array includes proteins linked with intrinsic as well as extrinsic cell death pathways, such as BAD, BID, BIM, CD40, CAS8, FAS, and HSP60.

Following treatment, cells were lysed using a RIPA buffer (Sigma-Aldrich, USA) containing a protease inhibitor cocktail (Thermo Fisher Scientific, USA). The protein concentration in the lysates was calculated through utilizing a protein assay kit of bicinchoninic acid (BCA) (Pierce, Thermo Fisher Scientific, USA). Incubation of equal protein concentration (200 µg per sample) with the protein array membrane was set following to protocol of manufacturer. Subsequently, washing and incubation of the membrane with a biotin-conjugated secondary antibody followed by streptavidin–horseradish peroxidase (HRP) solution. Signal detection was performed using an enhanced chemiluminescence (ECL) reagent (GE Healthcare, USA), and the membrane was imaged using a ChemiDoc MP Imaging System (Bio-Rad, USA). Levels of expression of protein were quantified by analyzing the intensity of the signals on the array membrane using ImageJ software (National Institutes of Health, USA). Normalization of signal intensities were followed to reference standard spots provided on the array.

Analysis of gene expression of cell cycle proteins

To assess the effect of RAM and 5-Aza on cell cycle-related gene expression, quantitative real-time PCR (qRT-PCR) was employed to evaluate levels of mRNA MCM2, MCM3, cyclin B1, and CDK2 subsequent treatment for 24 h.

RNA Extraction and cDNA synthesis

RNeasy Mini Kit (Qiagen, Germany) was utilized to isolated total RNA from HuH-7 cells in accordance with manufacturer’s guidelines following a 24 h. drug exposure period (RAM: 50, 100 and 150 µg/mL); 5-Aza: 5 µM and their combinations). A NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) was used to measure the concentration and purity of RNA.). For cDNA synthesis, total RNA (1 µg) was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific, USA).

Quantitative Real-Time PCR (qRT-PCR)

Performance of qRT-PCR was done by utlizing Powerup SYBR Green Master Mix (Applied Biosystems, Thermo Fisher Scientific, USA) on an Applied Biosystems 7500 Real-Time PCR System. The designing of Gene-specific primers for MCM2, MCM3, cyclin B1, CDK2, HSPB6, GATA, PTX1, IL-1β, IL-6, COX1, COX2 and the housekeeping gene GAPDH were performed and validated. Each reaction consisted of SYBR Green Master Mix (10 µL), 1 µL of cDNA template, each primer (1 µL) (final concentration 0.5 µM), and nuclease-free water (8 µL) in 20 µL of final volume. The following cycling conditions were set: Firstly, denaturation was done at 95°C for two minutes, accompanied through forty denaturation cycles at 95°C for fifteen seconds, 30 s of annealing at 60°C, and 30 s of extension at 72°C.

Data normalization and statistical analyses

Computation of relative gene expression levels was done by utilizing 2 − ΔΔCt method, with GAPDH as the internal reference. Triplicate was used to perform all reactions in order to ensure accuracy. GraphPad Prism was used to analyzed the data statistically. (GraphPad Software, USA), and outcomes are represented as mean with standard deviation (SD). Differences in the levels of gene expression between treatment groups and standard was evaluated through one-way ANOVA accompanied via Tukey’s post hoc test, with P-values < 0.05 considered significant.

Data was statistically analyzed using SPSS software (ver.22; SPSS Inc., Chicago, IL, USA). The data presented as mean ± standard deviation (SD). Comparisons between treated cells and control has done by a two-tailed Student’s t-test or one-way ANOVA. Also, GraphPad Prism (GraphPad Software, USA) was used. Data is shown as triplicate and significance of data was considered if P -value was < 0.05 or higher.

Results

Cytotoxic effect of RAM and 5-Aza on HuH-7cell line

The first step into investigating the combine impact of RAM and 5-Aza was assessment of drug cytotoxicity separately/combination at different concentrations by NRU assay. The NRU assay utilizes to determine viability of cells via assessing cell’s ability to incorporate and retain the neutral red dye within their lysosomes. Viable cells absorb and retain the dye, which is then quantified spectrophotometrically to evaluate health of cell. As observed in our previous study, exposure of HuH-7 cells to RAM at concentrations of 50, 100 and 150 µg/mL caused the lowering of cell viability to increase in dose-dependent manner which corresponded with increase uptake of red neutral dye (Fig. 1) (Almasoud et al. 2024). A dose-dependent increase in cytotoxic effect of RAM was observed. The effect of cytotoxicity significantly reduced cell survival at all the concentrations compared with the unexposed control cells. When exposed to 5-Aza alone, the exposure considerably decreased the viability of cells compared to control cells. Exposure of the cells to combination of 5-Aza and RAM at the above concentrations also resulted in similar dose-dependent increase in cytotoxic effect of the combinational treatment when compared with unexposed controls.

Fig. 1.

Fig. 1

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NRU assay shows cell survival (%) in Huh-7 cells after subjected to Ramucirumab and 5-Aza for 24 h. Each value depicts mean ± SE (n = 3), (*p < 0.5,**p < 0.01,***p < 0.001,****p < 0.0001)in comparison to reference standard group

DNA damage induction by 5-Aza and RAM combination

Cell viability studies carried out above showed that individual and combinational exposure HuH-7 cell to 5-Aza and/or RAM resulted in induced dose-dependent cytotoxicity effect. We further proceeded to examine either this observed cytotoxicity was because of DNA-damage induced apoptosis or not. Our finding showed that RAM exposure alone can be able to produce considerable DNA damage at medium and high experimental concentration (Fig. 2). similarly, single exposure to 5-Aza alone induced significant level of cellular damage to DNA compared with unexposed controls. Also, combinational cellular testing with both 5-Aza and RAM resulted in significantly increased level of cellular damage to DNA compare with unexposed control cells. This finding suggests 5-Aza with RAM complement the cytotoxic effect of RAM to induce DNA damage.

Fig. 2.

Fig. 2

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The ratio of (FITC-dUTP + PI) Florescence intensity in HuH-7 cells after subjected to Ramucirumab and 5-Aza for 24 h as measured by TUNNEL assay. Each value depicts the mean SE ± (n = 3), (*P < 0.5, **P < 0.01) compared with control group

Expression of Apoptotic protein in HuH-7 cells treated to RAM and 5-Aza

The Tunnel assay carried out showed that DNA damage was induced upon cellular exposure to RAM and 5-Aza. However, a variety of potential downstream pathways may influence the DNA damage induced cell-death. We investigated that death of cell caused by apoptosis i.e., if the damage of DNA resulted in apoptosis. To do this, we employed protein array containing proteins in intrinsic and extrinsic pathways of apoptosis and evaluated the expression of these proteins in the cells upon exposure. Findings demonstrated that the protein expression in the extrinsic apoptosis pathway for example CD40, BAD, CAS8 and FAS as well as chaperone protein HSP60 were all significantly induced due to cellular exposure to RAM in dose-dependent manner (Fig. 3). Contrastingly, these proteins expressions were remarkably decreased in cells exposed to 5-Aza only or combined with RAM at 50 and 100 µg/mL in contrast to unexposed cells, suggesting different apoptotic pathways being activated in different drug exposure groups. However, cells exposed to combination of 5-Aza and 150 µg/mL RAM showed significantly elevated protein expression comparable to levels in those exposed to RAM only. As for BID, another protein in extrinsic pathway of apoptosis exhibited mixed expression pattern in the exposed groups. Expression of BIM was significantly higher at all concentrations for both treatment groups in comparison to unexposed cells, barring cells exposed to combination of 5-Aza and 100µg/mL RAM.

Fig. 3.

Fig. 3

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Equal quantity protein extracts from control and treated HuH-7 cells were examined using the antibody array. A The spots indicate protein expression in HUH-7 cells after treatments. B The chemiluminescent intensities were quantified by densitometry. Representative bar graph of upregulated and downregulated apoptotic proteins. A positive control was utilized to normalize results from different membranes. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001 and comparison was presented with control group

TNFR2 activates the extrinsic pathway of apoptosis similar to FAS receptor, when bound by TNF. Its expression in cells treated with RAM alone was comparable to what was found in unexposed cells. However, expression of TNFR2 suggestively decreased in cells upon treatment with 5-Aza alone or when co-treated with RAM at all concentrations. Taken together with data from FAS expression, it seems FAS is the likely receptor activating the apoptosis pathway in cells exposed to RAM.

As expected, expression of anti-apoptotic protein, BCL-2, was downregulated in all cells for each treatment divisions when matched with unexposed control cells. Contrastingly, executioner caspase, CAS3, expression was found to be considerably high in all exposed cell groups, excluding cells treated with combination of 100 µg/mL Ram and 5-Aza. In a similar pattern to CAS3, expression of p53 was greatly elevated in all exposed groups when contrasted with untreated cells, barring cells exposed to 100µg/mL RAM and 5-Aza combination.

Cytochrome C demonstrated an expression pattern that is similar to that of the proteins in intrinsic pathway of apoptosis. A substantial elevation in cytochrome c expression was witnessed in cells exposed to RAM only in contrast to untreated cells. Exposure to 5-Aza alone on combined with RAM resulted in diminished cytochrome C expression compared to controls, excepting cells exposed to 150 µg/mL RAM and 5-Aza.

Effect of exposure on cell cycle genes

Cell cycle genes assessment was undertaken to determine possible cell cycle perturbation prior to apoptosis. Gene expression of MCM2 and 3, cyclin B1, and CDK2 were determined 24 h after exposure. Interestingly, our data showed that all cell cycle genes were considerably downregulated 24 h after exposure of RAM and or 5-Aza at all concentrations trialled (Fig. 4). This finding suggests meaningful perturbation of cell cycle 24 h after exposure to the drugs.

Fig. 4.

Fig. 4

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Fold-change in Cell Cycle genes expression in HuH-7 cells as assessed by real-time PCR (qPCR). HuH-7 cell line was subjected to different doses of Ramucirumab or 5-Aza alone, and combined therapy of Ramucirumab and 5-Aza for 24 h. The genes measured were MCM2, MCM3, CycB1 and CDK2. Data is displayed as mean with SD of n = 3 experiments. Statistical significance is designated as follows: *P < 0.05, **P < 0.01, ***P < 0.001 and comparison was executed with control group

Effect on transcriptional modulators of hypomethylation genes

HSPB6, PTX1, and GATA are associated with cellular hypomethylation mechanism. Here we measured the effect of RAM alone, and combined with 5-Aza via transcriptional regulation of related genes. We evaluated the gene expression as low levels might be linked with the observed repression of cell cycle genes investigated above. As expected, the expression post-treatment to RAM only or combined with 5-Aza at all concentrations trialled, resulted in significant repression of their expression as opposed to control unexposed cells (Fig. 5). Conversely, expression of PTX1 was suggestively amplified post-exposure to 5-Aza only. This hints that repression of this gene might have caused reduced levels of cell cycle genes after exposure. Thus, downregulation of hypomethylation genes illustrated significant effect on RAM levels as shown in (Fig. 5).

Fig. 5.

Fig. 5

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Fold-change in Hypomethylation genes expression in HuH-7 cells as analyzed by real-time PCR (qPCR). HuH-7 cells were subjected to different doses of Ramucirumab or 5-Aza alone, and combined therapy with Ramucirumab and 5-Aza for 24 h. The genes measured were HSP6, PITX1 and GATA. Data is shown as mean ± SD of n = 3 experiments. Statistical significance is indicated as follows: *p < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and comparison was performed with control group

Induction of inflammatory cytokines

Inflammation is often accompanied by apoptosis (Xu et al. 2024). On the basis of above evidence, we explored possible induction of inflammatory genes in cells post-exposure. Evaluation of canonical inflammatory genes expression, IL-1β, IL-6, COX1 and COX2 suggested that expression levels of IL-1β, IL-6, and COX1 genes were significantly repressed at all concentrations evaluated for all exposure groups in contrast to control unexposed cells, with the exception of IL-6 expression in cells treated with combination of 5-Aza and RAM (Fig. 6). On the contrary, expression of COX2 was considerably increased for all treatment groups and drug combinations when contrasted with unexposed control cell group. However, cells that were exposed to 5-Aza only exhibited significantly repressed gene expression for COX2. Taken together, these findings suggest possible oxidative stress that may not be linked to inflammation post-exposure.

Fig. 6.

Fig. 6

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Fold-change in Inflammatory genes expression in HuH-7 cells as analyzed by real-time PCR (qPCR). HuH-7 cells were subjected to different doses of Ramucirumab or 5-Aza alone, and combined therapy with Ramucirumab and 5-Aza for 24 h. The genes measured were IL1B, IL.6, Cox1 and Cox2. Data is shown as mean ± SD of n = 3 experiments. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001 and comparison was presented with control group

Discussion

Ramucirumab, a human IgG1 monoclonal antibody with high specificity for VEGFR-2, works by blocking binding of VEGF A, C, and D (VEGF-A, VEGF-C, and VEGF-D) from binding their associated receptors (Clarke and Hurwitz 2013). Subsequently, this inhibits VEGFR-2 activation and related signaling streams critical for tumor angiogenesis. Recent evidence further highlights VEGFR-2’s role in advancing tumor and its promise as a drug target.

Ramucirumab is currently approved as monotherapy for HCC treatment in patients where α-fetoprotein (AFP) levels ≥ 400 ng/mL following progression on sorafenib, a multikinase inhibitor targeting the VEGF pathway among others (Syed 2020; Zhu et al. 2019). While clinical trials such as REACH-2 have demonstrated a modest overall survival benefit, the improvement is relatively limited, often raising concerns about its cost-effectiveness (Roviello et al. 2019). Additionally, ramucirumab’s toxicity profile, including hypertension, proteinuria, and risks of bleeding or hepatic decompensation, poses challenges, particularly in cirrhotic patients with compromised liver function (Clarke and Hurwitz 2013; Bruix et al. 2021). Resistance mechanisms, such as upregulation of alternative angiogenic pathways or VEGF-independent processes, further limit its efficacy in some tumors. The therapy’s eligibility restriction to high-AFP patients and the inherent heterogeneity of HCC tumors reduce its broader applicability. It does become apparent that a combinational therapy with a drug targeting alternative but close pathway might proffer a synergistic cancer cell killing property to circumvent these problems.

5-Aza is a hypomethylating agent that inhibits DNA methyltransferases, leading to the reactivation of silenced tumor suppressor genes and disruption of cancer cell epigenetics (Momparler 2005). Widely used in hematologic malignancies, it has shown promise in solid tumors, including hepatocellular carcinoma (HCC), by modulating the tumor microenvironment and reversing neoplastic epigenetic changes (Chen et al. 2020). We explored the synergistic effect of ramucirumab and 5-azacitidine HuH-7 HCC cell line. We have shown in a previous study that combined RAM and 5-Aza induced cancer cell killing on the HuH-7 cells through oxidative stress trigger and disturbance in mitochondrial integrity (Almasoud et al. 2024). Emerging evidence supports synergistic promise of co-administering angiogenesis inhibitors with epigenetic therapies, as angiogenesis and epigenetic regulation share interconnected roles in tumor growth and resistance mechanisms (Ribatti and Tamma 2020). This combinatorial approach could provide a dual mechanism for targeting the tumor microenvironment and reversing tumor-associated epigenetic alterations.

Current investigation probed the synergistic anticancer effects of RAM and 5-Aza in hepatocellular carcinoma (HCC) cells, focusing on cytotoxicity, DNA damage, apoptosis, and cell cycle modulation. The results provide compelling evidence for effectiveness of studied combination treatment in enhancing anticancer outcomes, with potential implications for improving the management of HCC.

The Neutral Red Uptake (NRU) assay revealed dose-dependent cytotoxicity for RAM, 5-Aza, and their combination. The combination treatment resulted in significantly enhanced cytotoxicity compared to either treatment alone. This aligns with our previous study where we the same result was replicated by MTT assay (Almasoud et al. 2024).

In addition, prior studies have also shown that hypomethylating agents like 5-Aza can sensitize cancer cells to cytotoxic agents by reactivating silenced tumor suppressor genes (Cheng et al. 2003; Shang et al. 2017). The DNA damage witnessed with combination treatment, as confirmed by the TUNEL assay, suggests a synergistic interaction between RAM and 5-Aza. While RAM alone did not substantially induce DNA damage, it complemented 5-Aza-induced damage, particularly at 50µg/mL of RAM. This synergy is likely owing to potential of RAM to increase apoptosis initiated by 5-Aza, resulting in increased death of neoplastic cells.

The apoptotic pathways activation provides a mechanistic basis for cytotoxic effects seen in this study. RAM treatment predominantly activated extrinsic pathway of apoptosis, as evidenced by the upregulation of FAS, CAS8, and CD40. This aligns with function of VEGFR-2 inhibitors in promoting tumor apoptosis by modulating death receptor pathways (Ntellas, et al. 2020). Conversely, 5-Aza induced mixed apoptotic responses, with variable expression of proteins in intrinsic and extrinsic apoptosis pathways. Interestingly, the combination treatment resulted in significantly high executioner caspase CAS3 and the tumor suppressor p53 at certain concentrations, suggesting a convergence of pathways of apoptosis signaling. This particular result is in harmony with studies indicating that combination treatment often induce robust apoptotic responses by simultaneously targeting multiple pathways (Li et al. 2024; Mokhtari et al. 2017). This finding also reiterates the reactivation of genes associated with tumour suppression by 5-Aza to induce apoptosis.

However, the inconsistent expression of BID and TNFR2 across treatment groups warrants further investigation. These variations may reflect the activation of compensatory mechanisms or off-target effects, as observed in other studies on combination therapies involving VEGFR inhibitors (Mokhtari et al. 2017). The reduction in expression of cytochrome c in cells exposed to 5-Aza only or combined with lower RAM concentrations further highlights the intricacy of apoptotic modulation as a consequence of these treatments. This finding along with decrease in expression levels of proteins in the extrinsic apoptotic pathway suggests that combined 5-Aza with RAM may be employing an alternative apoptosis pathway. However, the relationship between the influence of RAM exposure and its combination at 150 µg/mL with 5-Aza suggests a threshold dosage in combination might be needed to trigger cell death receptor signaling.

Significant downregulation of genes of cell cycle, including MCM2, MCM3, cyclin B1, and CDK2, was observed in all treatment groups. This highlights that cell cycle arrest precedes apoptosis, particularly in combined treatment. The repression of regulators of transcription such as HSP6 and GATA further supports this observation, as these genes play critical roles in modulating cell cycle succession (Tipping et al. 2009; Zhang et al. 2022). HSP6, is part of the HSP70 family. While it is not constitutively expressed under usual physiological settings in humans, its expression is influenced under conditions of stress, such as exposure to heat, oxidative stress, or other cellular insults (Ramirez et al. 2015). When taken together with findings in our previous study, the oxidative stress triggered by these treatments may have caused the repression of both HSP6 and GATA gene expressions halting cell cycle progression (Almasoud et al. 2024). In fact, earlier studies have exhibited that HSP6 expression correlates with malignancy (Wang, et al. 2020; Zhou 2022). The ability of RAM to disrupt cell cycle-related pathways, in addition to its antiangiogenic effects, underscores its potential as a multifaceted therapeutic agent (Mokhtari et al. 2017).

The repression of key pro-inflammatory cytokines (IL-6 and IL-1β) in most treatment groups, alongside the upregulation of COX2, indicates that the observed cytotoxicity is mediated through oxidative stress rather than classical inflammatory responses. This is consistent with reports indicating that COX2 overexpression in cancer cells may reflect a response to oxidative stress, promoting tumor cell apoptosis (Sun et al. 2009). Interestingly, the unique expression pattern of COX2 in 5-Aza-treated cells highlights potential differences in how these agents modulate stress responses. For deep understanding, future in vivo studies are recommended to investigate the synergistic effects of RAM and 5-Aza on animal models to assess; clinical efficacy, potential side effects, immune response to treatment. Additionally, it is important to evaluated different dosing regimens and timing of treatments to identify optimal patterns that maximize cytotoxic effects on cancer cells while minimizing adverse effects on healthy tissues. Finally, in vivo studies should assess the quality of life of animal models to understand the long-term effects of treatment, contributing to the development of new therapeutic strategies for advanced liver cancer.

Conclusion

This study provides ample evidence supporting combination treatment in oncology, particularly for aggressive tumors including HCC. By elucidating underlying mechanics for the synergistic effects of RAM and 5-Aza, this work lays the groundwork for developing more effective therapeutic strategies. While the findings demonstrate promising anticancer effects, several limitations should be addressed. The differential regulation of apoptotic proteins indicates the activation of alternative pathways that require further exploration. The study’s in vitro nature limits the direct translation of these results to clinical settings. Thua, future research should focus on in vivo validation using animal models and pharmacokinetic studies to assess the safety and efficacy of RAM and 5-Aza combination therapy. Investigating one type of HCC cells may limit the generalizability of the results to other types. Furthermore, no long-term studies were conducted to assess the effects of the treatment, which may impact the stability of the results.

Acknowledgements

This work was funded by Researchers Supporting Project number (RSP2025R26), King Saud University, Riyadh, Saudi Arabia.

Authors' contributions

Hadeel Almasoud and Fares A. Alzahrani, evaluated cytotoxicity, DNA damage, inflammatory markers, results analysis. Saud Alarifi and Badr Aldahmash assessed apoptotic protein expression. Bader Almutairi and Bashayer Aljuhani measured cell cycle. Khadijah Yaseen assessed the hypomethylation genes. Hadeel Almasoud and Abdullah AlKahtane performed protein expression assay. Saad Alkahtani wrote and edited the article. All authors have approved the final version article.

Funding

This work was funded by Researchers Supporting Project number (RSP2025R26), King Saud University, Riyadh, Saudi Arabia.

Data availability

The data generated or analyzed in this article are online publicly available without request.

Declarations

Ethics approval

Not applicable.

Consent to participate:

Not applicable.

Competing interests:

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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