Encapsulating Epigallocatechin-3-Gallate (EGCG) in Chitosan Nanoparticles Influenced Multiple Genes Controlling Oncostatic Signaling Pathways in HepG2 Cells

<jats:title>Abstract</jats:title><jats:p>The primary polyphenol in green tea, epigallocatechin-3-gallate (EGCG), is the primary active polyphenol in green tea that showed a remarkable anticancer effect in a variety of cancer types including liver cancer. However, its anticancer impact on gene expressions related to liver cancer proliferation and apoptosis is limited. The current study investigated the oncostatic influence of chitosan encapsulating EGCG (Chit-nanoEGCG) on human hepatocellular carcinoma HepG2 cells compared with its native form and the conventional anticancer drug cisplatin <jats:italic>in vitro</jats:italic>. The Chit-nanoEGCG caused a dose-dependent decrease in cell viability and significantly induced apoptosis compared with control. Flow cytometric analysis confirmed the results of RT-qPCR, displaying a significant increase in the expression of transcriptomes (P53, Bax, Caspase-3, Caspase-9, and PARP) with a significant decrease in the antiapoptotic Bcl-2 gene. Also, there was a significant decrease in transcription genes (OCT4 and SOX2) and receptor genes (CD133, NOTCH1, c-MET, and Ezrin) but an increase in CD95 expression. Furthermore, there was a highly significant decrease in oncogenes (mTOR, PI3K, RALA, and BMI) in HepG2 cells after Chit-nanoEGCG treatment when compared with the control group. The current findings indicate that Chit-nanoEGCG had a significant impact on a number of genes involved in controlling signaling pathways to inhibit the proliferation and development of HepG2 cells. In addition, encapsulating EGCG in chitosan nanoparticles increases its antitumor effectiveness compared to its native form, which encourages the use of various polyphenols in nanochemotherapy for cancer treatment. The finding that the nanoparticles (NPs) influenced the expression of genes involved in apoptosis and proliferation enables the development of medical chemotherapeutic agent for the treatment of hepatocellular carcinoma.</jats:p>                 <jats:p><jats:bold>Graphical Abstract</jats:bold></jats:p>

section_content: The most prevalent primary liver cancer is hepatocellular carcinoma (HCC), also known as hepatoma, and it begins in the hepatocyte, the main cell type in liver. Hepatocellular carcinoma is the main cancer type of the liver and constitutes more than 90% of initial liver tumors. Approximately 85% of people with cirrhosis develop hepatocellular carcinoma. After lung cancer, it continues to be the second main cause of death in men (Philips et al. 2021). Hepatocarcinogenesis is a particularly complex process that involves multiple genes. Numerous genes that regulate signaling pathways were discovered to be the main signaling that triggered carcinogenesis in HCC (Wang et al. 2020a ). These genes include apoptotic genes, receptor genes, transcription genes, and oncogenic driver genes (Hu et al. 2022). We anticipated that measuring gene expression at the mRNA level, one may investigate how these genes are regulated during transcription. We hypothesized that the mechanism underlying the anticancer effects of ( -)-epigallocatechin-3-gallate (1, EGCG) may be associated with key genes involved in cell proliferation and death (Tang et al. 2020). 

The balance between cell division and death is maintained by fundamental regulatory mechanisms primarily apoptosis. It is crucial to comprehend apoptotic pathways when creating new anticancer medications. Since cell death brought on by apoptosis results in minimal inflammation, the development of apoptosis-targeting anticancer drugs has recently attracted a lot of attention (Amer et al. 2022). 

Fresh Camellia sinensis (L. ) Kuntze, Theaceae, leaf tea is a pleasant beverage with nutritional benefits. There is an enormous amount of evidence from studies using cell culture and animal models that supports the anticancer properties of green tea and its bioactive polyphenols. Additionally, epidemiological research revealed that dietary consuming tea on a regular basis may help prevent and lower the risk of a variety of cancers, including liver cancer (Rady et al. 2018 ). The most prevalent and biologically active catechin in green tea is EGCG. Abundant articles showed various biological effects of EGCG including pro-apoptotic and antiproliferative influence (Li et al. 2023). 

The poor oral bioavailability of active agents and subsequent delivery to the tumor via a slow and sustained release of natural products such as EGCG are overcome using nanoformulation-based strategies, which may be one reason for their limited usefulness for human consumption. Chitosan nanoparticles encapsulating EGCG (Chit-nanoEGCG) provided a good platform for cancer nanotherapy. Chitosan's biodegradability and biocompatibility rule out any chance of systemic toxicity (Das et al. 2021). Traditionally, cisplatin is anticancer chemotherapy for HCC with poor therapeutic and many side effects (Sanna et al. 2017). The versatility of EGCG's functions, including its antioxidant and antiinflammatory properties as well as its modulation of various cell signaling pathways that control tumor growth and cell proliferation, can be attributed to its role in the management of diseases (Arab et al. 2016). Additionally, EGCG induces apoptosis, and cell cycle arrest reduces angiogenesis, inhibits invasion and metastasis, and boosts anti-tumor immunity (Wang et al. 2020b). Hepatocellular carcinoma is one of the cancers for which EGCG has a chemopreventive effect (Sojoodi et al. 2020). In a variety of preclinical cell culture and animal model systems, including HCC, EGCG has demonstrated significant anticancer effects. All of these results support research efforts to develop biocompatible EGCG nanoparticles for use in treating precancerous lesions in people, such as those of the prostate, breast, colon, and Barrett's esophagus. 

It is reported that EGCG modulates cancer signaling and metabolic pathways such as TNF, PI3K/Akt/mTOR, p53, p38 MAPK, and NF-κB pathways (Rady et al. 2018). Most of these studies were performed on the protein level. Nevertheless, it is still unclear how EGCG in nanoformulation affects the genes involved in preventing cancer. We anticipated that measuring gene expression at the mRNA level, one may investigate how these genes are regulated during transcription. This might make it possible to pinpoint alterations in gene regulation that contribute to the growth and death of cancer cells. The aim of this study is to evaluate the effects of Chit-nanoEGCG to that of cisplatin and its original form on genes involved in the development and death of HepG2 cells in vitro.
section_name: Introduction

section_name: Materials and Methods

section_content: Human hepatocellular cancer cell line (HepG2) was obtained from Vacsera, Egypt. Chitosan, PHR1333-1 g, and EGCG (Purity ≥ 95%; Lot number: 0000292373) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), pH 7. 4 phosphate-buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM), and penicillin-streptomycin were bought from Lonza Bioproducts in Belgium. Invitrogen provided the TRIzol reagent. Both the SensiFASTTM cDNA Synthesis Kit and SensiFASTTM SYBR No-ROX were purchased from Bioline in Australia.
section_name: Materials

section_name: Chitosan Nanoparticles

section_content: Chit-nanoEGCG particles were prepared using ionic gelation technique (Dube et al. 2010). Briefly, nanoparticles (NPs) were prepared by adding a dropwise tripolyphosphate (TPP) solution (0. 1%, w/v; pH 3) to a chitosan solution (0. 1% w/v in 0. 175% v/v acetic acid; pH 3) containing EGCG (0. 05% w/v), under magnetic stirring at ambient temperature (25 °C) for 10 min (700 rpm). Using a dialysis membrane, the solution containing EGCG nanoparticles was dialyzed to eliminate any contaminants. After that, the mixture was lyophilized to create a powdered nanoformulation, which was then redispersed in deionized water for further application.
section_name: Synthesis

section_content: The size and morphology of Chit-nanoEGCG nanoparticles were examined by TEM (JEOL JEM-2100 HRTEM, JEOL, Japan) which was attached to a CCD camera at an accelerating voltage of 200 kV. One drop of the Chit-nanoEGCG solution was mounted on a thin film of amorphous carbon deposited on a copper grid (300 mesh). The solution was air dried, and the sample was examined directly under the microscope or via the micrographs to measure the nanoparticles.
section_name: Transmission Electron Microscope

section_content: In order to determine the size distribution and zeta potential of Chit-nanoEGCG in aqueous dispersion solution, a Malvern Zetasizer (Nano-ZS90, USA) was used. Chit-nanoEGCG was examined in-depth at 25 °C, pH 3-4, and a scattering angle of 90°. For three parallel measurements, the mean and standard deviation of the Chit-nanoEGCG particles' diameter and zeta potential were expressed.
section_name: Dynamic Light Scattering

section_content: After the nanoparticles had been disintegrated down, the amount of EGCG was measured using liquid chromatography/mass spectrometry (LC/MS). This aids in figuring out how well EGCG is entrapped in the nanoformulation as well. An acetic acid solution was added to the redispersed nanoparticles, and they were then incubated in a 1% v/v solution for 30 min to dissolve them. Using centrifugation, the entire solution was run through a Millipore centrifugal filter with a 100-kDa cutoff for 15 min at a rate approximately 5000 × g to separate the EGCG. Using LC/MS, the concentration of the centrifugate containing EGCG was measured. The following formula was used to calculate the loading efficiency: Loading (entrapment) efficiency = [EGCG]f/[EGCG]t × 100, where [EGCG]f is the concentration of EGCG in the centrifugate and [EGCG]t is the theoretical concentration of EGCG (i. e., the total quantity of EGCG added initially).
section_name: Loading Efficiency

section_content: The Holding Company for Biological Products and Vaccines (vacsera, Egypt) provided the human HepG2 cells. Other media and reagents were supplied by Lonza Bioproducts, Belgium.
section_name: Cell Culture

section_content: HepG2 cells were given various concentrations of EGCG and Chit-nanoEGCG (0 to 10,000 g/ml) and allowed to sit in the wells for 48 h. The same amount of cisplatin was applied to positive control cells. Cells that had not been treated served as a negative control. Three times the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed with comparable outcomes. Cells (1 × 10 4 cells per well) were initially seeded in 96-well plates and then subjected to treatments with various concentrations. Drug-containing media were taken out and MTT-containing medium was added after 48 h. The formed formazan crystals were solubilized with dimethyl sulfoxide (DMSO) after 4 h at 37 °C of incubation. Using an ELISA microplate reader, the absorbance was found at 540 nm (BioTek, USA). After treatment, the percentage of viable cells was determined, and the untreated controls were judged to be 100% viable. Viability % = (Mean OD of test dilution/Mean OD of cell control) × 100, where OD is optical density, was used to calculate the viability of the cells. GraphPad Prism software's non-linear regression analysis was used to calculate inhibitory concentration of 50% (IC 50 ) of Chit-nanoEGCG, native EGCG, and cisplatin IC 50 values.
section_name: Cell Viability Assay

section_content: For 48 h, HepG2 cancer cells were given the IC50 dose of Chit-nanoEGCG, EGCG, and cisplatin or not at all. Following two PBS washes, cells were re-suspended in binding buffer and stained for 15 min at room temperature with annexin V and PI (BD Sciences). Using flow cytometry, the apoptosis rates of cells were evaluated (BD FACS-Caliber, USA). Instead of staining, cells were incubated with a specific antibody for the marker for 30 min in the following cases: P53 (detection kit, ab139565), Bax (detection kit, ab139543), Bcl-2 (detection kit, ab119113), PARP (detection kit, ab32138), caspase 9 (detection kit, ab65615), caspase 3 (detection kit, ab6561) (Miltenyi Biotec, Germany). The Accuri C6 Cytometer (BD Biosciences, San Jose, CA) was used to analyze the data after the cells had been incubated at room temperature for 30 min. The cells were then washed with PBS/BSA, centrifuged at 400 × g for 5 min, and then resuspended in 0. 5% paraformaldehyde in PBS/BSA (Becton Dickinson) (Masters and Harrison 2014).
section_name: Cytometric Analysis

section_content: RNA was extracted from samples of all HepG2 cell treatments, including Chit-nanoEGCG, native EGCG, cisplatin treatment, and control groups, in two separate experiments (Invitrogen, USA). Reverse transcription of RNA (400 ng) was performed using a 2720 thermal cycler and the SensiFAST™ cDNA Synthesis Kit (Bioline, Australia) in accordance with the manufacturer's instructions (Applied Biosystems, Singapore). Using the SensiFAST™ SYBR No-ROX Kit from Bioline and the following primers from Invitrogen and Life Technology, gene expression analysis was carried out (Table S1 ). The expression level of the housekeeping gene β-actin was used to normalize RT-qPCR. Gene expression was quantified using the comparative Ct method for relative quantification (2 -ΔΔCt ) after normalization to β-actin (Livak and Schmittgen 2001). Gene expression assessment was repeated five times for each sample.
section_name: RT-qPCR Test

section_content: GraphPad Prism 9. 0 software was used to analyze all statistical data after ensuring that each group's distribution followed a normal course. Means and standard deviation are used to represent the results (Mean ± SD). A one-way analysis of variance of variance (ANOVA) was used to assess statistical comparisons. In comparison to the control group, the percentage of changes in the treated groups was estimated.
section_name: Statistical Analysis

section_name: Results and Discussion

section_content: The Chit-nanoEGCG were synthesized by ionic gelation with TPP ions and the nanoparticles were characterized using TEM and dynamic light scattering. TEM micrograph of the CS-NPs was shown in Fig. S1A. The Chit-nanoEGCG showed a spherical shape with a particle size of 16-30 nm. A representative diagram of the size distribution of Chit-nanoEGCG by dynamic light scattering is shown in Fig. S1B. The sample was analyzed in triplicate to yield the average particle size (Z-Average) at a constant temperature of 25 °C. Z-Average of Chit-nanoEGCG NPs was 219. 7 ± 1. 96 diameter nanometer (d. nm) and the polydispersity index (PDI) of NPs, which indicates the size distribution, was 0. 4 in average. A nanoparticle system with PDI value in range of 0. 1-0. 4 indicated that the system has moderately disperse distribution. It is observed that the zeta potential value of the prepared Chit-nanoEGCG was found to be -51. 5 ± 5. 91 mV which indicated that NPs are moderately to highly dispersed (more than + 30 mV or less than -30 mV) with moderate to high stability and the conductivity was 0. 26 milliSiemens/cm at a constant temperature of 25 °C (Fig. S1C ). Chit-nanoEGCG showed the encapsulation efficiency from 32. 50 ± 7. 44 to 69. 00 ± 2. 92%.
section_name: Chit-nanoEGCG Synthesis and Characterization

section_content: The treatment of HepG2 cells with Chit-nanoEGCG, native EGCG, and cisplatin caused dose-dependent loss of cell viability after 48 h (Fig. 1A ). The mean concentration which inhibited 50% of cell growth (IC 50 ) was detected by averaging the individual results from three repeated experiments. The IC 50 doses of chit-nanoEGCG, native EGCG, and cisplatin in HepG2 cells were 340. 0, 228. 7, and 515. 8 µg/ml, respectively. FITC-Annexin V/PI analysis was performed on HepG2 cells after treatment with a 60% of IC 50 dose of Chit-nanoEGCG, native EGCG, and cisplatin for 48 h. Figure 1B shows highly significant apoptosis in the Chit-nanoEGCG group (early and late apoptosis are 18. 92 ± 0. 54 and 25. 03 ± 0. 63, respectively) when compared to the control group (early and late apoptosis are 0. 34 ± 0. 07and 0. 25 ± 0. 04, respectively) (p < 0. 001).
section_name: Cell Viability, Apoptosis, and Assessment of Efficacy of Chit-nanoEGCG

section_content: In HepG2 cells, Chit-nanoEGCG treatment significantly upregulated the expression of apoptotic proteins including P53 (Fig. 2A ), Bax (Fig. 2B ), caspase-3 (Fig. 2E ), caspase-9 (Fig. 2F ), and PARP (Fig. 2G ), compared to the untreated cells. In contrast, compared to native EGCG treatment, cisplatin treatment, and the untreated cells, the antiapoptotic protein Bcl-2 (Fig. 2C ) was significantly downregulated after Chit-nanoEGCG treatment.
section_name: Flow Cytometric Analysis

section_name: RT-qPCR

section_content: RT-qPCR shows a significant increase in P53 (Fig. 3A ) and Bax (Fig. 3B ) apoptotic genes after Chit-nanoEGCG treatment when compared with native EGCG treatment, cisplatin group, or control group in HepG2 cells. In contrast, the antiapoptotic protein Bcl-2 was significantly decreased after treatment with Chit-nanoEGCG when compared with native EGCG treatment, cisplatin treatment, and control group in HepG2 cells as shown in Fig. 3C.
section_name: Apoptotic Genes Expression

section_content: The current results demonstrated a significant decrease in OCT4 (Fig. 4A ) and SOX2 (Fig. 4B ) expression, while an insignificant expression in NANOG (Fig. 4C ) transcription genes after Chit-nanoEGCG treatment was observed when compared with control groups.
section_name: Transcription Genes Expression

section_content: When comparing the Chit-nanoEGCG treatment to the control, RT-qPCR reveals a substantial rise in CD95 (Fig. 5B ) but a significant decrease in CD133 (Fig. 5C ), NOTCH1 (Fig. 5D ), c-MET (Fig. 5E ), and Ezrin (Fig. 5F ). However, there was no significance in the expression of CD44 (Fig. 5A ) and ABCG2 (Fig. 5G ) genes.
section_name: Receptor and Transporter Genes Expression

section_content: When HepG2 cells were treated with Chit-nanoEGCG, native EGCG, and cisplatin or left untreated, the expressions of the oncogenes mTOR (Fig. 6A ), PI3K (Fig. 6B ), RALA (Fig. 6C ), and BMI (Fig. 6A ) were significantly decreased Hepatocellular carcinoma is one of the most serious malignancies worldwide with a high morbidity and mortality. A passible approach for managing cancer is chemoprevention employing naturally occurring phytochemicals that may inhibit the cancer initiation and progression. However, their bioavailability for medication restricts their application. In this study, we evaluated the efficacy of delivering EGCG to human HCC cells (HepG2) in an in vitro condition using chitosan nanoparticles. Several factors influenced our opinion to use EGCG and HepG2 cells in this study. This includes liver cancer as one of the most common malignancies in both men and women worldwide. Furthermore, in numerous cell culture and preclinical investigations, EGCG has demonstrated impressive chemopreventive potential (Li et al. 2023). The current study investigated the oncostatic influence of Chit-nanoEGCG nanoparticles on HepG2 hepatocellular carcinoma cells. The results demonstrated that the Chit-nanoEGCG induced apoptosis and suppressed the proliferation and the growth of cancer cells. Further proof that the Chit-nanoEGCG increased the expression of genes involved in apoptosis and decreased the expression of genes involved in proliferation is provided by the current investigation. This effect might be attributed to the targeting ability of the EGCG-loaded nanoparticles and the cellular uptake of EGCG by cancer cells. The cellular uptake of Chit-nanoEGCG needs further investigations. In another study, low doses of small nano-EGCG that activate the AMPK signaling pathway significantly suppressed the invasion, colony formation, and proliferation of human lung cancer cells (Chen et al. 2020 ). 

In the current study, ionic gelation with TPP was used to encapsulate EGCG in chitosan to produce Chit-nanoEGCG. Zeta potential and TEM imaging were used to characterize the prepared Chit-nanoEGCG which showed small, highly stable, and spherical nanoparticles, with quite accurate matching between the zeta and TEM image values. The present findings agree with recent work that ionic gelation-prepared chitosan-TPP nanoparticles enhance the antioxidant activities of astaxanthin in both in vitro and in vivo models (Kim et al. 2022). 

The main goal of cancer therapy is to induce apoptosis in cancer cells while limiting concurrent death in normal cells. In numerous cancer preclinical models, numerous reports have shown that EGCG has broad anticancer activities on cancer cell growth (Bonsignore et al. 2021 ). In agreement with our results, previous studies demonstrated a significantly increased anticancer effect of EGCG encapsulated in CSNPs than native EGCG, indicating that nanoformulations increased the stability and bioavailability of EGCG leading to better clinical results. HepG2 remained viable regardless of the CS-NP concentration. These findings support earlier research showing that, after 48 h of cell exposure, CS-NPs with a diameter of up to 150 nm had no less than 10% harmful effect on human liver cancer cells (hepG2) (Loutfy et al. 2016). Having the vehicle be inactive to the cells indicated that cell viability reduction is due to the active compound in the Chit-nanoEGCG and not the vehicle itself (Mohammed et al. 2017). Therefore, before drawing a clear conclusion and beginning its biological application, a more quantitative investigation at the molecular and protein levels is needed to verify the influence of chitosan size and duration on genotoxic effect. 

In this study, Chit-nanoEGCG showed dose-dependent loss of cell viability and inhibited cell growth at 48-h posttreatment with low IC 50 doses compared with native EGCG. The anticancer effect exerted by EGCG on HepG2 cells seems to be achieved by inhibiting cell growth and proliferation as evidenced in Fig. 2. Decreasing HepG2 cell viability by EGCG agrees with other previous findings in different cancer cells, including cervical carcinoma cells (Zhu et al. 2019), lung cancer cells (Jiang et al. 2018), and breast cancer cells (Chen et al. 2018). 

There is a lack of studies describing the effect of EGCG on HCCs, compared to other cancers. Our data demonstrated that HepG2 cells treated with Chit-nanoEGCG showed a significant increase in the apoptotic protein expression (P53, Bax, caspase-3, caspase-9, and PARP) with a concomitant decrease in the anti-apoptotic Bcl-2 proteins when compared with groups treated with natural EGCG or control group, and these results are confirmed by qRT-PCR analysis. So, a second possible anticancer mechanism of EGCG in HepG2 cells may be through the increased expression of apoptotic genes. P53 plays an essential part in cancer concerning cell cycle arrest and apoptosis. The fundamental role of p53 is its ability to induce apoptosis (Neamatallah et al. 2014). P53 performs its function by suppression of antiapoptotic Bcl-2 family proteins and upregulation of apoptotic proteins Bax and caspase-3 (Öhlinger et al. 2020). Upregulation of P53 increases the Bax/Bcl-2 ratio, which is an indicator of apoptosis in cancer cells (Pan et al. 2020). P53 can directly interact with Bax and stimulates the release of cytochrome C that aid in apoptosis. Bax is a protein that is involved in the intrinsic apoptotic pathway as a complex with Bak protein. This protein complex is inserted into the mitochondrial membrane and activates the caspase-3 signaling cascade, which leads to the subsequent release of cytochrome c and ATP from the mitochondrial membrane into the cytosol and finally apoptosis (Chi et al. 2014). Poly (ADP-ribose) polymerase (PARPs) induces cell death through the caspaseindependent pathway parthanatos (Peter et al. 2015). BCL-2 proteins have been implicated in the control of glucose homeostasis and metabolism in different cell types. So, a third possible anticancer mechanism of EGCG is via inhibition of the glycolytic pathway by suppression of Bcl2 (Wang et al. 2015). 

The HCC development is a complicated process characterized by the activation of numerous signaling pathways regulated by specific genes, which contributes to carcinogenesis in a complementary manner. OCT4 and SOX2 are crucial transcriptional regulators that keep cancer stem cells pluripotent and capable of self-renewal; their overexpression may lead to the development of a variety of malignancies (Kim et al. 2015). The current study showed a marked decrease in the expression of OCT4 and SOX2 genes in cells treated with Chit-nanoEGCG confirming a previous study that chemotherapy inhibited OCT4 and SOX2 overexpression in human neuroblastoma (Yang et al. 2012 ). These results associated with an insignificant change in the expression of the transcription gene, NANOG, in cells treated with Chit-nanoEGCG. The preservation of pluripotency in embryonic stem cells depends on numerous factors, including the transcription factors OCT4, SOX2, and NANOG (Saunders et al. 2013 ). SOX2 and NANOG also maintained cancer stem cells (CSCs) in solid tumors (Rady et al. 2018). As surface biomarkers for cancer cells that are resistant to chemotherapeutic drugs, CD44 and CD133 have both been identified (Liu et al. 2013). The results showed downregulation in CD44 and CD133 expression in HepG2 cells after treatment with Chit-nanoEGCG. By enlisting a large number of pro-apoptotic factors to create the death-inducing signaling complex, the surface receptor CD95 causes apoptosis. The NOTCH signaling pathway controlled stem cell self-renewal and differentiation. Although the function of the NOTCH signaling pathway in HCC is still unclear, NOTCH1 was found to be essential for maintaining the self-renewal of cancer-initiating cells in various malignancies (Sikandar et al. 2010 ). 

This study showed that NOTCH1 expression was downregulated after treatment with Chit-nanoEGCG. The activation of the proto-oncogene mesenchymal-epithelial transition (c-MET) signaling results in invasion and metastasis of cancer and also drug resistance (Kim and Kim 2017). Many studies showed that c-MET is highly expressed in different types of soft and solid cancers. Inhibition of c-MET expression has shown promising results in the inhibition of cancer cell growth (Frampton et al. 2015). Additionally, there is a strong correlation between the overexpression of the Ezrin gene and the development of portal vein thrombosis, invasion, and recurrence of HCC (Kang et al. 2010). According to the results of this study, Chit-nanoEGCG can stop the expression of Ezrin, a downstream effector molecule of Rho (the Ras homolog family), which stops the progression and metastasis of HCC (Ruan et al. 2013). The results showed the downregulated expression of ABCG2 after treatment with Chit-nanoEGCG. ABCG2 overexpression was associated with increased drug efflux, chemo-resistance, and invasion of the cell (Nomura et al. 2015). 

This study showed that Chit-nanoEGCG could inhibit the expression of mTOR, PI3K, RALA, and BMI oncogenes which play a pivotal role in HCC, leading to inhibiting the HCC progression and metastasis (Almatroodi et al. 2020). Another possible anticancer mechanism of EGCG in HepG2 cells may be via the DNA methyltransferase. Many tumor suppressor genes are inactivated by hypermethylation in many tumors including hepatocellular carcinoma (El-Bendary et al. 2020). EGCG inhibits the DNA methyltransferase of tumor cells causing an epigenetic reactivation of genes silenced during carcinogenesis (Negri et al. 2018). Figure 7 provides a mechanistic summary of the effects of Chit-nanoEGCG on HepG2 cells in the current study.
section_name: Oncogenes Expression

section_content: Our findings imply that nano-encapsulation of EGCG, particularly through its modulation of apoptotic genes, may have influenced several anticancer pathways in HepG2 cells. Compared to native EGCG, Chit-nanoEGCG had a much greater chemopreventive effect on liver cancer cells in vitro. Thus, the rapid development of nanoformulated natural products may provide promising approaches for overcoming several limitations of conventional anticancer therapies.
section_name: Conclusions

section_name: Acknowledgements

section_content: The authors express their sincere thanks to Faculty of Science and Faculty of Medicine, Mansoura University, Mansoura, Egypt, for providing the necessary research facilities.
section_name: Acknowledgements

section_name: Data Availability

section_content: The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
section_name: Data Availability

section_name: Supplementary Information

section_content: The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s43450-024-00538-3. 

Author Contribution MAE, MN, MMAS, AIO, SMS, and OAA contributed to methodology development and performing experiments. MAE and SMS are involved in data analysis and interpretation. SMS and OAA contributed to the study consultation, conceptualization of the manuscript, and overall writing, reviewing and editing of the manuscript. All authors discussed the contents and contributed to the final revised manuscript. 

Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).
section_name: Supplementary Information

section_content: Ethical Approval The study was designed and conducted according to ethical norms approved by Medical Research Ethics Committee of Mansoura University, Mansoura, Egypt. (Protocol Code No. R. 21. 11. 1514). 

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section_name: Declarations