The present study hypothesized that MH could be a potential candidate and a promising treatment option for HCC cell lines. Our results demonstrated that treatment of MH and particularly its synergetic effect with DOX-induced significant cytotoxicity and apoptosis induction in HCC cell lines, HepG2 and Hep3B, respectively in a concentration-dependent manner. It is worthy to note that Hep2G cells are significantly more sensitive to cell viability inhibition by MH or combined treatment than Hep3B cell line. Both HepG2 and Hep3B were derived from different hepatic progenitor origins and different stages of hepatocyte differentiation which in turn might explain the differences in MH or combined treatment-induced cytotoxicity and apoptosis between Hep3B and HepG2 cells. In addition, MH had no significant cytotoxic effect on the human normal neonatal liver cells. In the present study, we focused on the HepG2 cells that showed a high sensitivity for MH or combined treatment with DOX.
Recently, there is an increased focus directed towards the identification and characterization of novel natural products, such as polyphenols and flavonoids-rich honey . In addition, the new approaches to combined chemotherapies with polyphenols, or polyphenol-containing foods as honey becomes imperative to increase the effectiveness of the chemotherapy, also; this way can overcome the cancer cell resistance and minimize the adverse toxicity. Besides, the mixture of polyphenols existing in whole food is easily consumed and more effective when compared to the single or purified molecule in cancer prevention through synergistic and additive effects [41, 42].
Compared to other honey types, MH has been gaining traction in the research sector, since it is very rich in bioactive compounds such as phenolic and flavonoid compounds [27, 30, 31]. Afrin et al. has reported that MH is enriched with elevated levels of antioxidant compounds, at which the most abundant compounds are flavonols, for instance, quercetin represents 11.81%, luteolin (8.30%), and kaempferol (3.70%) of the total phenolic content  and these three compounds have been identified in MH by Alvarez-Suarez et al. . and by Marshall et al.  as well. In addition, phenolic compounds such as Gallic acid and syringic acid were the main components in MH, which represents 36.57% and 32.55%, respectively, of the total phenolic content, while the other phenolic acids such as.
4-hydroxybenzoic acid, apigenin, isorhammentin, and caffeic acid were presented in MH in low proportion . MH has been identified with its potential anticancer activity . The current study focused mainly on investigating the pro-apoptotic and anti-proliferative effects of MH in HepG2 and Hep3B cell lines. Moreover, our work was carried out, while taking into consideration a priorly conducted study, which demonstrated MH’s safety by showing unaltered hematological and clinical chemistry profiles of MH treated mice . Therefore, the anticancer effect of MH could potentially be specific to HepG2 and Hep3B cells since there is no cytotoxic effect on normal human neonatal liver cells, as opposed to that of the chemotherapy drug, doxorubicin, which was found to be toxic against the normal healthy cells.
Our MTT results proved the cytotoxic effects of MH and combined treatment with DOX on HepG2 and Hep3B cells after treatment with different concentrations of MH (1.25–20%) for 48 h, suggesting that MH is effective in inhibiting HepG2 and Hep3B cells proliferation. Fernandez et al. reported on the IC50 values of MH on three different cancer cell lines. Firstly, the murine melanoma cells (B16.F1) after 24 h, 48 h, and 72 h of exposure to MH (UMF10+) exhibited IC50 values of 2%, 1.3%, and 0.8%, respectively. Similarly, for colorectal carcinoma (CT26) cells, the IC50 values recorded at 24 and 72 h, were 2% and 1% respectively. Whereas, the IC50 values for human breast cancer (MCF-7) cells, were more than 5% and 4% at 24 and 72 h, respectively . Furthermore, a previous study also demonstrated that the antiproliferative effect of MH was associated with the activation of the caspase-9-dependent apoptotic pathway . Portokalakis et al. reported that MH showed cytotoxicity towards MCF-7 cells after 24 h treatment in a dose-dependent manner, and the concentration of MH, which produced the IC50 values was 2.2% for UMF 18 + honey and 4.7% for UMF 5 + honey . In the light of these findings, it has been noted that the IC50 values of MH vary among the different types of tested cell lines, according to their molecular, genetic characteristics, and progenitor origins. However, differences may also be attributed to the variations in the honey content, especially flavonoid and phenolic acids, which are responsible for the antitumor activities [26, 45]. According to the previous investigations, phenolic compounds such as quercetin, luteolin, kaempferol, gallic acid, and caffeic acid that are the major components in MH, play an important role in the suppression of cancer cell proliferation [26, 41, 45, 46]. On the other hand, according to a previous study, the IC50 value of Adriamycin (Doxorubicin) for HepG2 cells was 1.12 μg/ml, which is equivalent to 2 μM . It was previously reported that apigenin, a bioflavonoid present in honey) significantly reversed doxorubicin sensitivity in doxorubicin-resistant hepatocellular carcinoma cell line BEL-7402/ADM and induced caspase-dependent apoptosis .
The induction of apoptosis, along with targeting the pathways that regulate and coordinate cellular proliferation and differentiation, is regarded as an important targeted-therapy approach for cancer treatment. Cellular morphological changes are hallmarks of apoptosis and hence microscopic analysis is essential to observe these alterations . Our results showed that the MH treatment of HepG2 cells resulted in apoptotic morphological features. The most pronounced morphological changes occurred at concentrations (½ IC50 MH+½ IC50 DOX), combined treatment, compared to both the untreated cells and the positive control alone, hence confirming the synergistic effect of the combination treatment. Taken together with the data from the viability assays, this hinted towards the possibility of the potential involvement of an apoptosis-mediated, cellular proliferation inhibitory mechanism. Therefore, to investigate the inhibitory effects of MH or combined treatment, Annexin V assay was coupled with flow cytometry to quantify the MH and DOX treated HepG2 cells, undergoing early and late apoptosis. The Annexin V assay capitalizes on the apoptotic-characteristic early disruption of the plasma membrane and the consequent translocation of the membrane phospholipid, phosphatidylserine to the membrane outer surface. This change in locale facilitates the binding of the phospholipid-binding protein, Annexin V, to phosphatidylserine and thus allowing the detection of the cells undergoing early apoptosis. Whereas, the counterstain PI, is only capable of entering the cells once the integrity of their plasma membranes is greatly diminished, a feature exhibited by the cells undergoing late-stage apoptosis. In the present study, HepG2 or Hep3B cells were treated with MH, DOX, separately, or in combined treatment with MH and DOX for 48 h. MH did not have a significant effect on normal human neonatal liver cells, whereas, in HepG2 and Hep3B cells, the combination treatment synergistically decreased cell viability as seen in (Fig. 1). Furthermore, combined treatment-induced cell death revealed features of apoptosis. The percentage of cells with annexin-V-positive staining and the population of sub-G1 cells, representing apoptosis induction, were also increased in HepG2 and Hep3B, respectively (Fig. 3). Our data showed that the percentage of HepG2 cells undergoing early apoptosis markedly increased to 21%, 27%, and 54% following treatment with MH, DOX, and/or combined treatment, respectively, compared to the untreated cells or single treatment of MH or DOX. Moreover, the percentage of HepG2 cells at the late apoptosis stage exhibited an increase to 48%, 55%, and 81% after 48 h treatment with MH, DOX, or combined treatment, respectively, compared to untreated cells. Our data is in agreement with previous studies, where the mouse melanoma, B16.F1 cells, and human colon cancer, HCT-116 and LoVo, cells exhibited dose-dependent apoptosis induction, upon treatment with MH .
The synergistic pro-apoptotic effect attained by using a combination of MH and DOX was evident by the enzyme-linked immunosorbent apoptosis assay on HepG2 cells revealed a significant induction of apoptosis. Hence, we decided to further study this effect on the activity of major apoptotic triggers. Bcl-2 and caspase-3 family members are crucial for the regulation of apoptosis. Moreover, the observed morphological features could be induced by caspase-3 activation that is essential for the characteristic morphological alterations associated with apoptosis . Therefore, we studied the effect of MH on HepG2 cells and focused on caspase-3, a key mediator of the mitochondrial apoptosis events . Upon proteolytic activation by active initiator caspase (caspase-9), caspase-3 proceeds to cleave various substrates, such as poly (ADP-ribose) polymerase (PARP). The aforementioned cleavage of these substrates dictates the characteristic morphological and biochemical features evident in apoptosis . To investigate if MH or the combined treatment would be able to activate caspase-3 in the treated HepG2 and Hep3B cells, caspase-3 activity was assayed. Consistently with our earlier results, MH, or combined treatment showed a significant increase in caspase-3 activity in treated HepG2 and Hep3B cells compared to the untreated cells after 48 h. While the highest increase in caspase-3 activity occurred at concentrations (½ IC50 MH + ½ IC50 DOX) of the combined treatment compared to the single treatment with MH or DOX. The data of HepG2 cells of caspase-3 and apoptosis induction was higher than those of Hep3B cells. Moreover, western blot analysis confirmed the cleavage of PARP and pointed towards the induction of apoptosis, after MH, or combined treatment. All in all, our results suggested that MH, or combined treatment induced apoptosis of HepG2 and Hep3B cells via an extrinsic or an intrinsic apoptosis pathway. Thus, we evaluated whether MH or combined treatment would be able to induce apoptosis in HepG2 cells through the signaling mediated extrinsic apoptosis pathway, or the cell stress-mediated intrinsic apoptosis pathway. In this study, a reduction in Bcl-2 expression and an increase in Bax expression were observed in HepG2 cells after treatment with MH or combined treatment, and the highest induction was detected in combined treatment. Western blot analysis for the pro-apoptotic protein, Bax, was found to be a transcriptional target of p53, a crucial tumor suppressor protein . Moreover, p53, a transcriptional activator, was found to play a role in the induction of the transcription of many genes, including apoptosis-related ones. Therefore, the upregulation of Bax could be triggered via the p53 signaling cascade . Besides, Bax plays an important role in controlling the mitochondrial disruption-mediated cell death, which is marked by the release of cytochrome c into the cytosol . The Bax upregulation coincided with the increase in caspase-3 activity that leads to the activation of PARP cleavage and consequently cell death, suggesting that MH or combined treatment induced apoptosis in HepG2 cells via the activation of the intrinsic (mitochondrial) apoptosis pathway. Consistent with our results, a previous report also showed that MH triggered apoptosis in murine melanoma cells (B16.F1) via the activation of the intrinsic apoptosis pathway . Furthermore, it was reported that the antiproliferative effect of MH was brought about through the activation of the caspase-9 dependent apoptotic pathway, leading in turn to the activation of caspase-3, reduced Bcl-2 expression, DNA fragmentation, and finally cell death . Moreover, another study demonstrated that Chrysin, a common flavonoid of MH, induced apoptosis via the activation of the p53/Bcl-2/caspase-9 pathway in HCC (HepG2) cells . Furthermore, Im et al. reported that luteolin (the major phenolic compound of MH) activated caspase-8, − 9, and − 3, and cleaved PARP in human hepatocellular carcinoma SK-Hep-1 cells . Besides, it was reported that quercetin (common flavonoid of MH) remarkably inhibited HCC cell (HepG2 and SMCC-7721) proliferation and induced apoptosis by upregulating the expression of Bad and Bax and downregulating the expression of Bcl-2 in vitro .
RAF/MAPK/ERK signaling pathway is activated in HCC and was found to correlate with the advanced stage of the disease . Therefore, we evaluated the potential effect of MH on the RAS/ERK pathway by studying the protein expression of total ERK1/2 and their phosphorylation by western blot. The phosphorylation of ERK1/2 is essential for their activation, and hence studying their phosphorylation and relating it to the total protein abundance, sheds light on the potential effect of MH or combined treatment on ERK1/2 activity. The treatment of HepG2 cells with MH, or combined treatment for 48 h, elicited a significant decrease in the p-ERK1/2, despite the total ERK1/2 abundance exhibiting no marked changes. In addition, the most profound reduction in p-ERK1/2 and was recorded with the combined treatment of (½ IC50 MH + 1 μM DOX), which showed a 73% reduction in the p-ERK1/2, reaffirming the synergistic effect of the combination employed, suggesting the anticancer and antiproliferative effect of MH might be mediated through the RAS/ERK signaling pathway. Complementing our data, a priorly conducted study has revealed that Gelam honey alone and /or combined with ginger, induced early apoptosis, partly through the RAS/ERK pathway, in colorectal cancer cells . Furthermore, Ding et al. demonstrated that quercetin reduced cell viability, induced apoptosis, cleaved caspase-3, cleaved PARP, upregulated Bax protein expression, and downregulated p-ERK1/2 protein levels in HepG2 cells . Shao et al. demonstrated that phytochemicals, such as apigenin (one of the flavonoids present in MH), downregulates the activation of ERK and Akt in human colon cancer cells . Moreover, ERK1/2 has been previously implicated with β-catenin, mTOR, and cyclin D1 in cellular proliferation [39, 59]. Hence, studying the abundance of these proteins following the treatment of HepG2 cells with MH should help to better understand the underlying molecular mechanisms associated with the anti-proliferative and pro-apoptotic effect of the honey.
mTOR and its downstream effector, S6K, are important regulators of protein translation. Moreover, the mTOR signaling pathway was found to upregulated in several carcinomas, including HCC, where overexpression of S6K was recorded in nearly half of the studied HCCs . Furthermore, the Wnt/β-catenin pathway (Canonical Wnt signaling) has been found to enhance the m-TOR-mediated modulation of S6K . The crosstalk between the RAS/ERK and mTOR signaling pathways to positively and negatively regulate one another is well documented, while the simultaneous inhibition of both pathways induced an anti-proliferative effect on different cancer cells . The protein expression of both mTOR and S6K in HepG2 cells following treatment with MH, DOX, or combinations of MH and Dox showed inhibition in cell proliferation, where both proteins exhibited a significant downregulation, with the most marked inhibition being evident in the cells treated with combinations of MH and DOX. The present results may be attributed to quercetin (one of the major phenolic compounds of MH). Wu et al. reported that quercetin inhibited the proliferation of HCC cells via the downregulation of hexokinase-2 (HK2) protein level and suppression of the AKT/mTOR pathway . In addition, Ji et al. have demonstrated that quercetin induced autophagy via inhibiting the AKT/mTOR pathway in HCC . Another study reported that luteolin reduced cell viability and induced apoptosis in prostate cancer cells, besides, it downregulated AKT, ERK, mTOR, and P70S6K . Luteolin downregulated the protein expression levels of phosphorylated Akt, mTOR, p70S6K, and MAPK, also induced caspase and PARP cleavages in glioblastoma cells and promoting cell cycle arrest .
Moreover, we further studied the effect of MH on the oncogenic β-catenin expression, a transcription factor, which plays a critical role in HCC progression. β-catenin is a multifaceted protein, as it plays an integral role in the maintenance of the E-cadherin-catenin cell adhesion complex in the cellular junctions, through mediating the binding of cadherins to the actin cytoskeleton . In addition, β-catenin is also a key protein in the canonical Wnt signaling pathway . The activation of this pathway induces the cytoplasmic accumulation of β-catenin and its consequent translocation into the nucleus, thus triggering the transcription of downstream target genes . The Abnormal upregulation of the Wnt/β-catenin signaling pathway is the hallmark of many tumors, including liver cancers, where the upregulation of β-catenin plays an important role in the development and progression of HCC . Therefore, various approaches have been conceived and executed throughout the past years to target the Wnt/β-catenin pathway, as a means to develop novel therapies for HCC treatment . The treatment of HepG2 cells with MH or combined treatment resulted in a significant reduction in the abundance of β-catenin, with the most profound decrease being recorded under the combined treatment, comprising both MH and DOX.
In the light of our western blot results, we assumed that there is a connection between the decreased p-ERK1/2 and the decrease in β-catenin expression. This inhibition pattern of β-catenin expression correlates with the decreased p-ERK1/2, implying that MH may be down-regulating β-catenin through the Ras/ERK signaling pathway. Benn et al. revealed that around 50–70% of HCC patients exhibited increased p-ERK due to the activated RAS/ERK pathway. The activation of this pathway is associated with the inhibitory phosphorylation of Glycogen synthase kinase 3 beta (GSK-3β), which acts as a negative regulator of the canonical Wnt signaling, resulting in the inactivation of GSK-3β and the accumulation of β-catenin . The (½ IC50 MH plus 1 μM DOX) combined treatment markedly reduced the protein expression levels of β–catenin in HepG2 cells after 48 h, effectively validating the synergistic effect of the combined treatment MH and DOX. Consistent with our results, It was uncovered that MH possesses components, such as polyphenols and quercetin flavonoids, which were found to contribute to its antiproliferative properties, through reducing β-catenin/Tcf transcriptional activity and down-regulating the canonical Wnt signaling pathway in colon cancer cells . Moreover, the MH bioflavonoid Galangin was found to diminish the β-catenin response transcription (CRT), which is abnormally elevated in colorectal and liver cancers . This bioflavonoid acts by enhancing the degradation of the intracellular β-catenin, as well as restraining the β-catenin/T-cell factor-dependent gene expressions, such as cyclin D1 and c-myc. Thus, it consequently exerts an anti-proliferative effect on the CRT-positive cancer cells . In conclusion, the phytochemicals present in MH suppressed β-catenin in HepG2 cells, thus contributing to the MH’s anti-cancer effect .
Previous studies demonstrated that the overexpression of cyclin D1 could be attributed to β-catenin activation and the RAS/ERK signaling pathway [74, 75]. Cyclin D, a crucial regulator of the cell cycle progression, is overexpressed in different cancers, especially in HCC . Moreover, it is a downstream target of β-catenin and RAS/ERK signaling pathway in HCC, where the increased p-ERK1/2, results in elevated expression of cyclin D1 and the inhibitory phosphorylation of the tumor suppressor protein, pRb . Besides, the overexpression of cyclin D1 was found to be involved in HCC tumor cell differentiation, therefore targeting cyclin D1, is regarded as an alternative approach in cancer therapy . In the present study, the cyclin D1 expression in MH treated HepG2 cells, revealed a significant downregulation with the most significant decrease being exhibited by the cells treated with combined treatment. Our data demonstrated that cyclin D1 might be one of the downstream targets of β-catenin and ERK1/2 in HepG2. Consistent with our results, MH contains high amounts of quercetin, which can profoundly inhibit the growth and proliferation of HepG2 cells by reducing the expression of cyclin D1 .