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Manuka honey enhanced sensitivity of HepG2, hepatocellular carcinoma cells, for Doxorubicin

and induced apoptosis through inhibition of Wnt/β-catenin and ERK1/2

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Abstract

Background

Recently, there is increasing awareness focused on the identification of naturally occurring

anticancer agents derived from natural products. Manuka honey (MH) has been recognized for

its biological properties as antimicrobial, antioxidant, and anticancer properties. However, its

antiproliferative mechanism in hepatocellular carcinoma is not investigated. The current study

focused mainly on investigating the molecular mechanism and synergistic effect of anticancer

properties of MH on Doxorubicin (DOX)-mediated apoptotic cell death, using two different p53

statuses (HepG2 and Hep3B) and one non-tumorigenic immortalized liver cell line.

Results

MH treatment showed a proliferative inhibitory effect on tested cells in a dose-dependent

manner with IC50 concentration of (6.92 ± 0.005%) and (18.62 ± 0.07%) for HepG2 and Hep3B

cells, respectively, and induced dramatic morphological changes of Hep-G2 cells, which

considered as characteristics feature of apoptosis induction after 48 h of treatment. Our results

showed that MH or combined treatments induced higher cytotoxicity in p53-wild type, HepG2,

than in p53-null, Hep3B, cells. Cytotoxicity was not observed in normal liver cells. Furthermore,

the synergistic effect of MH and Dox on apoptosis was evidenced by increased annexin-V-

positive cells and Sub-G1 cells in both tested cell lines with a significant increase in the

percentage of Hep-G2 cells at late apoptosis as confirmed by the flow cytometric analysis.

Consistently, the proteolytic activities of caspase-3 and the degradation of poly (ADP-ribose)

polymerase were also higher in the combined treatment which in turn accompanied by

significant inhibitory effects of pERK1/2, mTOR, S6K, oncogenic β-catenin, and cyclin D1 after

48 h. In contrast, the MH or combined treatment-induced apoptosis was accompanied by

significantly upregulated expression of proapoptotic Bax protein and downregulated expression

of anti-apoptotic Bcl-2 protein after 48 h.

conclusion:

Our data showed a synergistic inhibitory effect of MH on DOX-mediated apoptotic cell death in

HCC cells. To our knowledge, the present study provides the first report on the anticancer

activity of MH and its combined treatment with DOX on HCC cell lines, introducing MH as a

promising natural and nontoxic anticancer compound.

Background

Hepatocellular carcinoma (HCC) is one of the most malignant tumors emanating from

hepatocytes. It is the highest prevalent primary liver cancer, representing the sixth most

common cancer worldwide [1]. Moreover, HCC-related mortality ranks second globally, with a

higher prevalence in males than females [1, 2]. In developing countries, particularly those in

East Asia and sub-Saharan Africa, the incidence of HCC and the consequent mortality are

significantly higher than in their developed counterparts [1]. Egypt has been facing a growing

incidence of HCC, which represents the highest leading cause of local death among all other

cancers [3]. This is mostly due to the high prevalence of endemic viral hepatitis, caused by

hepatitis B (HBV) and hepatitis C viruses (HCV), compared to other risk factors [4, 5]. Both

viruses are associated with the progression of a series of events, from chronic hepatitis to

cirrhosis, then finally to HCC [1]. Since HCC has a very poor prognosis at the early stages, with

rapid growth and a high rate of metastasis, most HCC patients are diagnosed at the advanced

stages [6].

Currently, the conventional treatments for HCC, such as liver transplantation or resection [7],

radiotherapy, and chemotherapy, have severe side effects. As drug resistance and adverse

effects remain two critical hurdles [8], the necessity of beefing up the development of new

anticancer agents has become apparent. For a substance to be utilized as a chemopreventive

and chemotherapeutic agent in cancer treatment, it should exhibit high efficiency in hampering

tumor growth, induce low side effects and alleviate the effect of carcinogenic agents [9].

Nowadays, significant awareness has been raised to identify naturally occurring anticancer

agents derived from food and natural products. Among these natural products is honey, which

has a potent anticancer effect, as it can suppress carcinogenesis by modulating or interfering

with the molecular events of the initiation [10], proliferation [11], and progression stages [12].

Honey has a series of medicinal properties, such as anti-inflammatory [13], wound healing [14],

anti-oxidative [15, 16], anti-diabetic [16, 17], antimicrobial [18], antibacterial [19, 20],

antihyperlipidemic [21], antiproliferative, antimetastatic and antitumor properties [22]. These

medicinal effects can be attributed to the various pharmacologically active constituents of

honey, especially flavonoids and phenolic components [23, 24]. Therefore, kinds of honey

possessing high phenolic and flavonoid content, such as Manuka Honey (MH) are deemed very

medicinally attractive [24].

MH is monofloral honey obtained from the Manuka tree (Leptospermum scoparium), belonging

to the family Myrtaceae, and collected by honey bees called (Apis Mellifera) in New Zealand

and the Eastern region of Australia [25]. Isolation and characterization of the bioactive fraction

of MH by using HPLC demonstrated that it comprises a complex mixture of carbohydrates, fatty

acids, proteins, vitamins, and minerals containing various kinds of phytochemicals rich in

polyphenols and flavonoids, that have been identified with potent ROS scavenging activity

[25,26,27,28,29]. The major flavonoids are pinobanksin and pinocembrin, representing

approximately 36% and 23% of the total flavonoid content, respectively followed by the

presence of quercetin (11.81%), luteolin (8.30%), kaempferol (3.70%), chrysin, and galangin.

Leptosin derivatives and methyl syringate were characterized as the major compounds in MH,

representing approximately 35.5% and 43.87% of the total phenolic content, respectively [30,

31]. Moreover, MH contains other various phenolic compounds, such as apigenin,

isorhammentin, 4-hydroxybenzoic acid, and caffeic acid. This dark honey has recently garnered

a lot of attention and consideration for its biological properties, especially its antimicrobial

effects, antioxidant efficacy, and potential role in wound healing [27, 32].

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Manuka honey enhanced sensitivity of HepG2, hepatocellular carcinoma cells, for Doxorubicin and induced apoptosis through inhibition of Wnt/β-catenin and ERK1/2 Metrics details Abstract Background Recently, there is increasing awareness focused on the identification of naturally occurring anticancer agents derived from natural products. Manuka honey (MH) has been recognized for its biological properties as antimicrobial, antioxidant, and anticancer properties. However, its antiproliferative mechanism in hepatocellular carcinoma is not investigated. The current study focused mainly on investigating the molecular mechanism and synergistic effect of anticancer properties of MH on Doxorubicin (DOX)-mediated apoptotic cell death, using two different p53 statuses (HepG2 and Hep3B) and one non-tumorigenic immortalized liver cell line. Results MH treatment showed a proliferative inhibitory effect on tested cells in a dose-dependent manner with IC50 concentration of (6.92 ± 0.005%) and (18.62 ± 0.07%) for HepG2 and Hep3B cells, respectively, and induced dramatic morphological changes of Hep-G2 cells, which considered as characteristics feature of apoptosis induction after 48 h of treatm ...
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