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. 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. 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.

中文翻译：

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麦卢卡蜂蜜通过抑制 Wnt/β-catenin 和 ERK1/2 增强了肝细胞癌细胞 HepG2 对阿霉素的敏感性并诱导细胞凋亡

最近，人们越来越关注从天然产物中提取的天然抗癌剂的鉴定。麦卢卡蜂蜜 (MH) 因其抗菌、抗氧化和抗癌特性等生物学特性而得到认可。然而，尚未研究其在肝细胞癌中的抗增殖机制。目前的研究主要集中在研究 MH 抗癌特性对多柔比星 (DOX) 介导的细胞凋亡的分子机制和协同作用，使用两种不同的 p53 状态（HepG2 和 Hep3B）和一种非致瘤性永生化肝细胞系。MH 处理对受试细胞显示出剂量依赖性的增殖抑制作用，对 HepG2 和 Hep3B 细胞的 IC50 浓度分别为 (6.92 ± 0.005%) 和 (18.62 ± 0.07%)，并诱导Hep-G2细胞发生剧烈的形态变化，这被认为是处理48小时后诱导凋亡的特征。我们的结果表明，MH 或联合治疗在 p53 野生型 HepG2 中比在 p53 缺失型 Hep3B 细胞中诱导更高的细胞毒性。在正常肝细胞中未观察到细胞毒性。此外，MH 和 Dox 对细胞凋亡的协同作用通过两种测试细胞系中膜联蛋白 V 阳性细胞和 Sub-G1 细胞的增加以及晚期细胞凋亡的 Hep-G2 细胞百分比显着增加来证明，如流式细胞术分析。一致地，联合治疗中 caspase-3 的蛋白水解活性和聚（ADP-核糖）聚合酶的降解也较高，这反过来又伴随着 pERK1/2、mTOR、S6K、48 小时后致癌 β-连环蛋白和细胞周期蛋白 D1。相比之下，MH 或联合治疗诱导的细胞凋亡伴随着 48 小时后促凋亡 Bax 蛋白的显着上调和抗凋亡 Bcl-2 蛋白的下调表达。我们的数据显示 MH 对 HCC 细胞中 DOX 介导的凋亡细胞死亡具有协同抑制作用。据我们所知，本研究首次报道了 MH 的抗癌活性及其与 DOX 联合治疗 HCC 细胞系，将 MH 作为一种有前途的天然无毒抗癌化合物。我们的数据显示了MH对HCC细胞中DOX介导的凋亡细胞死亡的协同抑制作用。据我们所知，本研究首次报道了 MH 的抗癌活性及其与 DOX 联合治疗 HCC 细胞系，将 MH 作为一种有前途的天然无毒抗癌化合物。我们的数据显示 MH 对 HCC 细胞中 DOX 介导的凋亡细胞死亡具有协同抑制作用。据我们所知，本研究首次报道了 MH 的抗癌活性及其与 DOX 联合治疗 HCC 细胞系，将 MH 作为一种有前途的天然无毒抗癌化合物。