Protective effects of Manuka honey on LPS-treated RAW 264.7 macrophages. Part 2: Control of oxidative stress induced damage, increase of antioxidant enzyme activities and attenuation of inflammation.

The redox-system is altered by oxidative stress that is initiated by oxidative agents such as lipopolysaccharides (LPS) and reactive oxygen species (ROS), which are strongly involved in chronic inflammation. Even if Manuka honey (MH) is a good source of polyphenol rich antioxidants, its antioxidant and anti-inflammatory effects are still elusive. The aim of the present work was to explore the protective effects of MH against E.coli LPS stimulated oxidative stress and inflammatory condition and the underlying mechanisms on murine RAW 264.7 macrophages. Pre-treatment with MH markedly inhibited LPS induced ROS and nitrite accumulation and increased the protection against cellular biomolecules such as lipids, proteins, and DNA. Stimulation by LPS suppressed both antioxidant enzyme activities and expressions, and Keap1-Nrf2 signaling pathway which was significantly (p < 0.05) increased in the presence of MH. The pro-inflammatory cytokines, such as TNF-α, IL-1ß and IL-6, and other inflammatory mediators (iNOS) were enhanced after LPS treatment, whereas MH suppressed the expression of these inflammatory markers. Moreover, MH also inhibited the expression of TLR4/NF-кB via IкB phosphorylation in LPS-stressed RAW 264.7 macrophages. In conclusion, MH acted as a natural agent for preventing oxidative and inflammatory-related diseases.

Manuka honey synergistically enhances the chemopreventive effect of 5-fluorouracil on human colon cancer cells by inducing oxidative stress and apoptosis, altering metabolic phenotypes and suppressing metastasis ability.

The development of chemo-sensitizers is urgently needed to overcome 5-fluorouracil (5-FU) therapeutic resistance and adverse toxicity in colorectal cancer. This work aims to evaluate the synergic effects of 5-FU and Manuka honey (MH), a rich source of bioactive compounds, in enhancing the anticancer effects of this drug on human colon cancer HCT-116 and LoVo cells. Compared to 5-FU alone, MH synergistically enhanced the chemotherapeutic effects of 5-FU, by reducing cell proliferation through the suppression of EGFR, HER2, p-Akt and p-mTOR expression, and promoting apoptosis by the modulation pro-apoptotic (p53, Bax, Cyto c, FasL caspase-3, -8, -9 and cleave-PARP) and anti-apoptotic (Bcl-2) markers. The activations of p-p38MAPK and p-Erk1/2 pathways and ROS production were also involved in this process. Downregulation of transcription factor (NF-κB and Nrf2) and antioxidant enzyme activity (SOD, catalase, glutathione peroxidase and glutathione reductase) and expression (SOD, catalase and HO-1) were more evident after the combined treatment, leading to more cell death by oxidative stress. Moreover, additive effects were also observed by increasing lipid and protein oxidation and arresting cell cycle. All the parameters of mitochondrial respiration and glycolysis function decreased and both cells entered the quiescent stage after the combined treatments. MH also influenced the anti-metastasis effects of 5-FU by decreasing migration ability, suppressing the expression of MMP-2, MMP-9 and increasing N-cadherin and E-cadherin. In conclusion, MH could be a useful preventive or adjuvant agent in the treatment of colorectal cancer with 5-FU.

The inhibitory effect of Manuka honey on human colon cancer HCT-116 and LoVo cell growth. Part 1: the suppression of cell proliferation, promotion of apoptosis and arrest of the cell cycle.

Numerous investigations have been made on plant phenolic compounds and cancer prevention in recent decades. Manuka honey (MH) represents a good source of phenolic compounds such as luteolin, kaempferol, quercetin, gallic acid and syringic acid. The aim of this work was to evaluate the chemopreventive effects of MH on human colon cancer HCT-116 and LoVo cells. Both cells were exposed to different concentrations of MH (0-20 mg mL-1 for HCT-116 cells and 0-50 mg mL-1 for LoVo cells) for 48 h to measure apoptosis and cell cycle arrest as well as apoptosis and cell cycle regulatory gene and protein expression. MH exhibited profound inhibitory effects on cellular growth by reducing the proliferation ability, inducing apoptosis and arresting the cell cycle in a dose-dependent manner. Interestingly, MH treatment in non-malignant cells did not exert any significant toxicity at similar concentrations. The apoptosis event was associated with the increasing expression of p53, cleaved-PARP and caspase-3 and with the activation of both intrinsic (caspase-9) and extrinsic (caspase-8) apoptotic pathways. MH induced cell cycle arrest in the S phase in HCT-116 cells, and simultaneously, in LoVo cells, it occurred in the G2/M phase through the modulation of cell cycle regulator genes (cyclin D1, cyclin E, CDK2, CDK4, p21, p27 and Rb). The expression of p-Akt was suppressed while the expression of p-p38MAPK, p-Erk1/2 and endoplasmic stress markers (ATF6 and XBP1) was increased for apoptosis induction. Overall, these findings indicate that MH could be a promising preventive or curative food therapy for colon cancer.

Purification, characterization and cytotoxicity assessment of Ageritin: The first ribotoxin from the basidiomycete mushroom Agrocybe aegerita.

BACKGROUND: Several species belonging to Ascomycota phylum produce extracellular ribonucleases, known as ribotoxins, which exhibit RNase activity through the cleavage of a single phosphodiester bond, located at the universally conserved sarcin/ricin loop of the large rRNA leading to inhibition of protein biosynthesis. Clarifying the structure-function relationship in ribotoxins is interesting for their use in human tumour therapy and in construction of pest resistant transgenic plants. RESULTS: The ribotoxin Ageritin has been isolated for the first time from the Basidiomycetes class. The enzyme, characterized by means of its amino acid composition, N-terminal sequence and a circular dichroism, structurally differs from Ascomycota ribotoxin prototype, although it was able, as α-sarcin, to release a specific α-fragment. However, it does not display aspecific ribonucleolytic activity. Ageritin exerts cytotoxicity and cell death promoting effects towards CNS model cell lines (SK-N-BE(2)-C, U-251 and C6), as vinblastine, a plant alkaloid used in cancer therapy. Moreover, our results indicate that Ageritin initially activates caspase-8, whereas caspase-9 cleavage was not detected, demonstrating the involvement of an extrinsic apoptotic pathway. CONCLUSIONS: Our findings show that Ageritin is the earliest diverging member of the Ascomycota ribotoxin family, suggesting that ribotoxins are more widely distributed among fungi than previously believed. GENERAL SIGNIFICANCE: Ageritin, structurally different from the widely known Ascomycota ribotoxins, with promising anti-cancer properties vs. aggressive brain tumours, has been found from the basidiomycete fungus Agrocybe aegerita. Finally, this finding highlights that the ribotoxin family has divergent members in Basidiomycota phylum, whose structural and functional characterization can give new information on ribotoxin or ribonuclease superfamilies.