Investigating the Anti-tumour Properties of Iraqi Propolis in vitro and in vivo

The present study was conducted to evaluate anti-oxidant, anti-mutagenic and anti-tumour potentials of propolis samples collected during the period 20-42007 to 1-7-2008 from five
different geographical locations in Iraq (Baghdad1;B1, B2, Dahuk; D, Mosul; M and Salah ad-Din; S), and/or their phenolic compounds (caffeic acid; CA, chrysin; CH and caffeic acid phenethyl ester; CAPE) in vitro using one normal (human dermal fibroblast; HuFb) and two
cancer (human promyeloid leukemia; HL-60 and human colon carcinoma; HCT116) cell  lines. Additionally, chemical analyses of propolis samples were carried out. Phenolic compounds (flavonoids and phenolic acids and their ester) concentrations were estimated using high performance liquid chromatography that was coupled to electrospray mass spectrometry (HPLC-ESI/MS) analysis. Such compounds were further determined spectro-photometrically using FolinCiocalteu reagent . Accordingly, 38 different compounds were characterized and 33 of them were polyphenols. The others included four clerodane diterpenoids that have not been reported in temperate zones, while one compound was considered unknown. Quantitative assay of flavonoids showed that phenolic acids and their
esters were the predominant constituents in propolis extracts, followed by
flavones and flavonols, and then flavanones and dihydroflavonols.The highest concentrations of these compounds were observed in D and M samples. The free radical scavenging activity of propolis was evaluated by 2, 2diphenyl-1-picrylhydrazyl (DPPH) assay. The results revealed that propolis extracts exhibited strong free radical scavenging
activity, but D and M samples had the highest activity, while CAPE has the highest anti-radical activity than other investigated compounds (CA or CH). The results of in vitro growth inhibitory (GI) and growth inhibitory rate (GIR) against HL-60, HCT-116 and HuFb cells demonstrated that a treatment of cell cultures with B2, D, M or CAPE decreased the growth of cells significantly (P ≤ 0.05) as compared to control cultures (untreated), and the effect was dose-, as well as, time-dependent. The M sample was less toxic against HuFb cells, while CH had no toxic effects against HL-60 cells. The cell death was evaluated in vitro in tumour cell lines after a treatment with propolis extracts or their phenolic compounds through four types of assays, which were morphological aspects, gel electrophoresis, Annexin V/PI Flow cytometry and regulation of Bcl-2/Bax gene expression. Results revealed that the tested substances showed a potent inhibitory cytotoxic effect against the proliferation of HL-60 and HCT-116 cells through either apoptosis or necrosis,
respectively. The apoptotic effect was associated with down-regulation of Bcl-2 and activation of Bax expression and the effect was markedly at 10 hours of treatment (22% and 33%, respectively). Such evaluations were further extended for M sample only and included p53 expression (HCT-116 cells), cell cycle perturbations through monoparametric and biparametric DNA evaluations and assessment of γ-H2AX-histone formation (HL-60 and HCT-116). With respect to p53 gene, the HCT-116 cells, a slight increased expression
was observed after three hours of M sample treatment (4.80%), and such increase was also detected at six hours (5.57%), but after 10 hours it was reduced to reach no expression. However, after 24 hours, the expression started to increase again, and 27.98% of the cells were positive, but at 48 hours, it was decreased to reach 5.14%. After 72 hours, the positive expression was increased to reach 11.31%. The monoparametric cell cycle distribution assay revealed that HL-60 cells treated with 5 µg ml -1  of M sample at 48 and 72 hours exhibited an increase in proportion of cells at S phase and a decrease in G1 phase, while no changes in cell cycle phases were observed at 24 hours. In cells treated with 15 or 25 µg m  at 24 and 48 hours, there was an accumulation of cells in the S phase too, while a G2/M accumulation was apparent only after a treatment with 25 µg ml  at 72 hours. Furthermore, a sub-G1 peak was observed after a treatment with 15 or 25 µg ml -1  at different time intervals. On the other hand, most accumulations of HCT-116 cells occurred in G1 or G2/M phase in a time- and dose-dependent manner, while no sub-G1 peak was observed. In biparametric evaluation, the cells (HL-60 and HCT-116) progressed in the S phase more slowly than untreated cells, and in HL-60 cells, a fraction of new G1 positive BrdUrd cells was detected after 10 hours post-treatment. In both cell lines, the cells that were in G1 phase (negative BrdUrd) at the start of treatment remained blocked in this phase for less than 24 hours, and started to show S phase cell populations only after 24 hours. The expression of γ-H2AX in HL-60 cells was clearly increased in a timedependent manner, showing a fraction of cells with an increased γ-H2AX for the six investigated periods (3, 6, 10, 24, 36 and 48 hours post-treatment), but such increase was not observed in HCT-116 cells. The anti-tumour evaluation (histopathologically and immunohistochemically for p53 and Ki-67 gene expressions) was further extended in vivo on tumours obtained from nude mice xenografted with HCT-116 tumour cells and treated orally (p.o.) or intrapertoneally (i.p.) with two doses (500 and 1000 mg kg-1 ) of M sample for 20 (i.p.) or 27 days (p.o.). The M sample was significantly effective in reducing the tumour size in a dose-dependent manner, but the i.p. treatment was associated with adverse side effects. Histopathological examinations of tumour sections from p.o. treated mice revealed a decreased percentage of mitosis and increased endoreduplications of cells, as well as, necrosis, ulceration, degeneration and inflammatory cells infiltration. The p53 gene showed a positive expression in 16% of vehicle tumour cells (control), while it showed an increased expression in tumour sections of animal treated orally with the dose 500 mg kg -1  (33.5%) and a further increase (40.8%) at the-1 dose 1000 mg kg-1 . With respect to Ki-67, the reverse picture was observed, inwhich, the gene was expressed in 85% of vehicle tumour section cells, while itwas expressed in 47.0 and 46.8% of tumour sections obtained from mice treated with the dose 500 or 1000 mg kg-1 , respectively.