Tumor-suppressing potential of stingless bee propolis in in vitro and in vivo models of differentiated-type gastric adenocarcinoma

Abstract

The protective property of propolis across a wide spectrum of diseases has long been realized, yet the anti-tumor efficacy of this bioactive substance from Philippine stingless bees has remained poorly understood. Here, we showed the tumor-suppressing potential of crude ethanolic extract of Philippine stingless bee propolis (EEP) in in vitro models of gastric cancer highlighting the first indication of remarkable subtype specificity towards differentiated-type human gastric cancer cell lines but not the diffuse-type. Mechanistically, this involved the profound modulation of several cell cycle related gene transcripts, which correlated with the prominent cell cycle arrest at the G0/G1 phase. To reinforce our data, a unique differentiated-type gastric cancer model, A4gnt KO mice, together with age-matched 60 week-old C57BL/6 J mice were randomly assigned to treatment groups receiving distilled water or EEP for 30 consecutive days. EEP treatment induced significant regression of gross and histological lesions of gastric pyloric tumors that consistently corresponded with specific transcriptional regulation of cell cycle components. Also, the considerable p21 protein expression coupled with a marked reduction in rapidly dividing BrdU-labeled S-phase cells unequivocally supported our observation. Altogether, these findings support the role of Philippine stingless bee propolis as a promising adjunct treatment option in differentiated-type gastric cancer.

Figure 1

EEP acts in vitro through modulation of the cell cycle and apoptotic machineries. (A) Real time PCR profile of selected genes associated in cell cycle regulation and apoptosis in four human GC cell lines following 48 hour-incubation with either culture medium alone or EEP whose concentration approximated the determined IC₅₀ value (@ 48 h) for each respective cell lines. Data shown as mean ± SD are representative of three independent experiments with each experiment run in duplicates. ^#P < 0.05 and ^**P < 0.01 using Independent sample t-test, P < 0.05 using Mann-Whitney U test. (B) Cell cycle analysis of AGS cells using propidium iodide staining depicting the representative raw data of the untreated control and EEP-treated cells (IC₅₀ value @ 48 h) (left) and graphical analysis of the cell cycle distribution in the sub-G1, G0/G1, S, G2/M, and multi-nuclear phases (right). Data are shown as mean ± SD taken from triplicates of two independent experiments. NS- not significant, ^*P < 0.01 using Independent sample t-test. (C) Validation of DNA fragmentation-initiated apoptosis using TUNEL assay in three sensitive human GC cell lines after application of either culture medium alone or EEP at respective IC₅₀ concentration at 48 h (above). Positive control cells were subjected to DNase I treatment prior to TdT labeling while negative control cells were incubated in TdT solution in lieu of TdT reagent (below). Scale bar: 100 μm (D) Comparison of the number of cells undergoing apoptosis expressed as % apoptotic cells between untreated and EEP-treated groups in three human GC cell lines. ^*P < 0.05 using Independent sample t-test.

Figure 2

EEP supplementation induces promising anti-tumor response in mice model of differentiated-type gastric adenocarcinoma. (A) Representative stomach tissues of 60-week old C57BL/6 J and A4gnt KO mice reflecting the gross mucosal elevation of the pyloric antrum following oral administration of respective treatments for 30 consecutive days. (B) H&E sections of the mouse pyloric mucosa among the different treatment groups. Scale bar: 100 μm (C) T-lymphocyte infiltration of the pyloric mucosa among the different treatment groups as depicted by CD3 immunostaining. Scale bar: 100 μm (D) Comparison of the gross mucosal elevation score among the different treatment groups. 0 - none/healthy mucosa, 1 - mildly, 2 – moderately, 3 - markedly. ^*P < 0.05 using Independent-sample t-test. (E) Comparison of the gastric mucosal thickness among the different treatment groups expressed as mean measurement (μm) taken from at least three different areas of the pyloric mucosa. Means with different letter are significant at P < 0.05 using ANOVA with Tukey-HSD posttest. (F) Comparison of the number of CD3-positive T-lymphocytes among the different treatment groups expressed as mean count per 100 μm area taken from at least three different points with the highest cell density. Means with different letter are significant at P < 0.05 using ANOVA with Tukey-HSD posttest. i – Wildtype + dH₂0, ii – Wildtype + EEP, iii – A4gnt KO + dH₂0, iv - A4gnt KO + EEP.

Figure 3

EEP consistently affects cell cycle process in vivo to confer efficient anti-tumor action. (A) mRNA expression levels of several genes related to cell cycle regulation and apoptosis among the different treatment groups using real time PCR. Data are shown as an average of two independent experiments with each analysis run in duplicates. Means with different letter are significant at P < 0.05 using ANOVA with Tukey-HSD posttest. ^**P < 0.05 using Kruskal-Wallis test. (B) Representative sections of gastric pyloric mucosa depicting p21 immunoreaction between untreated and EEP-treated A4gnt KO mice. Scale bar: 100 μm (C) Comparison of the number of p21-positive cells between untreated and EEP-treated A4gnt KO animals expressed as mean counts per 100 μm taken from at least three different areas with the highest cell density. ^*P < 0.05 using Independent sample t-test. (D) Representative sections of gastric pyloric mucosa among the different treatment groups showing BrdU labeling of actively dividing cells in the synthesis (S) phase of the cell cycle. Scale bar: 100 μm (E) Comparison of the number of BrdU-positive cells among the different treatment groups expressed as mean counts per 100 μm area taken from at least three different points with the highest cell density. Means with different letter are significant at P < 0.05 using ANOVA with Tukey-HSD posttest. (F) Representative sections of pyloric mucosa demonstrating cells undergoing apoptosis between untreated and EEP-treated A4gnt KO mice as revealed by TUNEL assay. Scale bar: 100 μm (G) Comparison of the number of FITC-dUTP-positive cells expressed as mean counts per 100 μm area taken from at least three different points with the highest cell density. P < 0.05 using Independent sample t-test. i – Wildtype + dH₂0, ii – Wildtype + EEP, iii – A4gnt KO + dH₂0, iv - A4gnt KO + EEP.