Nutr Res Pract.  2016 Apr;10(2):139-147. 10.4162/nrp.2016.10.2.139.

Loquat (Eriobotrya japonica) leaf extract inhibits the growth of MDA-MB-231 tumors in nude mouse xenografts and invasion of MDA-MB-231 cells

• You MK
• Kim MS
• Jeong KS
• Kim E
• Kim YJ
• Kim HA

• KMID: 2313910
• DOI: http://doi.org/10.4162/nrp.2016.10.2.139

Abstract

BACKGROUND/OBJECTIVES
The present study was conducted to examine the inhibitory effect of loquat leaves on MDA-MB-231 cell proliferation and invasion.
MATERIALS/METHODS
Female athymic nude mice were given a subcutaneous (s.c.) inoculation of MDA-MB-231 cells and randomly grouped to receive a s.c. injection of either 500 mg/kg ethanol, water extract or vehicle five times a week. Tumor growth, mitotic rate and necrosis were examined. MDA-MB-231 cells were cultured with DMSO or with various concentrations of loquat water or ethanol extract. Proliferation, adhesion, migration, invasion and matrix metalloproteinase (MMP) activity were examined.
RESULTS
Tumor growth of xenograft nude mouse was significantly reduced by loquat extracts. The results of mitotic examination revealed that loquat extracts reduced tumor cell division. Both ethanol and water extracts significantly inhibited MDA-MB-231 cell proliferation. The protein expression of ErbB3 was significantly down-regulated by loquat leaf extracts. Loquat leaf extracts increased apoptosis of MDA-MB-231 cells following 24 hour incubation and the ethanol extract was more potent in inducing apoptosis than the water extract. Furthermore, loquat extracts inhibited adhesion, migration and invasion of MDA-MB-231 cells. MMP activity was significantly inhibited by loquat extracts.
CONCLUSION
Our results show that extracts of loquat inhibit the growth of tumor in MDA-MB-231 xenograft nude mice and the invasion of human breast cancer cells, indicating the inhibition of tumor cell proliferation and invasion.

Keyword

Loquat; nude mice; MDA-MB-231 cells; tumor growth; invasion

MeSH Terms

Animals
Apoptosis
Breast Neoplasms
Cell Division
Cell Proliferation
Dimethyl Sulfoxide
Eriobotrya*
Ethanol
Female
Heterografts*
Humans
Mice
Mice, Nude*
Necrosis
Water
Dimethyl Sulfoxide
Ethanol
Water

Figure

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  Fig. 1 HPLC chromatograms of the ursolic standard (A) and the Eriobotrya japonica water (B) and ethanol (C) extracts.

• 

  Fig. 2 Effect of Eriobotrya japonica on the body weight (A), food intake (B) and tumor volume (C) of athymic nude mice. Three weeks after inoculation, nude mice were randomly grouped to receive s.c. injection of either 500mg/kg ethanol (ME), water extract (MH) or vehicle (MC) for 4 weeks, five times a week. The weight, food intake and tumor volume were measured weekly. MC: PBS s.c., ME: 50% ethanol extract (500 mg/kg) s.c., MH: water extract (500 mg/kg) s.c.. Means with the same letter are not significantly different based on "one-way ANOVA followed" by Duncan's multiple range test (P < 0.05)

• 

  Fig. 3 Effect of Eriobotrya japonica on the mitotic figures (A) and the necrosis (B) of tumors in MDA-MB231 xenograft nude mice. Formalin-fixed, paraffin-embedded tumor tissues were sectioned at 4µmand stained with H&E for microscopic findings. MC: PBS s.c., ME: 50% ethanol extract (500 mg/kg) s.c., MH: water extract (500 mg/kg) s.c. arrow: mitotic figure

• 

  Fig. 4 Effect of Eriobotrya japonica on the cell availability (A) and proliferation (B) of MDA-MB-231 cells. (A) Cells were seeded at a density of 1 × 103cells/well in 96 well plates and treated with different concentration of ME or MH for 24h. After completion of the treatment, the cell viability was determined by MTT assay. (B) Cells were seeded in 6 well plates at a concentration of 6 × 104cells/well. Complete medium with PBS, ME or MH was replaced every other day. Cells were harvested on day 5 after treatment and were counted. Each assay was performed in triplicate and repeated in three independent experiments. ME: 50% ethanol extract, MH: water extract Means with the same letter are not significantly different based on "one-way ANOVA followed" by Duncan's multiple range test (P < 0.05)

• 

  Fig. 5 Effect of Eriobotrya japonica on the apoptosis of MDA-MB-231 cells. (A), (B) Cells were treated with PBS, ME or MH for 24h. Cells were lysed and determined protein expression of ErbB2(A) and ErbB(B) by western blotting analysis. (C) MDA-MB-231 cells were incubated for 24h and cell apoptosis was evaluated by Muse Cell Analyzer. ME: 50% ethanol extract, MH: water extract Means with the same letter are not significantly different based on "one-way ANOVA followed" by Duncan's multiple range test (P < 0.05)

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  Fig. 6 Effect of Eriobotrya japonica on the adhesion of MDA-MB-231 cells. Cells were added to the matrigel coated plates and incubated for 90min in the presence of PBS. ME or MH and attached cells were harvested and counted. Each assay was performed in triplicate and repeated in three independent experiments. ME: 50% ethanol extract, MH: water extract Means with the same letter are not significantly different based on "one-way ANOVA followed" by Duncan's multiple range test (P < 0.05)

• 

  Fig. 7 Effect of Eriobotrya japonica on the MMP activities (A) and the migration (B) of MDA-MB-231 cells. (A) MDA-MB-231 cells (2 × 105/ well) were seeded in 6-well plates. After treatment with DMSO or 75, 100, 150, 200 or 250 µg /ml of extract for 24 hours, the supernatants were collected and subjected to gel electrophoresis. (B) MDA-MB-231 cells were seeded into 6 well plates and cultured to near confluence. Confluent monolayer was carefully wounded and washed cellular debris gently. The wounded monolayer was incubated in 10% FBS DMEM containing 20 µg/ml fibronectin and DMSO, ME or MH for 24h. ME: 50% ethanol extract, MH: water extract

• 

  Fig. 8 Effect of Eriobotrya japonica on the invasion of MDA-MB-231 cells. Boyden chamber was used for the invasion assay. MDA-MB-231 cells (2 × 105) were seeded and DMSO , ME or MH was carefully added to the upper chamber. 20 µg of fibronectin was added to the lower chamber. The Boyden chamber was incubated for 24 hours at 37℃ and 5% CO2. Following incubation, the cells that transversed the Matrigel and attached to the lower surface of the insert were fixed and stained. ME: 50% ethanol extract, MH: water extract Means with the same letter are not significantly different based on "one-way ANOVA followed" by Duncan's multiple range test (P < 0.05)

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