Modulation of expression of heat shock proteins and apoptosis by Flueggea leucopyrus (Willd) decoction in three breast cancer phenotypes

Abstract

Background: During the past few years, there has been an increasing interest among the Traditional and Folk medical practitioners of Sri Lanka in the use of a decoction prepared from Flueggea leucopyrus (Willd.) for treating various cancers including breast cancer. In the present study, the cytotoxicity of this decoction and its effects on Heat Shock Protein (HSP) expression and apoptosis were compared in three breast cancer phenotypes, to scientifically evaluate if a decoction prepared from F. leucopyrus (Willd.) is useful for the treatment of breast cancer.

Methods: Cytotoxic potential of the F. leucopyrus decoction was determined by evaluating its effects in MCF-7, MDA-MB-231 and SKBR-3 breast cancer cell lines, and MCF-10A (non-cancerous) breast cell line, by use of the Sulphorhodamine (SRB) assay. The effect of the decoction on HSP gene expression in the above cells was evaluated by (a) Real time reverse transcription PCR (RT-PCR) and (b) Immunofluorescence analysis of HSP protein expression. Effects of the decoction on apoptosis were evaluated by (a) fluorescent microscopic examination of apoptosis related morphological changes and (b) DNA fragmentation (c) Caspase 3/7 assay.

Results: F. leucopyrus decoction can mediate significant cytotoxic effects in all three breast cancer cells phenotypes (IC50 values: 27.89, 99.43, 121.43 μg/mL at 24 h post incubation periods, for MCF-7, MDA-MB-231, SKBR-3 respectively) with little effect in the non-cancerous breast cell line MCF-10A (IC50: 570.4 μg/mL). Significant (*P <0.05) inhibitions of HSP 90 and HSP 70 expression were mediated by the decoction in MCF-7 and MDA-MB-231, with little effect in the SKBR-3 cells. Clear apoptotic morphological changes on Acridine orange/Ethidium bromide staining and DNA fragmentation were observed in all three breast cancer cell lines. Caspase 3/7 were significantly (*P <0.05) activated only in MDA-MB-231 and SKBR-3 cells indicating caspase dependent apoptosis in these cells and caspase independent apoptosis in MCF-7 cells.

Conclusions: Modulation of HSP 90 and HSP 70 expressions is a possible mechanism by which the decoction of F. leucopyrus mediates cytotoxic effects MCF-7 and MDA-MB-231 cells. This effect appears to correlate with enhanced apoptosis in these cells. In SKBR-3 cells, mechanisms other than HSP inhibition may be utilized to a greater extent by the decoction to mediate the observed cytotoxic effects. Overall findings suggest that the decoction has the potential to be exploited further for effective treatment of breast cancer.

Fig. 1

Cell survival (by SRB assay) of all three breast cancer cells (MCF-7, MDA- MB-231 & SKBR-3) and normal breast cell line (MCF-10A). 1.1 Treated with the F. leucopyrus decoction for 24 h. Data values are expressed as mean ± SD of eight replicates. 1.2 Treated with Paclitaxel for 24 h (Positive control). Data values are expressed as mean ± SD of eight replicates

Fig. 2

Effects of the F. leucopyrus decoction on HSP 90 expression in (a) MCF-7, (b) MDA-MB-231 & (c) SKBR-3 breast cancer cells. 2.1 Real time PCR results of HSP 90 gene expression. Data are representative of three independent experiments (mean ± SD of three replicates). 2.2 Qualitative Immunofluorescence results, Scale bar: 100 μm. 2.3 Quantitative Immunofluorescence results. Data are representative of five independent experiments (mean ± SD of five replicates)

Fig. 3

Effects of the F. leucopyrus decoction on HSP 70 expression in (a) MCF-7, (b) MDA-MB-231 & (c) SKBR-3 breast cancer cells. 3.1 Real time PCR results of HSP gene expression. Data are representative of three independent experiments (mean ± SD of three replicates). 3.2 Qualitative Immunofluorescence results, Scale bar: 100 μm. 3.3 Quantitative Immunofluorescence results. Data are representative of five independent experiments (mean ± SD of five replicates)

Fig. 4

Effects of the decoction on HSP 70 and HSP 90 expression in MCF 10a cells treated with F. leucopyrus decoction for 24 h. Quantitative Immunofluorescence results of HSP 70 and HSP 90 gene expression in MCF 10a cells mediated by the different concentrations of the decoction, (as analyzed by One way ANOVA with Dunnett’s post test) Data values are expressed as mean ± S.D

Fig. 5

Effects of the F. leucopyrus decoction on apoptosis in the three breast cancer cell phenotypes. Fluorescent microscopic observations of morphological changes in a MCF-7, b MDA-MB-231 or c SKBR-3 cells (magnification 200×), following the treatment with different concentrations of the decoction, by AO/EB staining

Fig. 6

DNA fragmentations in MCF-7, MDA-MB-231 or SKBR-3 breast cancer cells treated with F. leucopyrus decoction. Lane 1- 100 bp ladder, Lane 2- MCF-7 Control, Lane 3- MCF-7 Paclitaxel 5 μg/mL, Lane 4-MCF-7 Decoction 40 μg/mL, Lane 5-MCF-7 Decoction 100 μg/mL Lane 6- 100 bp ladder, Lane 7-MDA-MB-231 Control, Lane 8- MDA-MB-231 Paclitaxel 5 μg/mL, Lane 9- MDA-MB-231 Decoction 40 μg/mL, Lane 10- MDA-MB-231 Decoction 100 μg/mL, Lane 11- 100 bp ladder, Lane 12- SKBR-3 Control, Lane 13- SKBR-3 Paclitaxel 5 μg/mL, Lane 14- SKBR-3 Decoction 40 μg/mL, Lane 15- SKBR-3 Decoction 100 μg/mL, Lane 16- 100 bp ladder, Lane 17- MCF 10A Control, Lane 18- MCF10A Decoction 100 μg/mL

Fig. 7

Activation of Caspase 3/7 in breast cancer cells 24 h after treatment with the decoction. ***p <0.05. 7.1 MCF-7, 7.2 MDA-MB-231 cells, 7.3 SKBR-3 cells. Data are representative of three independent experiments (mean ± SD of three replicates)