Polyphenolic extract of InsP 5-ptase expressing tomato plants reduce the proliferation of MCF-7 breast cancer cells

Abstract

In recent years, by extensive achievements in understanding the mechanisms and the pathways affected by cancer, the focus of cancer research is shifting from developing new chemotherapy methods to using natural compounds with therapeutic properties to reduce the adverse effects of synthetic drugs on human health. We used fruit extracts from previously generated human type I InsP 5-ptase gene expressing transgenic tomato plants for assessment of the anti-cancer activity of established genetically modified tomato lines. Cellular assays (MTT, Fluorescent microscopy, Flow Cytometry analysis) were used to confirm that InsP 5-ptase fruit extract was more effective for reducing the proliferation of breast cancer cells compared to wild-type tomato fruit extract. Metabolome analysis of InsP 5-ptase expressing tomato fruits performed by LC-MS identified tomato metabolites that may play a key role in the increased anti-cancer activity observed for the transgenic fruits. Total transcriptome analysis of cancer cells (MCF-7 line) exposed to an extract of transgenic fruits revealed a number of differently regulated genes in the cells treated with transgenic extract compared to untreated cells or cells treated with wild-type tomato extract. Together, this data demonstrate the potential role of the plant derived metabolites in suppressing cell viability of cancer cells and further prove the potential application of plant genetic engineering in the cancer research and drug discovery.

Fig 1. Overview of applied research strategy.

Total metabolites of transgenic tomato fruits were analyzed for their potential anti-cancer properties by application of the total metabolite mixture to MCF-7 cancer cells followed by cell viability assays and transcriptome analysis of the cancer cells. The total tomato wild-type and transgenic fruit extracts were scanned for the potential metabolite with antioxidant and anti-cancer properties by using LC-MS.

Fig 2. Total phenolic content in mature (red) InsP 5-ptase expressing and wild-type tomato fruits.

(A) Standard curve using galic acid (0–1000 mg) as a standard reagent. (B) Total phenolic content in the mature tomato fruits of InsP 5-ptase transgenic lines (L6 and L7) and control lines (WT and EV). The total phenolic content is expressed as mg/g galic acid equivalent. The different letters (a,b) means statistically different groups (p<0.01) using Tukey Post-ANOVA test. Error bars represent standard error value.

Fig 3. Analysis of metabolome of wild-type (WT), empty vector control (EV) and InsP 5-ptase expressing tomato fruits (L6, L7) using LC-MS.

(A) Chromatogram built by MZmine. The chromatogram includes one fruit sample from each line analyzed: Line 6 (blue), Line 7 (green), Empty Vector (red), and Wild-Type (pink). Multiple compounds have been highlighted. Some minor retention shifts were corrected by the program. (B) Dendrogram created by Metaboanalyst from the list of cursorily identified peaks, showing the clustering of samples. (C) Metaboanalyst completed an ANOVA analysis on all samples with a p value < 0.01. Green dots are a representation of metabolites that were not significantly different. Red dots are a representation of metabolites that were significantly different between at least two samples.

Fig 4. Relative differences in concentrations between control (WT, EV) and transgenic tomato samples (L6, L7) for eight identified flavonoid metabolites.

The relative increase of concentration was based on average intensity fold change between samples. Separate images were created with Metaboanalyst using data generated by LC-MS.

Fig 5. Assay of cell viability of MCF-7 breast cancer cells treated with tomato extracts originated from control (WT, EV) and transgenic fruits (L6, L7).

(A) Fluorescent microscopy images demonstrating the Ethidium bromide/Acridine orange (EB-AO) staining assay on MCF-7 cancer cells. The cells were treated with total metabolite extract (1μg/ml) from transgenic (L6 and L7), and control (WT and EV) tomato fruits. The incubation time was 24 hrs at 37°C with 5% CO₂. The green fluorescence demonstrates the nucleus of the viable cells and the red fluorescence represents the nucleus of the apoptotic cells. The image is representative of the two independent experiments. (B) Statistical analysis of the EB-AO staining assay demonstrating the viability of the MCF-7 cells after incubation with total metabolite extract of tomato fruits. (C) MTT cell viability assay performed on MCF-7 cells using the same conditions to confirm the results of (EB-AO) assay. The different letters (a,b,c) means statistically different groups (p<0.01) using Tukey Post-ANOVA test. Error bars represent standard error value.

Fig 6. Data of flow cytometry analysis of MCF-7 breast cancer cells treated with tomato extracts originated from control (WT, EV) and transgenic fruits (L6, L7).

(A) Flow Cytometry analysis on MCF-7 cancer cell line after treatment with total metabolite extract of transgenic and control fruits. The results are presented as a percentage of live, necrotic and apoptotic cells (100% total). The cells treated with 1μg/ml of the total metabolite extract of control (WT and EV) and transgenic (L6 and L7) tomato fruit are compared to the non-treated cells (NC). Three biological replicates were used for this experiment. (B) Flow Cytometry analysis graph for representatives of the non-treated MCF-7 cells and the cells treated with total metabolite extract of tomato fruits. The figures show live cells (bottom left quadrant), apoptotic cells (bottom right quadrant) and necrotic cells (top quadrants).

Fig 7. Microarray heat-map analysis of MCF-7 breast cancer cells treated with tomato fruit extracts originated from control and transgenic tomato lines.

The analysis shows the genes that demonstrate changes in expression after treatment with total metabolite extract from transgenic lines (L6 and L7) and control line (WT) compared to non-treated cells (NC). The MCF-7 cells were incubated with the tomato fruits extracts (1μg/ml) for 24 hrs, after which the cells were harvested and the total RNA was extracted. The genes with significant change in expression were selected after statistical analysis using ANOVA statistical test using a p-value <0.001 and Tukey post-hoc test. Only the genes with identified functions are displayed. The range of fold change of identified genes is from -0.5 to 0.5.