Investigating the effect of the alcoholic extract of Pterocarya fraxinifolia (Poir) spach leaves on inhibiting the proliferation and expression of genes involved in apoptosis in human breast cancer cell line

Cancer is one of the most serious causes of death, and breast cancer is the most common malignancy among women. Medicinal plants can play a vital role in cancer treatment. Many effective cancer drugs today are derived from natural plant products. This study evaluates the cytotoxic effect of Pterocarya fraxinifolia leaf methanolic extract. It also evaluates its effect on the expression of the P21, BID, BCL-2, RB1, and MDM2 genes in the MCF-7 breast cancer cell line.

Extraction was done from 20 grams of dried and powdered leaves exposed to air by the soaking (maceration) method using pure methanol and after 24 hours in an incubator shaker at a speed of 120 rpm, temperature of 25°C and in the dark. After filtering and drying the extract, 5 mg of the resulting dry substance was dissolved in 1 ml of RPMI-1640 medium. After re-filtration, it was stored as a stock to prepare different concentrations. MCF-7 breast cancer cell lines and HGF-1 as a normal cell line were cultured in RPMI1640 medium containing FBS 10% (w/v), penicillin antibiotics, streptomycin 1% (w/v), and at 37°C temperature and 5% carbon dioxide pressure in an incubator. These cells were exposed to various concentrations of methanolic extract from Pterocarya fraxinifolia leaves for 24 hours. Cell survival rate was assessed with MTT colorimetric assay, and expression of genes involved in apoptosis (P21, BID, BCL-2, RB1, MDM2) in cancer cells treated with IC25 concentration of plant extract was evaluated by real-time PCR technique. RNA extraction from MCF-7 and HGF-1 cells was performed according to the RNX-plusTM kit instructions. cDNA synthesis was performed using Fermentase Company's kit (RevertAid First Strand cDNA Synthesis Kit) and according to its instructions. In this study, the GAPDH gene was used as an internal control.

The results of the MTT assay showed that the Pterocarya fraxinifolia (Poir) spach leaf methanolic extract had a concentration-dependent cytotoxic effect on MCF-7 breast cancer cells, and during the experiment, with increasing drug concentration, the effect of cytotoxicity increased in both cancer and normal lines and high inhibition was observed at concentrations of 1000 and 1200 μg.ml-1. The IC50 of Pterocarya fraxinifolia methanol extract against MCF-7 and HGF-1 cell lines was 452.1 and 479.2 μg.ml-1, respectively. Real-time PCR results showed that treatment with the Pterocarya fraxinifolia plant extract enhanced the expression of the P21 gene, while expression was nearly constant in extract-treated normal cells. The BID gene expression was increased in cancer cells treated with the plant extract. In contrast, normal cells under the influence of the extract showed a slight decrease in gene expression. The plant extract decreased the expression of the BCL-2 gene in cancer cells, whereas the expression of this gene in normal cells did not change significantly under the extract. The RB1 gene expression was not significantly altered in healthy cells after plant extract treatment but increased in the cancer cell line MCF-7. MDM2 gene expression in cancer cells treated with plant extract remained unchanged, whereas it slightly increased in healthy cells treated with extract.

This study provides an overview of how Pterocarya fraxinifolia extract can inhibit cancer cell growth. This study confirms the inhibitory activity of the plant's methanolic extract on breast cancer cells. With further investigation, the plant compounds may one day be used to treat cancer.