Scrophularia Atropatana Extract Reverses TP53 Gene Promoter Hypermethylation and Decreases Survivin Antiapoptotic Gene Expression in Breast Cancer Cells

Abstract

Background: In many cases of breast cancer, the aberrant methylation of TP53 gene leads to uncontrolled cell proliferation and apoptosis inhibition. Moreover, expression of oncogenes which are under the control of P53 protein could be altered. Survivin as a conspicuous example of this category plays important roles in tumorigenesis, drug resistance and apoptosis inhibition. The present study was done to reveal the effects of Scrophularia atropatana extract on epigenetic situation of TP53 gene promoter and the expression levels of anti-apoptotic gene, survivin and its potential for production of cancer epi-drugs. Methods: Cytotoxic effect of dichloromethane extracts of Scrophularia plant on MCF-7 cell line was assessed in our previous study. Cell death ELISA (enzyme-linked immunosorbent assay) and TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) tests were used to investigate the occurrence of apoptosis in the treated cells. Methylation Specific PCR (MSP) was employed to assess the changes in methylation status of the TP53 gene promoter. Furthermore, quantitative real time PCR was utilized to evaluate the resulting changes in TP53 and survivin genes expression. Results: Cell death ELISA and TUNEL assays confirmed the occurrence of apoptosis. MSP test revealed a significant change in the methylation status of TP53 promoter. QRT-PCR showed an increased TP53 gene expression in the treated cells while a significant decrease in survivin mRNA was evident. Conclusions: According to the outcomes, dichloromethane extract of S. atropatana returned the TP53 gene promoter hypermethylation to normal state. This plant could be a promising source for production of epi-drugs due to its apoptotic effects and reversal of TP53 epigenetic alterations.

Keywords: BIRC5; Epigenetics; P53; Scrophularia atropatana; survivin.

Figure 1

Apoptotic Effects of SADCM on MCF-7 Cells are Observed by TUNEL Assay. A, Non Treated Cells; B, MCF-7 Cells Treated with SADCM. Arrows Indicate the Apoptotic Cells.

Figure 2

Comparison of Apoptosis and Necrosis between Untreated MCF-7 Cells and the Cells Treated with 200 and 400 mg/ml Concentrations of SADCM in 24 hours (P < 0.01).

Figure 3

MSP with Methylated Primer Pairs. The amplicon in 166 bp size, represents a methylated status. N --, control DNA extracted from peripheral blood (neither methylated nor treated with SBS); N ++, control DNA extracted from peripheral blood (methylated using M.SssI CpG Methyltransferase enzyme and treated with SBS); M -, untreated MCF-7 cells; M +, MCF-7 cells treated with SADCM, L: 100 bp Ladder.

Figure 4

N --, Control DNA Extracted from Peripheral Blood (neither methylated nor treated with SBS); M -, untreated MCF-7 cells; M +, MCF-7 cells treated with SADCM; L, 100 bp Ladder.

Figure 5

mRNA Expression Levels in Untreated Cells Versus MCF-7 Cells Treated with 200 and 400 mg/ml Concentrations of SADCM in 24 h; A, TP53 and B, survivin (P < 0.01).