Mutational changes in aromatase gene of breast cancer patients and cytotoxic effect of anthocyanins extracted from grape seeds (Vitis venifera)‎ in vitro

Other Title(s)

التغيرات التطفيرية في جين الأروماتيز لمريضات سرطان الثدي و التأثيرات السمية الخلوية للأنثوسيانين المستخلص من بذور العنب الأسود (Vitis venifera)‎ في الزجاج

Dissertant

al-Amiri, Rahim Fadil Husayn

Thesis advisor

Nada, Sad Muhammad
Shawkat, Muayyad Sabri

University

University of Baghdad

Faculty

College of Science

Department

Biotechnology Department

University Country

Iraq

Degree

Ph.D.

Degree Date

2014

English Abstract

One hundred blood samples of Women with breast cancer were collected from Early Cancer Detection Center in Baghdad, Medical City from 01/02/2012 to 15/08/2012.

In addition Blood samples were taken from 50 healthy individual (without breast cancer) as a control group.

Blood samples were subjected to DNA isolation and molecular detection for mutation in first position of codon 39 (T-C substitution) by using CTPP-PCR techniques.

A mutation was found for the first time in Iraqi population in the first position of codon 39 in aromatase gene which led to aromatase gene appears with two alleles: the first allele which is mutant allele (C allele) contains C base in the first position in codon 39 and normal allele (T allele) that contains T base in the first position in codon 39 in aromatase gene.

This mutation was approved by DNA sequencing for PCR fragment for mutant and normal alleles; the point mutation in the first position of codon 39 was compared with the normal aromatase sequence on NCBI web site.

This mutation caused aromatase gene appeared in three genotypes namely TT, TC and CC with percentage frequencies 79, 18 and 3% respectively in breast cancer women while the percentage frequencies for this genotype were 82, 18 and 0% in healthy women.

In addition results showed that T- and C alleles frequencies were 88 and 12% for diseased women, 92 and 8% for healthy women.

Furthermore, the aromatase concentration in diseased women, plasma was higher than healthy women.

Anthocyanins were extracted from grape (Vitis venifera) seed and were examined as a growth inhibitor for MCF7 and MDAMB231 breast cancer cells lines.

It was found that anthocyanins induced growth inhibitor for both cells in dose dependent manner which is 25μg/ml, when cells were treated with anthocyanins at the concentrations 10, 25, 50, 75 and 100μg/ml, the cells viability Summary II percentage of MCF7 cells were 95.6, 34.3, 24, 21.8 and 18% respectively and the cells viability for MDAMB231 cells were 97.05, 25.58, 21.94, 16.29 and 6.77% respectively, also the concentration 25 μg/ml showed dose dependent effect on both cells.

Anthocyanins have cytotoxicity effect against MCF cells, and this effect was confirmed by flow cytometeric analysis.

When MCF7 cells were treated with anthocyanins at concentrations 10, 25, 50, 75 and 10μg/ml respectively, the percentage of G2/M phase were 10.9, 14.3, 17.6, 20.8 and 23.7% respectively.

Results showed an increase in cells in G2/M phase, these events were accompanied with G2 in MCF7 cells, accumulating cells in G2 phase, this suggesting that anthocyanins block MCF7 cells in transition from G2 to M phase.

On the other hand, anthocyanins induced apoptosis in MCF7 cells upon treatment with anthocyanins concentration 10, 25, 50, 75 and 100μg/ml respectively for 16 hrs.and the mean values for MCF7 cells in late apoptotic population were 4.8, 5.6, 6.5, 9.4 and 11.4 respectively however, treatment with the same concentrations for 24 hrs.

gave the mean values for MCF7 cells 23, 29.7, 32.2, 44 and 49 respectively, these results led revealed anthocyanins ability to drive MCF7 cells to apoptosis after treatment with anthocyanins, this was confirmed by western blotting method for investigating proteins participate in apoptosis development.

Results showed that increasing anthocyanins led to increase p53, p21 and tublin proteins which play a crucial role in apoptosis progress, also the results showed that anthocyanins have the ability to affect on aromatase protein by inhibiting its expression when treating MCF7 cells with anthocyanins.

Furthermore aromatase gene expression in cancer and normal breast tissues were studied by using quantitive polymerase chain reaction (qPCR) for breast tissue specimens, a qPCR analyzing using oligonucliotide primer derived from exon I of human aromatase gene revealed that aromatase mRNA was detected in Summary III 31 tissue specimens, however no aromatase gene expression was found in 4 tissues specimens while 5 cases exhibited aromatase gene expression less than normal state.

Main Subjects

Biology

Topics

• Genetics
• Iraq
• Health aspects

No. of Pages

150

Table of Contents

• APA
• MLA
• AMA

Language

English

Data Type

Arab Theses

Record ID

BIM-601463