[Traditional medicine CDP and promoter demethylation in breast cancer cell lines].
OBJECTIVE
To investigate the demethylation effect of CDP on P16 and E-CADHERIN genes.


METHODS
Breast cancer cell lines T47D and MDA-MB-435 were treated with CDP and DNA methyltransferase inhibitor 5-azacytidine (5-aza-C). The methylation of P16 and E-CADHERIN gene promoters were measured by methylation-specific PCR (MSP). The RNA transcription was determined by reverse transcription-PCR(RT-PCR).


RESULTS
1) The methylation-specific fragments of P16 gene promoter existed in T47D cells after 25, 50 and 75 micromol/L of CDP treatment for 6 days. An absolute demethylation on P16 gene occurred after treatment with 100 micromol/L of CDP. The unmethylation-specific fragments appeared in T47D cells after being treated with 25, 50, 75 and 100 micromol/L of CDP for 6 days. The RNA expression of P16 was detected after treatment with 75 and 100 micromol/L of CDP. 2) After being treated with 50 micromol/L of CDP, the methylation-specific fragments of CpG island in P16 gene promoter still existed in T47D cells. The unmethylation-specific fragments in T47D cells started to appear after 24 hours of treatment and lasted until 144 hour of treatment. The RNA expression was detected after 144 hours of treatment. 3) The demethylation on E-CADHERIN gene and genomic DNA or RNA transcription were not detected in MDA-MB-435 cells.


CONCLUSIONS
CDP has concentration- and time-dependent demethylation effect on P16 gene in T47D cells, but not on E-CADHERIN gene in MDA-MB-435 cells, which indicates that CDP has substantial diversity in molecular activities.
Chai
Xin-Juan
XJ
Department of Biochemistry and Molecular Biology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, China.
Luan
Ju
J
Yang
Wen-Li
WL
Zhang
Ying-Chun
YC
Ge
Ya-Jun
YJ
Qin
Yang
Y
D001374:Azacitidine / Q000494:pharmacology; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D015820:Cadherins / Q000235:genetics*; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects*; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D019942:Genes, p16; D006801:Humans
D015820:Cadherins; D004365:Drugs, Chinese Herbal; D001374:Azacitidine