[Triptolide evaluates DNA methylation level of matrix metalloproteinase 9 gene in human fibrosarcoma HT-1080 cells].
OBJECTIVE
The effect of triptolide on the DNA methylation level of MMP-9 gene and the mRNA expression of tissue inhibitors of met-alloproteinases (TIMPs) were examined in human fibrosarcoma HT-1080 cells to explore the molecular mechanisms involved in the anticancer activity of triptolide.


METHODS
HT-1080 cells were cultured in MEM containing 10% newborn calf serum and 1% penicillin-streptomycin. Triptolide was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 goL-1 and stored at -20 degrees C. Triptolide was freshly diluted with culture medium perior to use and directly added to cell cultures at the indicated concentration, and incubated for 72 hours at 37 degrees C in a humidified atmosphere with 5% CO2, with changes of reagents every 24 hours. Methylation specific PCR (MSP)was applied to assess the methylation status of MMP-9 gene promoter, and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was employed to measure the mRNA expression of tissue inhibitors of metalloproteinases (TIMPs) in human fibrosarcoma HT-1080 cells after 72 hours of treatment with 6 nmol x L(-1), 12 nmol x L(-1) or 18 nmol x L(-1) triptolide, respectively.


RESULTS
The methylation index of MMP-9 gene promoter was statistically elevated in HT-1080 cells after 72 hours of treatment with 18 nmol L(-1) triptolide, compared with those in controls (0.61 +/- 0.10 vs 0.39 +/- 0.10, P < 0.05), while no significant difference was noted between 6 nmol x L(-1) or 12 nmol x L(-1) triptolide treated HT-1080 cells and controls (0.40 +/- 0.15 vs 0.39 +/- 0.10, 0.46 +/- 0.20 vs 0.39 +/- 0.10, respectively, both P > 0.05). The mRNA expression of TIMP-1, -2, -3 or -4 was not significantly changed in HT-1080 cells after 72 hours of treatment with the indicated concentrations of triptolide, respectively compared with those in controls (all P > 0.05).


CONCLUSIONS
The results demonstrated that triptolide upregulates the methylation level of MMP-9 gene in HT-1080 cells in vitro.
Yang
Shengbo
S
Department of Dermatology and Venereology, Second Xiangya Hospital, Central South University, Changsha 410011, China.
Wen
Haiquan
H
Zhang
Guiying
G
Zhao
Sha
S
Luo
Yongqi
Y
Lu
Qianjin
Q
D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000187:drug effects*; D004224:Diterpenes / Q000494:pharmacology*; D004852:Epoxy Compounds / Q000494:pharmacology; D005354:Fibrosarcoma / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000235:genetics* / Q000378:metabolism; D010616:Phenanthrenes / Q000494:pharmacology*; D019714:Tissue Inhibitor of Metalloproteinases / Q000235:genetics / Q000378:metabolism
D018906:Antineoplastic Agents, Alkylating; D004224:Diterpenes; D004852:Epoxy Compounds; D010616:Phenanthrenes; D019714:Tissue Inhibitor of Metalloproteinases; C001899:triptolide; D020780:Matrix Metalloproteinase 9