Chemopreventive mechanism of polypeptides from Chlamy Farreri (PCF) against UVB-induced malignant transformation of HaCaT cells.
To investigate polypeptide from Chlamy Farreri (PCF)'s protective effect against skin cancer, we used a cellular model of ultraviolet B (UVB)-induced malignant transformation. The human keratinocyte cell line HaCaT was repeatly exposed to UVB (10 mJ/cm(2), 20 times) and malignant transformation was confirmed by Gimesa staining, cell cycle analysis and various assays [anchorage independent growth, matrix metalloproteinase-9 (MMP9) activity, plating efficiency]. The malignant transformation was found to be effectively prevented by PCF pretreatment (2.84mM for 2h prior to each UVB exposure). We investigated the mechanism of PCF-mediated action by determining its effect on DNA methylation status of the tumour suppressor genes [P16 and ras association domain family 1 A (RASSF1A)] in the UVB-transformed cells. Both genes were found to be hypermethylated by chronic UVB exposure. The expression levels of P16, RASSF1A, DNA methyltransferases (DNMTs) and DNA damage inducible protein a (GADD45a) were measured by reverse transcriptase-polymerase chain reaction and western blotting. While chronic UVB exposure was found to suppress the expression of P16 and RASSF1A, it enhanced the expression of DNMT3b. In the early phase of UVB-induced malignant transformation, the GADD45a expression was increased, however, it declined with a continued irradiation of the cells. The UVB-induced DNA hypermethylation of P16 and RASSF1A and subsequent gene silencing was reversed by PCF treatment. The inhibition of DNMTs expression suggested that PCF blocked DNA methylation and thereby the silencing of tumour suppressor genes. Furthermore, the PCF-mediated substantial increase in GADD45a expression indicated that PCF promoted demethylation of tumour suppressor genes via GADD45a induction.
Qingdao University Medical College, 308 Ningxia Road, Qingdao 266071, Shandong, China hanyt19@126.com.
Yantao
Y
Han
Qingdao University Medical College, 308 Ningxia Road, Qingdao 266071, Shandong, China.
Huihui
H
Zhao
Qingdao University Medical College, 308 Ningxia Road, Qingdao 266071, Shandong, China.
Qixiao
Q
Jiang
Qingdao University Medical College, 308 Ningxia Road, Qingdao 266071, Shandong, China.
Hui
H
Gao
Qingdao University Medical College, 308 Ningxia Road, Qingdao 266071, Shandong, China.
Chunbo
C
Wang
D000818:Animals; D018797:Cell Cycle Proteins / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000187:drug effects* / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D019175:DNA Methylation / Q000187:drug effects / Q000528:radiation effects; D015870:Gene Expression / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000528:radiation effects*; D009363:Neoplasm Proteins / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D009381:Neoplasms, Radiation-Induced / Q000235:genetics / Q000517:prevention & control*; D009687:Nuclear Proteins / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D049897:Pectinidae; D010455:Peptides / Q000494:pharmacology*; D020011:Protective Agents / Q000494:pharmacology; D012878:Skin Neoplasms / Q000235:genetics / Q000517:prevention & control*; D025521:Tumor Suppressor Proteins / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D014466:Ultraviolet Rays; D000097584:DNA Methyltransferase 3B; D000097642:GADD45 Proteins
D018797:Cell Cycle Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004248:DNA (Cytosine-5-)-Methyltransferases; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D010455:Peptides; D020011:Protective Agents; D025521:Tumor Suppressor Proteins; D000097584:DNA Methyltransferase 3B; C000614131:CDKN2A protein, human; C439070:GADD45A protein, human; D000097642:GADD45 Proteins; C411762:RASSF1 protein, human