All other chemicals were of reagent grade. == Cell proliferation assay == Cell proliferation was measured by the MTT assay. gene and p53 protein levels via the inhibition of HPV-16 E6 expression. Keywords:nordihydroguaiaretic acid, cervical cancer, histone acetylation, p21, p53 == Introduction == Nordihydroguaiaretic acid (NDGA) is a non-specific inhibitor of lipoxygenase. NDGA and its derivatives exhibit anti-cancer effect on various types of cancer, including cervical cancer (13). High-risk human papillomavirus (HPV) infection is strongly associated with cervical cancer. The persistent expression of HPV oncogenes E6 and E7 is crucial for the induction and maintenance of malignant phenotypes (4). HPV E6 and E7 are well-known for their ability to inhibit the activity of tumor suppressors p53 and pRb, respectively (5). Studies have shown that derivatives of NDGA inhibit the growth of cervical cancer Ebastine cells by stabilizing p53 as a result of down-regulated E6 expression, which may be partially responsible for apoptosis induction Ebastine or cell cycle arrest at the G1phase found in NDGA-treated cells (1,6). p21 is a key mediator of G1/S transition, the expression of which is frequently absent or at low levels in cervical cancer (7), and the restoration of p21 expression suppresses the growth of cervical cancer cells (8,9). It is known that regulation of p21 expression occurs primarily at the transcription level in p53-dependent or p53-independent manners (10) and acetylation of histone H3 within the p21 promoter is also a key event for effective p21 transcription (11,12). With respect to cervical cancer, the results of certain studies showed that the p53 state is correlated with p21 expression (13) and that restoration of p53 function, either by RNAi or drug treatment against HPV E6, induces p21 expression (14,15). This study aimed to investigate the effect of NDGA on the growth Ebastine ID1 and p21 expression of cervical cancer SiHa cells. Moreover, the effect of NDGA on levels of histone H3 acetylation, p53 protein and other factors was investigated in order to determine the mechanism by which NDGA regulated the p21 gene expression. == Materials and methods == == Cell line == The human cervical cancer cell line SiHa was obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai Institute of Cell Biology, Chinese Academy of Sciences, China), cultured in Dulbeccos modified Eagles medium (DMEM; 4.5 g/l glucose; Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 g/ml streptomycin in a 5% CO2incubator at 37C. == Reagents == NDGA (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) and the stock solution was diluted to the required Ebastine concentrations in DMEM prior to being added to Ebastine the cell culture medium. Final DMSO concentrations were 0.2%. Antibodies against Ac-Histone H3 (Lys 9/14) and -actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against p53 were purchased from Signalway Antibody (Pearland, TX, USA). All other chemicals were of reagent grade. == Cell proliferation assay == Cell proliferation was measured by the MTT assay. SiHa cells were seeded into 96-well culture plates at 5103cells per well. Each group consisted of six parallel wells and was subsequently treated with various concentrations of NDGA for 24, 48 and 72 h. The control cells were treated with 2% DMSO in culture medium. Following treatment, the cells were incubated with MTT reagent (0.5 mg/ml) at 37C for 4 h. The resulting formazan crystals were solubilized by adding 150 l DMSO to each well. The optical density (OD) at 492 nm was measured and cell viability was determined using the formula: cell viability (%) = (absorbance of the treated wells – absorbance of the blank control wells)/(absorbance of the negative control wells – absorbance of the blank control wells) 100%. The MTT experiments were repeated at least three times. == Flow cytometry analysis == The cell cycle was analyzed using flow cytometry. Briefly, cells (1106) were collected and washed twice in phosphate-buffered saline (PBS), then fixed in 70% alcohol for 30 min at 4C. After being washed in cold PBS three times, the cells were resuspended in 1 ml.