Anacardic acid (AA) is a mixture of 2-hydroxy-6-alkylbenzoic acid homologs. It is widely regarded as a non-specific histone acetyltransferase inhibitor of p300. The effects and the mechanisms of AA in LNCaP cells (prostate cancer cells) remain unknown. To investigate the effect of AA on LNCaP cells, we had carried out several experiments and found that AA inhibits LNCaP cell proliferation, induces G1/S cell cycle arrest and apoptosis of LNCaP cell. The mechanisms via which AA acts on LNCaP cells may be due to the following aspects. First, AA can regulate p300 transcription and protein level except for its mechanisms regulating function of p300 through post-translational modification in LNCaP cells. Second, AA can activate p53 through increasing the phosphorylation of p53 on Ser15 in LNCaP cells. AA can selectively activate p21 (target genes of p53). Third, AA can down-regulates androgen receptor (AR) through supressing p300. Our study suggests that AA has multiple anti-tumor activities in LNCaP cells and warrants further investigation. Chin J Cancer Res 2012;24(4):275-283 www.thecjcr.org culture medium when necessary. DMEM, growth factorreduced matrigel and fetalbovine serum (FBS) were bought from BD Corporation (San Jose, CA). The Lipofectamine™ 2000 and the Total RNA Extraction kit were purchased from Invitrogen (Carlsbad, CA). The MTT Cell Proliferation and Cytotoxicity Assay kit and the Annexin V-FITC & PI Apoptosis Detection kit were purchased from KeyGen Biotech (Nanjing, China). SYBR Green PCR Master Mix were purchased from Fermentas (Burlington, Ontario, Canada). The Total Protein Extraction kit was purchased from ProMab (Changsha, China). Primary antibodies for p300, p21, p53, AR, cyclin D1 and siRNA specifically for p300 were bought from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibody for phospho-p53 (Ser 15) was purchased from Cell Signaling Technology. RPMI 1640 was purchased from GIBCO.
Cell lines and cell cultureLNCaP was purchased from Yinrun (Changsha, China). LNCaP is a classical metastatic prostate adenocarcinoma cell line, derived from metastatic lymph nodes. LNCaP was cultured with both RPMI 1640 and 10% FBS, and subcultured weekly (37 ℃, 5% CO 2 ).
p300 siRNA transfectionT h e L n c a p c e l l s w e r e s e e d e d i n t o 6 -w e l l p l a t e s (4×10 4 -5×10 4 cells/well) and cultured in 2 mL basic culture medium containing 10% FBS overnight until the cells were 70% confluent. Cells were transiently transfected with a validated scrambled control siRNA, or p300 siRNA by using InterferinTM transfection reagent. The mixture of siRNA and InterferinTM transfection reagent was incubated for 10 min, added to each well of the 6-well plates and incubated at 37 ℃ for 24 h before drug treatment.
Detecting the inhibition of cell growth using MTT assaysLncap cells were treated with AA or transfected with p300 siRNA before drug treatment. Cells were incubated at various concentrations (0, 5, 25, and 125 μmol/L) at a series of time points (0, 4, 24 and 28 h). Then, 5 mg/mL of MTT so...