Pseudolaric acid B inhibits inducible cyclooxygenase-2 expression via downregulation of the NF-κB pathway in HT-29 cells.
OBJECTIVE
Pseudolaric acid B (PAB) is a diterpene acid isolated from the root and trunk bark of Pseudolaric kaempferi Gordon. Previous work has found that PAB has anti-inflammatory and anti-tumor effects in xenograft models of human hepatocellular carcinoma. The aim of this study is to evaluate the correlation between anti-cancer and anti-inflammatory effects of PAB and its molecular mechanisms on HT-29 cells.


METHODS
Production of prostaglandin E2 (PGE2) in HT-29 cells was evaluated by ELISA. mRNA of cyclooxygenase-2 (COX-2) was analyzed by RT-PCR assay. High-content screening (HCS) method was adopted to detect the cytokine mixture (CM)-induced transcription activity of NF-κB and STAT3. Western blotting was used to evaluate the protein expression levels of inflammatory mediators induced by CM. After treatment with PAB in various concentrations, the inhibition rate of cell proliferation was measured with sulforhodamine B assays. For the in vivo studies, tumor-bearing models xenografted with HT-29 cells were developed in nude mice, and following oral administration with PAB, tumor inhibition rate was calculated.


RESULTS
PAB inhibited the PGE2 production in HT-29 cells significantly (P < 0.05) with similar results detected at the COX-2 mRNA level. Furthermore, PAB suppressed the COX-2 protein expression and significant nuclear translocation of NF-κB and STAT3 induced by CM, which correlated with a concomitant degradation of I-κB and a decrease in constitutive STAT3 phosphorylation (P < 0.05). Moreover, various concentrations of PAB inhibited the proliferation of HT-29 cells in a dose- and time-dependent manner. In vivo, after treatment with PAB for 17 days, the tumor weight of the 50 and 100 mg/kg treated groups was 0.62 ± 0.15 and 0.54 ± 0.06 g, respectively. When compared to the control group (0.82 ± 0.16 g), the inhibition rate of tumor weight was 24.2% at 50 mg/kg (P < 0.05) and 34.7% at 100 mg/kg (P < 0.001).


CONCLUSIONS
PAB shows potential anti-cancer activity in HT-29 cells, and its molecular mechanisms are related to the anti-inflammatory action.
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38, Xueyuan Road, Haidian District, Beijing, 100191, China.
Li
L
Hou
Bo
B
Xu
Wei
W
Guo
Fu-Xiang
FX
Ran
Jing-Tao
JT
Liu
Xia
X
Yuan
Hong-Zheng
HZ
Fu
Jing-Rong
JR
Cui
D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D051546:Cyclooxygenase 2 / Q000235:genetics*; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology*; D015232:Dinoprostone / Q000378:metabolism; D004224:Diterpenes / Q000494:pharmacology*; D015536:Down-Regulation; D004365:Drugs, Chinese Herbal / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D019073:HT29 Cells / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D016328:NF-kappa B / Q000378:metabolism*; D009374:Neoplasms, Experimental / Q000188:drug therapy; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
D052246:Cyclooxygenase 2 Inhibitors; D004224:Diterpenes; D004365:Drugs, Chinese Herbal; D016328:NF-kappa B; D012333:RNA, Messenger; C058391:pseudolaric acid B; D051546:Cyclooxygenase 2; D015232:Dinoprostone