Pseudolaric Acid Mechanism Of Apoptosis Induced By Tumor Cells

Pseudolaric acid B is a diterpene acid isolated from the root bark of Pseudolarix Kaempferi Gorden, and exerts potent antifungal, antimicrobial and antifertility activities. The mechanisms of pseudolaric acid B-induced cell death in human cervical carcinoma HeLa and human melanoma A375-S2 in vitro were reported in this dissertation.The studies demonstrate that pseudolaric acid B inhibited the proliferation of HeLa and A375-S2 cells in a time- and dose-dependent manner. MTT assay, photomicroscopical observation, fluorescence microscopical observation, TUNEL and DNA agarose gel electrophoresis showed that pseudolaric acid B induced cell apoptosis in HeLa and A375-S2 cells.In 5 μM. pseudolaric acid B-treated HeLa cells, caspase-3 proenzyme decreased followed by the degradation of caspase-3 substrates, ICAD (inhibitor of caspase dependent DNase) and PARP (poly-(ADP-ribose) polymerase). Caspase-3 inhibitor, z-DEVD-fmk, partially reduced pseudolaric acid B-induced HeLa cell death, which indicates caspase-3 was activated. Pseudolaric acid B increased the expression of pro-apoptotic protein, Bax and decreased the expression of anti-apoptotic mitochondrial protein, Bcl-2, but not BcI-X_l. Pseudolaric acid B increased the expression of p53 protein at 24 h and peaked at 36 h. Mitogen-activated protein kinase (MAPK) is a kind of Ser/Thr protein kinase, which exerts different effects in signal transduction. Pseudolaric acid B increased phosphorylation of JNK and JNK MAPK inhibitor (SP600125) apparently inhibited pseudolaric acid B-induced cell death. SP600125 also inhibited pseudolaric acid B-induced DNA fragmentation. Furthermore, pseudolaric acid B reversed the up-regulation of p53 and down-regulation of p-ERK. Pseudolaric acid B increased the activity of PKC and staurosporine, PDTC, genistein and manumycin A partially inhibited pseudolaric acid B-induced cell death.