Studies On The Mechanisms Of Oridonin-induced Tumor Cell Death

This dissertation reports that oridonin isolated from Rabdosia rubescens, induced cell death through different mechanisms in A375-S2, HeLa, and L929 cells.The studies demonstrate that oridonin significantly inhibited proliferation of several lines of tumor cells, including human malignent melanoma A375-S2 (IC50: 15.1 1.2 mol/L), chronic myelogenous leukemic cell K562 (IC50: 16.2 1.2 mol/L), human histocytic lymphoma U937 (IC50: 27.1 2.3 mol/L), promyelocytic cell HL-60 (IC50: 20.4 3.6 mol/L), and mouse fibrosarcoma L929 (IC50: 35.6 4.2 mol/L). Moreover, treatment with oridonin 34.3 mol/L for 12 h significantly inhibited A375-S2 cell growth, and showed minor cytotoxicity against PBMC, suggesting that oridonin showed selective antitumor activity and had less side effects on human normal cells.In oridonin-treated A375-S2 cells for 12 h, cysteine aspartic specific protease: caspase-3, -8 were activated at early stages, apoptotic bodies were formed, nuclear damage was observed by Hoechst 33258 staining and DNA fragmentation was exhibited. Oridonin increased the expression of the apoptosis inducer, Bax, decreased the expression of the anti-apoptotic protein, Bcl-xL, promoted the release of cytochrome c without affecting Bcl-2 expression, and activated down-stream caspase-9 in mitochondrial pathway. These observations indicated that appropriate dose of oridonin envisioned an initial premitochondrial phase that involved the Bcl-2 family of proapoptotic protein Bax that required the participation of caspase-9 and caspase-3. However, treatment with oridonin 137.4 mol/L for 12 h, the majority of A375-S2 cell underwent necrosis measured by LDH activity-based assay. Twelve hours after treatment with 34.3 mol/L oridonin, the ratio of Bax/ Bcl-xL protein expression was increased and release of cytochrome c were decreased by extracellular signal-regulated kinase (ERK) MARK inhibitor (PD98059) and phosphoinositide 3-kinases (PI3-K) inhibitor, wortmannin. Mitochondrial permeability transition (MPT) inhibitor, decylubiquinone, suppressed the release of cytochrome c without affectingBax expression. The activation of tumor suppression factor p53 by oridonin was also blocked by wortmannin. Taken together, Oridonin induced A375-S2 cell apoptosis by activating parallel p53, ERK and p38 pathways, increasing the ratio of Bax/Bcl-xL protein expression, and promoting the release of cytochrome c into cytosol, resulting in apoptotic cell death.The growth-inhibitory activity of oridonin against L929 cells is in time- and dose-dependent manner. Treatment with various concentrations of oridonin for 12 h, the majority of L929 cells underwent apoptosis as measured by LDH activity-based assay. Although apoptotic bodies were observed in oridonin-treated L929 cells, DNA fragmentation as a hallmark of apoptosis was not found. Pan-caspase inhibitor, z-VAD and caspase-3 inhibitor, z-DEVD sensitized L929 cells to oridonin instead of inhibition of apoptosis, however, PARP inhibitor (DPQ) effectively blocked oridonin-induced cell death. At 12 h treatment, PARP proenzyme was significantly cleaved. This result indicated that oridonin-induced L929 cell death required PARP degradation in caspases-independent manner. In addition, MEK/ERK inhibitor (PD98059) markedly blocked oridonin-induced cell death, whereas p38 inhibitor (SB203580) and JNK inhibitor (SP600125) weakly protected the cells against death. Treatment with 41.2 fonol/L oridonin for 12 h significantly induced a persistent ERK activation and p38 inactivation in L929 cells without evident changes in the protein levels. Moreover, oridonin increased the ratio of Bax/Bcl-2 protein expression, whereas it had no effect on the expression of Bcl-xL. These results indicated that regulation of Bcl-2 and MAPK families may be the effector mechanisms of oridonin-induced L929 cell death, independent of caspase pathway.In HeLa cells, oridonin induced oligonucleosomal fragmentation of DNA and increased caspase-3 activity, on the other hand, reduced the expression of inhibitor of caspase-3-a...