Studies On The Mechanisms Of EMD-621 And N-demethyl-clarithromycin-induced Cell Apoptosis

The mechanisms of EMD-621 -induced cell death in human cervical carcinoma HeLa, gastric adenocarcinoma SGC-901, and mechanism of N-demethyl-clarithromycin-induced HeLa cell death were studied in this dissertation.It showed that EMD-621 had potent antitumor activity in vivo, and it could inhibit the proliferation of SGC-7901, HeLa, K562 and MCF-7 in a dose- and time-dependent manner in vitro. Results of MTT assay, photomicroscopical observation, DNA agarose gel electrophoresis, LDH release, flowcytometric analysis cell-cycle distribution and Annexin V binding to the membrane phospholipid phosphatidylserine showed that EMD-621 could induce cell apoptosis in HeLa and SGC-7901 cells.After treatment with EMD-621, the caspase-3 of HeLa cells was activated, followed by the degradation of caspase-3 substrates, ICAD (inhibitor of caspase dependent DNase) and PARP (poly-(ADP-ribose) polymerase). Caspase family inhibitor, caspase-1, -3, -8 inhibitors could reduce EMD-621-induced HeLa cell death, and the caspase 10 inhibitor almost had no effect, while the caspase-9 inhibitor could increase the cell death. FasL (Fas ligand) and FADD (Fas-associated with death domain) expressions were up-regulated, which suggested that Fas/FasL pathway was activated in EMD-621-induced cell apoptosis. The drug decreased the expression of anti-apoptotic mitochondrial protein Bcl-2, not BcI-X_l, and increased the expression of pro-apoptotic protein Bax. It also could cause the loss of mitochondrial membrane potential. EMD-621 could increase the expression of pro-apoptotic protein p53 and p21 proteins and down-regulated the expression of anti-apoptotic protein SIRT1. The cell death was partially reduced by p38 MAPK inhibitor (SB 203580) and increased by ERK inhibitor (PD98059). At the same time, phosphorylation of ERK was down-regulated and that of p38 was up-regulated indicating different roles of these MAPK at different stages of cell death. Simultaneously, the activation of protein kinase B (PKB/Akt) was also decreased in EMD-621-treated HeLa cell.EMD-621-enhanced engulfment was partially blocked by a nuclear factor (NF-kB) inhibitor PDTC or proteasome inhibitor MG132. Further studies revealed that EMD-621 could induce IκBα(inhibitorκB) degradation. Tyrosine kinase inhibitor genistein also increased the cell death. As a whole, EMD-621 could induce apoptosis in HeLa cells via accumulation of p53, altered Bax/Bcl-2 ratio, activation of caspases and p38 MAPK.In EMD-621-treated SGC-7901 cells, caspase family inhibitor (z-VAD-fmk), caspase-1 inhibitor (Ac-YVAD-cmk), caspase-3 inhibitor (z-DEVD-fmk) and caspase-10 inhibitor (z-AEVD-fmk) partially augmented the cell death, while caspase-8 inhibitor (z-IETD-fmk) reversed the cell death and caspase-9 inhibitor (z-LEHD-fmk) almost had no effect. Caspase-3 was activated followed by the degradation of caspase-3 substrates, ICAD and PARP, and the caspase family inhibitor failed to inhibit PARP cleavage. This result suggested that there might have an unknown signal pathway from the mitochondrial to the downstream protein PARP, which is cleaved in a caspase-independent manner.ORAC (Oxygen Radical Absorbance Capacity) assay was used to detect the change of radical. EMD-621 almost had no effect on scavenging hydroxyl radical. FasL expression was up-regulated in 12 h and began to decrease in 36 h, while FADD expression was down-regulated suggesting that there might exist new target in EMD-621-induced cell apoptosis. EMD-621 up-regulated the expression ratio of Bax/Bcl-2 and significantly increased the expression of p53 protein. It also caused the loss of mitochondrial membrane potential. The cell death was partially reduced by p38 MAPK inhibitor (SB 203580), while ERK inhibitor (PD98059) augmented the cell death; ERK phosphorylation was down-regulated and p38 phosphorylation was up-regulated. Moreover, PI3K inhibitor wortmannin reduced the cell viability, EMD-621 induced IκBαdegradation and tyrosine kinase inhibitor, genistein, also augmented the cell death. Generally, EMD-621 could induce apoptosis in SGC-7901 cells via accumulation of p53, altered Bax/Bcl-2 ratio, and p38/JNK MAPK.N-demethyl-clarithromycin was the main metabolite of clarithromycin in vivo, and it had been reported that N-demethyl-clarithromycin had potent anti-inflammatory activity. There had no report on its anticancer activity. Therefore, for the first time the apoptotic effect of N-demethyl-clarithromycin on human cervical cancer HeLa cells was investigated in this study. This study showed that N-demethyl-clarithromycin could inhibit the proliferation of HeLa cells. Based on morphologic observation, the DNA fragmentation suggested that N-dernethyl-clarithromycin could induce HeLa cell death involved in a mechanism of apoptosis. The caspase inhibitors z-VAD-frnk, z-DEVD-fmk, z-LEHD-fmk effectively enhanced N-demethyl-clarithromycin-induced cell viability, while the z-IETD-fmk almost had no effect. Caspase-3 was activated followed by the degradation of caspase-3 substrates, ICAD and PARE N-demethyl-clarithromycin up-regulated the expression ratio of mitochondrial proteins Bax/Bcl-2 and significantly increased the expression of p53 protein. It also could cause the lost of mitochondrial membrane potential and down-regulated SIRT1 protein expression. ERK, JNK and p38 MAPK were activiated and the cell death were partially increased by ERK inhibitor (PD98059), JNK MAPK inhibitor (SP600125) and p38 MAPK inhibitor (SB203580). Simultaneously, the activation of protein kinase B (PKB/Akt) was also decreased in N-demethyl-clarithromycin-treated HeLa ceils. Moreover, EMD-621 induced IκBαdegradation and tyrosine kinase inhibitor genistein also augmented the cell death. These results indicated that N-demethyl-clarithromycin-induced apoptosis of HeLa cells might involve a mitochondrial-related caspase pathway.