[Effects of Emodin Derivative on Cell Cycle, Apoptosis and NF-κB Pathway in Burkitt Lymphoma Cells].

OBJECTIVE: To investigate the effect and mechanism of a novel emodin derivative YX-18 on Burkitt lymphoma (BL) cells. METHODS: MTT assay was used to detect the effect of YX-18 on the proliferation of BL cell lines CA46 and Raji. Annexin V-PE/7-AAD double staining assay was used for detecting the effect of YX-18 on the apoptosis of CA46 and Raji cells. PI/RNase staining was used to test the effect of YX-18 on CA46 and Raji cell cycle. JC-1 method was used to measure the changes of mitochondrial membrane potential after YX-18 treatment, and DAPI staining was used to detect the morphology of apoptotic cells. Western blot was used to analyze the distribution changes of NF-κB pathway protein (P65, P-P65, IκB, P-IκB) in the cytoplasm and cell nucleus, and also the expression changes of cyclin-related protein P21, CDK2, P-CDK2, Cycling D1, Cycling E1, and the apoptosis-related protein Caspase-3, Caspase-8, Caspase-9 and the proliferation-related protein C-MYC, BCL-2 by YX-18. Real-time fluorescence-quantitative PCR was used to evaluate the effects of YX-18 on mRNA levels of C-MYC and Ki-67 genes in CA46 and Raji cells, and EBNA-1 and EBER genes of EBV in Raji (EBV+) cells. RESULTS: Novel Emodin derivative YX-18 could effectively inhibit the proliferation of BL cell lines CA46 and Raji, showing a time-dependent effect (24, 48 and 72 h: rCA46=0.89, 0.75, 0.75, rRaji=0.87, 0.73, 0.64). IC50 of CA46 cells and Raji cells treated with YX-18 for 24 h was 1.77±0.04 µmol/L and 1.97±0.22µmol/L, respectively. CA46 cells and Raji cells were treated with YX-18 at concentration of 2.0 and 4.0 µmol/L for 24 h. Compared with the control group, both strains of cells showed a very significant apoptosis at the concentration of 2.0 and 4.0 µmol/L (P<0.01), showing a concentration-dependent effect (rCA46=0.99, rRaji=0.92). Moreover, the cleavaged Caspase-3, 8 and 9 proteins were activated by YX-18 into verious degrees in both two cell lines. Both the two cell lines displayed by YX-18 cell cycle arrest at G0/G1 phase (P<0.01) after exposed to YX-18 for 24 hours at the concentration of 1.0, 2.0 µmol/L in CA46 cells and at 0.5 and 1 µmol/L in Raji cells, respectively. YX-18 decreased expression level of cyclin D1, cyclin E1, CDK2, p-cdk2 proteins and increased p21Waf1/Cip1 level in CA46 and Raji cells. YX-18 significantly declined mitochondrial membrane potential in both cells at the concentration of 2.0 and 4.0 µmol/l (P<0.01) with concentration-dependent manner (rCA46=-0.96, rRaji=-0.99). Western blot tests indicated that YX-18 down-regulated nucleus P65 and intracellular cytoplasm P65, P-IκB, P-P65 protein, and upregulated intracellular IκB level with dose-dependent manner. Meanwhile, the expression level of the cell proliferation-related molecules C-MYC and BCL-2 was decreased significantly. YX-18 suppressed mRNA levels of C-MYC and Ki-67 in both cell lines, and EBNA-1 in EBV-positive Raji cells in a concentration-dependent way. CONCLUSION: The novel emodin derivative YX-18 can significantly inhibit the proliferation of Burkitt lymphoma cells, and induce the cell apoptosis and cycle arrest. The inhibitory effect of YX-18 on the proliferation of Burkitt lymphoma cells may be related with the effect of Caspase apoptosis pathway, the proliferation and apoptosis-related molecules, such as C-MYC and Ki-67, and also to the inhibition of NF-κB pathway.

Plasminogen activator, tissue type regulates germinal vesicle breakdown and cumulus expansion of bovine cumulus-oocyte complex in vitro†.

Plasminogen activator, tissue type (PLAT) and its inhibitor serpin family E member 1 (SERPINE1) cooperatively regulate PLAT activity in various reproductive processes. However, it is unknown whether this includes bovine oocyte maturation. We addressed this question in the present study by evaluating PLAT and SERPINE1 protein localization in immature cumulus-oocyte complexes (COCs), as well as PLAT mRNA and protein expression in cultured COCs after 0, 8, 16, and 24 h of in vitro maturation (IVM). We also examined the effects of PLAT and SERPINE1 on germinal vesicle breakdown (GVBD) and oocyte cyclic 3' 5' adenosine monophosphate (cAMP) levels, cumulus expansion index, and expansion-related gene expression in oocytes derived from bovine COCs cultured for 4, 8, and 12 h and in COCs cultured for 16 h. Both PLAT and SERPINE1 localized in cumulus cells but only the latter was detected in oocytes. PLAT and SERPINE1 transcript levels increased during IVM; however, from 8 to 16 h, the levels of PLAT remained stable whereas those of SERPINE1 increased, resulting in a decline in PLAT concentration. Additionally, PLAT delayed GVBD, increased oocyte cAMP levels, and blocked cumulus expansion and associated gene expression, which was reversed by SERPINE1 supplemented. Thus, PLAT delays bovine oocyte GVBD by enhancing oocyte cAMP levels during the first 8 h of IVM; suppression of PLAT activity via accumulation of SERPINE1 in COCs results in cumulus expansion from 8 to 16 h of IVM. These findings provide novel insights into the molecular mechanisms underlying in vitro bovine oocyte maturation.