| Section 1 BBD9 inhibited cell growth and induced apoptosis In lymphoma cellsObjective:To study the effects of BBD9 on lymphoma cells.Methods:Cell growth was measured by MTT assay in lymphoma cell lines and primary cells from patients with lymphocytic leukemia. Cell morphology was observed by Wright-Giemsa stain. AV-PI staining and DNA ladder was used for detecting apoptosis.Results:The cell viabilities of four lymphoma cell lines, including Raji, L428, Namalwa and Jurkat, were significantly inhibited by BBD9 at the concentration of 0-4μg/ml. The cell survival rate was decreased with the increasing concentration of BBD9. Furthermore, BBD9 inhibited cell growth in all four cell lines in a time dependent manner. When treated at the same concentration of BBD9, fewer cells would survive with increasing time. The 24h IC50 of BBD9 on Raji, L428, Namalwa and Jurkat were 1.5μg/ml,1.89μg/ml,1.83μg/ml,1.59μg/ml, respectively. Primary cells from two patients with lymphoma were treated with BBD9 for 24h, and also showed growth inhibition in a dose dependent manner. The IC50 for primary cells were 2.09μg/ml and 2.43μg/ml, respectively. The cell growth inhibition induced by BBD9 was also measured in mononuclear cells from normal bone marrow by MTT assay. The results showed that the cell growth of normal mononuclear cells was inhibited, in a dose dependant manner, by BBD9 at the concentration of 0-4μg/ml. However, normal mononuclear cells were less sensitive to BBD9 treatment than primary lymphoma cells were, and the 24h IC50 was 3.46μg/ml. After treated by BBD9 for 24h, Raji and L428 cells, observed by Wright-Giemsa stain, showed a typical apoptotic morphology, nuclear fragmentation and apoptotic body. Typical DNA ladders were also seen in Raji and L428 cells treated by BBD9 for 24h. With the concentration of BBD9 increasing, the DNA ladder became more significant. Further study revealed that BBD9 could induce apoptosis in lymphoma cells, as measured by AV-PI assay. After treated by 0,1, 2,3μg/ml BBD9 for 24h, the Raji cells in early apoptosis were 2.87±0.35%,9.36±2.10%,11.69±0.89%,46.96±3.58% respectively. Meanwhile, the L428 cells in early apoptosis were 4.45±0.97%.9.02±0.64%,17.62±2.33%,35.87±4.44%, respectively. Furthermore, caspase-3,-8,-9 and PARP were cleaved and activated, as measured by western blot, in Raji cells and L428 cells treated by BBD9.Conclusions:(1) BBD9 inhibited cell growth of four lymphoma cell lines(2) BBD9 inhibited cell growth of primary cells, but inhibited less in bone marrow normal mononuclear cells.(3) BBD9 induced apoptosis in lymphoma cells. Section 2 BBD9 affected some signaling pathway in lymphoma cellObjective:To reveal the alteration of proteins involved in mitochondria apoptosis; To reveal the expression of proteins involved in PI3K/Akt signaling pathway; To study the mechanism of PTEN protein upregulated in Raji cells after cells being treated by BBD9. To reveal the alteration of proteins involved in NF-κB signaling pathway, and to better understand the mechanism of BBD9 effecting on lymphoma cells.Methods:Western blot was used for detecting BCL-2 family proteins, IAP family proteins and PI staining was used for cell cycle analysis; Western blot was also used for assaying the expression of proteins involved in cell cycle regulating proteins, assaying the expression of proteins involved in PI3K/Akt and NF-κB signaling pathway. Real-time PCR was messured to detect the mRNA of PTEN and expression of the mir-17-5p and mir-20a. Immunofluorescence detection was messured to detect the expression of the NF-κB and IκB in cytosplasm and intranuclear.Results:After treated by BBD9, the expression of Raji cells'pro-apoptotic protein, Bak, Bax pBad and Bim were up-regulated and anti-apoptotic protein, Mcl-1 were down-regulated. BBD9 also inhibited the expression of IAP family proteins, such as XIAP, CIAP1 and Survivin. In addition, the results of cell cycle analysis showed that BBD9 induced cell cycle arrest in G2/M phase. After being treated by 2μg/ml BBD9 for Oh,4h,8h,12h,24h, the Raji cells arrested in G2/M phase were 8.41±3.62%. 11.51±2.44%,17.36±6.11%,23.09±2.40%,25.21±9.04%, respectively. Cell cycle regulators cyclin A, cyclin B1 and CDK1 were all down-regulated as measured by western blot assay, in Raji cells treated by BBD9 for 24h.After inhibited with 0-3μg/ml BBD9 for 24h, the expression of total Akt of Raji cell was not change, the pAkt and PI3K were decreased while PTEN was obiviously increased. Compared with the untreated control group, the expressions of PTEN mRNA were 3.76,7.13 and 19.75 folds. Furthermore, microRNA-miR-17-5p, miR-20a were significantly down regulated in Raji cells treated with BBD9.After inhibited with 0-3μg/ml BBD9 for 24h, the expression of cytoplasmic IκB of Raji cell increased, while the pIκB was decreased, meanwhile the expression of intranuclear NF-κB the was downregulated. Immunofluorescence detection revealed that the untreated control group, the red fluorescent light in the location of nuclear was bright, and weak in the cytoplasm which means the expression of NF-κB in nuclear was low. After treated with 2μg/ml BBD9 for 24h, the red fluorescent light in the blue fluorescent light location, which indicate the location of nuclear, was weak, that means the expression fo NF-κB in nuclear was downregulated and BBD9 inhibited the NF-κB transfer to nuclear.Conclusions:(1) BBD9 up-regulated pro-apoptotic protein such as Bak, Bax, pBad and Bim and down-regulated anti-apoptotic protein Mcl-1; and inhibited XIAP, CIAP1 and Survivin proteins; BBD9 also induced cell cycle arrested in G2/M phase through down-regulation of cyclin A, cyclin B1 and CDK1. By these signal pathways, BBD9 induce apoptosis of lymphoma cells so that inhibited the proliferation of lymphoma cells.(2) BBD9 inhibited PI3K/Akt signaling pathway which maybe the important mechanism that BBD9 induced apoptosis of lymphoma cells.(3) BBD9 downregulated miR-17-5p, miR-20a, and upregulated PTEN mRNA so as to upreglated the expression of PTEN protein, which inhibiter PI3K/Akt signaling pathway. (4) BBD9 inhibited pIκB, inhibited NF-κB transfer to nuclear so as to inhibit NF-κB signaling pathway, which maybe another important mechanism that BBD9 induced apoptosis of lymphoma cells.Summary:BBD9 inhibited cell growth in lymphoma cell lines and primary cells from patients through inducing apoptosis, which was time-and dose-depedent to some extent. BBD9 may induce Pro-apoptotic protein such as Bak, Bax pBad and Bim up-regulated and anti-apoptotic protein Mcl-1 down-regulated. IAP family proteins, include XIAP, CIAP1 and Survivin was downregulated by BBD9. Furthermore, BBD9 also can induce cell cycle arrested in G2/M phase through down-regulation of cyclin A cyclin B1 and CDK1. In addition, BBD9 increased the expression of mRNA of PTEN, and increase the expression of PTEN protein so as to inhibited PI3K/Akt signal pathway. BBD9 inhibited PI3K/Akt signal pathway, which maybe an important mechanism that BBD9 induced apoptosis of lymphoma cells. BBD9 also inhibited pIκB, inhibited NF-κB transfer to nuclear so as to inhibit NF-κB signaling pathway, which maybe another important mechanism that BBD9 induced apoptosis of lymphoma cells. |
PDF Full Text Request