Study Of Apoptosis Effect Of Berberine In Pediatric Cancer Cells

Objective:The oncoprotein MDM2 plays critical roles in cancer initiation, progression and the development of resistance to therapy. We studied pediatric cancer patients, including those with acute lymphoblastic leukemia (ALL) and neuroblastoma (NB), finding that patients with a poor prognosis commonly had cancer cells that expressed constitutively highlevels of MDM2. Our preliminary data supports a previously unrecognized mechanism of action for Berberine:It specifically downregulates MDM2 in cancer cells. An important function of MDM2 is the ubiquitination (as an E3 ligase) and degradationof the tumor suppressor p53, due to interactions with DAXX and HAUSP. Interestingly, disruption of these interactions can result in self-ubiquitination of MDM2 and p53 activation. So, we want to elucidate the following questions:1. Effect of berberine on the proliferation and apoptosis of pediatric tumor cells.2. Effect of berberine on the MDM2,p53 expression of pediatric tumor cells and its underlying mechanism.3. To test the hypothesis that regulation of MDM2 by DAXX inhibition is the major mechanism by which Berberine differs from Dox in exerting its anti-cancer effect. Methods:1.20 ALL cell lines were selected as study model. Proliferation inhibition of berberine on those cell lines were determined by WST assay and calculate IC50 of each cell line. Analyze the relationship between IC50 and the expression of MDM2,p53 on each cell line.2. The neuroblastoma cell lines(NB-1691 and NB-1643) and the conventional chemotherapeutic drug doxorubicin-resistant acute lymphoblastic leukemia (ALL) cell lines (EU-1,REH) were selected as study model. Western-blot was used to detect the protein expression changes of MDM2,p53,Bcl-2 of each cell line in different dose or time treatment of berberine.3. The conventional chemotherapeutic drug doxorubicin-resistant ALL cell line EU-1 was selected as study model. RT-PCR was used to detect the mRNA expression changes of MDM2,p53,p21,PUMA,GAPDH of of EU-1 cells treated by constant concentration of berberine for various time.4. The conventional chemotherapeutic drug doxorubicin-resistant ALL cell line EU-1 was selected as study model. Western-blot was used to detect the protein expression changes of p53 downstream targets:p21 and PUMA of EU-1 cells treated by various concentration of berberine for 24h.5. The protein synthesis inhibitor cycloheximide (CHX) were utilized to treat cells. Western-blot was used to detect the protein half-life changes of MDM2,p53,Bcl-2 of EU-1,UOC-B1 cells treated by berberine.6. The conventional chemotherapeutic drug doxorubicin-resistant ALL cell line EU-1 was selected as study model. Western-blot was used to check wheatear the apoptosis associated protein caspase 3 and its substrate PARP of EU-1 cells were cleaved after berberine treatment.7. Western-blot was used to detect the constitutional expression of DAXX,MDM2 and HAUSP in 8 cell lines including 4 ALL cell lines and 4 NB cell lines. Western-blot was used to detect the protein changes of DAXX and MDM2 in EU-1 cells treated by various concentration of berberine for 24h. Analyze the relationships between DAXX,MDM2 and HAUSP expression and the proliferation inhibition of berberine on each cell line.8. The NB-1691 cell line was selected as study model. DAXX-siRNA was transfected into NB-1691 cells to knock down DAXX expression. Western-blot was used to detect the changes of DAXX,MDM2 and HAUSP expression after DAXX-siRNA transfection.9. Co-immunoprecipitation was used to detect the ubiquitination changes of MDM2 of EU-1 cells treated by various concentration of berberine for 24h.10. Construct MDM2 without ubiquitin E3 ligase activity plasmid C464A and transfect the C464A plasmid, wt-MDM2 plasmid and the empty vector in to EU-4 cells.western-blot was used to detect the MDM2 expression of those transfected cells followed by berberine treatment.11. Annexin V apoptosis detection kit was used to detect the apoptosis of tumor cells in different dose or time treatment of berberine.Results:1. BBR inhibits MDM2 expression, regardless of p53 status in different ALL cells2. Induction of p53 by BBR in wt-p53/MDM2-expressing cells3. BBR activates p53 function by inducing its transcriptional targets4. MDM2 transcription is not repressed by BBR5. BBR promotes degradation of the MDM2 protein and p53 stabilization6. The expression of DAXX is absolutely required for BBR to down-regulate MDM2 and induce cell death7. Cytotoxicity of BBR is positively associated with MDM2 expression level in ALL cell lines, regardless of their p53 status.8. BBR-induced cytotoxicity associated with MDM2 expression9. BBR strongly induces apoptosis in ALL cells with wt-p53 and MDM2 overexpression Conclusoins:Berberine downregulates MDM2, inducing potent apoptosis of human cancer cells with wt-p53/MDM2 overexpression1. Expression of TRAIL receptors and its significance in children with acute leukemiaObjective:To explore the expression of TRAIL receptors (TR1-R4) and its significance in clinic classification and prognosis of children with acute leukemia.Method:Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of TRAIL receptors on leukemic cells of 22 children with acute leukemia.10 cases of children with no-malignant blood diseases were served as control group.Results:The mRNA expression of DR4 and DR5 in AL group was dramatically higher than that in control group (p<0.05). However, the mRNA expression of DcR1 and DcR2 in AL group was significantly lower than that in control group (p<0.05). DR5 was expressed in both AL group and control group, but its mRNA relative amount was markedly higher than that of DR4 in AL group. No significant differences of TR1-R4 expressions were observed in different clinical sub-types.Conclusion:There are significant differences between TRAIL receptors TR1-R4 in children with acute leukemia. TRAIL and its receptors play an important role in TRAIL and its receptors mediated apoptosis of leukemic cells. 2. Berberine Augments TRAIL-induced Apoptosis of Leukemic CellsObjective:To investigate the apoptosis-inducing effect of TRAIL(TNF Related Apoptosis-Inducing Ligand) and berberine on the leukemic cell line Molt-4 and the relationship between the NF-κB/P65 expression and the synergistic effect.Method:MTT assays were used to measure the cytotoxic effects of TRAIL used alone or combined with the Chinese herb berberine. The apoptosis was detected by flow cytometry and microscopy. Western blotting was used to detect the expressions of caspase3,caspase8 and NF-κB/P65 in different chemotherapeutic groups.Results:(1) The MTT assay suggested berberine augments TRAIL-induced apoptosis in Molt-4 cells in a dose and time dependent way(P<0.05).(2)The Apoptosis can be detected by flow cytomery; Morphological observation also showed the typical apoptotic changes.(3) Western blot showed that the activity of caspase3 and 8 were up regulated when treated with either TRAIL alone or TRAIL combined with berberine. However, in the combinational use of berberine the caspase3, caspase8 activity was up regulated much more than in the absence of berberine. When treated with TRAIL alone, the expression of NF-κB/P65 was increased in a dose and time-dependent way. When berberine was added (50mg/L), the NF-κB/P65 activation was suppressed.Conclusion:(1)TRAIL combined with berberine can induce apoptosis of Molt-4 cells in a synergistic effect.(2)The suppression of NF-κB/P65 expression, caspase3 and caspase8 activation were involved in the mechanism of the synergistic effect.