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Study of the Effect and Mechanism of Proteasome Inhibitor (PS-341) on Neuroblastoma Cells

Author: LinMeiLi
Tutor: HeQiaoJun;YangBo
School: Zhejiang University
Course: Pharmacology
Keywords: PS-341 ATRA Apoptosis Differentiation Neuroblastoma
CLC: R96
Type: Master's thesis
Year: 2009
Downloads: 11
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Abstract

Aims:PS-341 (named as Bortezomib or Velcade), a kind of selectively and reversibly proteasome inhibitor, which has been approved by U.S. FDA for the treatment of patients with multiple myeloma. The aim of this study was to investigate the function and mechanisms of PS-341-induced apoptosis as well as the synergistic activity of PS-341 plus ATRA on inducing differentiation of neuroblastoma cells.Methods:1) The cytotoxic activity of PS-341 was tested on CHP126 cells by using MTT assay, and the concentration of the drug resulting in 50% inhibition of cell growth (IC50) was calculated using the software of Dose-Effect Analysis with Microcomputers. Cell growth curve was drawn according to the results of counting cells each day; Flow cytometry analysis was performed to detect cell apoptosis and cell cycle progression. DAPI staining was used to detect the DNA fragmentation in CHP126 cells; Western blot was used to detect the protein expression level of NFκB pathway, p21 and apoptotic related proteins (Cleaved caspase-3、PARP、Bcl-2、Bax). Real-time quantitative PCR assay was performed to detect mRNA expression level of p21.2) CHP126 and SH-SY5Y cells were used to detect the sensitivity of PS-341 and ATRA, and the concentrations that caused no more than 5% apoptotic cells compared with the negative control group were determined as needed. Cells were treated with PS-341 and ATRA alone or in combination, and cell growth curve was performed with typan blue staining. For differentiation assay, the morphological changes and neuritis outgrowth of cells were photographed by microscopic, and the relative length of the neuritis and the ratio of differentiated cells were calculated by Image Pro5.0 software. Western blot and Immunofluorescence were used to detect the expression of human neuroblastoma differentiation marker Tau andβ-ⅢTubulin in CHP126 and SH-SY5Y cells. Western blot was performed to detect the expression of RARa, RARβ, p27 and JNK. Through culturing the hippocampus neurons from new born mouse, PI and Annexin V staining was used to detect the apoptosis when cells were treated with 5 nM PS-341,20 nM PS-341,10 nM ATRA,5μM ATRA or 5 nM PS-341 plus 10 nM ATRA for 72 h, and Western blot was performed to detect the expression of apoptotic associated proteins (Bax, Bcl-2, capse3 and caspse9).Results:1) PS-341 inhibited the proliferation of CHP126 cells at 72 h with mean IC50 value of 11.25 nM. CHP126 cells were treated with 0,5,10 and 20 nM PS-341 respectively, and cells were harvested at 0,24,48 and 72 h, and cell growth curve was obtained. The results revealed that PS-341 could inhibit cell proliferation in a time-and concentration-dependent manner. Flow cytometry analysis revealed that PS-341 failed to influence the cell cycle distribution of CHP126 cells, but could induce CHP126 cell apoptosis. There was 14.6%, 39.6% and 58.2% apoptosis in CHP126 cells when treated with 5,10,20 nM PS-341 for 72 h, together with 15.3%,17.9%,18.3%,55.1% and 58.2% apoptosis when treated with 20 nM PS-341 for 6,12,24,48 and 72 h. DAPI staining confirmed that PS-341 treatment caused DNA fragmentation and apoptotic bodies in a time and concentration-dependent manner in CHP126 cells. Western blot results revealed the conventional NFκB pathway was not involved in PS-341 induced apoptosis in CHP126 cells, but caused a dramatic promotion of p21 expression both in mRNA and protein level. At the same time, PS-341 decreased the expression of Bcl-2 with a promotion of Bax expression, subsequently with caspase 3 cleavage which resulting in 85 kd PARP cleavage, so as to induce apoptosis in CHP126 cells.2) CHP126 and SH-SY5Y cells were treated with different concentrations of PS-341 and ATRA for 72 h, and typan blue staining was used to detect the apoptosis of the cells. The concentrations of PS-341 and ATRA that caused no more than 5% apoptotic cells compared with the negative control group were 5 nM and 10 nM respectively. Through the proliferation assay, the results exhibited that PS-341 had a synergistic activity with ATRA on suppressing cell proliferation both in CHP126 cells and SH-SY5Y cells, as well as inducing the neuritis outgrowth. And the ratio of differentiated cells with the treatment of ATRA was 28.0%, and the PS-341 plus ATRA was promoted to 50.0%. And the Immunofluorescence and Western blot results demonstrated that the neuroblastoma differentiation marker Tau andβ-ⅢTubulin were both up-regulated when treated with PS-341 plus ATRA in CHP126 and SH-SY5Y cells. Furthermore, ATRA decreased RARa expression, and PS-341 could accumulate the expression of RARa. In addition, PS-341 promoted ATRA induced RARβ, p27 and JNK expression. Then, the hippocampus neurons from new born mouse were cultured and treated with different concentrations and compatibility, and the flow cytomentry analysis results illustrated that 5 nM PS-341,10 nM ATRA and 5 nM PS-341 plus10 nM ATRA failed to induce apoptosis in hippocampus neurons,5μM ATRA and 20 nM PS-341 could induce 24.02% and 58.77% hippocampus neurons apoptosis. Furthermore,20 nM PS-341 could promote Bax expression; both 5μM ATRA and 20 nM PS-341 could decrease Bcl-2, caspase9 and caspase3 expression.Conclusions:As referred above, we got the conclusion that:1) PS-341 induced human neuroblastoma CHP126 cells apoptosis through up-regulating p21 expression, so as to promoting Bax, decreasing Bcl-2, and subsequently activating caspase 3, as well as PARP cleavage.2) PS-341 had a synergistic activity with ATRA on inducing differentiation of neuroblastoma CHP126 and SH-SY5Y cells. And the results indicated that PS-341 could prevent the ATRA induced decrease of RAR a, so as to increase the sensitivity of ATRA, which was characterized with an increase in the expression of RARβ, and subsequently with a promotion of p27 and JNK. Through treating the hippocampus neurons with different strategy, the results indicated that low concentration of drug combination could decrease the toxicity of the drugs on normal cells. However, further research was needed for searching the detailed mechanism. All the study above could offer the experimental basis for the clinical treatment strategy of PS-341.

Study of the Effect and Mechanism of Proteasome Inhibitor (PS-341) on Neuroblastoma Cells

Abstract

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