摘要
目的
泛素-蛋白酶体通路(ubiquitin-proteasome pathway,UPP)是生物体内蛋白质选择性降解的重要途径,可影响基因表达调控、细胞周期、氧化应激反应等多种细胞活动。蛋白酶体活性的异常改变,可引起多种蛋白质降解异常,导致细胞功能紊乱,使肿瘤细胞持续生长,是肿瘤发生的标志之一。蛋白酶体抑制剂特异性地抑制蛋白酶体活性,阻止肿瘤生长、粘附及血管生成，可以作为抗肿瘤治疗的有效靶点。硼替佐米(Bortezomib,商品名万坷,Velcade)是高选择性的,可逆性的蛋白酶体抑制剂,通过抑制多种蛋白质的降解,可以稳定p21,p27和p53蛋白,转录因子(如c-myc、c-fos、c-jun), IκB,细胞周期调节蛋白和一些Bcl-2家族成员(如Bak and Bax)。在生理情况下NF-κB在胞质中与抑制蛋白IκB结合,硼替佐米通过抑制IκB的降解,下调NF-κB的活性,不但可以增加肿瘤细胞的凋亡,还可以增加肿瘤细胞对化疗、放疗、免疫治疗的敏感性。X连锁凋亡抑制蛋白(X chromosome linked inhibitor of apoptosis protein, XIAP)是内源性凋亡抑制蛋白家族(inhibitor of apoptosis, IAPs)中效力最强的蛋白,它主要通过BIR3结构域抑制caspase-9, BIR2结构域抑制caspase-3和-7,达到抑制凋亡的作用。XIAP的表达受多条信号转导通路的影响,NF-κB, PI3K和MAPK等均可间接调节XIAP的转录。有资料显示XIAP基因在多个白血病细胞株及急性白血病原代细胞中高表达,这提示XIAP可能与白血病的发生、病程演变及预后等密切相关。本研究观察硼替佐米对K562细胞增殖、凋亡及XIAP基因和蛋白表达的影响,研究硼替佐米对白血病细胞的作用及其与XIAP之间的关系,为其做为新的抗白血病药物提供实验依据。
方法
1.水溶性四唑盐光吸收(WST-1)法检测细胞增殖：终浓度为5nmol/L, 10nmol/L,30nmol/L,50nmol/L,100nmol/L硼替佐米分别作用于K562细胞12h、24h、36h、48h, WST-1法检测细胞的增殖情况,计算增殖抑制率,并找出硼替佐米作用于K562细胞的最适时间及作用浓度。
2.瑞氏染色观察细胞形态：终浓度为30nmol/L硼替佐米处理K562细胞24h后,瑞氏染色观察细胞生长状态及形态学变化。
3.流式细胞术检测细胞凋亡：终浓度为5nmol/L, 10mol/L,30nmol/L, 50nmol/L, 100nmol/L硼替佐米分别作用于K562细胞24h,流式细胞仪AnnexinⅤFITC/PI双标检测细胞凋亡率。
4.原位末端转移酶标记(TUNEL)技术检测细胞凋亡：终浓度为30nmol/L硼替佐米处理K562细胞24h后,TUNEL技术检测细胞的凋亡情况。
5. RT-PCR法检测XIAP mRNA的表达：终浓度为5nmol/L,10nmol/L 30nmol/L,50nmol/L,100nmol/L硼替佐米分别作用于K562细胞24h,RT-PCR法检测硼替佐米对K562细胞XIAP mRNA表达的影响。
6.免疫组化法检测XIAP蛋白的表达：终浓度为30nmol/L硼替佐米作用于K562细胞24h,免疫组化法检测硼替佐米对K562细胞XIAP蛋白表达的影响。
7.统计分析：所得结果应用SPSS13.0统计学软件处理,对计量资料,同一时间点各实验组与对照组之间比较用两样本t检验,计数资料采用χ2检验,统计学数据用均数±标准差(x±s)表示。以α=0.05为检验水准,P<0.05为差异有显著性。
结果
1.WST-1结果显示：终浓度为5nmol/L、10nmol/L、30nmol/L、50nmol/L、100nmol/L的硼替佐米分别作于K562细胞24小时,细胞增殖抑制率分别为(13.6±0.15)%、(28.7±0.49)%、(55.4±1.11)%、(68.1±1.12)%、(81.4±0.13)%,分别与空白对照组比较存在显著差异(P<0.01)；硼替佐米组间两两比较,具有显著性差异(P<0.05),其24小时IC50为24.6 nmol/L。30nmol/L硼替佐米分别作用于K562细胞12小时、24小时、36小时、48小时,细胞增殖抑制率为(29.1±0.92)%、(55.4±1.11)%、(57.8±0.84)%、(59.8±1.18)%,分别与空白对照组相比,有显著性差异(P<0.05)；组间两两比较,硼替佐米12h与24h对K562细胞的抑制率有显著性差异(P>0.05),但24h与36h、48h的抑制率无显著性差异(P>0.05)。
2.细胞形态、TUNEL及流式细胞术结果显示：终浓度为30nmol/L硼替佐米处理K562细胞24h,瑞氏染色,光学显微镜下示硼替佐米组细胞呈现凋亡形态学改变,表现为染色质浓缩,核固缩、核边集、核碎裂,胞质中见大量空泡及凋亡小体形成；正常对照组细胞形态正常。TUNEL法计数300个细胞中阳性细胞数为147,阳性率为49.0%,与空白对照组(阳性细胞数7,阳性率2.3%)比较差异有统计学意义(P<0.01)。浓度为5nmol/L、10nmol/L、30nmol/L、50nmol/L、100nmol/L的硼替佐米分别作于K562细胞24小时,AnnexinV FITC/PI双标结果显示凋亡率分别为：(15.36±0.7)%,(32.21±1.2)%,(53.87±1.3)%,(77.85±1.0)%,(81.25±2.8)%。均高于对照组(0.32±0.6%),P<0.01。进一步行组间两两比较,有显著统计学差异(P<0.05)。
3. RT-PCR和免疫组化结果显示：浓度为5nmol/L、10nmol/L、30nmol/L、50nmol/L、100nmol/L的硼替佐米分别作于K562细胞24小时后,RT-PCR扩增结果显示,XIAP mRNA灰度比值分别为0.89±0.029,0.79±0.022,0.59±0.031,0.34±0.037,0.24±0.042,与空白对照组(0.96±0.020)相比有显著差异,p<0.01；不同浓度硼替佐米之间XIAP mRNA灰度比值相比有显著差异,p<0.05。终浓度为30nmol/L硼替佐米处理细胞24h后,经免疫组化法染色法检测结果显示：K562细胞中XIAP蛋白阳性表达率在硼替佐米组为(13.33±2.71)%,与空白组(55.90±1.65)%相比显著降低(P<0.01)。硼替佐米组XIAP蛋白表达阳性平均积分为42.00±8.54,较空白组248.33±20.74有显著性差异(P<0.01)。
结论
1.硼替佐米能有效抑制K562细胞的增殖,其抑制作用具有时间和浓度依赖性。
2.硼替佐米能明显诱导K562细胞凋亡,随时间增加凋亡率增加。
3.硼替佐米能明显下调K562细胞XIAP mRNA及XIAP蛋白的表达,从而增加细胞凋亡。
4.蛋白酶体抑制剂可以有效诱导白血病K562细胞凋亡,这种效应可能通过硼替佐米下调NF-κB活性,降低XIAP基因的转录,从而使caspase活性增高来发挥,以此为白血病的治疗提供了新的策略及新的治疗思路。
Objective
The ubiquitin-proteasome pathway(UPP), an important intracellular pathway of selective protein degradation, has an effect on multiple cell functions, such as transcriptional regulation, cell cycle, oxidation and stess reaction and so on. Abnormal proteasome activity can lead to cell dysfunction through pathological protein degradation, which can cause continuous growth of tumor cells. So it is one of the important tumor markers. Proteasome inhibitors specifically inhibit tumor growth, adhesion and angiogenesis by modulating proteasome activity, which can serve as an effective target for tumor treatment.
Bortezomib (Velcade), a highly selective, reversible proteasome inhibitor, can stabilize p21, p27, p53, IκBα, cyclins, transcriptional factors (e.g.c-myc, c-fos, and c-jun), and Bcl-2 family members (e.g., Bak and Bax) by inhibiting multiple protein degradation. It blocks the activation of NF-κB by preventing proteasome degradation of NF-κB inhibitor,IκBα.Through inhibition of NF-κB, bortezomib not only promotes apoptosis of cancer cells but also sensitizes them to chemotherapy, radiation, or immunotherapy.
X-linked inhibitor of apoptosis protein (XIAP), as a member of endogenous inhibitors of apoptosis(IAPs), is considered to be a key physiological regulator of apoptosis, which inhibits caspase-9 by BIR3 region, and caspase-3 or caspase-7 by BIR2 region. Various signaling pathways, including NF-kB, PI3K and MAPK, indirectly modulate XIAP gene transcription. Data shows that XIAP gene were highly expressed in leukemia cell lines and leukemia cells of patients with acute leukemia, which suggests that XIAP may be associated with leukemia incidence, evolution and prognosis. In our experiments, proliferation and apoptosis of the K-562 cells were observed before and after cells treated with bortezomib, and the expression of XIAP mRNA and protein in K-562 cells was assayed. In this article, the possibility that bortezomib can be used in blastic phase of chronic myeloid leukemia was explored.
Method
1. WST-1 assay cell proliferation:K562 cell was cultured and treated with 5 nmol/L, 10nmol/L,30nmol/L,50nmol/L, 100nmol/L of bortezomib for 12h,24h,36h and 48h., The proliferation of cells were examined by WST-1 assay. Then the growth inhibitory rates were counted and the optimal concentration and time was found.
2. Wright staining observe cell morphology:30nmol/L of Bortezomib treated K562 cells for 24h, morphological changes and growth state of cell were observed under the microscope by Wright staining.
3. Flow cytometry detect cell apoptosis:K562 cells was treated with 5 nmol/L, 10nmol/L,30nmol/L,50nmol/L, 100nmol/L of Bortezomib for 24h,applicate of flow cytometry Annexin V FITC/PI double-labeled to detect cell apoptosis.
4. TUNEL staining analyzed cell apoptosis:The cell apoptosis was analyzed by TUNEL staining when the K562 cells were treated with 30nmol/L Bortezomib for 24h. The positive cell was defined as brown pellet in cytoplasm.300 cells in each slide were randomly counted with positive and negative cells. Positive rate of cells was calculated based on formula:positive rate= positive cells/300×100%.
5. RT-PCR assessed the expression of the XIAP mRNA:The expression of the XIAP gene was assessed by RT-PCR in K562 cells treated with 5 nmol/L, 10nmol/L, 30nmol/L,50nmol/L, 100nmol/L of Bortezomib for 24h.
6. Immuno-histochemistry assayed the expression of XIAP protein:The expression of the XIAP protein was assayed by immuno-histochemistry in K562 cells treated with 30nmol/L of Bortezomib for 24h.
7. Data analyzed:The were using software SPSS 13.0. The counting data were analyzed with theχ2 test. The quantative data were presented as mean±standard difference. Taking a=0.05 as the significant standard of test.
Results
1. Growth inhibitory effect of Bortezomib on K-562 cells
After the K562 cells were treated with 5nmol/L, lOnmol/L,30nmol/L, 50nmol/L,100nmol/L Bortezomib for 24h, cells growth were significantly inhibited with inhibition rates at(13.6±0.15)%、(28.7±0.49)%、(55.4±1.11)%、(68.1±1.12)%、(81.4±0.13)%, respectively. Then Compared with the inhibitory effect of different Bortezomib groups there were significant difference (P<0.05). Data analysis revealed that Bortezomib could suppress the proliferation of K562 cells in dose-dependent manners. The IC50 was 24.6 nmol/L of Bortezomib treated K562 cell 24h.Then K562 cells were treated with 30 nmol/L Bortezomib for 12h,24h,36h,48h, inhibition rates at(29.1±0.92)%、(55.4±1.11)%、(57.8±0.84)%、(59.8±1.18)%, respectively. Bortezomib could suppress the proliferation of K562 cells in time-dependent manners, but there was no statistical difference between 24h group,36h group and 48h group, the optimal time was 24h.
2. Apoptosis of K562 cells induced by Bortezomib
After the K562 cells were treated with 30 nmol/L Bortezomib for 24h, Wright staining, optical microscopy showed bortezomib treated cells showed morphological changes of apoptosis, apoptotic cells showed chromatin condensation, nuclear condensation, nuclear margination, nuclear fragmentation, cytoplasmic vacuoles and see a large number of apoptotic body formation; control cells with normal morphology. The apoptotic cells increased significantly as detected by TUNEL staining. The positive rate was 83.67% in Bortezomib treated group. Compared with untreated group(positive rate 2.33%), the differences had statistical significance (P<0.01). Concentration 5nmol/L,10nmol/L,30nmol/L,50nmol/L,100nmol/L of bortezomib were treated with K562 cells 24 hours, Annexin v FITC/PI double labeling showed that apoptotic rates were:(15.36±0.7)%, (32.21±1.2)%, (53.87±1.3)%, (77.85±1.0)%, (81.25±2.8)%. Higher than the control (0.32±0.6%), P <0.01.There was a significant difference between groups with different concentrations of bortezomib groups, (P<0.05).
3. Expression of XIAP mRNA and protein
K562 cells were treated with 5nmol/L,10nmol/L,30nmol/L,50nmol/L, 100nmol/L of Bortezomib for 24h, expression of XIAP mRNA was assayed by RT-PCR:The expression of XIAP mRNA was decreased by a dose-dependent manner. There were significant difference between different concentrations of Bortezomib groups and blank control group (P<0.01). Then Compared with the inhibitory effect of different Bortezomib groups there were significant difference (P<0.05).
When the K562 cells were treated with 30nmol/L of Bortezomib for 24h, the result of immuno-histochemistry showed that the mean score of XIAP protein in Bortezomib treated cells was 42.00±8.54, which was significantly lower than that in untreated cells 248.33±20.74 (P<0.01).
Conclusion
1. Bortezomib can obviously inhibit the cell survival of K562 cells in time-and dose-dependent manners with the optimal time at 24h.
2. GEM induces apoptosis of HL-60 cells in dose-dependent manners.
3. Bortezomib can significantly down-regulate expression of XIAP mRNA and protein in K562 cells.
4. Proteasome inhibitors can induce apoptosis of leukemia K562 cells, this effect may be played by bortezomib reduced NF-κB activity, XIAP gene transcription,then increased caspase activity, this is new strategies and new treatment ideathe for therapy of leukemias.
引文
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