| ObjectiveThe 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.Method1. 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.Results1. Growth inhibitory effect of Bortezomib on K-562 cellsAfter 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 BortezomibAfter 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 proteinK562 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).Conclusion1. 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. |
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