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Objective·To establish a cell-based screening system for identification of compounds with activity in regulating retinoic acid receptor (RARα) stability. Methods·The modified pMSCV plasmid constructs, named as RARα-EGFP-IRES-DsRed, consists of enhanced green fluorescent protein (EGFP) fusing to RARα and red fluorescent protein (DsRed) as internal references incorporating the internal ribosome entry site (IRES) as interval sequence. The RARα-EGFP-IRES-DsRed plasmid was stably transfected into NB4 cells which were named as NB4-pMGIR-RARα. Fluorescence signals of EGFP and DsRed indirectly reflecting the expression of RARα, were detected by flow cytometry in cells that were treated with all-trans retinoic acid, sodium valproate, cytarabine, lenalidomide, etoposide, montelukast and gambogic acid, respectively. Effects of these compounds on the expression of RARα protein were further examined by Western blotting. Results·A double fluorescence reporter system for screening compounds that can increase the stability of RARα protein was successfully established, and sodium valproate was identified as a potent compound to promote the stability of RARα. Conclusion·The double fluorescence reporter system can be used to screen compounds regulating the stability of RARα protein, which can be further used to identify compounds regulating the stability of other proteins.
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Objective To explore the effects of lentivirus-mediated RNA interference targeting HOXA10 gene on the proliferation, apoptosis and drug resistance of leukemic cell line NB4.Methods NB4 cells were divided into three groups: interference group, negative control group and untreated group.The infection efficiency of lentivirus for NB4 cells was detected by flow cytometry, and the expression of HOXA10 gene of NB4 cells at mRNA and protein level was detected by real-time PCR and Western blot.Cell survival was determined by MTF assay, and apoptosis and necrosis rates were detected by flow cytometry.Western blot was used to detect the influence of down-regulation HOXA10 gene on the multi-drug resistance-1 (MDR-1) protein.Results The ratio of GFP positive cells was up to 90 ％.HOXA10 gene mRNA and protein levels were decreased in interference group compared with control group.The inhibition rate of interference group was (52.12±4.02) ％, the apoptosis rate of interference group was (30.0±2.7) ％, and their differences in the interference group and in control groups (negative control group and untreated group) were significant (P ＜ 0.05).Western blot results showed that interfering HOXA10 gene significantly reduced the resistance gene MDR-1 expression level and reverse the drug-resistant of leukemia cells.Conclusions Lentivirns-shHOXA10 can steadily reduce the expression level of HOXA10, inhibit the leukemic cells proliferation, promote apoptosis and reverse drug-resistant.HOXA10 gene is expected to become a new target for reversing leukemia drug resistance.
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Objective To investigate the effects of simvastatin (SV) on the proliferation,differentiation and apoptosis of human promyelocytic leukemia cell line NB4.Methods NB4 cells were incubated with SV at different concentration with or without all-trans retinoic acid (ATRA),and NB4 cells without any treatment were taken as normal control.Cells of different groups were collected at 24 h,48 h and 72 h after incubation for further detection.Morphological changes by Wright stain were performed.MTT method was used to assay the growth inhibition rate and flow cytometry was used to detect the surface CD11b expression levels,the early stage apoptosis ratio and cell necrosis ratio.Results Treated with 15 μ mol/L SV,10 μ mol/L SV and 5 μ mol/L SV respectively,with the NB4 cells growth,the cell inhibition rates gradually increased (F =7.15,P =0.000),as well as CD11b expression levels (F =3.41,P =0.014) and AnnexinVexpression levels (F =43.38,P =0.000).Furthermore the NB4 cells treated with 15 μ mol/L SV exhibited the most significant changes with cell inhibition rate of 0.96±0.02,CD11b expression level increased to (62.41±6.37) ％ and AnnexinV expression level increased to (87.38±2.94) ％ after 72 h incubation.Combination of 15 μmol/L SV with 0.5 μmol/L ATRA displayed obvious interaction for increasing CD11b expression levels (F =4.093,P =0.025),while no significant interaction for cell inhibition rates and Annexin V expression levels were observed.After 72 h incubation,the CD11b expression levels (89.46±9.13) ％ in NB4 cells treated with 15 μ mol/L SV in combination with 0.5 μ mol/L ATRA were significantly higher than those treated with ATRA (71.27±7.27) ％ and SV (62.41±6.37) ％ (t =2.71,P =0.054; t =4.37,P =0.017)' solely.Conclusion Simvastatin in vitro inhibits NB4 cell proliferation,promotes cell apoptosis,and synergistically induces cell differentiation with ATRA dose-dependently in vitro,which indicates that SV may have the effect of synergistic anti-promyelocytic potency with ATRA.
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Objective To explore the effects and the possible molecular mechanism of flavonoids of puerarin (PR) on chronic myelogenous leukemia (CML) cell line K562 and acute promyelocytic leukemia (APL) cell line NB4 in vitro. Methods MTT assays were used to detect the inhibitory effects of cell proliferation. The apoptosis of K562 and NB4 cells was detected by flow cytometry marked with Annexin V/PI. The expression of bcr-abl, p53, bcl-2, Fas/FasL in K562 cells and JNK, PARP, bcl-2 and Caspase 3 in NB4 cells at protein level was detected by Western blot. Results PR could inhibit the proliferation of K562 and NB4 cells in a time-dose dependent manner. The expression of protein levels of bcr-abl fusion gene declined, while the p53 protein otherwise increased, and both were in a dose-dependent manner (F = 18.74, P <0.05). The application of PR had no effect on bcl-2 and Fas/FasL protein expression in K562 cells. The JNK, PARP and Caspase3 proteins were upregulated in NB4 cells, while bcl-2 was downregulated with the increasing concentrations of PR (F=42.32, P <0.05). Conclusion PR could inhibit leukemic cell proliferation, induce cell cycle block, and increase cell apoptosis through different molecular mechanisms. It suggestes that PR might potentially be a kind of broad spectrum anti-leukemia agent.
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Objective To explore the role of the change of the CD11b expression in hyperleukocytosis and acute promyelocytic leukemia(APL) differentiation syndrome, we observed the effects of all-trans retinoie acid(ATRA) and trioxide arsenic(ATO) alone or the combination together with daunorubicin (DNR) on CD11b expression of human APL cell line NB4 cells. Methods By using the mono-antibody of CD11b FITC-conjugated, we monitored the level of CD11b expression of NB4 cells by flow cytometry. Results With the length of the 1 μmol/L ATRA exposure,the CD11b expression was increased. Exposed to ATRA for 24 h, 48 h, 72 h and 168 h;the CD11b expression of NB4 cell was (33.34±3.15)%, (55.59±5.13)%, (86.08±5.12)% and (90.69±2.69)%, respectively which were higher than control group and the difference was statistically significant (P0.05), but was significantly lower than that in ATRA exposure(P0.05). Conclusion By avoiding the increase of CD11b expression of promyelocytic leukemia cells, ATRA combined with ATO or two drugs together with DNR could play a role in eliminating incidence of hyperleukocytosis and retinoic acid syndrome.
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AIM: To explore the different effect and mechanism of arsenic sulfide on telemorase activity and hTERT-mRNA expression in CML cell lines-KS62 and APL cell lines-NB4. METHODS: Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). The expression of hTERT-mRNA was analyzed by semi-quantitative RT-PCR. Flow cytometry was used to analyze the cell cycle and apoptosis. RESULTS: 0.15-0.6 mg/L arsenic sulfide (72 h)can induce apoptosis and inhibit telomerase activity and hTERT-mRNA expression in NB4 cell. The concentration of arsenic sulfide with the same effect on K562 cell was 0.3-3 mg/U 0.3 mg/L arsenic sulfide (72 h) can cause the proportion of the NB4 cell in G2/M phase increased, but for K562 cell, The concentration of arsenic sulfide was 1.5 mg/L. CONCLUSION: Telomerase system may be one.of the pathway for arsenic sulfide inducing apoptosis of NB4 and K562 cell; G2/M phrase arrest may have correlation with decrease of telomerase activity; The sensitivity of NB4 and K562 call for arsenic sulfide is different, the mechanism of it need to study more.
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Objective To explore molecular mechanisms of apoptosis induced by STI571 in human acute promyelocytie 1eukemia cell lines NB4.nethods The expression of Annexin-V,Fas,Caspase-3 and bcl-2 in NB4 cells were detected by FCM after the treatment of STI571 at(0.5,1.0,5.0 μmol/L)ranging for 24 h,48 h and 72 h.Results With the increasing dose of STI571,the expression of bcl-2,Caspase-3,Annexin-V,Fas in NB4 changed from(10.22±0.62)declining to (5.82±0.52),from(42.21±1.02)ascending to(52.35±0.83),from(25.A2±1.21)ascending to(37.84±0.63),from(18.21±0.81)to(21.41±1.02)respectively.With the dealing time increasing(24,48,72 h),the expression of bcl-2,Caspase-3,Annexin-V,Fas in NB4,changed from (5.81±0.52)declining to(2.51±0.43),from(52.31±0.83)ascending to(69.51±1.12),from(37.81±0.93)ascending to(78.62±0.83),from(23.41±0.73)to(26.53±1.02)respectively.Conclusion STI571 can enhance the apoptosis program to Ni4 in a time-dependence and dose-dependence manner,but no change to Fas was observed.
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Objective To investigate the mechanisms of oridonin inducing apoptosis on acute leukeamia NB4 cells and its mechanism. Methods NB4 cells in culture medium in vitro were given with different concentrations (8, 16, 24, and 32 μmol/L) of oridonin. The inhibitory rate of the cells was measured by MTT assay, cell apoptotic rate was detected by flow cytometry (FCM), morphology of apoptosis was observed by Hoechst 33258 fluorescence staining, DNA fragmentation was assayed by agarose gel electrophoresis, caspase-3 expression was detected by Western blotting, and caspase-3 activity was assayed with colorimetric assay kit before and after apoptosis occurred. Results Oridonin (over 16 μmol/L) could inhibit the growth of NB4 cells and cause apoptosis significantly, the suppression was both in a timeand dose-dependent manner. Marked changes of apoptosis including condensation of chromatin and nuclear fragmentation were observed very clearly by Hoechst 33258 fluorescence staining and a characteristic "ladder" of DNA fragments was elicited by agarose gel electrophoresis; Western blot analysis revealed that caspase-3 was activated by the loss of caspase-3 proenzyme (32 kDa) and the appearance of its 20 kDa subunit, and that along with the apoptotic process caspase-3 activity was increased concurrently. Conclusion Oridonin can induce apoptosis in NB4 cells via activation of caspase-3. These results will provide laboratory evidence for the clinical treatment of acute leukemia with oridonin.
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BACKGROUND: Recently, inorganic arsenic trioxide (As2O3) was reported to induce complete remission in a high proportion of patients with refractory acute promyelocytic leukemia (APL). To illustrate cellular and molecular mechanisms of As2O3 in the treatment of APL, many experimental studies were performed on APL-derived cell lines in vitro. Previous studies showed that As2O3 inhibited proliferation and induced apoptosis in the APL-derived cell lines. This study was done to clarify the in vitro mechanisms of As2O3-induced apoptosis in APL-derived NB4 cell lines. METHODS: To determine the effects of As2O3 in the various concentrations, NB4 cells were cultured with 0.1 to 2micro M/L of As2O3. To assay the apoptosis in NB4 cell lines, DNA fragmentation assay and TUNEL were performed. To find out the molecular change of As2O3- induced apoptotic NB4 cell lines, RT-PCR and Western blot analysis for PML-RARalpha chimeric protein expression and flow cytometry for bcl- 2/bax expression were performed. To clarify the caspase activation pathway, Western blot analysis and flow cytometry for procaspase expression were performed. RESULTS: As2O3 induces apoptosis on NB4 cells in relatively high concentration (0.5 to 2 micro M/L) for 2 days. After 2 days of culture the PML-RARalpha chimeric protein expression decreased rapidly by Western blot and RT-PCR analysis and bcl-2 expression also decreased by flow cytometry. The expression of bax by flow cytometry showed a marked increase in high concentration (2micro M/L) but there was no change in low concentration (0.5micro M/L). In the Western blot analysis, the amount of pro`enzyme of caspase-3 was significantly decreased in the cells with high concentration (2micro M/L) compared with that in the cells with low concentration (0.5micro M/L). As2O3 induces proteolytic processing of pro-caspase 7 but not pro-caspase 9 and 8. CONCLUSION: Apoptosis of APL-derived NB4 cell lines was induced by As2O3 and progressed rapidly in higher concentrations. During apoptosis, activation of caspase-7 pathway and degradation of PML-RARalpha chimeric protein, decrease in bcl-2 and increase in bax were shown.
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Humans , Apoptosis , Arsenic , Blotting, Western , Caspase 3 , Caspase 7 , Cell Line , DNA Fragmentation , Flow Cytometry , In Situ Nick-End Labeling , Leukemia, Promyelocytic, AcuteABSTRACT
OBJECTIVE:To study the mechanism that apoptosis of NB4 cells were induced by artesunate. METHODS: The apoptosis of NB4 cell induced by artesunate was determined by Annexin V-FITC FCM analysis and cytomorphology and the activity of Caspase-3 was determined by ELISA assay. RESULTS: Artesunate could induce apoptosis of NB4 cell with the activity of Caspase-3 increasing. CONCLUSIONS: The apoptosis of NB4 cell induced by artesunate is achieved by activating Caspase-3.
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Aim To investigate the effect of Trichostatin A(T SA)on histone deacetylase (HDAC1) and P21 WAF1/CIP1,cyclin dependent ki nase inhibitor,in NB4 cells for exploring the underlying mechanism of TSA in an ti-leukemia.Methods The total proteins and P21 WAF1/CIP1 mRN A were extracted from acute promyelocytic leukemia NB4 cells treated with or wit hout TSA of different concentrations at different time points,Western blot anal ysis was performed to determine the level of HDAC1 and P21 WAF1/CIP1 protei n,respectively,and RT-PCR was performed to detect the level of P21 WAF1/CI P1 mRNA.Results (1)The level of HDAC1 was obviously inhibited by TSA,and had decreased at 4 hours at IC 50 and lasted for 48 h. The inhibi tion of HDAC1 was significant at the TSA concentration of 37.5 nmol?L -1 , but there was no difference between 75~300 nmol?L -1.(2)The levels of P21 WAF1/CIP1 mRNA and protein were up-regulated by TSA in dose-and ti me-dependent manner,and mRNA increased in 8 h,but the P21 WAF1/CIP1 prot ein was detected at 12 hours and lasted for 48 hours.Conclusion TSA could inhibit the level of HDAC1 and enhance the expression of P21 WAF1/CIP1 protein and mRNA,and the results suggest that inhibiting HDAC1 and increasin g P21 WAF1/CIP1 may be one of the possible mechanisms of anti-leukemia by TSA.