Antitumor mechanism of danusertib in human AML HL-60 cells

AIM: To investigate the effects of danusertib, a pan-inhibitor of Aurora kinases, on the viability, cell cycle, apoptosis and autophagy of human acute myelocytic leukemia (AML) HL-60 cells.METHODS: The effect of danusertib on the viability of HL-60 cells was examined by MTT assay. The effect of danusertib on apoptosis of HL-60 cells was quantitated by the flow cytometry using an Annexin V/7-AAD apoptosis detection kit. The effect of danusertib on autophagy in the HL-60 cells was assessed by flow cytometry and confocal microscopic analysis. The levels of various proteins related to the cell cycle, apoptosis and autophagy were determined by Western blot.RESULTS: Danusertib decreased the viability of human AML HL-60 cells and induced the cell cycle arrest in G₂/M phase. Danusertib also induced mitochon-drium-dependent apoptosis by activation of caspase-3 and autophagy in the HL-60 cells via inhibition of PI3K/Akt/mTOR signaling pathway.CONCLUSION: Danusertib shows effective antitumor ability for promising AML treatment.     

Cytochrome C induces apoptosis in HL-60 cells through inhibiting bcl-2,bcl-xl mRNA expression

AIM:To study the effect of cytochrome C on HL-60 cells in vitro and the expression of relevant apoptotic genes.METHODS:HL-60 cells were treated with different concentrations of cytochrome C for 24 h.The suppressing rate was assayed by MTT.The morphology of cell was observed by microscope and fluorescence microscope.The apoptosis was assayed by flow cytometry (FCM) and DNA electrophoresis.The expression changes of bcl-2 and bcl-xl mRNA was examined by RT-PCR.RESULTS:The suppressing rate increased with the increase in the cytochrome C concentrations.When treated with 0-37.5 mg/L cytochrome C for 24 h,the percentage of apoptotic HL-60 cells increased in a dose-dependent manner,and the typical cells and the appearance of apoptotic DNA ladder were observed.At the same time,within this range of concentration,the expression of bcl-2 and bcl-xl mRNA decreased gradually.When treated with cytochrome C at concentration higher than 37.5 mg/L,the percentage of apoptotic HL-60 cells did not increase,but decreased,while the cell necrosis was observed.CONCLUSIONS:It suggested from the results that at certain range of concentration,cytochrome C induces apoptosis or necrosis in HL-60 cells.The percentage of apoptosis,the changes of expression of bcl-2 and bcl-xl depend on the dose of cytochrome C.The mechanism that cytochrome C induces apoptosis in HL-60 cells may be related to suppressing the expression of bcl-2 and bcl-xl.     

Effect of G-CSF on the apoptosis induced by Ara-c in HL-60 leukemic cells

AIM: To observe the effects of methionine-induced hyperhomocysteinemia on protein C(PC), antithrombin-Ⅲ (AT-Ⅲ) and von willebrand factor (vWF).METHODS:The proliferat ion of HL-60 leukemia cell was observed by hemopoiet ic cell culture.Apoptosis was measured by the morphology of apoptosis cell, the quantitation of DNA fragmentation with the diphenylamine reaction.The change in drug sensitivity was measured by MTT.RESULTS:In group M, the levels of methionine(29.97±5.34 μmol/L) and homocysteine(13.30±2.19 μmol/L) in serum were signifficantly higher than those(14.48±1.97 μmol/L and 5.36±1.19 μmol/L, respectively, P＜0.01) of group C.The levels of AT-Ⅲ and PC of group M were signifficantly lower than those of group C (P＜0.01). The level of vWF in plasma of group M was higher than that of group C (P＜0.01). Immunohistochemistry showed that vWF expression in endothelial cells of aorta was decreased. CONCLUSION:Methionine-induced hyperhomocysteinemia had promotive effects on coagulation and inhibiting effects on antioagulation.     

CpG-oligodeoxynucleotides induce differentiation and apoptosis in HL60 cells

AIM: To investigate the differentiative and apoptotic effect of CpG-oligodeoxynucleotides on HL60 cells and its mechanism. METHODS: After HL60 cells were exposed to synthetic CpG-oligodeoxynucleotides,non-CpG-oligodeoxynucleotides or ZpG-oligodeoxynucleotides for 72 hours, respectively, the inhibition of HL60 cells were detected using MTT method, NBT test was used and CD14 expression were determined.,Apotosis of HL60cells were mensurated with flow cytometry and transmission electron microscope, and caspase 3，Bcl-2 and Bax expres-sion of HL60 cells treated with oligodeoxynucletides were detected using immunohistochemistry RESULTS: Treatment with CpG-oligodeoxynucleotides induced the differentiation and apoptosis in HL60 cells, but non-CpG-oligodeoxynucleotide and ZpG-oligodeoxynucleotide had no effect on HL60 cells. CONCLUSION: CpG-oligodeoxynucleotides can induce the differentiation and apoptosis in HL60 cells. It may provide a new approach for the immunological treatment of leukemia.     

Enhanced anti-leukemic activity of decitabine to leukemia HL-60 cells by anti-miR-21 oligonucleotide

AIM: To investigate the role of anti-miR-21 oligonucleotide (AMO) in the anti-leukemic activity of decitabine (DCA) in vitro.METHODS: AMO and scramble oligonucleotide (SCR) were constructed and transfected into HL-60 cells. The miR-21 expression was analyzed by real-time PCR to identify the transfection efficiency. The cells were treated with DCA at gradient concentrations (0.5, 2.0 and 4.0 μmol/L) for 48 h. The mRNA expression of human period circadian protein 3 (hPer3) was detected by real-time PCR. The early apoptotic rates were determined by flow cytometry with Annexin V/PI staining. Mean fluorescence intensities (MFI) of CD117 and CD11b were also measured by flow cytometry. RESULTS: The miR-21 relative expression level in AMO group was significantly lower than that in blank group and SCR group (P<0.01). IC₅₀ of DCA in AMO group was significantly lower than that in blank group and SCR group (P<0.01).With the same concentration of DCA, the early apoptotic rate, the mRNA expression of hPer3 and the MFI of CD11b in AMO group were significantly higher than those in blank group and SCR group (P<0.01). The MFI of CD117 in AMO group were significantly lower than those in blank group and SCR group (P<0.01). CONCLUSION: Activation of hPer3 expression plays an important role in enhanced anti-leukemic activity of decitabine by AMO in vitro.     

Effects of alkyl-lysophospholipids on proliferation and differentiation in HL-60cells

AIM: To explore the effect of alkyl-lysophospholipids (ALP) on the proliferation, apoptosis and differentiation of HL-60cells. METHODS: Proliferative potential was measured by colony formation assays. Apoptotic cells were detected by morphology, DNA gel electrophoresis and flow cytometry analysis. Both morphological criteria and NBT dye reduction were utilized to determine the extent of differentiation. RESULTS: After 9 h of incubation wit15 mg/L of ALP, apoptotic cells, identified by condensened and fragmented nuclei, were present. After 6 days of incubation wit1 mg/L of ALP, the NBT reduction rate in HL-60cells increased to 84.2±2.6%. CONCLUSION: ALP can induce apoptosis and differentiation, and inhibit growth in HL-60cells.     

Role of calcium ion in the cyotoxicity and DNA fragment induction in HL-60 cells by 6F isolated from Pteris semipinnata L.

AIM: To investigate the changes of cytosolic free calcium concentration([Ca²⁺]_i) and expression of Bcl-2 in HL-60 cells treated by 6F isolated from Pteris semipinnata L.(PSL), and to discuss the relations between calcium ion and cytotoxicity and DNA fragment induction effects of 6F. METHODS: HL-60 cells were used as in vitro model. [Ca²⁺]_i was measured on fluorescent spectrophotometry using Fura-2/AM as Ca²⁺ indicator. Bcl-2 expressing level was measured by flow cytometry. Tetrazolium salt(MTT) and diphenylamine staining methods were applied for cytotoxicity assay and DNA fragmentation detection, respectively. RESULTS: [Ca²⁺]_i increased obviously in a dose and time dependent manner after treated HL-60 cells with 6F. 6F decreased the expressing level of Bcl-2. Adding 2 mmol/L Ca²⁺ to the medium, or 1 mmol/L EDTA to chelate Ca²⁺, or 4 μmol/L calcium ionophore A 23187 to increase the concentration of cytosolic Ca²⁺, the DNA fragment induction by 6F was not affected, whereas the cytotoxicity of 6F was enhanced. 250 μmol/L Zn²⁺ attenuated the DNA fragment induction, and the cytotoxicity of 6F against HL-60 cells was enhanced significantly. CONCLUSION: It was speculated that the decreased expressing of Bcl-2 by compound 6F was related to increased [Ca²⁺]_i in HL-60 cells, and DNA fragment induction was possibly catalyzed by Ca²⁺ - independent DNase.     

Effect of lentinan on apoptosis and PI3K/AKT signaling pathway in leukemic HL-60 cells in vitro

AIM:To study of the regulatory effect of lentinan on human leukemic HL-60 cell apoptosis and its effect on PI3K/AKT signaling pathway in HL-60 cells in vitro.METHODS:Lentinan at concentrations of 0 mg/L, 15 mg/L, 30 mg/L and 45 mg/L was applied to HL-60 cells cultured to the logarithmic phase in vitro, and the inhibitory effect of lentinan on the viability of HL-60 cells was measured by MTT assay after 24 h, 48 h and 72 h. The apoptosis induced by lentinan was analyzed by flow cytometry. The protein levels of cleaved PARP, cleaved caspase-9, cleaved caspase-3, cleaved caspase-8, cytochrome C, PI3K, AKT and p-AKT were determined by Western blot. After treatment with PI3K inhibitor LY294002 at 5 mg/L for 72 h, the apoptosis of HL-60 cells was analyzed by flow cytometry. RESULTS:The viability of HL-60 cells was inhibited after treatment with lentinan at concentrations of 15 mg/L, 30 mg/L and 45 mg/L for 24 h, 48 h and 72 h in concentration-dependent and time-dependent manners (P<0.05). The apoptosis of HL-60 cells was promoted after treatment with lentinan (15 mg/L, 30 mg/L and 45 mg/L) for 72 h in a concentration-dependent manner (P<0.05). The protein levels of cleaved PARP, cleaved caspase-9, cleaved caspase-3 and cytoplasmic cytochrome C in the HL-60 cells induced by 30 mg/L lentinan were increased significantly with the increase in the treatment time (P<0.05), but caspase-8 did not show any change. The protein levels of PI3K, AKT and p-AKT were decreased obviously with the increase in the lentinan concentration (P<0.05). Treatment of HL-60 cells with LY294002, a PI3K pathway inhibitor, produced apoptosis-inducing effect similar to lentinan (P<0.05). CONCLUSION:Lentinan induces HL-60 cell apoptosis by inhibiting PI3K/AKT signaling pathway.     

Transition of WT1 isoforms inhibits proliferation and promotes apoptosis of human leukemia cell line HL-60

AIM: To study the potential effects of exogenous Wilms tumor 1 (WT1) isoforms on the proliferation and apoptosis of human leukemia cell line HL-60. METHODS: WT1 (17AA－/KTS－) gene obtained by RT-PCR was cloned into a PCDH1-MCS1-EF1-copGFP plasmid. The recombinant plasmid was confirmed by enzyme digestion and sequencing, and was transfected into HL-60 cells by LipofectamineTM 2000. The stable transformants were selected by G418 screening. WT1(17AA－/KTS－) expression was identified by real-time fluorescence quantitative RT-PCR and Western blotting. The proliferation of the cells was measured by MTT assay. The apoptosis of the cells was determined by morphological observation and flow cytometry analysis. RESULTS: The eukaryotic expression vector PCDH1-MCS1-EF1-copGFP-WT1 (17AA－/KTS－) was successfully constructed. The recombinant cells exhibited high mRNA and protein levels of WT1(17AA－/KTS－). The growth of recombinant cells was slower than that of HL-60 cells transfected with control vector and normal HL-60 cells. After exposed to As2O3 at 2 μmol/L for 48 h, both recombinant cells and control cells exhibited the morphological characteristics of apoptosis, but the former was more typical than the latter. The apoptosis was enhanced in the recombinant cells after the cells were exposed to As2O3 for 24 h. CONCLUSION: Exogenous WT1(17AA-/KTS-) isoform inhibits the proliferation and promotes the apoptosis of leukemic cells.     

Inhibitory effects of Co-SZ eye drops on apoptosis of lens epithelial cells and their signal transduction mechanisms

AIM: To investigate the inhibitory effect of Co-SZ eye drops on apoptosis of lens epithelial cells (LEC) induced by H₂O₂ and to study the cellular and molecular mechanisms.METHODS: (1) All lenses of Sprague-Dawley rats were incubated with H₂O₂ and Co-SZ eye drops.The apoptosis rates of LEC were determined by TUNEL method.The changes of LEC ultrastructure and the formation of apoptotic body were observed by electron microscopy.(2) Bovine LEC were incubated with H₂O₂ and Co-SZ eye drops.The inhibitory of LEC apoptosis was detected by MTT after incubation.The changes of fractional DNA content in LEC were detected by flow cytometry (FCM).［Ca2+］i , cAMP and cGMP of LEC were determined by spectrofluoremeter and radioimmunoassay, respectively.RESULTS: The LEC apoptosis rates in Co-SZ eye drops group were decreased significantly compared with H₂O₂ group by TUNEL.The ultrastructure changes in LEC of Co-SZ eye drops group were lighter than that in H₂O₂ group.The LEC apoptosis rates of Co-SZ eye drops group were dose-dependently decreased significantly compared with H₂O₂ groups via MTT assay.LEC apoptosis induced by H₂O₂ was inhibited by Co-SZ eye drops, and showing dose-dependent.The DNA contents in LEC of Co-SZ group were increased.The ［Ca2+］i and cAMP in Co-SZ group were decreased obviously.The cGMP was increased.CONCLUSION: The LEC apoptosis induced by H₂O₂ was inhibited by Co-SZ eye drops.The mechanism of apoptosis inhibition by Co-SZ eye drops maybe contribute to the increase in DNA content.The signal transduction mechanisms are related to the decrease in ［Ca2+］i and cAMP and the increase in cGMP.     

Primary study on the differentiation mechanism of embryonic stem cells to epidermal like cells induced by human amnion

AIM: To investigate the mechanism by which human amnion induced mouse embryonic stem (ES) cells to differentiate into epidermal like cells. METHODS: ES- BALB/ c cells were cocultured with human amnionintranswells for 4 - 5 days , andthose cultured alone without amnion were taken as control group. The morphological differentiation were observed . The committed differentiation of EScells into epidermal like cells were detected by integrin-β₁ , CK19 , CK15 andinvolucrin immunohistochemistry , respectively .RESULTS: After 4-5 days of coculture, ES cells differentiated into single layer of epidermal like cells, fitted tightly, with polyhedral in shape. The immunohistochemical staining results showed that, most of the cells were integin-β₁ positive, only a few cells were CK19 and CK15 positively stained. Most of the cells in control group died, the survived ones were different in morphological shapes, and no integrin-β₁, CK19 and CK15 positive cells were found. CONCLUSION: Soluble substances secreted by human amnion may play an important role in inducing the differentiation of mouse ES cells into epidermal like cells.     

Effects of melatonin on apoptosis of lymphocytes induced by ionizing radiation in mice

AIM: To explore the effect of melatonin (MLT) on the apoptosis of thymocytes and splenocytes in mice induced by ionizing radiation and its mechanism. METHODS: The percentages of apoptotic bodies and the DNA lytic rates of thymocytes and splenocytes in mice in vitro and in vivo were detected with flow cytometry and fluorospectrophotometry, respectively. RESULTS: The apoptosis of mouse thymocytes and splenocytes in vitro increased with significant dose-dependence in 0.5-6.0 Gy X-irradiation. When MLT of 2 mmol·L^-1 was added into thymocytes or splenocytes in vitro before irradiation with 0.5-6.0 Gy X-rays, the percentages of apoptotic bodies and the DNA lytic rates all decreased significantly as compared with those in the irradiation group. The percentages of apoptotic bodies in these two kinds of cells were 86.25% and 89.22% of those in the irradiation group, respectively, and the DNA lytic rates were 87.23% and 89.16%, respectively. When MLT was injected into intraperitonium in mice 60 min before whole-body irradiation with 2 Gy X-rays, the percentages of apoptotic bodies and the DNA lytic rates were significantly lower than those in the irradiation group, and near or lower than those in the sham-irradiation group. MLT of 0.1-2.5 mg/kg decreased the lymphocyte apoptosis, but without significant dose-dependence. CONCLUSION: The protective effects of MLT on mouse lymphocytes damaged by irradiation in vivo were obvious than those in vitro.     

Inhibition of FoxM1 sensitizes leukemia K562 cells to homoharringtonine

AIM: To study whether inhibition of forkhead box protein M1(FoxM1) sensitizes leukemia K562 cells to homoharringtonine (HHT). METHODS: K562 cells were incubated with HHT at different concentrations (0μmol/L, 0.015μmol/L, 0.030μmol/L and 0.045μmol/L) for different time (0 h, 24 h, 48 h and 72 h). The mRNA and protein levels of FoxM1 were detected by real-time PCR and Western blot. FoxM1 siRNA was transfected into K562 cells with 0.015μmol/L HHT after 6 h. After 72 h incubation, the cell proliferation was detected by cell counting and soft agar assay, and the proportion of apoptotic K562 cells was determined by flow cytometry. The expression of c-Myc and Sp1 were detected by real-time PCR and Western blot. RESULTS: FoxM1 expression was reduced time-dependently and dose-dependently, suggesting that HHT mediated the downregulation of FoxM1 in K562 cells. In K562 cells, treatment with FoxM1 siRNA and HHT inhibited the cell proliferation and promoted the apoptosis significantly. Therefore, inhibition of FoxM1 sensitized leukemia K562 cells to HHT. The expression of c-Myc and Sp1 was positively regulated by FoxM1. CONCLUSION: HHT inhibits Forkhead box protein M1 expression in K562 cells. Inhibition of FoxM1 sensitizes K562 cells to HHT.     

Changes in the expression of Bcl-2 and Fas in HL-60 cells treated with cyclosporine A

AIM: To investigate the changes of the expression of Bcl-2 and Fas protein and the apoptosis in HL-60 cells induced by cyclosporine A. METHODS: The expression of Bcl-2 and Fas protein and apoptosis in HL-60 cells were measured by immunohistochemistry analysis and flow cytometric analysis. RESULTS: There was strong expression of Bcl-2 in HL-60 cells, treatment with cyclosporine A (CsA) for 8-10 h down-regulated the expression of Bcl-2. Fas protein expression in HL-60 cells was very low, CsA induced apoptosis of HL-60 cells, but didn't induce Fas protein expression. CONCLUSION: CsA induces apoptosis in HL-60 cells by down-regulating Bcl-2 expression.     

Stat5b signaling pathway regulates the expression of Survivin and promotes apoptosis in human colon cancer cells

AIM: The purpose of the study was to examine colon cancer cell lines to determine whether Stat5b/Survivin plays an important role in the process of apoptosis in colon cancer cells. METHODS: Protein lysates were extracted from colon cancer cells. Human colon cancer cell line HT29 was transfected with Stat5b antisense oligonucleotide mediated by liposome. MTT assay was used to measure the proliferation. Flow cytometry was applied to analyze the cell cycle and apoptosis. EMSA was used to detect the activity of Stat5. Western blotting was applied to measure the expression of Stat5, p-Stat5, cyclin D1, Survivin, Bcl-2 and Bcl-x L. RESULTS: Targeting of Stat5 using antisense oligonucleotide against the translation site resulted in apoptosis and downregulaed the expressions of Stat5, p-Stat5, cyclin D1 and Survivin, but not Bcl-2 and Bcl-xL. CONCLUSION: Constitutive activation of Stat5 is associated with the carcinogenesis of colon cancer cells. Blocking of Stat5 signaling inhibits the expression of Survivin and induces apoptosis in colon cancer cells.     

Changes and significance of Fas and cFLIP in apoptosis of human rhabdomyosarcoma cells induced by combination of TRAIL and cisplatin

AIM: To investigate the synergistic induction of apoptosis in rhabdomyosarcoma cells by the combination of TRAIL or TRAIL gene with cisplatin. METHODS: Rhabdomyosarcoma cells were treated with TRAIL, Ad／GT-TRAIL, cisplatin, respectively or the combination for 3 days. The cytotoxicity was observed by MTT assay. The apoptotic rates and the expression rates of Fas protein were measured by flow cytometry (FCM). The expression of cFLIP mRNA was determined by RT-PCR. RESULTS: Rhabdomyosarcoma cells were treated with Ad／ GT-TRAIL and TRAIL (100.0 μg/L), the cytotoxicity index were 52.5% and 43.5%, the percentage of apoptotic cells were 12.95% and 10.26%, respectively. Combined with cisplatin, the cytotoxicity index and the percentage of apoptotic cells were increased significantly (P<0.05). The expression of Fas protein in rhabdomyosarcoma cells was up-regulated and the expression of cFLIP was down-regulated with cisplatin, which were paralleled by the apoptotic rates. CONCLUSION: Combinatiion of Ad／GT-TRAIL or TRAIL and cisplatin has synergistic apoptosis-inducing effects on rhabdomyosacoma cells.     

Ethyl acetate extract of Pleione bulbocodioides (Franch.) Rolfe induces apoptosis of human leukemia K562 and HL-60 cells through intrinsic mitochondrial apoptosis pathway

AIM:To investigate the effects of ethyl acetate (EtOAc) extract of Pleione bulbocodioides (Franch.) Rolfe on proliferation and apoptosis of human leukemia K562 and HL-60 cells and the possible apoptosis pathway. METHODS:Human leukemia cell lines were treated with EtOAc extract of Pleione bulbocodioides at different concentrations. XTT method was used to evaluate the viability of K562 cells and HL-60 cells. The cell growth inhibition was calculated by Trypan blue exclusion test. The percentage of apoptotic cells was determined by flow cytometry, and 4',6-diamidino-2-phenylindole (DAPI) was used to observe morphological changes of the cells. The cell cycle was observed by propidium iodide (PI) staining. The protein expression of Bcl-2, Bax, cleaved poly(ADP-ribose) polymerase (PARP), cleaved caspase-3, cytochrome C and apoptosis-inducing factor (AIF) wase determined by Western blot. RESULTS:The cell viability and proliferation were inhibited by EtOAc extract of Pleione bulbocodioides with IC₅₀ of (42.14±2.54) mg/L for HL-60 cells and (51.28±3.12) mg/L for K562 cells at 24 h. The results of Annexin V-FITC/PI and DAPI staining showed that EtOAc extract of Pleione bulbocodioides induced cell apoptosis in a dose-dependent manner. The apoptotic rate was increased compared with control group (P<0.05). The G₂ phase increased with typical cell apoptosis-induced morphological changes. The levels of pro-apoptotic proteins Bax, cleaved PARP and cleaved caspase-3 were increased, while Bcl-2 was down-regulated (P<0.05). Cytochrome C and AIF in cytosol, characteristic proteins of intrinsic mitochondrial apoptosis pathway, also increased with the concentration of EtOAc extract of Pleione bulbocodioides increasing (P<0.05). CONCLUSION:EtOAc extract of Pleione bulbocodioides significantly inhibits cell proliferation and induces cell apoptosis in human leukemia cell lines HL-60 and K562 through intrinsic mitochondrial apoptosis pathway.