OBJECTIVETo study the effect of ZGDHu-1 on proliferation, differentiation and apoptosis in SHI-1 human leukemia cell line and explore its possible mechanism. Methods SHI-1 cells were cultured with different concentration of ZGDHu-1 and for different time. The cell proliferation was analysed by cell counting, alive cell count, MTT assay and Brdu-ELISA. Cell apoptosis was analysed by morphology, DNA content, Annexin-V/PI and Hoechst 33258 labeling method. Cell differentiation were assayed by morphology,expression of CD11b,CD14 and CD64 and NBT reduction. The expressions of phosphorylated p38MAPK or STAT3 were analysed by flow cytometry.

RESULTSZGDHu-1 inhibited SHI-1 cell proliferation in a time and dose dependent manner, the IC50- 48 h and IC50- 72 h were 250 ng/ml and 85 ng/ml, respectively. The majority of SHI-1 cells were arrested in G2/M phase. 48h after treated with 200 ng/ml ZGDHu-1, and those in G2/M phase accounted for (48.4 +/- 2.1)%. The SHI-1 cells apoptosis was increased with a time- and does-dependent manner. The morphology of SHI-1 cells cultured with 2-50 ng/ml ZGDHu-1 for three days become more mature with higher NBT positivity and up-regulated expressions of CD11b,CD14 and CD64. The expression of phosphor-p38MAPK was increased and phosphor-STAT3 down-regulated by the treatment of ZGDHu-1.

CONCLUSIONZGDHu-1 can inhibit SHI-1 cell proliferation and induce the cell differentiation and apoptosis. The mechanism may associate with its up-regulation of phosphor-p38MAPK and down-regulation phosphor-STAT3.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Formamides ; pharmacology ; Heterocyclic Compounds, 1-Ring ; pharmacology ; Humans ; Leukemia ; pathology ; Phosphorylation ; STAT3 Transcription Factor ; biosynthesis ; p38 Mitogen-Activated Protein Kinases ; biosynthesis

OBJECTIVETo explore the inhibition pathway of the EBV-immortalized cells (CD23(+)) in children with infectious mononucleosis (IM) caused by Epstein-Barr virus.

METHODSThe expressions of CD23, CD19, CD95, Bcl-2 and the co-expressions of CD23CD95, CD19CD23 on peripheral blood mononuclear cell (PBMC) were analyzed by flow cytometry (FCM) during acute phase, early convalescent phase and convalescent phase of 34 EBV-IM children and compared with that of 24 healthy donors.

RESULTS(1) The levels of CD23(+) and CD23(+)CD19(+) cells decreased and CD95(+), CD95(+)CD23(+), Bcl-2(+) cells increased markedly in IM patients in acute phase [CD95(+) cells (19.43 +/- 8.46)%; CD95(+)CD23(+) cells (1.81 +/- 1.71)%; Bcl-2(+) cells (23.41 +/- 26.47)%] and early convalescent phase [CD95(+) cells (12.94 +/- 5.05)%; CD95(+)CD23(+) (1.05 +/- 1.20)%; Bcl-2(+) cells (10.54 +/- 9.68)%], as compared with those of healthy controls [CD95(+) cells (10.39 +/- 2.90)%; CD95(+)CD23(+) cells (0.50 +/- 0.46)%; Bcl-2(+) cells (7.25 +/- 2.88)%]. The earlier the course of IM, the more abnormal the expressive levels. All the abnormal results returned to normal in convalescent phase. (2) Positive relationships were observed between the expressions of CD95(+)CD23(+) cells and that of CD23(+) cells, CD23(+)CD19(+) cells during acute and early convalescent phase, the expressions of Bcl-2(+), CD3(+) cells and CD23(+), CD23(+)CD19(+) cells during acute phase, the expressions of CD95(+)CD23(+) cells and Bcl-2(+) cells during acute phase, and the expressions of CD95(+)CD23(+) cells and CD95(+) cells during convalescent phase.

CONCLUSIONThe results indicate that CD95L-CD95 mediated apoptosis plays an important role in eliminating EBV-immortalized cells, which is counteracted partly by Bcl-2.

Antigens, CD19 ; blood ; Cell Transformation, Viral ; Cells, Cultured ; Child ; Child, Preschool ; Female ; Herpesvirus 4, Human ; Humans ; Infant ; Infectious Mononucleosis ; blood ; pathology ; virology ; Male ; Receptors, IgE ; blood ; bcl-Associated Death Protein ; blood ; fas Receptor ; blood

This study was aimed to investigate the activation of P38MAPK/STAT3 and expression of telomerase reverse transcriptase during sodium nitroprusside (SNP) inducing apoptosis of human leukemia cell line K562 and to explore the molecular mechanisms of SNP-inducing apoptosis in K562 cells. The K562 cell were treated with different concentrations of SNP and were cultured for different time. Cell apoptosis was analysed by cell morphology, DNA agarose gel electrophoresis, DNA content, and Annexin-V/PI labeling method. The TdT-mediated dUTP nick end labeling (TUNEL) assay was used to quantitate the in situ cell apoptosis. The expressions of phosphorylated p38MAPK or STAT3 were analysed by flow cytometry, while the expression of hTERT mRNA in transcriptional level was measured by fluorescence quantitative RT-PCR. The results showed that SNP inhibited K562 cell growth. The K562 cell apoptosis was confirmed by typical cell morphology and DNA fragment, peak of sub-G1 phase, TUNEL and Annexin-V/PI labeling. A majority of K562 cells were arrested in G0/G1 phase. After treatment with SNP at 0.5-3.0 mmol/L, the expression of phosphorylated-P38MAPK and phosphorylated-STAT3 increased first and decreased afterwards. Incubation of K562 cell with SNP (2 mmol/L) could increase the expression of phosphorylated-P38MAPK and phosphorylated-STAT3 at 12 hours and 24 hours respectively, and down-regulated at 72 hours and 48 hours. SNP could decrease the expression of hTERT-mRNA in time-and dose-dependent manner. It is concluded that SNP can significantly induce K562 cells apoptosis, its mechanism may be related to the activation of P38MAPK and suppression of phosphorylated-STAT3 and hTRET-mRNA.
Apoptosis ; drug effects ; Humans ; K562 Cells ; Nitroprusside ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; STAT3 Transcription Factor ; genetics ; metabolism ; Telomerase ; biosynthesis ; genetics ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism

To investigate the possible mechanisms of nitric oxide (NO)-induced apoptosis in leukemia cell line HL-60, HL-60 cells in vitro were incubated with sodium nitroprusside (SNP), the in situ cell apoptosis quantitatively was assayed by TdT-mediated dUTP nick end labeling (TUNEL), the cell cycle DNA and proteins expression of Bcl-2, Bax, mitochondrial membrane protein (APO2.7) were analyzed by flow cytometry. The results showed that SNP induced HL-60 cell apoptosis in a dosage- and time-dependent manner. After exposure to SNP at the concentration of 1.0 mmol/L for 48 hours, the percentage of apoptosis HL-60 was (42.2 +/- 3.5)% for subG1 and (52.5 +/- 7.6)% for TUNEL respectively, and they are significantly higher than those in control and potassium ferricyanide (PFC) groups as same concentration. During the apoptosis process, it showed a decrease of Bcl-2 protein and an increase of Bax protein and mitochondrial membrane protein in HL-60 cell, proteins of Bcl-2, Bax and mitochondrial membrane were expressed in a dosage- and time-dependent manner too. In conclusion, during the process of SNP induced apoptosis in HL-60 cell, the expression of mitochondrial membrane protein was increased, Bcl-2 and Bax proteins may be important regulators.
Apoptosis ; drug effects ; HL-60 Cells ; Humans ; Membrane Proteins ; analysis ; Mitochondrial Proteins ; analysis ; Nitric Oxide Donors ; pharmacology ; Nitroprusside ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; bcl-2-Associated X Protein

OBJECTIVETo study the effect of macrocalyxin A (MA) on proliferation, differentiation and apoptosis in HL-60 cells and explore its possible mechanisms.

METHODSDifferent concentration of MA were used to treat HL-60 cells. Proliferation inhibition was analyzed by Trypan blue staining and MTT assay, cell apoptosis by cell morphology, DNA content, cell cycle analysis, Annexin-V/PI and Hoechst 33258 fluorescence staining. The differentiation of HL-60 cells was evaluated by cell morphology, NBT tests and expression of CD11b, CD13, CD14. The expressions of bcl-2, bax, Fas, P53, mitochondrial transmembrane-potential (DeltaPsim) and mitochondrial membrane protein were analyzed by flow cytometry.

RESULTSMA could inhibit HL-60 cells proliferation capacity in a time-and dose-effect, with a 24 h IC50 value of 8.76 microg/ml, 48 h of 7.17 microg/ml and 72 h of 7.14 microg/ml. The HL-60 cells apoptosis was confirmed by cell morphology, sub-G1 phase and Annexin-V/PI labeling in a time and dose dependent manner. The more mature HL-60 cells were a with higher positivity of NBT and expressions of CD11b than those cultured without MA. The expression of bax was increased, while bcl-2, P53, Fas were unchanged on the MA treatment. MA could increase the expression of mitochondrial membrane protein in a dose-dependent manner while the DeltaPsim was reduced.

CONCLUSIONMA can inhibit proliferation and induce differentiation and apoptosis of the HL-60 cells. The mechanism may be related with up-regulating bax, opening the mitochondrial permeability transition pore and reducing DeltaPsim.

Apoptosis ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Diterpenes ; pharmacology ; HL-60 Cells ; Humans

This study was purposed to investigate the inhibitory effect of macrocalin A (MA) on proteasome of multiple myeloma U266 cells in vitro and molecular mechanism of MA-inducing apoptosis. U266 cells in vitro were incubated with different concentrations (2, 4, 8 µg/mL) of MA, the Hochest staining and Annexin-V/PI double staining were used to detect the apoptosis of U266 cells. The expressions of protein β1, β1i, β2, β2i, β5, β5i, ubiquitous, 19S subunit S6', and BAD,BCL-2, FAS, FAS-L,MAPK, PARP, Pro-caspase 3, cleaved-caspase 3 were detected by Western blot technique. The results showed that along with time prolonging and dose increasing of MA, the small and compact fluorescent particles were observed in cytoplasm and nucleus of U266 cells stained with Hoechst 33258, the Annexin V(+)/PI(-) cells and the total apoptosis cells (Annexin V(+)/PI(-) and Annexin V(+)/PI(+)) increased. MA could elevate the ubiquitylation level in U266 cells, suppress the expression of β1i,β2, β5i and 19S subunit S6', meanwhile the expression of BCJ-2, MAPK, PARP and pro-caspase 3 were down-regulated along with increasing of drug concentrations, but the expressions of BAD, FAS, FAS-L cleaved-caspase 3 were enhanced. It is concluded that MA can inhibit the effect of proteasome, and the mitochondrial pathway and death receptor pathway may play important roles in apoptosis of U266 cells induced by MA.
Apoptosis ; drug effects ; Cell Line, Tumor ; Diterpenes ; pharmacology ; Humans ; Multiple Myeloma ; pathology ; Proteasome Endopeptidase Complex ; metabolism ; Proteasome Inhibitors ; pharmacology