This study was aimed to explore the effect of bortezomib and low concentration cytarabine (Ara-C) on proliferation and apoptosis in U937 cell line and its mechanism. The proliferation and apoptosis of U937 cells treated with bortezomib (10 nmol/L) and(or) Ara-C (50 nmol/L) were observed by cell count, cell morphology, flow cytometry and Western blot. The results showed that bortezomib and Ara-C alone inhibited U937 cell proliferation. The inhibitory effect was enhanced by combination of these two drugs, the inhibitory rates of U937 cell proliferation were (55.00 ± 2.81)% and (70.02 ± 3.33)% after treatment for 24 h and 48 h, respectively. Bortezomib and Ara-C synergistically induced apoptosis and decreased mitochondrial membrane potential in U937 cells. The percentage of Rhodamin123 positive cells was (38.70 ± 1.54)%. Bortezomib and Ara-C also synergistically induced activation of caspase-9, caspase-8 and caspase-3. It is concluded that the bortezomib and low concentration Ara-C synergistically induced apoptosis in U937 cells, mainly through mitochondrial pathway, and possibly through death receptor pathway.
Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Cycle ; drug effects ; Cytarabine ; pharmacology ; Humans ; Pyrazines ; pharmacology ; U937 Cells

OBJECTIVE: To investigate the effect and mechanism of artesunate (ARTS) combined with cytarabine(Ara-C) and/or daunorubicin (DNR) on the proliferation and apoptosis of MV4-11 human mixed-lineage leukemia rearranged（MLL-r） acute myeloid leukemia (AML) cell line. METHODS: CCK-8 assay was used to detect the proliferation effect of individual or in combination of ARTS, DNR, Ara-C on MV4-11 cells. The IC₅₀ of ARTS, DNR and Ara-C was calculated separately. The cell apoptosis and expression of receptors DR4 and DR5 were detected by flow cytometry. Western blot was used to detect the expression of Caspase-3 and Caspase-9 in each groups. RESULTS: The inhibition effect of ARTS, Ara-C and DNR on the proliferation of MV4-11 were all dose-dependently (r=0.99, 0.90 and 0.97, respectively). The IC₅₀ of ARTS, Ara-C and DNR on MV4-11 for 48 hours were 0.31 μg/ml, 1.43 μmol/L and 22.47 nmol/L, respectively. At the dose of ARTS 0.3 μg/ml， Ara-C 1.0 μmol/L and DNR 15 nmol/L, the proliferation rate for 48 hours of the tri-combination treatment was significantly lower than that of the bi-combination treatment, while both were significantly lower than that of the individual treatment (all P＜0.05). In terms of bi-combination treatment, the cells proliferation rate for 48 hours of the ARTS+Ara-C group was significantly lower than that of the ARTS+DNR group, while both were significantly lower than that of the Ara-C+DNR group (all P＜0.05). The cooperativity index (CI) of bi- and tri-combination treatment were all less than 1. After 48 hours of drug action, the cell apoptosis rate of the ARTS+DNR+Ara-C group was significantly higher than that of the Ara-C+DNR group, while both were significantly higher than that of the ARTS+DNR group (all P＜0.05). Meanwhile, the was no statistical difference between the cells apoptotic rate of the ARTS+DNR+Ara-C group and the ARTS+Ara-C group (P>0.05). The expression of DR4 and DR5 also showed no difference between control group and drug group. Compared with the DNR+Ara-C group, the expressions of Caspase-3 were significantly down-regulated in both the ARTS+DNR+Ara-C group and the ARTS+Ara-C group (all P＜0.05). The down-regulation of Caspase-3 expression was the most significantly in the combination group of three drugs, while the Caspase-9 expressions in different groups showed no apparent change. CONCLUSION The in vitro study showed that tri-combination of ARTS+Ara-C+DNR and bi-combination of ARTS+Ara-C could inhibit the proliferation and promote apoptosis of MV4-11 cell line. The inhibition effect of these two combinations were significantly superior to that of the traditional Ara-C+DNR treatment. The mechanism underlying this finding may be identified by the down regulation of Caspase-3, while no altered expression was observed of Caspase-9, DR4 and DR5.
Humans ; Cytarabine/pharmacology* ; Daunorubicin/pharmacology* ; Caspase 3 ; Caspase 9 ; Artesunate/pharmacology* ; Leukemia, Myeloid, Acute ; Apoptosis ; Cell Line

OBJECTIVE: To construct cytarabine-resistant acute myeloid leukemia (AML) cell lines, and explore the correlation between Sirt1, PGC-1α expression levels and drug resistance. METHODS: Human acute promyelocytic leukemia Kasumi-1 cells were induced by the method of gradually increasing the concentration of Ara-C drug. The IC₅₀ value of Kasumi-1 cells before and after drug addition was detected by CCK-8 method, so as to construct Ara-C resistant cell lines. The expression levels of Sirt1 and PGC-1α mRNA in Kasumi-1 drug-resistant cell lines and their parental cell lines were detected by real-time fluorescence quantitative PCR, and the expression levels of Sirt1 and PGC-1α protein in kasumi-1 drug-resistant cell lines and their parental cell lines were detected by Western blot. RESULTS: The constructed Kasumi-1 cell line had common morphological characteristics of drug-resistant cell lines under microscope, and the drug resistance index was greater than 5, indicating that Kasumi-1 drug-resistant cells had good drug resistance after the construction. The RT-qPCR and Western blot assays showed that the expression levels of Sirt1 and PGC-1α mRNA and protein in the drug-resistant cell lines were higher than those of the parental cell lines (P<0.001). CONCLUSION AML cell lines resistant to Ara-C can be successfully induced by the method of gradually increasing the concentration, and the co-high expression of Sirt1 and PGC-1α may mediate the drug resistance of AML cells.
Cell Line ; Cytarabine/pharmacology* ; Drug Resistance ; Humans ; Leukemia, Myeloid, Acute/genetics* ; RNA, Messenger/genetics* ; Sirtuin 1

This study was aimed to investigate the changes of mitochondrial membrane potential (DeltaPsim) of HL-60 cells induced by cytarabine and the correlation between mitochondrial membrane potential and apoptosis of HL-60 cells. HL-60 cells were stained with Rhodamine 123; change of mitochondrial membrane potential of HL-60 cells was detected by flow cytometry. AO/EB staining and flow cytometry were used to examine the apoptosis of HL-60 cells. The results showed that the levels of HL-60 cell DeltaPsim in experimental groups decreased after cultured for 6 hours. In Ara-C 0.05 mg/ml group, rhodamine 123 fluorescence intensity in mitochondria of HL-60 cells at 6, 12, 24 hours were 117.9+/-7.6, 100.9+/-7.7, 87.6+/-10.7, respectively, there was significant difference between the different culture groups (p<0.05). In Ara-C 0.1 mg/ml group, rhodamine 123 fluorescence intensity in mitochondria of HL-60 cells at 6, 12, 24 hours were 111.9+/-10.1, 86.6+/-9.2, 68.4+/-12.2, respectively, there was significant difference between the different culture groups (p<0.05); rhodamine 123 fluorescence intensity was significantly different between the two groups at 12, 24 hours (p<0.05). In Ara-C 0.05 mg/ml group, the apoptosis rate of HL-60 cells at 6, 12, 24 hours were (41.2+/-3.0)%, (53.7+/-5.1)%, (65.8+/-2.6)% respectively, there was significant difference between the different culture groups (p<0.01); In Ara-C 0.1 mg/ml group, the apoptosis rate of HL-60 cells at 6, 12, 24 hours were (45.7+/-4.1)%, (58.2+/-4.3)%, (70.1+/-2.3)% respectively, there was significant difference between the different culture groups (p<0.01); the apoptosis rates showed no significantly difference between the two groups at same time. The changes of mitochondrial membrane potential and apoptosis rate of HL-60 cells were significantly negatively correlated. In Ara-C 0.05 mg/ml group, r was -0.89, p<0.01, while in Ara-C 0.1 mg/ml group, r was -0.76, p<0.01. It is concluded that the mitochondrial membrane potential on HL-60 cells decrease at HL-60 cells apoptosis induced by Ara-C, therefore the reduction of mitochondrial membrane potential may be one of the important mechanisms at the induced apoptosis.
Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Cytarabine ; pharmacology ; HL-60 Cells ; Humans ; Membrane Potential, Mitochondrial ; drug effects

OBJECTIVETo explore whether daunorubicin (DNR) combined with cytosine arabinoside (Ara-C) and DNR alone have similar effect on acute promyelocytic leukemia (APL) cell line NB4 and acute myeloblastic leukemia cell line HL-60 in vitro.

METHODSCell morphology, cells viability, and cell apoptosis (Annexin-V by flow cytometry assay) were analysed.

RESULTSAfter incubation with DNR plus Ara-C for 24 hours,NB4 cell viability [(36.75 +/- 3.82)%] (n = 6) and cell apoptosis rate [(21.24 +/- 5.82)%] (n = 3) did not change significantly compared to that treated with DNR alone for 24 hours [(35.73 + 6.28 )%, (22.55 +/- 3.26)%, respectively] (P > 0.05). However, HL-60 cell viability [(67.17 +/- 2.07)%] and cell apoptosis rate [(48.05 +/- 0.92)%] changed significantly in DNR plus Ara-C group compared with DNR alone [(63.31 +/- 1.80)% ,(41.51 +/- 0.89)%, respectively] (P < 0.01 and < 0.05, respectively).

CONCLUSIONDNR plus Ara-C and DNR alone have similar effect on NB4 cells, but have different effect on HL-60 cells.

Apoptosis ; drug effects ; Cytarabine ; pharmacology ; Daunorubicin ; pharmacology ; HL-60 Cells ; drug effects ; Humans ; Leukemia, Promyelocytic, Acute ; pathology

This study was aimed to explore the influence of anti-CXCR4 monoclonal antibody 12G5 on killing effect of cytosine arabinoside (Ara C) to HL-60 cell, and to assess its therapeutic value in marrow residual disease. HL-60 cells were cultured and co-cultured with leukemic stromal cells, and SDF-1 activity was inhibited with 10 microg/ml 12G5, then, killing effects of Ara C on HL-60 cells were investigated by MTT and morphology assay. Curves by MTT assay revealed that in the test group of 20 microg/ml Ara C, A(540) values decreased slowly but straightly, however, in control group A(540) values decreased markedly for the first two days, and increased from day 3 or 4. In the test group of 40 microg/ml Ara C, although increasing at constricted range of 7 - 9 days, A(540) values decreased in whole observing period of 12 days, while in control group A(540) values decreased markedly at day 0-3, and increased from day 4. Furthermore, two curves go across each other at day 5, and continue the increasing tendency. Morphology results showed that in both treated groups, the number of HL-60 cell decreased markedly and increased gradually in control group, but just contrary to test group. It is concluded that 12G5 may weaken the killing effect of Ara C on HL60 cell in earlier period, but reinforce the total killing effect and delay the occurrence of drug resistance simultaneously. Thus 12G5 has the therapeutic potential on marrow residual disease.
Antibodies, Monoclonal ; pharmacology ; Antimetabolites, Antineoplastic ; pharmacology ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cytarabine ; pharmacology ; Dose-Response Relationship, Drug ; Drug Synergism ; HL-60 Cells ; Humans ; Receptors, CXCR4 ; immunology

To investigate the effects of rhG-CSF on apoptosis of HL-60 cell induced by cytarabine (Ara-C), HL-60 cell were cultured with Ara-C, with or without rhG-CSF, and the changes of HL-60 cells were detected by morphology, including Wright's and acridine orange (AO) and ethidium bromide (EB) staining, and MTT assay. The results showed that comparing with Ara-C group, the apoptosis level of rhG-CSF + Ara-C group was enhanced (P < 0.01), and the OD value of MTT assay declined (P < 0.01). It is concluded that rhG-CSF can induce the apoptosis of HL-60 cells and enhance the apoptosis induced by Ara-C.
Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Cell Survival ; drug effects ; Cytarabine ; pharmacology ; Drug Synergism ; Granulocyte Colony-Stimulating Factor ; pharmacology ; HL-60 Cells ; Humans ; Recombinant Proteins

The purpose of this study was to investigate the effect of salidroside on human bone marrow mesenchymal stem cell (hBMMSC) apoptosis induced by cytarabine C (Ara-C) and its mechanism, hBMMSC were cultured in vitro and isolated by Fircoll density gradient centrifugation; cell surface antigens were measured by flow cytometry; the osteogenic and adipogenic differentiation of MSC was tested and evaluated by specific staining methods. The proliferation and apoptosis of cells exposed to Ara- C were detected by MTT and flow cytometry respectively. The experiments were divided into 4 groups: control group, Ara-C group, salidroside group and Ara-C+salidroside group. The mRNA expression of BCL-2 and BAX was assayed by RT-PCR. The results showed that the adherent cells displayed spindle and fibroblast cell-like shape; the hBMMSC expressed CD44, CD71 and HLA-ABC, not expressed CD34, CD45 and HLA-DR; the hBMMSC successfully differentiated into osteogenic and adipogenic lineages, which showed mineralization with von Kossa staining. Furthermore, liquid vacuoles were detected by oil red O staining; Ara- C exhibited a less inhibitory effect on the proliferation of hBMMSC treated with salidroside. The apoptosis of hBMMSC treated with salidroside were significantly higher as compared with control group (P < 0.05); RT-PCR results demonstrated that the BCL-2 expression was significantly down regulated but BAX mRNA expressions was up-regulated in Ara- C group as compared with those in the control group. Salidroside significantly inhibited the apoptosis of MSC and reversed the mRNA expression of BCL-2 and BAX. It is concluded that salidroside can inhibit the apoptosis of hBMMSC induced by Ara-C, its mechanism may be related with the regulation of BCL-2/BAX expression.
Apoptosis ; drug effects ; Bone Marrow Cells ; cytology ; drug effects ; Cells, Cultured ; Cytarabine ; pharmacology ; Glucosides ; pharmacology ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Phenols ; pharmacology

This study was purposed to investigate the impact of human umbilical cord-derived mesenchymal stem cells (hUC-MSC) on the sensitivity of HL-60 cells to therapeutic drugs so as to provide more information for exploring the regulatory effect of hUC-MSC on leukemia cells. Transwell and direct co-culture systems of HL-60 and hUC-MSC were established. The apoptosis and cell cycle of HL-60 cells were detected by flow cytometry. RT-PCR and Western blot were used to detect the mRNA and protein levels of Caspase 3, respectively. The results showed that the apoptosis of HL-60 induced by cytarabine (Ara-C) decreased significantly after direct co-cultured with hUC-MSC cycle mRNA (P < 0.05). The similar phenomenon was observed in transwell co-culture system. Cell cycle of HL-60 cells were arrested at G0/G1 phase and did not enter into S phase (P < 0.05) and the expression of Caspase-3 mRNA and protein in HL-60 cells were reduced (P < 0.05). It is concluded that hUC-MSC protected HL-60 from Arc-C induced apoptosis through regulating the cell cycle and down-regulating expression of Caspase 3 in HL-60 cells. In addition, this effect is caused by the soluble factors from hUC-MSC.
Apoptosis ; Caspase 3 ; metabolism ; Coculture Techniques ; Cytarabine ; pharmacology ; HL-60 Cells ; Humans ; Mesenchymal Stromal Cells ; cytology ; Umbilical Cord ; cytology

In order to investigate the potential role of human ERMAP gene in erythropoiesis, the ERMAP-dsDNA was designed, ERMAP-shRNA expressing plasmids was constructed, and ERMAP-shRNA/K562 cell was established. Cell morphology, biphenylamine staining, expression of cell surface antigens as well as quantitative level of human ERMAP gene were observed during K562 cells differentiating toward erythroid lineage induced by Ara-C. The results showed that at 72 hours after Ara-C treatment, ERMAP-shRNA/K562 cell size became large with increasing cytoplasm content. The percentage of biphenylamine positive cells increased from 1.17% to 2.04% (p < 0.05), but still lower than that in group K562 + Ara-C. The percentage of CD36(-)/CD235a(+) increased from 8.83% to 11.28%, CD36(+)/CD235a(+) increased from 1.23% to 2.64%, and CD36(+)/CD235a(-) increased from 0.59% to 1.47% respectively, which were all lower than that in group K562 + Ara-C at either time point. At the same time, the level of ERMAP expression increased slowly from 2.52 x 10(-3) to 4.53 x 10(-3), which was also significantly lower than that of group K562 + Ara-C. It is concluded that the ERMAP-shRNA inhibits the Ara-C-induced erythroid differentiation of K562 cells, which further suggests that there is relationship between hERMAP and erythroid differentiation and development.
Blood Group Antigens ; genetics ; Butyrophilins ; Cell Differentiation ; drug effects ; Cytarabine ; pharmacology ; Erythropoiesis ; drug effects ; Humans ; K562 Cells ; RNA, Messenger ; RNA, Small Interfering ; pharmacology