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This study was purposed to evaluate whether the safe concentration of magnetic nanoparticles of Fe₃O₄(MNPs-Fe₃O₄) for monocytes could induce the SKM-1 cell apoptosis. The average size and Zeta potential of MNPs-Fe₃O₄were determined by transmission electron microscopy and the Malvern Zetasizer 3000 HS, respectively. The cell viability after being exposed to MNPs-Fe₃O₄for 12, 24, 48, and 72 hours was detected by using cell count Kit-8. The cell apoptosis was evaluated by flow cytometry with Annexin V/PI double staining and Wright-Giemsa staining. The cell cycle was measured by flow cytometry. The levels of active caspase-3, survivin and bcl-rambo in cells treated with MNPs-Fe₃O₄and/or trolox for 48 hours were detected with Western blot. The results showed that the cell viability decreased in SKM-1 cells after exposure to 50 µmol/L and 100 µmol/L MNPs-Fe₃O₄(P < 0.05), but did not in monocytes (P > 0.05), compared with that of each non-MNPs-Fe₃O₄-treated group. This exposure also induced the SKM-1 cells to be arrested in G0/G1. Annexin V/PI staining assay showed that cell apoptotic rate induced by 100 µmol/L MNPs-Fe₃O₄was significantly high in SKM-1 cells while not so high in monocytes, and the pretreatment with trolox could attenuate the apoptosis. Moreover, the active caspase-3 increased in SKM-1 cells after the exposure to MNPs-Fe₃O₄, while that was not in monocytes, and the increased expression of BCL-rambo and the decreased expression of survivin involved in the process were also observed. It is concluded that MNPs-Fe₃O₄can induce the caspase 3-dependent SKM-1 cell apoptosis by increasing the BCL-rambo expression and decreasing the survivin expression, but this cytotoxic effect can not be observed in monocyte's.

The src homology 2 (SH2)-domain containing inositol-5-phosphatase (SHIP) is another recently identified lipid phosphatase after phosphatase and tensin homology deleted on chromosome ten gene (PTEN). It plays an important role in negatively regulating the proliferation of hematopoietic cells. The relationship between SHIP and the inhibition of tumor proliferation is rarely reported. The purpose of this study is to evaluate the apoptosis induced by SHIP gene in K562 cell line and to explore the involved signaling pathway. The K562 cells were transfected with human SHIP gene by using the lentiviral vector containing SHIP, and the transfection was verified by fluorescent quantitative PCR (FQ-PCR) and Western blot. Then the effects of SHIP protein expression on cell growth and apoptosis were measured. The levels of p-Akt, bcl-2 family, caspase and the activity of NFkappaB were assayed by Western blot and ELISA, respectively. The results are as follows: (1) Human leukemia cell line K562 was SHIP-negative; (2) Transfection with SHIP gene led to the re-expression of SHIP mRNA and protein in K562, as shown by FQ-PCR and Western blot; (3) The expression of SHIP protein inhibited cell growth and significantly increased apoptosis in K562 cells; (4) Compared to that in control group, the expression level of p-Akt-308 and p-Akt-473 in SHIP-expressing cell group decreased significantly (P<0.01); SHIP activated caspase-9, caspase-3, up-regulated protein levels of bad, p27, down-regulated expression of bcl-xL, while it had no effect on the expression of bcl-2 and bax. Furthermore, the inhibition of NF-kappaB was achieved along with the inactivation of Akt. These data suggest that SHIP gene has potential abilities to inhibit K562 leukemic cell proliferation and induce its apoptosis via inactivating PI3K/Akt pathway. The loss of SHIP might be the explanation of aberrant high-level p-Akt in human leukemia. It may be at least one of the mechanisms by which the loss of SHIP expression contributes to leukemia progression.

The aim of this study was to investigate the regulation of 5-aza-CdR on transcription of SHP-1 gene and effects on the proliferation and apoptosis of K562 cells. Methylation-specific PCR (MSP) was used to detect CpG island methylation in SHP-1 promoter. MTT and flow cytometry were used to detect the growth and apoptosis of K562 cells after treatment with different concentration of 5-aza-CdR. The expressions of SHP-1 mRNA and protein were determined by FQ-PCR and Western blot. The expression of p-JAK2 was assayed by Western blot. The result showed that methylation of SHP-1 gene promoter was detected in K562 cells, and the SHP-1 mRNA and protein were expressed again in K562 cells after treatment with 5-aza-CdR, meanwhile the expression of phosphorylated P-JAK2 was down-regulated; 5-aza-CdR significantly inhibited the cell growth in dose and time dependent manners. AG490 inhibited the cell proliferation. 5-aza-CdR increased the apoptosis rate of K562 cells also in dose- and time-dependent manners. The apoptosis rates of K562 cells treated with 5-aza-CdR for 1, 3 and 5 days were 9.3%, 24.2% and 37.7% respectively. After treatment with 2 micromol/L 5-aza-CdR for 24 hours, cells in G(0)/G(1) phase increased gradually, cells in G(2)/M phase decreased gradually, cells were arrested in G(0)/G(1) phase. The cell ratios in G(2)/M phase at 1, 3 and 5 days after treatment with 5-aza-CdR were 30.7%, 23.45 and 19.3% respectively. It is concluded that the 5-aza-CdR, inhibitor of specific methylation transferase, can re-express the silent SHP-1 gene in K562 cells, inhibits the proliferation of leukemia cells and induces cell apoptosis by activating JAK/STAT pathway.

Objective: To investigate the effect of the wild type phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor-suppressor gene on the proliferation and apoptosis of human chronic myeloid leukemia (CML) cells line (K562) in vitro and explore the influence of PTEN-FAK signaling pathway on invasion and metastasis of leukemia cells., Methods: The recombinant Ad-PTEN gene containing green fluorescent protein gene (Ad-PTEN-GFP) or the empty vector (Ad-GFP) was transfected into K562 cells and fresh leukemia cells from CML patients in blast crisis. The growth of K562 cells was assayed by MTT assay; the apoptosis rate was assessed by flow cytometry (FCM). PTEN and FAK mRNA levels were detected by real-time fluorescent relative- quantification reverse transcriptional PCR (FQ-PCR) and its protein levels by Western blot. The metastasis and invasive ability was examined by transwell chamber assay., Results: The growth of K562 cells was suppressed markedly when Ad-PTEN-GFP was transfected into K562 cells at the 200 multiplicity of infection (MOI). The maximum growth inhibition rate was 35.2%. Transwell results showed the number of cells entered the lower chamber in Ad-GFP group was 9.1 fold more than that in Ad-PTEN-GFP group;The ability of metastasis and invasion of fresh leukemia cells was also suppressed after transfection with Ad-PTEN-GFP. FAK and p-FAK proteins were down-regulated by 0.72 and 0.16 fold lower after transfected with Ad-PTEN-GFP compared with Ad-GFP group., Conclusions: PTEN gene might inhibit the proliferation, metastasis and invasive ability of leukemia cells via down-regulating FAK expression.

This study was aimed to investigate the effect of recombinant mutant human TNF-related apoptosis-inducing ligand (rmhTRAIL) combined with As(2)O(3) on inducing apoptosis of adriamycin-resistant leukemia cell line K562/A02 (mdr-1(+)). The morphologic changes of cells treated with rmhTRAIL were observed by inverted microscope, taking adriamycin-sensitive cell line K562 (mdr-1(-)) as control; the inhibitory rate of cell proliferation after being treated with rmhTRAIL, As(2)O(3) alone or combined was assayed by MTT method; the apoptosis peaks of K562/AO2 and K562 were quantitatively detected by flow cytometry with PI staining after being treated with rmhTRAIL, As(2)O(3) alone or in combination. The results indicated that the inhibition effect of rmhTRAIL and As(2)O(3) in combination on K562/AO2 and K562 cells was higher than that of riTRAIL and As(2)O(3) alone (p < 0.01), rmhTRAIL combined with As(2)O(3) had synergistic effect in killing K562/AO2 and K562 cells by king's formula. The apoptosis rates of K562/AO2 and K562 cells were 34.93 +/- 0.10% and 10.53 +/- 0.16% (p < 0.01), as well as 5.95 +/- 0.07%, and 3.50 +/- 0.01% (p < 0.05), 50.95 +/- 0.91% and 20.75 +/- 0.95% (p < 0.05) respectively when their cells were treated by rmhTRAIL and As(2)O(3) alone. The apoptosis rate in K562/AO2 group was higher than that in K562 group. It is concluded that rmhTRAIL can induce K562/A02 and K562 cell apoptosis; rmhTRAIL combined As(2)O(3) had synergistic effects; the efficacy of on rmhTRAIL or As(2)O(3) inducing K562/AO2 cell apoptosis is higher than that on their parental cell line K562.