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Byline: Su-Yun. Wang, Ning. Yang, Li-Jun. Zhang, Zhi-Yong. Cheng Essential thrombocythemia (ET) is a type of myeloproliferative neoplasm (MPN) that is negative for the BCR-ABL1 fusion gene.[1] It is characterized [...]

Objectives: To investigate the effect of Ruxolitinib on the expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 α (HIF-1α) in HEL cells., Methods: he HEL cells were treated with Ruxolitinib in different concentrations (1 nmol/L, 5 nmol/L, 10 nmol/L, 50 nmol/L, 100 nmol/L, 500 nmol/L). The growth inhibition of Ruxolitinib on HEL cells was detected by CCK-8 assay;the mRNA expression level of JAK2 were measured by RT-PCR and the protein level of p-JAK2, VEGF, HIF-1α were observed by Western blot after treated with Ruxolitinib for 24,48,72 h. Chick chorioallantoic membrane (CAM) test was used to testify the effect of Ruxolitinib on angiogenesis., Results: Ruxolitinib with different concentrations could inhibit HEL cells proliferation. RT-PCR showed that the mRNA level of JAK2 decreased in a concentration-dependent manner and Western blot demonstrated that the expression levels of p-JAK2, VEGF and HIF-1α were lower in Ruxolitinib treatment groups than those in control group ( P <0.05) after HEL cells were treated with different concentrations of Ruxolitinib for 24,48,72 h. Ruxolitinib significantly suppressed blood vessels'formation in CAM., Conclusions: Ruxolitinib can inhibit VEGF, HIF-1α expression and angiogenesis of HEL leukemia cells by inhibiting JAK2 pathway.

Jauns kinase (JAK)/transducer and activator of transcription（STAT） pathway is a classical approach to study the rapid changes of the gene expression in specific target cells by a variety of extracellular signals. The JAK and STAT transfer cytokine receptor signaling plays a unique role in multiple cellular and molecular biological changes.The abnormal signal of JAK/STAT pathway will lead to the hematopoietic abnormalities.Studies had shown that the abnormal activation of JAK2/STAT signaling pathway are in many kinds of malignant hematological diseases, such as in acute lymphoblastic/myeloid leukemia, chronic myeloid leukemia, lymphoma, myelodysplastic syndromes, myeloprofilerative neoplasm, especially in the patients of myeloproliferative neoplasm(MPN) with JAK gene mutation(JAK2V617F), this mutation has an important value for MPN diagnosis. At present, the effect of the specific inhibitors of JAK2 has showed good perspective, which had been applied to clinic treatment and achieved remarkable curative effect. In this review, the JAK2/STAT signaling transduction, the JAK2 signal and hematologic malignancies, the kagulation of signaling pathway and the inhibitors of JAK2/STAT signaling pathway are summarized.

This study was aimed to investigate the effects of proteasome inhibitor bortezomib on proliferation, apoptosis and the SHIP expression of K562 cells. K562 cells were treated with bortezomib of different concentrations. Cell proliferation was analyzed by MTT assay, cell apoptosis was detected by flow cytometry and SHIP mRNA expression was assayed by RT-PCR.The results showed that after being treated with 10, 20, 50 and 100 nmol/L bortezomib for 24 h, the inhibitory rates of K562 cells were (5.76 ± 1.47)%, (10.55 ± 1.59)%, (17.14 ± 2.05)% and (27.69 ± 3.57)% respectively, and were higher than that in control (1.30 ± 0.10); when K562 cells were treated with 20 nmol/L bortezomib for 24, 48 and 72 h, the inhibitory rates of cell proliferation were (10.55 ± 1.59)%, (16.33 ± 2.53)% and (19.78 ± 1.56)% respectively, there was statistic difference of cell proliferation rate between 24 h group and 48 h group (P < 0.05). After being treated with 10,20,50,100 nmol/L bortezomib for 24 h, the apoptotic rates of K562 cells were (12.7 ± 0.6)%, (26.9 ± 0.9)%, (32.6 ± 1.2)% and (72.5 ± 1.5)% respectively,and all higher than that in control (1.0 ± 0.5)% (P < 0.05). According to results of RT-PCR detection, the expression level of SHIP mRNA was obviously up-regulated after treatment with bortezomib, and showed statistical difference in comparison with control. It is concluded that bortezomib inhibits proliferation of K562 cells in time and concentration-dependent manner and induces apoptosis through up-regulation of SHIP gene.

Aim: To investigate the effect of tyrosine kinase inhibitor imatinib mesylate on the PTEN signaling pathway and the cell invasion in K562 cells., Methods: K562 cells were treated with different concentrations of imatinib mesylate. After different time periods, the mRNA levels of BCR/ABL, PTEN and FAK were detected by real-time fluorescent quantitative PCR (FQ-PCR) to analyze their relationships. The protein level of FAK was detected by immunocytochemistry. The cell invasive ability was examined by Transwell (Boyden chamber) assay., Results: In the initial 36 h, the expression level of PTEN mRNA was up-regulated and the FAK mRNA was down-regulated with the reduction of BCR/ABL fusion gene expression and the cell invasive ability of K562 cells was inhibited by 2 μg/mL imatinib mesylate. 48 h later, the PTEN mRNA expression level decreased and the FAK mRNA expression level was elevated with the restore of BCR/ABL fusion gene. BCR/ABL mRNA level presented a positive correlation with PTEN mRNA expression level, and a negative correlation with FAK mRNA., Conclusion: Tyrosine kinase inhibitor imatinib mesylate can regulate PTEN/FAK pathway and inhibit the leukemia K562 cell invasive ability via restraining BCR/ABL fusion gene.

Objective: To explore the effects of tumor-suppressing gene wild type PTEN on the cell proliferation, apoptosis and the possible regulations of apoptosis-related molecules Survivin, Xiap and Smac gene in human chronic myeloid leukemia (CML) and cell line K562 cells., Methods: (1) The recombinant adenovirus containing green fluorescent protein (GFP) and PTEN (Ad-PTEN-GFP) or empty vector (Ad-GFP) was transfected into K562 cells. The growth of K562 cells was observed by MTT assay while cell cycle and apoptotic rate were assessed by flow cytometry (FCM). PTEN, Survivin, Xiap and Smac mRNA levels were detected by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR) while PTEN protein levels analyzed by Western blot. (2) The expression levels of PTEN, Survivin, Xiap and Smac mRNA were detected in 10 chronic myelogenous leukemia (CML) patients in chronic phase (CML-CP), 10 CML patients in blast crises (CML-BC) and 10 normal control marrow mononuclear cells (MMNC)., Results: The growth of K562 cells was suppressed markedly. And the maximal growth inhibition rate was 38.6% after the transfection of PTEN. Survivin, Xiap, Smac mRNA expression levels were down-regulated by around 6.14, 7.44 and 2.95 folds respectively (0.0700 ± 0.0059, 0.0089 ± 0.0006, 0.0600 ± 0.0039 vs 0.4370 ± 0.0790, 0.0661 ± 0.0072, 0.1580 ± 0.0078 vs 0.4530 ± 0.0810, 0.0700 ± 0.0079, 0.1770 ± 0.0085, all P < 0.01). The mRNA expression level of PTEN in CML-BC patients was lower than that in CML-CP patients and normal control. But Survivin, Xiap, Smac mRNA expression levels were higher in CML-BC patients than those in CML-CP and normal control., Conclusion: The over-expression of PTEN gene may inhibit the proliferation of K562 cells and promote cell apoptosis via the regulation of Survivin, Xiap and Smac genes.

Invasion and metastasis are both the main biological characteristics in malignant tumor which are influence tumor therapeutic effect and prognosis. The tumor cells interact with vascular endothelial cells and cell matrix, penetrate vascular endothelial and degrade the extracellular matrix, and metastasis to the local and distant by the interactions of a variety of signaling molecules. PTEN protein has protein phosphatase and lipid phosphatase dual activity which is produced by PTEN gene. As a tumor suppressor gene, regulates the cell signal pathways to sustain the normal physiological functions, negatively regulates of tumor cell growth and cell cycle, induces apoptosis, and inhibits invasion, infiltrating and metastasis of tumor cells. This article is reviewed about how PTEN participates in inhibiting tumor cell invasion and metastasis.

Pten gene is the first antioncogene with dual phosphatase activity discovered so far, pten gene regulates the cell cycle progress, apoptosis, metastasis and invasion of the tumor cells through negatively regulating the multiple signaling transduction pathways. Multiple myeloma (MM) is a malignant tumor occurring in terminal stage of B cell differentiation. The genetic changes are considered as the important factors in MM pathogenesis, among which the deletion of antioncogene is a critical genetic change. However, little is known about the genetic change of pten in MM. This review summarizes the research advance on pten in MM including structure of pten, mechanism of pten effect and correlation of pten with MM in order to provide some references for the investigating new gene target to treat the MM.

This study was aimed to investigate the effect of beta-elemene on proliferation and apoptosis of human multiple myeloma cell line RPMI-8226 and its mechanism. The effect of beta-elemene on the growth of human multiple myeloma cell line RPMI-8226 was detected by MTT. The effect of beta-elemene on the apoptosis of RPMI-8226 cells was determined by flow cytometry with Annexin-V/PI staining. The effects of beta-elemene on the expression of BCL-2, caspase-3, DR-4 and NF-kappaB P65 proteins were analyzed by Western blot. The results showed that the beta-elemene obviously inhibited the proliferation of RPMI-8226 cells in both time- and dose-dependent manners. Treatment with 10 - 80 micromol/L beta-elemene for 48 hours induced apoptosis of RPMI-8226 cells in a dose-dependent manner. The expression of caspase-3 and DR-4 proteins in RPMI-8226 cells treated with beta-elemene increased in a time-dependent manner, while expressions of BCL-2 and NF-kappaB P65 proteins decreased. It is concluded that the beta-elemene can inhibit the proliferation of RPMI-8226 cells by inducing the cell apoptosis. Activating the mitochondrial and death receptor pathways of apoptosis and inhibiting the anti-apoptosis pathway may involve in the beta-elemene-induced apoptosis.

Objective: To investigate the effect of tumor-suppressing gene,wild type PTEN gene, mediated by adenovirus vector on the cell proliferation, apoptosis and the influence on apoptosis key factor Bcl-2 and Caspase family on human chronic myeloid leukemia (CML) cell line K562 in vitro., Methods: The recombinated Ad-PTEN gene containing green fluorescent protein gene (Ad-PTEN-GFP) or the empty vector (Ad-GFP) was transfected into K562 cells. The growth of K562 cells was evaluated by MTT assay; the transfection efficiency of Ad-PTEN-GFP, apoptosis rate and proliferation index (PI) were assessed by flow cytometry (FCM). Morphological characteristics of transfected cells under light and transmission electron microscope were applied to demonstrated the apoptosis; DNA ladder and fluorescent staining were also tested; the PTEN, Bcl-2 mRNA levels were detected by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR); PTEN and Bcl-2 protein levels were detected by Western Blotting; and Caspase-3/7 and -9 protein activity were detected by corresponding kits., Results: The 200 multiplicity of infection (MOI) of Ad-PTEN-GFP was applied to transfect K562 cells. The maximum growth inhibiting ratio was 37.1%. The early and advanced apoptosis rates were higher than Ad-GFP group and untransfected group (P<0.05). After 3 days transfaction of PTEN gene the Bcl-2 mRNA and protein were 0.27 fold and 0.58 fold respectively; and the Caspase-3/7 and -9 protein activity increased in time-depentend manner after transfected with PTEN gene at the first 3 days compared with Ad-GFP group and untransfected group., Conclusion: Over expression of PTEN gene can inhibit K562 cells proliferation and promote cell apoptosis probability via inhibiting Bcl-2 expression and up-regulating the Caspase-3/7 and -9 ability.

This study was purposed to investigate the effect of rapamycin on proliferation, apoptosis, cell cycle progression and the regulation of chemokine receptor CXCR4 on RPMI8226 cells. Different concentrations of rapamycin were used to treat the multiple myeloma cell line RPMI8226 for different times. The proliferation of the cells was detected by MTT assay; the apoptosis rate and cell cycle were determined by flow cytometry (FCM); apoptosis of cells was observed by inverted microscopy; the cylin D1, CXCR4 and mTOR mRNA expressions were detected by RT-PCR or FQ-PCR after treating RPMI8226 cells with different concentrations of rapamycin. The results indicated that the rapamycin could inhibit the proliferation of RPMI8226 cells and induce their apoptosis. The cell cycle was arrested at the G(0)/G(1) phase. PCR results showed the down-regulation of mTOR, cyclin D1 and mTOR mRNA expressions after treating RPMI8226 cells with different concentrations of rapamycin for 24 hours. It is concluded that the rapamycin significantly inhibits the growth of RPMI8226 cells in a dose-and time-dependent mannes and induce cell apoptosis. Cell cycle arrests at the G(0)/G(1) phase, may be due to the down-regulation of the mTOR and cyclin D1 expressions. In additions, the down-regulation of CXCR4 mRNA expression is correlated with the reduction of adhesion between myeloma cells and stromal cells.

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.