[Effect of Etoposide on Elimination of Chronic Myeloid Leukemia Stem Cells by Imatinib in Vivo].

OBJECTIVE: To investigate the effect of etoposide (ETO) on elimination of chronic myeloid leukemia (CML) stem cells by imatinib mesylate(IM) in vivo. METHODS: SCL-tTA/BCR-ABL mice were used as CML animal model. Flow cytometry was used to assess the effect of ETO alone or in combination with IM on the number of leukemia stem cell （LSC） in bone marrow and spleen, and peripheral blood neutrophils in CML mice and normal control FVB mice. RESULTS: The results showed that in CML mice, the number and proportion of LSC in bone marrow and the proportion of neutrophils in peripheral blood decreased significantly after ETO and IM combined treatment, and the degree of decrease was more significant than that of both alone. While in wild type FVB mice, the combination of ETO and IM showed no significant effect on the number and proportion of LSK cells in bone marrow and the proportion of neutrophils in spleen. CONCLUSION: ETO can selectively enhance elimination of CML LSC by IM in vivo.

Synthesis and biological evaluation of aminoalcohol rheinate as anti-osteosarcoma agents / 药学学报

Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is the primary anthraquinone in the roots of rhubarb. A recent study showed that rhein can inhibit tumor cell proliferation and induce apoptosis in human tumor cells. However, the clinical application of rhein has been hampered by its poor bioavailability, low aqueous solubility and gastrointestinal disorders. In current study, twenty-four target compounds were designed and synthesized by coupling various hydrophilic alkanolamines to the 2-carboxyl of rhein, and their structures were established by IR, HR-MS, 1H NMR spectra. Solubility test showed that all compounds were 10.04 to 15.08 mg·mL-1 in water, which was 220 to 330-fold better than that of rhein (0.045 6 mg·mL-1). All of rhein derivatives displayed more potent anti-tumor activity than rhein, and most of them were comparable to adriamycin, particularly, compound 4t exhibited IC50 value of 2.08 μmol·L-1, more effective than adriamycin (IC50=2.35 μmol·L-1). Hydroxyapatite adsorption experiment suggests that compound 4t has a better bone affinity than that of tetracycline.

Selective and effective targeting of chronic myeloid leukemia stem cells by topoisomerase II inhibitor etoposide in combination with imatinib mesylate in vitro.

Imatinib mesylate (IM) and other BCR-ABL tyrosine kinase inhibitors (TKIs) have improved chronic myeloid leukemia (CML) patient survival markedly but fail to eradicate quiescent CML leukemia stem cells (LSCs). Thus, strategies targeting LSCs are required to induce long-term remission and achieve cure. Here, we investigated the ability of topoisomerase II (Top II) inhibitor etoposide (Eto) to target CML LSCs. Treatment with Eto combined with IM markedly induced apoptosis in primitive CML CD34+ CD38- stem cells resistant to eradication by IM alone, but not in normal hematopoietic stem cells, CML and normal mature CD34- cells, and other leukemia and lymphoma cell lines. The interaction of IM and Eto significantly inhibited phosphorylation of PDK1, AKT, GSK3, S6, and ERK proteins; increased the expression of pro-apoptotic gene Bax; and decreased the expression of anti-apoptotic gene c-Myc in CML CD34+ cells. Top II inhibitors treatment represents an attractive approach for targeting LSCs in CML patients undergoing TKIs monotherapy.

Silencing of miR-21 sensitizes CML CD34+ stem/progenitor cells to imatinib-induced apoptosis by blocking PI3K/AKT pathway.

BCR-ABL tyrosine kinase inhibitor imatinib fails to eradicate leukemia stem cells (LSCs), the underlying mechanisms maintaining CML LSCs remain poorly understood. Here, we showed that transient inhibition of miR-21 by antagomiR-21 markedly increased imatinib-induced apoptosis in CML, but not normal CD34+ stem/progenitor cells. Furthermore, PI3K inhibitors also significantly sensitized CML CD34+ cells to imatinib-induced apoptosis. MiR-21 or PI3K inhibitor in combination with imatinib treatment significantly decreased AKT phosphorylation and c-Myc expression than either agent did alone, but did not affect Bim and Bcl-6 expresssion. These findings indicate that miR-21 is required for maintaining the imatinib-resistant phenotype of CML CD34+ cells through PI3K/AKT signaling pathway, thus providing the basis for a promising therapeutic approach to eliminate CML LSCs.

Targeting miR-21 sensitizes Ph+ ALL Sup-b15 cells to imatinib-induced apoptosis through upregulation of PTEN.

Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) cells are insensitive to BCR-ABL tyrosine kinase inhibitor imatinib, the underlying mechanisms remain largely unknown. Here, we showed that imatinib treatment induced significant upregulation of miR-21 and downregulation of PTEN in Ph+ ALL cell line Sup-b15. Transient inhibition of miR-21 resulted in increased apoptosis, PTEN upregulation and AKT dephosphorylation, whereas ectopic overexpression of miR-21 further conferred imatinib resistance. Furthermore, knockdown of PTEN protected the cells from imatinib-induced apoptosis achieved by inhibition of miR-21. Additionally, PI3K inhibitors also notably enhanced the effects of imatinib on Sup-b15 cells and primary Ph+ ALL cells similar to miR-21 inhibitor. Therefore, miR-21 contributes to imatinib resistance in Ph+ ALL cells and antagonizing miR-21 demonstrates therapeutic potential by sensitizing the malignancy to imatinib therapy.

[Gleevec induces apoptosis in K562 cells through activating caspase-3].

The present study is to elucidate the mechanisms underlying Gleevec-induced apoptosis of chronic myeloid leukemia (CML) K562 cells in vitro. The apoptotic cell death and cell cycle distribution after Gleevec treatment and the effect of PDCD4 siRNA on Gleevec-induced apoptosis of K562 cells were analyzed by flow cytometry. The effect of Gleevec on p-Crkl, caspase-3, PARP and PDCD4 protein levels, and the knockdown efficacy of PDCD4 siRNA were detected by Western blotting. The results showed that Gleevec dramatically suppressed the phosphorylation level of Crkl in a dose-dependent manner and induced significant apoptosis and G0/G1 cell cycle arrest of K562 cells in time- and dose-dependent manners. In addition, Gleevec activated caspase-3 and its downstream substrates PARP, and the caspase pan inhibitor Z-VAD-FMK (50 micromol x L(-1)) markedly reduced Gleevec-induced apoptosis from 47.97% +/- 10.56% to 31.05% +/- 9.206% (P < 0.05). Moreover, Gleevec significantly increased the protein expression of programmed cell death 4 (PDCD4). PDCD4 knockdown by siRNA reduced Gleevec-induced apoptosis from 46.97% +/- 14.32% to 42.8% +/- 11.43%. In summary, Gleevec induced apoptosis in K562 cells via caspase-3 activation.

Curcumin induces FasL-related apoptosis through p38 activation in human hepatocellular carcinoma Huh7 cells.

AIM: The aim of this study is to explore the underlying molecular mechanism of curcumin-induced apoptosis in human hepatocellular carcinoma (HCC) Huh7 cells. MAIN METHODS: Fas and FasL mRNA expression was analyzed by reverse transcription PCR. Western blot was applied to detect the protein expression of Bcl-2 family members, MAPK family members, c-Jun, c-Fos, ATF-2, caspase-3, PARP, TNF receptor family members and the respective ligands. Apoptotic cells were assayed with annexin V/PI double staining and flow cytometry. KEY FINDINGS: Curcumin treatment resulted in a fast and significant increase of Fas and Fas ligand (FasL) along with activation of caspase-3 and cleavage of PARP in Huh7 cells. Inhibition of caspase-3 activity by the specific inhibitor Z-DEVD-FMK rescued Huh7 cells from curcumin-induced apoptosis. Neutralization of FasL significantly protected the cells from curcumin-induced caspase-3 activation and apoptosis in a dose-dependent manner. Moreover, p38 was rapidly activated in response to curcumin, and inactivation of p38 by pharmacologic inhibitor SB203580 dramatically suppressed curcumin-induced FasL expression and apoptosis. SIGNIFICANCE: Our results demonstrated that curcumin induces apoptosis through p38-denpendent up-regulation of FasL in Huh7 cells.

Imidazo [4,5f][1,10] phenanthroline derivatives as inhibitor of c-myc gene expression in A549 cells via NF-κB pathway.

1,10-Phenanthroline has been shown to exhibit anticancer activity. Here, a series of imidazo [4,5f][1,10] phenanthroline derivatives 1-10 were synthesized and their biological activities were further elucidated. We found that 2-(4-Brominephenyl)-imidazo [4,5f][1,10] phenanthroline (compound 3) possessed potent antiproliferation activities again a variety of tumor cell lines using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Flow cytometric analysis revealed that compound 3 induced both through apoptosis and necrosis in human lung adenocarcinoma cell line, A549. Moreover, compound 3 treatment led to up-regulation of IκBα and down-regulation of p65 and c-myc in A549 cells. Taken together, these results suggested that compound 3 inhibited cell proliferation by suppression of NF-κB activity and down-regulation of c-myc gene expression and may be a candidate for further evaluation as a chemopreventive and chemotherapeutic agent for human cancers, especially for lung cancer.

Study on the anti-wear performance of Ni-base composite coating sucker joint that contains nano-diamond and nano-polytetrafluoroethylene.

With the development of oilfields, the problem of eccentric wear between casing and sucker rod in rod-pumped wells operation is more and more severe. Investigations on the eccentric wear show that the abrasion of sucker rod joint is more serious than the sucker rod itself. A new method of producing the Ni-base composite coating that contains nano-diamond and nano-polytetrafluoroethylene (PTFE) on sucker joint obtained by electrodeposition is presented in this paper. The test results show that the anti-wear performance and hardness of the sucker rod improve significantly with the increase of nano-diamond. The addition of nano-PTFE particle is useful in reducing the friction factor. Field tests demonstrate that the life of the sucker rod joint is increased and the maintenance cycle of the rod-pumped well is prolonged.

[Effect of p21-targeted shRNA on curcumin-induced apoptosis of human hepatoma Huh7 cells].

In the present study, shRNA plasmid of pSi-p21 targeting p21 mRNA was constructed and the effect of p21 shRNA on curcumin-induced apoptosis of human hepatoma Huh7 cells was investigated. The effect of curcumin on the expression of p21 mRNA and protein and the silence efficiency of pSi-p21 were detected with RT-PCR and Western blotting. The effect of pSi-p21 on curcumin-induced apoptosis of Huh7 cells was evaluated with DAPI staining. The results showed that curcumin significantly upregulated p21 mRNA and protein expression, which was knocked down by pSi-p21 of Huh7 cells. DAPI staining results showed that pSi-p21 significantly decreased curcumin-induced apoptosis of Huh7 cells. The data suggested that curcumin induced apoptosis of Huh7 cells via upregulation of p21 expression.

[Effect of curcumin on JAK-STAT signaling pathway in hepatoma cell lines].

The effect of curcumin on JAK-STAT signaling pathway was investigated in hepatoma cell lines Huh7 and Hep3B. Curcumin inhibited cell proliferation and induced apoptosis of both cell lines, but Huh7 cells were more sensitive to curcumin than Hep3B cells. Curcumin (50 micromol x L(-1)) significantly increased phosphorylations of p38 (T180/Y182) and STAT-1 (S727) in Huh7 and Hep3B cells, and caused relocalization of phosphorylated-STAT-1 (Y701) from cytoplasm to nucleus in Hep3B cells. In addition, curcumin (25 and 50 micromol x L(-1)) dramatically suppressed the phosphorylation level of STAT-1 (Y701) and resulted in a significant reduction of nuclear phosphorylated-STAT-1 (Y701) in Huh7 cells.

Abrogation of G2/M arrest sensitizes curcumin-resistant hepatoma cells to apoptosis.

In this study, we showed that curcumin treatment resulted in activation of Chk1-mediated G2 checkpoint, which was associated with the induction of G2/M arrest and the resistance of cancer cells to curcumin-induced apoptosis. Further investigation revealed that inhibition of Chk1 significantly abrogated G2/M arrest and sensitized curcumin-resistant cells to apoptosis via upregulation of Bad and in turn the loss of mitochondrial membrane potential. These results indicate that Chk1-mediated G2/M arrest may serve as a mechanism for curcumin resistance and Chk1 represents a potential target for the reversal of this resistance. Our findings should be helpful for clinical application of curcumin.