[Decitabine Enhances the Sensitivity of Leukemia Stem Cell to Allo-NK Cell-Mediated Killing].

OBJECTIVE: To investigate the allo-NK cell-mediated killing effect enhanced by decitabine on leukemia stem cells(LSC) and the underlying mechanisms. METHODS: LSC were separated from KG1a cells by using immunomagnetic beads. Allo-NK cells were isolated and purified from PBMC of healthy donors. Cytotoxicity of allo-NK cells against LSC were measured by LDH releasing assay. The apoptosis induced by allo-NK cells in LSC and the expressions of NKG2D ligands including MICA/B and ULBP1-3 on LSC were detected by flow cytometry. RESULTS: The killing rate of allo-NK cells to LSC treated with 10 µmol/L decitabine for 24 hours was significant higher than that to LSC without treatment(60.52%±3.52% vs 22.08%±2.07%, 73.93%±2.33% vs 28. 99%±3.13%, 83.08%±1.32% vs 36.44%±2.40%, respectively)at the effector-target ratios of 5:1, 10:1, 20:1 (P<0.05). At the effector-target ratio of 10:1, decitabine significantly enhanced the apoptosis of LSC induced by allo-NK cells (7.84%±0.34% vs 3.33%±0.64%)(P<0.05). The expressions of NKG2D ligands(MICA/B,ULBP1,ULBP2,ULBP3) on LSC treated with decitabine 10 µmol/L for 24 hours were significantly increased (P<0.05). CONCLUSION: Decitabine may enhance the allo-NK cell-mediated killing effects on LSC by up-regulation of the expressions of NKG2D ligands on LSC.

Manumycin induces apoptosis in prostate cancer cells.

BACKGROUND: Manumycin exhibits an antitumor effect in a variety of cancer cell lines, including prostate cancer cell lines (DU145 and PC-3). Our previous studies demonstrated that manumycin induced the apoptosis of anaplastic thyroid cancer cells and leukemia cells via the intrinsic apoptosis pathway. In the current study, we further evaluated the effect of manumycin in two prostate cancer cell lines (LNCaP and 22Rv1), and here we elucidate some of the underlying mechanisms. MATERIALS AND METHODS: The cell viability of prostate cancer cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after treatment with manumycin for 48 hours. Apoptosis was detected by flow cytometry using annexin V and propidium iodide. The expressions of B-cell lymphoma (Bcl)-2 family members and the activations of caspase-9 and caspase-3 were detected by Western blotting. RESULTS: Manumycin treatment resulted in significant decreases in the viabilities of the two prostate cancer cell lines in a dose-dependent manner through apoptosis, and this apoptosis involved caspase-9 activation. A specific inhibitor of caspase-9 protected cells from caspase-3 activation, apoptosis, and cytotoxicity induced by manumycin. We also found that manumycin downregulated Bcl-2 expression and upregulated Bax expression. CONCLUSION: Our data suggest that manumycin induces apoptosis in prostate cancer cells through regulation of the Bcl-2 family involving caspase-9 activation. These results suggest that manumycin may be beneficial for the treatment of prostate cancer.

[Drug resistance of leukemic stem cells mediated by hedgehog signaling pathway].

Drug resistance and relapse are the major challenge for current treatment of acute leukemia. It is critical for ultimately curing leukemia to overcome chemoresistance of leukemic stem cells (LSC) and to eradicate LSC. Recent studies have found that abnormal activated Hedgehog (HH) signaling pathway plays an important role in a wide variety of tumors and regulates multi-drug resistance of LSC. This review briefly summarizes the molecular mechanism of HH signal pathway inducing drug resistance of LSC and leading to novel strategies for eradicating LSC.

Expression of sonic hedgehog signaling components in hepatocellular carcinoma and cyclopamine-induced apoptosis through Bcl-2 downregulation in vitro.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Aberrant activation of sonic hedgehog (Shh) signaling pathway plays important roles in tumorigenesis and progression of several tumors. Cyclopamine, an important inhibitor of Shh signaling pathway, can induce cell apoptosis. However, the mechanisms underlying cyclopamine-induced apoptosis are not well understood. The aim of this study is to determine the expression of the Shh signaling pathway components in HCC and to investigate the mechanisms underlying cyclopamine-induced apoptosis in HCC cells. METHODS: Shh signaling components (Shh, Ptch, Smo and Gli-1) expression levels were evaluated by immunohistochemistry on tissue microarrays containing 98 HCCs with paired adjacent noncancerous liver tissues. The relationships between sonic hedgehog signal pathway and clinicopathological factors were analyzed in HCC. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis was detected by flow cytometry. mRNA and protein levels were analyzed by RT-PCR and Western blot, respectively. RESULTS: Shh, Ptch, Smo and Gli-1 were overexpressed in HCC tissues compared with paired adjacent noncancerous liver tissue. Activated Shh signaling pathway was associated with tumor size, capsular invasion and vascular invasion in HCC. Cyclopamine remarkably decreased cell viability, induced apoptosis and downregulated Bcl-2 expression in HCC cells. CONCLUSIONS: Shh signaling pathway plays an important role in HCC tumorigenesis and progression, indicating that Shh signaling pathway is a potential therapeutic target for HCC. Cyclopamine induces apoptosis through downregulating Bcl-2 in HCC.

[Effects of manumycin combined with methoxyamine on apoptosis in myeloid leukemia U937 cells].

BACKGROUND & OBJECTIVE: Repair of DNA damage is important to cell survival. Our previous study showed DNA damage response induced by manumycin in cancer cells. We hypothesized that methoxyamine, an inhibitor of base-excision repair, can enhance the antineoplastic effect of manumycin. This study was to investigate apoptosis induced by manumycin combined with methoxyamine in myeloid leukemia cell line U937, and to explore the role of mitochondrial apoptotic pathway in apoptosis induction of the two drugs. METHODS: U937 cells were treated with various concentrations of manumycin and/or methoxyamine for 48 h. The cell viability was analyzed by MTT assay. Colony formation was evaluated by soft agar clonogenic assay. Cell apoptosis was investigated by flow cytometry. Protein expressions of cytochrome c, caspase-9, and poly ADP-ribose polymerase (PARP) were determined by Western blot. RESULTS: The dose-response curve of manumycin was shifted to the left after addition of methoxyamine. The combination index (CI) was less than 1 (P<0.05) in U937 cells (P<0.05), indicating a synergistic effect of manumycin and methoxyamine. Rates of colony formation of U937 cells treated with 1 micromol/L manumycin, or 5 mmol/L methoxyamine, or the combination of the two were 0.3641+/-0.0463, 0.7541+/-0.0379, and 0.0473+/-0.0024, respectively compared with that of control cells (P<0.05). Moreover, the drug combination resulted in enhanced apoptosis in U937 cells. The apoptotic rates of the control, manumycin, methoxyamine and combination group were (2.34+/-0.30)%, (8.80+/-0.95)%, (2.21+/-0.19)%, and (13.37+/-0.91)%, respectively. The combination of manumycin with methoxyamine also promoted the release of cytochrome c from mitochondria into the cytosol, activated caspase-9, and led appearance of specific cleavage of PARP in U937 cells. CONCLUSION: Methoxyamine enhances manumycin-induced apoptosis in U937 myeloid leukemia cells.

[Cytotoxicity of allogenetic natural killer cells against CD34+ acute myelogenous leukemia cells].

OBJECTIVE: To study the cytotoxic effect of allogenetic natural killer (NK) cells in vitro on human CD34+ acute myelogenous leukemia cells. METHODS: CD34 expression on acute myelogenous leukemia KG1a cells was detected by flow cytometry. KG1a cells were co-cultured at different effector-to-target (E:T) ratios with NK cells isolated from 5 healthy individuals using magnetic cell sorting. Lactate dehydrogenase (LDH) release assay was employed to examine the cytolysis of KG1a cells in the co-culture, and the inhibition rate of the KG1a cell colony formation in methylcellulose was determined with K562 cells sensitive to NK cells as the control. RESULTS: A expression rate as much as (98.0-/+1.1)% was detected for CD34 antigen on KG1a cells, and the isolated NK cells (CD3(-)CD16+CD56+ cells) had a purity of (93.2-/+3.7)% after magnetic cell sorting. Allogenetic NK cells exhibited obvious cytotoxicity and colony inhibition in vitro against KG1a cells at different E:T ratios, and the effects were significantly enhanced as the E:T ratios increased (P<0.05). At the same E:T ratio, the cytotoxicity and colony inhibition rate of allogenetic NK cells against KG1a cells was lower than those against K562 cells (P<0.05). CONCLUSION: Allogenetic NK cells exhibit obvious cytotoxicity and colony formation against CD34+ acute myelogenous leukemia cells.

Gli-1 siRNA induced apoptosis in Huh7 cells.

AIM: To investigate the effects of Gli-1 small interference RNA (siRNA) on Huh7 cells, and the change of Bcl-2 expression in Huh7 cells. METHODS: Human hepatocellular carcinoma cells Huh7 were used. Cell viability was analyzed by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The expressions of Gli-1 and Bcl-2 family members were detected by RT-PCR and Western blot. Apoptosis was detected by Flow cytometry using propidium iodide, measured by Hoechst 33258 staining using Advanced Fluorescence Microscopy and caspase-3 enzymatic assay. Cell growth was analyzed after treatment with Gli-1 siRNA and 5-fluorouracil (5-Fu). RESULTS: Inhibition of Gli-1 mRNA in Huh7 cells through Gli-1 siRNA reduced cell viability. Gli-1 siRNA treatment also induced apoptosis by three criteria, increase in the sub-G1 cell cycle fraction, nuclear condensation, a morphologic change typical of apoptosis, and activation of caspase-3. Gli-1 siRNA was also able to down-regulate Bcl-2. However, Gli-1 siRNA resulted in no significant changes in Bcl-xl, Bax, Bad, and Bid. Furthermore, Gli-1 siRNA increased the cytotoxic effect of 5-Fu on Huh7 cell. CONCLUSION: Down-regulation of Bcl-2 plays an important role in apoptosis induced by Gli-1 siRNA in HCC cells. Combination Gli-1 siRNA with chemotherapeutic drug could represent a more promising strategy against HCC. The effects of the strategies need further investigation in vivo and may have potential clinical application.

[Involvement of mitochondria apoptotic pathway in the manumycin inducing apoptosis of U937 and HL-60].

OBJECTIVE: To investigate the apoptosis induced by manumycin in U937 and HL-60 cell lines, and to explore the role of mitochondria apoptotic pathway in manumycin-inducing apoptosis. METHODS: Leukemic cells line U937 and HL-60 were treated by manumycin at 2 micromol/L for different time. Apoptosis of leukemia cells was detected by flow cytometry. The cytosolic proteins were extracted using a digitonin buffer. The protein expression of cytochrome C, caspase-9, caspase-8, and caspase-3 were determined by western blot. Mitochondrial membrane potential was detected by JC-1. RESULTS: In U937 and HL-60 cells, manumycin induced mitochondrial depolarization after 6 h treatment. The average red/green fluorescence ratios at 6 h were significantly (P < 0.01) lower than those at time 0, being 0.51 +/- 0.07 and 0.41 +/- 0.06 for control group respectively. Manumycin induced cytochrome C release from the mitochondria into the cytosol after 6 h treatment, and activated caspase-9, caspase-8, and caspase-3 after a 16h treatment. The broad-spectrum caspase-inhibitor Z-VAD-fmk at 50 micromol/L was able to inhibit caspase cleavage completely, but only reduced the manumycin-induced apoptosis rates by 51.69% and 56.47% in U937 and HL-60, respectively. CONCLUSION: Manumycin induced apoptosis in U937 and HL-60 cell lines via mitochondria apoptotic pathway.

Requirement of reactive oxygen species generation in apoptosis of leukemia cells induced by 2-methoxyestradiol.

AIM: To investigate the effects of 2-methoxyestradiol (2-ME) on 2 myeloid leukemia cell lines HL-60 and U937, and to explore its mechanisms. METHODS: Human myeloid leukemia cells HL-60 and U937 were used. Measurement of mitochondrial membrane potential (Dym) was performed using 5,5',6,6'-Tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide ( JC-1). Apoptosis and cellular nitric oxide (NO) were detected by flow cytometry using Annexin V and NO sensor dye. Superoxide anion was measured with a fluorescent plate reader by dihydroethidium (DHE). Cytotoxicity was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay. RESULTS: 2-ME resulted in viability decrease in a dose-dependent manner. 2-ME treatment also generated reactive oxygen species (ROS), including NO and superoxide anions, which resulted in mitochondria damage. 2-ME-induced apoptosis was correlated with an increase in ROS. The quenching of ROS with N-acetyl-L-cysteine protected leukemia cells from 2-ME cytotoxicity and prevented apoptosis induction by 2-ME. Furthermore, the addition of manumycin, a farnesyltransferase inhibitor, significantly enhanced apoptosis induced by 2-ME. CONCLUSION: Cellular ROS generation plays an important role in the cytotoxic effect of 2-ME. It is possible to use ROS generation agents, such as manumycin, to enhance the antileukemic effect. The combination strategy needs further in vivo justification and may have potential clinical application.