Eps8 vaccine exerts prophylactic antitumor effects in a murine model: a novel vaccine for breast carcinoma.

Cancer vaccines are an effective way to prevent the occurrence of cancer. Epidermal growth factor receptor pathway substrate 8 (Eps8) is a novel tumor-associated antigen, which is overexpressed in the majority of tumor types. In the present study, the Eps8 protein was cloned and characterized, and its feasibility as an antitumor agent in murine breast carcinoma was investigated. The results revealed that the Eps8 protein increased the secretion of interleukin (IL)-12 in the culture supernatant of dendritic cells (DCs). The Eps8 protein­pulsed DCs induced significant cytotoxic T lymphocyte (CTL) responses, T-cell proliferation and a higher level of interferon (IFN)-γ in the culture supernatant of the splenocytes ex vivo. Additionally, when the mice were immunized with the Eps8 vaccine, this resulted in a regression of 4T1 breast tumors and significantly prolonged survival time in the tumor­bearing mice compared with that in the phosphate-buffered saline (PBS) control group. The Eps8 vaccine induced higher CTL responses in the splenocytes of mice vaccinated against the 4T1 cells; the ratio of CD4+/CD8+ T cells was increased in the Eps8 group; and the percentage of CD4+CD25+ FoxP3+ regulatory T (Treg) cells in the Eps8 group was significantly lower compared with that of the PBS group. The results suggested that the Eps8 vaccine was able to stimulate antitumor effects against 4T1 breast cancer cells in vitro and in vivo, and it may provide a potential immunotherapeutic agent for the treatment of breast cancer.

[NVP-BEZ235 inhibits proliferation and colony-forming capability of CD34(+)CD38(-) human acute myeloid leukemia stem cells].

This study was aimed to explore the effect of NVP-BEZ235, a dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor, on proliferation, cell cycle and colony forming capability of CD34(+)CD38(-) human acute myeloid leukemia (AML) KG1a cells. Flow cytometry was used to detect expression of CD34 and CD38 on the surface of human AML KG1a cells; Trypan blue assay was used to analyze the effect of NVP-BEZ235 at various concentrations on proliferation of KG1a cells; flow cytometry was performed to examine the cell cycle of KG1a cells after NVP-BEZ235 treatment; Soft agar colony-forming experiment was used to detect the colony forming ability of KG1a cells treated with NVP-BEZ235 at various concentrations. The results indicated that the percentage of CD34(+)CD38(-) AML KG1a cells was (98.02 ± 0.72)%. NVP-BEZ235 (0.125 - 1 µmol/L) inhibited the proliferation of KG1a cells in a time-and dose-dependent manner (P < 0.05) and the 50% inhibition concentrations (IC50) at 24 h and 48 h were 0.597 µmol/L and 0.102 µmol/L, respectively. KG1a cells were arrested at G0/G1 phase after treating with 0.5 µmol/L NVP-BEZ235 for 24 h, it was significantly higher than that of control group (83.2 ± 3.80)% vs (43.47 ± 9.60)% (P < 0.05). KG1a cells treated with NVP-BEZ235 (0 - 1 µmol/L) for 14 d and 21 d, the number of colony decreased respectively from (375.67 ± 21.46) per 2500 KG1a cells and (706.33 ± 87.31) per 2500 KG1a cells to 0, with statistical significance (P < 0.05). It is concluded that NVP-BEZ235 can inhibit proliferation and colony-forming capability of CD34(+)CD38(-) human AML KG1a cells.

[Expression of NKG2D ligands on dendritic cells at different development stages and its effect on cytotoxicity of NK cells].

AIM: To investigate the expression of NKG2D ligands on dendritic cells(DC) at different development stages and its effect on cytotoxicity of natural killer(NK) cells. METHODS: The monocytes were cultured into immature dendritic cells(iDC) and mature dendritic cells(mDC) with cytokines. NK cells were obtained from normal peripheral blood by CD56 antibody magnetic isolation.The expression of NKG2D ligands (MICA/B, ULBP1-3) was detected by flow cytometry. Cytotoxicity of NK cells and the NK cells blocked with anti-NKG2D mAbs against iDC and mDC was tested using LDH-releasing method. RESULTS: IDC and mDC were of typical morphology and phenotypes. MICA, MICB, ULBP1, and ULBP3 were expressed on iDC and their expression rate was (32.39+/-8.30)%, (17.75+/-3.40)%, (26.71+/-6.48)%, (38.37+/-6.89)%, respectively. MICA and ULBP3 were expressed on mDC and their expression rate was (7.81+/-3.33)% and (8.36+/-2.42)%, respectively, which was lower than that on mDC (P<0.01). At the each E:T ratio cytotoxicity of NK cells against iDC was stronger than that against mDC (P<0.01). cytotoxicity of NK cells blocked with anti-NKG2D mAb against iDC was decreased compared with that of NK cells unblocked (P<0.05) while cytotoxicity of NK cells blocked with anti-NKG2D mAb against mDC showed no decrease compared with that of NK cells unblocked (P>0.05). CONCLUSION: The expression of NKG2D ligands on iDC is higher than that on mDC, which plays an important role in the cytotoxic effect of NK cells against iDC, but has no effect on that against mDC. NKG2D-NKG2D ligands shows one of the molecular mechanisms that NK cells kill iDC selectively.

[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.

Angiogenic potential difference between two types of endothelial progenitor cells from human umbilical cord blood.

The hierarchy of endothelial progenitor cells (EPCs) in human umbilical cord blood has been disclosed. In this study we compare, for the first time, the angiogenic potential difference between two types of EPCs. We cultured mononuclear cells (MNCs) isolated from human umbilical cord blood using endothelial cell-conditioned medium and obtained two types of EPCs, referred to as circulating angiogenic cells (CACs) and high proliferative potential endothelial progenitor cells (HPP-EPCs). Both types of cells possess characteristics of EPCs, including expressing CD31, VE-cadherin, KDR and von Willebrand factor, uptake of Ac-LDL and binding to lectin. However, unlike CACs, which express CD14 but not CD133, HPP-EPCs express CD133 but not CD14. Also, unlike CACs, HPP-EPCs display stronger proliferation and clonogenic potential in vitro and show stronger ability to promote vascular growth in the hind-limb model of ischemia in mice (BALB/C-nu) in vivo.