Yi Zhang

Endothelial progenitor cells (EPCs) have the ability to differentiate into mature endothelial cells. Inhibition of EPC proliferation and migration may be a new method for anti-tumor therapy. Medroxyprogesterone acetate (MPA) may act on tumor angiogenesis by impacting biological functions of EPCs. The aim of this work was to study the effect of different concentrations of MPA combined with 17β-estradiol (17β-E2) on proliferation, migration, and apoptosis of EPCs in vitro. Proliferation tests (MTT analysis) and migration assay of EPCs, isolated from bone marrow of canine, were performed to detect their response to different concentrations of MPA combined with 17β-E2 (1 × 10(-8) mol/L). The growth curves were drawn every 24 h for 7 consecutive days. The cell cycle and apoptosis of EPCs were analyzed by flow cytometry. 17β-E2 (1 × 10(-8) mol/L) increased EPC proliferation, while lower concentration of MPA (≤ 10(-5) mol/L) partially inhibited it. The higher concentration of MPA (≥ 10(-4) mol/L) combined with 17β-E2 had a significant inhibitory effect on EPC growth, arresting it in the S phase. It also increased the apoptosis rate and damaged the migration ability of EPCs. Low concentration of MPA partially inhibited the function of 17β-E2 that promotes the proliferation of EPCs. However, high concentration of MPA combined with 17β-E2 inhibited a variety of biological functions of EPCs. So, the MPA has a bidirectional effect combined with 17β-E2 on the cell biology of EPCs.

The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. ECs are constantly subjected to shear stress resulting from blood flow and are able to convert mechanical stimuli into intracellular signals that affect cellular behaviors and functions. The aim of this study is to elucidate the effects of Rac1, which is the member of small G protein family, on EC migration under different laminar shear stress (5.56, 10.02, and 15.27 dyn/cm2). The cell migration distance under laminar shear stress increased significantly than that under the static culture condition. Especially, under relative high shear stress (15.27 dyn/cm2) there was a higher difference at 8 h (P < 0.01) and 2 h (P < 0.05) compared with static controls. RT-PCR results further showed increasing mRNA expression of Rac1 in ECs exposed to laminar shear stress than that exposed to static culture. Using plasmids encoding the wild-type (WT), an activated mutant (Q61L), and a dominant-negative mutant (T17N), plasmids encoding Rac1 were transfected into EA.hy 926 cells. The average net migration distance of Rac1Q61L group increased significantly, while Rac1T17N group decreased significantly in comparison with the static controls. These results indicated that Rac1 mediated shear stress-induced EC migration. Our findings conduce to elucidate the molecular mechanisms of EC migration induced by shear stress, which is expected to understand the pathophysiological basis of wound healing in health and diseases.

Endoplasmic reticulum (ER) stress induces both autophagy and apoptosis yet the molecular mechanisms and pathways underlying the regulation of these two cellular processes in cells undergoing ER stress remain less clear. We report here that eukaryotic elongation factor-2 kinase (EEF2K) is a critical controller of the ER stress-induced autophagy and apoptosis in tumor cells. DDIT4, a stress-induced protein, was required for transducing the signal for activation of EEF2K under ER stress. We further showed that phosphorylation of EEF2K at Ser398 was essential for induction of autophagy, while phosphorylation of the kinase at Ser366 and Ser78 exerted an inhibitory effect on autophagy. Suppression of the ER stress-activated autophagy via silencing of EEF2K aggravated ER stress and promoted apoptotic cell death in tumor cells. Moreover, inhibiting EEF2K by either RNAi or NH125, a small molecule inhibitor of the enzyme, rendered tumor cells more sensitive to curcumin and velcade, two anticancer agents that possess ER stress-inducing action. Our study indicated that the DDIT4-EEF2K pathway was essential for inducing autophagy and for determining the fate of tumor cells under ER stress, and suggested that inhibiting the EEF2K-mediated autophagy can deteriorate ER stress and lead to a greater apoptotic response, thereby potentiating the efficacy of the ER stress-inducing agents against cancer.

BACKGROUND: Intense nanosecond pulsed electric fields (nsPEFs) have been known to promote apoptosis without physically changing membrane structure or damaging morphology of tumor cells. To determine the contribution of centrosome to the progression of apoptosis by nsPEFs, HeLa cells were exposed to high intensity (6 kV/cm) nsPEFs (8-32 ns) in normal culture condition and cell biology and molecular parameters of cells were investigated. MATERIALS AND METHODS: Apoptotic cell death was identified by TUNEL assay after being exposed to the nsPEFs with various pulse durations, while immunofluorescent staining was performed to detect the number and distribution of centrosomes. To clarify whether nsPEFs-induced centrosome over-duplication is the consequence of DNA damage, we used comet assay to detect simultaneous DNA damage. And additionally Western Blot was used to detect PLK1 protein level to explore the correlation between apoptotic cell death and nsPEFs-induced centrosome over-duplication. Correlation between nsPEFs and molecular parameters was statistically analyzed. RESULTS: NsPEFs induced a clear apoptosis reaching a maximum at 24ns, 24h after exposure (p < 0.05), where DNA fragmentation and over-duplicated centrosomes were observed. This apoptosis may be promoted in a time- and pulse duration-dependent manner. Polo-like kinase (PLK1) protein levels were significantly decreased by such nsPEFs (p < 0.05). Control treatment without the nsPEFs did not cause any damage to the cultured HeLa cells. CONCLUSIONS: Intense nsPEFs promote cell apoptosis through a centrosome-mediated pathway involving a reduction in the level of PLK1, which may provide new therapeutic targets for human cancer treatment.

Our recent study revealed a new role of nucleus accumbens-1 (NAC1), a transcription factor belonging to the BTB/POZ gene family, in regulating autophagy. Moreover, we found that the high-mobility group box 1 (HMGB1), a chromatin-associated nuclear protein acting as an extracellular damage associated molecular pattern molecule (DAMP), is the downstream executor of NAC1 in modulating autophagy. In response to stress such as therapeutic insults, NAC1 increases the expression, cytosolic translocation and release of HMGB1; elevated level of the cytoplasmic HMGB1 leads to activation of autophagy. The NAC1-HMGB1 partnership may represent a previously unrecognized pathway that regulates autophagy in response to various stresses such as chemotherapy.