zhen Guo

IONIZING RADIATION PROMOTES MIGRATION AND INVASION OF CANCER CELLS THROUGH TRANSFORMING GROWTH FACTOR-BETA-MEDIATED EPITHELIAL-MESENCHYMAL TRANSITION

PURPOSE To examine whether ionizing radiation enhances the migratory and invasive abilities of cancer cells through transforming growth factor (TGF-β)-mediated epithelial-mesenchymal transition (EMT). METHODS AND MATERIALS Six cancer cell lines originating from different human organs were irradiated by 60Co γ-ray at a total dose of 2 Gy, and the changes associated with EMT, including morphology, EMT markers, migration and invasion, were observed by microscope, Western blot, immunofluorescence, scratch assay, and transwell chamber assay, respectively. Then the protein levels of TGF-β in these cancer cells were detected by enzyme-linked immunosorbent assay, and the role of TGF-β signaling pathway in the effect of ionizing radiation on EMT was investigate by using the specific inhibitor SB431542. RESULTS After irradiation with γ-ray at a total dose of 2 Gy, cancer cells presented the mesenchymal phenotype, and compared with the sham-irradiation group the expression of epithelial markers was decreased and of mesenchymal markers was increased, the migratory and invasive capabilities were strengthened, and the protein levels of TGF-β were enhanced. Furthermore, events associated with EMT induced by IR in A549 could be reversed through inhibition of TGF-β signaling. CONCLUSIONS These results suggest that EMT mediated by TGF-β plays a critical role in IR-induced enhancing of migratory and invasive capabilities in cancer cells.

HIF-1 and NDRG2 contribute to hypoxia-induced radioresistance of cervical cancer Hela cells

Hypoxia inducible factor 1 (HIF-1), the key mediator of hypoxia signaling pathways, has been shown involved in hypoxia-induced radioresistance. However, the underlying mechanisms are unclear. The present study demonstrated that both hypoxia and hypoxia mimetic cobalt chloride could increase the radioresistance of human cervical cancer Hela cells. Meanwhile, ectopic expression of HIF-1 could enhance the resistance of Hela cells to radiation, whereas knocking-down of HIF-1 could increase the sensitivity of Hela cells to radiation in the presence of hypoxia. N-Myc downstream-regulated gene 2 (NDRG2), a new HIF-1 target gene identified in our lab, was found to be upregulated by hypoxia and radiation in a HIF-1-dependent manner. Overexpression of NDRG2 resulted in decreased sensitivity of Hela cells to radiation while silencing NDRG2 led to radiosensitization. Moreover, NDRG2 was proved to protect Hela cells from radiation-induced apoptosis and abolish radiation-induced upregulation of Bax. Taken together, these data suggest that both HIF-1 and NDRG2 contribute to hypoxia-induced tumor radioresistance and that NDRG2 acts downstream of HIF-1 to promote radioresistance through suppressing radiation-induced Bax expression. It would be meaningful to further explore the clinical application potential of HIF-1 and NDRG2 blockade as radiosensitizer for tumor therapy.

EMP-induced alterations of tight junction protein expression and disruption of the blood-brain barrier.

The blood-brain barrier (BBB) is critical to maintain cerebral homeostasis. In this study, we examined the effects of exposure to electromagnetic pulse (EMP) on the functional integrity of BBB and, on the localization and expression of tight junction (TJ) proteins (occludin and ZO-1) in rats. Animals were sham or whole-body exposed to EMP at 200 kV/m for 400 pulses. The permeability of BBB in rat cerebral cortex was examined by using Evans Blue (EB) and lanthanum nitrate as vascular tracers. The localization and expression of TJ proteins were assessed by western blot and immunofluorescence analysis, respectively. The data indicated that EMP exposure caused: (i) increased permeability of BBB, and (ii) altered localization as well as decreased levels of TJ protein ZO-1. These results suggested that the alteration of ZO-1 may play an important role in the disruption of tight junctions, which may lead to dysfunction of BBB after EMP exposure.

Mechanisms involved in the blood–testis barrier increased permeability induced by EMP

The blood-testis barrier (BTB) plays an important role in male reproductive system. Lots of environmental stimulations can increase the permeability of BTB and then result in antisperm antibody (AsAb) generation, which is a key step in male immune infertility. Here we reported the results of male mice exposed to electromagnetic pulse (EMP) by measuring the expression of tight-junction-associated proteins (ZO-1 and Occludin), vimentin microfilaments, and transforming growth factor-beta (TGF-beta3) as well as AsAb level in serum. Male BALB/c mice were sham exposed or exposed to EMP at two different intensities (200kV/m and 400kV/m) for 200 pulses. The testes were collected at different time points after EMP exposure. Immunofluorescence histocytochemistry, western blotting, laser confocal microscopy and RT-PCR were used in this study. Compared with sham group, the expression of ZO-1 and TGF-beta3 significantly decreased accompanied with unevenly stained vimentin microfilaments and increased serum AsAb levels in EMP-exposed mice. These results suggest a potential BTB injury and immune infertility in male mice exposed to a certain intensity of EMP.

TCTP overexpression is associated with the development and progression of glioma

Upregulation of translationally controlled tumor protein (TCTP) has been reported in a variety of malignant tumors. However, the impact of TCTP in glioma remains unclear. The objective of this study was to investigate the expression and prognostic value of TCTP in glioma patients. Western blot analysis was used to characterize the expression patterns of TCTP in 45 glioma and 22 normal brain tissues. Immunohistochemistry on a tissue microarray containing 127 cases of glioma was performed to analyze the association between TCTP expression and clinicopathological features. Compared with normal brain tissues, TCTP expression was significantly higher in glioma tissues (p <0.001). In addition, high TCTP expression in glioma was significantly associated with advanced pathological grade (p = 0.018). Kaplan–Meier analysis showed that patients with glioma and higher TCTP expression tend to have shorter overall survival time (p <0.001). In multivariate analysis, TCTP expression was proved to be an independent prognostic factor for patients with glioma (p <0.001). In conclusion, this study confirmed the overexpression of TCTP and its association with tumor progression in glioma. It also provided the first evidence that TCTP expression in glioma was an independent prognostic factor of patients, which might be a potential diagnostic and therapeutic target of glioma.

Paclitaxel-loaded poly(D,L-lactide-co-glycolide) nanoparticles for radiotherapy in hypoxic human tumor cells in vitro

Radioresistant hypoxic cells may contribute to the failure of radiation therapy in controlling certain tumors. Some studies have suggested the radiosensitizing effect of paclitaxel. The poly(D,L-lactide-co-glycolide)(PLGA) nanoparticles containing paclitaxel were prepared by o/w emulsification-solvent evaporation method. The physicochemical characteristics of the nanoparticles (i.e. encapsulation efficiency, particle size distribution, morphology, in vitro release) were studied. The morphology of the two human tumor cell lines: a carcinoma cervicis (HeLa) and a hepatoma (HepG2), treated with paclitaxel-loaded nanoparticles was photomicrographed. Flow cytometry was used to quantify the number of the tumor cells held in the G2/M phase of the cell cycle. The cellular uptake of nanoparticles was evaluated by transmission electronic microscopy. Cell viability was determined by the ability of single cell to form colonies in vitro. The prepared nanoparticles were spherical in shape with size between 200nm and 800nm. The encapsulation efficiency was 85.5％. The release behaviour of paclitaxel from the nanoparticles exhibited a biphasic pattern characterised by a fast initial release during the first 24 h, followed by a slower and continuous release. Co-culture of the two tumor cell lines with paclitaxel-loaded nanoparticles demonstrated that the cell morphology was changed and the released paclitaxel retained its bioactivity to block cells in the G2/M phase. The cellular uptake of nanoparticles was observed. The free paclitaxel and paclitaxel-loaded nanoparticles effectively sensitized hypoxic HeLa and HepG2 cells to radiation. Under this experimental condition, the radiosensitization of paclitaxel-loaded nanoparticles was more significant than that of free paclitaxel.Keywords: Paclitaxel；Drug delivery；Nanoparticle；Radiotherapy；Hypoxia；Human tumor cells；cellular uptake

Comparative Investigations on the Protective Effects of Rhodioside, Ciwujianoside-B and Astragaloside IV on Radiation Injuries of the Hematopoietic System in Mice

The aim of this study was to investigate the protective effects of three glycosides (rhodioside, ciwujianoside‐B and astragaloside IV) on the hematopoietic system in the mice exposed to γ‐rays, and to examine the possible mechanisms involved. Mice were pretreated with the glycosides (40 mg/kg, i.g.) daily for 7 days prior to radiation. The survival of mice pretreated with three glycosides after total body irradiation (6.0 Gy) was examined. Peripheral blood leucocytes and endogenous spleen colony counts, colony‐forming unit‐granulocyte macrophage assay, analysis of DNA content and apoptosis rate determination were performed to evaluate the effects of the three glycosides on hematogenesis. The fragmentation of double‐stranded DNA in lymphocytes was detected by the comet assay. The changes in cell cycle were analysed by flow cytometry. Furthermore, the expression levels of Bcl‐2, Bax and nuclear factor‐kappa B (NF‐κB) were measured by western blot and the electrophoretic mobility shift assay. The results showed that pretreatment with all of the glycosides improved survival time and increased the number of leucocytes, spleen colonies and granulocyte‐macrophage colonies in mice exposed to 6.0 Gy γ‐radiation. Rhodioside showed more protective efficacy than both ciwujianoside‐B and astragaloside IV. All three glycosides significantly increased the proliferation abilities of bone marrow cells, and decreased the ratio of cells in G0/G1 phase. Further analysis showed that these three glycosides were able to decrease DNA damage and the increment in the Bax/Bcl‐2 ratio induced by radiation. In summary, the three glycosides showed radioprotective effects on the hematopoietic system in mice, which was associated with changes in the cell cycle, a reduction in DNA damage, and down‐regulation of the ratio of Bax/Bcl‐2 in bone marrow cells exposed to radiation. Copyright

Overexpression of keratin 17 is associated with poor prognosis in epithelial ovarian cancer

The aim of this study was to investigate the association between keratin 17 (K17) expression and the clinicopathological features of patients with epithelial ovarian cancer (EOC). K17 expression was detected by real-time quantitative RT-PCR in EOC and adjacent noncancerous tissues. In addition, K17 expression was analyzed by immunohistochemistry in 104 clinicopathologically characterized EOC cases. The expression levels of K17 mRNA and protein in EOC tissues were both significantly higher than those in noncancerous tissues. In addition, positive expression of K17 correlated with the clinical stage (p = 0.001). Furthermore, Kaplan–Meier survival analysis showed that a high expression level of K17 resulted in a significantly poor prognosis of EOC patients. Multivariate analysis revealed that EOC expression level was an independent prognostic parameter for the overall survival rate of EOC patients. Our data are the first to suggest that increased K17 expression in EOC is significantly associated with aggressive progression and poor prognosis. K17 may be an important molecular marker for predicting the carcinogenesis, progression, and prognosis of EOC.

Synthetic gelatinases inhibitor attenuates electromagnetic pulse-induced blood-brain barrier disruption by inhibiting gelatinases-mediated ZO-1 degradation in rats.

Previously we found that exposure to electromagnetic pulse (EMP) induced an increase in blood-brain-barrier (BBB) permeability and the degradation of tight junction protein ZO-1 in rats. Matrix metalloproteinases (MMPs), in particular gelatinases (MMP-2 and MMP-9), play a key role in degradation of tight junction proteins, are known mediators of BBB compromise. We hypothesized that the degradation of ZO-1 by gelatinases contributed to EMP-induced BBB opening. To test this hypothesis, the mRNA level of ZO-1, protein levels of MMP-2, MMP-9 and tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2) were detected in rat cerebral cortex after exposing rats to EMP at 200 kV/m for 200 pulses. It was found that the mRNA level of ZO-1 was unaltered at different time points after EMP exposure. The protein levels of MMP-2 and MMP-9 significantly increased at 3 h and 0.5 h, respectively. However, TIMP-1 (inhibitor of MMP-9) and TIMP-2 (inhibitor of MMP-2) only moderately increased after EMP exposure. In addition, in situ zymography results showed that the gelatinase activity increased in cerebral microvessels at 3 h after EMP exposure. When rats were treated with gelatinases inhibitor (SB-3CT) before EMP exposure, the EMP-induced BBB opening was attenuated and the ZO-1 degradation was reversed. Our results suggested that EMP-induced BBB opening was related to gelatinase mediated ZO-1 degradation.

Knock-down of NDRG2 sensitizes cervical cancer Hela cells to cisplatin through suppressing Bcl-2 expression

BackgroundNDRG2, a member of N-Myc downstream regulated gene family, plays some roles in cellular stress, cell differentiation and tumor suppression. We have found that NDRG2 expression in cervical cancer Hela cells increases significantly upon stimulation with cisplatin, the most popular chemotherapeutic agent currently used for the treatment of advanced cervical cancer. This interesting phenomenon drove us to evaluate the role of NDRG2 in chemosensitivity of Hela cells.MethodsIn the present study, RNA interference was employed to down-regulate NDRG2 expression in Hela cells. RT-PCR and Western blot were used to detect expression of NDRG2, Bcl-2 and Bax in cancer cells. Real-time PCR was applied to detect miR-15b and miR-16 expression levels. Drug sensitivity was determined with MTT assay. Cell cloning efficiency was evaluated by Colony-forming assay. Apoptotic cells were detected with annexin V staining and flow cytometry.ResultsIn vitro drug sensitivity assay revealed that suppression of NDRG2 could sensitize Hela cells to cisplatin. Down-regulation of NDRG2 didn’t influence the colony-forming ability but promoted cisplatin-induced apoptosis of Hela cells. Inhibition of NDRG2 in Hela cells was accompanied by decreased Bcl-2 protein level. However, Bcl-2 mRNA level was not changed in Hela cells with down-regulation of NDRG2. Further study indicated that miR-15b and miR-16, two microRNAs targetting Bcl-2, were significantly up-regulated in NDRG2-suppressed Hela cells.ConclusionsThese data suggested that down-regulation of NDRG2 could enhance sensitivity of Hela cells to cisplatin through inhibiting Bcl-2 protein expression, which might be mediated by up-regulating miR-15b and miR-16.