Xie Zhang

Exogenous Basic Fibroblast Growth Factor Inhibits ER Stress–Induced Apoptosis and Improves Recovery from Spinal Cord Injury

To investigate the mechanism of endoplasmic reticulum (ER) stress–induced apoptosis as well as the protective action of basic fibroblast growth factor (bFGF) both in vivo and in vitro.

Aberrant human leucocyte antigen‐G expression and its clinical relevance in hepatocellular carcinoma

The clinical relevance of human leucocyte antigen‐G (HLA‐G) has been postulated in malignancies. Hepatocellular carcinoma (HCC) is a major contributor to cancer incidence and mortality worldwide; however, potential roles of HLA‐G in HCC remain unknown. In the current study, HLA‐G expression in 219 primary HCC lesions and their adjacent non‐tumourous samples was analysed with immunohistochemistry. Correlations among HLA‐G expression and various clinical parameters were evaluated. Meanwhile, functional analysis of transfected cell surface HLA‐G expression on NK cell cytolysis was performed in vitro. HLA‐G expression was observed in 50.2% (110/219) of primary HCC lesions, and undetectable in corresponding adjacent normal liver tissues. HLA‐G expression was found in 37.8%, 41.9% and 71.4% of stage I, II and III HCC lesions, respectively. Data revealed that HLA‐G expression in HCC was strongly correlated to advanced disease stage (I versus II, P= 0.882; I versus III, P= 0.020; II versus III, P= 0.037). HLA‐G expression was also more frequently observed in elder patients (≥median 52 years, 57.5%versus 43.4%, P= 0.004). Meanwhile, plasma soluble HLA‐G in HCC patients was significantly higher than that in normal controls (median, 92.49U/ml versus 9.29U/ml, P= 0.000). Functional assay showed that HLA‐G expression in transfected cells could dramatically decrease the NK cell cytolysis (P= 0.036), which could be markedly restored by the blockade of HLA‐G (P= 0.004) and its receptor ILT2 (P= 0.019). Our finding indicated that HLA‐G expression was strongly correlated to advanced disease stage, and more frequently observed in elder patients. Its relevance to HCC progression might be result from the inhibition of NK cell cytolysis.

Clinical relevance and functional implications for human leucocyte antigen-g expression in non-small-cell lung cancer.

HLA‐G has been documented both in establishment of anti‐tumour immune responses and in tumour evasion. To investigate the clinical relevance of HLA‐G in non‐small‐cell lung cancer (NSCLC), expression status and potential significance of HLA‐G in NSCLC were analysed. In this study, HLA‐G expression in 101 NSCLC primary lesions and plasma soluble HLA‐G (sHLA‐G) from 91 patients were analysed with immunohistochemistry and ELISA, respectively. Correlations between HLA‐G status and various clinical parameters including survival time were evaluated. Meanwhile, functional analysis of transfected cell surface HLA‐G expression and plasma sHLA‐G form NSCLC patients on natural killer (NK) cell cytolysis were performed. Data revealed that HLA‐G was expressed in 41.6% (42/101) NSCLC primary lesions, while undetectable in adjacent normal lung tissues. HLA‐G expression in NSCLC lesions was strongly correlated to disease stages (P= 0.002). Plasma sHLA‐G from NSCLC patients was markedly higher than that in normal controls (P= 0.004), which was significantly associated with the disease stages (I versus IV, P= 0.025; II versus IV, P= 0.029). Patient plasma sHLA‐G level (≥median, 32.0 U/ml) had a significantly shorter survival time (P= 0.044); however, no similar significance was observed for the lesion HLA‐G expression. In vitro data showed that both cell surface HLA‐G and patient plasma sHLA‐G could dramatically decrease the NK cell cytolysis. Our findings indicated that both lesion HLA‐G expression and plasma sHLA‐G in NSCLC is related to the disease stage and can exert immunosuppression to the NK cell cytolysis, indicating that HLA‐G could be a potential therapeutic target. Moreover, plasma sHLA‐G in NSCLC patients could be used as a prognosis factor for NSCLC.

Nerve growth factor improves functional recovery by inhibiting endoplasmic reticulum stress-induced neuronal apoptosis in rats with spinal cord injury.

BackgroundEndoplasmic reticulum (ER) stress-induced apoptosis plays a major role in various diseases, including spinal cord injury (SCI). Nerve growth factor (NGF) show neuroprotective effect and improve the recovery of SCI, but the relations of ER stress-induced apoptosis and the NGF therapeutic effect in SCI still unclear.MethodsYoung adult female Sprague-Dawley rats’s vertebral column was exposed and a laminectomy was done at T9 vertebrae and moderate contusion injuries were performed using a vascular clip. NGF stock solution was diluted with 0.9% NaCl and administered intravenously at a dose of 20 μg/kg/day after SCI and then once per day until they were executed. Subsequently, the rats were executed at 1d, 3 d, 7d and 14d. The locomotor activities of SCI model rats were tested by the 21-point Basso-Beattie-Bresnahan (BBB) locomotion scale, inclined plane test and footprint analysis. In addition, Western blot analysis was performed to identify the expression of ER-stress related proteins including CHOP, GRP78 and caspase-12 both in vivo and in vitro. The level of cell apoptosis was determined by TUNEL in vivo and Flow cytometry in vitro. Relative downstream signals Akt/GSK-3β and ERK1/2were also analyzed with or without inhibitors in vitro.ResultsOur results demonstrated that ER stress-induced apoptosis was involved in the injury of SCI model rats. NGF administration improved the motor function recovery and increased the neurons survival in the spinal cord lesions of the model rats. NGF decreases neuron apoptosis which measured by TUNEL and inhibits the activation of caspase-3 cascade. The ER stress-induced apoptosis response proteins CHOP, GRP78 and caspase-12 are inhibited by NGF treatment. Meanwhile, NGF administration also increased expression of growth-associated protein 43 (GAP43). The administration of NGF activated downstream signals Akt/GSK-3β and ERK1/2 in ER stress cell model in vitro.ConclusionThe neuroprotective role of NGF in the recovery of SCI is related to the inhibition of ER stress-induced cell death via the activation of downstream signals, also suggested a new trend of NGF translational drug development in the central neural system injuries which involved in the regulation of chronic ER stress.

Preparation and characterisation of bFGF-encapsulated liposomes and evaluation of wound-healing activities in the rat

OBJECTIVE The basic fibroblast growth factor (bFGF) has a very short half-life in vivo, and this limits its therapeutic value for frequent administration. Liposome technology was used to improve the stability of bFGF and to prolong its effects in vivo. METHODS bFGF-encapsulated liposomes (bFGF-lip) were prepared using the pH gradient method. Four critical factors were investigated including concentration of citric acid solution, incubation time of blank liposomes, incubation temperature and sonication time. The pharmacodynamics of bFGF-lip was investigated by establishing a deep second-degree burns model in rats. RESULTS The optimal bFGF-lip were characterised by high entrapment efficiency (79.88 ± 3.37%), good physical stability (K(E): 1.02 ± 0.413%) and high bioactivity ((6.147 ± 0.769) × 10(5) IU ml(-1)). The middle dose of bFGF-lip (60 IU ml(-1)) demonstrated the fastest tissue collagen generation as well as the earliest and highest tumour growth factor (TGF)-β1 and dermal cell proliferation (proliferating cell nuclear antigen (PCNA)) expression as compared with other treatments. CONCLUSION Results of the present study imply that bFGF-lip have promising prospects for application in wound-healing therapies.

Characterization of HLA-G expression in renal cell carcinoma

Previous studies showed that human leukocyte antigen (HLA)-G is specifically upregulated in renal cell carcinoma (RCC). However, a larger cohort of RCC patients are necessary to obtain more information. In this study, 109 RCC primary lesions (clear cell, n = 95; chromophobe, n = 4; papillary, n = 4; collecting duct, n = 6) and corresponding adjacent tumor-negative renal tissues (n = 34) were analyzed for the HLA-G expression by immunohistochemistry (IHC). Meanwhile, plasma soluble HLA-G (sHLA-G) from 16 RCC patients and 144 sex- and age-matched normal individuals was detected by enzyme-linked immunosorbent assay. Correlations between lesion HLA-G expression and various clinical parameters were evaluated. Receiver-operating characteristic (ROC) curve analysis was used to determine the feasibility of HLA-G protein staining and sHLA-G as a diagnosis marker for RCC. IHC data showed that HLA-G was observed in 49.5% of clear cell, 50% of either chromophobe or collecting duct RCC lesions but undetectable in papillary RCC and tumor-negative renal tissues. This finding was consistent with the western blot results. sHLA-G was pronouncedly increased in RCC patients when compared with normal controls (median: 39.5 vs 19.2 U/ml, P = 0.002). However, no correlation was observed between HLA-G expression and various clinical parameters. We found that the area under ROC curve for HLA-G expression and sHLA-G was 0.739 [95% confidence interval (95% CI): 0.659-0.816, P = 0.000] and 0.733 (95% CI: 0.619-0.847, P = 0.002), respectively. Our findings indicated that, except the papillary RCC, other types of RCC could express HLA-G. Furthermore, both lesion HLA-G expression and plasma sHLA-G level might be a useful preoperative biomarker for diagnosis.

Alteration of HLA-F and HLA I antigen expression in the tumor is associated with survival in patients with esophageal squamous cell carcinoma.

Alteration of human leukocyte antigen (HLA) expression, such as decreased HLA I (HLA‐A, ‐B and ‐C) antigens and elevated nonclassical HLA I antigens (HLA‐E, ‐F and ‐G), was reported to have an unfavorable prognosis in various cancers. In our study, HLA‐F expression in 105 primary esophageal squamous cell carcinoma (ESCC) lesions and 62 case‐matched adjacent normal tissues, and HLA I antigens among 68 cases were analyzed by immunohistochemistry. Data revealed that HLA‐F expression was observed in 58.1% (61/105) of the ESCC lesions and in 54.8% (34/62) of the normal esophageal tissues. Among the 62 case‐matched samples, HLA‐F expression (lesion vs. normal tissue) was upregulated, unchanged and downregulated in 13 (21.0%), 6 (9.6%) and 43 (69.4%) cases, respectively. Patients with HLA‐F positive had a worse survival than those with HLA‐F negative (p = 0.040). Patients with upregulated HLA‐F expression (lesion vs. normal tissue) had significantly worse survival than those with HLA‐F unchanged and downregulated (p = 0.010). Furthermore, decreased HLA I expression was observed in 41.2% (28/68) patients and was with worse prognosis in comparison to those with preserved HLA I expression (p = 0.001). Multivariate analysis using Coxs proportional hazards model revealed that upregulated HLA‐F expression (p = 0.026) and downregulated HLA I expression (p = 0.013) could be an independent unfavorable prognostic factor. In conclusion, our study provided the evidence that alteration of HLA I and HLA‐F antigen expression was associated with survival in patients with ESCC.

A synthetic compound, 1,5-bis(2-methoxyphenyl)penta- 1,4-dien-3-one (B63), induces apoptosis and activates endoplasmic reticulum stress in non-small cell lung cancer cells

Endoplasmic reticulum (ER) stress‐induced cancer cell apoptosis has become a novel signaling target for the development of therapeutic drugs for cancer treatment. Curcumin, a dietary phytochemical, exhibits growth‐suppressive activity against cancer cells via multitarget mechanisms. However, the low stability and poor pharmacokinetics significantly limit its clinical applications. Thus, we designed and synthesized a novel monocarbonyl analog of curcumin, 1,5‐bis(2‐methoxyphenyl) penta‐1,4‐dien‐3‐one (B63). This compound exhibited a higher chemical stability in cultural medium and a better intracellular profile than curcumin. Treatment with B63 potently induced apoptosis of human non‐small cell lung cancer (NSCLC) cells in a dose‐responsive manner, while exhibiting no cytotoxicity in normal lung fibroblast cells. Its antitumor effect was associated with the ER stress‐mediated apoptotic pathway and, ultimately, the activation of the caspase cascades. However, curcumin at the same concentrations did not cause ER stress in H460 cells. Further, C/EBP homologous protein knockdown by siRNA attenuated B63‐induced cell apoptosis, indicating that the apoptotic pathway is ER stress‐dependent. In vivo, the volume and weight of the tumor were reduced significantly by pretreating the H460 tumor cells with B63 before implantation. Taken together, these insights on the novel compound B63, from both chemical and biological perspectives, may provide a novel anticancer candidate for the treatment of NSCLC.

A novel mono-carbonyl analogue of curcumin induces apoptosis in ovarian carcinoma cells via endoplasmic reticulum stress and reactive oxygen species production

The aim of the present study was to investigate the apoptosis of human ovarian cancer cell lines, A2780 and CP70, induced by a novel curcumin analogue, B19. The proliferation of cells was detected with methyl thiazolyl tetrazolium (MTT) assay and apoptosis was examined by flow cytometry. Reactive oxygen species (ROS) were assessed by the fluorescent indicator DCF-DA. The protein expression of the endoplasmic reticulum (ER) stress pathways, GRP78, XBP-1, ATF-4 and CHOP, was examined with Western blotting. A growth inhibitory effect was observed after treatment with B19 in a dose-dependent manner and with more potential than curcumin. At 20 µM, B19 induced significant apoptosis in CP70 cells. Furthermore, B19 induced the ER stress response, while curcumin had no effect on ER stress. These results suggest that B19 has more effective antitumor properties than curcumin, and is associated with the activation of ER stress and ROS in ovarian cancer cells.

Regulation of Caveolin-1 and Junction Proteins by bFGF Contributes to the Integrity of Blood-Spinal Cord Barrier and Functional Recovery.

The blood–spinal cord barrier (BSCB) plays important roles in the recovery of spinal cord injury (SCI), and caveolin-1 is essential for the integrity and permeability of barriers. Basic fibroblast growth factor (bFGF) is an important neuroprotective protein and contributes to the survival of neuronal cells. This study was designed to investigate whether bFGF is beneficial for the maintenance of junction proteins and the integrity of the BSCB to identify the relations with caveolin-1 regulation. We examined the integrity of the BSCB with Evans blue dye and fluorescein isothiocyanate–dextran extravasation, measured the junction proteins and matrix metalloproteinases, and evaluated the locomotor function recovery. Our data indicated that bFGF treatment improved the recovery of BSCB and functional locomotion in contusive SCI model rats, reduced the expression and activation of matrix metalloproteinase-9, and increased the expressions of caveolin-1 and junction proteins, including occludin, claudin-5, p120-catenin, and β-catenin. In the brain, in microvascular endothelial cells, bFGF treatment increased the levels of junction proteins, caveolin-1 small interfering RNA abolished the protective effect of bFGF under oxygen–glucose deprivation conditions, and the expression of fibroblast growth factor receptor 1 and co-localization with caveolin-1 decreased significantly, which could not be reversed by bFGF treatment. These findings provide a novel mechanism underlying the beneficial effects of bFGF on the BSCB and recovery of SCI, especially the regulation of caveolin-1.