Pigment epithelial-derived factor (PEDF)-triggered lung cancer cell apoptosis relies on p53 protein-driven Fas ligand (Fas-L) up-regulation and Fas protein cell surface translocation.

Pigment epithelium-derived factor (PEDF), a potent antiangiogenesis agent, has recently attracted attention for targeting tumor cells in several types of tumors. However, less is known about the apoptosis-inducing effect of PEDF on human lung cancer cells and the underlying molecular events. Here we report that PEDF has a growth-suppressive and proapoptotic effect on lung cancer xenografts. Accordingly, in vitro, PEDF apparently induced apoptosis in A549 and Calu-3 cells, predominantly via the Fas-L/Fas death signaling pathway. Interestingly, A549 and Calu-3 cells are insensitive to the Fas-L/Fas apoptosis pathway because of the low level of cell surface Fas. Our results revealed that, in addition to the enhancement of Fas-L expression, PEDF increased the sensitivity of A549 and Calu-3 cells to Fas-L-mediated apoptosis by triggering the translocation of Fas protein to the plasma membrane in a p53- and FAP-1-dependent manner. Similarly, the up-regulation of Fas-L by PEDF was also mediated by p53. Furthermore, peroxisome proliferator-activated receptor γ was determined to be the upstream regulator of p53. Together, these findings uncover a novel mechanism of tumor cell apoptosis induced by PEDF and provide a potential therapeutic strategy for tumors that are insensitive to Fas-L/Fas-dependent apoptosis because of a low level of cell surface Fas.

Plasminogen kringle 5 induces endothelial cell apoptosis by triggering a voltage-dependent anion channel 1 (VDAC1) positive feedback loop.

Human plasminogen kringle 5 (K5) is known to display its potent anti-angiogenesis effect through inducing endothelial cell (EC) apoptosis, and the voltage-dependent anion channel 1 (VDAC1) has been identified as a receptor of K5. However, the exact role and underlying mechanisms of VDAC1 in K5-induced EC apoptosis remain elusive. In the current study, we showed that K5 increased the protein level of VDAC1, which initiated the mitochondrial apoptosis pathway of ECs. Our findings also showed that K5 inhibited the ubiquitin-dependent degradation of VDAC1 by promoting the phosphorylation of VDAC1, possibly at Ser-12 and Thr-107. The phosphorylated VDAC1 was attenuated by the AKT agonist, glycogen synthase kinase (GSK) 3ß inhibitor, and siRNA, suggesting that K5 increased VDAC1 phosphorylation via the AKT-GSK3ß pathway. Furthermore, K5 promoted cell surface translocation of VDAC1, and binding between K5 and VDAC1 was observed on the plasma membrane. HKI protein blocked the impact of K5 on the AKT-GSK3ß pathway by competitively inhibiting the interaction of K5 and cell surface VDAC1. Moreover, K5-induced EC apoptosis was suppressed by VDAC1 antibody. These data show for the first time that K5-induced EC apoptosis is mediated by the positive feedback loop of "VDAC1-AKT-GSK3ß-VDAC1," which may provide new perspectives on the mechanisms of K5-induced apoptosis.

Microfluidic chip capillary electrophoresis coupled with electrochemiluminescence for enantioseparation of racemic drugs using central composite design optimization.

Optimization based on central composite design (CCD) for enantioseparation of anisodamine (AN), atenolol (AT), and metoprolol (ME) in human urine was developed using a microfluidic chip-CE device. Coupling the flexible and wide working range of microfluidic chip-CE device to CCD for chiral separation of AN, AT, and ME in human urine, a total of 15 experiments is needed for the optimization procedure as compared to 75 experiments using the normal one variable at a time optimization. The optimum conditions obtained are found to be more robust as shown by the curvature effects of the interaction factors. The developed microfluidic chip-CE-ECL system with adjustable dilution ratios has been validated by satisfactory recoveries (89.5-99% for six enanotiomers) in urine sample analysis. The working range (0.3-600 µM), repeatability (3.1-4.9% RSD for peak height and 4.0-5.2% RSD for peak area), and detection limit (0.3-0.6 µM) of the method developed are found to meet the requirements for bedside monitoring of AN, AT, and ME in patients under critical conditions. In summary, the hyphenation of CCD with the microfluidic chip-CE device is shown to offer a rapid means for optimizing the working conditions on simultaneous separation of three racemic drugs using the microfluidic chip-CE device developed.

Nerve growth factor-beta expression is associated with lymph node metastasis and nerve infiltration in human hilar cholangiocarcinoma.

BACKGROUND: Angiogenesis and lymphangiogenesis are important processes in the progression of malignant tumors. Previous studies have shown that nerve growth factor-beta (NGF-beta) can promote the initiation and progression of many tumors. In addition, vascular endothelial growth factor-C (VEGF-C) has become recognized as the most important lymphangiogenic factor. In the present study, the expression of NGF-beta in human hilar cholangiocarcinoma and its relationship with lymphangiogenesis, lymph node metastasis, nerve infiltration, and VEGF-C expression was investigated. METHODS: Nerve growth factor-beta and VEGF-C expression were investigated by immunohistochemistry in samples from 28 cases of hilar cholangiocarcinoma. The lymphatic vessel density (LVD) in the tumor tissue that indicated lymphangiogenesis were calculated by immunostaining with the lymphendothelial-specific antibody D2-40. The relationship between NGF-beta expression and VEGF-C expression, lymphangiogenesis, lymph node metastasis, and nerve infiltration was evaluated. RESULTS: The overexpression of NGF-beta and VEGF-C occurred in 57.1% (16/28) and 46.4% (13/28) of tumor samples, respectively. Nerve growth factor-beta overexpression was highly correlated with VEGF-C overexpression (P = 0.005), LVD (P < 0.001), lymph node metastasis (P = 0.021), nerve infiltration (P = 0.019), and tumor stage (P = 0.040). Furthermore, VEGF-C overexpression was highly correlated with LVD (P < 0.001) and lymph node metastasis (P < 0.001). However, there was no statistic significance in the relation between NGF-beta expression and sex (P = 0.185), age (P = 0.387), maximal tumor size (P = 0.736), Bismuth classification (P = 0.627) as well as histological grade (P = 0.203). CONCLUSIONS: Nerve growth factor-beta might promote lymph node metastasis and nerve infiltration in human hilar cholangiocarcinoma.

[Effects of mutant kringle 5 eye drops on the survival time of corneal allografts in rat].

OBJECTIVE: To investigate the preventative effect of mutant kringle 5 (mK5) eye drops on corneal allograft rejection. METHODS: It was a experimental study. The outbred strain F344 and Lewis rats were used as donors and recipients respectively. Sixty Lewis rats were randomly divided into B, C, D and E Group; Group A, F344 rats autograft control; Group B, allograft control (the control groups were given normal sodium only); Group C and D, allograft groups, were treated with 5 mg/L and 10 mg/L mK5 eye drops respectively; Group E, allograft group, was treated with 0.1% dexamethasone eye drops. The eye drops were applied one drop four times per day for two weeks, the occurrence and development of corneal allograft rejection and corneal neovascularization (CNV) was observed every other day by slit-lamp microscope, the grafts were evaluated clinically by means of Holland's scoring system and the area of CNV was calculated. Nine rats per group were killed on the 14th day, and the corneas were taken for histopathological examinations. Analysis of variance was used to analyze the outcomes. RESULTS: The average graft survival time of Group B, C, D and E was (9.3 +/- 2.1), (21.1 +/- 7.3), (23.5 +/- 10.8) and (28.2 +/- 19.1) d respectively, Compared with Group B, Group C and D had a statistically significant prolongation of survival time (q = 10.24, 13.47; P < 0.05). Though treated with 0.1% dexamethasone eye drops (Group E) prolonged transplant survival time as compared with mK5 eye drops, but the difference was not statistically significant (q = 2.54, 1.49; P > 0.05). The occurrence of CNV in Group A was (3.1 +/- 0.8) d, Group B (2.6 +/- 0.5) d, Group C (6.4 +/- 0.5) d, Group D (7.8 +/- 0.7) d and Group E (5.3 +/- 1.0) d. Significant difference (q = 31.58, 51.21, 19.98; P < 0.05) was found between groups C, D, E and Group A. There were also significant difference between groups C, D, E and Group B (q = 43.87, 67.14, 24.53; P < 0.05). The CNV areas of Group C and Group D were also smaller than Group B (q = 30.76, 62.14; P < 0.05). The results was similar compared with Group E (q = 15.20, 25.64; P < 0.05). Fewer inflammatory cells and CNV were found in the cornea of the groups treated with mK5 eye drops. CONCLUSION: Topical application of mK5 eye drops can prevent corneal graft rejection and corneal neovascularization in rats.

Selective inhibition of CDK7 ameliorates experimental arthritis in mice.

Cyclin-dependent kinases (CDKs) have emerged as anti-inflammatory targets. The purpose of this study was to explore the therapeutic effects of a selective CDK7 inhibitor, BS-181, on mice with established collagen-induced arthritis (CIA). CIA mice were administered intraperitoneally with BS-181 (10 mg/kg) twice daily for 2 weeks. Control mice received vehicle only. Arthritis severity and joint histopathology were examined. The proinflammatory cytokines and anti-type II collagen antibodies (anti-CII) were determined by ELISA. IkB kinase (IKK)-ß/NF-κB activation in the arthritic joints was assessed by Western blot. The ratio of Th17 cells was determined by flow cytometry. In vitro, splenocytes from mice with established CIA were stimulated with CII in the presence or absence of BS-181 and cytokines were detected. BS-181 treatment reduced the clinical score and histological damage in CIA mice. The serum proinflammatory cytokines (IL-6, IL-1ß and IL-17) and anti-CII IgG2a levels were also decreased by BS-181 administration. Moreover, IKK-ß/NF-κB signaling pathway was inhibited in arthritic joints. BS-181 administration also decreased the ratio of Th17 cells. In addition, CIA splenocytes pretreated with BS-181 produced less proinflammatory cytokines in vitro. These findings indicate that CDK7 inhibition by BS-181 is effective in the treatment of CIA, which might be mediated by suppression of IKK-ß/NF-κB activation and Th17 cell response.

[High-dose glucose induces human retinal endothelial cell apoptosis].

OBJECTIVE: To examine the direct effect of high glucose levels on primary cultured human retinal capillary endothelial cells (HRCEC). METHODS: HRCECs were isolated from donated eyes and cultured for 6 days in the media containing 5 or 25 mmol/L glucose. The cell viability was determined by trypan blue exclusion assay and cell cycle analyzed by flow cytometry, with the cell apoptosis assayed by TUNEL method. RESULTS: The cell viability was significantly decreased after exposure to 25 mmol/L glucose, and the number of apoptotic cells determined by flow cytometry and TUNEL was significantly increased in response to high-dose glucose treatment. CONCLUSION: High-dose glucose induces apoptosis in HRCEC, which may contribute to the development of diabetic retinopathy.

[A deletion mutant of plasminogen kringle 5 inhibits retinal capillary endothelial cell proliferation].

OBJECTIVE: To obtain purified deletion mutant of plasminogen kringle 5 (K5) using gene mutation and genetic recombination methods and assess its anti-angiogenic activity in vitro. METHODS: A deletion mutant of K5 was obtained by deleting 15 amino acids from K5 while retaining all the 3 disulfide bonds. This K5 mutant (Mut1) was expressed in E. coli and affinity purified. The inhibition effect of K5 Mut1 on primary retinal capillary endothelial cells and pericytes from the same origin was assessed by MTT assay. RESULTS: The K5 Mut1 inhibited the proliferation of primary retinal capillary endothelial cells in a concentration-dependent manner, with an apparent half-inhibition concentration (EC(50)) of approximately 35 nmol/L, which was 2-fold more potent than intact K5. In the same concentration range, this peptide did not inhibit pericytes from the same origin, suggesting an endothelial cell-specific inhibition. CONCLUSION: This K5 deletion mutant is a more potent angiogenic inhibitor than K5 and may have therapeutic potential in the treatment of such disorders with abnormal neovascularization as diabetic retinopathy, age-related macular degeneration and solid tumor.

Dual inhibition of plasminogen kringle 5 on angiogenesis and chemotaxis suppresses tumor metastasis by targeting HIF-1α pathway.

We had demonstrated that plasminogen kringle 5 (K5), a potent angiogenic inhibitor, inhibited retinal neovascularization and hepatocellular carcinoma growth by anti-angiogenesis. The current study investigated the effects and the underlying mechanisms of K5 on both tumor growth and spontaneous pulmonary metastasis in Lewis lung carcinoma (LLC) implanted mouse model. Similarly, K5 could decrease expression of VEGF in LLC cells and grafted tissues and suppress tumor angiogenesis and growth. K5 had no direct effect on proliferation and apoptosis of LLC. However, K5 could significantly inhibit SDF-1α-induced chemotaxis movement of LLC cells and resulted in a great reduction of surface metastatic nodules and micrometastases in the lungs of LLC tumor-bearing mice. K5 also decreased expression of chemokine (C-X-C motif) receptor 4 (CXCR4) in LLC cells and grafted tissues. Furthermore, K5 down-regulated SDF-1α expression in metastatic lung tissues of LLC-bearing mice. Therefore, K5 may suppress tumor pulmonary metastasis through inhibiting SDF-1α-CXCR4 chemotaxis movement and down-regulation of VEGF. Moreover, the role of hypoxia inducible factor-1α (HIF-1α), a crucial transcriptional factor for both VEGF and CXCR4 expression, was evaluated. The siRNA of HIF-1α attenuated expression of VEGF and CXCR4 and inhibited LLC migration. K5 decreased HIF-1α protein level and impaired nuclear HIF-1α accumulation. These results showed for the first time that K5 inhibits LLC growth and metastasis via the dual effects of anti-angiogenesis and suppression of tumor cell motility by targeting the pivotal molecule, HIF-1α.

Pigment epithelium-derived factor gene loaded in cRGD-PEG-PEI suppresses colorectal cancer growth by targeting endothelial cells.

Pigment epithelium-derived factor (PEDF) recombinant protein has been investigated in many kinds of solid tumors due to its potent antiangiogenic activity. However, the complexity of protein purification, instability of recombinant protein and requirement of repeated injections are obstacles for the recombinant PEDF therapy for solid tumors. We successfully synthesized polyethyleneglycol-polyetherimide (PEG-PEI) and cRGD-PEG-PEI which was coupled with a cyclic RGD peptide, a special ligand for integrin αvß3 receptor, as the vehicle for PEDF gene therapy in this study. In vitro, the competitive binding assay showed that cRGD contributed to the enhanced gene transfection efficiency of PEG-PEI in human umbilical vein endothelial cells (HUVECs). PEDF gene delivered by cRGD-PEG-PEI apparently suppressed growth of tumor with a 67.4% reduction and decreased microvessel density in nude mice bearing SW620 human colorectal xenografts. Accordingly, SW620 tumors from cRGD-PEG-PEI/PEDF-pcDNA3.1 (+)-treated mice expressed more PEDF than that of the control groups. Our study demonstrated that cRGD-PEG-PEI transported the PEDF gene into endothelia cells more efficiently than PEG-PEI, resulting in more effective inhibitory effects on tumor growth by anti-angiogenesis. Therefore, for the first time, we have explored an effective non-viral vehicle for PEDF gene therapy by targeting endothelial cells.

Novel method for expression and purification of human pigment epithelium-derived factor with biological activities in Escherichia coli.

We developed a novel method for the expression and purification of recombinant human PEDF in Escherchia coli, and proved it to be simple, convenient, and cheap to obtain this protein with biological activity intact. Human PEDF gene, amplified by PCR from human retinal cNDA library, was cloned into the prokaryotic expression vector pET-22b(+). The recombinant pET-22b(+)/PEDF was expressed in E. coli strain BL21(DE3). The recombinant protein showed a molecular weight of about 50 kDa and was mainly in the form of inclusion bodies according to SDS-PAGE and Western blot analysis. The insoluble rPEDF was solublized from inclusion bodies by denaturation using 6 M urea, purified by His-tag affinity chromatography, and renatured to natural structure by dialysis in the presence of DTT. The rPEDF could cell-type-specifically inhibit HRCEC proliferation in a dose-dependent manner and induce HRCEC apoptosis.