Oxidative stress affects retinal pigment epithelial cell survival through epidermal growth factor receptor/AKT signaling pathway.

AIM: To investigate the cross-talk between oxidative stress and the epidermal growth factor receptor (EGFR)/AKT signaling pathway in retinal pigment epithelial (RPE) cells. METHODS: Human RPE cell lines (ARPE-19 cell) were treated with different doses of epidermal growth factor (EGF) and hydrogen peroxide (H2O2). Cell viability was determined by a methyl thiazolyl tetrazolium assay. Cell proliferation was examined by a bromodeoxyuridine (BrdU) incorporation assay. EGFR/AKT signaling was detected by Western blot. EGFR localization was also detected by immunofluorescence. In addition, EGFR/AKT signaling was intervened upon by EGFR inhibitor (erlotinib), PI3K inhibitor (A66) and AKT inhibitor (MK-2206), respectively. H2O2-induced oxidative stress was blocked by antioxidant N-acetylcysteine (NAC). RESULTS: EGF treatment increased ARPE-19 cell viability and proliferation through inducing phosphorylation of EGFR and AKT. H2O2 inhibited ARPE-19 cell viability and proliferation and also suppressed EGF-stimulated increase of RPE cell viability and proliferation by affecting the EGFR/AKT signaling pathway. EGFR inhibitor erlotinib blocked EGF-induced phosphorylation of EGFR and AKT, while A66 and MK-2206 only blocked EGF-induced phosphorylation of AKT. EGF-induced phosphorylation and endocytosis of EGFR were also affected by H2O2 treatment. In addition, antioxidant NAC attenuated H2O2-induced inhibition of ARPE-19 cell viability through alleviating reduction of EGFR, and phosphorylated and total AKT proteins. CONCLUSION: Oxidative stress affects RPE cell viability and proliferation through interfering with the EGFR/AKT signaling pathway. The EGFR/AKT signaling pathway may be an important target in oxidative stress-induced RPE cell dysfunction.

Phage display selection of peptides that home to atherosclerotic plaques: IL-4 receptor as a candidate target in atherosclerosis.

Imaging or drug delivery tools for atherosclerosis based on the plaque biology are still insufficient. Here, we attempted to identify peptides that selectively home to atherosclerotic plaques using phage display. A phage library containing random peptides was ex vivo screened for binding to human atheroma tissues. After three to four rounds of selection, the DNA inserts of phage clones wer sequenced. A peptide sequence, CRKRLDRNC, was the most frequently occurring one. Intravenously injected phage displaying the CRKRLDRNC peptide was observed to home to atherosclerotic aortic tissues of low-density lipoprotein receptor-deficient (Ldlr-/-) mice at higher levels than to normal aortic tissues of wild-type mice. Moreover, a fluorescein- or radioisotope-conjugated synthetic CRKRLDRNC peptide, but not a control peptide, homed in vivo to atherosclerotic plaques in Ldlr(-/-) mice, while homing of the peptide to other organs such as brain was minimal. The homing peptide co-localized with endothelial cells, macrophages and smooth muscle cells a mouse and human atherosclerotic plaques. Homology search revealed that the CRKRLDRNC peptide shares a motif of interleukin-receptor (IL-4) that is critical for binding to its receptor. The peptide indeed co-localized with IL-4 receptor (IL-4R) at atherosclerotic plaques. Moreover, the peptide bound to cultured cells expressing IL-4R on the cell surface and the binding was inhibited by the knock-down of IL-4R. These results show that the CRKRLDRNC peptide homes to atherosclerotic plaques through binding to IL-4R as its target and may be a useful tool for selective drug delivery and molecular imaging of atherosclerosis.

Enhanced therapeutic effects on the multi-drug resistant human leukemia cells in vitro and xenograft in mice using the stealthy liposomal vincristine plus quinacrine.

The multi-drug resistance (MDR) could be caused by the over-expression of adenosine triphosphate binding cassette transporters such as p-glycoprotein, thereby resulting in the efflux of anti-cancer drugs from the cells. An anti-resistant stealthy liposomal vincristine plus quinacrine was defined in this study. Human chronic myelogenous leukemia K562 and MDR K562 cells were included for comparisons. Anti-tumor activity studies were performed on female BALB/c nude mice with MDR K562 cell xenografts. Results showed that quinacrine was effective in reversing the resistance in the MDR K562 cells, and enhanced the anti-tumor effect of vincristine in K562 cells. The caspase-9 and -3 activities in the MDR K562 and K562 cells were increased with the dose rise of quinacrine. In the MDR K562 cell xenografts in mice, the anti-resistant tumor effect of the stealthy liposomal vincristine plus quinacrine was evidently observed. The enhanced anti-tumor effects of vincristine by quinacrine in the resistant/non-resistant K562 cells could be because of the direct injury and the potentiating apoptotic effect of vincristine via activating the initiator caspase-9 and subsequently the effector caspase-3, and the long circulatory effect of stealthy liposomes. The stealthy liposomal encapsulation of vincristine plus quinacrine could be a potential therapeutic approach for resistant human leukemia.

Identification of a peptide ligand recognizing dysfunctional endothelial cells for targeting atherosclerosis.

Targeting ligands to dysfunctional or activated endothelial cells overlying atherosclerotic plaques could provide tools for selective drug delivery to atherosclerotic lesions. To identify peptides selectively targeting dysfunctional endothelial cells, a phage library was screened against tumor necrosis factor-alpha (TNF-alpha) activated bovine aortic endothelial cells (BAECs). Five rounds of biopanning were carried out and the phage clones selected were examined for their DNA inserts. A phage clone displaying the CLWTVGGGC sequence occurred most frequently and was found to bind specifically to TNF-alpha activated BAECs over untreated cells. On the other hand, bindings of the phage clone to human umbilical vein endothelial cells, lymphatic endothelial cells, and epithelial cells were minimal. Flow cytometric and fluorescence microscopic studies showed the preferential binding of the CLWTVGGGC peptide to TNF-alpha activated BAECs compared to untreated cells. In vivo studies demonstrated selective homing and co-localization of the CLWTVGGGC peptide to dysfunctional endothelial cells overlying atherosclerotic plaques in low-density lipoprotein receptor-deficient mice. These results demonstrate that the CLWTVGGGC peptide could specifically recognize dysfunctional endothelial cells at atherosclerotic plaques and be used as a targeting ligand for drug delivery and imaging of atherosclerosis.

Detection of apoptosis in a rat model of focal cerebral ischemia using a homing peptide selected from in vivo phage display.

Focal cerebral ischemia, known as stroke, is caused by a sudden interruption in the blood supply to the brain. We attempted to identify peptides that can home to ischemic stroke tissue and detect the apoptosis of cells. A phage library displaying random peptides was screened for homing peptides to ischemic stroke tissue in a rat transient middle cerebral artery (MCA) occlusion model. After three rounds of in vivo screening, a phage clone displaying the most frequently occurring CLEVSRKNC sequence was selected. The CLEVSRKNC-phage preferentially homed to ischemic stroke tissue after intravenous administration into the MCA occlusion rats. The fluorescein-labeled synthetic CLEVSRKNC peptide, but not a scrambled control peptide, homed to ischemic stroke tissue with a lack of homing to non-ischemic brain tissue. The CLEVSRKNC peptide co-localized with a portion of neuronal cells, rather than with astrocytes, undergoing apoptosis at the penumbra region of stroke lesions. In autoradiographic studies, the uptake of the (131)I-labeled CLEVSRKNC peptide into an ischemic lesion increased at the first day and peaked at the third day after the injury. These results demonstrate that the CLEVSRKNC peptide can home to ischemic stroke tissue, while detecting apoptotic neuronal cells, and suggest it has applications as a targeting moiety for molecular imaging and selective drug delivery to stroke tissue.

A new atherosclerotic lesion probe based on hydrophobically modified chitosan nanoparticles functionalized by the atherosclerotic plaque targeted peptides.

We developed a new imaging probe for atherosclerotic lesion imaging by chemically conjugating an atherosclerotic plaque-homing peptide (termed the AP peptide) to hydrophobically modified glycol chitosan (HGC) nanoparticles. The AP peptide was previously discovered by using an in vivo phage display screening method. HGC nanoparticles were labeled with the near-infrared (NIR) fluorophore Cy5.5, yielding nanoparticles 314 nm in diameter. The binding characteristics of nanoparticles to cytokine (TNF-alpha)-activated bovine aortic endothelial cells (BAECs) were studied in vitro under static conditions and in a dynamic flow environment. AP-tagged HGC-Cy5.5 nanoparticles (100 microg/ml, 2 h incubation) bound more avidly to TNF-alpha-activated BAECs than to unactivated BAECs. Nanoparticles were mostly located in the membranes of BAECs, although some were taken up by the cells and were visible in the cytoplasm, suggesting that the AP peptides in HGC nanoparticles retained target selectivity for activated BAECs. Binding selectivity of AP-tagged HGC-Cy5.5 nanoparticles was also studied in vivo. NIR fluorescence imaging demonstrated that AP-tagged HGC-Cy5.5 nanoparticles bound better to atherosclerotic lesions in a low-density lipoprotein receptor-deficient (Ldlr(-/-)) atherosclerotic mouse than to such lesions in a normal mouse. These results suggest that the newly designed AP-tagged HGC-Cy5.5 nanoparticles may be useful for atherosclerotic lesion imaging, and may also be employed to elucidate pathophysiological changes, at the molecular level, on atherosclerotic endothelium.

Targeting bladder tumor cells in vivo and in the urine with a peptide identified by phage display.

Bladder cancer is one of the most common tumors of the genitourinary tract. Here, we use phage display to identify a peptide that targets bladder tumor cells. A phage library containing random peptides was screened for binding to cells from human bladder tumor xenografts. Phage clones were further selected for binding to a bladder tumor cell line in culture. Six clones displaying the consensus sequence CXNXDXR(X)/(R)C showed selective binding to cells from primary human bladder cancer tissue. Of these, the CSNRDARRC sequence was selected for further study as a synthetic peptide. Fluorescein-conjugated CSNRDARRC peptide selectively bound to frozen sections of human bladder tumor tissue, whereas only negligible binding to normal bladder tissue was observed. When the fluorescent peptide was introduced into the bladder lumen, in a carcinogen-induced rat tumor model, it selectively bound to tumor epithelium. Moreover, when the peptide was intravenously injected into the tail vein, it homed to the bladder tumor but was not detectable in normal bladder and control organs. Next, we examined whether the peptide can detect tumor cells in urine. The fluorescent peptide bound to cultured bladder tumor cells but not to other types of tumor cell lines. Moreover, it bound to urinary cells of patients with bladder cancer, while showing little binding to urinary cells of patients with inflammation or healthy individuals. The CSNRDARRC peptide may be useful as a targeting moiety for selective delivery of therapeutics and as a diagnostic probe for the detection of bladder cancer.

Pharmacokinetics and anti-asthmatic potential of non-parenterally administered recombinant human interleukin-1 receptor antagonist in animal models.

The objectives of this study were to define the pharmacokinetics of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and its effects on allergic asthma, cell adhesion molecules, and upper respiratory tract following non-parenteral administration in animals. Pharmacokinetics and immunomodulating effects of rhIL-1ra were investigated in Sprague-Dawley rats and asthmatic guinea pigs, respectively. Effects on the upper respiratory tract following the applications of rhIL-1ra were investigated on the ex vivo nasal mucosa of Sprague-Dawley rats and in situ in the upper palate of Chinese toads. Absolute bioavailabilities after intratracheal and intranasal administrations of rhIL-1ra were 94.3% and 24.8%, respectively. After administration of rhIL-1ra solution as ultrasonic spraying, the asthmatic symptom in guinea pigs was obviously attenuated. The plasma soluble intercellular cell adhesion molecule (sICAM-1) and P-selectin levels in asthmatic guinea pigs were each dose-dependently reduced with the increase of rhIL-1ra dose. The rhIL-1ra solution after administration via the airway seemed to have no impact on the integrity of nasal mucosa and mucocilia clearance in the upper respiratory tract. The present study provides evidence that rhIL-1ra effectively suppresses allergen-induced asthmatic symptoms through spraying, which corresponds to nasal and pulmonary absorption or both, and the efficacy is associated with downregulation of sICAM-1 and P-selectin expressions.

A novel stealth liposomal topotecan with amlodipine: apoptotic effect is associated with deletion of intracellular Ca2+ by amlodipine thus leading to an enhanced antitumor activity in leukemia.

The objectives of the present study were to define whether amlodipine induces apoptosis and what mechanism is involved in the process in human resistant and non-resistant leukemia cells following co-administration of stealth liposomal topotecan with amlodipine, a novel antiresistant liposomes developed by our institution. In three leukemias, K562, HL-60, and multidrug resistant (MDR) HL-60, cytotoxicity of topotecan was potentiated by amlodipine, while topotecan alone was resistant to MDR HL-60 cells. In two selected K562 or MDR HL-60 cells, the apoptotic effects were increased by addition of amlodipine, showing a dose-dependent manner. The activities of caspase 3 and 7 (marked as caspase 3/7), and caspase 8 were significantly activated by topotecan with amlodipine co-treated as the stealth liposomes. The deletions of intracellular Ca2+ stores induced by amlodipine correlated with the activated activities of caspase 3/7, or 8, respectively. In xenograft model with MDR HL-60 in nude mice, antitumor activity of stealth liposomal topotecan with amlodipine was significantly enhanced as compared to that of stealth liposomal topotecan or topotecan alone. In conclusion, apoptotic effect is associated with deletion of intracellular Ca2+ by amlodipine through activation of caspase 8 and then 3/7 activities. The enhanced antitumor activities by stealth liposomal topotecan with amlodipine are mainly due to the potentiating apoptotic effect and reversing the resistance by amlodipine. Stealth liposomal encapsulation of anticancer agent with a modulator may provide a novel strategy for improving the chemotherapeutic effects.

Cloning and Expression of Human CD20 Gene on NIH-3T3 Cell Membrane.

CD20 is a specific antigen expressed on normal and neoplastic B cells exclusively. Recent researches showed that in B cell leukemia, CD20 was over-expressed. Therefore monoclonal antibody (McAb) to CD20 may be of clinical value in diagnosis and treatment of some leukemias and lymphomas. In this study, the full length gene of CD20 cDNA were cloned from total RNA of Raji cells, inserted into an eukaryotic expression vector pcDNA3.1, forming a recombinant plasmid pcDNA3.1/CD20. NIH-3T3 cells were transfected with pcDNA3.1/CD20 and selected with G418 for the transfected cells. Alkaline phosphatase against alkaline phosphatase assay(APAAP) experiments showed that the selected cells could express the human CD20 onto its surface. Balb/c mice were immunized with CD20( ) NIH-3T3 cells once three weeks for 3 shuts. Indirect immunofluorescence experiments were done with the Raji cells and the serum of the immunized mice, and the results showed that the spleen of the immunized mice could be used to prepare the McAb to CD20.