A novel water-soluble Cu(II) gluconate complex inhibits cancer cell growth by triggering apoptosis and ferroptosis related mechanisms.

Metal copper complexes have attracted extensive attention as potential alternatives to platinum-based anticancer drugs due to their possible different modes of action. Herein, a new copper(II) gluconate complex, namely [Cu(DPQ)(Gluc)]·2H2O (CuGluc, DPQ = pyrazino[2,3-f][1,10]phenanthroline), with good water-solubility and high anticancer activity was synthesized by using D-gluconic acid (Gluc-2H) as an auxiliary ligand. The complex was well characterized by single-crystal X-ray diffraction analysis, elemental analysis, molar conductivity, and Fourier transform infrared spectroscopy (FTIR). The DNA-binding experiments revealed that CuGluc was bound to DNA by intercalation with end-stacking binding. CuGluc could oxidatively cleave DNA, in which 1O2 and H2O2 were involved. In addition, CuGluc was bound to the IIA subdomain of human serum albumin (HSA) through hydrophobic interaction and hydrogen bonding, showing a good affinity for HSA. The complex showed superior anticancer activity toward several cancer cells than cisplatin in vitro. Further studies indicated that CuGluc caused apoptotic cell death in human liver cancer (HepG2) cells through elevated intracellular reactive oxygen species (ROS) levels, mitochondrial dysfunction, cell cycle arrest, and caspase activation. Interestingly, CuGluc also triggered the ferroptosis mechanism through lipid peroxide accumulation and inhibition of glutathione peroxidase 4 (GPX4) activity. More importantly, CuGluc significantly inhibited tumor growth in vivo, which may benefit from the combined effects of apoptosis and ferroptosis. This work provides a promising strategy to develop highly effective antitumor copper complexes by coordinating with the glucose metabolite D-gluconic acid and exploiting the synergistic effects of apoptosis and ferroptosis mechanisms.

Synthesis, characterization and biological activity of novel copper complexes containing a ß-carboline derivative and amino acids.

Copper complexes have long been considered as a promising class of anticancer or antibacterial therapeutics. In this paper, two novel copper(II) complexes containing a ß-carboline derivative and amino acids, namely [Cu(1-Im-ßc)(L-Val)]ClO4·0.5H2O (Cu1) and [Cu(1-Im-ßc)(L-Phe)]ClO4·0.5H2O (Cu2), where 1-Im-ßc = 1-(2-imidazolyl)-ß-carboline, L-Val = L-valine, and L-Phe = L-phenylalanine, were designed and synthesized. The complexes were characterized by elemental analysis, infrared spectroscopy, molar conductivity measurements, and mass spectrometry to determine their spatial structures and compositions. Both complexes bind to DNA by insertion. The complexes also show a good affinity for human serum albumin (HSA). In addition, the antitumor activity of the two complexes against lung cancer cells (A549), cervical cancer cells (HeLa), and breast cancer cells (MBA-MD-231) is significantly superior to that of the traditional antitumor drug, cisplatin. Finally, the anticancer mechanism results show that the complexes can induce apoptosis in HeLa cells, which is associated with mitochondrial damage, oxidative stress caused by reactive oxygen species (ROS) production, and activation of the caspase protein family. This study demonstrates that the introduction of aromatic heterocyclic alkaloid ligands with a broad spectrum of biological activities and water-soluble amino acid ligands into copper complexes can regulate their amphiphilic properties and biological activity, so as to obtain highly efficient copper-based therapeutics.

Synthesis, structural studies, interaction with DNA/HSA and antitumor evaluation of new Cu(II) complexes containing 2-(1H-imidazol-2-yl)pyridine and amino acids.

Copper complexes are considered as potential candidates for anticancer therapy and medical applications. In this paper, three new Cu(II) complexes, [Cu(IPY)2](ClO4)2·H2O (CuI1), [Cu(IPY)(L-Phe)H2O]ClO4·0.5H2O (CuI2) and [Cu(IPY)(L-Val)H2O]ClO4 (CuI3) (where IPY = 2-(1H-imidazol-2-yl)pyridine, L-Phe = L-phenylalanine, and L-Val = L-valine), with good amphipathic properties were synthesized and characterized. Their single crystal X-ray diffraction results revealed that CuI1 was four-coordinated, while CuI2 and CuI3 both adopted a five-coordinated tetragonal pyramidal configuration. Multi-spectral methods, viscosity experiment and molecular docking technique showed that the three complexes interacted with DNA through insertion. The results of the gel electrophoresis experiments indicated that DNA was oxidatively cleaved by all the complexes in a concentration-dependent manner. Moreover, singlet oxygen (1O2), hydrogen peroxide (H2O2) and superoxide anion radicals (˙O2-) were associated with the oxidative cleavage of DNA. All the complexes also had good binding affinity with human serum albumin (HSA). The MB degradation assay revealed that all complexes could react with H2O2 to form ˙OH through Fenton-like processes. The complexes displayed good antiproliferative activity against the tested human cancer cells in vitro, including cervical carcinoma cells (HeLa), liver cancer cells (HepG2 and BEL-7402) and gastric adenocarcinoma cells (SGC-7901), but showed lower toxicity to normal liver cells (LO2). The anticancer mechanism research revealed that CuI1, CuI2 and CuI3 arrested the cell cycle at the S phase, elevated intracellular reactive oxygen species (ROS) levels and induced loss of mitochondrial membrane potential (MMP). The results indicated that these Cu(II) complexes could induce DNA damage and ROS-mediated mitochondrial dysfunction, leading to cancer cell apoptosis. Our work provides a theoretical basis for the design of new low-toxicity and highly efficient anticancer Cu(II) complexes by incorporating biological metabolites and aromatic heterocyclic ligands.

Corneal alteration and pathogenesis in diabetes mellitus.

The incidence of diabetes mellitus (DM) and its complications have increased considerably worldwide. Diabetic keratopathy is the major complication of the cornea characterized by delayed corneal wound healing, decreasing corneal epithelial sensitivity, and recurrent corneal ulcers. There is accumulating evidence that diabetic keratopathy is correlated with the hyperglycemic state. Different corneal components may produce different alterations under hyperglycemia. In addition, diabetic nerve alteration may become a novel biomarker of early-stage DM. Abnormalities of the corneal nerve plexus have been associated with diabetic inflammatory states. There is rapidly growing evidence based on investigations of diabetic corneal nerves through in vivo confocal microscopy. Understanding the molecular pathogenesis caused by hyperglycemia may assist in the identification of novel biomarkers, as well as therapeutic targets for early treatment. This review mainly summarizes recent findings on corneal alteration and pathogenesis in DM.

Reactive oxygen species mediates a metabolic memory of high glucose stress signaling in bovine retinal pericytes.

AIM: To investigate the role of reactive oxygen species (ROS) and antioxidant mechanism underlying the metabolic memory of bovine retinal pericytes (BRPs) induced by high glucose. METHODS: Effects of high glucose levels and culture time on BRPs viability were evaluated by CCK-8. BRPs were grown in high-glucose media (30 mmol/L) for 4d followed by culture in normal glucose condition (5.6 mmol/L) for 4d in an experimental group. In contrast, in negative and positive control groups, BRPs were grown in either normal-glucose media or high-glucose media for 8d, respectively. The ROS levels, apoptosis, the expression and activity of manganese superoxide dismutase (MnSOD) in BRPs, as well as the protective effect of adeno-associated viral (AAV)-mediated over expression of MnSOD were determined separately by DCHFA, ELISA and Western blot. RESULTS: Comparing the result of cells apoptosis, activity and protein expression of MnSOD and caspase-3, the cell culture system that exposed in sequence in 30 mmol/L and normal glucose for 4d was demonstrated as a suitable model of metabolic memory. Furthermore, delivery of antioxidant gene MnSOD can decrease BRPs apoptosis, reduce activated caspase-3, and reverse hyperglycemic memory by reducing the ROS of mitochondria. CONCLUSION: Increased ROS levels and decreased MnSOD levels may play important roles in pericyte loss of diabetic retinopathy. BRPs cultured in high glucose for 4d followed by normal glucose for 4d could be an appropriate model of metabolic memory. rAAV-MnSOD gene therapy provides a promising strategy to inhibit this blinding disease.

Efficacy and necessity of prophylactic vitrectomy for acute retinal necrosis syndrome.

AIM: To compare the efficacy of prophylactic vitrectomy for acute retinal necrosis syndrome(ARN) with routine treatment in Chinese patients, thereby investigate the necessity of prophylactic vitrectomy for ARN. METHODS: Thirty patients (37 eyes) were retrospectively included in this study. The eyes were divided into 2 groups by treatment, including routine treatment, which consisted of antiviral medication and vitrectomy after retinal detachment (RD) (n=21), and prophylactic vitrectomy, which consisted of antiviral medication and vitrectomy for the prevention of RD performed during the active inflammatory phase (n=16). The extent of necrosis was determined by fundus photographs at the time of presentation (for eyes with mild vitreous opacity) or the drawings in the operation records. Necrosis of the 37 eyes was divided into 3 grades, including peripheral, middle-peripheral and extensive. The follow-up period ranged from 8 to 57 months. Differences in visual acuity and necrosis between groups were identified using independent samples t-test. RESULTS: Necrosis was more extensive in the routine treatment group than in the prophylactic vitrectomy group (P<0.05). In the routine treatment group, conservative treatment improved necrosis and prevented RD in 6 eyes (29%). Seven eyes (33%) obtained anatomical success, but retinal redetachment occurred in 8 eyes (57%). There were also 5 eyes (24%) developed ocular hypotony or atrophy. Ten eyes (48%) achieved equal or increased visual acuity. In the prophylactic vitrectomy group, RD occurred in 2 eyes (13%). Twelve eyes (75%) were completely anatomically successful, and 10 eyes underwent silicone oil removal. Only one eye (6%) became ocular hypotony. Fourteen eyes (88%) achieved equal or increased visual acuity. The prophylactic vitrectomy group achieved better vision trends than the routine treatment group (P<0.05). Eyes with peripheral necrosis had better visual outcomes than those with mid-peripheral (P<0.05) or extensive (P<0.05) necrosis. However, there was no significant difference between eyes with mid-peripheral and extensive necrosis (P=0.3008) CONCLUSION: Prophylactic vitrectomy can prevent RD and improve the prognosis of ARN, making it an option for cases with rapidly progressing necrosis despite antiviral treatment and cases with moderate to extensive necrosis and severe vitreous opacity.

Survivin and p53 expression in primary and recurrent pterygium in Chinese patients.

AIM: To assess the expression of anti-apoptotic protein survivin and tumor suppressor p53 protein in primary and recurrent pterygium and to investigate the relationship between them. METHODS: Survivin was assessed immunohistochemically using rabbit polyclonal antibody and p53 using mouse monoclonal antibody in a study sample of 20 cases of primary pterygium, 10 cases of recurrent pterygium and 10 cases of normal conjunctiva. RESLULTS: In our study, 35% of primary (7 of 20) and 40% of recurrent (4 of 10) pterygium specimens were positive for survivin staining; 45% of primary (9 of 20) and 50% of recurrent (5 of 10) pterygium specimens were positive for p53 expression; and all normal conjunctiva showed no staining of either survivin or p53. The p53 and survivin immunoreactivity in primary and recurrent pterygium groups was greater than those in normal conjunctiva group (P<0.05). There were no differences in p53 and survivin immunoreactivity between groups of primary and recurrent pterygium (P>0.05). The expression of survivin clearly segregated with p53-positive pterygium as compared with p53-negative cases [8 of 14 cases (57.1%) vs 3 of 16 cases (15.2%)]. The Fisher's exact test analysis confirmed a highly statistically significant correlation between survivin and p53 expression (P<0.05). CONCLUSION: The survivin and p53 are overexpressed with correlation between them in primary and recurrent pterygium.

Inhibitory effect of paclitaxel on endothelial cell adhesion and migration.

The long-term success of percutaneous coronary interventions has been limited by restenosis. Therefore, local delivery of paclitaxel, an antiproliferative agent, using drug-eluting stents has been applied to prevent in-stent restenosis. However, paclitaxel not only inhibits smooth muscle cell proliferation, but also delays re-endothelialization of the damaged site, which may cause potentially life-threatening cardiovascular adverse events, especially late and very late stent thrombosis. We investigated the role of paclitaxel in endothelial cell line ECV304 adhesion and migration. Accordingly, changes in vasodilator-stimulated phosphoprotein protein (VASP) phosphorylation and cAMP-dependent protein kinase activity during ECV304 cell detachment and reattachment were investigated as well. The results showed that the decrease in VASP phosphorylation paralleled the inhibition of cAMP-dependent protein kinase (PKA) activity in the presence of paclitaxel (10 microg/l). Cell adhesion assay and two- and three-dimensional cell migration assays were performed to determine the effect of paclitaxel on the adhesion and migration of ECV304 cells. Paclitaxel significantly suppresses the adhesion (p < 0.05) and migration of ECV304 cells (p < 0.05). These data suggest that the inhibitory effect of paclitaxel may be produced by decreasing the phosphorylation of VASP via inhibition of PKA activity during ECV304 cell adhesion and migration.

[The effects of caspase-3 in apoptosis of cultured retinal microvascular pericytes induced by advanced glycation end products].

OBJECTIVE: One of the earliest changes observed in the development of diabetic retinopathy (DR) is the selective loss of pericytes and acellular capillaries. We tested the hypothesis that advanced glycation end products (AGE) might be involved in the disappearance of retinal pericytes by apoptosis and further investigated the activity and effect of caspase-3 at the same time. METHODS: Cultured bovine retinal microvascular pericytes (BRPs) were exposed to various concentrations of advanced glycation end products-bovine serum albumin (AGE, 0.47, 1.88, 7.50 micromol/L) for 4 days. We assayed the degree of pericytes apoptosis by fluorescence activated cell sorting, and further measured the caspase-3 activity and the effect of selective caspase-3 inhibitor Z-DEVD-fmk on apoptosis and the of ratio Bcl-2/Bax expression. RESULTS: The results showed that AGE could induce significantly the apoptosis of BRPs in a dose-dependent manner compared with controls (r = 0.867, P < 0.01), associated with an increase in intracellular caspase-3 activity. Selective caspase-3 inhibitor Z-DEVD-fmk inhibited pericyte apoptosis induced by AGE. CONCLUSION: These data suggest that the pericyte loss in DR involves an apoptotic process, and that activation of caspase-3 are associated with apoptotic process, which can provide new therapeutic perspectives in diabetic retinopathy.

Advanced glycation end-products induce apoptosis involving the signaling pathways of oxidative stress in bovine retinal pericytes.

One of the histopathologic hallmarks of early diabetic retinopathy is the selective loss of pericytes. Evidences suggest that the pericyte loss in vivo is mediated by apoptosis. However, the underlying cause of pericyte apoptosis is not fully understood. This study investigated the effect of advanced glycation end products (AGEs) on apoptotic cell death in bovine retinal pericytes (BRPs). After incubation of BRPs with 0.47, 1.88, 7.5, 30 microM of AGE-bovine serum albumin (BSA) for 4 days, we assayed the pericytes apoptosis by FACS (fluorescence activated cell sorting), and further measured the signaling pathway involved. The results showed that AGE-BSA could induce significantly the apoptosis of BRPs in a dose-dependent manner compared with controls, associated with an increase in intracellular malondialdehyde level and caspase-3 activity; a decrease in intracellular catalase, SOD activities and Bcl-2/Bax ratio. SOD and selective caspase-3 inhibitor Z-DEVD-fmk can inhibit pericyte apoptosis induced by AGE-BSA. These data suggest that the pericyte loss in diabetic retinopathy involves an apoptotic process, and that elevated AGE observed in diabetes may cause apoptosis in BRPs through an oxidative stress mechanism. The decreased Bcl-2/Bax ratio and activation of caspase-3 are associated with apoptotic process.

[Advanced glycation end products induce apoptosis and expression of apoptotic genes in cultured bovine retinal capillary pericytes].

OBJECTIVE: The purpose of the present study was to examine whether advanced glycation end products (AGE) contribute to the development of apoptosis and expression of apoptotic genes in cultured bovine retinal capillary pericytes (BRPs), in order to investigate retinal microvascular pathologic process during early stage of diabetic retinopathy. METHODS: After a 4 day incubation with various concentrations of AGE (8, 32, 125, 500, 2,000 mg/L) which were prepared in vitro by incubating bovine serum albumin (BSA) with glucose, we studied the degree of apoptosis and the expressions of apoptotic genes (Bax and bcl-2) in BRPs by staining with the Annexin V-FITC and propidium iodide (PI), terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and immunohistochemistry. RESULTS: (1) After a 4 day incubation with various concentrations of AGE, BRPs showed typical changes of apoptosis, i.e. shrunken cell size, nuclear condensation associated with DNA fragmentation, a relatively intact cell membrane, and loss of cell viability eventually. (2) AGE could induce significant apoptosis of BRPs in a dose-dependent manner as compared with normal cultures or the albumin group not treated by glucose (r = 0.878, P < 0.01). (3) After a 4 day incubation, the level of pro-apoptotic gene Bax was upregulated (r = 0.855, P < 0.01), whereas the level of pro-survival gene bcl-2 was downregulated by AGE in a dose-dependent manner (r = -0.850, P < 0.01). (4) There was a positive correlation between apoptotic rate and Bax-bcl-2 ratio of BRPs induced by AGE (r = 0.808,P < 0.01). CONCLUSIONS: AGE can induce a significant apoptosis of BRPs in a dose-dependent manner and the rate of apoptosis was determined by the Bax/bcl-2 ratio. These results suggest that the selective loss of pericytes in diabetic retinopathy involves an apoptotic process.

[Two-dimensional gel electrophoresis of matrix metalloproteinases in subretinal fluids].

In this work, the experimental conditions for two-dimensional gel electrophoresis of subretinal fluids (SRF) matrix metalloproteinases were established. The conditions tested included the composition of lysis solution and lysis method, the composition of rehydration solution and isoelectric focusing program (IEF), the composition of equilibration buffer and equilibration process and the composition of incubation solution and incubation methods. The main equipments used were IPGphor isoelectric focusing system from Amersham pharmacia and PROTEAN II xi cell from Bio-Rad, the gel strips used were the 18 cm long, pH 3 - 8 Linear immobiline DryStrips. Among the 9 samples analyzed, 2 were PVR-A, 2 were PVR-C1, 2 were PVR-C2, 2 were PVR-C3 and the remaining one could not be classified definitely. The new 2-DE MMPs method is better than Gelatin SDS-PAGE zymograhpy method, as it is higher in resolution, sensitivity and reproducibility. The experimental results suggested that the four types of MMPs expressed differently at different stages of PVR. Two of the MMPs isomers have same molecular weight (MW) but different in isoelectic points (pI). The four MMPs are determined to be MMP-1, MMP-2, MMP-9 and MMP-9, with MMP-9 has two active forms. In addition, MMP-9 and MMP-1 may be present in PVR-A samples but not in PVR-C samples, whereas MMP-2 is present in PVR-C but not in PVR-A samples. These results revealed the complex profiles of MMPs' expression in PVR. The new method can be applied to test MMPs expression in tissues, cells and other types of samples with a little modification in the protocol, and can be followed by mass spectroscopic analysis of MMPs.