Cyclen-based cationic lipids for highly efficient gene delivery towards tumor cells

Background

Gene therapy has tremendous potential for both inherited and acquired diseases. However, delivery problems limited their clinical application, and new gene delivery vehicles with low cytotoxicity and high transfection efficiency are greatly required.

Methods

In this report, we designed and synthesized three amphiphilic molecules (L1–L3) with the structures involving 1, 4, 7, 10-tetraazacyclododecane (cyclen), imidazolium and a hydrophobic dodecyl chain. Their interactions with plasmid DNA were studied via electrophoretic gel retardation assays, fluorescent quenching experiments, dynamic light scattering and transmission electron microscopy. The in vitro gene transfection assay and cytotoxicity assay were conducted in four cell lines.

Results

Results indicated that L1 and L3-formed liposomes could effectively bind to DNA to form well-shaped nanoparticles. Combining with neutral lipid DOPE, L3 was found with high efficiency in gene transfer in three tumor cell lines including A549, HepG2 and H460. The optimized gene transfection efficacy of L3 was nearly 5.5 times more efficient than that of the popular commercially available gene delivery agent Lipofectamine 2000™ in human lung carcinoma cells A549. In addition, since L1 and L3 had nearly no gene transfection performance in normal cells HEK293, these cationic lipids showed tumor cell-targeting property to a certain extent. No significant cytotoxicity was found for the lipoplexes formed by L1–L3, and their cytotoxicities were similar to or slightly lower than the lipoplexes prepared from Lipofectamine 2000™.

Conclusion

Novel cyclen-based cationic lipids for effective in vitro gene transfection were founded, and these studies here may extend the application areas of macrocyclic polyamines, especially for cyclen.     

Activation of gelatinases by fibrin is PA/plasmin system-dependent in human glomerular endothelial cells

Evidence suggests that fibrin deposit is related to severity of glomerulonephropathy. Fibrin is considered to play an active role beyond a haemostatic plug or temporary matrix in response to injury. We have reported that fibrin induced specific morphological changes and up-regulated intercellular adhesion molecule-1 expression of glomerular endothelial cells (GECs). Changes of gelatinases activity have been implicated playing a prominent role in glomerular diseases involving matrix turnover. This study examined whether overlying fibrin influences the expression of gelatinase A and B in cultured human GECs and mechanism underlying the activation. No gelatinase activity was detectable in supernatant of cultured GECs; however, physiological concentration of fibrin (0.5–2.0 mg/ml) induced a dramatic expression of activated MMP-2 and MMP-9 at both mRNA and protein level in a dose and time dependent manner. Increased mRNA level of membrane-type 1 matrix metalloproteinases (MT1-MMPs) was also found. Interestingly, we observed that fibrin also induced the expression of tissue type plasminogen activator (tPA), urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 by casein zymographic and reverse zymographic analysis. Fibrin plate assay revealed the net activity was PA predominant. Serine protease inhibitor aprotinin blocked the conversion of pro-gelatinase A and B to their active forms. The results demonstrate that overlying fibrin increased the secretion of gelatinase A and B from GECs. PA/plasmin proteolytic pathways contributed to the activation of gelatinases.     

Safety evaluation of DT388IL3, a diphtheria toxin/interleukin 3 fusion protein, in the cynomolgus monkey

We developed a fusion toxin, DT₃₈₈IL3, consisting of the catalytic and translocation domains of diphtheria toxin (DT₃₈₈) linked to interleukin 3 (IL3) for the treatment of patients with acute myeloid leukemia (AML). Our goal in this study was to estimate a range for the maximum tolerated dose (MTD) and to evaluate the dose-limiting toxicity (DLT) of DT₃₈₈IL3 in cynomolgus monkeys (Macaca fasicularis), which possess cross-reactive IL3 receptors. In our previous study, we administered up to six infusions of DT₃₈₈IL3 at 40, 60, or 100 g/kg every other day to three pairs (one male monkey and one female monkey) of young adult monkeys. In five of six monkeys, results showed a dose-dependent increase in malaise and anorexia but no consistent abnormalities in serum chemistries or blood counts. There was no evidence of organ damage by blood tests or histopathology. However, the female treated at 100 g/kg, died of moderate to severe vasculitis of multiple tissues. Based on these findings, this study repeated the 100 g/kg group and added a group that received 150 g/kg in an effort to confirm a dose response. Two female monkeys were treated with up to six infusions of DT₃₈₈IL3 at 100 g/kg or 150 g/kg every other day. One additional female monkey was treated as a negative control. Monkeys in the 100 g/kg group showed moderate malaise and anorexia, but no consistent abnormalities in blood counts or serum chemistries. Moderate elevations of liver enzymes were noted in the 150 g/kg group in addition to severe malaise and anorexia. No significant findings were revealed at gross necropsy. The histopathological findings revealed regenerative myeloid hyperplasia and hepatic degeneration and regeneration in the 150 g/kg group. Similar lesions of less severity were detected in the 100 g/kg group. DT₃₈₈IL3 plasma half-life was approximately 20 min with a peak concentration of approximately 2 g/ml (30,000 pM). The IC₅₀ for AML blasts in vitro was 6 pM. Collectively, our results suggest that DT₃₈₈IL3 can be tolerated at doses up to 100 g/kg in a nonhuman primate, which is higher than previously reported for other AML directed diphtheria toxin fusion proteins, and should in principle allow for dose escalation with reduced toxic side effects. Based on these findings a phase I clinical trial has recently been initiated with DT₃₈₈IL3 for the treatment of AML.     

Effects of gender and hypovolemia on sympathetic neural responses to orthostatic stress

We tested the hypothesis that women have blunted sympathetic neural responses to orthostatic stress compared with men, which may be elicited under hypovolemic conditions. Muscle sympathetic nerve activity (MSNA) and hemodynamics were measured in eight healthy young women and seven men in supine position and during 6 min of 60 degrees head-up tilt (HUT) under normovolemic and hypovolemic conditions (randomly), with approximately 4-wk interval. Acute hypovolemia was produced by diuretic (furosemide) administration approximately 2 h before testing. Orthostatic tolerance was determined by progressive lower body negative pressure to presyncope. We found that furosemide produced an approximately 13% reduction in plasma volume, causing a similar increase in supine MSNA in men and women (mean +/- SD of 5 +/- 7 vs. 6 +/- 5 bursts/min; P = 0.895). MSNA increased during HUT and was greater in the hypovolemic than in the normovolemic condition (32 +/- 6 bursts/min in normovolemia vs. 44 +/- 15 bursts/min in hypovolemia in men, P = 0.055; 35 +/- 9 vs. 45 +/- 8 bursts/min in women, P < 0.001); these responses were not different between the genders (gender effect: P = 0.832 and 0.814 in normovolemia and hypovolemia, respectively). Total peripheral resistance increased proportionately with increases in MSNA during HUT; these responses were similar between the genders. However, systolic blood pressure was lower, whereas diastolic blood pressure was similar in women compared with men during HUT, which was associated with a smaller stroke volume or stroke index. Orthostatic tolerance was lower in women, especially under hypovolemic conditions. These results indicate that men and women have comparable sympathetic neural responses during orthostatic stress under normovolemic and hypovolemic conditions. The lower orthostatic tolerance in women is predominantly because of a smaller stroke volume, presumably due to less cardiac filling during orthostasis, especially under hypovolemic conditions, which may overwhelm the vasomotor reserve available for vasoconstriction or precipitate neurally mediated sympathetic withdrawal and syncope.     

DGGE analysis of 16S rDNA of ammonia-oxidizing bacteria in chemical–biological flocculation and chemical coagulation systems

Microbial community DNA was extracted from activated sludge samples taken from a chemical bioflocculation process and a chemical coagulation process in Shanghai, China. 16S rDNA of ammonia-oxidizing bacteria (AOB)was amplified by nested polymerase chain reaction and fingerprinted by denaturing gradient gel electrophoresis for microbial structure analysis. The Shannon diversity index of each sample was determined. The results indicated that the microbial structure of AOB in chemical bioflocculation process was comparable at two operational conditions. The ammonia-oxidizing bacterial communities were similar in three channels of the chemical bioflocculation process and in three serial tanks in the chemical coagulation process at the same condition. The diversity of microbial structures in the chemical bioflocculation process was higher than in the chemical coagulation process, in which the microbial structure was similar to that in the influent. Although the microbial study provides insights to the nitrification removal, higher microbial diversity of AOB does not necessarily mean higher ammonia oxidization. Molecular analysis should be combined with chemical assays to optimize operational conditions.     

Mass spectrometric characterization of crustacean hyperglycemic hormone precursor-related peptides (CPRPs) from the sinus gland of the crab, Cancer productus

Crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs) are produced during the proteolytic processing of CHH preprohormones. Currently, the physiological roles played by CPRPs are unknown. Due to their large size, direct mass spectrometric sequencing of intact CPRPs is difficult. Here, we describe a novel strategy for sequencing Cancer productus CPRPs directly from a tissue extract using nanoflow liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry. Four novel CPRPs were characterized with the aid of MS/MS de novo sequencing of 27 truncated CPRP peptides. Extensive modifications (methionine oxidation and carboxy-terminal methylation) were identified in both the full-length and truncated peptides. To investigate the origin of the modifications and truncations, a full-length CPRP was synthesized and subjected to the same storage and extraction protocols used for the characterization of the native peptides. Here, some methionine oxidation was seen, however, no methylation or truncation was evident suggesting much of the chemical complexity seen in the native CPRPs is unlikely due to a sample preparation artifact. Collectively, our study represents the most complete characterization of CPRPs to date and provides a foundation for future investigation of CPRP function in C. productus.     

DNA-PKcs, but not TLR9, is required for activation of Akt by CpG-DNA

CpG-DNA and its related synthetic CpG oligodeoxynucleotides (CpG-ODNs) play an important role in immune cell survival. It has been suggested that Akt is one of the CpG-DNA-responsive serine/threonine kinases; however, the target protein of CpG-DNA that leads to Akt activation has not been elucidated. Here, we report that ex vivo stimulation of bone marrow-derived macrophages (BMDMs) from mice lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) results in defective phosphorylation and activation of Akt by CpG-DNA. Unexpectedly, loss of the Toll-like receptor 9 has a minimal effect on Akt activation in response to CpG-DNA. Further in vitro analysis using purified DNA-PK and recombinant Akt proteins reveals that DNA-PK directly induces phosphorylation and activation of Akt. In addition, in BMDMs, DNA-PKcs associates with Akt upon CpG-DNA stimulation and triggers transient nuclear translocation of Akt. Thus, our findings establish a novel role for DNA-PKcs in CpG-DNA signaling and define a CpG-DNA/DNA-PKcs/Akt pathway.     

Roles of the Ras-MEK-mitogen-activated protein kinase and phosphatidylinositol 3-kinase-Akt-mTOR pathways in Jaagsiekte sheep retrovirus-induced transformation of rodent fibroblast and epithelial cell lines

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a transmissible lung cancer of sheep. The virus can induce tumors rapidly, and we previously found that the JSRV envelope protein (Env) functions as an oncogene, because it can transform mammalian and avian fibroblast cell lines. (N. Maeda, Proc. Natl. Acad. Sci. USA 98:4449-4454, 2001). The molecular mechanisms of JSRV Env transformation are of considerable interest. Several reports suggested that the phosphatidylinositol 3-kinase/Akt pathway is important for transformation of mammalian fibroblasts but not for chicken fibroblasts. In this study, we found that Akt/mTOR is involved in JSRV transformation of mouse NIH 3T3 fibroblasts, because treatment with the mTOR inhibitor rapamycin reduced transformation. We also found that H/N-Ras inhibitor FTI-277 and MEK1/2 inhibitors PD98059 and U0126 strongly inhibited JSRV transformation of NIH 3T3 fibroblasts, suggesting that the H/N-Ras-MEK-mitogen-activated protein kinase (MAPK) p44/42 pathway is necessary for the transformation. In RK3E epithelial cells, the MEK1/2 inhibitors also eliminated transformation, but FTI-277 only partially inhibited transformation. It was noteworthy that p38 MAPK inhibitors enhanced JSRV transformation in both fibroblasts and epithelial cells. Treatment of transformed cells with p38 inhibitors both increased levels of phospho-MEK1/2 and phospho-p44/42 and induced rapid enhancement of the transformed phenotype. Immunohistochemical staining of tumor tissues from naturally and experimentally induced OPA and naturally occurring enzootic nasal adenocarcinoma revealed strong activation of MAPK p44/42 in all cases examined. However, p38 activation was not generally observed. These results indicate that signaling through two pathways (in particular, H/N-Ras-MEK-MAPK and, to a lesser extent, Akt-mTOR) is important for JSRV-induced transformation and that p38 MAPK has a negative regulatory effect on transformation, perhaps via MEK1/2 and p44/42.     

Intersubgenomic heterosis in seed yield potential observed in a new type of Brassica napus introgressed with partial Brassica rapa genome

This paper reports the observation on the intersubgenomic heterosis for seed yield among hybrids between natural Brassica napus (AⁿAⁿCⁿCⁿ) and a new type of B. napus with introgressions of genomic components of Brassica rapa (A^rA^r). This B. napus was selected from the progeny of B. napus × B. rapa and (B. napus × B. rapa) × B. rapa based on extensive phenotypic and cytological observation. Among the 129 studied partial intersubgenomic hybrids, which were obtained by randomly crossing 13 lines of the new type of B. napus in F₃ or BC₁F₃ to 27 cultivars of B. napus from different regions as tester lines, about 90% of combinations exceeded the yield of their respective tester lines, whereas about 75% and 25% of combinations surpassed two elite Chinese cultivars, respectively. This strong heterosis was further confirmed by reevaluating 2 out of the 129 combinations in a successive year and by surveying hybrids between 20 lines of the new type of B. napus in BC₁F₅ and its parental B. napus in two locations. Some DNA segments from B. rapa were identified with significant effects on seed yield and yield components of the new type of B. napus in BC₁F₅ and intersubgenomic hybrids in positive or negative direction. It seems that the genomic components introgressed from B. rapa contributed to improvement of seed yield of rapeseed.