Bromoenol Lactone Attenuates Nicotine-Induced Breast Cancer Cell Proliferation and Migration

Objectives

Calcium independent group VIA phospholipase A₂ (iPLA₂β) and Matrix Metalloproteinase-9 (MMP-9) are upregulated in many disease states; their involvement with cancer cell migration has been a recent subject for study. Further, the molecular mechanisms mediating nicotine-induced breast cancer cell progression have not been fully investigated. This study aims to investigate whether iPLA₂β mediates nicotine-induced breast cancer cell proliferation and migration through both in-vitro and in-vivo techniques. Subsequently, the ability of Bromoenol Lactone (BEL) to attenuate the severity of nicotine-induced breast cancer was examined.

Method and Results

We found that BEL significantly attenuated both basal and nicotine-induced 4T1 breast cancer cell proliferation, via an MTT proliferation assay. Breast cancer cell migration was examined by both a scratch and transwell assay, in which, BEL was found to significantly decrease both basal and nicotine-induced migration. Additionally, nicotine-induced MMP-9 expression was found to be mediated in an iPLA₂β dependent manner. These results suggest that iPLA₂β plays a critical role in mediating both basal and nicotine-induced breast cancer cell proliferation and migration in-vitro. In an in-vivo mouse breast cancer model, BEL treatment was found to significantly reduce both basal (p<0.05) and nicotine-induced tumor growth (p<0.01). Immunohistochemical analysis showed BEL decreased nicotine-induced MMP-9, HIF-1alpha, and CD31 tumor tissue expression. Subsequently, BEL was observed to reduce nicotine-induced lung metastasis.

Conclusion

The present study indicates that nicotine-induced migration is mediated by MMP-9 production in an iPLA₂β dependent manner. Our data suggests that BEL is a possible chemotherapeutic agent as it was found to reduce both nicotine-induced breast cancer tumor growth and lung metastasis.     

The Role of Individual Domains and the Significance of Shedding of ATP6AP2/(pro)renin Receptor in Vacuolar H+-ATPase Biogenesis

The ATPase 6 accessory protein 2 (ATP6AP2)/(pro)renin receptor (PRR) is essential for the biogenesis of active vacuolar H⁺-ATPase (V-ATPase). Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD) and transmembrane domain (TM) of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M) and causes X-linked mental retardation Hedera type (MRXSH) and X-linked parkinsonism with spasticity (XPDS) in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.     

PI3K/Akt Signal Pathway Involved in the Cognitive Impairment Caused by Chronic Cerebral Hypoperfusion in Rats

Chronic cerebral hypoperfusion (CCH) is a common pathophysiological state that usually occurs in conditions such as vascular dementia and Alzheimer''s disease, both of which are characterized by cognitive impairment. In previous studies we found that learning capacity and memory were gradually impaired with CCH, which altered the expression of synaptophysin, microtubule associated protein-2, growth associated protein-43, brain-derived neurotrophic factor, nerve growth factor, N-methyl-D-aspartate receptor subunit 1, cAMP response element-binding protein and tau hyperphosphorylation in the hippocampus. However, the molecular basis of cognitive impairment in CCH remains obscure. Here we explore the hypothesis that the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signal pathway is involved in this type of cognitive impairment. In order to determine if the expression of PI3K, Akt and phosphorylated Akt (p-Akt) proteins are altered at different stages of CCH with differing levels of cognitive impairment. we performed permanent, bilateral occlusion of the common carotid arteries (2-VO) to induce CCH. Adult male SD rats were randomly divided into sham-operated group, 2-VO 1 week group, 2-VO 4 weeks group and 2-VO 8 weeks group. Behavior tests were utilized to assess cognitive abilities, while western blots were utilized to evaluate protein expression. Rats in the 2-VO groups spent less time exploring novel objects than those in the sham-operated group, and the discrimination ratio of the 2-VO 8 weeks group and the sham-operated group were higher than chance (0.50). Escape latencies in the Morris water maze task in the 2-VO 1 week group were longer than those in the sham-operated group on day 4 and day 5, while escape latencies in the 2-VO 4 weeks group were longer than those in the sham-operated group from day 3 to day 5. Escape latencies in 2-VO 8 weeks group were longer than those in the sham-operated group from day 2 to day 5. NE (northeast) square swimming times in the 2-VO 1 week group, 2-VO 4 weeks group and 2-VO 8 weeks group were shorter than that in the sham-operated group. Western blotting showed that the PI3K expression in the 2-VO 1 week group was lower than that in sham-operated group, while p-Akt expression in the 2-VO 8 weeks group was higher than that in the sham-operated group. There was a linear relationship between the PI3K expression and the discrimination ratio, as well as a linear relationship between the PI3K and NE square swimming time. Thus, we propose that the PI3K/Akt signal pathway is an important cell pathway that is associated with the cognitive impairment following CCH.     

Antitumor activity of Pulsatilla chinensis (Bunge) Regel saponins in human liver tumor 7402 cells in vitro and in vivo

Pulsatilla chinensis (Bunge) Regel is a Chinese medicinal herb for "blood-cooling" and detoxification. Now it is used for the treatment of malignant tumor, but the antitumor mechanisms and toxic side effects of P. chinensis are unclear. The present study was undertaken to investigate if P. chinensis saponins (PRS) possesses anticancer effects and toxic side effects in human liver tumor 7402 cells in vitro and vivo. 7402 cells were treated with different concentrations of PRS for 24h. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis was assessed by flow cytometry. The in vivo effect of PRS on 7402 tumor cells transplanted in athymic nude mice was investigated. 15 saponins were isolated and identified from PRS. PRS inhibited the proliferation of human liver tumor 7402 cells in vitro by apoptosis. 19 days after administration of PRS (100, 200mg/kg), the weight of tumor mass was markly decreased in nude mice. The anti-tumor effect of PRS in vivo was associated with a significant increase in the 7402 apoptosis rate. Although PRS inhibited the weight of mice, it showed almost no effect on leukocyte number, liver and spleen weight index. Light microscopic histopathological examination showed that PRS had no specific lesion in organ. These results suggested that P. chinensis saponins exert potential anticancer activity in treating tumors in nude mice and no toxic side effects.     

人群预存免疫力是2009甲型H1N1流感大流行表现温和的重要原因

历史上最具杀伤力的1918年西班牙流感大流行由H1N1亚型流感病毒引起[1],随后H1N1亚型流感继续在人群中流行,并且在20世纪20年代到50年代又引起了数次暴发[2-3]。1957年,H1N1流     

Characterization of Syrian hamster adapted prions derived from L-type and C-type bovine spongiform encephalopathies

Atypical forms of bovine spongiform encephalopathy (BSE) may be caused by different prions from classical BSE (C-BSE). In this study, we examined the susceptibility of mice overexpressing mouse and hamster chimeric prion protein (PrP) to L-type atypical BSE (L-BSE). None of the transgenic mice showed susceptibility to L-BSE, except mice overexpressing hamster PrP. We also examined the transmission properties of L-BSE in hamsters. The incubation period of hamsters intracerebrally inoculated with L-BSE was 576.8 days, and that of the subsequent passage was decreased to 208 days. Although the lesion and glycoform profiles and relative proteinase K resistant core fragment of the abnormal isoform of PrP (PrPcore) of L-BSE were similar to that of C-BSE, the deposition of the abnormal isoform of PrP (PrP^Sc) and the molecular weight of PrPcore of L-BSE was different from than that of C-BSE. In hamster models, some prion strain characteristics of L-BSE were indistinguishable from those of C-BSE.Key words: prion, atypical, L-BSE, PrPcore, hamster, transmission     

Apolipoprotein A-V association with intracellular lipid droplets

Apolipoprotein A-V (apoA-V) plays a key role in the regulation of triglyceride (TG) metabolism. Given the very low concentration of apoA-V in plasma, we hypothesized that apoA-V may influence plasma TG levels by affecting the assembly and/or secretion of apoB-containing lipoproteins. When apoA-V was overexpressed in cultured Hep3B cells, neither the amount of apoB secreted nor the density distribution of apoB-containing lipoproteins was affected. Fluorescence microscopy and cell lysate immunoprecipitation studies revealed that apoA-V is not associated with apoB intracellularly, yet immunoprecipitation of apoA-V from the cell culture medium resulted in coprecipitation of apoB. These data suggest that the apoA-V association with apoB-containing lipoproteins is a postsecretory event. Confocal fluorescence microscopy revealed the presence of apoA-V in distinct cellular structures. Based on Nile Red staining, we identified these structures to be intracellular lipid droplets. These data suggest that apoA-V has a unique association with cellular lipids and, therefore, may be involved in the storage or mobilization of intracellular lipids.     

Freezing shortens the lifetime of DNA molecules under tension

DNA samples are commonly frozen for storage. However, freezing can compromise the integrity of DNA molecules. Considering the wide applications of DNA molecules in nanotechnology, changes to DNA integrity at the molecular level may cause undesirable outcomes. However, the effects of freezing on DNA integrity have not been fully explored. To investigate the impact of freezing on DNA integrity, samples of frozen and non-frozen bacteriophage lambda DNA were studied using optical tweezers. Tension (5–35 pN) was applied to DNA molecules to mimic mechanical interactions between DNA and other biomolecules. The integrity of the DNA molecules was evaluated by measuring the time taken for single DNA molecules to break under tension. Mean lifetimes were determined by maximum likelihood estimates and variances were obtained through bootstrapping simulations. Under 5 pN of force, the mean lifetime of frozen samples is 44.3 min with 95% confidence interval (CI) between 36.7 min and 53.6 min while the mean lifetime of non-frozen samples is 133.2 min (95% CI: 97.8–190.1 min). Under 15 pN of force, the mean lifetimes are 10.8 min (95% CI: 7.6–12.6 min) and 78.5 min (95% CI: 58.1–108.9 min). The lifetimes of frozen DNA molecules are significantly reduced, implying that freezing compromises DNA integrity. Moreover, we found that the reduced DNA structural integrity cannot be restored using regular ligation process. These results indicate that freezing can alter the structural integrity of the DNA molecules.     

Genetic Linkage Mapping and Analysis of Muscle Fiber-Related QTLs in Common Carp (Cyprinus carpio L.)

A genetic linkage map of common carp (Cyprinus carpio L.) was constructed using Type I and Type II microsatellite markers and a pseudo-testcross mapping strategy. The microsatellite markers were isolated from microsatellite-enriched genomic libraries and tested for their segregation in a full-sib mapping panel containing 92 individuals. A total of 161 microsatellite loci were mapped into 54 linkage groups. The total lengths of the female, male and consensus maps were 2,000, 946, and 1,852?cM, with an average marker spacing of approximately 13, 7, and 11?cM, respectively. Muscle fiber-related traits, including muscle fiber cross-section area and muscle fiber density, were mapped to the genetic map. Three QTLs for muscle fiber cross-section area and two QTLs for muscle fiber density were identified when considering both significant and suggestive QTL effects. The QTLs with largest effects for muscle fiber cross-section area and muscle fiber density were 21.9% and 18.9%, and they were located in LG3, respectively.