Autophagy induced by a sulphamoylated estrone analogue contributes to its cytotoxic effect on breast cancer cells

Background

Autophagy can either be protective and confer survival to stressed cells, or it can contribute to cell death. The antimitotic drug 2-ethyl-3-O-sulpamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) is an in silico-designed 17-β-estradiol analogue that induces both autophagy and apoptosis in cancer cells. The aim of the study was to determine the role of autophagy in ESE-15-ol-exposed human adenocarcinoma breast cancer cells; knowledge that will contribute to future clinical applications of this novel antimitotic compound. By inhibiting autophagy and determining the cytotoxic effects of ESE-15-ol-exposure, deductions could be made as to whether the process may confer resistance to the drug, or alternatively, contribute to the cell death process.

Methods and results

Spectophometrical analysis via crystal violet staining was used to perform cytotoxicity studies. Morphology studies were done using microscopic techniques namely polarization-optical transmitted light differential interference light microscopy, fluorescent microscopy using monodansylcadaverine staining and transmission electron microscopy. Flow cytometry was used to quantify the autophagy inhibition and assess cell viability. Results obtained indicated that 3-methyladenine inhibited autophagy and increased cell survival in both MCF-7 and MDA-MB-231 cell lines.

Conclusion

This in vitro study inferred that autophagy inhibition with 3-methyladenine does not confer increased effectiveness of ESE-15-ol in inducing cell death. Thus it may be concluded that the autophagic process induced by ESE-15-ol exposure in MCF-7 and MDA-MB-231 cells plays a more significant role in cell death than conferring survival.

     

功能矫形前伸青春期大鼠下颌后浅层嚼肌细胞凋亡的研究

目的：研究功能矫形前伸大鼠下颌后浅层嚼肌细胞凋亡的变化规律,探讨功能矫形的肌肉改建机理。方法：选用50只5周龄Sprague-Dawley（SD）雄性大白鼠,随机分为实验组和对照组各25只。实验组大鼠戴自制上颌功能矫治嚣,引导下颌前伸,并打开咬合。利用RT-PCR方法检测两组大鼠浅层嚼肌Bcl-2和Bax基因表达情况,利用TUNEL方法检测浅层嚼肌细胞凋亡情况。结果：①Bcl-2和Bax基因表达随大鼠戴用矫治器时间的延长而升高,至第3周开始下降但仍高于对照组,但Bax的表达高于Bcl-2。Bax/Bcl-2比值随大鼠戴用矫治器时间的延长而升高,至第4周开始下降。②TUNEL实验结果显示浅层嚼肌细胞在戴用矫治器1天后,开始出现凋亡,随着时间延长而增加,至第3周达到顶峰,第4周开始下降。结论：①Bax/Bcl-2比值升高促进浅层嚼肌细胞凋亡。②功能矫形可引起浅层嚼肌细胞凋亡,导致肌肉的结构和功能发生适应性改建。     

功能矫形前伸青春期大鼠下颌后翼外肌MyoD、myogenin的表达

目的：探讨＂应力-生长（改建）＂在细胞水平上的体现,为功能矫形治疗和矫治效果的保持提供新思路和实验依据。方法：本实验选用20只4周龄,雄性SD大鼠随机分为8组。其中实验组大鼠经戊巴比妥麻醉后佩戴上颌斜面导板,对照组未佩用。依据时间不同又分为四组：1d,7d,14d,21d。采用RT-PCR技术分析各组大鼠翼外肌组织中肌分化相关基因MyoD、myogenin mRNA的表达变化。结果：未施加功能矫形力的大鼠翼外肌组织MyoD表达伴随其生长发育呈现递减趋势,实验组在第7 d出现表达上调。同时,力学刺激后实验组动物myogenin的表达与对照组相比较在14 d组出现明显上调。结论：功能矫形力作用于翼外肌组织可以诱导MyoD和myogenin的表达上调进而诱导成肌细胞的分化。     

基于钙黄绿素- 铜(Ⅱ)荧光体系测定乙酰半胱氨酸

目的:基于钙黄绿素-铜(Ⅱ)荧光体系测定乙酰半胱氨酸。方法:在pH=8.0的Na2HPO.412H2O-KH2PO4缓冲液中,以492 nm为激发波长,520 nm为发射波长测定乙酰半胱氨酸溶液的荧光强度。结果:在pH=8.0的Na2HPO.412H2O-KH2PO4缓冲液中,二价铜离子与钙黄绿素配位引起荧光猝灭。由于乙酰半胱氨酸中巯基上的硫离子与Cu2+的亲和力很强,可从钙黄绿素-铜(Ⅱ)的络合物中夺取铜离子而使钙黄绿素游离出来,从而使体系的荧光得以恢复,并且荧光恢复的程度与加入乙酰半胱氨酸的量在一定范围内成线性。结论:建立了一种测定乙酰半胱氨酸的荧光分析新方法,该方法的线性范围为6.0 10-6～1.4 10-5 mol/L,检出限为4.010-6 mol/L。     

The hedgehog receptor patched is involved in cholesterol transport

Background

Sonic hedgehog (Shh) signaling plays a crucial role in growth and patterning during embryonic development, and also in stem cell maintenance and tissue regeneration in adults. Aberrant Shh pathway activation is involved in the development of many tumors, and one of the most affected Shh signaling steps found in these tumors is the regulation of the signaling receptor Smoothened by the Shh receptor Patched. In the present work, we investigated Patched activity and the mechanism by which Patched inhibits Smoothened.

Methodology/Principal Findings

Using the well-known Shh-responding cell line of mouse fibroblasts NIH 3T3, we first observed that enhancement of the intracellular cholesterol concentration induces Smoothened enrichment in the plasma membrane, which is a crucial step for the signaling activation. We found that binding of Shh protein to its receptor Patched, which involves Patched internalization, increases the intracellular concentration of cholesterol and decreases the efflux of a fluorescent cholesterol derivative (BODIPY-cholesterol) from these cells. Treatment of fibroblasts with cyclopamine, an antagonist of Shh signaling, inhibits Patched expression and reduces BODIPY-cholesterol efflux, while treatment with the Shh pathway agonist SAG enhances Patched protein expression and BODIPY-cholesterol efflux. We also show that over-expression of human Patched in the yeast S. cerevisiae results in a significant boost of BODIPY-cholesterol efflux. Furthermore, we demonstrate that purified Patched binds to cholesterol, and that the interaction of Shh with Patched inhibits the binding of Patched to cholesterol.

Conclusion/Significance

Our results suggest that Patched may contribute to cholesterol efflux from cells, and to modulation of the intracellular cholesterol concentration. This activity is likely responsible for the inhibition of the enrichment of Smoothened in the plasma membrane, which is an important step in Shh pathway activation.     

An IGF-I promoter polymorphism modifies the relationships between birth weight and risk factors for cardiovascular disease and diabetes at age 36

BACKGROUND: Biochemical testing for pheochromocytoma by measurement of fractionated plasma metanephrines is limited by false positive rates of up to 18% in people without known genetic predisposition to the disease. The plasma normetanephrine fraction is responsible for most false positives and plasma normetanephrine increases with age. The objective of this study was to determine if we could improve the specificity of fractionated plasma measurements, by statistically adjusting for age. METHODS: An age-adjusted metanephrine score was derived using logistic regression from 343 subjects (including 33 people with pheochromocytoma) who underwent fractionated plasma metanephrine measurements as part of investigations for suspected pheochromocytoma at Mayo Clinic Rochester (derivation set). The performance of the age-adjusted score was validated in a dataset of 158 subjects (including patients 23 with pheochromocytoma) that underwent measurements of fractionated plasma metanephrines at Mayo Clinic the following year (validation dataset). None of the participants in the validation dataset had known genetic predisposition to pheochromocytoma. RESULTS: The sensitivity of the age-adjusted metanephrine score was the same as that of traditional interpretation of fractionated plasma metanephrine measurements, yielding a sensitivity of 100% (23/23, 95% confidence interval [CI] 85.7%, 100%). However, the false positive rate with traditional interpretation of fractionated plasma metanephrine measurements was 16.3% (22/135, 95% CI, 11.0%, 23.4%) and that of the age-adjusted score was significantly lower at 3.0% (4/135, 95% CI, 1.2%, 7.4%) (p < 0.001 using McNemar's test). CONCLUSION: An adjustment for age in the interpretation of results of fractionated plasma metanephrines may significantly decrease false positives when using this test to exclude sporadic pheochromocytoma. Such improvements in false positive rate may result in savings of expenditures related to confirmatory imaging.     

Ex vivo modulation of chemical-induced mutagenesis by subcellular liver fractions of rats treated with rooibos (Aspalathus linearis) tea, honeybush (Cyclopia intermedia) tea, as well as green and black (Camellia sinensis) teas

Male Fischer rats were given unprocessed (not oxidized) and processed (oxidized) rooibos and honeybush teas as well as green and black teas as a sole source of drinking fluid for 10 weeks, and sub cellular liver fractions were prepared. Cytosolic fractions of rats consuming the unprocessed herbal teas, green and black teas significantly (P < 0.05) protected against 2-acetylaminofluorene (2-AAF)-induced mutagenesis in the Salmonella mutagenicity test with strain TA 98, using Aroclor 1254-induced microsomes. A marginal or no protection was obtained with the processed herbal teas. The mutagenic response of aflatoxin B1 (AFB1) against Salmonella strain TA 100 was significantly (P < 0.05) inhibited by cytosolic fractions from rats treated with processed and unprocessed herbal teas, while no effect was obtained with the green and black teas. Microsomal fractions prepared from livers of rats treated with both the processed and unprocessed rooibos teas and the unprocessed honeybush tea, significantly (P < 0.05) reduced the activation of AFB1 while no protection was observed against 2-AAF-induced mutagenesis. In contrast, microsomal fractions from rats treated with the green, black and unprocessed honeybush teas significantly (P < 0.05) enhanced the mutagenic response of 2-AAF. None of the tea treatments significantly affected the concentration of the microsomal liver cytochrome P450.     

TNF-alpha and IFN-gamma inversely modulate expression of the IL-17E receptor in airway smooth muscle cells

The interleukin-17B receptor (IL-17BR) is expressed in a variety of tissues and is upregulated under inflammatory conditions. This receptor binds both its cognate ligand IL-17B and IL-17E/IL-25, a novel cytokine known to promote Th2 responses. The present study shows that airway smooth muscle cells express IL-17BR in vitro and that its expression is upregulated by TNF-alpha and downregulated by IFN-gamma. Our data indicate that TNF-alpha upregulates IL-17BR mainly through nuclear factor-kappaB as assessed with the IkappaB kinase 2 inhibitor AS-602868. In addition, both IFN-gamma and dexamethasone are able to antagonize a TNF-alpha-induced IL-17BR increase in mRNA expression. The mitogen-activated protein kinase kinase inhibitor U0126 totally reversed the inhibition observed with IFN-gamma, suggesting the involvement of the extracellular signal-regulated kinase pathway in this effect. In addition, on stimulation with IL-17E, airway smooth muscle cells increase their expression of ECM components, namely procollagen-alphaI and lumican mRNA. Furthermore, immunohistochemical analysis of biopsies from asthmatic subjects reveals that this receptor is abundant in smooth muscle layers. This is the first report showing IL-17BR receptor in structural cells of the airways. Our results suggest a potential proremodeling effect of IL-17E on airway smooth muscle cells through the induction of ECM and that its receptor is upregulated by proinflammatory conditions.     

Isolation and characterization of microsatellite markers from Pacific white shrimp (Litopenaeus vannamei)

We report the development of 11 polymorphic microsatellite loci in pacific white shrimp (Litopenaeus vannamei) using an unenriched genomic library. The number of the alleles ranged from two to 18 and observed hererozygosity ranged from 0.0286 to 0.9429, indicating that these markers will be useful for population studies and mapping in pacific white shrimp. Seven loci were detected deviated from Hardy–Weinberg, caused by deficiency of heterozygote, suggesting population genetic structure across the sampled population. No evidence for linkage disequilibrium was found.     

A comparative study on the antimutagenic properties of aqueous extracts of Aspalathus linearis (rooibos), different Cyclopia spp. (honeybush) and Camellia sinensis teas

Antimutagenic activity of aqueous extracts of the South African herbal teas, Aspalathus linearis (rooibos) and Cyclopia spp. (honeybush) was compared with that of Camellia sinensis (black, oolong and green) teas in the Salmonella mutagenicity assay using aflatoxin B(1) (AFB(1)) and 2-acetylaminofluorene (2-AAF) as mutagens. The present study presents the first investigation on antimutagenic properties of C. subternata, C. genistoides and C. sessiliflora. The herbal teas demonstrated protection against both mutagens in the presence of metabolic activation, with the exception of "unfermented" (green/unoxidised) C. genistoides against 2-AAF, which either protected or enhanced mutagenesis depending on the concentration. Antimutagenic activity of "fermented" (oxidised) rooibos was significantly (P<0.05) less than that of Camellia sinensis teas against AFB(1), while for 2-AAF it was less (P<0.05) than that of black tea and similar (P>0.05) to that of oolong and green teas. Antimutagenic activity of unfermented C. intermedia and C. subternata exhibited a similar protection as fermented rooibos against AFB(1). Against 2-AAF, fermented rooibos exhibited similar protective properties than unfermented C. intermedia and C. sessiliflora. Unfermented rooibos was less effective than the C. sinensis teas and fermented rooibos, but had similar (P>0.05) antimutagenicity to that of fermented C. sessiliflora against AFB(1) and fermented C. subternata against 2-AAF. Fermented C. intermedia and C. genistoides exhibited the lowest protective effect against 2-AAF, while fermented C. intermedia exhibited the lowest protection when utilising AFB(1) as mutagen. Aspalathin and mangiferin, major polyphenols in rooibos and Cyclopia spp., respectively, exhibited weak to moderate protective effects when compared to the major green tea catechin, (-)epigallocatechin gallate (EGCG). Antimutagenic activity of selected herbal tea phenolic compounds indicated that they contribute towards (i) observed antimutagenic activity of the aqueous extracts against both mutagens and (ii) enhancement of the mutagenicity of 2-AAF by unfermented C. genistoides. Antimutagenic activity of the South African herbal teas was mutagen-specific, affected by fermentation and plant material, presumably due to changes and variation in phenolic composition.