Inhibitory effect of 5F on development of lung cancer in A/J mice

The purpose of the study is to investigate the effect of ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) on the model of induced A/J mice lung cancer in A/J mice. The expressions of tumor-related molecules including P65 and Bcl-2 at protein level were examined using the immunohistochemical method (IHC). Side effects of 5F were also monitored. The results indicated that 5F significantly suppressed the development of B[a]P and NNK-induced lung cancer in vivo by facilitating cell apoptosis with minimal side effects. Compared to the expressions of P65 and Bcl-2 in model group, the levels were strongly attenuated both in blank and 5F injection groups. Moreover, P65 and Bcl-2 levels varied among different groups receiving 5F treatment. The expressions of P65 and Bcl-2 were much lower in groups receiving high-concentration 5F treatment than those with low-concentration 5F injection. Findings revealed that 5F inhibited the pathogenesis of lung cancer through accelerating apoptosis in a dose-dependent manner.     

Early manifestations of NNK-induced lung cancer: role of lung immunity in tumor susceptibility

A strong correlation exists between smoking and lung cancer; however, susceptibility to lung cancer among smokers is not uniform. Similarly, mice show differential susceptibility to the tobacco carcinogen nitrosamine 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which produces lung tumors in A/J but not in C3H mice. Host immunity may play a role in the susceptibility to cancer, and cigarette smoke/nicotine suppresses the immune system through activation of nicotinic acetylcholine receptors (nAChRs). Mammalian lungs express alpha7-nAChRs, and NNK is a high-affinity agonist for alpha7-nAChRs. To examine whether NNK differentially modulates lung immunity in susceptible and resistant mouse strains, A/J and C3H mice were treated with NNK and/or immunized with sheep red blood cells. Lung tissues and RNA of treated and untreated animals were analyzed by immunohistochemistry and RT-PCR for alpha7-nAChR and COX-2 expression. Spleen- and the lung-associated lymph node cells from control and immunized animals were assessed for immunologic responses, including anti-sheep red blood cell antibody plaque-forming cells, concanavalin A-induced T-cell proliferation, and the anti-CD3/CD28 antibody-induced rise in intracellular calcium. NNK strongly suppressed these responses in A/J but not in C3H mice. Similar NNK-induced immunologic changes were seen in another pair of carcinogen-sensitive (NGP) and relatively carcinogen-resistant (B10.A) mouse strains. Moreover, NNK stimulates a significantly higher expression of COX-2 and alpha7-nAChRs in A/J than in C3H lungs. These results suggest that the susceptibility to chemical carcinogenesis among various mouse strains might be influenced by their immunologic response to the carcinogen.     

人经血来源间充质干细胞对小鼠A549肺癌化疗效果的影响研究

目的：探讨人经血来源间充质干细胞(mesenchymal stem cells,MenSCs)对小鼠A549肺癌化疗效果的影响。方法：取20只BALB/C裸小鼠建立肺癌模型,随机分为实验组和对照组各10只;对照组仅给予化疗干预,实验组则于化疗期间将某机构赠予的MenSCs经DiI荧光标记后于鼠尾静脉(3×105/只)进行注射。肺癌造模后14 d,两组小鼠均断颈椎处死并收集其肿瘤,比较两组小鼠瘤体体积、瘤体质量、抑瘤率及双肺湿重、肿瘤转移率、肿瘤转移个数、抑制转移率差异。结果：20只BALB/C裸小鼠均进行统计,无脱落报告。20只小鼠接瘤后第7天在右前肢腋下均可触及瘤体长出,两组小鼠瘤体生长曲线比较,差异无统计学意义(P>0.05)。两组小鼠的平均瘤体体积和平均瘤体质量比较,差异均无统计学意义(P均>0.05);抑瘤率为8.4%。实验组小鼠双肺湿重、肿瘤转移率及肿瘤转移个数均显著低于对照组小鼠,差异有显著性(P<0.05);抑制转移率为71.8%。结论：MenSCs协同化疗在抑制小鼠肺癌肿瘤转移方面有一定效果。     

Replicative Legionella pneumophila lung infection in intratracheally inoculated A/J mice. A murine model of human Legionnaires' disease.

The role of host immune responses in the pathogenesis of Legionnaires' disease is incompletely understood, due in part to the current lack of an animal model that is both susceptible to replicative Legionella pneumophila-induced lung infection and for which species-specific immunological reagents are available. We have developed a model of replicative L. pneumophila lung infection in intratracheally inoculated A/J mice. L. pneumophila was obtained in the exponential growth phase and inoculated into the trachea of 6- to 8-week-old female A/J mice. Microbiological and histopathological evidence of infection was demonstrated in mice inoculated with 10(6) colony-forming units. Development of an acute pneumonia that resembled human Legionnaires' disease coincided with exponential growth of the bacteria in the lung 24 to 48 hours after intratracheal inoculation of L. pneumophila. This was associated with increased plasma levels of interferon-gamma at 24 hours after inoculation. After 48 hours, the bacteria were gradually eliminated from the lung over the next 5 days, corresponding with resolution of the inflammatory response in the lung, thereby mimicking the outcome frequently seen in the immunocompetent human host. Treatment of animals with anti-interferon-gamma antibody enhanced bacterial replication and disease progression, indicating an important role of host immune response in resolution of the infection. Because of the availability of murine-specific reagents, this model of replicative L. pneumophila lung infection in A/J mice after intrapulmonary inoculation of L. pneumophila potentially provides an important tool for future studies investigating the role of host immune responses in the pathogenesis of Legionnaires' disease in the immunocompetent host.     

5-氟尿嘧啶诱导人肺腺癌A549细胞自噬

目的 探讨5-氟尿嘧啶(5-FU)是否诱导人肺腺癌A549细胞发生自噬.方法 吖啶橙(AO)荧光染色和透射电镜观察自噬泡的变化;细胞免疫化学法测定LC3-Ⅱ的表达;流式细胞术及吖啶橙染色结合激光扫描共焦显微镜检测细胞凋亡;DNA电泳检测凋亡梯形条带的产生.结果 吖啶橙荧光染色和透射电镜观察结果显示,5-氟尿嘧啶处理细胞...     

柚皮苷对人肺癌顺铂耐药株A549/DDP细胞的逆转作用

目的:探究柚皮苷(naringin,NRG)对人肺癌A549/DDP细胞顺铂耐药性的影响及其可能的分子机制。方法:采用CCK-8法检测柚皮苷和顺铂对A549/DDP细胞的毒性作用,采用Chou-Talalay中效分析法对两药的联合效应进行评价;采用流式细胞术检测细胞凋亡;采用Western blot法检测P-糖蛋白(P-glycoprotein,P-gp)、多药耐药相关蛋白1(multidrug resistance-associated protein 1,MRP1)、p-Akt、CXC趋化因子受体4(CXC chemokine receptor 4, CXCR4)、Bcl-2、Bax和cleaved caspase-3的蛋白水平。结果:顺铂耐药株A549/DDP细胞中P-gp、MRP1、p-Akt和CXCR4蛋白的水平显著高于非顺铂耐药株A549细胞(P0.05);柚皮苷和顺铂可剂量依赖性地抑制A549/DDP细胞活力(P0.05),IC_(50)分别为36.92μmol/L和129.77μmol/L;当抑制率超过15%时,二者联用呈协同效应;柚皮苷与顺铂可诱导A549/DDP细胞凋亡(P0.05),并且两药联用组的细胞凋亡率高于顺铂组(P0.05);柚皮苷和顺铂可上调Bax和cleaved caspase-3的蛋白水平(P0.05),下调Bcl-2的蛋白水平(P0.05),同时柚皮苷还可剂量依赖性地下调P-gp、MRP1、p-Akt和CXCR4的蛋白水平(P0.05)。结论:柚皮苷可增强人肺癌A549/DDP细胞对顺铂的敏感性。这可能与柚皮苷上调Bax的蛋白水平,下调Bcl-2、P-gp、MRP1、p-Akt和CXCR4的蛋白水平有关。     

C5 deficiency in A/J mice is not associated with resistance to the development of secondary amyloidosis.

The aim of the study was to determine whether C5 deficiency in the mouse is associated with resistance to the development of secondary amyloidosis. Chronic inflammation was induced in the F2 progeny, derived from matings between amyloid-susceptible and amyloid-resistance mice, by daily injections of azocasein for thirty days. Using a restriction fragment length polymorphism generated by digestion of genomic DNA with the restriction enzyme HindIII, C5 sufficient and deficient DNA can be clearly differentiated. Eight mice were found to be C5 sufficient, 32 were heterozygotes and 14 were found to be C5 deficient. Grading of the splenic amyloid load from negative to 4+ was performed after staining tissue squashes with Congo red and viewing them under a polarizing microscope. Seventeen mice were noted to have negative to trace, 18 had moderate (1+ - 2+) and 19 had heavy (3+ - 4+) amyloid deposition. There was no correlation between splenic amyloid load and C5 deficiency. Based on these results it is clear that C5 deficiency and resistance to secondary amyloidosis are not associated.     

水通道蛋白5在人胎肺发育中的表达及意义

目的检测水通道蛋白5（AQP5）在胎儿正常肺组织和发育异常肺组织的表达,探讨AQP5对胎肺发育的影响及其在肺液代谢中的作用。方法应用RT-PCR检测AQP5mRNA在各胎肺中的表达以及real-timePCR检测AQP5在正常胎肺和发育异常胎肺中的基因表达差异。结果AQP5mRNA在各孕周胎肺均有表达,发育异常胎肺组织较正常胎肺组织表达量明显减少,差异有统计学意义（P〈0.05））。结论发育异常胎肺AQP5表达下降,其对胎肺的正常发育和肺液代谢可能有重要影响。     

Gender difference in bone metastasis of human small cell lung cancer,SBC-5 cells in natural killer-cell depleted severe combined immunodeficient mice

Lung cancer frequently develops multiple organ metastases, which thus makes this disease a leading cause of malignancy-related death worldwide. A gender difference is reported to affect the incidence and mortality of lung cancer; however, whether and how the gender difference is involved in lung cancer metastasis is unclear. This study evaluated the gender difference in multiple organ metastases in human small cell lung cancer (SBC-5) cells by using natural killer cell-depleted severe combined immunodeficient mice. Among multiple organ metastases, only bone metastasis formation significantly increased in female mice in comparison to males, while no significant difference was observed in the metastases to the liver and lungs. The suppression of androgen by castration or androgen receptor antagonist treatment in male mice also induced a significant increase of bone metastases. The number of osteoclasts in the bone metastatic lesions was greater in female mice and in mice with androgen suppression than in control male. However, there was no significant difference in the serum concentration of parathyroid hormone-related protein (PTHrP) associated with gender or androgen suppression. An in vitro study also indicated that sex steroid treatment had no effect on the proliferation or PTHrP production in SBC-5 cells. These results indicate that the balance of sex steroids therefore plays an important role in the formation of bone metastasis in small cell lung cancer, and suggests diverse mechanisms of interaction between cancer cells and host cells in the bone microenvironment.     

Cardiac failure in C5-deficient A/J mice after Candida albicans infection

The effect of a deficiency in the C5 component of complement on the pathophyisology of infection with the fungal pathogen Candida albicans was studied by using the A/J inbred mouse strain and the BcA17 congenic mouse strain. Acute infection caused by intravenous injection of C. albicans blastospores is associated with rapid fungal replication in the heart, brain, and, in particular, kidneys of C5-deficient mice. Histological studies and analysis of markers for tissue damage indicated that the heart is the organ that is most affected and that it ultimately fails in C5-deficient mice. In A/J and BcA17 mice, tissue damage is associated with (i) cellular infiltration in the heart, which is not seen in the kidney despite the higher fungal load in the latter organ, and (ii) a very strong inflammatory response, including elevated levels of many cytokines and chemokines. This results in cardiomyopathy, which is associated with elevated levels of creatine kinase and cardiac troponin I in the circulation. Damage to the cardiac muscle is associated with metabolic changes, including hypoglycemia, decreased lipid utilization resulting in elevated levels of cardiac triglycerides, and unproductive glucose utilization linked to a dramatic increase in the level of pyruvate dehydrogenase kinase 4 (Pdk4), a negative regulator of the pyruvate dehydrogenase complex.     

Detection of thrombomodulin in human lung cancer cells.

Thrombomodulin (TM), which usually exists in vascular endothelial cells and exerts an anticoagulant activity, was detected by Western blot analyses and immunocytochemical staining using three anti-TM monoclonal antibodies in cultured cell lines derived from a squamous cell carcinoma and an adenocarcinoma of the lung, but was not detected in a cell line derived from a small cell carcinoma. Functional assays indicated that TM detected in these cells was functionally active. The presence of TM in 22 specimens of surgically removed lung cancer tissue was also examined by an immunohistochemical method. TM was present along the cell membranes in 4 (36%) of 11 squamous cell carcinomas examined, but was not detected in 10 adenocarcinomas and 1 large cell carcinoma examined. Because TM is identical to fetomodulin, which modulates embryogenesis, the authors have concluded that TM is an oncodevelopmental antigen. The authors have also suggested that functionally active TM on lung cancer cells may modulate cancer cell behaviors in such ways as exhibiting anticoagulant activity.     

The characteristics of human NKT cells in lung cancer--CD1d independent cytotoxicity against lung cancer cells by NKT cells and decreased human NKT cell response in lung cancer patients

The activation of human Valpha24+Vbeta11+natural killer T cells (NKT) cells (Valpha24 NKT cells) induces effective antitumor responses with secondary immune effects through activation of conventional T cells and natural killer cells. In this study, we attempted to analyze the characteristics of human NKT cells in lung cancer patients. Valpha24 NKT cells stimulated with alpha-GalCer from healthy volunteers exhibited direct cytotoxic activity against two (RERF-LC-OK and PC-3) of seven human lung cancer cell lines studied. Cytotoxicity by Valpha24 NKT cells against human lung cancer cells was dependent on the perforin pathway and independent of Fas/FasL pathway. Intracellular adhesion molecule (ICAM)-1 expression on tumor cells was clearly associated with the cytotoxicity of Valpha24 NKT cells. On the other hand, the proportion of Valpha24 NKT cells in the patients with lung cancer was lower than that in the healthy volunteers. Furthermore, the proliferative response of Valpha24 NKT cells to alpha-GalCer was significantly lower in the peripheral blood mononuclear cells in the patients with lung cancer. Addition of granulocyte colony-stimulating factor moderately restored the low proliferative response of Valpha24 NKT cells in the patients with lung cancer, however the percentage by which the response was restored in these patients was still lower than the natural response in healthy volunteers. These results suggest that Valpha24 NKT cells may play a pivotal role or the antitumor response in lung cancer.     

纳米羟基磷灰石可介导转染人肺癌A549细胞

目的 探讨纳米羟基磷灰石（nHAP）介导带有增强型绿色荧光蛋白（EGFP）基因的pGenesil-1质粒转染人肺癌A549细胞的可行性。方法 用均相沉淀法制备nHAP;透射电镜观察纳米粒结构;经超声分散及碳酸钠预处理后,在pH值7.4环境下应用多聚赖氨酸（PLL）修饰nHAP;实验分为nHAP-PLL组、脂质体组及对照组,分别配制转染复合物并转染A549细胞;荧光显微镜观察EGFP的表达;流式细胞仪检测pGenesil-1的转染率。结果 透射电镜下nHAP呈针状颗粒,粒径较均匀,约（15~20）nm×（60~80）nm,分散程度良好;nHAP-PLL组和脂质体组均见EGFP表达阳性的A549细胞,对照组未见EGFP表达阳性的A549细胞;nHAP-PLL组pGenesil-1转染A549细胞的转染率为(31.8±1.9)%,与脂质体组的转染率 (33.4±3.7)%无差异（P>0.01）,对照组未见转染阳性的A549细胞。结论 nHAP经PLL表面修饰后可介导人肺癌A549细胞基因转染。     

人穿孔素N端肽段的放射诱导表达研究

目的：构建人穿孔素N端（hPFN—N）的真核放射诱导表达载体，并在放射诱导条件的刺激下研究hPFN-N的表达产物（rhPFN-N）在肺癌细胞SPC—A1中的分布及其对该细胞的细胞毒性作用。方法：以克隆有hPFN全长cDNA序列的载体pcDNA3．1（＋）／hPFN为模板，用PCR扩增hPFN-N，将该基因片段克隆到含放射诱导启动子的真核表达载体pcDNA3．1（＋）／Egr-1中，以重组体稳定转染SIC—A1细胞，经过X射线的放射诱导后，应用RT-PCR、细胞免疫化学法检测hPFN-N蛋白的表达，用MTT法测定该蛋白对靶细胞的杀伤活性。结果：成功构建了真核放射诱导表达载体pcDNA3．1（＋）／Egr-hPFN-N。以重组体转染SPC—A1细胞后，用RT-PCR检测到hPFN-N mRNA的表达。细胞免疫化学法检测结果呈阳性反应，MTT法检测结果为rhPFN-N对靶细胞的杀伤活力为29．2％。结论：构建了pcDNA3．1（＋）／Egr-hPFN-N真核放射诱导表达载体，在经过X射线的放射诱导后，hPFN-N能够在SPC—A1细胞的细胞膜上大量表达，表达产物rhPFN-N对靶细胞有明显杀伤活力。     

Serum amyloid A gene expression and AA amyloid formation in A/J and SJL/J mice

Serum amyloid A (SAA) gene expression and AA amyloid fibril formation were studied in A/J and SJL/J mice, two strains which have been reported to possess defects in AA fibril formation. Four types of inflammatory stimulation were employed: acute inflammation stimulated with lipopolysaccharide (LPS), chronic inflammation with casein in complete Freund's adjuvant, amyloidosis with injection of amyloid enhancing factor (AEF) together with casein in complete Freund's adjuvant, and non-amyloidogenic inflammation in the presence of AEF with injection of AEF together with LPS. Both A/J and SJL/J mice developed splenic amyloidosis 1 day after initiation of chronic inflammation in the presence of AEF. No amyloid deposits were detected during any of the other types of inflammation. Amyloidotic mice exhibited decreased amounts of SAA mRNA in liver and spleen concomitant with decreased amounts of SAA in serum. Alpha-I-acid glycoprotein mRNA was present in liver throughout the course of AEF accelerated amyloidosis, indicating that decreased SAA gene expression and AA fibril formation is not part of a general inhibitory effect of AEF on protein synthesis.     

Somatic mutation of the Caspase-5 gene in human lung cancer

Using cDNA array-based gene expression profiling, we previously found reduced expression of the Caspase-5 gene in highly metastatic subpopulations of a lung cancer cell line. The Caspase-5 gene contained poly(A) repeats in its coding region, an area that has been reported to be mutated in both endometrial and gastrointestinal tumors displaying evidence of microsatellite instability. In order to determine the contribution of Caspase-5 gene inactivation to lung cancer development and progression, the mutational status of the Caspase-5 poly(A) tract in 30 primary lung cancers with distant metastasis and 30 lung cancer cell lines was determined by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. Three somatic mutations of the Caspase-5 gene were found in two out of 30 lung cancer tissues, although no mutations were found in other genes that also contain small nucleotide repeats, such as hMSH3, hMSH6 and BAX. The results of the present study, combined with our prior cDNA array-based gene expression profiling data, suggest that Caspase-5 might be a suppressor gene of highly metastatic potential in lung cancer.     

Effects of salinomycin on cancer stem cell in human lung adenocarcinoma A549 cells

Lung cancer is a leading cause of death in human. Cancer stem cells have been regarded as basis for failure of current therapeutic options. Salinomycin was shown to kill these cancer stem cells in some types of cancer such as breast cancer and leukemia. The in vitro anticancer activities of salinomycin have been validated against the lung cancer cell line A549 via sulforhodamine B and colony formation assay. Salinomycin has been demonstrated to significantly rupture the in vitro lung cancer tumorospheres from ALDH positive A549 lung cells using flow cytometry. Expression of stem cell markers OCT-4, NANOG and SOX2 in ALDH positive A549 lung cells was decreased significantly by real-time RT-PCR analysis after 24 hour salinomycin treatment. Taken together, salinomycin may provide a promising approach for lung cancer chemotherapy.     

A study of mast cells in autoimmune NZB/W F1 mice: possible relationship between mast cells and increased vascular permeability in the thymus of NZB/W F1 mice.

We examined the possible relationship between thymic mast cells and increased vascular permeability in the thymus of autoimmune NZB/W F1 mice. Light-microscopic observation of tissue sections showed that non-autoimmune BDF1 mast cells increased with age. In contrast, autoimmune NZB/W F1 mast cells did not increase in the thymic parenchyma at the age of 9 weeks. However, NZB/W F1 mast cells resumed the age-associated increase from the age of 12 weeks and exceeded the number of BDF1 mast cells at the age of 30 weeks. Blood histamine levels of 9-week-old NZB/W F1 mice were higher than those of BDF1 mice of comparable age. Furthermore, peritoneal mast cells of NZB/W F1 mice were more sensitive to compound 48/80 than those of BDF1 mice. Increased blood histamine levels of NZB/W F1 mice seem to be due to the enhanced histamine release from mast cells. These results suggest a possible correlation between the high histamine levels by degranulation of mast cells and increased vascular permeability in the thymus of NZB/W F1 mice.     

Serum amyloid A gene expression and AA amyloid formation in A/J and SJL/J mice.

Serum amyloid A (SAA) gene expression and AA amyloid fibril formation were studied in A/J and SJL/J mice, two strains which have been reported to possess defects in AA fibril formation. Four types of inflammatory stimulation were employed: acute inflammation stimulated with lipopolysaccharide (LPS), chronic inflammation with casein in complete Freund''s adjuvant, amyloidosis with injection of amyloid enhancing factor (AEF) together with casein in complete Freund''s adjuvant, and non-amyloidogenic inflammation in the presence of AEF with injection of AEF together with LPS. Both A/J and SJL/J mice developed splenic amyloidosis 1 day after initiation of chronic inflammation in the presence of AEF. No amyloid deposits were detected during any of the other types of inflammation. Amyloidotic mice exhibited decreased amounts of SAA mRNA in liver and spleen concomitant with decreased amounts of SAA in serum. Alpha-I-acid glycoprotein mRNA was present in liver throughout the course of AEF accelerated amyloidosis, indicating that decreased SAA gene expression and AA fibril formation is not part of a general inhibitory effect of AEF on protein synthesis.