Behaviour of the new asbestos amphibole fluor-edenite in different lung cell systems

The aim of the present research was to determine whether the recently identified and characterized new fibrous amphibole fluoro-edenite may induce a cytopathic response in cultured cells. The final goal was to gain suggestions on the potentiality of fluoro-edenite to be harmful to human beings. Epidemiological studies, in fact, have shown an excess of developing mesothelioma among residents in Biancavilla, a town in eastern Sicily located in the Etna volcanic area. Therefore, we treated human lung fibroblasts, human lung alveolar epithelial cancer cell line A549 and monocyte-macrophage cell line J774 with fluoro-edenite or crocidolite; the latter used as a highly toxic amphibole asbestos reference. Our results show that fluoro-edenite may induce functional modifications and affects some biochemical parameters in tested cell cultures in a concentration and time dependent manner. However, the observed functional modifications induced by fluoro-edenite are generally less dramatic than those induced by crocidolite and more evident on human lung alveolar epithelial cancer cell line A549 with respect to those obtained on human lung fibroblasts or monocyte-macrophage cell line J774. The sequence of the damage is hypothesised to be as follows: at increasing fluoro-edenite concentrations, and/or treatment times, the increase in reactive oxygen species (ROS) production could trigger significant DNA damage in cell cultures, concomitantly with drop in cell metabolism and increase in lactic dehydrogenase release. In conclusion, according to our data, fluoro-edenite appears as a probable carcinogenic agent, responsible for the high incidence of malignant pleural mesothelioma in Biancavilla.     

Fluoro-edenite fibres induce lung cell apoptosis: an in vivo study

We previously showed that apoptosis in the lungs of sheep exposed to fluoro-edenite fibres is induced via the receptor pathway. The present study was performed to gain further insights into the mechanisms of activation of programmed cell death induced by the fibres. Fluoro-edenite fibres are similar in size and morphology to some amphibolic asbestos fibres. They have been found in benmoreitic lavas, in the local stone quarry, in building materials and in road paving at Biancavilla, a town in eastern Sicily (Italy), where epidemiological surveys revealed a cluster of mortality from pleural mesothelioma. Inhalation of asbestos fibres can cause chronic inflammation and carcinogenesis. Since fluoro-edenite has been shown to activate the apoptotic process, we set out to characterise the expression of apoptosis-regulating proteins in fluoro-edenite-exposed lung disease and sought to determine if apoptosis results from fluoro-edenite exposure. Lung tissue from apparently healthy sheep habitually grazing near Biancavilla was processed for immunohistochemical localisation of bcl-2 and bax. Results showed epithelial and interstitial bax overexpression, especially in cells directly in contact with the fibres, and negative bcl-2 immunoexpression. TUNEL-positive cells were detected in alveoli and connective tissue. The integrity of alveolar epithelium and alveolar apoptosis are critical determinants in the pathways that initiate fibrogenesis in the lung and fibroblastic foci are usually found close to abnormal or denuded alveolar epithelium. Our results are consistent with the hypothesis that apoptosis is an important mechanism for removing cells with irreparable fluoro-edenite-induced genetic changes that predispose them to a neoplastic evolution.     

小檗胺衍生物（EBB）体外抑制肺癌细胞增殖机制的初探

本文研究并发现了新型半天然钙调素(CaM)拮抗剂--O-4-乙氧基丁基小檗胺(O-4-ethoxy-butyl-berbamine,EBB),具有选择性抑制肺巨细胞癌(PG)的增殖和降低细胞内CaM水平的能力,对人胚肺细胞(HEL)的增殖和细胞内CaM的水平影响较小.同时观察到EBB引起肿瘤细胞内CaM水平降低的原因,是对CaM基因的转录产物(mRNA)和翻译产物(CaM)两方面作用的结果.此外,EBB还能部分降低PG细胞内原癌基因和突变的抑癌基因转录产物mRNA的水平.EBB抑制肿瘤细胞增殖的机制,除了对CaM基因的表达水平和CaM活性调控外,可能还直接或间接地影响了相关的原癌基因和突变的抑癌基因的表达.     

Regulation of interlocking gene regulatory network subcircuits by a small molecule inhibitor of retinoblastoma protein (RB) phosphorylation: Cancer cell expression of HLA-DR

The induction of the major histocompatibility (MHC), antigen-presenting class II molecules by interferon-gamma, in solid tumor cells, requires the retinoblastoma tumor suppressor protein (Rb). In the absence of Rb, a repressosome blocks the access of positive-acting, promoter binding proteins to the MHC class II promoter. However, a complete molecular linkage between Rb expression and the disassembly of the MHC class II repressosome has been lacking. By treating A549 lung carcinoma cells with a novel small molecule that prevents phosphorylation-mediated, Rb inactivation, we demonstrate that Rb represses the synthesis of an MHC class II repressosome component, YY1. The reduction in YY1 synthesis correlates with the advent of MHC class II inducibility; with loss of YY1 binding to the promoter of the HLA–DRA gene, the canonical human MHC class II gene; and with increased Rb binding to the YY1 promoter. These results support the concept that the Rb gene regulatory network (GRN) subcircuit that regulates cell proliferation is linked to a GRN subcircuit regulating a tumor cell immune function.     

P16^INK4、Rb基因表达在肺癌发生中协调性研究

采用mRNA原位双杂交和免疫组织化学方法对31例非小细胞肺癌组织进行P16^INK、Rb基因、Rb基因表达水平及其相关性的研究。结果显示,以Dig-碱性磷酸酶－NBT／BCIP系统检测P16^INK4基因转录,阳性结果呈蓝色,阴性杂交率为22．6％（7／31）;以Bio－辣根过氧化物酶－AEC系统检测Rb基因转录,阳性结果为红色,阴性率为16．1％（5／31）。免疫组织化学检测显示P16^INK4蛋白质阴性率为42％（13／31）;Rb蛋白表达阴性率为19．4％（6／31）。Rb、P16^INK4基因在非小细胞肺癌发生中起协同调控作用,以P16^INK4基因表达异常为主。     

Knockdown of the Sodium-Dependent Phosphate Co-Transporter 2b (NPT2b) Suppresses Lung Tumorigenesis

The sodium-dependent phosphate co-transporter 2b (NPT2b) plays an important role in maintaining phosphate homeostasis. In previous studies, we have shown that high dietary inorganic phosphate (Pi) consumption in mice stimulated lung tumorigenesis and increased NPT2b expression. NPT2b has also been found to be highly expressed in human lung cancer tissues. The association of high expression of NPT2b in the lung with poor prognosis in oncogenic lung diseases prompted us to test whether knockdown of NPT2b may regulate lung cancer growth. To address this issue, aerosols that contained small interfering RNA (siRNA) directed against NPT2b (siNPT2b) were delivered into the lungs of K-ras ^LA1 mice, which constitute a murine model reflecting human lung cancer. Our results clearly showed that repeated aerosol delivery of siNPT2b successfully suppressed lung cancer growth and decreased cancer cell proliferation and angiogenesis, while facilitating apoptosis. These results strongly suggest that NPT2b plays a role lung tumorigenesis and represents a novel target for lung cancer therapy.     

Expression of Fas antigen in the cells from bronchoalveolar lavage fluid (BALF)

Fas antigen is a cell surface receptor protein that mediates apoptosis expressed in various cells. In this study Fas expression was examined in cells of patients with lung diseases in which changes in the lung immunology were documented. We have performed bronchoalveolar lavage (BAL) in 24 patients with sarcoidosis (8), lung fibrosis (9), primary lung cancer (7), and we compared expression of Fas in BALF cells from all groups and healthy volunteers (6). Fas protein was detected by immunocytochemistry using APAAP technique with an LSAB 2 kit (Dako). Positive reactions for Fas were found in the cytoplasm of epithelial cells, macrophages, neutrophils and lymphocytes (according to the intensity). There were some differences in proportion of positive cells and intensity of reaction between patients with interstitial lung diseases, healthy volunteers as well as patients with lung cancer. Higher expression of Fas in alveolar macrophages was observed in patients with sarcoidosis, lower in patients with lung cancer, lung fibrosis and the lowest in healthy persons. The analysis of Fas antigen expression in the BALF cells may be useful in evaluation of the role of apoptosis in lung homeostasis and pathology.     

Knockdown of response gene to complement 32 (RGC32) induces apoptosis and inhibits cell growth,migration, and invasion in human lung cancer cells

Response gene to complement 32 (RGC32) is a novel protein originally identified as a cell cycle activator and has been demonstrated to be overexpressed in a variety of human malignancies, including lung cancer. However, the potential role of RGC32 in lung cancer initiation and progression remains to be elucidated. In the present study, RNA interference mediated by plasmid expressing RGC32 short-hairpin RNA (shRNA) was utilized to knockdown RGC32 expression in human lung cancer LTE cells. We found that the mRNA and protein expression levels of RGC32 were significantly decreased in RGC32-specific shRNA-transfected cells in comparison with the untransfected and control shRNA-transfected cells. Furthermore, knockdown of RGC32 dramatically reduced cell proliferation, colony formation, and invasion and migration capacities of LTE cells in vitro. Specific down-regulation of RGC32 caused G0/G1 cell cycle arrest and eventual apoptosis. Meanwhile, Western blot analysis indicated that cells with stably knockdown of RGC32 showed decreased expression levels of Cyclin D1, Cyclin E, Bcl-2, matrix metalloproteinase (MMP)-2, and MMP-9, but increased expression levels of activate caspase-3, Bax, and cleaved poly (ADP-ribose) polymerase (PARP) in comparison with control shRNA-transfected cells. Taken together, our data suggest that RGC32 is involved in tumorigenesis of human lung cancer and may serve as a promising therapeutic target for lung cancer.     

The Expression of the Ubiquitin Ligase SIAH2 (Seven In Absentia Homolog 2) Is Increased in Human Lung Cancer

Objectives

Lung cancer is the leading cause of cancer-related deaths worldwide. Overall 5-year survival has shown little improvement over the last decades. Seven in absentia homolog (SIAH) proteins are E3 ubiquitin ligases that mediate proteasomal protein degradation by poly-ubiquitination. Even though SIAH proteins play a key role in several biological processes, their role in human cancer remains controversial. The aim of the study was to document SIAH2 expression pattern at different levels (mRNA, protein level and immunohistochemistry) in human non-small cell lung cancer (NSCLC) samples compared to surrounding healthy tissue from the same patient, and to analyse the association with clinicopathological features.

Materials and Methods

One hundred and fifty-two samples from a patient cohort treated surgically for primary lung cancer were obtained for the study. Genic and protein expression levels of SIAH2 were analysed and compared with clinic-pathologic variables.

Results

The present study is the first to analyze the SIAH2 expression pattern at different levels (RNA, protein expression and immunohistochemistry) in non-small cell lung cancer (NSCLC). We found that SIAH2 protein expression is significantly enhanced in human lung adenocarcinoma (ADC) and squamous cell lung cancer (SCC). Paradoxically, non-significant changes at RNA level were found, suggesting a post-traductional regulatory mechanism. More importantly, an increased correlation between SIAH2 expression and tumor grade was detected, suggesting that this protein could be used as a prognostic biomarker to predict lung cancer progression. Likewise, SIAH2 protein expression showed a strong positive correlation with fluorodeoxyglucose (2-deoxy-2(¹⁸F)fluoro-D-glucose) uptake in primary NSCLC, which may assist clinicians in stratifying patients at increased overall risk of poor survival. Additionally, we described an inverse correlation between the expression of SIAH2 and the levels of one of its substrates, the serine/threonine kinase DYRK2.

Conclusions

Our results provide insight into the potential use of SIAH2 as a novel target for lung cancer treatment.     

The retinoblastoma protein (Rb) as an anti-apoptotic factor: expression of Rb is required for the anti-apoptotic function of BAG-1 protein in colorectal tumour cells

Although the retinoblastoma-susceptibility gene RB1 is inactivated in a wide range of human tumours, in colorectal cancer, the retinoblastoma protein (Rb) function is often preserved and the RB locus even amplified. Importantly, we have previously shown that Rb interacts with the anti-apoptotic Bcl-2 associated athanogene 1 (BAG-1) protein, which is highly expressed in colorectal carcinogenesis. Here we show for the first time that Rb expression is critical for BAG-1 anti-apoptotic activity in colorectal tumour cells. We demonstrate that Rb expression not only increases the nuclear localisation of the anti-apoptotic BAG-1 protein, but that expression of Rb is required for inhibition of apoptosis by BAG-1 both in a γ-irradiated Saos-2 osteosarcoma cell line and colorectal adenoma and carcinoma cell lines. Further, consistent with the fact that nuclear BAG-1 has previously been shown to promote cell survival through increasing nuclear factor (NF)-κB activity, we demonstrate that the ability of BAG-1 to promote NF-κB activity is significantly inhibited by repression of Rb expression. Taken together, data presented suggest a novel function for Rb, promoting cell survival through regulating the function of BAG-1. As BAG-1 is highly expressed in the majority of colorectal tumours, targeting the Rb–BAG-1 complex to promote apoptosis has exciting potential for future therapeutic development.     

Expression of human MutT homologue (hMTH1) protein in primary non-small-cell lung carcinomas and histologically normal surrounding tissue

In situ, oxidation of deoxyguanosine yields 8-hydroxy-2'-deoxyguanosine (8-oxo-dG), which is mutation prone and results in a G:C --> T:A transversion following DNA replication. Another pathway to the formation of DNA containing 8-oxo-dG is by the misincorporation of 8-oxo-dGTP via DNA polymerase. Human MutT homologue (hMTH1), an 8-oxo-dGTPase, prevents misincorporation of this oxidized nucleotide by hydrolyzing 8-oxo-dGTP to 8-oxo-dGMP. Previous studies have shown that hMTH1 mRNA is overexpressed in human renal cell carcinomas and breast tumors. Elevated levels of hMTH1 protein have also been detected in brain tumors. In the current study, we determined whether hMTH1 protein is overexpressed in primary non-small-cell lung carcinomas as compared to adjacent histologically normal lung tissue. Twenty matched human lung tumor/normal pairs were examined by Western analysis for expression of hMTH1 protein. Overexpression in the tumors was detected in 4/8 (50%) adenocarcinomas, 4/4 (100%) adenocarcinomas with bronchioalveolar (BAC) features, 2/2 (100%) BACs, and 3/6 (50%) squamous cell carcinomas. The data from Western analysis were validated by immunohistochemical staining for hMTH1 protein. The results of this study indicate that hMTH1 protein may be a potential marker for the detection of persistent oxidative stress in lung cancer.     

TRPM7的表达水平与肺癌发生发展的关系

为了解TRPM7在肺癌中的表达及其与肺癌进展的关系,本研究检测了TRPM7在非小细胞肺癌患者肺癌组织样本和相邻正常肺泡组织样本中的表达,以及TRPM7在人肺腺癌A549细胞系和人支气管上皮细胞系16HBE中的表达。通过转染shRNA敲低肺癌细胞中的TRPM7,并应用TRPM7拮抗剂Waixenicin A处理细胞。免疫组化染色和Western blotting分析显示,与正常肺泡组织样本中的TRPM7表达相比,TRPM7在肺癌样本中显著高表达。TRPM7的表达水平与癌症分期有关,分期越高,TRPM7的表达水平越高。TRPM7在A549细胞中的表达强度显著高于16HBE细胞。细胞集落形成测定结果显示,沉默TRPM7会显著抑制细胞集落形成的能力。SRB细胞活力测定显示,沉默TRPM7会显著抑制细胞活力。沉默TRPM7显著降低了肺癌细胞的迁移(-68.94%)和侵袭(-68.84%)能力。沉默TRPM7显著抑制了热休克蛋白90α(HSP90α)、尿激酶型纤溶酶原激活剂(uPA)和基质金属蛋白酶2 (MMP2)的表达。Waixenicin A显著抑制了肺癌细胞的活力及Hsp90α/uPA/MMP2信号分子的表达。另外,Waixenicin A显著降低了肺癌细胞的迁移(-65.35%)和侵袭(-71.85%)能力。本研究表明,TRPM7的异常表达通过激活Hsp90α/uPA/MMP2信号通路来提高人肺癌细胞的活力和转移能力。研究结果表明,靶向TRPM7的抑制剂可能是治疗肺癌的有效药物。     

Expression of mdm-2 oncoprotein in the primary and metastatic sites of mammary tumor (GI-101) implanted athymic nude mice

The expression of mdm-2 oncoprotein (p90) was determined in a human breast tumor xenograft line (GI-101) that was derived from a 57 year old female cancer patient with recurrent, infiltrating ductal adenocarcinoma (Stage IIIa, T3N2MX). Immunoprecipitation coupled western blot analysis of the primary tumors that have been obtained from xenograft implanted athymic nude mice, using mdm-2 (Ab-1) mouse monoclonal antibody, primarily revealed high level expression of a 90 kD full length mdm-2 protein. In the GI-101 tumor the level of full length mdm-2 (p90) protein expression increased with the increase in the size of the tumor (100 to 2,000 mm(3)) and a maximum expression was detected in 2,000 mm(3) size tumors. In addition to the expression in the primary site, a significantly high level expression of mdm-2 protein (p90) was detected in the lung and liver tissues also, which are the known metastatic sites for GI-101 xenograft tumors. However, the level of mdm-2 protein expression was undetectable in the lung and liver tissues obtained from control mice. A cell line (GI-101A) derived from the GI-101 xenograft tumor also showed a high level expression of mdm-2 protein after several generations of cell passage. When the GI-101A cells were treated with DES (Diethylstilbestrol) the mdm-2 protein expression increased after 10 min treatment and reached a peak level at 40 min. Interestingly, DES (10 and 20 microM) treatment increased the total cell number also after 96 hr treatment compared to the non-treated cells. It appears that mdm-2 (p90) may have a significant role in supporting the tumor cell growth as well as the metastatic process of the GI-101A cells.     

WT1 Promotes Cell Proliferation in Non-Small Cell Lung Cancer Cell Lines through Up-Regulating Cyclin D1 and p-pRb In Vitro and In Vivo

The Wilms’ tumor suppressor gene (WT1) has been identified as an oncogene in many malignant diseases such as leukaemia, breast cancer, mesothelioma and lung cancer. However, the role of WT1 in non-small-cell lung cancer (NSCLC) carcinogenesis remains unclear. In this study, we compared WT1 mRNA levels in NSCLC tissues with paired corresponding adjacent tissues and identified significantly higher expression in NSCLC specimens. Cell proliferation of three NSCLC cell lines positively correlated with WT1 expression; moreover, these associations were identified in both cell lines and a xenograft mouse model. Furthermore, we demonstrated that up-regulation of Cyclin D1 and the phosphorylated retinoblastoma protein (p-pRb) was mechanistically related to WT1 accelerating cells to S-phase. In conclusion, our findings demonstrated that WT1 is an oncogene and promotes NSCLC cell proliferation by up-regulating Cyclin D1 and p-pRb expression.     

The Aqueous Extract of Ficus religiosa Induces Cell Cycle Arrest in Human Cervical Cancer Cell Lines SiHa (HPV-16 Positive) and Apoptosis in HeLa (HPV-18 Positive)

Natural products are being extensively explored for their potential to prevent as well as treat cancer due to their ability to target multiple molecular pathways. Ficus religiosa has been shown to exert diverse biological activities including apoptosis in breast cancer cell lines. In the present study, we report the anti-neoplastic potential of aqueous extract of F. religiosa (FR_aq) bark in human cervical cancer cell lines, SiHa and HeLa. FR_aq altered the growth kinetics of SiHa (HPV-16 positive) and HeLa (HPV-18 positive) cells in a dose-dependent manner. It blocked the cell cycle progression at G₁/S phase in SiHa that was characterized by an increase in the expression of p53, p21 and pRb proteins with a simultaneous decrease in the expression of phospho Rb (ppRb) protein. On the other hand, in HeLa, FR_aq induced apoptosis through an increase in intracellular Ca²⁺ leading to loss of mitochondrial membrane potential, release of cytochrome-c and increase in the expression of caspase-3. Moreover, FR_aq reduced the migration as well as invasion capability of both the cervical cancer cell lines accompanied with downregulation of MMP-2 and Her-2 expression. Interestingly, FR_aq reduced the expression of viral oncoproteins E6 and E7 in both the cervical cancer cell lines. All these data suggest that F. religiosa could be explored for its chemopreventive potential in cervical cancer.