Silencing the Metallothionein‐2A Gene Induces Entosis in Adherent MCF‐7 Breast Cancer Cells

The presence of a live cell cohabiting within another cell has fascinated scientists for many decades. Far from being a spurious event, many have attempted to uncover the molecular mechanism underlying this phenomenon. In this study, we observed anchorage‐dependent MCF‐7 cells internalizing neighboring epithelial cells (entosis) after siRNA‐mediated silencing of the Metallothionein‐2A (MT‐2A) gene. MTs belong to a family of low‐molecular weight proteins, which bind metal ions endogenously and its over‐expression has been reported in a variety of cancers that include breast, prostate, and colon. We provide microscopic evidence at light and ultrastructural levels of the occurrence of entosis after altering MT expression in a subpopulation of MCF‐7 breast cancer cells by silencing the MT‐2A gene. Our results demonstrate that adheren junctions may play important roles in the formation of cell‐in‐cell cytostructure after MT‐2A gene downregulation and the entotic process does not appear to involve genes associated with autophagy. Interiorized cells often underwent lysosomal degradation within the cytoplasmic body of the engulfing cell. It would appear that a subset of breast cancer cells could die via entosis after MT‐2A gene silencing. Anat Rec 293:1685–1691, 2010. © 2010 Wiley‐Liss, Inc.     

Involvement of Nuclear JAK2 Signaling in AG490‐Induced Apoptosis of Gastric Cancer Cells

Although JAK2 inhibitors can result in antitumor activity against various tumors, some tumors have showed insensitivity or resistance to the inhibitors. To investigate the possible mechanisms underlying responses of gastric cancer (GC) cells to AG490, a specific JAK2 inhibitor, human GC cell lines SGC7901 and AGS were used. AG490 did not significantly induce apoptosis in SGC7901 cells, but it did in AGS cells. Interestingly, in SGC7901 cells, AG490 led to increased nuclear translocation of total JAK2 proteins, accompanied with initial inactivation but later reactivation of JAK2. However, in AGS cells, AG490 led to decreased nuclear localization of total JAK2 proteins, accompanied with sustained inactivation of JAK2. Moreover, silencing of human homolog of Drosophila Hairy and enhancer of split (Hes) 1 with siRNA partly blocked AG490‐induced nuclear translocation of JAK2, and enhanced AG490‐induced apoptosis in SGC7901 cells. The results collectively suggested that nuclear JAK2 signaling pathway may act as an escape way from JAK2 inhibitors in some GC cells. Anat Rec, 296:1865–1873, 2013. © 2013 Wiley Periodicals, Inc.     

胃癌中EZH2的表达及临床评估

目的：探讨EZH2在胃癌中的表达及其临床意义。方法：采用逆转录荧光PCR方法检测胃癌组织和配对的正常组织中EZH2的表达,分析EZH2与临床病理参数之间的关系。结果：53例标本中34例（64.15%,34/53）癌组织中EZH2表达增高,EZH2在胃癌组织中的表达明显高于配对正常组织（P〈0.05）。EZH2表达在低分化胃癌中表达明显高于高-中分化胃癌（P〈0.01）;EZH2表达在T3-T4期表达增高,明显高于T1-T2期（P〈0.05）;而EZH2的表达与性别、年龄等因素无关。结论：EZH2在胃癌组织表达增高,与分化程度及TNM分期有关,提供胃癌的进展和肿瘤形成;并预测胃癌治疗的预后方面,可能是一种新的生物标志物。     

Coordinated expression of REG4 and aldehyde dehydrogenase 1 regulating tumourigenic capacity of diffuse‐type gastric carcinoma‐initiating cells is inhibited by TGF‐β

Aldehyde dehydrogenase 1 (ALDH1) has been shown to serve as a marker for cancer‐initiating cells (CICs), but little is known about the regulation of the CIC functions of ALDH1+ cancer cells. We isolated ALDH1+ cells from human diffuse‐type gastric carcinoma cells and characterized these cells using an Aldefluor assay. ALDH1+ cells constituted 5–8% of the human diffuse‐type gastric carcinoma cells, OCUM‐2MLN and HSC‐39; were more tumourigenic than ALDH1? cells; and were able to self‐renew and generate heterogeneous cell populations. Using gene expression microarray analyses, we identified REG4 (regenerating islet‐derived family, member 4) as one of the genes up‐regulated in ALDH1+ cells, and thus as a novel marker for ALDH1+ tumour cells. Induced expression of REG4 enhanced the colony‐forming ability of OCUM‐2MLN cells, while knockdown of REG4 inhibited the tumourigenic potential of ALDH1+ cells. We further found that TGF‐β signalling reduces the expression of ALDH1 and REG4, and the size of the ALDH1+ cell population. In human diffuse‐type gastric carcinoma tissues, the expression of ALDH1 and REG4 correlated with each other, as assessed by immunohistochemistry, and ALDH1 expression correlated inversely with Smad3 phosphorylation as a measure of TGF‐β signalling. These findings illustrate that, in diffuse‐type gastric carcinoma, REG4 is up‐regulated in ALDH1+ CICs, and that the increased tumourigenic ability of ALDH1+ cells depends on REG4. Moreover, TGF‐β down‐regulates ALDH1 and REG4 expression, which correlates with a reduction in CIC population size and tumourigenicity. Targeting REG4 in ALDH1+ CICs may provide a novel strategy in the treatment of diffuse‐type gastric carcinoma. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Transforming Growth Factor‐β Suppressed Id‐1 Expression in a smad3‐Dependent Manner in LoVo Cells

TGF‐β plays an important role in regulating cell differentiation and proliferation in human cancers such as colorectal cancer. Id‐1 has been identified as a marker in colorectal cancer progression. The aim of this study was to investigate the role of TGF‐β in regulating Id‐1 in LoVo cells. siRNA was used to silence smad2, smad3, and p38 MAPK gene expression in Lovo cells. Interference efficiency and the role of TGF‐β on Id‐1 expression were analyzed using a luciferase reporter assay, RT‐PCR, and Western blotting. Cell viability was determined using the MTT assay. In this study, we demonstrated that TGF‐β1 downregulated Id‐1 protein expression in LoVo cells. Smad2 and smad3 siRNA inhibited TGF‐β1‐induced 4×SBE luciferase reporter activity. p38 MAPK siRNA inhibited TGF‐β1‐induced 3×AP‐1 luciferase reporter activity. However, the suppression of Id‐1 by TGF‐β1 was recovered by smad3 siRNA but not smad2 or p38 MAPK siRNA. Moreover, TGF‐β1 stimulated cellular proliferation and p21^Waf1 protein expression, which might be mediated by suppressing Id‐1 expression. In conclusion, this study demonstrated that TGF‐β1 suppressed Id‐1 expression in a smad3‐dependent manner in LoVo cells using RNAi technology. These results provide new insight into the mechanisms of TGF‐β function in colorectal cancer cells. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

COX‐2‐Mediated Regulation of VEGF‐C in Association With Lymphangiogenesis and Lymph Node Metastasis in Lung Cancer

The mechanisms underlying the effects of COX‐2 on tumor lymphangiogenesis remain largely undefined. Here, the human lung cancer cell lines A549, 95D, Anip973, and AGZY83‐a with different metastatic capacities were investigated by immunostaining, western blotting, and real‐time RT‐PCR. We observed increased expressions of COX‐2 and VEGF‐C in the three highly metastatic cell lines compared with the less metastatic AGZY83‐a cell line. The COX‐2‐specific inhibitor Celecoxib suppressed VEGF‐C expression whereas the main COX‐2 metabolite PGE₂ elevated VEGF‐C expression in Anip973 and AGZY83‐a cells in positive and negative experiments. To determine the functional link to COX‐2 more specifically and elucidate the mechanistic pathway, we used a siRNA to knock down the high COX‐2 expression in Anip973 cells and transfected a COX‐2 cDNA to enhance the low COX‐2 expression in AGZY83‐a cells, and then treated the cells with EP1/EP4 agonists or antagonists, respectively. The results revealed that the EP1/EP4 agonists significantly increased VEGF‐C production in the COX‐2‐knockdown Anip973 cells. In contrast, the EP1/EP4 antagonists diminished VEGF‐C production in the COX‐2‐overexpressing AGZY83‐a cells. Furthermore, animal models provided evidence that Celecoxib decreased VEGF‐C expression, lymphangiogenesis, and lymph node metastases in Anip973 cells, whereas PGE₂ treatment increased the same factors in the parental AGZY83‐a cells. A positive correlation between COX‐2 and VEGF‐C was also confirmed in vivo. The present data suggest that COX‐2 regulates VEGF‐C expression via the PGE₂ pathway, and that EP1/EP4 receptors are involved in PGE₂‐mediated VEGF‐C production. Thus, COX‐2 may represent a candidate gene for blocking tumor lymphangiogenesis and lymph node metastasis. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Preferential up‐regulation of heparanase and cyclooxygenase‐2 in carcinogenesis of Barrett’s oesophagus and intestinal‐type gastric carcinoma

Sonoda R, Naomoto Y, Shirakawa Y, Fujiwara Y, Yamatsuji T, Noma K, Tanabe S, Takaoka M, Gunduz M, Tsujigiwa H, Nagatsuka H, Ohara N, Yoshino T, Takubo K, Vieth M & Tanaka N
(2010) Histopathology 57 , 90–100
Preferential up‐regulation of heparanase and cyclooxygenase‐2 in carcinogenesis of Barrett’s oesophagus and intestinal‐type gastric carcinoma Aims: Metaplastic changes secondary to chronic inflammation at the gastro–oesophageal junction and at the pyloric antrum are recognized as the premalignant conditions of Barrett’s oesophageal adenocarcinoma and intestinal‐type gastric carcinoma (GC), respectively. Heparanase (HPSE) and cyclooxygenase (COX)‐2 have been proved to play critical roles in inflammation as well as in cancer. The aim was to examine the meaning of their expression in inflammation‐related carcinogenesis. Methods and results: First, expression of HPSE and COX‐2 in 78 clinical tissues of Barrett’s oesophagus was examined by immunohistochemistry and in situ hybridization. Their expression was increased during the metaplasia–dysplasia sequence with increased neovascularization. Successively, their expression in Barrett’s dysplasia was compared with that of GC (22 cases of diffuse‐type and 10 of intestinal‐type). Interestingly, the expression pattern in Barrett’s dysplasia was similar to that in intestinal‐type GC, which mainly arises from chronic inflammation. Furthermore, cultured cell lines isolated from differentiated GC tissues, which are often found to be of intestinal‐type, revealed up‐regulated mRNA expression of HPSE and COX‐2. Conclusions: HPSE and COX‐2 are preferentially up‐regulated in Barrett’s oesophagus and intestinal‐type GC. These molecules may play an important role during the development of inflammation‐related adenocarcinoma of the upper gastrointestinal tract.     

Identification of novel RP2 mutations in a subset of X‐linked retinitis pigmentosa families and prediction of new domains

X‐linked Retinitis Pigmentosa (XLRP) shows a huge genetic heterogeneity with almost five distinct loci on the X chromosome. So far, only two XLRP genes have been identified, RPGR (or RP3) and RP2, being mutated in approximately 70% and 10% of the XLRP patients. Clinically there is no clearly significative difference between RP3 and RP2 phenotypes. In the attempt to assess the degree of involvement of the RP2 gene, we performed a complete mutation analysis in a cohort of patients and we identified five novel mutations in five different XLRP families. These mutations include three missense mutations, a splice site mutation, and a single base insertion, which, because of frameshift, anticipates a stop codon. Four mutations fall in RP2 exon 2 and one in exon 3. Evidence that such mutations are different from the 21 RP2 mutations described thus far suggests that a high mutation rate occurs at the RP2 locus, and that most mutations arise independently, without a founder effect. Our mutation analysis confirms the percentage of RP2 mutations detected so far in populations of different ethnic origin. In addition to novel mutations, we report here that a deeper sequence analysis of the RP2 product predicts, in addition to cofactor C homology domain, further putative functional domains, and that some novel mutations identify RP2 amino acid residues which are evolutionary conserved, hence possibly crucial to the RP2 function. Hum Mutat 18:109–119, 2001. © 2001 Wiley‐Liss, Inc.     

Association between the Interleukin‐6 Gene −572 C/G Polymorphism and the Risk of Type 2 Diabetes Mellitus: A Meta‐Analysis of 11,681 Subjects

The association between the interleukin‐6 (IL‐6) gene ?572 C/G (rs1800796) polymorphism and type 2 diabetes mellitus (T2DM) risk remains controversial. Thus, we performed this meta‐analysis by searching PubMed, Embase, Web of Science, CBMdisc and CNKI databases until January 30, 2012. In addition, hand searching of the references of identified articles was performed. A total of 10 case–control studies including 11,681 subjects were selected to evaluate the possible association. Our results showed evidence for significant association between the IL‐6 gene ?572 C/G polymorphism and T2DM risk (for G allele vs. C allele: odds ratio [OR] = 1.29, 95% confidence interval [CI] = 1.09–1.52, P = 0.002, P = 0.008 after Bonferroni testing; for G/G vs. C/C: OR = 1.89, 95% CI = 1.51–2.37, P < 0.00001, P < 0.00004 after Bonferroni testing; for GG vs. G/C + C/C: OR = 1.75, 95% CI = 1.20–2.56, P = 0.004, P = 0.016 after Bonferroni testing; for G/G + G/C vs. C/C: OR = 1.32, 95% CI = 1.11–1.57, P = 0.001, P = 0.004 after Bonferroni testing). In addition, similar results were obtained in the subgroup analysis based on ethnicity. In summary, the present meta‐analysis suggests a significant association between the IL‐6 gene ?572 G allele and increased risk of T2DM.     

Expression of ezrin,Bcl‐2, and Ki‐67 in chondrosarcomas

Söderström M, Palokangas T, Vahlberg T, Böhling T, Aro H, Carpen O. Expression of ezrin, Bcl‐2, and Ki‐67 in chondrosarcomas. APMIS 2010; 118: 769–76. The aim of the present study was to investigate whether the expression of ezrin, a membrane‐cytoskeleton linker and regulator of cellular signaling, is associated with clinical features of chondrosarcoma. For this purpose, we studied the expression of ezrin in 54 chondrosarcomas by immunohistochemistry and correlated the expression with other tumor characteristics, markers of proliferation, apoptosis and with clinical parameters. The intensity of ezrin staining increased with the histologic grade, and a significant positive association existed between the tumor grade and ezrin expression (p = 0.0475). In addition, there was a positive correlation between the expression of ezrin and Bcl‐2, an anti‐apoptotic protein (r = 0.83, p < 0.0001), as well as between ezrin expression and increased proliferation as measured by Ki‐67 index (r = 0.70, p < 0.0001). The positive correlation of ezrin expression with Bcl‐2 and Ki‐67 as well as with tumor grade suggests that an aggressive behavior of chondrosarcoma may be related to activation of ezrin and that ezrin inhibitors could provide a much needed adjuvant therapy in chondrosarcomas. In conclusion, our results indicate that high ezrin expression correlates with aggressive features of chondrosarcomas. Further analyses on the pathways downstream of ezrin are warranted.     

Intratumoural heterogeneity of intestinal expression reflects environmental induction and progression‐related loss of induction in undifferentiated‐type gastric carcinomas

Aims: Gene expression in tumours is regulated by environmental as well as genetic/epigenetic factors. This study assessed the environmental factors in intestinal expression of gastric cancers. Methods and results: We immunohistochemically examined intratumoural heterogeneity in the expression of Cdx2, MUC2, MUC5AC and MUC6 in 39 intramucosal and 49 extramucosally invasive undifferentiated‐type gastric carcinomas (UGCs), consisting of signet ring cell carcinomas showing a layered structure (LS) in the mucosa and dedifferentiated tubular adenocarcinomas without LS and with minor tubular components (TC). The LS retains mucosal vertical polarity with superficial MUC5AC expression. Loss of this polarity was independent of intestinal expression and associated with extramucosal invasion. In LS⁺ UGCs, intestinal expression was enhanced as the size of mucosal spread increased and was significantly reduced with deeper extramucosal invasion, whereas, in LS^?/TC⁺ UGCs, intestinal expression was frequent and predominant in the mucosa and was insignificantly reduced with deeper extramucosal invasion. Conclusions: In LS⁺ UGCs, intestinal expression showed dynamic alteration probably by environmental induction and progression‐related loss of induction, whereas it was relatively stable in LS^?/TC⁺ UGCs. Thus, intestinal expression in UGCs is not useful as a marker of tumour progression because it is also affected by environmental factors and genetic lineage.     

Unusual Effect of Diethyl Zinc and Triisobutylaluminium in Ethylene/1‐Hexene Copolymerisation using an MgCl2‐Supported Ziegler‐Natta Catalyst

Summary: The use of triisobutylaluminium and diethyl zinc as cocatalyst and chain transfer agent respectively in ethylene/1‐hexene copolymerisation, carried out with a Ziegler‐Natta catalyst of type MgCl₂/TiCl₄/diisobutyl phthalate, has been found to give significantly higher comonomer incorporation than was obtained when triethylaluminium was used in combination with diethyl zinc. Triisobutylaluminium reacts readily with diethyl zinc, yielding metallic zinc, most likely via the formation of intermediate hydride species. In contrast, the use of 2,6‐dimethylpyridine as external donor in this system has been found to have relatively little effect on comonomer incorporation and distribution, indicating that a change from tendentially isospecific to syndiospecific propagation in propene polymerisation is not accompanied by a major improvement in comonomer distribution in ethylene/1‐hexene copolymerisation.

¹H NMR spectrum of the liquid product of the reaction between triisobutylaluminium and diethyl zinc.     

Screening for insulinoma antigen 2 and zinc transporter 8 autoantibodies: a cost‐effective and age‐independent strategy to identify rapid progressors to clinical onset among relatives of type 1 diabetic patients

In first‐degree relatives of type 1 diabetic patients, we investigated whether diabetes risk assessment solely based on insulinoma antigen 2 (IA‐2) and zinc transporter 8 (ZnT8) antibody status (IA‐2A, respectively, ZnT8A) is as effective as screening for three or four autoantibodies [antibodies against insulin (IAA), glutamate decarboxylase 65 kDa (GAD) glutamate decarboxylase autoantibodies (GADA) and IA‐2A with or without ZnT8A] in identifying children, adolescents and adults who progress rapidly to diabetes (within 5 years). Antibodies were determined by radiobinding assays during follow‐up of 6444 siblings and offspring aged 0–39 years at inclusion and recruited consecutively by the Belgian Diabetes Registry. We identified 394 persistently IAA⁺, GADA⁺, IA‐2A⁺ and/or ZnT8A⁺ relatives (6·1%). After a median follow‐up time of 52 months, 132 relatives developed type 1 diabetes. In each age category tested (0–9, 10–19 and 20–39 years) progression to diabetes was significantly quicker in the presence of IA‐2A and/or ZnT8A than in their joint absence (P < 0·001). Progression rate was age‐independent in IA‐2A⁺ and/or ZnT8A⁺ relatives but decreased with age if only GADA and/or IAA were present (P = 0·008). In the age group mainly considered for immune interventions until now (10–39 years), screening for IA‐2A and ZnT8A alone identified 78% of the rapid progressors (versus 75% if positive for ≥ 2 antibodies among IAA, GADA, IA‐2A and ZnT8A or versus 62% without testing for ZnT8A). Screening for IA‐2A and ZnT8A alone allows identification of the majority of rapidly progressing prediabetic siblings and offspring regardless of age and is more cost‐effective to select participants for intervention trials than conventional screening.     

Growth‐Inhibitory Activity of Melatonin on Murine Foregastric Carcinoma Cells In Vitro and the Underlying Molecular Mechanism

Melatonin (MLT) is an indolic hormone produced mainly by the pineal gland. Recent human and animal studies have shown that MLT exerts obvious oncostatic activity both in vitro and in vivo. The purpose of this study was to investigate the antiproliferative effect of MLT on the murine foregastric carcinoma (MFC) cell and to determine the underlying molecular mechanism. Cell viability was determined using the Cell Counting Kit‐8 (CCK‐8) and the results revealed that MLT exhibited a dose‐ and time‐dependent inhibitory effect on MFC cell growth. Our studies also demonstrated upregulation of p21 and Bax and downregulation of Bcl‐2 at both the mRNA and the protein levels in response to MLT treatment of MFC cells. These changes in the expression of these molecules were consistent with the results of the CCK‐8. Furthermore, the mRNA and protein expression of membranous MLT receptors was also upregulated. Taken together, these results confirm the oncostatic effect of MLT in MFC cells and the expression of membranous MLT receptors is a potential approach to tumor cells in gastric cancer therapeutic treatment. Anat Rec, 296:914–920, 2013. © 2013 Wiley Periodicals, Inc.