Role of COX-2 in carcinogenesis of colorectal cancer and its relationship with tumor biological characteristics and patients' prognosis

AIM: Recent clinical epidemiological studies havedemonstrated the preventive effect of non-steroidal anti-inflammatory drugs (NSAIDs) against colorectal cancer.The underlying mechanism might be the inhibition of rate-limiting enzyme cyclooxygenase-2 (COX-2) in metabolismof arachidonic acid. The role of COX-2 in carcinogenesisof colorectal cancer and its relationship with tumorbiological characteristics and patients′ prognosis stillremain unclear. This study was to investigate the role ofCOX-2 expression in carcinogenesis of colorectal cancerand its relationship with tumor biological characteristicsand patients′ prognosis.METHODS: A total of 139 colorectal cancers and 19adenomas surgically treated in School of Oncology, PekingUniversity, from January 1993 to September 2001 wereretrospectively studied. COX-2 expression was detected withtissue microarray (TMA) and immunohistochemistry (IHC)procedure. The association between COX-2 expression andclinicopathological features and its influence on patients′prognosis were studied.RESULTS: COX-2 expression was strong in colorectal cancer,moderate in adenoma and weak in normal mucosa, whichdemonstrated statistically significant difference (x2=46.997,P＜0.001). COX-2 expression had no association withclinicopathological features such as gross type,differentiation, invasion depth, vessel emboli and TNMstaging. Cox proportional hazards modeling analysis and Logrank test revealed no prognostic role of COX-2 expressionin colorectal cancer patients.CONCLUSION: COX-2 may play an important role in theearly stage of carcinogenesis, and its expression in colorectalcancer is not associated with clinicopathological features and patients′ prognosis.

COX-2 expression and tumor angiogenesis in colorectal cancer

AIM: Cyclooxygenase-2 (COX-2) is one of the rate-limiting enzymes in metabolism of arachidonic acid, and COX-2 inhibitors demonstrate preventive effects on cancer,especially on colorectal cancer. The underlying mechanism remains unclear. The aim of this study was to illustrate the relationship between angiogenesis and COX-2 in carcinogenesis of colorectal cancer.METHODS: One hundred and seventy patients with colorectal cancer were enrolled in our study from January 1993 to September 2001 in School of Oncology, PekingUniversity. COX-2 and VEGF expression were detected with the immunohistochemistry (IHC) technique. IHC assays were carried out with the aid of tissue microarray (TMA)procedure. Specimens from 35 of these patients were examined with reverse transcriptase PCR (RT-PCR).RESULTS: COX-2 and VEGF expressions were stronger in colorectal cancer than those in the corresponding normal tissues, at both protein and mRNA levels. One hundred patients were eligible for analysis after IHC assay of COX-2 and VEGF. The positive rate of VEGF was much higher in COX-2 positive group (47/85) than in COX-2 negative group (x^2 = 4.181, P= 0.041). The result was further verified by the result of RT-PCR (x^2 = 8.517, P = 0.003). Correlation coefficient was 0.409 after Spearman correlation analysis (P=0.015).CONCLUSION: COX-2 may be involved in the course of tumor angiogenesis of colorectal cancer and acts through VEGF.

Expression of ECRG4,a novel esophageal cancer-related gene,downregulated by CpG island hypermethylation in human esophageal squamous cell carcinoma

AIM: To study the mechanisms responsible for inactivation of a novel esophageal cancer related gene 4 (ECRG4) in esophageal squamous cell carcinoma (ESCC). METHODS: A pair of primers was designed to amplify a 220 bp fragment, which contains 16 CpG sites in the core promoter region of the ECRG 4 gene. PCR products of bisulfite-modified CpG islands were analyzed by denaturing high-performance liquid chromatography (DHPLC), which were confirmed by DNA sequencing. The methylation status of ECRG 4 promoter in 20 cases of esophageal cancer and the adjacent normal tissues, 5 human tumor cell lines (esophageal cancer cell line-NEC, EC109, EC9706; gastric cancer cell line- GLC; human embryo kidney cell line-Hek293)and 2 normal esophagus tissues were detected. The expression level of the ECRG 4 gene in these samples was examined by RT-PCR. RESULTS: The expression level of ECRG 4 gene was varied.Of 20 esophageal cancer tissues, nine were unexpressed,six were lowly expressed and five were highly expressed compared with the adjacent tissues and the 2 normal esophageal epithelia. In addition, 4 out of the 5 human cell lines were also unexpressed. A high frequency of methylation was revealed in 12 (8 unexpressed and 4 lowly expressed)of the 15 (80%) downregulated cancer tissues and 3 of the 4 unexpressed cell lines. No methylation peak was observed in the two highly expressed normal esophageal epithelia and the methylation frequency was low (3/20) among the 20 cases in the highly expressed adjacent tissues. The methylation status of the samples was consistent with the result of DNA sequencing. CONCLUSION: These results indicate that the inactivation of ECRG 4gene by hypermethylation is a frequent molecular event in ESCC and may be involved in the carcinogenesis of this cancer.

Anti-Helicobacterpyloriimmunoglobulin G(IgG)and IgA antibody responses and the value of clinical presentations in diagnosis of H.pyloriinfection in patients with precancerous lesions

AIM: To determine the prevalence of Helicobacter pylori (H. pylon) infection, the serum anti-H. pylori immunoglobulin G (IgG) and IgA antibody responses, and the value of clinical presentations in diagnosis of H. pylori infection in patients with gastric atrophy, intestinal metaplasia and dysplasia.METHODS: H. pylori infection was detected by histology in 209 patients with mild chronic atrophic gastritis (CAG, n=76),severe CAG (n=22), mild intestinal metaplasia (IM, n=22),severe IM (n=58), or dysplasia (DYS, n=31). Serum antiH. pylori IgG and IgA were double sampled and evaluated by enzyme-linked immunoadsordent assays. 35 clinical presentations were observed and their relationship with H.pylori infection was analyzed by the k-means cluster method.RESULTS: Both IgG and IgA levels in H. pylori positive patients were significantly higher than those negative for H.pylori(P＜0.001-0.01). The prevalence of H. pylori was highest in severe IM (84.5%), and lowest in mild CAG (51.3%)(P＜0.01). They were similar in severe CAG (68.2%), mild IN (72.7%), and DYS (67.7%). In H.pylori positive patients,the IgG levels in severe CAG were significantly higher than those in mild CAG (P＜0.01). In H.pylori negative patients,both IgG and IgA levels increased remarkably in severe IM,compared to those in mild IM (P＜0.01-0.05).H. pylori infection exhibited no association with patient′s gender (62.1% in males; 71.7 % in females) and age (r=0.0814, P=0.241).The diagnostic accuracy based on 35 clinical presentations was 65.7 %. It could be improved by 5.7% when only the assemblage of digestive symptoms were engaged, or by 8.6 % when the pathogenic factors, general status and grossoscopy were combined. The diagnostic accuracy could be decreased when only the general symptoms were engaged, or when the pathogenic factors were accompanied with some common digestive symptoms.CONCLUSION: H. pylori infection is a major risk factor for the process from atrophy, IM to DYS of gastric mucosa.Serum IgG and IgA are good indicators to evaluate this progress with a certain arrearage. Investigation on the effective assemblages of clinical presentations may provide a better understanding in the pathogenesis, diagnosis and treatment for H. pylori infection.

PPARγ pathway activation results in apoptosis and COX-2 inhibition in HepG2 cells

AIM: To investigate whether troglitazone (TGZ), theperoxisome proliferator-activated receptor (PPAR) gammaligand, can induce apoptosis and inhibit cell proliferation inhuman liver cancer cell line HepG2 and to explore themolecular mechanisms. METHODS: [3-(4,5)-dimethyithiazol-2-yl]-2,5-diphenyltetrazolium bromide (NTT), [3H] Thymidine incorporation,Hochest33258 staining, DNA ladder, enzyme-linkedimmunosorbent assay (ELISA), RT-PCR, Northern and Western blotting analyses were employed to investigate the effect of TGZ on HepG2 cells and related molecular mechanisms.RESULTS: TGZ was found to inhibit the growth of HepG2cells and to induce apoptosis. During the process, the expression of COX-2 mRNA and protein and Bcl-2 protein was down-regulated, while that of Bax and Bak proteins was up-regulated, and the activity of caspase-3 was elevated.Furthermore, the level of PGE2 was decreased transiently after 12 h of treatment with 30 gM troglitazone. CONCLUSION: TGZ inhibits cell proliferation and induces apoptosis in HepG2 cells, which may be associated with the activation of caspase-3-like proteases, down-regulation of the expression of COX-2 mRNA and protein, Bcl-2 protein,the elevation of PGE2 levels, and up-regulation of the expressions of Bax and Bak proteins.

Peroxisome proliferator-activated receptor gamma ligands inhibit cell growth and induce apoptosis in human liver cancer BEL-7402 cells

AIM: To investigate the characteristics of PPAR gamma ligands induced apoptosis in liver cancer cells.METHODS: The effects of ligands for each of the PPAR gamma ligands on DNA synthesis and cell viability were examined in BEL-7402 liver cancer cells. Apoptosis was characterized by Hochest33258 staining, DNA fragmentation,TUNEL and ElISA, and cell cycle kinetics by FACS. Modulation of apoptosis related caspases expression by PPAR gamma ligands was examined by Western blot.RESULTS: PPARgamma ligands, 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) and troglitazone (TGZ), suppressed DNA synthesis of BEL-7402 cells. Both 15d-PGJ2 and TGZ induced BEL-7402 cell death in a dose dependent manner, which was associated with an increase in fragmented DNA and TUNEL-positive cells. At concentrations of 10 and 30 μM,15d-PGJ2 or troglitazone increased the proportion of cells with G0/G1 phase DNA content and decreased those with S phase DNA content. There was no significant change in the proportion of cells with G2/M DNA content. The activities of Caspases-3, -6, -7 and -9 were increased by 15d-PGJ2and TGZ treatment, while the activity of Caspase 8 had not significantly changed.CONCLUSION: The present results suggest the potential usefulness of PPAR gamma ligands for chemoprevention and treatment of liver cancers.

Detection of human papillomavirus in Chinese esophageal squamous cell carcinoma and its adjacent normal epithelium

AIM: To investigate the putative role of human papillomavirus (HPV) infection in the carcinogenesis of esophageal squamous cell carcinoma in China.METHODS: Twenty-three esophageal squamous cell carcinoma samples and the distal normal epithelium from Shanxi Province, and 25 more esophageal squamous cell carcinoma samples from Anyang city, two areas with a high incidence of esophageal cancer in China, were detected for the existence of HPV-16 DNA by PCR, mRNA in situ hybridization (ISH) and immunohistochemistry (IHC) targeting HPV-16 E6 gene. RESULTS: There were approximately 64 % (31/48) patients having HPV-16 DNA in tumor samples, among them nearly twothirds (19/31) samples were detected with mRNA expression of HPV-16 E6. However, in the normal esophageal epithelium from cancer patients, the DNA and mRNA of HPV-16 were found with much less rate: 34.7 % (8/23) and 26.1% (6/23) respectively.In addition, at protein level detected by IHC assay, 27.1% (13/48) tumor samples had virus oncoprotein E6 expression, while only one case of normal epithelium was found positive.CONCLUSION: HPV infection, especially type 16, should be considered as a risk factor for esophageal malignancies in China.

Detection of frameshift mutations of RIZ in gastric cancers with microsatellite instability

AIM. To study the frameshift mutations of the retinoblastoma protein-interacting zinc finger gene RIZin gastric cancer with microsatellite instability, and to identify two coding polyadenosine tracts of RIZ.METHODS: Frameshift mutations at (A)8 and (A)9 tracts of RIZ were detected in 70 human gastric cancer (HGC)specimens by DHPLC and DNA sequencing. Microsatellite instability (MSI) status was assessed by two mononucleotide markers, BAT26 and BAT25, by means of denaturing highperformance liquid chromatography (DHPLC).RESULTS: In 70 HGC samples, 8 (11.4%) were found positive for instabilities at BAT26 and BAT25. In 7 of the 8 cases with instabilities at both BAT26 and BAT25 (MSI-H), 1 was unstable at BAT26 but stable at BAT25. Frameshift mutations were identified in 4 (57.1%) of the 7 samples with MSI-H in the (A)9 tract of RlZwithout mutations in the (A)8 tract.In contrast, frameshift mutations were found in neither of the polyadenosine tracts in 63 samples of MSI-L or MSI stable tumors. Pro704 LOH detection in 4 cases with frameshift mutations did not find LOH in these cases.CONCLUSION: Frameshift mutations of RIZmay play an important role in gastric cancers with MSI.

Immunocytochemical detection of HoxD9 and Pbx1 homeodomain protein expression in Chinese esophageal squamous cell carcinomas

AIM: To evaluate the expression pattern of two novel oncofetal antigens, the HoxD9 and Pbx1 homeoproteins in esophageal squamous cell carcinomas (ESCCs) to determine what role they would play in the carcinogenesis of ESCC.METHODS: We obtained tissue samples of ESCC from 56 patients who underwent esophagectomy but not preoperative chemotherapy or radiotherapy. The diagnosis of ESCC was established and confirmed by staff pathologists. We used a highly sensitive, indirect,immunocytochemical method to detect HoxD9 and PbX1 proteins. We qualitatively and quantitively evaluated cells that exhibited and staining using a light microscope.RESULTS: In all observed carcinoma tissue samples, more than 60% of neoplastic cells stained lightly or strongly for HoxD9, and more than 50% of neoplastic cells stained lightly or strongly for Pbx1.CONCLUSION: Our data suggest that HoxD9 and Pbx1 are inappropriately expressed in most human esophageal squamous cell carcinoma. Understanding the role of Hox genes in esophageal epithelial cell carcinogenesis may not only augment early detection but also offer new avenues for treatment of this disease.

Differences in biological features of gastric dysplasia, indefinite dysplasia, reactive hyperplasia and discriminant analysis of these lesions

AIM: To investigate the differences in biological features of gastric dysplasia (Dys), indefinite dysplasia (IDys) and reactive hyperplasia (RH) by studying the biomarker alterations in cell proliferation, cell differentiation, cell cycle control and the expression of house-keeping genes, and further to search for markers which could be used in guiding the pathological diagnosis of three lesions. METHODS: Expressions of MUC5AC, MUC6, adenomatous polyposis coli (APC), p53, Ki-67, proliferation cell nuclear antigen (PCNA) and EGFR were studied by immunohistochemistry with a standard Envision technique in formalinfixed and paraffin-embedded specimens from 43 RH, 35 IDys, 35 Dys and 36 intestinal type gastric carcinomas (IGC). In addition, Bayes discriminant analysis was used to investigate the value of markers studied in differential diagnosis of RH, IDys, Dys and IGC. RESULTS: The MUC5AC and MUC6 antigen expressions in RH, IDys, Dys and IGC decreased gradually (MUC5AC:86.04%, 77.14%, 28.57%, 6.67%; MUC6: 65.15%, 54.29%, 20.00%, 25.00%, respectively). The expressions of the two markers had no significant difference between RH and IDys, but were all significantly higher than those ofthe other two lesions (MUC5AC: x2 = 27.607, 38.027 and 17.33, 26.092; MUC6: x2= 16.54, 12.665 and 9.282, 6.737, P＜0.01). There was no significant differencebetween RH and IDys, Dys and IGC in MUC6 expression. The APC gene expression in the four lesions had a similar decreasing tendency (RH 69.76%, IDys 68.57%, Dys39.39%, IGC 22.86%), and it was significantly higher in the first two lesions than in the last two (x2 = 7.011,16.995 and 14.737, 19.817, P＜0.05). The p53 expressionin RH, IDys, Dys and IGC was 6.98%, 20%, 57.14% and 50%, respectively. There was no significant differencebetween RH and IDys or Dys and IGC, but the p53 expression in RH and IDys was significantly lower than that in Dys and IGC (x2 = 7.011, 16.995 and 14.737, 19.817, P＜0.01).The Ki-67 label index was significantly different among four lesions (RH: 0.298±8.92%, IDys: 0.358±9.25%,Dys: 0.498±9.03%, IGC: 0.620±10.8%, P＜0.001). Positive immunostaining of PCNA was though observed in all specimens, significant differences were detected among four lesions (F= 95.318, P＜0.01). In addition, we used Bayes discriminant analysis to investigate molecular pathological classification of the lesions, and obtained the best result with the combination of MUC5AC, Ki-67 and PCNA. The overall rate of correct classification was67.4% (RH), 68.6% (IDys), 70.6% (Dys) and 84.8% (IGC), respectively.CONCLUSION: Dys has neoplastic biological characteristics, while RH and IDys display hyperplastic characteristics. MUC5AC and proliferation-related biomarkers (Ki-67, PCNA) are more specific in distinguishing Dys from RH and IDys.

Quantitative detection of common deletion of mitochondrial DNA in hepatocellular carcinoma and hepatocellular nodular hyperplasia

AIM: To study the deletion of mitochondiral DNA in hepatocellular carcinoma and hepatocellular nodular hyperplasia and its significance in the development of cancer. METHODS: Deleted mtDNA (CD-mtDNA) and wild type mtDNA (WT-mtDNA) were quantitatively analyzed by using real-time PCR in 27 hepatocellular carcinomas (HCC) and corresponding noncancerous liver tissues and 27 hepatocellular nodular hyperplasiae (HNH). RESULTS: A novel CD (4 981 bp) was detected in 85% (23/27) and 83%(22/27) of HCC and HNH tumor tissues, respectively, which were significantly higher than that in paired noncancerous liver tissues (57%, 15/27) (P<0.05). The CD/WT-mtDNA ratio in HCC tumors was 0.00092 (median, interquartile range, 0.0001202-0.00105), which was significantly higher than that in paired noncancerous liver tissues (median, 0.000, quartile range, 0-0) (P=0.002, Mann-Whitney Test), and was 25 of times of that in HNH tissues (median, 0.0000374, quartile range, 0-0.0004225) (P=0.002, Mann-Whitney test). CONCLUSION: CD-mtDNA mutation plays an important role in the development and progression of HCC.

Gene expression profiles of hepatocytes treated with La(NO_3)_3 of rare earth in rats

AIM: To compare the gene expression between La(NO3)3-exposed and control rats in vivo. METHODS: Rats were fed La(NO3)3 once daily at a dose of 20 mg/kg for one month by gavage. Gene expression of hepatocytes was detected using mRNA differential display (DD) technique and cDNA microarray and compared between treated and control groups. RESULTS: Six differentially expressed sequence tags were cloned by DD, of which five were up regulated and one was down regulated in treated rats. Two sequences were determined. One band was novel. The other shared 100% sequence homology with AU080263 Sugano mouse brain mncb Mus musculus cDNA clone MNCb-5435 5'. With DNA microarray, 136 differentially expressed genes were identified including 131 over-expressed genes and 5 under-expressed genes. Most of these differentially expressed genes were cell signal and transmission genes, genes associated with metabolism, protein translation and synthesis. CONCLUSION: La(NO3)3 could change the expression levels of some kinds of genes. Further analysis of the differentially expressed genes would be helpful for understanding the wide biological effect spectrum of rare earth elements.

Molecular profiling of hepatocellular carcinomas by cDNA microarray

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. Conventional diagnosis and treatment of this malignancy have been dismal and should be complemented by novel tools. The development and progress of HCC are believed to be caused by the accumulation of genetic changes resulting in altered expression of thousands of cancer-related genes, which can be measured by globe genetic analysis. Gene expression profiling of HCC has been employed to elucidate hepatocarcinogenesis and disclose molecular mechanisms underlying complex clinical features.Identifying phenotype-associated genes/profiles has impacts on current diagnosis and management strategy of HCC. In spite of some pitfalls of this technology and challenges in improving the research process, scrutinous validation of profiling data of HCC combined with other approaches will eventually benefit the patients.

Studies on the role of microtubules in myofibrillogenesis

Co-localization of mierotubule (MT) and muscle myosin (MHO) myofibril immunofluoresoence in developing myotubes of chicken' skeletal muscle cultures was observed by using double staining of tubulin and MHO indirect immunofluorescence. 12-o-tetradeeanoyl-phorbol-13-acetate (TPA) selectively and reversibly blocks mycfibrillogenesis and alters the morphology of myotubes into myosacs where MTs are present in radiating pattern,when the arrested myogenic cells recover and start myofibrillogenesis after released from TPA, prior to the emergence of myofibrils, the pre-existing MTs become bipolarly aligned ooincidently with the tubular restoration of cell shape. Single nascent myofibrils overlapping with MTs extend into the base of growth tips where MTs go farther to the end of the tips. That MT might act as scaffold in guiding the bipolar elongation of the growing myofibrils was suggested. Taxol and ooloemid disturbed MT polymerization and disposition, and interfered with the normal spatial assembly of myofibrils in developing myotubes.

Effect of Diallyl Trisulfide on Induction of UDS by Mutagenic Drugs in Primary Rat Hepatocytes

Mosl of anticancer drugs are mutagenic. A possible exeeption is diallyl .trisulfide(DAT ), a component of garlic. It is an antimutagenic anticunccr chemical although it ismainly uscd as antibiotic. Its modifying effeci on induction of UDS by mutagenicmitomycin C (MMC), cyclophosphamide (CP) and cis-diamine dichloroplatin (DDP) was invcstigiltcd with the UDS assay in the primary cultures of Wistar rat hepatocytes (hpc)using the autoradiographic technique. Resultsshowed that 1.0-4.0 nmol/ml of DAT didnot inducc UDS and that MMC, CP and DDP resulted in a significant induction ofdosc-dependent UDS. DAT enhanced induction of UDS by these drugs. A dose-effectrclationship was observed betwecn dose of DAT and enhancement of induction of UDS.Howcvcr, thc mcchanism of the enhancement is not clear.