Therapeutic potential of FLANC, a novel primate-specific long non-coding RNA in colorectal cancer.

OBJECTIVE: To investigate the function of a novel primate-specific long non-coding RNA (lncRNA), named FLANC, based on its genomic location (co-localised with a pyknon motif), and to characterise its potential as a biomarker and therapeutic target. DESIGN: FLANC expression was analysed in 349 tumours from four cohorts and correlated to clinical data. In a series of multiple in vitro and in vivo models and molecular analyses, we characterised the fundamental biological roles of this lncRNA. We further explored the therapeutic potential of targeting FLANC in a mouse model of colorectal cancer (CRC) metastases. RESULTS: FLANC, a primate-specific lncRNA feebly expressed in normal colon cells, was significantly upregulated in cancer cells compared with normal colon samples in two independent cohorts. High levels of FLANC were associated with poor survival in two additional independent CRC patient cohorts. Both in vitro and in vivo experiments demonstrated that the modulation of FLANC expression influenced cellular growth, apoptosis, migration, angiogenesis and metastases formation ability of CRC cells. In vivo pharmacological targeting of FLANC by administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoparticles loaded with a specific small interfering RNA, induced significant decrease in metastases, without evident tissue toxicity or pro-inflammatory effects. Mechanistically, FLANC upregulated and prolonged the half-life of phosphorylated STAT3, inducing the overexpression of VEGFA, a key regulator of angiogenesis. CONCLUSIONS: Based on our findings, we discovered, FLANC as a novel primate-specific lncRNA that is highly upregulated in CRC cells and regulates metastases formation. Targeting primate-specific transcripts such as FLANC may represent a novel and low toxic therapeutic strategy for the treatment of patients.

TNFα gene/protein in tumorigenesis of sporadic colon adenocarcinoma.

PURPOSE: Inherited polymorphisms in immunomodulatory genes such as cytokines may contribute to variation in immunological response and genetic susceptibility for complex diseases, including cancer. TNFα can mediate tumor progression by inducing proliferation, invasion and metastasis of tumor cells. The aim of our study was to examine the allelic frequencies of TNFα promoter SNPs, -1031 T/C, -857 C/T, -308 G/A and -238 G/A, in patients with sporadic colon adenocarcinoma in order to investigate the possible role of these SNPs in susceptibility to sporadic colon cancer. Another aim of this study was to examine the influence of TNFα SNPs on TNFα mRNA and protein expression in colon tumors and their possible role in the development and progression of this type of tumor. RESULTS: The distribution of all four TNFα SNP genotypes in patients showed no significant difference compared to controls. No statistically significant difference in TNFα mRNA expression in tumors and corresponding normal mucous tissue was found (p=0.14). A statistically significant (p=0.028) difference was found in TNFα mRNA expression between histological grade 1 and histological grade 2 and 3 tumors. Additionally, a statistically significant correlation (p=0.03) was found between TNFα-857 C/T genotypes and TNFα mRNA expression in tumor tissue. TNFα mRNA expression was significantly higher in the tumor tissue of patients with -857 CT and -857 TT genotypes. Most of the tumors (78.26%) were positive for TNFα protein. No correlation was found between the TNFα protein expression and clinicopathological characteristics as well as TNFα genotypes. However, patients with TNFα protein negative tumors had longer survival but the result was not statistically significant (p=0.365). CONCLUSION: Our results suggest the role of TNFα as one of the immunomodulatory genes in the progression of sporadic colon cancer.

Reduced FHIT expression is associated with tumor progression in sporadic colon adenocarcinoma.

PURPOSE: Tumor supressor gene FHIT was identified at chromosome 3p14.2 spanning the FRA3B fragile site and is very often inactivated in different types of cancer. The aim of this study was to examine the frequency of FHIT gene LOH as well as FHIT mRNA and protein expression in sporadic colon adenocarcinoma. METHODS: The results of LOH, real-time qRT-PCR and imunohistochemical analyses were correlated with clinico-pathological characteristics of patients and their tumors in order to evaluate the role of FHIT gene/protein in sporadic colon adenocarcinoma tumorigenesis. RESULTS: One hundred and thirty one (96.3%) samples were informative for both markers and 33/131 (25.2%) demonstrated LOH. Expression of FHIT mRNA was significantly decreased in colon tumors relative to that in corresponding normal tissue (p = 7.2×10(-6)). Most of the samples (54.0%) were negative for FHIT protein, 26.4% adenocarcinomas showed a weak to moderate immunostaining and 19.6% adenocarcinomas showed strong FHIT immunostaining. No correlation was found between FHIT gene LOH status, mRNA expression or FHIT protein immunostaining and clinico-pathological characteristics. Expression of FHIT mRNA was significantly decreased in FHIT LOH positive tumors (p = 0.027). Patients with LOH negative tumors or FHIT protein positive tumors had longer survival but this findings were not statistically significant. CONCLUSIONS: Our overall results suggest that reduced expression of FHIT gene may be associated with the progression of these malignant tumors.

Analysis of MSH2 Loss of Heterozygosity, Expression, and IVS10+12G>A Polymorphism in Sporadic Colon Cancer.

BACKGROUND: mutS homolog 2 (MSH2) deficiency may be involved in the development of microsatellite instability found in certain sporadic colorectal tumors. In addition to mutations or loss of heterozygosity resulting in complete loss of MSH2 function, polymorphisms affecting MSH2 expression have been also identified. Therefore, the aim of this study was to examine MSH2 status in sporadic colon cancer. MATERIALS AND METHODS: MSH2 status was examined at the DNA, RNA and protein levels through loss of heterozygosity (LOH) analysis, quantitative real-time PCR and immunohistochemistry. MSH2 IVS10+12A>G polymorphism was examined by real-time single nucleotide polymorphism genotyping. RESULTS: MSH2 LOH was more frequent in tumors larger than 5 cm (p=0.032), mRNA expression was also significantly lower and the same expression pattern was present in the corresponding normal mucosa of the same patient (p=0.013 and p=0.008, respectively). No association was found between IVS10+12A>G polymorphism and susceptibility to sporadic colon cancer. CONCLUSION: Altered MSH2 expression detected in sporadic colon tumors pointing to its role in colorectal tumorigenesis without a hereditary component.

[Laparoscopic surgery in acute abdominal trauma]. / Laparoskopska kirurgija kod akutne ozljede trbuha.

In the past few years, laparoscopy has been increasingly used in patients with acute abdominal trauma. This metod in experienced hands enables direct eye visualisation of abdominal cavity, ensures accurate and early diagnosis and leads to prompt decision on the therapy. Reviewing the literature, laparoscopy in acute abdominal trauma is a safe and accurate procedure to yield diagnosis, enable laparoscopic interventions and help avoid unnecessary laparotomy. In acute abdominal trauma, laparoscopy is mostly used as a diagnostic method, not so often as a therapeutic method, but an expanding range of therapeutic interventions is available. We show our early results with diagnostic and therapeutic laparoscopy from January 1st 2003 to March 31st 2004, stressing therapeutic laparoscopy. We performed 79 laparoscopic explorations in patients with diagnosis of acute abdomen. 7 of them were explored because of acute abdominal trauma. In 3 patients there was no need for intervention after laparoscopic exploration, in 1 patient we electrocoagulates smaller bleeding from the spleen and in other 3 patients we made conversion to open surgery procedure, because of III-IV grade liver or spleen injury. In our group we didn't have injuries of any other internal abdominal organs. Laparoscopic treatment of acute abdominal trauma with spleen or liver injury is applicable in I-III grade liver injury and I-II grade spleen injury, while in greater injuries it is necessary to make conversion to laparotomy.

[Doxycycline induced esophageal ulcers: report of two cases and review of the literature]. / Vrijedovi jednjaka prouzroceni doksiciklinom: prikaz dvaju bolesnika i pregled literature.

Doxycycline-induced esophageal ulcers (DIEU) are rarely observed in endoscopy units. On the other hand doxycycline itself has been claimed as an offending drug in 1/4 out of about 1000 cases of drug induced esophageal ulcers reported in the literature so far. In this article we present two patients with typical history and endoscopic features of esophageal ulcers as an consequence of doxycycline therapy. The therapy consisted of doxycycline withdrawal along with treatment with proton pump inhibitors and sucralfate which had led to disappearance of chest discomfort within one week period. Fourteen days of the tratment beginning complete recovery of the mucosal defects has occurred in both patients. In conclusion, in case of chest pain and painful swallowing occurring in a person who takes doxycycline, DIEU has to be considered followed by the esophagoscopy which would confirm clinical suspicious with high specificity leading to correct diagnosis and treatment of this condition.

Loss of NF2/Merlin expression in advanced sporadic colorectal cancer.

PURPOSE: NF2/Merlin was first identified through its association with neurofibromatosis type 2 (NF2). However, accumulating evidence suggests a more general involvement in tumorigenesis and, in particular, a broader role in tumor suppression. The aim of this study was to examine NF2/Merlin involvement in sporadic colorectal cancer. METHODS: This study is the first to examine the role of NF2/Merlin in sporadic colorectal cancer through LOH analysis at the NF2 locus and mRNA expression analysis via quantitative RT-PCR of total NF2, NF2 isoform I and II. In addition, Merlin protein expression was assessed by immunohistochemistry and Western blotting. RESULTS: NF2 LOH was detected in 20.0 % of heterozygous cases and was found to be more frequent in tumors larger than 5 cm in diameter (p = 0.041) and in tumors with a less differentiated phenotype (p = 0.027). No differences were observed in total NF2 and NF2 isoform I/isoform II mRNA expression between the tumors and their corresponding normal mucous tissues. NF2 isoform II was the most predominant isoform in all samples analyzed. mRNA expression levels of total NF2 and isoforms I and II were significantly lower in poorly differentiated tumors (p = 0.033, p = 0.036 and p = 0.044, respectively). Weak Merlin immunostaining was more frequent in poorly differentiated tumors (p = 0.034) and tumors classified as Dukes' C (p = 0.023). A distinct pattern of Merin phosphorylation was observed in tumors compared to normal mucous tissues. CONCLUSION: Our data indicate that NF2/Merlin may serve as a potential target in the management of colorectal cancer.

Gallbladder cancer.

Gallbladder cancer is the fifth most common cancer involving gastrointestinal tract, but it is the most common malignancy of the biliary tract, accounting for 80-95% of biliary tract cancers. This tumor is a highly lethal disease with an overall 5-year survival of less than 5% and mean survival mere than 6 months. An early diagnosis is essential as this malignancy progresses silently with a late diagnosis. The percentage of patients diagnosed to have gallbladder cancer after simple cholecystectomy for presumed gallbladder stone disease is 0.5-1.5%. Patients with preoperative suspicion of gallbladder cancer should not be treated by laparoscopy. Epidemiological studies have identified striking geographic and ethnic disparities-inordinately high occurrence in American Indians, elevated in Southeast Asia, yet quite low elsewhere in the Americas and the world. Environmental triggers play a critical role in eliciting cancer developing in the gallbladder, best exemplified by cholelithiasis and chronic inflammation from biliary tract and parasitic infections. Improved imaging modalities and improved radical aggressive surgical approach in the last decade has improved outcomes and helped prolong survival in patients with gallbladder cancer. The overall 5-year survival for patients with gallbladder cancer who underwent R0 curative resection was from 21% to 69%. In the future, the development of potential diagnostic markers for disease will yield screening opportunities for those at risk either with ethnic susceptibility or known anatomic anomalies of the biliary tract.

Interleukin-6-174 G/C polymorphism is not associated with IL-6 expression and susceptibility to sporadic colon cancer.

Interleukin-6 (IL-6) has been implicated in tumorigenesis; however, its role is still far from being clearly defined. Regulation of IL-6 expression is highly complex, and additional complexity is introduced by single-nucleotide polymorphisms in the IL-6 gene. These single-nucleotide polymorphisms might influence mRNA transcription, which might in turn result in increased susceptibility to certain tumors. The aim of this study was to analyze IL-6 mRNA and protein expressions in sporadic colon cancer. Influence of IL-6-174 G/C polymorphism on IL-6 mRNA expression and sporadic colon cancer susceptibility was evaluated as well. The frequency of IL-6-174 G/C was analyzed by polymerase chain reaction-restriction fragment length polymorphism analysis. IL-6 mRNA and protein expressions were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. No statistically significant difference in IL-6 mRNA expression in tumor tissue compared with the corresponding normal tissue was observed (p = 0.116). No correlation was found between IL-6 mRNA and protein expressions and clinicopathological features of sporadic colon tumors. There was no association of IL-6-174 G/C genotypes with IL-6 mRNA expression in colon tumors and corresponding normal mucous tissue (p = 0.355; p = 0.152). Finally, there was no association of IL-6-174 G/C with susceptibility to sporadic colon cancer.

Association of H-ras polymorphisms and susceptibility to sporadic colon cancer.

High incidence of colon cancer worldwide indicates the importance of studying genetic alterations that lead to its carcinogenesis. Two polymorphisms in H-ras gene, hexanucleotide tandem repeats in the first intron and SNP 81T>C in the first exon, that might be connected with susceptibility to cancer have been described. The aim of our study was to investigate these loci in Croatian population and to determine if any of them is connected with susceptibility to colon cancer in our population. Two hundred healthy volunteers and 200 colon cancer patients were genotyped using PCR and RFLP methods. We noted statistically significant difference in genotype distribution at hexanucleotide locus between healthy population and colon cancer patients (p=0.013) but not in allelic distribution. At SNP 81 T>C statistically significant difference in distribution of both genotypes (p=9.15x10(-6)) and alleles (p=2.77x10(-6)) was found. No differences were found between genders.

Vascular endothelial growth factor polymorphisms -1154 G/A and -460 C/T are not associated with VEGF mRNA expression and susceptibility to sporadic colon cancer.

Vascular endothelial growth factor (VEGF) is important mediator of angiogenesis, and its expression in colorectal tumors is related to tumor progression. VEGF expression has been detected in normal mucosa, primary colon cancers, and metastatic tumors, and patients with low VEGF expression have a better survival rate. In addition, anti-VEGF monoclonal antibody improves overall survival when used in combination with existing metastatic colorectal cancer therapy. Therefore, prediction of VEGF production based on individual genetic background might be important for predicting the course of the disease and the efficacy of anticancer treatment. The number of studies evaluating the influence of VEGF polymorphisms on cancer susceptibility is growing; however, their results are often conflicting. In addition, these studies are rarely accompanied with the expression analysis examining the influence of these polymorphisms on mRNA expression in tumor tissue. In this study, we have examined the influence of VEGF polymorphisms -1154 G/A and -460 C/T on VEGF mRNA expression and susceptibility to sporadic colon cancer by real-time PCR-SNP and mRNA expression analysis. The study included population control group consisting of 160 unrelated volunteers and a group of 160 patients with sporadic colon cancer. According to our results, -1154 G/A and -460 C/T do not influence VEGF mRNA expression in colorectal tumors and susceptibility to sporadic colon cancer, although the role of other polymorphisms cannot be excluded.