SLCO1B1 Polymorphisms are Associated With Drug Intolerance in Childhood Leukemia Maintenance Therapy.

BACKGROUND: Therapy discontinuations and toxicities occur because of significant interindividual variations in 6-mercaptopurine (6-MP) and methotrexate (MTX) response during maintenance therapy of childhood acute lymphoblastic leukemia (ALL). 6-MP/MTX intolerance in some of the patients cannot be explained by thiopurine S-methyl transferase (TPMT) gene variants. In this study, we aimed to investigate candidate pharmacogenetic determinants of 6-MP and MTX intolerance in Turkish ALL children. METHODS: In total, 48 children with ALL who had completed or were receiving maintenance therapy according to Children's Oncology Group (COG) protocols were enrolled. Fifteen single-nucleotide polymorphisms in 8 candidate genes that were related to drug toxicity or had a role in the 6-MP/MTX metabolism (TPMT, ITPA, MTHFR, IMPDH2, PACSIN2, SLCO1B1, ABCC4, and PYGL) were genotyped by competitive allele-specific PCR (KASP). Drug doses during maintenance therapy were modified according to the protocol. RESULTS: The median drug dose intensity was 50% (28% to 92%) for 6-MP and 58% (27% to 99%) for MTX in the first year of maintenance therapy, which were lower than that scheduled in all patients. Among the analyzed polymorphisms, variant alleles in SLCO1B1 rs4149056 and rs11045879 were found to be associated with lower 6-MP/MTX tolerance. CONCLUSIONS: SLCO1B1 rs4149056 and rs11045879 polymorphisms may be important genetic markers to individualize 6-MP/MTX doses.

Anti-proliferative and anti-invasive effects of ferulic acid in TT medullary thyroid cancer cells interacting with URG4/URGCP.

Ferulic acid (4-hydroxy-3-methoxycinnamic acid; FA), a common dietary plant phenolic compound, is abundant in fruits and vegetables. The aim of present study is to investigate the effects of FA on cell cycle, apoptosis, invasion, migration, and colony formation in the TT medullary thyroid cancer cell line. The effect of FA on cell viability was determined by using CellTiter-Glo assay. IC50 dose in the TT cells was detected as 150 µM. URG4/URGCP (upregulated gene-4/upregulator of cell proliferation) is a novel gene in full-length mRNA of 3.607 kb located on 7p13. It was determined that FA caused a decrease in the expression of novel gene URG4/URGCP, CCND1, CDK4, CDK6, BCL2, MMP2, and MMP9, a significant increase in the expression of p53, PARP, PUMA, NOXA, BAX, BID, CASP3, CASP9, and TIMP1 genes in TT human thyroid cancer cell line by using real-time PCR. It was found that FA in TT cells suppressed invasion, migration, and colony formation by using matrigel invasion chamber, wound healing, and colony formation assay, respectively. In conclusion, it is thought that FA indicates anticarcinogenesis activity by affecting cell cycle arrest, apoptosis, invasion, migration, and colony formation on TT cells.

Temozolomide may induce cell cycle arrest by interacting with URG4/URGCP in SH-SY5Y neuroblastoma cells.

Temozolomide (TMZ) is an alkylating drug used usually in glioma treatment by inducing the apoptosis in glioma cell. The aim of the study is to investigate the anticancer mechanism of TMZ in SH-SY5Y human neuroblastoma cell line. Cytotoxic effects of TMZ were determined by using XTT assay. IC50 doses in the SH-SY5Y were detected as 5 mM. Expression profiles of novel genes URG4/URGCP, CCND1, CCND2, CDK4, and BCL2 were determined by real-time PCR. The apoptotic effects of TMZ were evaluated with TUNEL method. Furthermore, effects of TMZ on colony formation and invasion were investigated in this study. It was observed that TMZ in SH-SY5Y cell line caused a significant decrease in the gene expressions of URG4/URGCP, CCND1, CCND2, CDK4, and BCL2. According to TUNEL assay results, TMZ markedly induced apoptosis in SH-SY5Y cell line. It was found that TMZ in SH-SY5Y cell line suppressed invasion and colony formation using matrigel invasion chamber and colony formation assay, respectively. To conclude, it is thought that TMZ demonstrates anticarcinogenesis activity by affecting cell cycle arrest, apoptosis, invasion, and colony formation on SH-SY5Y cells. TMZ may be an effective agent for treatment of neuroblastoma as a single or in combination with other drugs.

Valproic acid inhibits the proliferation of SHSY5Y neuroblastoma cancer cells by downregulating URG4/URGCP and CCND1 gene expression.

Valproic acid (VPA), used for the treatment of epilepsy and bipolar disorder, regulates several signaling pathways in brain cells. The up-regulated gene 4 (URG4/URGCP) is a novel gene located on 7p13. URG4/URGCP stimulates cyclin D1 (CCND1) mRNA expression, and URG4/URGCP silencing diminishes CCND1 mRNA expression in HepG2 cells. This study was performed to investigate the anti-cancer mechanism of action of VPA by analyzing the expression of novel gene URG4/URGCP, CCND1, p21, p53, p65 (RelA), Bax, and Bcl-2 in SHSY5Y neuroblastoma (NB) cancer cells. Cytotoxic effects of VPA in SHSY5Y were noticed in time and dose dependent manner with the IC50 doses within the range of 0.5-10 mM. IC50 doses in the SHSY5Y were detected as 7.5 mM. Expression profiles were determined by semi quantitative RT-PCR and URG4/URGCP protein change by western blot analysis. Our results suggest that VPA induces cell cycle arrest in SHSY5Y due to the decrease in URG4/URGCP, CCND1 gene expression and the increase in p65. To conclude, VPA may be a prospective agent for the treatment of NB as a single agent or in combination with other drugs. Thus, more studies should be designed to find a safe dose with the best effects of VPA.

Regulation of URG4/URGCP and PPARα gene expressions after retinoic acid treatment in neuroblastoma cells.

Neuroblastoma (NB), originating from neural crest cells, is the most common extracranial tumor of childhood. Retinoic acid (RA) which is the biological active form of vitamin A regulates differentiation of NB cells, and RA derivatives have been used for NB treatment. PPARα (peroxisome proliferator-activated receptor) plays an important role in the oxidation of fatty acids, carcinogenesis, and differentiation. URG4/URGCP gene is a proto-oncogene and that overexpression of URG4/URGCP is associated with metastasis and tumor recurrence in osteosarcoma. It has been known that URG4/URGCP gene is an overexpressed gene in hepatocellular carcinoma and gastric cancers. This study aims to detect gene expression patterns of PPARα and URG4/URGCP genes in SH-SY5Y NB cell line after RA treatment. Expressions levels of PPARα and URG4/URGCP genes were analyzed after RA treatment for reducing differentiation in SH-SY5Y NB cell line. To induce differentiation, the cells were treated with 10 µM RA in the dark for 3-10 days. Gene expression of URG4/URGCP and PPARα genes were presented as the yield of polymerase chain reaction (PCR) products from target genes compared with the yield of PCR products from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. SH-SY5Y cells possess small processes in an undifferentiated state, and after treatment with RA, the cells developed long neurites, resembling a neuronal phenotype. PPARα gene expression increased in RA-treated groups; URG4/URGCP gene expression decreased in SH-SY5Y cells after RA treatment compared with that in the control cells. NB cell differentiation might associate with PPARα and URG4/URGCP gene expression profile after RA treatment.

Leukemogenesis as a new approach to investigate the correlation between up regulated gene 4/upregulator of cell proliferation (URG4/URGCP) and signal transduction genes in leukemia.

The aim of the study is to the determine the profiles of cell cycle genes and a new candidate oncogene of URG4/URGCP which play role in leukemia, establishing the association between the early prognosis of cancer and the quantitation of genetic changes, and bringing a molecular approach to definite diagnosis. In this study, 36 newly diagnosed patients' with ALL-AML in the range of 0-18 years and six control group patients' bone marrow samples were included. Total RNA was isolated from samples and then complementary DNA synthesis was performed. The obtained cDNAs have been installed 96 well plates after prepared appropriate mixtures and assessed with LightCycler(®) 480 Real-Time PCR quantitatively. CHEK1, URG4/URGCP, CCNG1, CCNC, CDC16, KRAS, CDKN2D genes in the T-ALL group; CCND2, ATM, CDK8, CHEK1, TP53, CHEK2, CCNG2, CDK4, CDKN2A, E2F4, CCNC, KRAS genes in the precursor B-ALL group and CCND2, CDK6 genes in the AML group have shown significant increase in mRNA expression level. In the featured role of acute leukemia the regulating signaling pathways of leukemogenesis partially defined, although identification of new genetic markers in acute leukemia subgroups, will allow the development of early diagnostic and new treatment protocols.

Expression of URG4/URGCP, Cyclin D1, Bcl-2, and Bax genes in retinoic acid treated SH-SY5Y human neuroblastoma cells.

Retinoic acid (RA) plays important roles in development, growth, and differentiation by regulating the expression of its target genes. The pro-apoptotic Bax gene may form channels through oligomerization in the mitochondrial membrane and facilitate the cytosolic release of cytochrome c. The anti-apoptotic Bcl-2 gene can inhibit this process. Up-regulated gene 4/Upregulator of cell proliferation (URG4/URGCP) is a novel gene located on 7p13. URG4/URGCP also stimulates cyclin D1 (CCND1) mRNA expression, and RNAi-mediated URG4/URGCP silencing diminishes CCND1 mRNA expression in HepG2 cells. In this study, the effects of RA treatment on URG4/URGCP, CCND1, Bcl-2 and Bax gene expression changes in undifferentiated and differentiated SHSY5Y neuroblastoma cells was analyzed. SHSY5Y cells were cultured in the appropriate conditions. To induce differentiation, the cells were treated with 10 micromolar RA in the dark for 3-10 days. SHSY5Y cells possess small processes in an undifferentiated state, and after treatment with RA, the cells developed long neurites, resembling a neuronal phenotype. Total RNA was isolated with Tri-Reagent. Expression profiles of the target genes were determined by semi-quantitative RT-PCR. According to the results, Bcl-2 and CCND1 gene expression levels were increased, while URG4/URGCP and Bax gene expression was decreased in RA treated cells compared to the control cells. Our preliminary results suggest that RA may induce cell proliferation and escape apoptosis using a novel pathway by the URG4/URGCP gene. Further investigations are needed to clarify more direct transcriptional targets of RA signaling and the interaction of RA pathways with other pro-regenerative signals.

Investigation of microRNA expression changes in HepG2 cell line in presence of URG4/URGCP and in absence of URG4/URGCP suppressed by RNA interference.

Hepatocellular carcinoma (HCC) originates from liver cells and is one of the most common malignant cancers in the world. microRNAs (miRNA), are single strand non-coding RNA molecules with the length of 18-25 nucleotides. miRNAs play an important role in the development of HCC, i.e., miRNAs have a significant impact on multistep hepatocellular carcinogenesis including cellular migration and invasion. URG4/URGCP (up-regulated gene-4/upregulator of cell proliferation) is up-regulated in the presence of HBxAg and has been identified and characterized by Satiroglu-Tufan et al. The full-length URG4/URGCP is 3.607 kb. Overexpression of URG4/URGCP in the presence of HBV X protein may function as a putative oncogene that significantly contributes to multi-step hepatocarcinogenesis. In this study, we aimed to investigate potential miRNA expression changes in HepG2 cell line model system in the presence of URG4/URGCP and in the absence of URG4/URGCP, which was suppressed by RNA interference. To functionally characterize URG4/URGCP, independent cultures of HepG2 cells were stably transfected with pcDNA3 or pcDNA3-URG4/URGCP. Relative quantification of whole genome miRNAs was analyzed by RT-PCR using human whole genome miRNA qPCR profiling kits. Among the 1,034 human miRNAs investigated by the arrays, 77 miRNAs were up-regulated and nine miRNAs were down-regulated in the presence of URG4/URGCP. In conclusion, we have analyzed miRNA profiles in HepG2 cells in presence or absence of URG4/URGCP gene using RNA interference. Some of these miRNAs may play roles in URG4/URGCP gene related disease development through the regulation of different signaling pathways.

Detection of deleted in malignant brain tumors 1 and runt-related transcription factor 3 gene expressions in bladder carcinoma.

Bladder cancer is the fifth most commonly diagnosed cancer in the United States, where the majority of tumors are transitional cell carcinoma. Deleted in malignant brain tumors 1 (DMBT1) gene is located at chromosome 10q25.3-q26.1. DMBT1 gene expression has yet to be investigated in patients with bladder cancer. Runt-related transcription factor 3 (RUNX3) is a candidate tumor suppressor gene which is localized on the chromosome 1p36. RUNX3 gene expression in bladder carcinogenesis is particularly unknown. We aimed to evaluate DMBT1 and RUNX3 gene expression profiles in bladder cancer and how their expressions could be related to carcinogenesis in the bladder and their correlation with clinicopathological parameters. Fifty-six paraffin embedded specimens of transitional cell carcinoma of the urinary bladder were used. Total RNA was extracted from bladder specimens and cDNA was synthesized. The quantification of DMBT1 and RUNX3 mRNAs were succeeded according to the manufacturers' instructions by using RT-PCR. DMBT1 and RUNX3 gene expressions were identified in 100% of bladder carcinoma samples. No significant association was found in these genes expression levels when compared to sex and age. RUNX3 gene expression was decreased non-significantly in high-grade tumors. When DMBT1 gene expression was compared to tumor grades, a significant decrease was detected between grade I and III (P = 0.028). Disruption of expression in relation to tumor suppressors like DMBT1 and RUNX3 genes was associated with bladder cancer. Furthermore, detailed studies including these genes should be performed in protein levels and used more patient specimens in a large scale study.

The expression of URGCP gene in prostate cancer cell lines: correlation with rapamycin.

Molecular targets in prostate cancer are continually being explored, for which there are currently few therapeutic options. Rapamycin (RPM) is an antifungal macrolide antibiotic isolated from Streptomyces hygroscopicus which can inhibit the G1 to S transition. URGCP (upregulator of cell proliferation) is a novel gene located on chromosome 7p13. We aimed to investigate the role of URGCP gene expression changes in PC3, DU145, and LNCAP cell lines with/out RPM. Average cell viability and cytotoxic effect of rapamycin were investigated at 24 h intervals for three days by using Trypan blue dye exclusion test and XTT assay. Cytotoxic effects of rapamycin in DU145, PC3 and LNCAP cells were detected in time and dose dependent manner with the IC(50) doses within the range of 1-100 nM. As the results were evaluated, IC(50) doses in the DU145, PC3, and LNCaP cells were detected as 10, 25, and 50 nM, respectively. The mean relative ratios of URGCP gene expression in DU145, LNCAP and PC3 cells were found as -1.48, 6.59 and -13.00, respectively, when compared to rapamycin-free cells. The False Discovery Rate adjusted p value in DU145, LNCAP and PC3 were 1.25 × 10(-5), 2.20 × 10(-8) and 6.20 × 10(-9), respectively. When the URGCP gene expression level is compared between the dose and control group, we found that URGCP gene expression was significantly decreased in dose groups of DU145 and PC3 cells.

Evaluation of deleted in malignant brain tumors 1 (DMBT1) gene expression in bladder carcinoma cases: preliminary study.

This study was undertaken to evaluate the expression of DMBT1 in bladder cancer and its correlation with clinico-pathological parameters analyzed in bladder carcinoma patients. We investigated DMBT1 in 56 paraffin embedded specimens of transitional cell carcinoma of the urinary bladder. We assessed DMBT1 gene expression at mRNA level by RT-PCR. Our results show 100% expression of DMBT1 in bladder carcinoma samples. Due to this preliminary results; gene expression was compared to tumor grade, and a significant difference was detected between grade 1 and 3 (p = 0.028). The down-regulation of DMBT1 gene expression in carcinomas suggests the possible role in bladder cancer.

The EEC syndrome and SHFM: report of two cases and mutation analysis of p63 gene.

The p63 gene is a transcription factor and a member of the p53 family. Heterozygote mutation of the p63 gene is suggested in a number of human syndromes including limb development and/or ectodermal dysplasia. The EEC syndrome, consisting of ectrodactyly (E), ectodermal dysplasia (E) and cleft lip (C) with or without cleft palate, is the prototype of these syndromes with the presence of heterozygote mutation in the p63 gene in most of the patients. Nonsyndromic split hand/foot malformation (SHFM) is one of the EEC-like syndromes, and the p63 gene mutation was reported in only a few patients. Five different loci have been mapped to date, but the etiology is yet to be explained in the rest of the patients. Here, we report two cases. Case 1, diagnosed with EEC syndrome, had type 2 urogenital sinus and a new heterozygous mutation of 934G>A (D312N) in exon 8 of the p63 gene. Case 2 was diagnosed as SHFM with no mutation in the p63 gene. Genotype and phenotype correlation of these two cases among the reported patients is discussed in this report.

The expression of novel gene URG4 in osteosarcoma: correlation with patients' prognosis.

AIMS: Up-regulated gene 4 (URG4) is a novel gene that may be associated with the onset of tumorigenesis and cell cycle regulation. The present study examined for the first time the expression of URG4 in osteosarcoma, which is one of the most rapidly growing sarcomas, and investigated its prognostic value in both disease-free and overall survival of the patients. METHODS: The expression of URG4 in osteosarcoma tissues was examined by immunohistochemistry in 46 patients who underwent surgical operation for osteosarcoma; the correlation of URG4 with proliferating cell nuclear antigen index (PCNA) and microvessel count (MVC) was analysed, and the prognostic value of URG4 in patients was investigated. RESULTS: URG4 was highly expressed in 40 of 46 (86.96%) osteosarcoma specimens with cytoplasmic staining, and also increased in the specimens with recurrence (p < 0.05) and metastasis (p < 0.05). The mean disease-free survival and overall survival were 50.25 and 54.08 months for patients with over-expressed URG4, compared with 69.54 and 70.01 months for those with low expression. URG4 was also found to be highly related with PCNA, while no significant relationship was found between URG4 and MVC. CONCLUSIONS: URG4 may play important roles in the development of osteosarcoma, and might be a useful molecular marker for predicting the prognosis of osteosarcoma.

Serum or plasma cartilage oligomeric matrix protein concentration as a diagnostic marker in pseudoachondroplasia: differential diagnosis of a family.

Pseudoachondroplasia (PSACH) is an autosomal-dominant osteochondrodysplasia due to mutations in the gene encoding cartilage oligomeric matrix protein (COMP). Clinical diagnosis of PSACH is based primarily on family history, physical examination, and radiographic evaluation, and is sometimes extremely difficult, particularly in adult patients. Genetic diagnosis based on DNA sequencing, on the other hand, can be expensive, time-consuming, and intensive because COMP mutations may be scattered throughout the gene. However, there is evidence that decreased plasma COMP concentration may serve as a diagnostic marker in PSACH, particularly in adult patients. Here, we report the serum and/or plasma COMP concentration-based differential diagnosis of a family with affected adult members. The mean serum and/or plasma COMP concentrations of the three affected family members alive (0.69+/-0.15 and/or 0.81+/-0.08 microg/ml, respectively) were significantly lower than those of an age-compatible control group of 21 adults (1.52+/-0.37 and/or 1.37+/-0.36 microg/ml, respectively; P<0.0001). Bidirectional fluorescent DNA sequencing-based genetic diagnosis of these patients revealed a heterozygous mutation for the nucleotide change 1532A>G in exon 14 of the COMP gene, resulting in a substitution of amino acid 511 from aspartic acid to glycine in COMP. Thus, serum and/or plasma COMP concentration may be suggested as an additional diagnostic marker to aid clinical and radiographic findings in suspected cases of PSACH.

Apoptotic cell death and its relationship to gastric carcinogenesis.

AIM: To investigate the apoptotic process of cells within the intestinal metaplasia areas co-localizing with chronic gastritis and gastric carcinomas and to analyze the involvement of proteins regulating apoptosis in the process of intestinal metaplasia related gastric carcinogenesis. METHODS: Forty-two gastric carcinoma and seventeen chronic gastritis cases were included in this study. All cases were examined for the existence of intestinal metaplasia. Ten cases randomly selected from each group were processed for TUNEL assay. TUNEL positive cells within the intestinal metaplasia areas, co-localizing either to gastric carcinoma or chronic gastritis, were counted and converted to apoptotic indices. In addition, p53, bcl-2 and bax expression patterns within these tissues were analyzed on the basis of immunohistochemistry. RESULTS: Twenty-eight of the cases were intestinal and 14 of the cases were diffuse type adenocarcinomas. 64% (27/42) of the gastric carcinoma cases had intestinal metaplasia. Intestinal metaplasia co-localized more with intestinal type carcinomas compared with diffuse type carcinomas [75% (21/28) vs 42% (6/14), respectively; P

A Concise Review on the Use of Mesenchymal Stem Cells in Cell Sheet-Based Tissue Engineering with Special Emphasis on Bone Tissue Regeneration.

The integration of stem cell technology and cell sheet engineering improved the potential use of cell sheet products in regenerative medicine. This review will discuss the use of mesenchymal stem cells (MSCs) in cell sheet-based tissue engineering. Besides their adhesiveness to plastic surfaces and their extensive differentiation potential in vitro, MSCs are easily accessible, expandable in vitro with acceptable genomic stability, and few ethical issues. With all these advantages, they are extremely well suited for cell sheet-based tissue engineering. This review will focus on the use of MSC sheets in osteogenic tissue engineering. Potential application techniques with or without scaffolds and/or grafts will be discussed. Finally, the importance of osteogenic induction of these MSC sheets in orthopaedic applications will be demonstrated.

Plasma Amino-Terminal Propeptide of C-Type Natriuretic Peptide Concentration in Normal-Weight and Obese Children.

OBJECTIVE: In studies on the relationship between amino-terminal propeptide of C-type natriuretic peptide (NT-proCNP) concentration and height velocity in children, CNP has been implicated as an emerging new growth marker during childhood. It has been reported that besides its well-studied role in growth, plasma CNP levels are reduced in overweight and/or obese adolescents, suggesting CNP as a potential biomarker in childhood obesity. The primary goal of this study was to test this hypothesis in a Turkish population. METHODS: Consent was taken from 317 children [ages 0-18 (158 girls, 159 boys)] and their parents. All subjects were physically examined; anthropometric measurements were obtained. Body mass index was calculated. During routine blood work, 1 mL extra blood was taken. Plasma NT-proCNP concentration was measured by enzyme-linked immunosorbent assay. RESULTS: Results confirmed the previously described relationship between plasma NT-proCNP concentration and growth velocity. Plasma NT-proCNP concentration showed a negative correlation with age, weight, and height in children. Gender was not a factor that alters the age-dependent plasma NT-proCNP concentration until puberty. CONCLUSION: Unlike previous reports, plasma NT-proCNP concentration of overweight/obese children was not significantly lower than that of children with normal weight in age groups analyzed in a Turkish population. Thus, it is too early to conclude that CNP is a potential biomarker in childhood obesity. Further studies are necessary to address this question.

Investigation of HER-2 codon 655 single nucleotide polymorphism frequency and c-ErbB-2 protein expression alterations in gastric cancer patients.

AIM: To investigate both whether the risk of gastric cancer is associated with the Ile/Val single nucleotide polymorphism (SNP) of human epidermal growth factor receptor-2 (HER-2) transmembrane domain-coding region at codon 655 and the suggested existence of HER-2 expression in gastric cancer cases in a Turkish patient group. METHODS: Polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) strategy was used to analyze the presence of HER-2 SNP at codon 655. c-erbB-2 expression pattern was analyzed by immunohistochemistry. The results were compared between gastric carcinoma group and chronic gastritis group, as well as between clinicopathological parameters and carcinoma. RESULTS: Results showed that Ile/Val genotype accounted for 20% within the Turkish gastric carcinoma group, and none in chronic gastritis group, and this genotyping was associated with stage IV gastric cancers (P=0.04). Positive membranous HER-2 immunoreactivity, on the other hand, accounted for 24% within the Turkish gastric carcinoma group and none from chronic gastritis cases; further, it was correlated with intestinal type carcinomas (P=0.007), and stage III-IV carcinomas (P=0.004). CONCLUSION: These observations imply that the tested HER-2 SNP may participate in the development and progression of gastric cancer. Thus, after confirming these results with large sample groups, HER-2 codon 655 SNP and/or c-erbB-2 overexpression may also be used as a poor prognostic indicator for gastric carcinomas.

The chick embryo chorioallantoic membrane as a model system for the study of tumor angiogenesis, invasion and development of anti-angiogenic agents.

Angiogenesis, the formation of new blood vessels, is essential for tumor growth, progression and metastasis. The development of agents that target tumor vasculature is ultimately dependent on the availability of appropriate preclinical screening assays. The chorioallantoic membrane (CAM) assay is well established and widely used as a model to examine angiogenesis, and anti-angiogenesis. This review 1) summarizes the currently used angiogenesis assays and the importance of CAM model among them; 2) summarizes the current knowledge about the development and structure of the CAM's capillary bed; 3) reports findings regarding the role played by molecular signaling pathways in angiogenesis process; 4) discusses the use, advantages and limitations of the CAM as a model for studying tumor angiogenesis and invasiveness, as well as development of angiogenic and/or anti-angiogenic agents; 5) discusses the importance of standardization of the major methodologies for all aspects of the use of the CAM in angiogenesis-related studies; 6) and finally, summarizes major findings regarding the agents developed by the use of CAM model in the study of tumor angiogenesis, invasion and development of anti-angiogenic agents.