- Email: [email protected]
Evaluation of K-ras mutations in colorectal carcinoma (CRC) patients of East-North of Iran
S200
Abstracts
Introuduction: Squamous cell carcinoma is the most common malignancy in oral cavity. Approximately more than 90% of oral malignancies are squamous cell carcinoma, which have unknown etiology. Recently increased oxidative stress has been implicated in the pathogenesis of various diseases, consist of SCC. The aim of this study is evaluation of stress oxidative activity and oxidative DNA damage of saliva in SCC and in comparison with normal groups. Material and method: Twenty six patients with SCC (mean age 63.5±2.6), and 46 normal ones (48.4±1.2) were enrolled in this study. This study was conducted at the Clinic of Oral Medicine of Tehran University of Medical Sciences in 2009. The unstimulated whole saliva malondialdehyde (MDA), was assayed as an indicator of lipid peroxidation. TAC levels were assayed by thiobarbituric acid and ferric reducing antioxidant potential (FRAP), and 8-OH-dG as an indicator of DNA damage, respectively, in both groups. Results: Level of saliva MDA as a lipid peroxidation marker but not antioxidant level in patients with SCC was significantly higher than that of control group and level of 8-OH-dG as an oxidative DNA damage marker in comparison with control group was lower in patients with SCC, but it was not significant. Conclusion: Results of this study suggested that increased lipid peroxidation and oxidative DNA damage, and decline in antioxidant defence are involved in pathogenesis of SCC. Keywords: Stress oxidative, antioxidant, 8-OH-dG, squamous cell carcinoma, saliva doi:10.1016/j.clinbiochem.2011.08.490
Poster – [A-10-666-1] Effects of lead, nickel and cobalt on chromatin proteins in rat alveolar macrophages and hepatocyte nuclei Rabbani Chadegani Azra, Shahmir Nosrat, Babaei Masoome Department of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran E-mail addresses: [email protected] (R.C. Azra), [email protected] (S. Nosrat), [email protected] (B. Masoome) Introduction: Heavy metals are environmental pollutants, although we can't ignore the side effects of these metals, their application in developing technology and citizens is increased. Methods: In the present study the effects of lead, nickel and cobalt on chromatin proteins in alveolar macrophages and hepatocyte nuclei were investigated, using SDS and agarose gel electrophoresis, UV–Vis spectroscopy and western blotting techniques. Alveolar macrophages were prepared from rat lung by lavage and incubated with various concentrations of metals. Results: The results showed that viability of the cells considerably decreased as metal concentrations were increased. The electrophoresis of histone and non-histone proteins showed that the histone proteins remain unchanged in the presence of metals although some minor effects were observed. Analysis of DNA extract from the treated cells and controls on agarose gel and also using fluorescent dyes revealed that all of these metals, at low concentration proceed the cells into apoptosis, as chromatin condensation and DNA fragmentation occurred in some cells. Whereas higher concentrations of metals induced necrosis. Studies on the effect of metals on nuclei represented that the absorbance at 260, 230 and 210 nm is decreased when metal concentration was increased. SDS gel electrophoresis and western blot also confirmed the results indicating that interaction of metals with the nuclei decreases the extractability of the histone proteins of the chromatin. Conclusion: It is finally concluded that lead, nickel and cobalt bind to chromatin proteins, however, the extent of binding was different among the metals.
Keywords: Lead, nickle, cobalt, chromatin
doi:10.1016/j.clinbiochem.2011.08.491
E.Poster – [A-10-718-2] Association between Cox-2 promoter −765G>C polymorphism and gastric adenocarcinoma in Iranian patients Kadivar Mehdia, Rostami Massomehb a Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran b Science and Research Branch, Islamic Azad University, Tehran, Iran E-mail addresses: [email protected] (K. Mehdi), [email protected] (R. Massomeh) Introduction: Gastric cancer (GC) is the second cause of cancerrelated death and its frequency is increasing in general populations especially in Asian countries. The development of GC is assumed to be a complex interaction between genetics, heredity, dietary factors and infections especially with H. pylori infection. As up-regulation of cyclooxigenase-2 (Cox-2) has been observed in inhibition of apoptosis, tumor growth, and angiogenesis, we investigated single nucleotide polymorphism of −765G>C in Cox-2 gene promoter. Materials and methods: Five ml of peripheral blood was collected from 100 patients with GC and 100 controls. DNA extraction was performed using standard salting-out method. Genomic DNA samples were amplified by PCR using specific primers for Cox-2 gene promoter. Subsequently, PCR products were digested with Bsh1236I restriction endonuclease and results were observed in %3 agarose gel stained with Ethidium bromide. Results: The results showed that the frequency of CC, CG and GG genotypes were 4%, 32% and 64%, respectively in normal controls and 5%, 44% and 51% in patients with gastric adenocarcinoma. The statistical analysis revealed significant differences between genotypes (P value C polymorphism in Iranian population. Conclusion: We revealed that subjects with C carriers of Cox-2 gene are more susceptible to develop Gastric cancer. As the frequency of this polymorphism varies among different ethnic populations, it is important to identify each genetic polymorphism in each population. Keywords: Cox-2 gene, gastric adenocarcinoma, RFLP-PCR doi:10.1016/j.clinbiochem.2011.08.492
Poster – [A-10-731-1] Evaluation of K-ras mutations in colorectal carcinoma (CRC) patients of East-North of Iran Lary Sara, Ghaffarzadegan kamran, Afsharnezhad sima, Hoseinkhani Saman, Mohammadi Ghazaleh E-mail addresses: [email protected] (L. Sara), [email protected] (G. kamran), [email protected] (A. sima), [email protected] (H. Saman), [email protected] (M. Ghazaleh) Introduction: Colon cancer is the third most common of digestive cancers in Iran after the stomach and esophagus cancer. K-ras mutations, one of the earliest events observed in colorectal carcinogenes, are a key step in colorectal cancer progression. K-ras mutations are mostly found in codons 12 and 13, and less frequently in codon 61. The aim of this study was identification of K-ras gene mutations in CRC patients among the East-North of Iran, and to assess whether they are linked with the clinicopathological parameters. Materials and methods: Tumor samples were collected from a consecutive series of 54 patients undergoing respective surgery for CRC.
Abstracts
S201
DNA was extracted from tumor. RFLP-PCR was applied for both codon 12 and 13 mutation detection. Results: Amongst colorectal patients, 35.2% (19 of 54) presented with mutations in K-ras. 47.36% (9 of 54) of the mutations occurred in codon 12, 42.10% (8 of 54) in codon 13 and 10.52% (2 0f 54) contained mutations in both codons. In our study K-ras mutations were significantly associated with CRC stage (P> 0.05). No significant correlations were found between the mutations and sex, age, grade and tumor location. Conclusion: Mutation of the K-ras gene is one of the most common genetic changes in the development of human CRC, but it occurs in a rather low frequency in East-North of Iran. However, in conclusion, we can say in this study that K-ras mutation plays an important role in the development, progression and stage of CRC.
Keywords: CRC, APC, FAP, Mutation
Keywords: Colorectal cancer, stage, K-ras, RFLP-PCR
Introduction: Bombesin, a 14-amino acid peptide, shows high affinity for BN/GRP receptors, which are over expressed by a variety of cancers, including prostate, breast, pancreas, gastrointestinal, and small cell lung cancer. In this study we synthesised and evaluated a 99mTc-bombesin analogue as an imaging agent for GRP receptorpositive tumors. Methods: Synthesis of the HYNIC peptide was carried out on Rink Amide MBHA (4-Methylbenzhydrylamine) resin. 99mTc labeling was performed in the presence of Coligand Tricine/EDDA. Radiochemical evaluation was carried out by Reversed phase HPLC and ITLC-SG. Invitro internalization was tested using human prostate cancer PC-3 cells with blocked and non-blocked receptors. Biodistribution was determined in rats. Results: 99mTc-EDDA-HYNIC-GABA-Bombesin (7–14) was obtained with radiochemical purities >95%. Results of in-vitro studies demonstrated a high stability in serum and suitable internalization (14.7% ± 1.2% at 4 h). Biodistribution data showed a rapid blood clearance, with renal excretion and binding towards GRP receptorpositive tissues such as pancreas (1.4 ± 0.3%ID/g after 4 h). Conclusion: We designed and characterized a new HYNIC-based peptide for diagnostic application. In view of these preclinical data, this novel bombesin conjugate with 99mTc, seems to be a promising candidate in the diagnostic imaging of tumors expressing GRP receptors.
doi:10.1016/j.clinbiochem.2011.08.493
E.Poster – [A-10-736-1] Novel mutation and polymorphism of the APC gene in patient with familial adenomatous polyposis Akbari Zahraa, Yaghoob Taleghani Mohammada, Montazer Haghighi Mahdia, Fatemi Seyyed Rezaa, Damavand Behzada, Irani Shemirani Atenaa, Zali Mohammad Rezab a Taleghani Hospital, Velenjak Street, Tehran, Iran b Taleghani Hospital, Tehran, Iran E-mail addresses: [email protected] (A. Zahra), [email protected] (Y.T. Mohammad), [email protected] (M.H. Mahdi), [email protected] (F.S. Reza), [email protected] (D. Behzad), [email protected] (I.S. Atena), [email protected] (Z.M. Reza) Introduction: Colorectal cancer which has become prevalent in developed countries.Familial adenomatous polyposis (FAP) is an autosomal dominant disease characterized by hundreds of adenomatous polyps in the colon and rectum. Mutation of the adenomatous polyposis coli (APC) gene, a tumor suppressor, is though to be an early event in patients with FAP. Method: This study was conducted on15patients with FAP. DNA was extracted with standard salting out protocol.Whole coding region of the gene was scanned using bidirectional sequencing analysis. Results: We studied the APC gene and identified two novel missense mutations and one polymorphism.The mutations were including a C > G (TCA → TGA) at nucleotide6676 in exon15. This alteration substitute an stop codon to the normal serin at residue2207 (Ser2207stop codon). The other mutation, C > G (CCC → CGC) at nucleotide6811 in exon 15. This alteration replaces proline with arginine at residue 2252 (Pro2252Arg) in another proband. Moreover a novel polymorphism was determined in exon15, A > G (CAA → CGA) at nucleotide 6850. It causes replacement of glutamine with arginine at residue2265 (Glu2265Arg).These mutations have not been reported previously in the patient with FAP. Conclusions: Our findings showed that probably the mutations have been located in these regions are responsible for some cases of FAP deficiency. The S2207 stop codon mutation encoded a truncated protein leading to a complete loss of function and a consequential deficiency of the APC protein making this a pathogenic mutation. Proline and glutamine are both hydrophilic polar amino acid with uncharged side chain while arginine is a hydrophobic polar amino acid with positive charge side chain. So the P2252R mutation and Q2265R polymorphism might affect the three dimensional structure of the protein. Therefore, we could consider the mutations in order to predict individuals at risk of FAP in our population.
doi:10.1016/j.clinbiochem.2011.08.494
Poster – [A-10-764-3] Preparation and evaluation of a new radiolabeled bombesin analogue for diagnosis of bombesin receptor expressing tumors Erfani Mostafa, Goudarzi Mostafa, Shirmardi Seyed Pejman Tehran, Iran E-mail addresses: [email protected] (E. Mostafa), [email protected] (G. Mostafa), [email protected] (S.S. Pejman)
Keywords: Bombesin, GRP receptor, tumour doi:10.1016/j.clinbiochem.2011.08.495
Poster – [A-10-764-4] Preparation and in Vitro evaluation of a somatistatin 2 recptor selective analog for radio-targeting Erfani Mostafa, Shirmardi seyed Pejman, Goudarzi Mostafa Tehran, Iran E-mail addresses: [email protected] (E. Mostafa), [email protected] (S. Pejman), [email protected] (G. Mostafa) Introduction: Somatostatin is a cyclic peptide hormone that occurs naturally in two bioactive molecular forms: somatostatin 14 and 28. It exerts different biological effects in different parts of the body such as brain, pituitary, pancreas, the gut and some component of the immune system. The effect includes inhibition of hormone secretion and modulation of neurotransmission and cell proliferation. Today five different somatostatin receptor subtypes have been characterized and cloned (sstr 1–5). As some of these receptors are over-expressed in several human tumors, especially neuroendocrine tumors and their metastases, these tumors can be visualized in vivo by radio metal chelator conjugated somatostatin analogues like Octreoscan. Radiolabeled somatostatin analogues have been success-
Abstracts
Introuduction: Squamous cell carcinoma is the most common malignancy in oral cavity. Approximately more than 90% of oral malignancies are squamous cell carcinoma, which have unknown etiology. Recently increased oxidative stress has been implicated in the pathogenesis of various diseases, consist of SCC. The aim of this study is evaluation of stress oxidative activity and oxidative DNA damage of saliva in SCC and in comparison with normal groups. Material and method: Twenty six patients with SCC (mean age 63.5±2.6), and 46 normal ones (48.4±1.2) were enrolled in this study. This study was conducted at the Clinic of Oral Medicine of Tehran University of Medical Sciences in 2009. The unstimulated whole saliva malondialdehyde (MDA), was assayed as an indicator of lipid peroxidation. TAC levels were assayed by thiobarbituric acid and ferric reducing antioxidant potential (FRAP), and 8-OH-dG as an indicator of DNA damage, respectively, in both groups. Results: Level of saliva MDA as a lipid peroxidation marker but not antioxidant level in patients with SCC was significantly higher than that of control group and level of 8-OH-dG as an oxidative DNA damage marker in comparison with control group was lower in patients with SCC, but it was not significant. Conclusion: Results of this study suggested that increased lipid peroxidation and oxidative DNA damage, and decline in antioxidant defence are involved in pathogenesis of SCC. Keywords: Stress oxidative, antioxidant, 8-OH-dG, squamous cell carcinoma, saliva doi:10.1016/j.clinbiochem.2011.08.490
Poster – [A-10-666-1] Effects of lead, nickel and cobalt on chromatin proteins in rat alveolar macrophages and hepatocyte nuclei Rabbani Chadegani Azra, Shahmir Nosrat, Babaei Masoome Department of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran E-mail addresses: [email protected] (R.C. Azra), [email protected] (S. Nosrat), [email protected] (B. Masoome) Introduction: Heavy metals are environmental pollutants, although we can't ignore the side effects of these metals, their application in developing technology and citizens is increased. Methods: In the present study the effects of lead, nickel and cobalt on chromatin proteins in alveolar macrophages and hepatocyte nuclei were investigated, using SDS and agarose gel electrophoresis, UV–Vis spectroscopy and western blotting techniques. Alveolar macrophages were prepared from rat lung by lavage and incubated with various concentrations of metals. Results: The results showed that viability of the cells considerably decreased as metal concentrations were increased. The electrophoresis of histone and non-histone proteins showed that the histone proteins remain unchanged in the presence of metals although some minor effects were observed. Analysis of DNA extract from the treated cells and controls on agarose gel and also using fluorescent dyes revealed that all of these metals, at low concentration proceed the cells into apoptosis, as chromatin condensation and DNA fragmentation occurred in some cells. Whereas higher concentrations of metals induced necrosis. Studies on the effect of metals on nuclei represented that the absorbance at 260, 230 and 210 nm is decreased when metal concentration was increased. SDS gel electrophoresis and western blot also confirmed the results indicating that interaction of metals with the nuclei decreases the extractability of the histone proteins of the chromatin. Conclusion: It is finally concluded that lead, nickel and cobalt bind to chromatin proteins, however, the extent of binding was different among the metals.
Keywords: Lead, nickle, cobalt, chromatin
doi:10.1016/j.clinbiochem.2011.08.491
E.Poster – [A-10-718-2] Association between Cox-2 promoter −765G>C polymorphism and gastric adenocarcinoma in Iranian patients Kadivar Mehdia, Rostami Massomehb a Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran b Science and Research Branch, Islamic Azad University, Tehran, Iran E-mail addresses: [email protected] (K. Mehdi), [email protected] (R. Massomeh) Introduction: Gastric cancer (GC) is the second cause of cancerrelated death and its frequency is increasing in general populations especially in Asian countries. The development of GC is assumed to be a complex interaction between genetics, heredity, dietary factors and infections especially with H. pylori infection. As up-regulation of cyclooxigenase-2 (Cox-2) has been observed in inhibition of apoptosis, tumor growth, and angiogenesis, we investigated single nucleotide polymorphism of −765G>C in Cox-2 gene promoter. Materials and methods: Five ml of peripheral blood was collected from 100 patients with GC and 100 controls. DNA extraction was performed using standard salting-out method. Genomic DNA samples were amplified by PCR using specific primers for Cox-2 gene promoter. Subsequently, PCR products were digested with Bsh1236I restriction endonuclease and results were observed in %3 agarose gel stained with Ethidium bromide. Results: The results showed that the frequency of CC, CG and GG genotypes were 4%, 32% and 64%, respectively in normal controls and 5%, 44% and 51% in patients with gastric adenocarcinoma. The statistical analysis revealed significant differences between genotypes (P value C polymorphism in Iranian population. Conclusion: We revealed that subjects with C carriers of Cox-2 gene are more susceptible to develop Gastric cancer. As the frequency of this polymorphism varies among different ethnic populations, it is important to identify each genetic polymorphism in each population. Keywords: Cox-2 gene, gastric adenocarcinoma, RFLP-PCR doi:10.1016/j.clinbiochem.2011.08.492
Poster – [A-10-731-1] Evaluation of K-ras mutations in colorectal carcinoma (CRC) patients of East-North of Iran Lary Sara, Ghaffarzadegan kamran, Afsharnezhad sima, Hoseinkhani Saman, Mohammadi Ghazaleh E-mail addresses: [email protected] (L. Sara), [email protected] (G. kamran), [email protected] (A. sima), [email protected] (H. Saman), [email protected] (M. Ghazaleh) Introduction: Colon cancer is the third most common of digestive cancers in Iran after the stomach and esophagus cancer. K-ras mutations, one of the earliest events observed in colorectal carcinogenes, are a key step in colorectal cancer progression. K-ras mutations are mostly found in codons 12 and 13, and less frequently in codon 61. The aim of this study was identification of K-ras gene mutations in CRC patients among the East-North of Iran, and to assess whether they are linked with the clinicopathological parameters. Materials and methods: Tumor samples were collected from a consecutive series of 54 patients undergoing respective surgery for CRC.
Abstracts
S201
DNA was extracted from tumor. RFLP-PCR was applied for both codon 12 and 13 mutation detection. Results: Amongst colorectal patients, 35.2% (19 of 54) presented with mutations in K-ras. 47.36% (9 of 54) of the mutations occurred in codon 12, 42.10% (8 of 54) in codon 13 and 10.52% (2 0f 54) contained mutations in both codons. In our study K-ras mutations were significantly associated with CRC stage (P> 0.05). No significant correlations were found between the mutations and sex, age, grade and tumor location. Conclusion: Mutation of the K-ras gene is one of the most common genetic changes in the development of human CRC, but it occurs in a rather low frequency in East-North of Iran. However, in conclusion, we can say in this study that K-ras mutation plays an important role in the development, progression and stage of CRC.
Keywords: CRC, APC, FAP, Mutation
Keywords: Colorectal cancer, stage, K-ras, RFLP-PCR
Introduction: Bombesin, a 14-amino acid peptide, shows high affinity for BN/GRP receptors, which are over expressed by a variety of cancers, including prostate, breast, pancreas, gastrointestinal, and small cell lung cancer. In this study we synthesised and evaluated a 99mTc-bombesin analogue as an imaging agent for GRP receptorpositive tumors. Methods: Synthesis of the HYNIC peptide was carried out on Rink Amide MBHA (4-Methylbenzhydrylamine) resin. 99mTc labeling was performed in the presence of Coligand Tricine/EDDA. Radiochemical evaluation was carried out by Reversed phase HPLC and ITLC-SG. Invitro internalization was tested using human prostate cancer PC-3 cells with blocked and non-blocked receptors. Biodistribution was determined in rats. Results: 99mTc-EDDA-HYNIC-GABA-Bombesin (7–14) was obtained with radiochemical purities >95%. Results of in-vitro studies demonstrated a high stability in serum and suitable internalization (14.7% ± 1.2% at 4 h). Biodistribution data showed a rapid blood clearance, with renal excretion and binding towards GRP receptorpositive tissues such as pancreas (1.4 ± 0.3%ID/g after 4 h). Conclusion: We designed and characterized a new HYNIC-based peptide for diagnostic application. In view of these preclinical data, this novel bombesin conjugate with 99mTc, seems to be a promising candidate in the diagnostic imaging of tumors expressing GRP receptors.
doi:10.1016/j.clinbiochem.2011.08.493
E.Poster – [A-10-736-1] Novel mutation and polymorphism of the APC gene in patient with familial adenomatous polyposis Akbari Zahraa, Yaghoob Taleghani Mohammada, Montazer Haghighi Mahdia, Fatemi Seyyed Rezaa, Damavand Behzada, Irani Shemirani Atenaa, Zali Mohammad Rezab a Taleghani Hospital, Velenjak Street, Tehran, Iran b Taleghani Hospital, Tehran, Iran E-mail addresses: [email protected] (A. Zahra), [email protected] (Y.T. Mohammad), [email protected] (M.H. Mahdi), [email protected] (F.S. Reza), [email protected] (D. Behzad), [email protected] (I.S. Atena), [email protected] (Z.M. Reza) Introduction: Colorectal cancer which has become prevalent in developed countries.Familial adenomatous polyposis (FAP) is an autosomal dominant disease characterized by hundreds of adenomatous polyps in the colon and rectum. Mutation of the adenomatous polyposis coli (APC) gene, a tumor suppressor, is though to be an early event in patients with FAP. Method: This study was conducted on15patients with FAP. DNA was extracted with standard salting out protocol.Whole coding region of the gene was scanned using bidirectional sequencing analysis. Results: We studied the APC gene and identified two novel missense mutations and one polymorphism.The mutations were including a C > G (TCA → TGA) at nucleotide6676 in exon15. This alteration substitute an stop codon to the normal serin at residue2207 (Ser2207stop codon). The other mutation, C > G (CCC → CGC) at nucleotide6811 in exon 15. This alteration replaces proline with arginine at residue 2252 (Pro2252Arg) in another proband. Moreover a novel polymorphism was determined in exon15, A > G (CAA → CGA) at nucleotide 6850. It causes replacement of glutamine with arginine at residue2265 (Glu2265Arg).These mutations have not been reported previously in the patient with FAP. Conclusions: Our findings showed that probably the mutations have been located in these regions are responsible for some cases of FAP deficiency. The S2207 stop codon mutation encoded a truncated protein leading to a complete loss of function and a consequential deficiency of the APC protein making this a pathogenic mutation. Proline and glutamine are both hydrophilic polar amino acid with uncharged side chain while arginine is a hydrophobic polar amino acid with positive charge side chain. So the P2252R mutation and Q2265R polymorphism might affect the three dimensional structure of the protein. Therefore, we could consider the mutations in order to predict individuals at risk of FAP in our population.
doi:10.1016/j.clinbiochem.2011.08.494
Poster – [A-10-764-3] Preparation and evaluation of a new radiolabeled bombesin analogue for diagnosis of bombesin receptor expressing tumors Erfani Mostafa, Goudarzi Mostafa, Shirmardi Seyed Pejman Tehran, Iran E-mail addresses: [email protected] (E. Mostafa), [email protected] (G. Mostafa), [email protected] (S.S. Pejman)
Keywords: Bombesin, GRP receptor, tumour doi:10.1016/j.clinbiochem.2011.08.495
Poster – [A-10-764-4] Preparation and in Vitro evaluation of a somatistatin 2 recptor selective analog for radio-targeting Erfani Mostafa, Shirmardi seyed Pejman, Goudarzi Mostafa Tehran, Iran E-mail addresses: [email protected] (E. Mostafa), [email protected] (S. Pejman), [email protected] (G. Mostafa) Introduction: Somatostatin is a cyclic peptide hormone that occurs naturally in two bioactive molecular forms: somatostatin 14 and 28. It exerts different biological effects in different parts of the body such as brain, pituitary, pancreas, the gut and some component of the immune system. The effect includes inhibition of hormone secretion and modulation of neurotransmission and cell proliferation. Today five different somatostatin receptor subtypes have been characterized and cloned (sstr 1–5). As some of these receptors are over-expressed in several human tumors, especially neuroendocrine tumors and their metastases, these tumors can be visualized in vivo by radio metal chelator conjugated somatostatin analogues like Octreoscan. Radiolabeled somatostatin analogues have been success-