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Opuntiol and opuntioside-I an anti-atherogenic agent interfering with chemotaxis via IPLA2B-dependent F-actin polymerization in human monocyte
S116
Pathology (2014), 46(S2)
PATHOLOGY 2014 ABSTRACT SUPPLEMENT
We report 7 novel KRAS gene mutations discovered while retrospectively studying the prevalence and pattern of KRAS mutations in cancerous tissue obtained from 56 Saudi colorectal cancer patients from the Eastern Province. Methods: Genomic DNA was extracted from paraffinembedded, formalin-fixed cancerous and noncancerous colorectal tissues. A nested PCR approach was implemented. Successful and specific PCR products were then bi-directionally sequenced. The functional impact of the novel mutations on the K-ras protein was assessed using bioinformatics tools and molecular modeling. Results: KRAS gene mutations were detected in the cancer tissue of 24 out of 56 cases studied. Of these, 11 were in exon 4 (19.64%). The 11 cases with exon 4 aberrations harbored 8 different mutations. All of these except two altered the K-ras protein amino acid sequence and all except one were novel as revealed by COSMIC database. The detected novel mutations were found to be somatic. The molecular modeling data fit with the prediction from Polyphen-2 (polymorphism phenotyping v2) and SIFT (sorting intolerant from tolerant tools), as well as conservation data. One mutation is predicted to be benign and modeling also showed little predicted effect on protein structure. The remaining mutations are predicted to cause substantial changes in the protein structure in line with the predicted damaging effect by polyphen-2 software. Of these, the Q150X nonsense mutation is the second truncating mutation to be reported in colorectal cancer in the literature.1 Conclusions: Our discovery of novel exon 4 KRAS mutations that are, so far, unique to Saudi patients from the Eastern Province may be attributed to environmental factors and/or racial/ethnic variations due to genetic differences. Alternatively, it may be related to the paucity of clinical studies on mutations other than those in codons 12, 13, 61 and 146. Further KRAS testing on a large number of patients, particularly beyond the most common hotspot alleles in exons 2 and 3, is needed to assess the prevalence and explore the exact prognostic and predictive significance of the discovered novel mutations as well as their possible role in colorectal carcinogenesis.
Reference
1. Palmirotta R, Savonarola A, Formica V, et al. A novel K-ras mutation in colorectal cancer. A case report and literature review. Anticancer Res 2009; 29: 3369–74.
Molecular Pathology: Poster#247 OPUNTIOL AND OPUNTIOSIDE-I AN ANTIATHEROGENIC AGENT INTERFERING WITH CHEMOTAXIS VIA IPLA2B-DEPENDENT F-ACTIN POLYMERIZATION IN HUMAN MONOCYTE Talat Roome1, Saeed Khan1, Anam Razzak1, Ayaz Ahmed1, Rafiq Khanani1, Ahsana Dar2, Shaheen Faizi2, Lubna Abidi2, Lubna2 and Martha Cathcart3 1Molecular Pathology, Department of Pathology, Dow diagnostic Reference and Research Laboratory, Dow International Medical College, Dow University of Health Sciences Karachi, Pakistan, 2H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Pakistan, and 3Department of Cell Biology, Cleveland Clinic Lerner Research Institute, Cleveland Clinic Foundation, OH, USA
Opuntia dilleni (KER-GAW) HAW belongs to the genus Opuntia (Fam. Cactaceae) and has shown remarkable effects as an anti-inflammatory agent. In this study we evaluate the effect of opuntiol and opuntioside-I against MCP-1 dependent chemotaxis. Monocytes Chemoattractant Protein-1 (MCP-1) being a b-chemokine exhibits chemotactic activity via activating signal transducing phospholipases (PLA2) calcium independent iPLA2b and calcium dependent cPLA2a.. We discovered earlier that MCP-1 induces association of iPLA2b with F-actin and actin polymerization is significantly reduced due to inhibition of iPLA2b or its reduced expression. In the present investigation we identified opuntiol and opuntioside-I reduced human monocyte migration (50–90%) dose dependently (2.5–10 mM) against MCP-1 in microchamber chemotaxis assay. Laser scanning confocal microscopy demonstrated opuntiol and opuntioside-I interfered with iPLA2b translocation to cell membrane in MCP-1 stimulated migrating cells and suppressed migration under agarose assay. Furthermore, MCP-1 induced- and iPLA2b dependent-actin polymerization was also inhibited in cells treated with 10 mM of opuntioside-I. F-actin polymerization was studied using phalloidin staining of filaments. In adoptive transfer mouse model, opuntiol and opuntioside-I (10 mg/mL) inhibited cell migration by 80% during thioglycolate induced peritonitis. These compounds (20 mg/kg) were also found to be effective upon oral treatment in mouse peritonitis model. Our results suggest that opuntiol and opuntioside-I interfere with phospholipase A2 signaling in MCP-1 stimulated monocyte via inhibiting iPLA2b activity. We can conclude that these compounds are potential therapeutic agents to reduce MCP-1 dependent monocyte migration and its related immune and inflammatory diseases, e.g., atherosclerosis.
Molecular Pathology: Poster#248 P53 CODON 72 GENE POLYMORPHISM STUDIES AND P53 EXPRESSION BY IHC IN ORAL LESIONS AS RISK FACTOR FOR MALIGNANCY Anand Narain Srivastava1, Nishi Tandon1, Tasleem Raza2 and Vijay Kumar3 1Department of Pathology, 2Department of Biotechnology, Era’s Lucknow Medical College & Hospital, Lucknow, India, and 3Department of Surgical Oncology, KG Medical University, Lucknow, India Background: Genetic mutation of p53 gene is common in several head and neck cancers, usually associated with smoking and HPV infection. In India, instead of HPV, tobacco/Pan Masala chewing are more commonly associated with oral cancer. Accordingly, the present study was planned. Materials and method: A total 41 cases of oral lesions which comprise 6 cases of leukoplakia and 35 cases of oral SCC were studied. The patients were between 30 and 60 years age and were tobacco/Pan Masala chewers. Analysis of p53 codon 72 gene polymorphisms was performed by PCR RFLP for Arg/Arg, Arg/Pro and Pro/Pro. Tissue expression of p53 was done by IHC. Results: Genotype frequencies of 35 carcinoma cases of p53 Arg/ Arg, Arg/Pro and Pro/Pro were 22.86%, 57.15% and 20%, respectively, and 6 leukoplakia cases of p53 Arg/Arg, and Arg/ Pro genotype were 50% and 50%. Out of 21 oral SCC biopsies, 14 (66.66%) showed p53 protein staining, 3 (14.28%) had weak
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
Pathology (2014), 46(S2)
PATHOLOGY 2014 ABSTRACT SUPPLEMENT
We report 7 novel KRAS gene mutations discovered while retrospectively studying the prevalence and pattern of KRAS mutations in cancerous tissue obtained from 56 Saudi colorectal cancer patients from the Eastern Province. Methods: Genomic DNA was extracted from paraffinembedded, formalin-fixed cancerous and noncancerous colorectal tissues. A nested PCR approach was implemented. Successful and specific PCR products were then bi-directionally sequenced. The functional impact of the novel mutations on the K-ras protein was assessed using bioinformatics tools and molecular modeling. Results: KRAS gene mutations were detected in the cancer tissue of 24 out of 56 cases studied. Of these, 11 were in exon 4 (19.64%). The 11 cases with exon 4 aberrations harbored 8 different mutations. All of these except two altered the K-ras protein amino acid sequence and all except one were novel as revealed by COSMIC database. The detected novel mutations were found to be somatic. The molecular modeling data fit with the prediction from Polyphen-2 (polymorphism phenotyping v2) and SIFT (sorting intolerant from tolerant tools), as well as conservation data. One mutation is predicted to be benign and modeling also showed little predicted effect on protein structure. The remaining mutations are predicted to cause substantial changes in the protein structure in line with the predicted damaging effect by polyphen-2 software. Of these, the Q150X nonsense mutation is the second truncating mutation to be reported in colorectal cancer in the literature.1 Conclusions: Our discovery of novel exon 4 KRAS mutations that are, so far, unique to Saudi patients from the Eastern Province may be attributed to environmental factors and/or racial/ethnic variations due to genetic differences. Alternatively, it may be related to the paucity of clinical studies on mutations other than those in codons 12, 13, 61 and 146. Further KRAS testing on a large number of patients, particularly beyond the most common hotspot alleles in exons 2 and 3, is needed to assess the prevalence and explore the exact prognostic and predictive significance of the discovered novel mutations as well as their possible role in colorectal carcinogenesis.
Reference
1. Palmirotta R, Savonarola A, Formica V, et al. A novel K-ras mutation in colorectal cancer. A case report and literature review. Anticancer Res 2009; 29: 3369–74.
Molecular Pathology: Poster#247 OPUNTIOL AND OPUNTIOSIDE-I AN ANTIATHEROGENIC AGENT INTERFERING WITH CHEMOTAXIS VIA IPLA2B-DEPENDENT F-ACTIN POLYMERIZATION IN HUMAN MONOCYTE Talat Roome1, Saeed Khan1, Anam Razzak1, Ayaz Ahmed1, Rafiq Khanani1, Ahsana Dar2, Shaheen Faizi2, Lubna Abidi2, Lubna2 and Martha Cathcart3 1Molecular Pathology, Department of Pathology, Dow diagnostic Reference and Research Laboratory, Dow International Medical College, Dow University of Health Sciences Karachi, Pakistan, 2H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Pakistan, and 3Department of Cell Biology, Cleveland Clinic Lerner Research Institute, Cleveland Clinic Foundation, OH, USA
Opuntia dilleni (KER-GAW) HAW belongs to the genus Opuntia (Fam. Cactaceae) and has shown remarkable effects as an anti-inflammatory agent. In this study we evaluate the effect of opuntiol and opuntioside-I against MCP-1 dependent chemotaxis. Monocytes Chemoattractant Protein-1 (MCP-1) being a b-chemokine exhibits chemotactic activity via activating signal transducing phospholipases (PLA2) calcium independent iPLA2b and calcium dependent cPLA2a.. We discovered earlier that MCP-1 induces association of iPLA2b with F-actin and actin polymerization is significantly reduced due to inhibition of iPLA2b or its reduced expression. In the present investigation we identified opuntiol and opuntioside-I reduced human monocyte migration (50–90%) dose dependently (2.5–10 mM) against MCP-1 in microchamber chemotaxis assay. Laser scanning confocal microscopy demonstrated opuntiol and opuntioside-I interfered with iPLA2b translocation to cell membrane in MCP-1 stimulated migrating cells and suppressed migration under agarose assay. Furthermore, MCP-1 induced- and iPLA2b dependent-actin polymerization was also inhibited in cells treated with 10 mM of opuntioside-I. F-actin polymerization was studied using phalloidin staining of filaments. In adoptive transfer mouse model, opuntiol and opuntioside-I (10 mg/mL) inhibited cell migration by 80% during thioglycolate induced peritonitis. These compounds (20 mg/kg) were also found to be effective upon oral treatment in mouse peritonitis model. Our results suggest that opuntiol and opuntioside-I interfere with phospholipase A2 signaling in MCP-1 stimulated monocyte via inhibiting iPLA2b activity. We can conclude that these compounds are potential therapeutic agents to reduce MCP-1 dependent monocyte migration and its related immune and inflammatory diseases, e.g., atherosclerosis.
Molecular Pathology: Poster#248 P53 CODON 72 GENE POLYMORPHISM STUDIES AND P53 EXPRESSION BY IHC IN ORAL LESIONS AS RISK FACTOR FOR MALIGNANCY Anand Narain Srivastava1, Nishi Tandon1, Tasleem Raza2 and Vijay Kumar3 1Department of Pathology, 2Department of Biotechnology, Era’s Lucknow Medical College & Hospital, Lucknow, India, and 3Department of Surgical Oncology, KG Medical University, Lucknow, India Background: Genetic mutation of p53 gene is common in several head and neck cancers, usually associated with smoking and HPV infection. In India, instead of HPV, tobacco/Pan Masala chewing are more commonly associated with oral cancer. Accordingly, the present study was planned. Materials and method: A total 41 cases of oral lesions which comprise 6 cases of leukoplakia and 35 cases of oral SCC were studied. The patients were between 30 and 60 years age and were tobacco/Pan Masala chewers. Analysis of p53 codon 72 gene polymorphisms was performed by PCR RFLP for Arg/Arg, Arg/Pro and Pro/Pro. Tissue expression of p53 was done by IHC. Results: Genotype frequencies of 35 carcinoma cases of p53 Arg/ Arg, Arg/Pro and Pro/Pro were 22.86%, 57.15% and 20%, respectively, and 6 leukoplakia cases of p53 Arg/Arg, and Arg/ Pro genotype were 50% and 50%. Out of 21 oral SCC biopsies, 14 (66.66%) showed p53 protein staining, 3 (14.28%) had weak
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.