Mustafa Akkiprik

Multifunctional roles of insulin-like growth factor binding protein 5 in breast cancer.

The insulin-like growth factor axis, which has been shown to protect cells from apoptosis, plays an essential role in normal cell physiology and in cancer development. The family of insulin-like growth factor binding proteins (IGFBPs) has been shown to have a diverse spectrum of functions in cell growth, death, motility, and tissue remodeling. Among the six IGFBP family members, IGFBP-5 has recently been shown to play an important role in the biology of breast cancer, especially in breast cancer metastasis; however, the exact mechanisms of action remain obscure and sometimes paradoxical. An in-depth understanding of IGFBP-5 would shed light on its potential role as a target for breast cancer therapeutics.

Functional roles and clinical values of insulin-like growth factor-binding protein-5 in different types of cancers.

Insulin-like growth factor-binding proteins (IGFBPs) are critical regulators of the mitogenic activity of insulin-like growth factors (IGFs). IGFBP5, one of these IGFBPs, has special structural features, including a nuclear transport domain, heparin-binding motif, and IGF/extracellular matrix/acid-labile subunit-binding sites. Furthermore, IGFBP5 has several functional effects on carcinogenesis and even normal cell processes, such as cell growth, death, motility, and tissue remodeling. These biological effects are sometimes related with IGF (IGF-dependent effects) and sometimes not (IGF-independent effects). The functional role of IGFBP5 is most likely determined in a cell-type and tissue-type specific manner but also depends on cell context, especially in terms of the diversity of interacting proteins and the potential for nuclear localization. Clinical findings show that IGFBP5 has the potential to be a useful clinical biomarker for predicting response to therapy and clinical outcome of cancer patients. In this review, we summarize the functional diversity and clinical importance of IGFBP5 in different types of cancers.

Melatonin protects against ischemic heart failure in rats

Ischemic injury, which occurs as a result of sympathetic hyperactivity, plays an important role in heart failure. Melatonin is thought to have antiatherogenic, antioxidant, and vasodilatory effects. In this study, we investigated whether melatonin protects against ischemic heart failure (HF). In Wistar albino rats, HF was induced by left anterior descending (LAD) coronary artery ligation and rats were treated with either vehicle or melatonin (10 mg/kg) for 4 weeks. At the end of this period, echocardiographic measurements were recorded and the rats were decapitated to obtain plasma and cardiac tissue samples. Lactate dehydrogenase, creatine kinase, aspartate aminotransferase, alanine aminotransferase, and lysosomal enzymes (β‐D‐glucuronidase, β‐galactosidase, β‐D‐N‐acetyl‐glucosaminidase, acid phosphatase, and cathepsin‐D) were studied in plasma samples, while malondialdehyde and glutathione levels and Na+, K+‐ATPase, caspase‐3 and myeloperoxidase activities were determined in the cardiac samples. Sarco/endoplasmic reticulum calcium ATPase (SERCA) and caveolin‐3 levels in cardiac tissues were evaluated using Western blot analyses. Furthermore, caveolin‐3 levels were also determined by histological analyses. In the vehicle‐treated HF group, cardiotoxicity resulted in decreased cardiac Na+, K+‐ATPase and SERCA activities, GSH contents and caveolin‐3 levels, while plasma LDH, CK, and lysosomal enzyme activities and cardiac MDA and Myeloperoxidase (MPO) activities were found to be increased. On the other hand, melatonin treatment reversed all the functional and biochemical changes. The present results demonstrate that Mel ameliorates ischemic heart failure in rats. These observations highlight that melatonin is a promising supplement for improving defense mechanisms in the heart against oxidative stress caused by heart failure.

Mitochondrial DNA common deletion is not associated with thyroid, breast and colorectal tumors in Turkish patients

Recently, efforts have been focused on mitochondrial DNA changes and their relation to human cancers. Among them, a 4977 bp deletion of mitochondrial DNA, named “common deletion”, has been investigated in several types of tumors, with inconsistent results. In this study, we investigated the presence of the common deletion in tissues from 25 breast, 25 colorectal and 50 thyroid tumors and in the adjacent healthy tissues from Turkish patients. Samples from healthy volunteers were also evaluated for comparison. Two PCR-based methods were used for the detection of the common deletion. First, two pairs of primers were used to amplify wild-type and deleted mtDNA. Then, a highly sensitive nested-PCR was performed, to determine low amounts of deleted genomes. By the first method, wild-type mtDNAs were observed in all samples, but a deletion was observed in only six thyroid samples, by using the nested-PCR method. In conclusion, the mitochondrial common deletion was very rare in our study group and did not appear to be not related with cancer.

Treatment with either obestatin or ghrelin attenuates mesenteric ischemia-reperfusion-induced oxidative injury of the ileum and the remote organ lung.

To evaluate the effects of exogenous ghrelin or obestatin on intestinal injury and accompanying pulmonary injury, intestinal ischemia-reperfusion (I/R) was induced in rats by obstructing the superior mesenteric artery for 60min, whereas laparotomy was performed in the sham group. At the beginning of the 90-min reperfusion period, the rats were injected with obestatin (100μg/kg), ghrelin (10ng/kg), or saline intravenously (iv). At the end of reperfusion, the blood, ileum, and lung samples were taken for the histological and biochemical assays. In the saline-treated I/R group, the increased serum interleukin (IL)-1β level, high damage scores, and elevated tissue malondialdehyde level and collagen content in both tissues were significantly reduced by obestatin or ghrelin. Increased ileal myeloperoxidase activity of the saline-treated I/R group was reduced by treatment with obestatin or ghrelin, whereas increased pulmonary myeloperoxidase activity was reduced with administration of obestatin. Increased DNA fragmentation in the ileum of the saline-treated I/R group was reduced by both peptides. Elevated luminol-lucigenin chemiluminescence levels and nuclear factor kappa B (NF-κB) messenger RNA (mRNA) expression in the ileum of the saline-treated-I/R group were significantly decreased by obestatin or ghrelin treatment. I/R-induced depletion of the antioxidant glutathione in both ileal and pulmonary tissues was prevented with either obestatin or ghrelin treatment. Administration of either obestatin or ghrelin exerts similar protective effects against I/R-induced ileal and pulmonary injury, thus warranting further investigation for their possible use against ischemic intestinal injury.

Clinical significance of miR-140-5p and miR-193b expression in patients with breast cancer and relationship to IGFBP5

The functional role of IGFBP5 in breast cancer is complicated. Experimental and bioinformatics studies have shown that IGFBP5 is targeted by miR-140-5p and miR-193b, although this has not yet been proven in clinical samples. The aim of this study was to evaluate the expression of miR-140-5p and miR-193b in breast cancer and adjacent normal tissue and assess its correlation with IGFBP5 and the clinicopathological characteristics of the tumors. IGFBP5 protein expression was analyzed immunohistochemically and IGFBP5, miR-140 and miR-193b mRNA expression levels were analyzed with real-time RT-PCR. Tumor tissue had higher miR-140-5p expression than adjacent normal tissue (p = 0.015). Samples with no immunohistochemical staining for IGFBP5 showed increased miR-140-5p expression (p = 0.009). miR-140-5p expression was elevated in invasive ductal carcinomas (p = 0.002), whereas basal-like tumors had decreased expression of miR-140-5p compared to other tumors (p = 0.008). Lymph node-positive samples showed an approximately 13-fold increase in miR-140-5p expression compared to lymph node-negative tissue (p = 0.049). These findings suggest that miR-140-5p, but not miR-193b, could be an important determinant of IGFBP5 expression and clinical phenotype in breast cancer patients. Further studies are needed to clarify the expressional regulation of IGFBP5 by miR-140-5p.

R72P Polymorphism of TP53 in Ulcerative Colitis Patients is Associated with the Incidence of Colectomy, Use of Steroids and the Presence of a Positive Family History

P53 tumor suppressor protein is one of the pivotal regulators for genome integrity, cell cycle and apoptosis. The most commonly and extensively studied single nucleotide polymorphism (SNP) of p53 is Arg>Pro substitution on codon 72 (R72P). Although we know that the SNP has unique functional effects on the protein, its clinical significance is not clearly identified yet. Aim of the study was to access the relationship between R72P genotype distribution and clinical variables in patients with ulcerative colitis (UC) and colorectal cancer (CRC). Genomic DNA samples were extracted from 95 UC, 50 CRC, and 219 healthy controls. R72P genotype analysis was carried out with polymerase chain reaction following by restriction enzyme digestion. We observed that Pro allele carriage is a strong risk factor for CRC (OR = 3.03; 95%CI = 1.91–2.40; p = 0.003), but only modest association with UC (OR = 1.61; 95%CI = 0.98–2.65; p = 0.059) (Pro/Pro and Pro/Arg genotypes vs. Arg/Arg genotype). We did not find any correlation between genotype distribution of the polymorphism and clinical parameters of CRC, but in UC, Pro/Pro genotype was significantly related to an inflammatory bowel disease family history (OR = 8.0; 95%CI = 1.68–38.08, p = 0.015), and Arg/Pro genotype was significantly associated with the history of disease-related colectomy (OR = 17.77; 95%CI = 0.98–323.34, p = 0.012) and steroid use (OR = 10.14; 95%CI = 2.63–39.12, p = 0.0002). Our data suggest that R72P variant seems to be associated with high risk for development of CRC but carries low risk for development of UC. R72P genotypes might be a useful predictive marker for surgical and medical treatment of UC.

Detection of PIK3CA Gene Mutations with HRM Analysis and Association with IGFBP-5 Expression Levels in Breast Cancer

Breast cancer is the second most common cancer and second leading cause of cancer deaths in women. Phosphatidylinositol-3-kinase (PI3K)/AKT pathway mutations are associated with cancer and phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene mutations have been observed in 25-45% of breast cancer samples. Insulin growth factor binding protein-5 (IGFBP-5) can show different effects on apoptosis, cell motility and survival in breast cancer. We here aimed to determine the association between PIK3CA gene mutations and IGFBP-5 expressions for the first time in breast cancer patients. Frozen tumor samples from 101 Turkish breast cancer patients were analyzed with high resolution melting (HRM) for PIK3CA mutations (exon 9 and exon 20) and 37 HRM positive tumor samples were analyzed by DNA sequencing, mutations being found in 31. PIK3CA exon 9 mutations (Q546R, E542Q, E545K, E542K and E545D) were found in 10 tumor samples, exon 20 mutations (H1047L, H1047R, T1025T and G1049R) in 21, where only 1 tumor sample had two exon 20 mutations (T1025T and H1047R). Moreover, we detected one sample with both exon 9 (E542Q) and exon 20 (H1047R) mutations. 35% of the tumor samples with high IGFBP-5 mRNA expression and 29.4% of the tumor samples with low IGFBP-5 mRNA expression had PIK3CA mutations (p=0.9924). This is the first study of PIK3CA mutation screening results in Turkish breast cancer population using HRM analysis. This approach appears to be a very effective and reliable screening method for the PIK3CA exon 9 and 20 mutation detection. Further analysis with a greater number of samples is needed to clarify association between PIK3CA gene mutations and IGFBP-5 mRNA expression, and also clinical outcome in breast cancer patients.

Identification of Differentially Expressed IGFBP5-Related Genes in Breast Cancer Tumor Tissues Using cDNA Microarray Experiments

IGFBP5 is an important regulatory protein in breast cancer progression. We tried to identify differentially expressed genes (DEGs) between breast tumor tissues with IGFBP5 overexpression and their adjacent normal tissues. In this study, thirty-eight breast cancer and adjacent normal breast tissue samples were used to determine IGFBP5 expression by qPCR. cDNA microarrays were applied to the highest IGFBP5 overexpressed tumor samples compared to their adjacent normal breast tissue. Microarray analysis revealed that a total of 186 genes were differentially expressed in breast cancer compared with normal breast tissues. Of the 186 genes, 169 genes were downregulated and 17 genes were upregulated in the tumor samples. KEGG pathway analyses showed that protein digestion and absorption, focal adhesion, salivary secretion, drug metabolism-cytochrome P450, and phenylalanine metabolism pathways are involved. Among these DEGs, the prominent top two genes (MMP11 and COL1A1) which potentially correlated with IGFBP5 were selected for validation using real time RT-qPCR. Only COL1A1 expression showed a consistent upregulation with IGFBP5 expression and COL1A1 and MMP11 were significantly positively correlated. We concluded that the discovery of coordinately expressed genes related with IGFBP5 might contribute to understanding of the molecular mechanism of the function of IGFBP5 in breast cancer. Further functional studies on DEGs and association with IGFBP5 may identify novel biomarkers for clinical applications in breast cancer.

A novel approach for rapid screening of mitochondrial D310 polymorphism

BackgroundMutations in the mitochondrial DNA (mtDNA) have been reported in a wide variety of human neoplasms. A polynucleotide tract extending from 303 to 315 nucleotide positions (D310) within the non-coding region of mtDNA has been identified as a mutational hotspot of primary tumors. This region consists of two polycytosine stretches interrupted by a thymidine nucleotide. The number of cytosines at the first and second stretches are 7 and 5 respectively, according to the GeneBank sequence. The first stretch exhibits a polymorphic length variation (6-C to 9-C) among individuals and has been investigated in many cancer types. Large-scale studies are needed to clarify the relationship between cytosine number and cancer development/progression. However, time and money consuming methods such as radioactivity-based gel electrophoresis and sequencing, are not appropriate for the determination of this polymorphism for large case-control studies. In this study, we conducted a rapid RFLP analysis using a restriction enzyme, BsaXI, for the single step simple determination of 7-C carriers at the first stretch in D310 region.Methods25 colorectal cancer patients, 25 breast cancer patients and 41 healthy individuals were enrolled into the study. PCR amplification followed by restriction enzyme digestion of D310 region was performed for RFLP analysis. Digestion products were analysed by agarose gel electrophoresis. Sequencing was also applied to samples in order to confirm the RFLP data.ResultsSamples containing 7-C at first stretch of D310 region were successfully determined by the BsaXI RFLP method. Heteroplasmy and homoplasmy for 7-C content was also determined as evidenced by direct sequencing. Forty-one percent of the studied samples were found to be BsaXI positive. Furthermore, BsaXI status of colorectal cancer samples were significantly different from that of healthy individuals.ConclusionIn conclusion, BsaXI RFLP analysis is a simple and rapid approach for the single step determination of D310 polymorphism of mitochondrial DNA. This method allows the evaluation of a significant proportion of samples without the need for sequencing- and/or radioactivity-based techniques.