Absence of GAPDH regulation in tumor-cells of different origin under hypoxic conditions in – vitro

<p>Abstract</p> <p>Background</p> <p>Gene expression studies related to cancer diagnosis and treatment are important. In order to conduct such experiment accurately, absolutely reliable housekeeping genes are essential to normalize cancer related gene expression. The most important characteristics of such genes are their presence in all cells and their expression levels remain relatively constant under different experimental conditions. However, no single gene of this group of genes manifests always stable expression levels under all experimental conditions. Incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of GAPDH expression regulation in Hep-1-6 mouse hepatoma and Hep-3-B and HepG2 human hepatocellular carcinoma cell lines as well as in human lung adenocarcinoma epithelial cell line (A-549) in addition to both HT-29, and HCT-116 colon cancer cell lines, under hypoxic conditions <it>in vitro </it>in comparison to other housekeeping genes like β-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches was comparatively examined <it>in vitro </it>on both protein and mRNA level, by western blot and semi quantitative RT-PCR, respectively.</p> <p>Findings</p> <p>No hypoxia-induced regulatory effect on GAPDH expression was observed in the cell lines studied <it>in vitro </it>that were; Hep-1-6 mouse hepatoma and Hep-3-B and HepG2 human hepatocellular carcinoma cell lines, Human lung adenocarcinoma epithelial cell line (A-549), both colon cancer cell lines HT-29, and HCT-116.</p> <p>Conclusion</p> <p>As it is the case for human hepatocellular carcinoma, mouse hepatoma, human colon cancer, and human lung adenocarcinoma, GAPDH represents an optimal choice of a housekeeping gene and/(or) loading control to determine the expression of hypoxia induced genes in tumors of different origin. The results confirm our previous findings in human glioblastoma that this gene is not an attractive target for tumor therapeutic approaches because of the lack of GAPDH regulation under hypoxia.</p

Influence of hypoxia and irradiation on osteopontin expression in head and neck cancer and glioblastoma cell lines

Background Tumor hypoxia is a known risk factor for reduced response to radiotherapy. The evaluation of noninvasive methods for the detection of hypoxia is therefore of interest. Osteopontin (OPN) has been discussed as an endogenous hypoxia biomarker. It is overexpressed in many cancers and is involved in tumor progression and metastasis. Methods To examine the influence of hypoxia and irradiation on osteopontin expression we used different cell lines (head and neck cancer (Cal27 and FaDu) and glioblastoma multiforme (U251 and U87)). Cells were treated with hypoxia for 24 h and were then irradiated with doses of 2 and 8 Gy. Osteopontin expression was analyzed on mRNA level by quantitative real-time RT-PCR (qPCR) and on protein level by western blot. Cell culture supernatants were evaluated for secreted OPN by ELISA. Results Hypoxia caused an increase in osteopontin protein expression in all cell lines. In Cal27 a corresponding increase in OPN mRNA expression was observed. In contrast the other cell lines showed a reduced mRNA expression under hypoxic conditions. After irradiation OPN mRNA expression raised slightly in FaDu and U87 cells while it was reduced in U251 and stable in Cal27 cells under normoxia. The combined treatment (hypoxia and irradiation) led to a slight increase of OPN mRNA after 2 Gy in U251 (24 h) and in U87 (24 and 48 h) cell lines falling back to base line after 8 Gy. This effect was not seen in Cal27 or in FaDu cells. Secreted OPN was detected only in the two glioblastoma cell lines with reduced protein levels under hypoxic conditions. Again the combined treatment resulted in a minor increase in OPN secretion 48 hours after irradiation with 8 Gy. Conclusion Osteopontin expression is strongly modulated by hypoxia and only to a minor extent by irradiation. Intracellular OPN homeostasis seems to vary considerably between cell lines. This may explain the partly conflicting results concerning response prediction and prognosis in the clinical setting

Inhibition of N-Myc down regulated gene 1 in in vitro cultured human glioblastoma cells

AIM: To study short dsRNA oligonucleotides (siRNA) as a potent tool for artificially modulating gene expression of N-Myc down regulated gene 1 (NDRG1) gene induced under different physiological conditions (Normoxia and hypoxia) modulating NDRG1 transcription, mRNA stability and translation. METHODS: A cell line established from a patient with glioblastoma multiforme. Plasmid DNA for transfections was prepared with the Endofree Plasmid Maxi kit. From plates containing 5 x 10(7) cells, nuclear extracts were prepared according to previous protocols. The pSUPER-NDRG1 vectors were designed, two sequences were selected from the human NDRG1 cDNA (5'-GCATTATTGGCATGGGAAC-3' and 5'-ATGCAGAGTAACGTGGAAG-3'. reverse transcription polymerase chain reaction was performed using primers designed using published information on -actin and hypoxia-inducible factor (HIF)-1 mRNA sequences in GenBank. NDRG1 mRNA and protein level expression results under different conditions of hypoxia or reoxygenation were compared to aerobic control conditions using the Mann-Whitney U test. Reoxygenation values were also compared to the NDRG1 levels after 24 h of hypoxia (P < 0.05 was considered significant). RESULTS: siRNA- and iodoacetate (IAA)-mediated downregulation of NDRG1 mRNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements bound by nuclear HIF-1 in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results. CONCLUSION: NDRG1 represents an additional diagnostic marker for brain tumor detection, due to the role of hypoxia in regulating this gene, and it can represent a potential target for tumor treatment in human glioblastoma. The siRNA method can represent an elegant alternative to modulate the expression of the hypoxia induced NDRG1 gene and can help to monitor the development of the cancer disease treatment outcome through monitoring the expression of this gene in the patients undergoing the different therapeutic treatment alternatives available nowadays

Upregulated Heat Shock Proteins After Hyperthermic Chemotherapy Point to Induced Cell Survival Mechanisms in Affected Tumor Cells From Peritoneal Carcinomatosis

In patients with peritoneal carcinomatosis cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) represents a promising treatment strategy. Here, we studied the role of hyperthermic chemotherapy on heat shock protein (HSP) expression and induction of tumor cell death and survival. HSP27, HSP70, and HSP90 combined with effects on tumor cell proliferation and chemosensitivity were analyzed in human colon cancer. Hyperthermic chemotherapy resulted in significant HSP27/HSP70 and HSP90 gene/protein overexpression in analyzed HT-29/SW480/SW620 colon cancer cells and peritoneal metastases from patients displaying amplified expression of proliferation markers, proliferating cell nuclear antigen and antiapoptotic protein Bcl-xL. Moreover, functionally increased chemoresistance against 5-fluorouracil/mitomycin C and oxaliplatin after hyperthermic chemotherapy points to induced survival mechanisms in cancer cells. In conclusion, the results indicate that intracellular HSP-associated antiapoptotic and proliferative effects after hyperthermic chemotherapy negatively influence beneficial effects of hyperthermic chemotherapy-induced cell death. Therefore, blocking HSPs could be a promising strategy to further improve the rate of tumor cell death and outcome of patients undergoing HIPEC therapy

Mycotic Ascending Aortic Pseudoaneurysm Causing a Large Mediastinal Abscess

WOS: 000267659000016PubMed ID: 19594821(ECHOCARDIOGRAPHY, Volume 26, July 2009)

Attachment and Psychopathology: Relationship between Adult Attachment and Depression, Panic Disorder, and Obsessive Compulsive Disorder

The objective of this study was to investigate the relationship between adult attachment dimensions and different types of psychopathologies. One hundred and four individuals who were diagnosed with depression, obsessive-compulsive disorder, or panic disorder; and 77 individuals who were not diagnosed with a psychopathology (i.e., control group) participated in the study. Participants completed self-report measures of adult attachment. All three disorder groups reported higher attachment anxiety as compared to the control group. Moreover, patients diagnosed with depression reported higher avoidant attachment as compared to the other disorder groups. A discriminant function analysis was conducted to test if adult attachment dimensions discriminate among different disorder groups and the control group. First function, which was defined by attachment anxiety, discriminated the control group from the three psychopathology groups and the second function, which was defined by attachment avoidance, discriminated the depression group from the other groups. These findings indicate that high attachment anxiety and avoidance emerge as risk factors to develop psychopathology. Possible mechanisms mediating the link between adult attachment and psychopathology are discussed in light of findings of the current study and cultural factors

Efficacy and Tolerability of Sertaconazole Nitrate in Mycotic Vaginitis

WOS: 000261815500007Objectives: In this research, single-dose of 300 mg sertaconazole nitrate ovule in mycotic vaginitis has been evaluated in terms of clinical and microbiological efficacy and safety. Patients and Methods: The study included 177 patients (mean age 31.8 +/- 7.8 years; range 15 to 53 years) who applied to our polyclinics with vaginitis complaints. Patients having cottage cheese-like discharge, vaginal pH<4.5, Whiff test (-) were accepted as mycotic vaginitis. To determine mycotic agents in vaginal discharge, samples were cultured in Sabouraud glucose agar. As a treatment, patients were administered a single-dose of 300 mg sertaconazole nitrate ovule. Its clinical and microbiological aspects have been evaluated in the first visit, a week after and finally one month after the first visit. Symptoms related to vaginitis, clinical and microbiological recovery rates and adverse effects have been noted. Results: All symptom scores were significantly lower in the second visit, and all except dysuria complaint in the third visit. Clinical recovery rates in the second and third visit were in 76.0% and 79.5%. According to mycotic culture test results, the microbiologic recovery rates were 88.8% in the second and 91.4% in the third visits. Conclusion: Single-dose sertaconazole nitrate ovule was evaluated as a convenient, symptom-relieving and safe treatment for mycotic vaginitis

Effect of Epidermal Growth Factor on In Vitro Maturation of Cat Oocytes Recovered from Ovaries at Follicular and Luteal Stages

This study aimed to determine the effect of different concentrations of Epidermal growth factor (EGF) on in vitro maturation (IVM) of cat oocytes collected from ovaries at follicular and luteal phases of reproductive cycle. A total of 612 cumulus oocytes complexes (COCs) were obtained from 42 queens at either follicular or luteal phases. Oocytes were cultured in: (1) TCM 199 (no EGF): (2) TCM 199 plus 10 ng/ml EGF; (3) TCM 199 plus 50 ng/ml EGF. in 5% CO2 aerobic condition for 48 hours According to the chromosomal analyses, each oocyte was placed into the following categories: germinal vesicle (GV), metaphase I (M I). metaphase II (M II) and degenerate. In the luteal phase. the number of oocytes that remained at the GV stage was higher in the EGF 50 group (41.7%; P < 0.05). In the follicular phase. the number of oocytes in M II stage was higher in EGF 10 group (37.9% P < 0.05). The percentage of matured oocytes that reached M II stage was higher in luteal phase (P < 0.05) than in follicular phase for all EGF treated groups. This concluded that. addition of low concentration of EGF (10 ng/ml) to maturation media has a positive effect on the oocytes in comparison with control group (0 ng/ml). In contrast, a high concentration of EGF (50 ng/ml) has a negative result on IVM of cat oocytes and oocytes collected from ovaries on luteal phase are more advisable than follicular phase