Engin Ulukaya

Apoptosis: why and how does it occur in biology?

The literature on apoptosis has grown tremendously in recent years, and the mechanisms that are involved in this programmed cell death pathway have been enlightened. It is now known that apoptosis takes place starting from early development to adult stage for the homeostasis of multicellular organisms, during disease development and in response to different stimuli in many different systems. In this review, we attempted to summarize the current knowledge on the circumstances and the mechanisms that lead to induction of apoptosis, while going over the molecular details of the modulator and mediators of apoptosis as well as drawing the lines between programmed and non‐programmed cell death pathways. The review will particularly focus on Bcl‐2 family proteins, the role of different caspases in the process of apoptosis, and their inhibitors as well as the importance of apoptosis during different disease states. Understanding the molecular mechanisms involved in apoptosis better will make a big impact on human diseases, particularly cancer, and its management in the clinics. Copyright

Interference by Anti-Cancer Chemotherapeutic Agents in the MTT-Tumor Chemosensitivity Assay

Background: One of the major goals of oncology is to predict the response of patients with cancer to chemotherapeutic agents by employing laboratory methods variously called ‘tumor chemosensitivity assays’, ‘drug response assays’, or ‘drug sensitivity assays’, in vitro. The MTT assay is one of the methods used to predict the drug response in malignancies. However, it may suffer from interference by the anticancer drugs with the MTT assay. Methods: The MTT assay, a colorimetric viability assay, was checked in a cell-free system in terms of its possible chemical interactions with 22 different anticancer drugs. Results: It was found that epirubicine, paclitaxel, doxetaxel, and cisplatin caused a relatively significant increase in absorbance values, resulting in the MTT assay giving rise to false results (untrue increase in viability) although most of the drugs tested did not seem to cause any significant change. Conclusion: It was concluded that before employing the MTT assay, drugs (or any kind of substances) to be included in the assay should be checked first in terms of possible chemical interactions with MTT, otherwise it may be impossible to evaluate the MTT viability assay results correctly.

The effect of melatonin on lipid peroxidation during radiotherapy in female rats.

BackgroundBecause radiotherapy is one of the causes of primary or secondary ovarian failure, protection of ovarian functions in the patients receiving total body or pelvic radiotherapy is of importance. In this study, we investigated the role of melatonin in the oxidative damage in both whole body and ovaries, which is caused by radiotherapy.Materials and MethodsEighteen female rats were divided into 3 groups, each of which consisted of 6 rats. First group was control group receiving no treatment, second group received total body radiotherapy (RT) by 2 × 360 cGy only and third group received radiotherapy plus melatonin. Malondialdehyde (MDA) levels in both blood and ovarian tissue were detected as the indicator of free radical (FR) damage. Levels of erythrocyte superoxide dismutase (SOD) and glutathion peroxidase (GPX) in blood were measured as the indicators of antioxidant level.ResultsRadiotherapy caused a significant increase in the levels of MDA in blood and ovarian tissue (p < 0.001). However, MDA levels decreased in the radiotherapy plus melatonin group (p < 0.05). SOD and GPX levels decreased insignificantly in the radiotherapy only group while they increased in the radiotherapy plus melatonin group significantly (p < 0.01 and p < 0.05, respectively).ConclusionMelatonin, in rats, reduced the level of MDA, which is elevated by radiotherapy and increased the levels of SOD and GPX, which are involved in the antioxidant system.ZusammenfassungZielDa die Strahlentherapie eine der Ursachen der primären bzw. sekundären Ovarialinsuffienz ist, ist es wichtig, die ovariellen Funktionen der Patienten, die einer Beckenoder Gesamtkörperstrahlentherapie unterzogen werden, zu schützen. In dieser Studie untersuchten wir daher, ob Melatonin in der Lage ist, die oxidative Schädigung sowohl des Gesamtkörpers als auch der Eierstocke durch Strahlentherapie zu verringern.Material und Methode18 weibliche Ratten wurden in drei Gruppen unterteilt, von denen jede aus sechs Ratten bestand. Die erste Gruppe war die unbehandelte Kontrollgruppe, die zweite Gruppe empfing nur eine Strahlentherapie von 2mal 360 cGy, und die dritte Gruppe wurde strahlentherapiert und erhielt Melatonin (Radiotherapie + Melatonin). Der Gehalt an Malondialdehyd (MDA) im Blut und ovariellen Gewebe wurde als Indikator für die Schädigung durch freie Radikale bestimmt. Die Bestimmung von Superoxiddismutase (SOD) und von Glutathionperoxidase (GPX) diente als Indikator des antioxidativen Status.ErgebnisseStrahlentherapie verursachte eine bedeutende Zunahme des Gehalts von MDA im Blut und im ovariellen Gewebe (p < 0,001). In der Gruppe der Tiere, die Strahlentherapie und Melatonin erhielten, waren die MDA-Werte signifikant erniedrigt (p < 0,05), SOD und GPX waren in der Gruppe 2 (Radiotherapie) unverandert gegenüber der Kontrolle und erhöhten sich signifikant in der Gruppe 3 (Radiotherapie + Melatonin), (p < 0,01 für SOD und < 0,05 für GPX).SchlußfolgerungenMelatonin verringert bei Ratten den Gehalt an MDA nach Strahlentherapie und erhöht zudem den Gehalt an SOD und GPX, welche antioxidativ wirken.

Effect of Mobile Phone Exposure on Apoptotic Glial Cells and Status of Oxidative Stress in Rat Brain

The aim of this study was to investigate the effects of mobile phone exposure on glial cells in brain. The study carried out on 31 Wistar Albino adult male rats. The rat heads in a carousel exposed to 900 MHz microwave. For the study group (n:14), rats exposed to the radiation 2h per day (7 days in a week) for 10 months. For the sham group (n:7), rats were placed into the carousel and the same procedure was applied except that the generator was turned off. For the cage control (n:10), nothing applied to rats in this group. In this study, rats were euthanized after 10 months of exposure periods and brains were removed. Brain tissues were immunohistochemically stained for the active (cleaved) caspase-3, which is a well-known apoptosis marker, and p53. The expression of the proteins was evaluated by a semi-quantitative scoring system. However, total antioxidative capacity (TAC), catalase, total oxidant status (TOS), and oxidative stress index were measured in rat brain. Final score for apoptosis in the exposed group was significantly lower than the sham (p < 0.001) and the cage control groups (p < 0.01). p53 was not significantly changed by the exposure (p > 0.05). The total antioxidant capacity and catalase in the experimental group was found higher than that in the sham group (p < 0.001, p < 0.05). In terms of the TOS and oxidative stress index, there was no statistically significant difference between exposure and sham groups (p > 0.05). In conclusion, the final score for apoptosis, total antioxidant capacity and catalase in rat brain might be altered by 900 MHz radiation produced by a generator to represent exposure of global systems for mobile communication (GSM) cellular phones.

Decreased plasma levels of soluble receptor for advanced glycation endproducts (sRAGE) in patients with nonalcoholic fatty liver disease

OBJECTIVES Levels of soluble receptor for advanced glycation endproducts (sRAGE) have been linked to several components of the metabolic syndrome. We tested the hypothesis that plasma levels of sRAGE may be associated with non-alcoholic fatty liver disease. DESIGN AND METHODS We enrolled subjects with definite nonalcoholic steatohepatitis (NASH, n=40), borderline NASH (n=8), simple fatty liver (n=9) and healthy controls (n=14). Plasma levels of sRAGE were measured by ELISA. RESULTS Concentrations of sRAGE were significantly lower in patients with definite NASH (1080+/-392 pg/mL, P<0.01) and borderline NASH (1050+/-278 pg/mL, P<0.05) compared to controls (1480+/-387 pg/mL). Levels of sRAGE were significantly and inversely correlated with ALT (r=-0.30, P<0.05) and AST (r=-0.23, P<0.05). CONCLUSION Plasma levels of sRAGE are significantly reduced in definite and borderline NASH.

Response to Neoadjuvant Chemotherapy in Breast Cancer Could be Predictable by Measuring a Novel Serum Apoptosis Product, Caspase-Cleaved Cytokeratin 18: A Prospective Pilot Study

The M30-monoclonal antibody recognizes a neo-epitope of cytokeratin 18 which is formed after caspase-cleavage during apoptosis. Caspase-cleaved cytokeratin 18 is released from apoptotic cells into circulation. The aim of this study was to evaluate the relationship between M30-antigen level and chemotherapy response in neoadjuvant treatment of breast cancer. Forty-two patients with invasive breast carcinoma received 4 cycles of anthracycline based neoadjuvant chemotherapy. Serum samples were obtained for assessment of M30-antigen levels before the administration of first chemotherapy cycle (baseline), and then after 24 and 48 hours for determination of chemotherapy induced apoptosis. M30-antigen levels at 24 and 48 hours were found to be significantly higher than baseline (p < 0.001, p = 0.003, respectively). M30-antigen levels in responders showed statistically significant increases at 24 and 48 hours (p < 0.001; p = 0.004, respectively), while statistically significant increases were not observed in nonresponders. Percentage change of M30-antigen levels was significantly higher in responders than nonresponders at 24 hours (p = 0.020). In conclusion, our study revealed a significant relationship between increases of M30-antigen levels in serum and overall response to therapy.

Utilization of cytokeratin- based biomarkers for pharmacodynamic studies

Cytokeratin (CK)18 is a useful serum biomarker for the determination of cell death of epithelial-derived tumors (carcinomas). ELISAs are available for caspase-cleaved CK18 (M30) released from apoptotic cells, or total CK18 (M65) released by cells undergoing cell death by any cause. These assays have been demonstrated to have prognostic or predictive utility in various types of carcinomas. Encouraging data have been reported by different investigators with regard to the potential use of CK18 as a serum efficacy biomarker for monitoring therapy efficiency in carcinoma patients. The ratio of caspase-cleaved to total CK18 can be determined conveniently in serum or plasma using commercially available ELISA kits (M30-Apoptosense® and M65® ELISA, Peviva AB, Bromma, Sweden). M30:M65 ratios potentially provide information as to whether tumor cells undergo apoptosis or necrosis. However, as discussed in this review, M30:M65 ratios should be interpreted with caution and, preferably, only be applied to samples that contain significant levels of CK18. We conclude that M30 and M65 biomarkers provide both quantitative and qualitative information on carcinoma cell death.

trans-Dichloridopalladium(II) and platinum(II) complexes with 2-(hydroxymethyl)pyridine and 2-(2-hydroxyethyl)pyridine: synthesis, structural characterization, DNA binding and in vitro cytotoxicity studies.

Four trans-palladium(II)- and trans-platinum(II)-chlorido complexes, trans-[PdCl(2)(2-hmpy)(2)] (1), trans-[PtCl(2)(2-hmpy)(2)] (2), trans-[PdCl(2)(2-hepy)(2)] (3) and trans-[PtCl(2)(2-hepy)(2)] (4) (2-hmpy = 2-(hydroxymethyl)pyridine and 2-hepy = 2-(2-hydroxyethyl)pyridine), have been synthesized and characterized by elemental analysis, IR, NMR, and X-ray diffraction. The binding properties of these complexes with fish sperm DNA (FS-DNA) were investigated by UV titration, viscosity, thermal denaturation and electrophoresis measurements. The complexes can bind to FS-DNA and complex 4 exhibits the highest binding constant. Gel electrophoresis assay demonstrates that all the complexes can cleave the pCMV-βgal plasmid DNA to a different degree. The cytotoxic activities of the complexes were tested against four different cancer cell lines. In general, the platinum(II) complexes are more effective than the isostructural palladium(II) complexes. Complex 4 shows high anticancer activity, compared to transplatin, cisplatin, carboplatin and oxaliplatin.

Cell death-inducing effect of novel palladium(II) and platinum(II) complexes on non-small cell lung cancer cells in vitro

PurposeTreatment for lung cancer is still far from satisfying rates. Therefore, there is a need for novel anticancer agents. For this purpose, novel platinum and palladium complexes {[Pd(sac)(terpy)](sac)·4H2O (Complex 1), [Pt(sac)(terpy)](sac)·5H2O (Complex 2), [PdCl(terpy)](sac)·2H2O (Complex 3), [PtCl(terpy)](sac)·2H2O (Complex 4)} have been tested against three different non-small cell lung cancer cell lines (A549, H1299, PC-3).MethodsGrowth-inhibiting effects have been tested by the MTT and ATP viability assays. Apoptosis has been detected by the caspase-cleaved cytokeratin 18 (M30-antigen) assay. Necrosis has been detected by staining the cells with fluorescent dyes. Mitotic index has been calculated by counting the mitotic figures after staining with hematoxylin.ResultsThe complex 3 exhibited significant anti-growth effects, and its anti-growth effect was more powerful than that of cisplatin that is a standard chemotherapeutic agent for this type of cancer. The complexes did not induce apoptosis, while necrosis clearly took place.ConclusionsNovel Pd(II) complex ([PdCl(terpy)](sac)·2H2O) seems to represent a potentially active drug against non-small cell lung cancer cell lines, and further studies in vivo are warranted.

Serum fetuin A/α2HS-glycoprotein levels in patients with non-alcoholic fatty liver disease: relation with liver fibrosis

Background Serum concentrations of fetuin A/α2HS-glycoprotein (AHSG) have been linked to human metabolic alterations and can serve as an indicator of liver cell function. We assayed serum levels of AHSG in patients with non-alcoholic fatty liver disease (NAFLD), a hepatic manifestation of the metabolic syndrome, and examined their association with clinical, biochemical and histological phenotypes. Methods Serum AHSG levels were determined by enzyme linked immunosorbent assay in 99 patients with biopsy-proven NAFLD and 75 age- and gender-matched controls. Results Serum AHSG levels were significantly higher in patients with NAFLD (940 ± 120 μg/mL) compared with healthy controls (800 ± 130 μg/mL, Students t test, P < 0.001). Bivariate analyses (Spearmans rank correlation) in patients with NAFLD showed a statistically significant association between AHSG levels and insulin resistance as assessed by the HOMA (homeostasis model assessment) index (r = 0.31, P < 0.01) and the liver fibrosis score index (r = 0.36, P < 0.001). The association between AHSG and fibrosis remained statistically significant even after adjustment for potential confounders, including the HOMA index ([beta] = 1.65, t = 2.38, P < 0.05). Conclusion Serum AHSG levels are significantly increased in adult patients with biopsy-proven NAFLD and are associated with insulin resistance. Importantly, our pilot data indicate that serum AHSG levels may identify NAFLD patients with higher fibrosis scores.