Gulperi Oktem

Involvement of protein phosphatase 2A in interferon-α-2b-induced apoptosis in K562 human chronic myelogenous leukaemia cells

Interferon-alpha (IFN-alpha)-2b is known to have antiproliferative effects on hematological malignant cells, especially chronic myelogenous leukaemia (CML). However, it can induce cytogenetical remissions in a very small percentage of the patients. Also during interferon therapy, resistance can emerge in the CML clones. K562 is an in vitro model cell line transformed from a Ph positive CML patient. It can be induced to differentiate to granulocytic and/or monocytic lineages with certain molecules. IFN-alpha-2b generally exerts its effects on CML cells by Janus family kinases (Jak/Stat) pathway, mostly through tyrosine kinase system. However, there is almost no data on the relevance of serine/threonine (Ser/Thr) protein phosphatase (PP) system in the interferon induced signal transduction pathways. In this study, we investigated serine/threonine protein phosphatases in the IFN-alpha-2b induced K562 cytotoxicity. Trypan blue dye exclusion test and MTT assay were utilised for determining cytotoxicity. IC(50) of IFN-alpha-2b on K562 cells was found to be 600IU/ml. However, no differentiation was determined by analysis of cell surface antigen expressions. Serine/threonine protein phosphatase inhibitors calyculin A (Cal A) and okadaic acid (OKA) augmented the IFN-alpha-2b induced cytotoxicity. Apoptosis assay by the mono-oligonucleosome detection and acridine orange/propidium iodide dye revealed marked apoptosis underlying cytotoxicity. Phosphatase enzyme assay revealed a gradual increase in protein phosphatase 2A (PP2A) activity during interferon induced cytotoxicity. Conversely, immunoblots showed no change in the expression of PP2A catalytic and regulatory subunits. In conclusion, PP2A plays a role in IFN-alpha-2b induced apoptosis of K562 cells and should be investigated as a new window furthermore.

Resveratrol attenuates doxorubicin-induced cellular damage by modulating nitric oxide and apoptosis.

Although doxorubicin (DOX) is a commonly used chemotherapeutic agent its clinical use is restricted due to its organ toxicities. The present investigation relates to reducing DOX induced side effects to the liver, kidney and ileum by usage of the antioxidant, anti-inflammatory agent, resveratrol (RES) and to investigate the role of nitric oxide synthase (NOS) in the process. Wistar rats were divided into four groups: control (saline i.p); DOX (20 mg/kg i.p), RES (20 mg/kg i.p) and DOX (20mg/kg i.p)+RES (20 mg/kg i.p). Immunohistochemical activity of both iNOS and eNOS were evaluated after DOX treatment and ultrastructural changes such as cellular damage and mitochondrial degeneration were evaluated. Degenerative ultrastructural changes were demonstrated especially in the DOX treated group. Variations in biochemical marker levels of oxidative stress on ischemia in tissues were not observed. Our data indicate that RES may prevent cellular damage in the early phase of DOX induced toxicity. RES could be used with its beneficial effects during early cellular damage in organ toxicity after DOX treatment in cancer patients.

Assessment of effects of pinealectomy and exogenous melatonin administration on rat sciatic nerve suture repair: an electrophysiological, electron microscopic, and immunohistochemical study

SummaryBackground. Collagen scar formation at the cut end of a nerve, an important problem in clinical practice for neurosurgeons in peripheral nerve surgery, obstructs sprouting of axons into appropriate distal fascicles, and thereby limits nerve regeneration. Researchers attempt to control collagen accumulation in the formation of neuroma by various physical and chemical methods, but these have yielded only limited functional success. This is the first experimental study investigating the effects of melatonin (MLT) on nerve repair and neuronal regeneration in rat sciatic nerve suture repair.Methods. The hypothesis that exogenous MLT administration may inhibit the formation of neuroma in peripheral nerve surgery was investigated in rat sciatic nerve model. In this study, a total of 80 rats were used for control groups (Groups Ia, Ib, IIa, and IId), MLT group (Group Ic), surgical pinealectomy (Px) groups (Groups IIb and IIc), and group of MLT treatment following Px procedure (Group IIe). All animals underwent a surgical intervention consisting of bilateral sciatic nerve section and primary suture repair. At 8 weeks after repair, the animals were killed following completion of recording of nerve action potentials (NAPs). Then, unilateral sciatic nerve specimens including the suture repair region were carefully removed and the excised segments were processed for electron microscopy examination. Afterwards, contralateral sciatic nerve specimens from two animals from each group were removed and stained for immunohistochemical analysis.Results. Results of morphometric analysis revealed that Px procedure caused an elevation of collagen content of the sciatic nerve and macroscopic neuroma formation, and that there was a statistically significant reduction in collagen content of the same region in pinealectomized animals treated with MLT (p<0.001). Accordingly, electrophysiological findings demonstrated that the stimulus intensities required to excite a NAP response were increased in surgical Px group, but the presence of a reduced threshold response was found in the group treated with MLT following Px procedure (p<0.01). Immunohistochemical staining for Type I collagen and Type III collagen was markedly more intense in the epineurium of animals after Px. Virtually no or only weak staining was observed in animals in control groups and the MLT treatment group. Results of immunohistochemical analysis revealed that surgical Px procedure caused a strong immunoreactivity for Type I collagen and Type III collagen in all connective tissue planes of the nerve, especially in the epineurium, and there was a statistically significant reduction in immunoreactivity of the repair region in animals receiving MLT treatment after Px procedure (p<0.001).Conclusion. This study demonstrates that exogenous MLT administration significantly inhibits collagen accumulation in the formation of neuroma in the suture repair site and thereby improves nerve regeneration. From a clinical standpoint, the positive effect of MLT administration on neuroma formation and nerve regeneration seems a particularly attractive treatment option. Therefore, we believe that nerve repair with addition of MLT may be a worthwhile option in addition to other treatment modalities in case of MLT deficiency, such as aging. However, further experimental and clinical studies using functional analysis warranted to confirm this result in future.

Expression profiling of stem cell signaling alters with spheroid formation in CD133high/CD44high prostate cancer stem cells

Cancer stem cells (CSC) isolated from multiple tumor types differentiate in vivo and in vitro when cultured in serum; however, the factors responsible for their differentiation have not yet been identified. The first aim of the present study was to identify CD133high/CD44high DU145 prostate CSCs and compare their profiles with non-CSCs as bulk counterparts of the population. Subsequently, the two populations continued to be three-dimensional multicellular spheroids. Differentiation was then investigated with stem cell-related genomic characteristics. Polymerase chain reaction array analyses of cell cycle regulation, embryonic and mesenchymal cell lineage-related markers, and telomerase reverse transcriptase (TERT) and Notch signaling were performed. Immunohistochemistry of CD117, Notch1, Jagged1, Delta1, Sox2, c-Myc, Oct4, KLF4, CD90 and SSEA1 were determined in CSC and non-CSC monolayer and spheroid subcultures. Significant gene alterations were observed in the CD133high/CD44high population when cultured as a monolayer and continued as spheroid. In this group, marked gene upregulation was determined in collagen type 9 α1, Islet1 and cyclin D2. Jagged1, Delta-like 3 and Notch1 were respectively upregulated genes in the Notch signaling pathway. According to immunoreactivity, the staining density of Jagged1, Sox2, Oct4 and Klf-4 increased significantly in CSC spheroids. Isolated CSCs alter their cellular characterization over the course of time and exhibit a differentiation profile while maintaining their former surface antigens at a level of transcription or translation. The current study suggested that this differentiation process may be a mechanism responsible for the malignant process and tumor growth.

Cancer stem cell differentiation: TGFβ1 and versican may trigger molecules for the organization of tumor spheroids.

Cancer stem cells (CSCs) have the ability to self-renew similar to normal stem cells. This process is linked with metastasis and resistance to chemotherapy and radiotherapy. In the present study, we constructed an in vitro differentiation model for CSCs. CSCs isolated and proliferated for one passage were maintained as monolayers or spheroid-forming cells with serum included media for differentiation process. Differentiation of adhesion molecules and cellular ultrastructural properties were investigated and compared in both monolayer and spheroid cultures. CD133+/CD44+ cancer-initiating cells were isolated from DU-145 human prostate cancer cell line monolayer cultures and propagated as tumor spheroids and compared with the remaining heterogeneous cancer cell bulk population. Microarray-based gene expression analysis was applied to determine genes with differential expression and protein expression levels of candidates were analyzed by immunohistochemistry. Electron microscopy showed detailed analysis of morphology. TGFβ1 was found to be significantly upregulated in monolayer CSCs. High expression levels of VCAN, COL7A1, ITGβ3, MMP16, RPL13A, COL4A2 and TIMP1 and low expression levels of THBS1, MMP1 and MMP14 were detected when CSCs were maintained as serum-grown prostate CSC spheroids. Immunohistochemistry supported increased immunoreactivity of TGFβ1 in monolayer cultures and VCAN in spheroids. CSCs were found to possess multipotential differentiation capabilities through upregulation and/or downregulation of their markers. TGFβ1 is a triggering molecule, it stimulates versican, Col7A1, ITGβ3 and, most importantly, the upregulation of versican was only detected in CSCs. Our data support a model where CSCs must be engaged by one or more signaling cascades to differentiate and initiate tumor formation. This mechanism occurs with intracellular and extracellular signals and it is possible that CSCc themselves may be a source for extracellular signaling. These molecules functioning in tumor progression and differentiation may help develop targeted therapy.

Induced growth inhibition, cell cycle arrest and apoptosis in CD133+/CD44+ prostate cancer stem cells by flavopiridol

Flavopiridol is a flavone that inhibits several cyclin-dependent kinases and exhibits potent growth-inhibitory activity, apoptosis and G1-phase arrest in a number of human tumor cell lines. Flavopiridol is currently undergoing investigation in human clinical trials. The present study focused on the effect of flavopiridol in cell proliferation, cell cycle progression and apoptosis in prostate cancer stem cells (CSCs). Therefore, cluster of differentiation 133 (CD133)+high/CD44+high prostate CSCs were isolated from the DU145 human prostate cancer cell line. The cells were treated with flavopiridol in a dose- and time-dependent manner to determine the inhibitory effect. Cell viability and proliferation were analyzed and the efficiency of flavopiridol was assessed using the sphere-forming assay. Flavopiridol was applied to monolayer cultures of CD133high/CD44high human prostate CSCs at the following final concentrations: 100, 300, 500 and 1000 nM. The cultures were incubated for 24, 48 and 72 h. The half maximal inhibitory concentration (IC50) value of the drug was determined as 500 nM for monolayer cells. Dead cells were analyzed prior and subsequent to exposure to increasing flavopiridol doses. Annexin-V and immunofluorescence analyses were performed for the evaluation of apoptotic pathways. According to the results, flavopiridol treatment caused significant growth inhibition at 500 and 1000 nM when compared to the control at 24 h. G0/G1 analysis showed a statistically significant difference between 100 and 500 nM (P<0.005), 100 and 1000 nM (P<0.001), 300 and 1000 nM (P<0.001), and 500 and 1000 nM (P<0.001). Flavopiridol also significantly influenced the cells in the G2/M phase, particularly at high-dose treatments. Flavopiridol induced growth inhibition and apoptosis at the IC50 dose (500 nM), resulting in a significant increase in immunofluorescence staining of caspase-3, caspase-8 and p53. In conclusion, the present results indicated that flavopiridol could be a useful therapeutic agent for prostate CSCs by inhibiting tumor growth and malignant progression, and inducing apoptosis.

The effect of exogenous melatonin administration on trabecular width, ligament thickness and TGF-β1 expression in degenerated intervertebral disk tissue in the rat

Intervertebral disk (IVD) degeneration, a complex pathological condition of varying origins, causes low back pain. Degenerative changes in IVD tissue affect the adjacent vertebral structure, resulting in a decreased vertebral trabecular width. It has been suggested that transforming growth factor-beta 1 (TGF-beta(1)) may have a role in the repair of connective tissue, as it occurs in the IVD degeneration process. In this study, we investigated the effects of exogenous melatonin (MEL) administration on vertebral trabecular width, ligament thickness and TGF-beta(1) expression in degenerated IVD tissue. Fifteen adult male Swiss Albino rats were divided randomly into three groups; nonoperated control, operated degeneration, and MEL treatment groups. In the operated degeneration and MEL treatment groups, cuts were made parallel to the end plates in the posterior annulus fibrosus at the fifth and tenth vertebral segments of the tail to induce IVD degeneration. In each group, TGF-beta(1) immunoreactivity and morphometry of vertebral trabecular width and anterior and posterior ligament thickness were evaluated. Histologically, disorganisation and irregularity of collagen fibres was seen in the degenerated (operated) IVD. Increased TGF-beta(1) expression in multinuclear chondrocytes was also observed as was decreased vertebral trabecular width. Importantly, the reduction of trabecular width observed in the operated degenerated group was reversed after MEL administration (p<0.0001). Similarly, TGF-beta(1) expression in multinuclear chondrocytes was dramatically increased after exogenous MEL application. Thus, there was a regression in histopathological changes after MEL treatment, with disk appearances similar to those of the control group. Based on our findings, we suggest that MEL activates the recovery process in the degenerated IVD tissue, possibly by stimulating TGF-beta(1) activity. This is the first report investigating the involvement of the pineal hormone MEL in the repair of rat IVD.

Differential effects of doxorubicin and docetaxel on nitric oxide production and inducible nitric oxide synthase expression in MCF-7 human breast cancer cells.

Anthracyclines and docetaxel are frequently used agents in the chemotherapy of breast cancer. In this study we examined the role of inducible nitric oxide synthase (iNOS) expression during the cytotoxicity of these drugs in the MCF-7 breast cancer cell line. Cell viability and cytotoxicity were evaluated by the trypan blue dye exclusion method. Apoptosis and necrosis were determined by the acridine orange/ethidium bromide dye method. The percentage of apoptotic cells was significantly higher with doxorubicin. However, total cytotoxic cell numbers were higher in the docetaxel group compared with doxorubicin, with respect to the control. Most of the cells were seen to be necrotic with the dye method. Cell extracts during the apoptotic process were applied to immunoblot by anti-iNOS monoclonal antibodies. While there was an increase in iNOS expression during docetaxel induced-cytotoxicity, a significant decrease in iNOS expression was detected during doxorubicin-induced cytotoxicity. The Griess method was used for detection of nitrate levels. It was compatible with immunoblot results. These data open a window for further studies to understand the mechanism underlining the cytotoxicity of docetaxel and doxorubicin.

Determination of nitric oxide synthase activity and apoptosis of germ cells in different obstruction models.

We aimed to determine the changes of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) immunoreactivity and apoptosis after proximal and distal obstruction models on ipsilateral and contralateral testicular tissues. Male albino Wistar rats were randomly divided into three groups (n=30): a control group which underwent sham operations (n=10), a unilateral vasal ligation (n=10) and a unilateral epididymal ligation group (n=10). iNOS and eNOS distribution and apoptosis were studied in both ipsilateral and contralateral testes using quantitative immunohistochemistry. Nitric oxide synthase activity was significantly affected in ipsilateral and contralateral testes cells after vasal and epididymal ligation. eNOS immunoreactivity increased markedly after ipsilateral vasal ligation (ILVL). Degeneration-related changes were also associated with changes in apoptotic rate. Analysis using the terminal dUTP nick end-labeling TUNEL method revealed that apoptotic cell numbers significantly increased after ILVL. p53 and bcl-2 immunoreactivity increased in both experimental groups compared with the sham-operated group. Changes in iNOS and eNOS immunolocalisation were strongly associated with cell damage, because germ cell degeneration was more prominent in the ILVL group. Altered p53 immunolocalisation was also associated with cell degeneration, and a rise in bcl-2 immunoreactivity might be considered to reflect a protective mechanism in the testis. These cellular changes could enlighten understanding of the interaction between testicular functioning and damage.

Cancer stem cell and embryonic development-associated molecules contribute to prognostic significance in ovarian cancer.

Objectives Embryonic molecules and cancer stem cell signaling resemble each other, and they organize cancer modality. We hypothesized that similar immunohistochemical expressions between tumor spheroids and patients’ samples compared with clinical relevance would give an important clue in patients’ prognosis. Methods Immunohistochemical expression of c-kit, Notch1, Jagged1, and Delta1 in 50 cases of primary ovarian tumors (10 endometrioid, 10 serous, 10 mucinous adenocarcinoma, 10 borderline serous, and 10 borderline mucinous tumors) and MDAH-2774 spheroids were investigated. Results were compared in both spheroids and tumor samples with morphologic parameters (histological grade) and clinical data (age, stage, tumor size, and metastasis). Results High c-kit and Notch1 immunoreactivity was shown in spheroids, but interestingly immunoreactivity of these molecules in tumor samples was different from patients’ clinicopathological characteristics. In serous carcinoma, metastasis correlated with Notch1 immunoexpression; in mucinous carcinoma, Jagged1 immunohistochemistry correlated with grade, stage, and metastasis of tumor; in borderline serous and mucinous tumors, Jagged1 correlated with high grade. Moreover, Jagged1 correlated with stage and Notch1 with size in borderline mucinous tumor. Endometrioid carcinoma statistics showed that there was a correlation between age and Notch1 expression. Conclusion Notch1, Jagged1, and Delta1 expressions might be useful markers for clinical prognosis of ovarian carcinomas; and Notch pathway, one of the most intensively studied putative therapeutic targets, may be a useful marker for cancer. Consequently, Jagged1 could be a marker for tumor grades and Notch1 as a marker for metastases.