Oya Orun

Prognostic role of sensitive-to-apoptosis gene expression in rectal cancer

AIM To investigate the association between prognosis of rectal cancer treated with chemoradiotherapy (CRT) and expression of sensitive-to-apoptosis (SAG), B-cell lymphoma-extra large (Bcl-X(L)) and Bcl-2 homologous antagonist/killer (Bak). METHODS Real-time quantitative polymerase chain reaction was used to determine the expression of proteins of interest, namely SAG, Bcl-X(L), Bak and β-actin, in rectal carcinoma patients who had a follow-up period of 3 years after CRT. Biopsy specimens were excised from the rectal tumor preceding CRT. RESULTS SAG, Bcl-X(L) and Bak proteins showed significant correlations with each other. In multivariate analysis, patients with high vs low SAG expression showed a statistically significant difference in 2-year survival rates: 56% vs 73%, respectively (P = 0.056). On the other hand, there were no significant correlations between the expression levels of all three genes and metastatic rates or tumor responses to CRT. Mean overall survival in the patients with elevated SAG expression was 27.1 mo ± 3.9 mo [95% confidence interval (CI): 19.3-34.9], and in patients with reduced expression, it was 32.1 mo ± 2.5 mo (95% CI: 27.3-36.9). The corresponding values for Bcl-X(L) were 28.0 mo ± 4.1 mo (95% CI: 19.9-36.1) and 31.7 mo ± 2.9 mo (95% CI: 26.0-37.5), and those for Bak were 29.8 mo ± 3.7 mo (95% CI: 22.5-37.2) and 30.6 mo ± 2.4 mo (95% CI: 25.5-35.0), respectively. CONCLUSION Two-year survival rates significantly correlated with low SAG expression, and SAG may be a candidate gene for good prognosis, independent of therapeutic response of different individuals.

Morphological alterations and distribution of occludin in rat testes after bilateral vasectomy

The aim of study was to investigate the fate and the morphology of the cells which constitute the spermatogenic line, and to determine the distribution of occludin in the testis in adult vasectomized Wistar rats. The rats were divided into two groups: control group (sham-operated) and vasectomized group. One, 3 and 6 months after sham and vasectomy operations, testis samples were examined. The weight of the testes was found to be reduced 3 and 6 months after vasectomy. There was vacuolization in the seminiferous tubules one month after vasectomy. The tubules showed severe atrophy 3 and 6 months after vasectomy. The occludin immunolabeling in the 3- and 6-month groups was weak and diffuse, and the density of the protein was found to be decreased. The increase in the number of apoptotic cells was accompanied by a time-dependent decrease in the number of haploid, diploid and tetraploid cells. This study demonstrated that vasectomy causes degeneration in the seminiferous tubules with alterations in occludin distribution with a decrease in the number of spermatogenic cells. Moreover, these alterations increase in a time-dependent manner.

Synthesis of Tolmetin Hydrazide-Hydrazones and Discovery of a Potent Apoptosis Inducer in Colon Cancer Cells

Tolmetin hydrazide and a novel series of tolmetin hydrazide–hydrazones 4a–l were synthesized in this study. The structures of the new compounds were determined by spectral (FT‐IR, 1H NMR) methods. N′‐[(2,6‐Dichlorophenyl)methylidene]‐2‐[1‐methyl‐5‐(4‐methylbenzoyl)‐1H‐pyrrol‐2‐yl]acetohydrazide (4g) was evaluated in vitro using the MTT colorimetric method against the colon cancer cell lines HCT‐116 (ATCC, CCL‐247) and HT‐29 (ATCC, HTB‐38) to determine growth inhibition and cell viability at different doses. Compound 4g exhibited anti‐cancer activity with an IC50 value of 76 μM against colon cancer line HT‐29 (ATCC, HTB‐38) and did not display cytotoxicity toward control NIH3T3 mouse embryonic fibroblast cells compared to tolmetin. In addition, this compound was evaluated for caspase‐3, caspase‐8, caspase‐9, and annexin‐V activation in the apoptotic pathway, which plays a key role in the treatment of cancer. We demonstrated that the anti‐cancer activity of this compound was due to the activation of caspase‐8 and caspase‐9 involved in the apoptotic pathway. In addition, in this study, we investigated the catalytical effect of COX on the HT‐29 cancer line, the apoptotic mechanism, and the moleculer binding of tolmetin and compound 4g on the COX enzyme active site.

The association of apoptotic protein expressions sensitive to apoptosis gene, p73 and p53 with the prognosis of cervical carcinoma

Objective To evaluate the expressions of several apoptotic pathway proteins in relation to clinical parameters and survival in patients with cervical carcinoma. Methods A total of 20 patients with clinically advanced staged carcinoma of cervix (International Federation of Gynecology and Obstetrics [FIGO] stage IIB-IVA) aged from 40 to 75 years were included in this study. The expression profile of anti-apoptotic protein (sensitive to apoptosis gene [SAG]), mitochondrial apoptotic proteins (B-cell lymphoma-extra-large [Bcl-xL] and Bcl-2 homologous antagonist/killer [Bak]), and tumor suppressor proteins (p73 and p53) were examined by real-time polymerase chain reaction experiments along with their relation to clinical parameters and survival analyses during follow-up for 5 to 8 years. Results No significant difference was found in the expressions of SAG, Bcl-xL, Bak, p73 and p53 proteins with respect to stage and grade of tumor. A significant positive correlation was noted between SAG and Bcl-xL genes (r=0.752, P<0.001) and between SAG and Bak genes (r=0.589, P=0.006). Among genes determined to be significantly associated with overall survival in the univariate analysis (P=0.026 for SAG, P=0.002 for Bcl-xL, and P=0.027 for p53), only p53 was identified as the significant predictor in the multivariate analysis (hazard ratio: 8.53, 95% confidence interval: 1.34–54.2, P=0.023). Conclusion In conclusion, our findings demonstrated a reverse correlation of SAG, Bcl-xL, and p53 expressions with overall survival of patients. No association of apoptotic pathway proteins with clinicopathological characteristics of cervical carcinoma patients was noted. Low SAG, Bcl-xL, and p53 expression levels revealed to be useful as prognostic predictors in patients with cervical carcinoma.

The Role of G Protein β3 Subunit Polymorphisms C825T, C1429T, and G5177A in Turkish Subjects with Essential Hypertension

Hypertension is a multifactorial disorder that constitutes a major risk factor for the cardiovascular system. Heterotrimeric G-proteins, which couple receptors for diverse extracellular enzymes or ion channels, are correlated with disease mechanisms. Several studies have demonstrated an association between G protein polymorphisms and essential hypertension in some populations, although contradictive results also exist. In this study, we have investigated the potential role of the C825T, C1429T, and G5177A polymorphisms of the β3 subunit of G-proteins in essential hypertension in a group of Turkish subjects. Genomic DNA from 106 normotensive individuals (117.4 ± 13.1, 75.2 ± 10.5; systolic blood pressure (SBP) and diastolic blood pressure (DBP) levels, respectively) and 101 hypertensive subjects (152.3 ± 18.0, 92.5 ± 11.6; SBP and DBP levels, respectively) were studied by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing methods for these polymorphisms. Allele frequencies of the polymorphisms were consistent with Hardy Weinberg equilibrium, except for the C825T polymorphism (χ2 = 7.8). The frequencies of the 825T and 1429T variants were higher in hypertensive subjects compared to those of controls. Differences between hypertensives and controls were not statistically significant, though difference was very close to significance for C825T (p = 0.056 and 0.099 for 825T and 1429T, respectively). T allele frequency in overall population showed significant association with hypertension for C825T (0.0134). The prevalence of the 5177A-variant was very low and all subjects carrying it were heterozygotes in both groups.

Synthesis of pro-apoptotic indapamide derivatives as anticancer agents

Abstract 4-Chloro-3-({[(substitutedamino)carbonothioyl]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (1–20) and 4-chloro-3-({[3-(substituted)-4-oxo-1,3-thiazolidine-2-ylidene]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide derivatives (21–31) were synthesized from 4-chloro-N-(2-methyl-2,3-dihydroindol-1-yl)-3-sulfamoylbenzamide (indapamide). 4-Chloro-3-({[(4-chlorophenyl) amino) carbonothioyl]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide 12 demonstrated the highest proapoptotic activity among all synthesized compounds on melanoma cell lines MDA–MB-435 with 3.7% growth inhibition at the concentration of 10 µM. Compound 12 (SGK 266) was evaluated in vitro using the MTT colorimetric method against melanoma cancer cell line MDA–MB435 growth inhibition for different doses and exhibited anticancer activity with IC50 values of 85–95 µM against melanoma cancer cell line MDA–MB435. In addition, this compound was investigated as inhibitors of four physiologically relevant human carbonic anhydrase isoforms, hCA I, II, IX and XII. The compund inhibited these enzymes with IC50 values ranging between 0.72 and 1.60 µM.

Investigation of the Association Between Dopamine D1 Receptor Gene Polymorphisms and Essential Hypertension in a Group of Turkish Subjects

Dopamine has been shown to influence blood pressure by regulating renal sodium excretion through direct interaction with the dopamine receptors, especially with the Dopamine D1 receptor (DRD1). To better understand the role of polymorphisms in those effects, we investigated the association between two polymorphic sites in the DRD1 promoter region (A–48G, G–94A) and essential hypertension in the Turkish population. The DRD1 variants were genotyped by restriction fragment length polymorphism (RFLP) analysis. A total of 205 unrelated individuals were enrolled in the study. We found that genotype distributions and allele frequencies of the control and hypertensive subjects were very similar and did not show any significant difference with respect to blood pressure (BP) and hypertension. Contribution of the gene variances in BP or hypertension by sex differences and dependence on body mass index (BMI) were also evaluated. Distribution of genotypes and allele frequencies were found to be in line with previous reports. However, increments detected in hypertensive subjects were far from being statistically significant.

Partial knockdown of TRF2 increase radiosensitivity of human mesenchymal stem cells

Telomere repeat binding factor TRF2 is a member of shelterin complex with an important role in protecting and stabilizing chromosomal ends. In the present study, we investigated the effect of partial knockdown of TRF2 on radiosensitivity of telomerase immortalized human mesenchymal stem cells (hMSC-telo1), which have a higher radioresistance compared to non telomerized counterpart. Partial knockdown of the protein achieved 15-20% reduction in TRF2 protein levels. The study compared the effect of 2.5Gy radiation in two-four days after irradiation for hMSC-telo1 cells and the cells transfected with siTRF2 and null control vector. Radio-response of the cells were examined using senescence associated β-Gal assay (β-Gal), colony forming assay (CFU) and γ-H2AX phosphorylation. TRF2 deficiency substantially increased radiosensitivity of cells compared to controls in both proliferation and senescence assay (2.4 fold increase in β-Gal, 1.6 fold decrease in CFU). In addition, it increased the γ-H2AX foci as revealed by both immunfluorescence and Western blot analysis. Our data suggests that partial knockdown of TRF2 in hMSC-telo1 cells cause increased γ-H2AX foci which led to fail TRF2 to protect telomeres from radiation thus TRF2 deficiency led to a 1,5-2 fold increase in the radiosensitivity of hMSC-telo1 cells through telomere destabilization.

Apoptotic Effects of Etodolac in Breast Cancer Cell Cultures

Nonsteroidal anti‐inflammatory drugs (NSAIDs) are commonly used as anti‐inflammatory and analgesic agents. This family of drugs suppresses prostaglandin synthesis through inhibition of cyclooxygenase (COX) enzymes. Recent studies displayed that anti‐carcinogenic actions of these drugs are mediated by COX‐2 enzyme. Currently, there is intense research on COX‐2 inhibitors as therapeutic targets. Etodolac is not perfectly selective but shows ‘preferential selectivity’ for COX‐2. Here, in this study, we wanted to take gene expression snapshots of several apoptotic proteins under different conditions of drug exposure. The aim, therefore, focused to determine differential effects of etodolac on the regulation of apoptotic genes in hormone‐responsive MCF‐7 and triple‐negative MDA‐MB‐231 cancer cell lines. Our data suggest that MDA‐MB‐231 is more responsive to etodolac exposure. Cell proliferation and apoptosis consistently regulated upon drug addiction. Furthermore, COX‐2/HER2 was explicitly an up‐regulated, phosphorylated form of Bad accumulated and anti‐apoptotic proteins SAG and survivin increased in both transcriptional and translational levels. Changes in mitochondrial Bcl‐2 family proteins were moderate and pro‐ and anti‐apoptotic proteins showed similar levels of regulation in both cell lines. We believe that these findings would be supportive for future studies targeting etodolac‐based therapies, as it reveals apoptotic factors differentially regulated in hormone‐responsive and invasive cell lines.

Chromatin modifications of hTERT gene in hTERT-immortalized human mesenchymal stem cells upon exposure to radiation

Regulation of telomerase activity is thought to participate in the cellular response to ionizing radiation. Epigenetic mechanisms play a role in this regulation, as well as other mechanisms such as transcription, phosphorylation, etc. Here, we investigated chromatin modifications in telomerase promoter upon exposure to ionizing radiation in human mesenchymal stem cells (hMSC) and telomerase-immortalized hMSCs (hMSC-telo1) together with a hMSC-telo1 cell line in which TRF2 expression was partially repressed (siTRF2 hMSC-telo1). Histone methylations and acetylations were compared in all cell lines after exposure to various doses of ionizing radiation (0.1, 1, 2.5 and 15 Gy) using chromatin immunoprecipitation assay. hTERT gene was shown to be quickly regulated through H3, H4 acetylations, as well as with H3K4 and H3K9 methylations, following radiation exposure, although the kinetic of hMSC-telo1 cells were different, indicative of the higher radioresistivity of these cells. To the authors surprise, there was an upregulation of endogenous telomerase activity in the hMSC-telo1 cells, even though the cells had already expressed high levels of ectopic hTERT. Our results show that telomerase regulation is one of the primary actions in response to damage and epigenetic factors play a major role in this regulation. Our results also suggested that partial silencing of TRF2 enhances the radiosensitivity of these cells, and endogenous telomerase is upregulated upon radiation, even under ectopic expression of hTERT in these cells.