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

Expression of TRF2 and its prognostic relevance in advanced stage cervical cancer patients

BACKGROUND: Telomeres are protective caps consisted of specific tandem repeats (5′-TTAGGG-3′). Shortening of telomeres at each cell division is known as "mitotic clock" of the cells, which renders telomeres as important regulators of lifespan. TRF2 is one of the critical members of shelterin complex, which is a protein complex responsible from the preservation of cap structure, and loss or mutation of TRF2 results in DNA damage, senescence or apoptosis. Since cancer is frequently associated with aberrant cell cycle progression, defective DNA repair or apoptosis pathways, TRF2 could be one likely candidate for cancer therapy. Here we investigated the prognostic role of TRF2 levels in cervical cancer patients. Fold-induction rates were evaluated with respect to median values after real-time PCR analysis. Overall survival, distant disease-free and local recurrence-free survival rates were calculated using Kaplan-Meier long rank test. RESULTS: Both five year overall- and disease-free survival rates were longer in patients with higher TRF2 expression compared to lower expression, but results were not statistically significant (69.2% vs 28.9%, respectively). Mean local recurrence-free survivals (LRF) were very close ( 58.6, CI: 44.3-72.9 vs 54.5, CI: 32.1-76.9 months) for high and low expressions, respectively. Cumulative proportion of LRF at the end of five year period was 76.9% for high and 57.1% for low TRF2 expression (P = 0.75). Statistically significant difference was found between survival ratios and Bcl-xL and p53 gene expressions, but not with TRF2. A respectable correlation between TRF2 expression and apoptosis along with distant metastasis was noted (P = 0.045 and 0.036, respectively). Additionally, high TRF2 expression levels had a positive impact in five year survival rate of stage IIIB-IVA patients (P = 0.04). CONCLUSIONS: Our results support the role of TRF2 in apoptosis and imply a positive relation with distant metastases and survival in advanced stage patients. The remarkable difference in survival periods of patients with different TRF2 expressions suggest that TRF2 may be a candidate factor to estimate survival for cervical cancer, a preliminary observation which should further be verified with a larger cohort

Sinkrotron X-ışınları yöntemlerinin biyolojik makromolekül yapısı çözümlenmesinde kullanılması

Bu çalışmada sinkrotron X-ışın küçük açı saçılması (XSAS) ile çözeltideki proteinlerin yapısal özelliklerinin araştırılması amaçlandı. Deneyler için hücre sinyal ileti sisteminde temel rol oynayan heterotrimerik G-proteinlerinin sinir sisteminde en yaygın bulunan tipi olan Go-proteininin a-altbiriminin (Goa) rekombinant olarak E.coli'den saflaştırılması denendi. Rekombinant Goa, E.coli BL21(DE3) hücrelerinde pT7/Nde1/Goa vektörü kullanılarak sentezlendi. Ekspresyon seviyesi Western blot ve GTPgS bağlama yöntemleriyle belirlendi ve IPTG'den bağımsız olduğu saptandı. Protein izolasyonu çalışmalarına indüklenmemiş geniş ölçekli kültürlerde, Lee ve ark. tarafından verilen yöntem izlenerek başlandı. Saflaştırmanın her aşamasında , protein OD ölçümleri ile saptandı ve Goa'nın varlığı SDS-PAGE ve Goa'ya özgü bir antikor kullanılarak (NEN-Dupont) Western blot analizleriyle saptandı. Bazı kesimler GTPgS bağlama testleriyle de denendi. Bu çalışmalarda değişik aşamalarda Lee ve ark.'na uygun sonuçlar alınmadığından ve görülen safsızlıklar nedeniyle bazı değişiklikler yapıldı. Örneğin yüksek molekül ağırlığındaki proteinleri uzaklaştırmak amacıyla Gel Filtrasyon kromatografisi eklendi ve Q-Sefaroz kolonu Mono Q ile değiştirildi. Bu değişimler Goa ile aynı molekül ağırlığına sahip ve E.coli glisin parçalama enzim sisteminin bir üyesi olan T-proteininin de Goa ile birlikte saflaşmasına yol açtı. Goa saflaştırma çalışmalarına paralel olarak X-ışınları küçük açı saçılma deneyleri için T-protein kullanıldı. Bu deneyler proteinin girasyon yarıçapının 2.58 nm olduğunu ve asimetrik bir yapıya sahip bulunduğunu gösterdi. T-proteini için elde edilen X-ışınları saçılma deseni G-protein a-altbiriminden beklenilen desen ile karşılaştırıldı. Analiz sonuçları bu proteinin yapısı ile GDP bağlı, aktif olmayan yapıdaki heterotrimerik bir kimerik G-proteininin a-altbiriminin yapısı arasında yüksek benzerlik olduğunu ortaya koydu. Goa proteininin saflaştırma yöntemini optimize etme ve T-protein şeklinin belirlenmesi çalışmaları sürmektedir. tructural Analysis Of Biological Macromolecules By Using Synchrotron X-Ray Scattering Method SUMMARY Our aim in this study has been to determine structural properties of proteins using synchrotron X-ray small angle scattering (XSAS). Isolation of Goa which is the a subunit of Go (Goa) , a major type of heterotrimeric G-protein in the nervous system was attempted for the measurements. Recombinant Goa was expressed in E.coli BL21(DE3) cells transformed with pT7/Nde1/ Goa plasmid. Expression levels were determined by Western blot analysis and GTPgS binding tests and it was established that the level of expression was not dependent on IPTG concentration. Protein isolation studies were initially carried out according to the method described by Lee et al. using large scale cultures which were not induced. At every step of purification, protein was detected by OD measurements and the presence of Goa was monitored by SDS-PAGE and Western blot analysis using commercial Goa antibody (NEN-Dupont). Some fractions were also assayed for GTPgS binding activity. Due to the inconsistencies with the results of Lee et al. and due to inhomogeneities of the final product, some modifications were introduced to this procedure. For example, to remove large molecular weight contaminants a gel filtration chromatography step was added and the Q-Sepharose column was replaced with Mono Q. These modifications resulted in co-purification of an E.coli protein of nearly the same molecular weight, the T-protein of glycine cleavage system. Parallel to Goa purification studies, T-protein was used for XSAS measurements. These measurements yield 2.58 nm for the radius of gyration of T-protein and indicate that the protein has an assymetric shape in solution. The scattering pattern of T-protein was compared with that expected from a G-protein a subunit. Analyses indicate a high similarity between T-protein structure and the structure of GDP-bound inactive form of a-subunit of a chimeric heterotrimeric G-protein. Studies to optimize the purification of Goa and determination of T-protein shape are in progress

Role of TRF2 and TPP1 regulation in idiopathic recurrent pregnancy loss

Telomeres are the tandem repeats (TTAGGG) present at the ends of the chromosomes that ensure chromosome stability and protect chromosomes from degradation. Telomeres in somatic human cells shorten after every cellular division and are linked to the cellular senescence. In this study we have investigated telomere length and expression of shelterin genes in aborted fetus material from idiopathic recurrent pregnancy losses. Telomere length was measured using Telomere Restriction Fragment Length (TRF) analysis. The gene expression levels for important shelterin complex proteins (TRF1, TRF2, POT1, and TPP1) were determined by Real-time Quantitative Reverse Transcriptase PCR (qRT-PCR). Our results demonstrated down regulation of TRF2 and TPP1 and a strong decline in average telomere length in abort material from women suffering from idiopathic recurrent pregnancy loss. We suggest that shorter telomere length and downregulation of the major shelterin components TRF2 and TPP1 leading to telomere uncapping, might play a critical role in recurrent pregnancy loss. (C) 2019 Elsevier B.V. All rights reserved

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-XL) 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-XL, Bak and beta-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-XL 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-XL 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. (C) 2011 Baishideng. All rights reserved

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. (C) 2011 Elsevier GmbH. All rights reserved