Browsing by Subject "Apoptosis"

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Open Access
4,5-dianilinophtalimide protects neuroendocrine cells against serum deprivation-induced stress and apoptosis
(2013) Ergin V.; Erdogan, M.; Karasu Ç.; Menevşe, A.
OBJECTIVE: The aim of this study was to reveal the effects of 4,5-dianilinophthalimide (DAPH), which inhibits amyloid β fibrillization, against serum deprivation (SD)-induced apoptosis and the possible mechanisms in differentiated PC12 neuron cells. METHODS: Firstly, we evaluated whether DAPH protects cell viability exposed to SD by MTT assay. Next, we examined the changes of phospho-p38 MAPK (Thr180/Tyr182), phospho-HSP27 (Ser82), phospho-c-JUN (Ser73) and cleaved-CASP3 (Asp175) profiles by immunoblotting, in PC12 cells exposed to SD. Intracellular reactive oxygen species (ROS) level was also measured. RESULTS: SD induced apoptosis accompanied by up-regulation of phospho-p38 MAPK (Thr180/Tyr182), phospho-HSP27 (Ser82), phospho-c-JUN (Ser73), cleaved-CASP3 (Asp175) and intracellular ROS content. Co-treatment with nontoxic doses of DAPH prevented apoptosis by the attenuation of activated proteins and reduction of ROS level. These results suggest that serum deprivation-induced apoptosis inhibited by DAPH administration. CONCLUSION: We have provided for the first evidence that DAPH has a neuroprotective effect on SD-caused stress, probably via contributing the reestablishment of redox homeostasis. © 2013 Neuroendocrinology Letters.
Open Access
Acquired tolerance of hepatocellular carcinoma cells to selenium deficiency: a selective survival mechanism?
(American Association for Cancer Research, 2003) Irmak, M. B.; Ince, G.; Ozturk, M.; Cetin Atalay, R.
Selenium is essential to human health, and its deficiency is associated with different diseases including liver necrosis. Selenium is protective against viral hepatitis and hepatocellular carcinoma (HCC). The underlying molecular mechanisms of selenium effects are not well known. In this study, in vitro response of HCC-derived cell lines to selenium deficiency is examined alone or in conjunction with Vitamin E and copper/zinc. Here, we show that itt vitro selenium deficiency in a subset of HCC-derived cell lines causes oxidative stress and cytochrome c release with subsequent cell death by apoptosis. The oxidative stress and consequent cell death induced by selenium deficiency on these cells are reverted by the antioxidant effect of Vitamin E. However, most HCC cell lines (10 of 13) tolerate selenium deficiency. Consequently, they escape apoptosis. Moreover, nine of these tolerant cell lines have integrated hepatitis B Virus (HBV) DNA in their genomes, and some display p53-249 mutation, indicating past exposure to HBV or aflatoxins, established factors for oxidative stress and cancer risk in liver. An HBV-transfected clone (2.2.15) of the sensitive HepG2 cell line has gained tolerance to selenium deficiency. Our findings indicate that selenium deficiency induces apoptosis in some "hepatocyte-like" cells. However, most HCC cells, particularly HBV-related ones, tolerate selenium deficiency and escape its deadly consequences. Thus, as demonstrated by the gain of survival capacity of apoptosis-sensitive cell lines with Vitamin E, such malignant cells have acquired a selective survival advantage that is prominent under selenium-deficient and oxidative-stress conditions.
Open Access
The AKT inhibitor MK-2206 is cytotoxic in hepatocarcinoma cells displaying hyperphosphorylated AKT-1 and synergizes with conventional chemotherapy
(Impact Group, 2013) Simioni, C.; Martelli, A. M.; Cani, A.; Cetin-Atalay, R.; McCubrey, J. A.; Capitani, S.; Neri, L. M.
Hepatocellular carcinoma (HCC) is one of the most common potentially lethal human malignancies worldwide. Advanced or recurrent HCC is frequently resistant to conventional chemotherapeutic agents and radiation. Therefore, targeted agents with tolerable toxicity are mandatory to improve HCC therapy and prognosis. In this neoplasia, the PI3K/Akt signaling network has been frequently shown to be aberrantly up-regulated. To evaluate whether Akt could represent a target for treatment of HCC, we studied the effects of the allosteric Akt inhibitor, MK-2206, on a panel of HCC cell lines characterized by different levels of Akt-1 activation. The inhibitor decreased cell viability and induced cell cycle arrest in the G0/G1 phase of the cell cycle, with a higher efficacy in cells with hyperphosphorylated Akt-1. Moreover, MK-2206 induced apoptosis, as documented by Annexin V labeling, and also caused autophagy, as evidenced by increased levels of the autophagy marker LC3A/B. Autophagy was shown to be a protective mechanism against MK-2206 cytotoxicity. MK-2206 down-regulated, in a concentration-dependent manner, the phosphorylation levels of Akt-1 and its downstream targets, GSK3 α/β and FOXO3A. MK-2206 synergized with doxorubicin, a chemotherapeutic drug widely used for HCC treatment. Our findings suggest that the use of Akt inhibitors, either alone or in combination with doxorubicin, may be considered as an attractive therapeutic regimen for the treatment of HCC.
Open Access
Apoptotic vascular smooth muscle cell depletion via BCL2 family of proteins in human ascending aortic aneurysm and dissection
(Blackwell Publishing Ltd, 2012) Durdu, S.; Deniz, G. C.; Balci, D.; Zaim, C.; Dogan, A.; Can, A.; Akcali, K. C.; Akar, A. R.
Aims: This study investigates the expression patterns of BCL2 (B-cell CLL/lymphoma2) family of proteins and the extent of vascular smooth muscle cell (VSMC) apoptosis in thoracic aortic aneurysms (TAA), type-A aortic dissections (TAD), and nondilated ascending aortic samples. Methods: Aortic wall specimens were obtained from patients undergoing surgical repair for TAA (n = 24), TAD (n = 20), and normal aortic tissues from organ donors (n = 6). The expression pattern of BCL2, BCL2L1 (BCL2-like1), BAK1 (BCL2-antagonist/killer1), and BAX (BCL2-associated X protein) proteins was investigated by immunohistochemistry. Furthermore, colocalization of alpha smooth muscle actin (ACTA2) and caspase3 (CASP3) in aortic VSMCs was analyzed by double-immunofluorescence staining. Onset of DNA fragmentation was measured by TUNEL assay. Results: Apoptotic index was significantly increased in both TAD group (31.3 ± 17.2, P < 0.001) and TAA group (21.1 ± 12.7, P = 0.001) relative to control aortas (2.0 ± 1.2). Anti-CASP3 and ACTA2 double-immunostaining confirmed apoptosis in VSMCs in TAA and TAD groups but not in controls. Proapoptotic BAX expression was significantly elevated in VSMCs of TAA patients, compared with that of controls (OR = 20; P = 0.02; 95% CI, 16-250). In contrast, antiapoptotic BCL2L1 expression was higher in controls compared with that of TAA group (OR = 11.2; P = 0.049; 95% CI, 1.0-123.9). Furthermore, BAX/BCL2 ratio was significantly increased in both TAA (1.2 ± 0.7, P < 0.001) and TAD (0.6 ± 0.4, P = 0.05) groups relative to controls (0.2 ± 0.1, P < 0.001). Conclusions: Apoptotic VSMC depletion in human TAA/TAD is associated with disturbance of the balance between proapoptotic and antiapoptotic members of the BCL2 family proteins, which may have a role in the pathogenesis of vascular remodelling in aortic disease. In light of the future studies, targeting apoptotic pathways in TAA and TAD pathogenesis may provide therapeutic benefits to patients by slowing down the progression and even possibly preventing the TAD. © 2012 Blackwell Publishing Ltd.
Open Access
Biocompatibility studies on lanthanum oxide nanoparticles
(Royal Society of Chemistry, 2015) Brabu, B.; Haribabu, S.; Revathy, M.; Anitha, S.; Thangapandiyan, M.; Navaneethakrishnan, K. R.; Gopalakrishnan, C.; Murugan, S. S.; Kumaravel, T. S.
Lanthanum oxide nanoparticles (LONP), a rare earth metal oxide, have unique properties that make them a suitable candidate for several biomedical applications. We investigated certain key in vitro and in vivo biocompatibility endpoints on LONP. LONP were cytotoxic in in vitro assays and predominantly exerted their action via release of reactive oxygen species. These nanoparticles were neither irritants nor sensitizers in a rabbit model. LONP extracts did not exert any acute systemic toxicity effects in mice. On the other hand LONP exerted toxicity to the liver following oral administration, suggesting that these particles are absorbed from the gastrointestinal (GI) tract and deposited in the hepatobiliary system. LONP did not induce any mutation in the Ames test both in the presence or absence of S-9. These observations provide a base line biocompatibility and toxicity data on LONP. The current findings will also be useful in defining standards for nanoparticle containing devices. © The Royal Society of Chemistry.
Open Access
Characterization of programmed cell death induced by oxidative stress in selenium - deficient hepatocellular carcinoma cells
(1999) İnce, Gülayşe
Selenium (Se) plays an important role in eliminating the reactive oxygen species generated during oxidative stress. Se-containing selenocysteine is an essential amino acid required for the synthesis of many selenoproteins, such as glutathione peroxidase. Se-dependent glutathione peroxidase is the major en2yme that metabolizes organic and hydrogen peroxides in the cell. It appears that Se manifests its antioxidant effect through the selenoproteins. Se deficiency causes a wide range of pathological conditions. Keshan disease, characterized by cardiomyocyte and liver cell death, defined as "necrosis", was found to be associated with Se deficiency. Se deficiency is also observed in liver cirrhosis, alcoholic liver disease, HIV positive and AIDS patients, thyroid hormone abnormalities, etc. Moreover, epidemiological studies show that Se deficiency increases the risk of many cancer types, including hepatocellular carcinoma (HCC). The molecular mechanisms of the pathologies associated with Se deficiency are mostly unknown. In this study, the in vitro responses of human hepatocellular carcinoma cells, to Se deficiency have been examined. Apoptotic changes (nuclear condensation, positive annexin V staining, genomic DNA breaks) are detected in Huh7, HepG2, and Mahlavu cells under Se-deficient culture conditions. Hydrogen peroxide addition into the culture medium aggravates apoptosis. Such changes are prevented by the supplementation of Se. The Se-dependent glutathione peroxidase enzyme is shown to be reduced in cells grown under Se-deficient conditions, explaining the build up of intracellular oxidative stress. It is proposed that the cell death observed in many pathologies associated with Se deficiency, is the result of the programmed cell death, triggered by an increase in intracellular oxidative stress, as opposed to necrotic cell death. Hep3B, Hep3B-TR, Hep40, PLC/PRF/5 and BC1/R14 cell lines have not displayed cell death when grown in Se-deficient conditions and were resistant to hydrogen peroxide, as well. The presence of such cell lines and the differential sensitivities manifested by HepG2, Huh7 and Mahlavu suggest a complete or partial gained resistance for apoptosis, which may contribute to the onset or progression of HCC, consistent with the increased risk of HCC in Se deficiency.
Open Access
Discovery of novel agents for liver cancer therapeutics and characterization of their bioactivities on cellular pathways
(2015-12) Durmaz, İrem
Hepatocellular carcinoma is the second deadliest and fifth most common cancer type worldwide. Due to the limited therapy options, it is crucial to develop novel targeted therapeutic agents that provide better prognosis and enhance life quality of patients. The specific aim of this thesis was to identify and characterize novel compounds with anticancer properties in liver cancer. Three groups of molecules were investigated. First group were cardiac glycosides extracted and purified from Digitalis Ferruginea. Extensive analysis Glycoside Lanatoside C revealed that these molecules induced ROS accumulation in liver cancer cells with differential downstream targets in mesenchymal-like PTENdeficient drug-resistant Mahlavu and epithelial-like PTEN-adequate drug-sensitive Huh7 liver cancer cells. Xenograft models on nude mice also confirmed the anticancer activities of Lanatoside C in vivo with decreased tumor volume and weight. The second group of compounds were novel molecules that contains triazolothiadiazine and triazolothiadiazole scaffold, derived from known NSAIDs (ibuprofen, naproxen and flurbiprofen). Results indicated that SubG1/G1 cell cycle arrest is induced in treated cells. In addition, extensive molecular analysis disclosed oxidative stress induction and COX activity inhibition leading to ASK1 activation and Akt inhibition. The levels of downstream elements GSK3β, β-catenin and CyclinD1 were also altered. Apoptosis was characterized as the cell death mechanism that is triggered by these molecules in liver cancer cells. Novel nucleobase/nucleoside analogues were the third group of molecules explored in this study. 24 of 127 investigated compounds showed significant cytotoxicity during initial screening. 6 molecules were selected for further molecular analysis upon real-time cytotoxicity assay. It was observed that the molecules induced SubG1/G1 cell cycle arrest through Src pathway inhibition. CyclinE-cdk2 complex formation was prevented then the inhibition of Rb leading to a decrease in cell growth and proliferation and induction of apoptosis in liver cancer cells. This thesis disclosed the mode of action of three groups molecules, glycosides are pure examples of drug repurposing. NSAID represent the modified small molecule compounds for novel targets and finally nucleobase analogs are novel compounds as anti metabolites.
Open Access
The Effect of estrogen on bone Marrow-Derived rat mesenchymal stem cell maintenance: inhibiting apoptosis through the expression of bcl-x l and bcl-2
(Springer Science+Business Media, 2012) Ayaloglu-Butun, F.; Terzioglu-Kara, E.; Tokcaer-Keskin, Z.; Akcali, K. C.
Mesenchymal Stem Cells (MSCs) have high therapeutic value for regenerative medicine and tissue engineering due to their differentiation potential and non-immunogenic characteristics. They are also considered as an effective in vivo delivery agent because of their ability to migrate to the site of injury. A major roadblock in their use for cell-based therapies is their rareness in vivo. Therefore, it is important to obtain increased number of functional MSCs in vitro in order to have adequate numbers for therapeutic regiments. We aimed to investigate the role of estrogen and its mechanism in obtaining more MSCs. MSCs were isolated from female and ovariectomized rats and cultured in the presence and absence of 10 -7 M estrogen. In the presence of estrogen, not only their CFU-F activity increased but also apoptotic rate decreased as shown by TUNEL staining leading to obtain more MSCs. Also the number of the cells in the colonies increased upon estrogen treatment. To reveal the mechanism of this effect, we focused on Bcl-2 family of proteins. Our immunoblotting experiments combined with knockdown studies suggested a critical role for anti-apoptotic Bcl-x L and Bcl-2. Estrogen treatment up regulated the expression Bcl-x L and Bcl-2. When we knocked down the expression of bcl-x L and bcl-2, MSCs lacking these genes showed an increase in the apoptotic rate in contrast to normal MSCs following estrogen treatment. Therefore, estrogen treatment will be of great advantage for cell-based therapies in order to get more functional MSCs and may provide opportunities to develop new strategies for debilitating diseases. © 2011 Springer Science+Business Media, LLC.
Open Access
Effects of biological compound Turkish propolis extract on breast cancer cells
(2013) Uğurlu, Deniz
Propolis is a resinous compound which is collected from various plants then combined with wax and bee enzymes by worker bees. There are many studies conducted on propolis or its active components aiming to find new treatment possibilities in diverse research fields such as immunology, infectious diseases, allergy, diabetes, ulcers, and oncology. Chemical analysis indicated that propolis is a multicomponent mixture of various compounds with prevalence of flavonoids and phenolic acids. Therefore it is important to investigate the propolis extract mechanisms of action in order to predict possible cytotoxic and may be therapeutic effects for cancer. The most common propolis extract is ethanol extract of propolis (EEP) whereas Turkish researchers were able to extract the propolis with dimethyl sulfoxide (DMSO) which can maximize the penetration of compounds from propolis to the cells as well as DMSO is a good solvent for flavonols (one of the most common compound in propolis). There are many studies conducted on propolis or its active components for treatment of cancer which reveals the potential of this biological compound in the development of novel anti-cancerous agents. However, anti-cancer activity of DMSO extract of Turkish propolis (DEP) on human breast cancer has not been investigated yet. The aim of this study was to investigate the anti-cancer effects of DMSO extract of Turkish propolis (DEP) on cancer cells. Inhibitory effects of propolis extracts collected from different regions of Turkey were analyzed on the growth of the human breast carcinoma cells. Two different propolis extracts were used to determine their cytotoxic effects of breast carcinoma cell lines using SRB staining and IC50 values were determined. The results showed that propolis is cytotoxic in dose-dependent manner (IC50 value of diverse from 25 ug/ml to 123 ug/ml). Real time monitoring (xCELLigence system) of propolis treated cells confirmed the cytotoxic effect of propolis, since increasing concentrations of propolis decreased the cell number in a dose- and cell line- dependent way. Furthermore, propolis treatment induces apoptosis in breast carcinoma cell lines. Propolis treated cells changed their adherent morphology to round cells and detached from the surface. Hoechst 33258 staining of propolis treated cells revealed the increasing number of cells displays DNA condensation. PARP-1, a 116 kDa nuclear enzyme, is cleaved in fragments of 89 and 24 kDa during apoptosis. Western blot analysis was performed to detect the PARP-1 cleavage in propolis treated cells. Decrease in the full-length PARP-1 protein levels supports our hypothesis that propolis shows its cytotoxic effect at least partially through induction of apoptosis. The effect of propolis on cell cycle was analyzed with flow cytometer after staining the cells with Propidium iodide (PI). Increase in the G2/M cell cycle arrest was observed in propolis treated cells compare to control DMSO treated MDA-MB-231 cells. In addition to cytotoxic effects, in vitro wound healing assay revealed that propolis treated MDA-MB-231 cells shows delayed invasion of the cells to the denuded area when compared to the DMSO control cells. In conclusion, propolis showed a cytotoxic effect on breast carcinoma cell lines by inducing apoptosis, G2/M arrest as well as delaying the invasion capacity of the cells which makes it a potent anti-tumorigenic compound that may be useful in cancer chemoprevention or therapy.
Open Access
Effects of Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) RNAi on apoptosis, DNA damage response, drug sensitivity, and HSA-MIR-495-3P overexpression in breast cancer
(2018-12) Köker, Şahika Cıngır
Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) is associated with nicotine addiction and it has an important role in the prognosis of lung cancer. Despite its important cellular functions, its role in breast cancer remains to be elucidated. In this thesis, I aimed to identify the alterations in the important cancer signaling pathways occurring upon CHRNA5 depletion. Drug resistance is one of the major obstacles in breast cancer therapy. Heterogeneous nature of breast cancer necessitates identification of more biomarkers which aid in precise diagnosis and hence development of proper treatment options. In this study, by using more than one cell line which is representative of different subtypes of breast cancer, I showed the alterations occurred in cancer signaling pathways such as cell cycle and apoptosis upon CHRNA5 depletion, which could serve as a novel biomarker in breast cancer subtyping. Depending on mutation status of TP53, which is the gatekeeper protein during G1/S checkpoint, CHRNA5 depletion mostly exerted its effects over decreasing the levels of total CHEK1 and pCHEK1 (S345) which significantly altered the response of MCF7 cells to topoisomerase inhibitors in terms of enhanced drug sensitivity. Increases in apoptotic markers, such as BAX/BCL2 ratio along with increased FAS levels, further confirmed that this sensitization of MCF7 cells upon CHRNA5 depletion might have ended with apoptosis. So far in the literature, there is no study examining the regulation of CHRNA5 by small endogenous molecules such as miRNAs. Due to the predictive binding sites in 3’UTR of CHRNA5 and the importance of participating in tamoxifen resistance in breast cancer; I also examined the interplay between miR-15a family and CHRNA5 in MCF7 cells. I showed significant decrease in CHRNA5 levels upon using miR-15a mimic while demonstrating similar activity of miR-15a family mimics with CHRNA5 depletion using RT-qPCR. Another important implication of CHRNA5 depletion in MCF7 cells was the global change in miRNA expression prolife which was verified with independent microRNA arrays. Based on these in silico results, hsa-miR-495-3p appeared as the most downregulated miRNA which is known as a tumor suppressor miRNA. As stated in the literature, the role of miR-495 differs depending on the tumor type. Therefore, I tried to restore its expression by mimicking along with CHRNA5 depletion. The transcriptomic changes observed with CHRNA5 depletion was boosted with the restoration of miR-495 levels.
Open Access
Effects of PI3K/AKT/MTOR and VEGFR pathway inhibitors on liver cancer stem cells and bioactivities of novel pyrazolic chalcone derivatives on liver cancer
(2017-12) Kahraman, Deniz Cansen
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality, such that it the second most frequent cause of cancer death worldwide. Due to its heterogeneous composition and aggressive behavior, it is resistant to conventional therapies and also Sorafenib and Regorafenib which are FDA-approved multikinase inhibitors targeting pathways involved in angiogenesis and proliferation. The mechanisms behind the acquired resistance to Sorafenib were described as activation of compensatory pathways such as PI3K/Akt/mTOR, JAK-STAT, epithelial to mesenchymal transition (EMT), microenvironment and presence of cancer stem cells. Liver cancer stem cells originate from damaged and transformed hepatic progenitor cells (HPCs) which are found responsible for chemo-resistance, tumor relapse, and metastasis. For this reason, the effects of PI3K/Akt/mTOR inhibitors, Sorafenib and DNA intercalators on the enrichment of LCSCs were investigated. CD133+/EpCAM+ population from HCC cells were analyzed by flow cytometry after treatment with inhibitors, and effective inhibitors against LCSCs were further tested for their potential combinatorial effects together with Sorafenib. It was shown that upon treatment with Sorafenib or DNA intercalators the LCSCs were enriched, whereas Rapamycin (mTOR inhibitor), LY294002 (PI3K inhibitor) were able to inhibit the enrichment of LCSCs and reduced the CD133+/EpCAM+ population ratio. Combination studies revealed that when cells are treated initially with Rapamycin and then with Sorafenib, both the LCSC ratio and the sphere formation capacity of cells were reduced compared to cells treated with Sorafenib alone. To understand the alterations in gene expression induced by the inhibitors, a large panel of genes involved in regulation of cancer pathways were analyzed using Nanostring nCounter Technology. Systematic pathway analysis using Cytoscape Score Flow algorithm application allowed us to identify differential response genes involved in stemness. It was shown that genes involved in regulation of stem cells (Wnt and Notch pathway) were downregulated upon treatment with Rapamycin and DAPT (Notch pathway inhibitor), yet Sorafenib treatment resulted in differential regulation of these pathways, where JAG1 gene was found to be up-regulated. Interestingly, IL-8 expression was upregulated dramatically upon treatment with Sorafenib, but downregulated upon DAPT or Rapamycin treatment. Inhibition of IL-8 signaling resulted in reduction in both LCSC ratio and sphere formation capacity of HCC cells, which could be indicating the role of IL-8 signaling in the conservation of stemness features of LCSCs. For this reason, blockade of IL-8 signaling was suggested to be a promising therapeutic approach for HCC. Another topic in this thesis focuses on the potential of VEGFR2 TKIs and quinoids to inhibit both liver cancer cells and liver cancer stem cells. VEGFR TKIs such as Sorafenib, are widely studied for the treatment of many cancers, yet as mentioned above, there are many clinical studies providing the evidence that anti-VEGF or anti-VEGFR therapies lead to stable disease, which is then followed by disease progression in different cancer types. In recent years it has also been shown that antiangiogenic agents are increasing cancer stem cell population via generation of tumor hypoxia. Quinoids, on the other hand, are compounds that are selectively active in hypoxic conditions. Thus, the main aim of this study was to evaluate the bioactivities of compounds from each group on liver cancer cells and also to analyze their effects on the enrichment of LCSCs. Our results have shown that VEGFR2 TKIs were cytotoxic at lower concentrations compared to quinoids. However, it was shown that VEGFR2 TKIs are more likely to enrich LCSC population whereas some of the quinoids were able to reduce this ratio. With this information, a new concept called “aggressiveness factor”, which defines the potential of a compound to cause more aggressive cancer, was introduced. In the last part of this thesis, bioactivities of pyrazolic chalcone derivatives on HCC cell lines and their mechanism of action were investigated. Chalcones and pyrazolic structures are well known for their anti-cancer activities. Newly synthesized pyrazolic chalcone derivatives were tested against different cancer cells, and selection based on the IC50 values of compounds was made to analyze their effect on a panel of HCC cells. Results have shown that, compounds 39, 42, 49 and 52 were the most effective derivatives which had anti-proliferative activities in less than 5 μM concentrations. Further investigation of cell cycle progression and cell death mechanisms have revealed that compounds 42 and 52 caused cell cycle arrest at the G2/M phase and induced apoptotic cell death. Also, levels of cell cycle proteins, p21, CDK1, and phospho-CyclinB1 were shown to decrease upon treatment with these compounds.
Open Access
Elucidating the mechanisms of T-DM1 resistance in in vitro models of HER2 overexpressing breast cancer
(2016-09) Saatci, Özge
Despite the presence of plethora of anti-cancer therapeutics with a variety of different mechanisms of action, it is still not possible to completely eradicate cancer due largely to the occurrence of refractory tumors even years after completion of the treatment. Such “resistant” tumors are formed over time as few cells, which have gained some advantageous genomic alterations eventually populate the entire organ. A lot of in vitro and in vivo studies are currently being done in order to identify the ways by which cancer cells become resistant to given therapy. This would decipher the weaknesses of the resistant tumors and would provide a means to combat drug resistance. T-DM1 is an anti-HER2 therapeutics, being used in refractory HER2-positive breast cancer patients since 2013. It initially generated a huge excitement owing to the highly favorable clinical findings; however, resistance was developed rapidly after 5-6 months following the initial treatment. Currently; very little is known about the mechanisms of acquired resistance against T-DM1, and therefore, identification of novel targets for the treatment of T-DM1 refractory patients would be highly beneficial. In this thesis, I have developed and characterized the acquired T-DM1 resistance phenotypically, and demonstrated abrogation of drug induced mitotic arrest and apoptosis as two novel mechanisms of resistance. I have further analyzed the genomic landscape of resistance in terms of the enrichment of cancer related processes. Cell cycle was found to be the most significantly enriched process among genes deregulated in T-DM1 resistance as identified by next-generation RNA sequencing. Cell cycle was also shown to be activated in TCGA patients expressing high levels of the TDM1 resistance signature and further supported the importance of rewiring cell cycle for the acquisition of T-DM1 resistance in patients as well. I have further identified two important mitotic genes; PLK1 and TACC3 as the common mediators of resistance in different HER2-overexpressing models by a targeted siRNA screen. I have showed that their genomic or pharmacological inhibition confers sensitization to T-DM1 induced growth inhibition, partially through re-induction of apoptotic cell death. I further uncovered a BCL2 dependency in T-DM1 resistant models which was also found to be associated with T-DM1 resistance as inhibition of Bcl2 enhanced T-DM1 induced growth inhibition. Since T-DM1 refractory HER2-positive breast cancer patients are currently not curable, these pre-clinical findings might guide the future clinical test to improve the survival of this patient subgroup via the usage of PLK1 or BCL2 inhibitors in combination with T-DM1.
Open Access
Evaluation of ATAD2 as a potential target in hepatocellular carcinoma
(Springer, 2021-11-05) Gürsoy Yüzügüllü, Özge; Ekin, U.; Özen, C.; Korhan, P.; Bağırsakcı, E.; Yılmaz, F.; Uzuner, H.; Alotaibi, H.; Kırmızıbayrak, P. B.; Atabey, N.; Karakülah, G.; Öztürk, M.; Yüzügüllü, Haluk
Purpose Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide with lack of effec-tive systemic chemotherapy. In this study, we aimed to evaluate the value of ATPase family AAA domain-containing protein 2 (ATAD2) as a biomarker and potential therapeutic target for HCC.Methods The expression of ATAD2 was tested in different HCC patient cohorts by immunohistochemistry and comparative transcriptional analysis. The co-expression of ATAD2 and proliferation markers was compared during liver regeneration and malignancy with different bioinformatics tools. The cellular effects of ATAD2 inactivation in liver malignancy was tested on cell cycle, apoptosis, and colony formation ability as well as tumor formation using RNA interference. The genes affected by ATAD2 inactivation in three different HCC cell lines were identified by global gene expression profiling and bioinformatics tools.Results ATAD2 overexpression is closely correlated with HCC tumor stage. There was gradual increase from dysplasia, well-differentiated and poorly-differentiated HCC, respectively. We also observed transient upregulation of ATAD2 expres-sion during rat liver regeneration in parallel to changes in Ki-67 expression. ATAD2 knockdown resulted in apoptosis and decreased cell survival in vitro and decreased tumor formation in some HCC cell lines. However, three other HCC cell lines tested were not affected. Similarly, gene expression response to ATAD2 inactivation in different HCC cell lines was highly heterogeneous.Conclusions ATAD2 is a potential proliferation marker for liver regeneration and HCC. It may also serve as a therapeutic target despite heterogeneous response of malignant cells.
Open Access
Functional analysis of Erbin gene in breast cancer drug resistance
(2021-02) Sunar, Gizem
Erbin is an ERBB2 interacting protein with roles in many signaling pathways. Breast cancer is one of the types of cancer that is affected by Erbin regulation. However, it is unclear how Erbin regulates the biological behavior and drug resistance of breast cancer cells. Some studies have claimed that Erbin promotes tumorigenesis and demonstrates oncogenic features in breast cancer, whereas others have indicated that it inhibits breast cancer development. The main aim of this study was to explore the role of the Erbin gene in breast cancer drug resistance. Bioinformatic analyses of breast cancer patient datasets have shown that a high level of Erbin expression predicts better survival in breast cancer patients treated with chemotherapy or targeted therapies while the Erbin level does not change the survival rates of untreated breast cancer patients. These analyses lead us to hypothesize that the Erbin expression level could alter the effect of the drug treatment and a reduced level of Erbin expression could promote resistance against doxorubicin and tamoxifen. In vitro studies have demonstrated that the protein expressions were apparently lower in MDA-MB-231 doxorubicin resistant (DoxR) and MCF-7 tamoxifen resistant (TamR) cells compared to non-resistant cell line counterparts. When the expression level of Erbin was downregulated by si-RNA transfection, it was observed that the protein level of the anti-apoptotic markers increased whereas apoptotic markers decreased in MDA-MB-231 cells. Proteins that promote cell survival and proliferation increased in Erbin downregulated MDA-MB-231 and MCF-7 cells. Besides, when Erbin was reduced, the viability of the MDA-MB-231 cells against doxorubicin increased but there was no significant change for tamoxifen in MCF-7 cells. Lastly, breast cancer patients with high Erbin expression that were treated with tamoxifen, chemotherapy or trastuzumab have higher levels of DNA damage, apoptosis and cell cycle arrest-related genes. On the contrary, patients with low Erbin expression have higher levels of cyclins, CDKs and anti-apoptotic genes. In conclusion, Erbin could play an important role in the drug resistance of breast cancer cells since the reduction in Erbin expression can promote drug resistance in these cells.
Open Access
Functionally conserved effects of rapamycin exposure on zebrafish
(Spandidos Publications, 2016-03) Sucularli, C.; Shehwana, H.; Kuscu, C.; Dungul, D. C.; Ozdag, H.; Konu, O.
Mechanistic target of rapamycin (mTOR) is a conserved serine/threonine kinase important in cell proliferation, growth and protein translation. Rapamycin, a well-known anti-cancer agent and immunosuppressant drug, inhibits mTOR activity in different taxa including zebrafish. In the present study, the effect of rapamycin exposure on the transcriptome of a zebrafish fibroblast cell line, ZF4, was investigated. Microarray analysis demonstrated that rapamycin treatment modulated a large set of genes with varying functions including protein synthesis, assembly of mitochondrial and proteasomal machinery, cell cycle, metabolism and oxidative phosphorylation in ZF4 cells. A mild however, coordinated reduction in the expression of proteasomal and mitochondrial ribosomal subunits was detected, while the expression of numerous ribosomal subunits increased. Meta-analysis of heterogeneous mouse rapamycin microarray datasets enabled the comparison of zebrafish and mouse pathways modulated by rapamycin, using Kyoto Encyclopedia of Genes and Genomes and Gene Ontology pathway analysis. The analyses demonstrated a high degree of functional conservation between zebrafish and mice in response to rapamycin. In addition, rapamycin treatment resulted in a marked dose-dependent reduction in body size and pigmentation in zebrafish embryos. The present study is the first, to the best of our knowledge, to evaluate the conservation of rapamycin-modulated functional pathways between zebrafish and mice, in addition to the dose-dependent growth curves of zebrafish embryos upon rapamycin exposure.
Open Access
Immunization with UV-induced apoptotic cells generates monoclonal antibodies against proteins differentially expressed in hepatocellular carcinoma cell lines
(Mary Ann Liebert, Inc, 2007) Celikkaya, H.; Ciraci, C.; Oztas, E.; Avci, M. E.; Ozturk, M.; Yagci, T.
Early and differential diagnosis of hepatocellular carcinoma (HCC) requires sensitive and specific tissue and serum markers. On the other hand, proteins involved in tumorigenesis are extensively modelated on exposure to apoptotic stimuli, including ultraviolet (UVC) irradiation. Hence, we generated monoclonal antibodies by using UVC-irradiated apoptotic cells of an HCC cell line, HUH7, aiming to explore proteins differentially expressed in tumors and apoptosis. We obtained 18 hybridoma clones recognizing protein targets in apoptotic HUH7 cells, and clone 6D5 was chosen for characterization studies because of its strong reactivity in cell-ELISA assay. Subtype of the antibody was IgG3 (κ). Targets of 6D5 antibody were found to be abundantly expressed in all HCC cell lines except FLC4, which resembles normal hepatocytes. We also observed the secretion of 6D5 ligands by some of the HCC cell lines. Moreover, cellular proteins recognized by the antibody displayed a late upregulation in UVC-induced apoptotic cells. We concluded that 6D5 target proteins are modulated in liver tumorigenesis and apoptotic processes. We therefore propose the validation of our antibody in tissue and serum samples of HCC patients to assess its potential use for the early diagnosis of HCC and to understand the role of 6D5 ligands in liver carcinogenesis. © Mary Ann Liebert, Inc.
Open Access
Induction of apoptosis by overexpression of the DNA-binding and DNA-PK-activating protein C1D
(1999) Rothbarth, K.; Spiess, E.; Juodka, B.; Yavuzer, U.; Nehls, P.; Stammer, H.; Werner, D.
Apoptosis is induced in various tumor cell lines by vector-dependent overexpression of the conserved gene C1D that encodes a DNA-binding and DNA-PK-activating protein. C1D is physiologically expressed in 50 human tissues tested, which points to its basic cellular function. The expression of this gene must be tightly regulated because elevated levels of C1D protein, e.g. those induced by transient vector-dependent expression, result in apoptotic cell death. Cells transfected with C1D-expressing constructs show terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of DNA ends. Transfections with constructs in which C1D is expressed in fusion with the (enhanced) green fluorescent protein from A. victoria (EGFP) allow the transfected cells to be identified and the morphological changes induced to be traced. Starting from intense nuclear spots, green fluorescence reflecting C1D expression increases dramatically at 12-24 hours post-transfection. Expression of C1D-EGFP protein is accompanied by morphological changes typical of apoptotic cell death, e.g. cytoplasmic vacuolation, membrane blebbing and nuclear disintegration. Cell shrinkage and detachment from extracellular matrix are observed in monolayer cultures while suspension cells become progressively flattened. The facility to differentiate between transfected and non-transfected cells reveals that non-transfected cells co-cultured with transfected cells also show the morphological changes of apoptosis, which points to a bystander effect. C1D-dependent apoptosis is not induced in cells with non-functional p53. Accordingly, C1D-induced apoptosis is discussed in relation to its potential to activate DNA-PK, which has been considered to act as an upstream activator of p53.
Open Access
Induction of ROS, p53, p21 in DEHP-and MEHP-exposed LNCaP cells-protection by selenium compunds
(Elsevier, 2011-07) Erkekoglu, P.; Rachidi, W.; Yazgullu, O. G.; Giray, B.; Ozturk, M.; Favier, A.; Hincal, F.
This study was designed to investigate the hypothesis that the toxic effects of di(2-ethylhexyl)phthalate (DEHP), the most abundantly used plasticizer and ubiquitous environmental contaminant that cause alterations in endocrine and spermatogenic functions in animals is mediated through the induction of reactive oxygen species (ROS) and activation of nuclear p53 and p21 proteins in LNCaP human prostate adenocarcinoma cell line. Protective effects of two selenocompounds, sodium selenite (SS) and selenomethionine (SM) were also examined. It was demonstrated that 24. h exposure of the cells to 3. mM DEHP or its main metabolite, mono(2-ethylhexyl)phthalate (MEHP, 3 μM) caused strongly amplified production of ROS. Both SS (30. nM) and SM (10 μM) supplementations reduced ROS production, and p53 and p21 activation that induced significantly only by MEHP-exposure. The overall results of this study indicated that the induction of oxidative stress is one of the important mechanisms underlying the toxicity of DEHP and this is mainly through the effects of the metabolite, MEHP. Generated data also emphasized the critical role of Se in modulation of intracellular redox status, implicating the importance of the appropriate Se status in cellular response against testicular toxicity of phthalates. © 2011 Elsevier Ltd.
Open Access
Investigation of the role of cGAMP in differentiation of T lymphocytes
(2016-10) Yıldız, Begüm
STING is the pivotal mediator for the recognition of host and pathogenic cytosolic dsDNA as well as cyclic di-nucleotides metabolites from microbes. STING can either recognize DNA itself or sense the presence of cGAMP, which is converted from ATP and GTP upon DNA binding to cGAS enzyme. Not only strategy against intracellular pathogens makes STING an ideal target, but also the recognition of DNA from host cells has a significant role in tumor immunity. Previous studies demonstrated that DNA released from cancerous cells are internalized by innate immune cells such as macrophages and dendritic cells in tumor microenvironment and trigger the production of IFN-β and other pro-inflammatory cytokines including IL-6, TNF-α, and IL-12 through STING triggered signaling pathway. These cytokines then enhance cytotoxic activity of CD8+ T cells by further increasing IFNγ production. Since enhanced T cell immunity is the hallmark of vaccine adjuvants, cyclic di-nucleotides such as cGAMP become an important and effective vaccine adjuvants against intracellular pathogens and malignant cells. Although STING activating cyclic di-nucleotides are envisioned as novel and potent vaccine adjuvants, more thorough research is needed to unearth the mechanism of action of STING on different immune cells. Therefore, it will pave the way for the initiation of successful human trials. The important criteria while developing vaccine adjuvant are the magnitude, and the quality of an immune response and its toxic side effects. To identify these, members of the both innate and adaptive immune system should be taken into account. However, previous studies merely focus on the function and effect of cGAMP in innate immune cells such as macrophages, monocytes and dendritic cells. However, to date there is no explicit study investigating the effect of STING signaling cascade on T-cells. In the light of these findings, we aimed to investigate the direct effect and function of cGAMP on T lymphocytes. Since there were not any preliminary data, we firstly stimulated Pan T cells with cGAMP alone or together with various TLR ligands and then, checked the cytokine profiles and the viability of cells. Surprisingly, 2.5µg/ml dose of cGAMP had a toxic effect on T cell but not on bone marrow derived dendritic cells and macrophages. While cGAMP triggered cell death, interestingly IL-17 secretion from both CD4+ and CD8+ T cells was dramatically increased. Beside, cGAMP stimulation drastically increased CD4+ /CD8+ T cells ratio of Pan T cells population. Next, we sought to identify the source of IL-17. The IL17 inductive capacity of cGAMP was investigated on purified CD4+ T cells from mice. Unexpectedly, data revealed that cGAMP elicited apoptosis of CD4+ T cells. Moreover, there was no significant induction of IL-17 secretion. Next, we aimed to find a condition that will reduce the toxic effect of cGAMP, while maintaining IL-17 secretion. When Pan T cells were stimulated with cGAMP and R848 (a TLR7 ligand), the toxic action of cGAMP decreased while IL-17 secretion was enhanced. Lastly, the potency of T cells stimulated with cGAMP was investigated. According to our results, macrophages were activated in the presence of conditioned medium obtained from T cells stimulated with cGAMP. When taken together our findings point out that STING dependent direct activation of T-cells via cGAMP and its subsequent effect on macrophages might be utilized as an immunotherapeutic approach where IL17 induction is important and could be harnessed as vaccine adjuvants against mucosal infections or against cancer.
Open Access
The involvement of apoptotic regulators during in vitro decidualization
(Society of the European Journal of Endocrinology, 2003) Akcali, K. C.; Gibori, G.; Khan, S. A.
Objectives: The uterus responds to an implanting blastocyst by undergoing extensive tissue modification leading to decidualization. This modification includes differentiation and apoptosis of epithelial as well as stromal cell compartments. It is generally accepted that the decidual cell regression pattern is similar to the pattern of initial differentiation, suggesting that decidual cell death is the end point of timed differentiation. However, the molecular mechanisms controlling these events are not understood clearly. Therefore, we aimed to investigate the involvement of apoptotic factors using an in vitro cell culture system. Design: In order to assess the role of apoptotic factors during decidualization, we used a decidual cell line (GG-AD) that had been transformed with a temperature-sensitive SV-40 mutant. At the non-permissive temperature (39°C), these cells showed the characteristics of differentiated decidual cells. They dedifferentiated into stromal cells when the temperature was shifted back to 33°C. Methods: We performed Northern blot analysis for bax, bcl-XL and bcl-2 at both temperatures. The onset of apoptosis was examined by Annexin V staining. The expression of p53 protein was also determined by Western blot. Results: We found an increase in the expression of bax when GG-AD cells were grown at 39°C. We also showed apoptosis with Annexin V staining at 39°C. The p53 protein expression was also similar to that of the animal models, suggesting that the programmed cell death of the decidual cells occurred in a p53-independent manner. Conclusions: These data indicate that a parallelism exists between the increased expression of pro-apoptotic genes and decidual cell death, similar to animal models. Therefore, an in vitro model of GG-AD cells can be used to assess directly the relationship between apoptotic regulators and decidualization and could be used to study the mechanism of decidual cell regression.