Treatment with cucurbitacin B alone and in combination with gefitinib induces cell cycle inhibition and apoptosis via EGFR and JAK/STAT pathway in human colorectal cancer cell lines

Bayram, Cem/0000-0001-8717-4668; Elmazoglu, Zubeyir/0000-0003-4527-8834WOS: 000375574200007PubMed: 26183715The epidermal growth factor receptor (EGFR) associated with signaling pathways, such as Janus kinase (JAK)/signal transducer and activator of transcription (STAT), plays an important role in colorectal cancers (CRCs). Gefitinib (Gef) is an orally active inhibitor targeting the adenosine tri phosphate-binding domain of EGFR, and cucurbitacin B (CuB) is a selective inhibitor of JAK/STAT signaling with potent antitumor activity via suppression of STAT3 phosphorylation, but the underlying mechanism is not clear. We aimed to investigate the apoptotic and antiproliferative effects of CuB as a single agent and in combination with Gef on both HT-29 and HCT-116 cell lines. Cell proliferation, cell cycle distribution, and apoptosis were evaluated using viability assay, fluorescent microscopy, cytotoxicity assay, proliferation, DNA fragmentation, and cleaved caspase 3 levels. Real-time polymerase chain reaction and Western blot analyses were performed to determine the expression of relevant genes and proteins including antiapoptotic, proapoptotic, and cell cycle regulation. EGFR, phosphorylated EGFR (pEGFR), STAT3, and pSTAT3 proteins were evalutaed with Western blot analysis. Our results showed that, compared to CuB alone, CuB plus Gef treatment caused a significant growth and cell cycle inhibition and induced apoptosis in both cell lines. Also CuB plus Gef treatment decreased DNA synthesis rate more effectively than CuB alone. Treatment with CuB alone and in combination with Gef decreased the expression levels of B-Cell CLL/Lymphoma 2 (Bcl-2), BCL2-like 1 (BCL2L1), cyclin D1, pSTAT3, and pEGFR and increased the expression levels of Bcl-2-like protein 4, Bcl-2 homologous antagonist/killer, Bcl-2-associated death promoter, Bcl-2-like protein 11, and p27kip1 levels. Our results suggest that treatment with CuB alone and more likely in combination with Gef may be a considerable alternative therapeutic approach for CRC, at least in vitro.Gazi University Research FoundationGazi University [01/2011-56]The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by Gazi University Research Foundation, with the project code number 01/2011-56

Effect of API-1 and FR180204 on cell proliferation and apoptosis in human DLD-1 and LoVo colorectal cancer cells

Elmazoglu, Zubeyir/0000-0003-4527-8834WOS: 000385579200033PubMed: 27698814The activation of the phosphatidylinositol-3 kinase/v-akt murine thymoma viral oncogene homolog (Akt) and mitogen activated protein kinase kinase/extracellular signal-regulated kinase (ERK) pathways are implicated in the majority of cancers. Selective inhibition of Akt and ERK represents a potential approach for cancer therapy. Therefore, the present study aimed to investigate the apoptotic and anti-proliferative effects of the novel and selective Akt inhibitor 4-amino-5,8-dihydro-5-oxo-8--D-ribofuranosyl-pyrido[2,3-d]pyrimidine-6-carboxamide (API-1) and selective ERK1/2 inhibitor FR180204 (FR) alone and in combination on colorectal cancer (CRC) cells (DLD-1 and LoVo). In addition, the effects of API-1 and FR on Akt and ERK signaling pathways were also investigated. The effects of the agents on DLD-1 and LoVo cells were evaluated in terms of cell viability, cytotoxicity, DNA synthesis rate, DNA fragmentation and caspase-3 activity levels. In addition, quantitative reverse transcription-polymerase chain reaction and western blot analysis were performed to examine relevant mRNA and protein levels. The present study observed that the combination of FR with API-1 resulted in significant apoptosis and cytotoxicity compared with any single agent alone in a time-dependent manner in these cells. Also, treatment with FR and API-1 in combination decreased the expression levels of B-cell lymphoma-2 (BCL2), Bcl-2-like1, cyclin D1 and cMYC, and increased the expression levels of BCL2-associated X protein and BCL2 antagonist/killer via phosphorylated Akt and phosphorylated ERK1/2 downregulation. The combination of Akt and ERK1/2 inhibitors resulted in enhanced apoptotic and anti-proliferative effects against CRC cells. The present study hypothesizes that the combination of FR and API-1 in CRC cells may contribute toward potential anti-carcinogenic effects. Additional analyses using other cancer cell lines and animal models are required to confirm these findings in vitro and in vivo.Scientific and Technological Research Council of TurkeyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [111S054]The present study was supported by The Scientific and Technological Research Council of Turkey (grant number 111S054)

The effects of thymoquinone and genistein treatment on telomerase activity, apoptosis, angiogenesis, and survival in thyroid cancer cell lines

WOS: 000427369100013PubMed: 29516914Context: Thyroid cancers (TCs) are the most common endocrine malignancies. There were two problems with the current cancer chemotherapy: the ineffectiveness of treatment due to resistance to cancer cell, and the toxic effect on normal cells. Aims: This study was aimed to determine the effects of thymoquinone (TQ) and genistein (Gen) phytotherapeutics on telomerase activity, angiogenesis, and apoptosis in follicular and anaplastic thyroid cancer cells (TCCs). Materials and Methods: Cell viability, caspase-3 (CASP-3) activity, and messenger RNA (mRNA) expression levels of human telomerase reverse transcriptase (hTERT), phosphatase and tensin homolog (PTEN), nuclear factor-kappa B (NF-kB), cyclin-dependent kinase inhibitor 1 (p21), and vascular endothelial growth factor-A (VEGF-A) genes were analyzed. Results: It was found that TQ and Gen treatment on TCCs caused a statistically significant decrease of cell viability, and mRNA expression levels of hTERT, VEGF-A, and NF-kB genes, but a statistically significant increase of PTEN and p21 mRNA expression levels. In addition, TQ and Gen treatment also caused a statistically significant increase active CASP-3 protein level in TCCs. Moreover, our results demonstrated that, when compared with follicular TCCs, anaplastic TCCs were more sensitive to the treatment of TQ and Gen. Conclusions: Based on these results, two agents can be good options as potential phytochemotherapeutics against TCCs.Gazi University Scientific Research Projects Coordination Unit [01/2010-68]This study was supported by the Gazi University Scientific Research Projects Coordination Unit (Project No: 01/2010-68)

Evaluation of apoptotic caspase levels in estimation of the wound age

WOS: 000432533300004Objectives: We aimed to investigate the potential use of the expression of apoptotic signaling pathway genes of rat in skin wound age estimation. Material and methods: For this purpose, we formed cutting tool injuries using a scalpel in an experimental model. Then, we assessed Caspase 3, 8 and 9 mRNA levels by using quantitative real-time PCR and protein levels by using immunohistochemistry in rat skin wounds. In addition, we used TUNEL assay to detect apoptotic cells. Results: We observed that Caspase 3 mRNA level significantly increased (2.1 +/- 0.4 folds) on day 3 (p < 0.05) and Caspase 8 mRNA level significantly increased (1.8 +/- 0.2 folds) on day 5 (p < 0.05). Caspase 9 mRNA level increased (1.9 +/- 0.1 folds) on day 3 and (2.5 +/- 0.4 folds) on day 5 (p < 0.05). The percentage values of polymorphonuclear leukocytes (PMNLs) and inflammatory mononuclear cells (IMCs) were observed after immunohistochemical staining by Caspase 3, 8, 9 antibodies. Our immunohistochemistry results were found to be consistent with the mRNA results observed. We reported a statistically significant increase in Caspase 3, 8 and 9-positive cells on days 3 and 5 after immunohistochemical staining as well. Conclusion: Our results suggest that time-dependent features of apoptotic factors might offer a potential tool in estimating wound age.Gazi University Research FoundationGazi University [01/2010-78]This study was supported by Gazi University Research Foundation, with the Project code number 01/2010-78

Hdac inhibitors, Ms-275 and salermide, potentiates the anticancer effect of ef24 in human pancreatic cancer cells

, Handan/0000-0003-4967-2740; YILMAZ, AKIN/0000-0002-4368-0777WOS: 000374213400002PubMed: 27330528Histone deacetylases (HDACs) play a major role in the regulation of chromatin structure and gene expression by changing acetylation status of histone and non-histone proteins. MS-275 (entinostat, MS) is a well-known benzamide-based HDACI and Salermide (SAL), a reverse amide compound HDACI, have antiproliferative effects on several human cancer cells. In this study, we aimed to investigate the effects of HDACIs (MS and SAL) alone and/or combined use with EF24 (EF), a novel synthetic curcumin analog, on human pancreatic cancer cell line (BxPC-3). In vitro, BxPC-3 cells were exposed to varying concentrations of MS, SAL with or without EF, and their effects on cell viability, acetylated Histone H3 and H4 levels, cytotoxicity, and cleaved caspase 3 levels, and cell cycle distribution were measured. The viability of BxPC-3 cells decreased significantly after treatment with EF, MS and SAL treatments. MS and SAL treatment increased the acetylation of histone H3 and H4 in a dose dependent manner. MS and SAL alone or combined with EF were increased the number of cells in G1 phase. In addition, treatment with agents significantly decreased the ratio of cell in G2/M phase. There were significant dose-dependent increases at cleaved Caspase 3 levels after MS treatment but not after SAL treatment. Our results showed that HDAC inhibitors (MS and SAL), when combined with EF, may effectively reduce pancreatic cancer cell (BxPC-3) progression and stop the cell cycle at G1 phase. Further molecular analyses are needed to understand the fundamental molecular consequences of HDAC inhibition in pancreas cancer cells.Gazi University Research FoundationGazi University [01/2010-39]This study is partially supported by Gazi University Research Foundation (Project code 01/2010-39)

Investigation of Ocular Neovascularization-Related Genes and Oxidative Stress in Diabetic Rat Eye Tissues After Resveratrol Treatment

Changes in vascular endothelial growth factor (VEGF), angiotensin-converting enzyme (ACE), matrix metalloproteinase (MMP)-9, and endothelial nitric oxide synthase (eNOS) mRNA expression profiles and oxidative stress in the eye tissue microenviroment may have important roles in ocular neovascularization and permeability in proliferative diabetic retinopathy. The present study investigated the effects of resveratrol (RSV) treatment on the mRNA expression profile of VEGF, ACE, MMP-9, and eNOS, which are associated with vascular neovascularization, and glutathione, protein carbonyl, and nitrite-nitrate levels, which are markers of oxidative stress in eyes of diabetic rats. Twenty-four Wistar albino male rats were divided into four groups. After diabetes induction with streptozotocin (10 mg/kg/day) RSV was administered to the RSV and diabetes mellitus (DM) + RSV groups for 4 weeks. The mRNA levels were measured by quantitative real-time polymerase chain reaction assay, and biochemical estimations were determined with spectrophotometric assays in eye homogenates. The mRNA expression levels of VEGF, ACE, and MMP-9 were increased in the DM group compared with the control group, and RSV treatment decreased their mRNA levels. Expression of eNOS mRNA was increased in the RSV and DM groups and decreased in the DM + RSV group. Nitrite-nitrate levels and protein carbonyl content were increased and glutathione levels were decreased in the DM group compared with controls. Consequently, these data suggest that RSV suppressed the expression of eNOS, which is actively involved in the inflammation and healing process in chronic diabetes. Although oxidative stress was increased in eye tissue from diabetic rats, mRNA levels of VEGF, MMP-9, and ACE genes associated with vascular remodeling did not change in diabetic eyes

Association of rs10757274 and rs2383206 Polymorphisms on 9p21 locus with Coronary Artery Disease in Turkish Population

Background and Objectives: Genetic predisposition is an important risk factor for coronary artery disease (CAD). In this study, we aimed to evaluate the impact of rs10757274 and rs2383206 polymorphisms in chromosome 9p21 on presence and severity of CAD in a Turkish population. Subjects and Methods: A total of 646 patients who underwent coronary angiography were included in this study. Coronary vessel score and Gensini score were calculated to assess the angiographic severity of CAD. Alleles of AA, AG, and GG were determined for rs10757274 (polymorphism-1) and rs2383206 (polymorphism-2) polymorphisms located in chromosome 9p21 from the blood samples. Results: There was a significant difference between the alleles in polymorphism-1 in the presence of coronary artery disease (38.9% in AA, 48.0% in GG and 56.4% in AG, p=0.017). However, there was no difference between the alleles in polymorphism-2. According to vessel scores, there was a significant difference between the alleles in polymorphism-1 (AA 0.71 +/- 1.04, GG 0.88 +/- 1.07, AG 1.06 +/- 1.12, p=0.018). In polymorphism-2, vessel scores did not show a difference between the alleles. In polymorphism-1, there was a significant difference in Gensini score (p=0.041). Gensini scores did not differ between the alleles in polymorphism-2 (p>0.05 for all). In multivariate analyses, none of the alleles was an independent factor for presence of CAD. Conclusion: The presence of rs10757274 polymorphism including AG allele in chromosome 9p21 was related to CAD. However, this relationship was not independent of other cardiovascular risk factors