ABSTRACT
(-)-Epicatechin is a phenolic compound with antioxidant activity that is present in natural food and drinks, such as cocoa and red wine. Evidence suggests that (-)-epicatechin exhibits anticancer activity; however, its mechanism of action is poorly understood. Here, we investigated the anticancer effects of (-)-epicatechin and its mechanism of action in breast cancer cells. We assessed the anticancer activity by cell proliferation assays, apoptosis by DNA fragmentation and flow cytometry. The expression of proteins associated with apoptosis was analyzed by the human apoptosis array. MitoSOXTM Red and biomarkers of oxidative damage were used to measure the effect of (-)-epicatechin on mitochondrial reactive oxygen species (ROS) and cellular damage, respectively. (-)-Epicatechin treatment caused a decreasing in the viability of MDA-MB-231 and MCF-7 cells. This cell death was associated with DNA fragmentation and an apoptotic proteomic profile. Further, (-)-epicatechin in MDA-MB-231 cells upregulated death receptor (DR4/DR5), increased the ROS production, and modulated pro-apoptotic proteins. In MCF-7 cells, (-)-epicatechin did not involve death receptor; however, an increase in ROS and the upregulation of pro-apoptotic proteins (Bad and Bax) were observed. These changes were associated with the apoptosis activation through the intrinsic pathway. In conclusion, this study shows that (-)-epicatechin has anticancer activity in breast cancer cells and provides novel insight into the molecular mechanism of (-)-epicatechin to induce apoptosis.
Subject(s)
Apoptosis/drug effects , Breast Neoplasms/drug therapy , Catechin/pharmacology , Cell Proliferation/drug effects , Breast Neoplasms/genetics , Breast Neoplasms/pathology , DNA Fragmentation/drug effects , Female , Flow Cytometry , Gene Expression Regulation, Neoplastic/drug effects , Humans , MCF-7 Cells , Reactive Oxygen Species/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/geneticsABSTRACT
Spindle-shaped stromal cells, like carcinoma-associated fibroblasts and mesenchymal stem cells, influence tumor behavior and can serve as parameters in the clinical diagnosis, therapy, and prognosis of early breast cancer. Therefore, the aim of this study is to explore the clinicopathological significance of tumor necrosis factor-related apoptosis-induced ligand (TRAIL) receptors (Rs) 2 and 4 (TRAIL-R2 and R4), and interleukin-6 R (IL-6R) in spindle-shaped stromal cells, not associated with the vasculature, as prognostic determinants of early breast cancer patients. Receptors are able to trigger the migratory activity, among other functions, of these stromal cells. We conducted immunohistochemical analysis for the expression of these receptors in spindle-shaped stromal cells, not associated with the vasculature, of primary tumors from early invasive breast cancer patients, and analyzed their association with clinicopathological characteristics. Here, we demonstrate that the elevated levels of TRAIL-R2, TRAIL-R4, and IL-6R in these stromal cells were significantly associated with a higher risk of metastatic occurrence (p = 0.034, 0.026, and 0.006; respectively). Moreover, high expression of TRAIL-R4 was associated with shorter disease-free survival and metastasis-free survival (p = 0.013 and 0.019; respectively). Also, high expression of IL-6R was associated with shorter disease-free survival, metastasis-free survival, and overall survival (p = 0.003, 0.001, and 0.003; respectively). Multivariate analysis showed that IL-6R expression was an independent prognostic factor for disease-free survival and metastasis-free survival (p = 0.035). This study is the first to demonstrate that high levels of IL-6R expression in spindle-shaped stromal cells, not associated with the vasculature, could be used to identify early breast cancer patients with poor outcomes.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Receptors, Interleukin-6/metabolism , Stromal Cells/metabolism , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor , Breast Neoplasms/genetics , Breast Neoplasms/mortality , Female , Humans , Kaplan-Meier Estimate , Middle Aged , Neoplasm Grading , Neoplasm Metastasis , Neoplasm Staging , Prognosis , Receptors, Interleukin-6/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Stromal Cells/pathology , Tumor Burden , Tumor Necrosis Factor Decoy Receptors/genetics , Tumor Necrosis Factor Decoy Receptors/metabolismABSTRACT
Seizure models have demonstrated that neuroinflammation and neurodegeneration are preponderant characteristics of epilepsy. Considering the lack of clinical studies, our aim is to investigate the extrinsic pathway of apoptosis in pharmacoresistant temporal lobe epilepsy (TLE) associated with hippocampal sclerosis (HS) patients, TLE(HS). By a specific death receptor-mediated apoptosis array plate, 31 upregulated targets were revealed in the sclerotic hippocampus from TLE(HS) patients. Amongst them are the encoding genes for ligands (FASLG, TNF, and TNFSF10) and death receptors (FAS, TNFRSF1A, TNFRSF10A, and TNFRSF10B). In addition, we evaluated the hippocampal relative mRNA expression of the two TNF receptors, TNFRSF1A and TNFRSF1B, in patients, being both upregulated (n = 14; P < 0.01 and P < 0.04, resp.) when compared to the post mortem control group (n = 4). Our results have clearly suggested that three different death receptor apoptotic systems may be associated with the maintenance and progression of TLE-associated HS: (1) TNF-TNFRSF1A, (2) FASLG-FAS, and (3) TNFSF10-TNFRSF10A/B. Their effects on epilepsy are still scarcely comprehended. Our study points out to TNF and TNF receptor superfamily pathways as important targets for pharmacological studies regarding the benefits of an anti-inflammatory therapy in these patients.
Subject(s)
Epilepsy, Temporal Lobe/metabolism , Hippocampus/metabolism , Sclerosis/metabolism , Adolescent , Adult , Apoptosis/genetics , Apoptosis/physiology , Epilepsy, Temporal Lobe/genetics , Female , Humans , In Vitro Techniques , Male , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , Receptors, Tumor Necrosis Factor, Type II/genetics , Receptors, Tumor Necrosis Factor, Type II/metabolism , Sclerosis/genetics , Young AdultABSTRACT
We investigated the roles of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor death receptor 5 (DR5) in the onset of acute leukemia and changes in their expression during chemotherapy. Bone marrow samples from 16 patients newly diagnosed with acute leukemia were collected before chemotherapy. Bone marrow samples from patients with non-hematologic malignancies served as the control group. Peripheral blood samples of patients with acute leukemia were also collected before chemotherapy and at 1 and 3 days after chemotherapy. Mononuclear cells in the bone marrow and peripheral blood were isolated and used to detect the expression of TRAIL and DR5 by flow cytometry. Compared with mononuclear cells from the control group, mononuclear cells from newly diagnosed patients with acute leukemia showed no significant difference in the expression of TRAIL (P > 0.05) but showed significantly increased expression of DR5 (P < 0.05). TRAIL and DR5 expression in peripheral blood mononuclear cells after chemotherapy was significantly increased compared to expression before chemotherapy (P < 0.05). Patients showing high expression of DR5 had a higher remission rate. One of the mechanisms underlying the treatment of leukemia with chemotherapy drugs may be the induction of TRAIL and DR5, which may promote apoptosis in leukemia cells. TRAIL-mediated apoptosis is regulated by DR5 expression.
Subject(s)
Gene Expression , Leukemia/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , TNF-Related Apoptosis-Inducing Ligand/genetics , Acute Disease , Adolescent , Adult , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Female , Humans , Leukemia/diagnosis , Leukemia/drug therapy , Leukemia/metabolism , Male , Middle Aged , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Remission Induction , TNF-Related Apoptosis-Inducing Ligand/metabolism , Young AdultABSTRACT
Prostate carcinoma (PCa) is one of the most common cancers in men. Prostate-specific antigen (PSA) has been widely used to predict the outcome of PCa and screening with PSA has resulted in a decline in mortality. However, PSA is not an optimal prognostic tool as its sensitivity may be too low to reduce morbidity and mortality. Consequently, there is a demand for additional robust biomarkers for prostate cancer. Death receptor 5 (DR5) has been implicated in the prognosis of several cancers and it has been previously shown that it is negatively regulated by Yin Yang 1 (YY1) in prostate cancer cell lines. The present study investigated the clinical significance of DR5 expression in a prostate cancer patient cohort and its correlation with YY1 expression. Immunohistochemical analysis of protein expression distribution was performed using tissue microarray constructs from 54 primary PCa and 39 prostatic intraepithelial neoplasia (PIN) specimens. DR5 expression was dramatically reduced as a function of higher tumor grade. By contrast, YY1 expression was elevated in PCa tumors as compared with that in PIN, and was increased with higher tumor grade. DR5 had an inverse correlation with YY1 expression. Bioinformatic analyses corroborated these data. The present findings suggested that DR5 and YY1 expression levels may serve as progression biomarkers for prostate cancer.
Subject(s)
Biomarkers, Tumor/metabolism , Prostatic Intraepithelial Neoplasia/metabolism , Prostatic Neoplasms/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Disease Progression , Gene Expression , Humans , Male , Prostatic Intraepithelial Neoplasia/pathology , Prostatic Neoplasms/pathology , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Tissue Array Analysis , YY1 Transcription Factor/genetics , YY1 Transcription Factor/metabolismABSTRACT
OBJECTIVE: This study assesses TRAIL-R2 (TNF-related apoptosis-inducing ligand receptor 2) and BCL2 (B cell CLL/lymphoma 2) expression as well as CpG island methylation within the TRAIL-R2 promoter in ovarian serous tumors and primary and metastatic serous EOC (epithelial ovarian cancer). METHODS: RNA and DNA were obtained from women with normal ovarian tissues (n = 18), ovarian serous cystadenoma tumors (n = 11) and serous EOC (n = 16) using Trizol®. Quantitative PCR was performed to quantify the relative levels of TRAIL-R2 and BCL2. The methylation frequency of the TRAIL-R3 promoter was assessed using a methylation-specific PCR assay after DNA bisulfite conversion. Differences between the groups were evaluated using the χ (2), Mann-Whitney U or Kruskal-Wallis tests, as indicated. RESULTS: We identified TRAIL-R2 and BCL2 mRNA expressed in all ovarian tumor groups, and there were significant differences between the groups. Both genes had low expression levels in ovarian serous cystadenoma and primary EOC tumors when compared with metastatic EOC. Methylation of the TRAIL-R2 promoter was frequently observed in all groups; however, there were no statistically significant associations. CONCLUSIONS: Primary EOC is associated with lower TRAIL-R2 and BCL2 expression levels, while metastatic EOC is associated with higher expression of these genes. Promoter DNA methylation was not related to this finding, suggesting there are other mechanisms involved in transcriptional control.
Subject(s)
Cystadenoma, Serous/genetics , DNA Methylation , Epigenesis, Genetic , Gene Expression , Neoplasms, Glandular and Epithelial/genetics , Ovarian Neoplasms/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Adult , Aged , Aged, 80 and over , Apoptosis Regulatory Proteins/genetics , Carcinoma, Ovarian Epithelial , Cell Line, Tumor , Female , Humans , Middle Aged , Neoplasm Staging , Polymerase Chain Reaction , Promoter Regions, Genetic , Prospective Studies , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Statistics, NonparametricABSTRACT
INTRODUCTION: The nuclear factor-kappaB (NF-NF-κ) has been shown to upregulate pro-apoptotic mediators such as TRAIL-DR4/-DR5 receptors and the p53 transcription factor depending on the type of stimulus and the cell type involved. Previously, apple procyanidins (Pcy) have been shown to upregulate the expression of TRAIL-DR4/-DR5 and thereby overcoming the resistance of human colon cancer-derived metastatic SW620 cells to TRAIL. OBJECTIVES: NF-κB and p53 were investigated for their involvement in the Pcy-triggered apoptosis of human derived-metastatic colon cancer (SW620) cells. MATERIALS AND METHODS: Cell death, p53, TRAIL-DR4/-DR5 proteins were analyzed by flow cytometry. DR4/DR5 mRNA was analyzed by RT-PCR in real time. Activated p50/p65 and p53 forms were studied by ELISA and immunoblotting RESULTS: Pcy-triggered cell death was prevented by specific inhibitors of NF-κB and of p53: amino-4-(4-phenoxy-phenylethylamino) quinazoline (QNZ) and pifithrin α (Pα), respectively. QNZ and Pα inhibited the Pcy-dependent activation of TRAIL-DR4/-DR5 death receptors. However, the upregulation of TRAIL-DR4 by Pcy was significantly decreased only when NF-κB and p53 inhibitors were used in combination; this effect was not observed with a single inhibitor. This effect was not observed for TRAIL-DR5 and suggested that the expression of each TRAIL-death receptor may be regulated differently. CONCLUSIONS: These data suggested that NF-κB and p53 are partially required in Pcy-triggered apoptosis of SW620 cells by up-regulating the expression of TRAIL-DR4/-DR5. In addition, the ratio between TRAIL-DR4/-DR5 may be a determining factor in the activation of TRAIL-death receptor mediated apoptosis.
Subject(s)
Apoptosis/drug effects , Cell Line, Tumor/drug effects , Malus/chemistry , NF-kappa B/metabolism , Proanthocyanidins/pharmacology , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Tumor Suppressor Protein p53/metabolism , Animals , Benzothiazoles/pharmacology , Cell Line, Tumor/physiology , Humans , NF-kappa B/genetics , Quinazolines/pharmacology , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Toluene/analogs & derivatives , Toluene/pharmacology , Tumor Suppressor Protein p53/genetics , Up-Regulation/drug effectsABSTRACT
Cancer of the reproductive tract encompasses malignancies of the uterine corpus, cervix, ovary, Fallopian tube, among others and accounts for 15% of female cancer mortalities. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates apoptosis by binding to death receptors and offers a promising cancer treatment. The goal of this study was to investigate and characterize the effect of TRAIL in endometrial cancer cell lines and normal (non-cancerous) epithelial cells of endometrial origin. We also examined the effect of TRAIL in other primary cultured cancers and normal cells of the human female reproductive tract and evaluated if TRAIL mediated apoptosis correlated with death receptors and decoy receptors 1 and 2.Herein, we demonstrate that TRAIL at concentrations which kill cancerous cells, does not mediate apoptosis or alter cell viability in normal human endometrium, ovary, cervix or Fallopian tube. The partial inhibition by a caspase 9 inhibitor and the total inhibition by a caspase 8 inhibitor demonstrates the dependency on the extrinsic apoptotic pathway. The selective mortality does not correlate with the presence of death or decoy receptors. These results suggest that TRAIL may be an effective treatment for endometrial cancer and other female reproductive cancers, with minimal secondary effects on healthy tissue.