Oxidative stress and glyoxalase I activity mediate dicarbonyl toxicity in MCF-7 mamma carcinoma cells and a tamoxifen resistant derivative

BackgroundAcquired tamoxifen resistance is a significant problem in estrogen receptor positive breast cancer. In a cellular model, tamoxifen resistance was associated with increased sensitivity towards toxic dicarbonyls and reduced free sulfhydryl group content. We here analyzed the role of oxidative stress and glyoxalase I activity on dicarbonyl resistance and the significance of glyoxalase I expression for survival.MethodsReactive oxygen species were determined by 2,7-dihydrochlorofluorescein diacetate. Inhibitors for NADPH-oxidase (diphenyleneiodonium), p38 MAPK (SB203580) and ERK1/2 (UO126) were applied to investigate interactions of these signaling molecules. N-acetyl cysteine was used to evaluate the effect of oxidative stress on cell viability, which was assessed by the resazurin assay. Gene expression was analyzed by real time qRT-PCR. Glyoxalase activity was inhibited by the specific inhibitor CS-0683 and siRNA. The relevance of glyoxalase 1 mRNA abundance on survival of breast cancer patients was evaluated by the KM-plotter web interface.Resultsα-Oxo-aldehydes caused an immediate increase in reactive oxygen species where the tamoxifen resistant cell line (TamR) responded at lower concentrations than the MCF-7 parental cell line. Inhibitor studies placed ROS production by NADPH-oxidase downstream of p38 MAPK. The antioxidant N-acetyl cysteine (NAC) increased survival, whereas glyoxalase (GLO1) inhibition increased dicarbonyl toxicity. GLO1 mRNA abundance was correlated with unfavorable prognosis of breast cancer patients.ConclusionsDicarbonyl toxicity was mediated by oxidative stress and GLO1 activity determines aldehyde toxicity in tamoxifen resistant cells.General SignificanceGlyoxalases might be predictive biomarkers for tamoxifen resistance and a putative target for the treatment of tamoxifen resistant breast cancer patients.     

Premature senescence in human breast cancer and colon cancer cells by tamoxifen-mediated reactive oxygen species generation

Aims

Cellular senescence is an important tumor suppression process in vivo. Tamoxifen is a well-known anti-breast cancer drug; however, its molecular function is poorly understood. Here, we examined whether tamoxifen promotes senescence in breast cancer and colon cancer cells for the first time.

Main methods

Human breast cancer MCF-7, T47D, and MDA-MB-435 and colorectal cancer HCT116 cells were treated with tamoxifen. Cellular senescence was measured by SA-β-gal staining and based on the protein expression of p53 and p21^Cip1/WAF1. The production of reactive oxygen species (ROS) was determined by staining with CM-H₂DCFDA and dihydroethidium (DHE). CK2 activity was assessed with a specific peptide substrate.

Key findings

Tamoxifen promoted senescence phenotype and ROS generation in MCF-7 and HCT116 cells. The ROS scavenger, N-acetyl-l-cysteine (NAC), and the NADPH oxidase inhibitor, apocynin, almost completely abolished this event. Tamoxifen inhibited the catalytic activity of CK2. Overexpression of CK2α antagonized senescence mediated by tamoxifen, indicating that tamoxifen induced senescence via a CK2-dependent pathway. A well-known CK2 inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), also stimulated ROS production and senescence in MCF-7 cells. Finally, experiments using T47D (wild-type p53) and MDA-MB-435 (mutant p53) cell lines suggested that tamoxifen induces p53-independent ROS production as well as p53-dependent senescence in breast cancer cells.

Significance

These results demonstrate that tamoxifen promotes senescence through a ROS–p53–p21^Cip1/WAF1 dependent pathway by inhibiting CK2 activity in breast cancer and colon cancer cells.     

USP28 facilitates pancreatic cancer progression through activation of Wnt/β-catenin pathway via stabilising FOXM1

Ubiquitination is an important post-translational modification that can be reversed by a family of enzymes called deubiquitinating enzymes (DUBs). Ubiquitin-specific protease 28 (USP28), a member of the DUBs family, functions as a potential tumour promoter in various cancers. However, the biological function and clinical significance of USP28 in pancreatic cancer (PC) are still unclear. Here, we showed that PC tumours had higher USP28 expression compared with that of normal pancreatic tissues, and high USP28 level was significantly correlated with malignant phenotype and shorter survival in patients with PC. Overexpression of USP28 accelerated PC cell growth, whereas USP28 knockdown impaired PC cell growth both in vitro and in vivo. Further, we found that USP28 promoted PC cell growth by facilitating cell cycle progression and inhibiting apoptosis. Mechanistically, USP28 deubiquitinated and stabilised FOXM1, a critical mediator of Wnt/β-catenin signalling. USP28-mediated stabilisation of FOXM1 significantly promoted nucleus β-catenin trans-activation, which in turn led to the activation of the Wnt/β-catenin pathway. Finally, restoration of FOXM1 expression abolished the anti-tumour effects of USP28-silencing. Thus, USP28 contributes to PC pathogenesis through enhancing the FOXM1-mediated Wnt/β-catenin signalling, and could be a potential diagnostic and therapeutic target for PC cases.Subject terms: Pancreatic cancer, Cell growth     

FOXM1 induces a global methylation signature that mimics the cancer epigenome in head and neck squamous cell carcinoma

The oncogene FOXM1 has been implicated in all major types of human cancer. We recently showed that aberrant FOXM1 expression causes stem cell compartment expansion resulting in the initiation of hyperplasia. We have previously shown that FOXM1 regulates HELLS, a SNF2/helicase involved in DNA methylation, implicating FOXM1 in epigenetic regulation. Here, we have demonstrated using primary normal human oral keratinocytes (NOK) that upregulation of FOXM1 suppressed the tumour suppressor gene p16^INK4A (CDKN2A) through promoter hypermethylation. Knockdown of HELLS using siRNA re-activated the mRNA expression of p16^INK4A and concomitant downregulation of two DNA methyltransferases DNMT1 and DNMT3B. The dose-dependent upregulation of endogenous FOXM1 (isoform B) expression during tumour progression across a panel of normal primary NOK strains (n = 8), dysplasias (n = 5) and head and neck squamous cell carcinoma (HNSCC) cell lines (n = 11) correlated positively with endogenous expressions of HELLS, BMI1, DNMT1 and DNMT3B and negatively with p16^INK4A and involucrin. Bisulfite modification and methylation-specific promoter analysis using absolute quantitative PCR (MS-qPCR) showed that upregulation of FOXM1 significantly induced p16^INK4A promoter hypermethylation (10-fold, P<0.05) in primary NOK cells. Using a non-bias genome-wide promoter methylation microarray profiling method, we revealed that aberrant FOXM1 expression in primary NOK induced a global hypomethylation pattern similar to that found in an HNSCC (SCC15) cell line. Following validation experiments using absolute qPCR, we have identified a set of differentially methylated genes, found to be inversely correlated with in vivo mRNA expression levels of clinical HNSCC tumour biopsy samples. This study provided the first evidence, using primary normal human cells and tumour tissues, that aberrant upregulation of FOXM1 orchestrated a DNA methylation signature that mimics the cancer methylome landscape, from which we have identified a unique FOXM1-induced epigenetic signature which may have clinical translational potentials as biomarkers for early cancer screening, diagnostic and/or therapeutic interventions.     

α-Tocopherol supplementation does not affect monocyte endothelial adhesion or C-reactive protein levels but reduces soluble vascular adhesion molecule-1 in the plasma of healthy subjects

Abstract

Vascular monocyte retention in the subintima is pivotal to the development of cardiovascular disease and is facilitated by up-regulation of adhesion molecules on monocytes/endothelial cells during oxidative stress. Epidemiological studies have shown that cardiovascular disease risk is inversely proportional to plasma levels of the dietary micronutrients, vitamin C and vitamin E (α-tocopherol). We have tested the hypothesis that α-tocopherol supplementation may alter endothelial/monocyte function and interaction in subjects with normal ascorbate levels (> 50 μM), as ascorbate has been shown to regenerate tocopherol from its oxidised tocopheroxyl radical form in vitro. Healthy male subjects received α-tocopherol supplements (400 IU RRR-α-tocopherol/day for 6 weeks) in a placebo-controlled, double-blind intervention study. There were no significant differences in monocyte CD11b expression, monocyte adhesion to endothelial cells, plasma C-reactive protein or sICAM-1 concentrations post-supplementation. There was no evidence for nuclear translocation of NF-κB in isolated resting monocytes, nor any effect of α-tocopherol supplementation. However, post-supplementation, sVCAM-1 levels were decreased in all subjects and sE-selectin levels were increased in the vitamin C-replete group only; a weak positive correlation was observed between sE-selectin and α-tocopherol concentration. In conclusion, α-tocopherol supplementation had little effect on cardiovascular disease risk factors in healthy subjects and the effects of tocopherol were not consistently affected by plasma vitamin C concentration.     

Effects of aldehydes on CD36 expression

Introduction: During the oil frying process lipid peroxidation compounds are formed. These products can modulate gene expression and alter cellular behaviour. The cellular uptake of oxidized LDL, a key step in the development of atherosclerosis, is mediated by the CD36 scavenger receptor, whose expression is down-regulated by α-tocopherol.

Objective: To determine the effects of water-soluble aldehydes, obtained from thermally oxidized sunflower oil on the expression of CD36 scavenger receptor in human monocytes (THP-1 cells). We also wanted to study the effects of α-tocopherol on CD36 expression in the presence of water-soluble aldehydes.

Materials and Methods: Sunflower oil was heated in a frying pan, at 180–200°C for 40?min, water-soluble aldehydes were isolated, and the content of thiobarbituric acid reacting substances (TBARS) was determined. THP-1 monocytes were cultured in RPMI medium during 24?h and incubated with increasing concentrations of the water-soluble aldehydes (ranging from 0.05 to 1?μM) and with or without 50?μM of α-tocopherol. In parallel, THP-1 cells were cultured with the same volume of an extract obtained from non-oxidized oil or distilled water. The CD36 expression at the cell surface was studied with fluorescence-activated cell sorting (FACS).

Results: Monocytes incubated in a medium containing water-soluble aldehydes, showed a dose dependent increase in the expression of the CD36 protein on the cell surface, compared to with the control groups. When the cells were treated simultaneously with 50?μM of α-tocopherol a significant reduction in the expression of the CD36 protein was observed.

Conclusion: Water-soluble aldehydes, extracted from thermally oxidized culinary oil, increase the expression of CD36. This effect is partially decreased by the presence of α-tocopherol.     

Targeting GRB7/ERK/FOXM1 Signaling Pathway Impairs Aggressiveness of Ovarian Cancer Cells

Ovarian cancer is a highly lethal disease with poor prognosis and especially in high-grade tumor. Emerging evidence has reported that aberrant upregulation and activation of GRB7, ERK as well as FOXM1 are closely associated with aggresivenesss of human cancers. However, the interplay between these factors in the pathogenesis of human cancers still remains unclear. In this study, we found that GRB7 (P<0.0001), ERK phosphorylation (P<0.0001) and FOXM1 (P = 0.001) were frequently increased and associated with high-grade tumors, as well as a high tendency in association with advanced stage ovarian cancer by immunohistochemical analysis. Intriguingly, the expressions of GRB7 (P<0.0001), ERK phosphorylation (P<0.001) and FOXM1 (P<0.001) showed a significant stepwise increase pattern along Grade 1 to Grade 3 ovarian cancers. Biochemical studies using western blot analysis demonstrated that enforced expression or knockdown of GRB7 showed GRB7 could elevate the levels of ERK phosphorylation and FOXM1, whereas enforced expression of FOXM1 could not alter levels of GRB7 and ERK phosphorylation. But inhibition of ERK signaling by U0126 or PD98059 could reduce the level of FOXM1 in GRB7-overexpressing ovarian cancer cells, suggesting that GRB7, ERK and FOXM1 are regulated orderly. Moreover, inhibition of ERK activity by U0126 or PD98059, or decreased FOXM1 expression by Thiostrepton significantly inhibited cell migration/invasion, tumor growth in vitro and in vivo. Collectively, our findings confer that targeting GRB7/ERK/FOXM1 signaling cascade may be a promising molecular therapeutic choice in combating ovarian cancer.     

CSN5 promotes the invasion and metastasis of pancreatic cancer by stabilization of FOXM1

COP9 signalosome subunit 5 (CSN5) has been involved in the progression of diverse human cancers. MMP2 plays an important role in the metastasis of cancer cells. However, the roles and relationship of in pancreatic cancer (PC) is still unknown. Here, our data shown that both CSN5 and MMP2 were significantly upregulated in PC compared with the corresponding adjacent tissues, where a positive correlation in their expression and associated malignant characteristics were found. Further, silencing of CSN5 expression markedly inhibited PC invasion and metastasis in vitro and in vivo, accompanied by decreased MMP2 expression. Moreover, the anti-metastasis role of CSN5 silence was reversed by MMP2 overexpression, whereas knockdown of MMP2 decreased PC metastasis driven by upregulation of CSN5. Further investigation revealed that CSN5 regulated MMP2 expression via activation of FOXM1 in PC cells. Mechanistically, CSN5 directly bound FOXM1 and decreased its ubiquitination to enhance the protein stability of FOXM1. Taken together, the results indicate that CSN5 can contribute to PC invasion and metastasis through activation of FOXM1/MMP2 axis.     

MTA1 promotes the invasion and migration of pancreatic cancer cells potentially through the HIF-α/VEGF pathway

Abstract

The metastasis-associated gene 1 (MTA1) has previously been recognized as an oncogene, and abnormal MTA1 expression has been related to progression of numerous cancer types to the metastasis stage. However, the function of MTA1 in the regulation of pancreatic cancer progression and metastasis remains unclear. Western blot analysis was adopted to determine the expression of MTA1 in pancreatic cancer tissues and corresponding near normal tissues. Steady clone with MTA1-overexpression and MTA1-inhibitionweregenerated via lentivirus technology in BxPc-3 cells. Transwell assay was carried out for detecting the invasion of pancreatic cancer cells. The migration activity was assessed using the wound scratch assay. The effect of MTA1 in pancreatic cancer was evaluated in the mice xenografts. Western blot analysis was employed to determine the expression of hypoxia inducible factor-α (HIF-α) and vascular endothelial growth factor (VEGF) in vitro and in vivo. We observed that MTA1 overexpression enhanced migration and invasion ability of pancreatic cancer cells in vitro and increased HIF-α and VEGF protein levels in vitro and in vivo. MTA1 inhibition had the opposite effects. MTA1 protein level was positively related to HIF-α and VEGF protein levels. These results indicated that MTA1 potentially promoted pancreatic cancer metastasis via HIF-α/VEGF pathway. This research supplies a new molecular mechanism for MTA1 in the pancreatic cancer progression and metastasis. MTA1 may be an effective therapy target in pancreatic cancer.     

Capacity of fucoxanthin for scavenging peroxyl radicals and inhibition of lipid peroxidation in model systems

Abstract

Carotenoids act as physiological antioxidant by scavenging reactive-free radicals as well as quenching singlet oxygen. Fucoxanthin is one of the abundant carotenoids found in edible brown seaweeds. The assessment of radical scavenging capacity of carotenoids has been the subject of extensive studies, which, however, gave inconsistent results. In the present study, the capacity of fucoxanthin for scavenging peroxyl radicals, chain carrying species of lipid peroxidation, was assessed quantitatively by measuring the effect of α-tocopherol on the decay of fucoxanthin induced by peroxyl radicals. It was found that α-tocopherol was 7.1 times more reactive than fucoxanthin in heptane solution, but interestingly fucoxanthin exerted 1.6 times higher reactivity than α-tocopherol in methanol solution. In SDS micelles, the relative reactivity of fucoxanthin and α-tocopherol depended on the site of peroxyl radical formation. The efficacy of lipid peroxidation inhibition by fucoxanthin was much less than that of α-tocopherol.     

Circumventing senescence is associated with stem cell properties and metformin sensitivity

Most cancers arise in old individuals, which also accumulate senescent cells. Cellular senescence can be experimentally induced by expression of oncogenes or telomere shortening during serial passage in culture. In vivo, precursor lesions of several cancer types accumulate senescent cells, which are thought to represent a barrier to malignant progression and a response to the aberrant activation of growth signaling pathways by oncogenes (oncogene toxicity). Here, we sought to define gene expression changes associated with cells that bypass senescence induced by oncogenic RAS. In the context of pancreatic ductal adenocarcinoma (PDAC), oncogenic KRAS induces benign pancreatic intraepithelial neoplasias (PanINs), which exhibit features of oncogene‐induced senescence. We found that the bypass of senescence in PanINs leads to malignant PDAC cells characterized by gene signatures of epithelial‐mesenchymal transition, stem cells, and mitochondria. Stem cell properties were similarly acquired in PanIN cells treated with LPS, and in primary fibroblasts and mammary epithelial cells that bypassed Ras‐induced senescence after reduction of ERK signaling. Intriguingly, maintenance of cells that circumvented senescence and acquired stem cell properties was blocked by metformin, an inhibitor of complex I of the electron transport chain or depletion of STAT3, a protein required for mitochondrial functions and stemness. Thus, our studies link bypass of senescence in premalignant lesions to loss of differentiation, acquisition of stemness features, and increased reliance on mitochondrial functions.     

Identification of phosphoenolpyruvate carboxykinase 1 as a potential therapeutic target for pancreatic cancer

Pancreatic cancer is the third leading cause of cancer-related mortalities and is characterized by rapid disease progression. Identification of novel therapeutic targets for this devastating disease is important. Phosphoenolpyruvate carboxykinase 1 (PCK1) is the rate-limiting enzyme of gluconeogenesis. The current study tested the expression and potential functions of PCK1 in pancreatic cancer. We show that PCK1 mRNA and protein levels are significantly elevated in human pancreatic cancer tissues and cells. In established and primary pancreatic cancer cells, PCK1 silencing (by shRNA) or CRISPR/Cas9-induced PCK1 knockout potently inhibited cell growth, proliferation, migration and invasion, and induced robust apoptosis activation. Conversely, ectopic overexpression of PCK1 in pancreatic cancer cells accelerated cell proliferation and migration. RNA-seq analyzing of differentially expressed genes (DEGs) in PCK1-silenced pancreatic cancer cells implied that DEGs were enriched in the PI3K-Akt-mTOR cascade. In pancreatic cancer cells, Akt-mTOR activation was largely inhibited by PCK1 shRNA, but was augmented after ectopic PCK1 overexpression. In vivo, the growth of PCK1 shRNA-bearing PANC-1 xenografts was largely inhibited in nude mice. Akt-mTOR activation was suppressed in PCK1 shRNA-expressing PANC-1 xenograft tissues. Collectively, PCK1 is a potential therapeutic target for pancreatic cancer.Subject terms: Pancreatic cancer, Oncogenes     

Generation of ROS by CAY10598 leads to inactivation of STAT3 signaling and induction of apoptosis in human colon cancer HCT116 cells

Abstract

Prostaglandin E₂ (PGE₂) has been reported to play critical roles in cell fate decision by interacting with four types of prostanoid receptors such as EP1, EP2, EP3 and EP4. The present study was aimed at investigating the effect of the EP4-specific agonist CAY10598 in human colon cancer HCT116 cells. Our study revealed that treatment with CAY10598 significantly reduced the cell viability and induced apoptosis in HCT116 cells, as evidenced by the induction of p53 and Bax, release of cytochrome c, cleavage of caspase-9, -7, and -3, and PARP, and the inhibition of Bcl-2, Bcl-xL and survivin expression. Moreover, treatment with CAY10598 diminished the phosphorylation of JAK2, leading to the attenuation of STAT3 activation in HCT116 cells. CAY10598-induced apoptosis in cells which were transiently transfected with EP4 siRNA or treated with an EP4 antagonist prior to incubation with the compound remained unaffected, suggesting an EP4-independent mechanism of apoptosis induction by CAY10598. We found that treatment with CAY10598 generated reactive oxygen species (ROS) and pretreatment of cells with N-acetyl cysteine rescued cells from apoptosis by abrogating the inhibitory effect of CAY10598 on the activation of JAK2/STAT3 signaling. In conclusion, CAY10598 induced apoptosis in HCT116 cells in an EP4-independent manner, but through the generation of ROS and inactivation of JAK2/STAT3 signaling.