Downregulation of the Werner syndrome protein induces a metabolic shift that compromises redox homeostasis and limits proliferation of cancer cells

The Werner syndrome protein (WRN) is a nuclear protein required for cell growth and proliferation. Loss‐of‐function mutations in the Werner syndrome gene are associated with the premature onset of age‐related diseases. How loss of WRN limits cell proliferation and induces replicative senescence is poorly understood. Here, we show that WRN depletion leads to a striking metabolic shift that coordinately weakens the pathways that generate reducing equivalents for detoxification of reactive oxygen species and increases mitochondrial respiration. In cancer cells, this metabolic shift counteracts the Warburg effect, a defining characteristic of many malignant cells, resulting in altered redox balance and accumulation of oxidative DNA damage that inhibits cell proliferation and induces a senescence‐like phenotype. Consistent with these findings, supplementation with antioxidant rescues at least in part cell proliferation and decreases senescence in WRN‐knockdown cancer cells. These results demonstrate that WRN plays a critical role in cancer cell proliferation by contributing to the Warburg effect and preventing metabolic stress.     

Predicting selective drug targets in cancer through metabolic networks

The interest in studying metabolic alterations in cancer and their potential role as novel targets for therapy has been rejuvenated in recent years. Here, we report the development of the first genome‐scale network model of cancer metabolism, validated by correctly identifying genes essential for cellular proliferation in cancer cell lines. The model predicts 52 cytostatic drug targets, of which 40% are targeted by known, approved or experimental anticancer drugs, and the rest are new. It further predicts combinations of synthetic lethal drug targets, whose synergy is validated using available drug efficacy and gene expression measurements across the NCI‐60 cancer cell line collection. Finally, potential selective treatments for specific cancers that depend on cancer type‐specific downregulation of gene expression and somatic mutations are compiled.     

Mesenchymal stem cell-derived interleukin-6 and vascular endothelial growth factor promote breast cancer cell migration

Several different cytokines and growth factors secreted by mesenchymal stem cells (MSCs) have been hypothesized to play a role in breast cancer progression. By using a small panel of breast cancer cell lines (MCF‐7, T47D, and SK‐Br‐3 cells), we analyzed the role of interleukin‐6 (IL‐6) and vascular endothelial growth factor A (VEGF) in the cross‐talk between MSCs and breast cancer cells. We performed migration assays in which breast cancer cells were allowed to migrate in response to conditioned medium from MSCs (MSCs‐CM), in absence or in presence of the anti‐VEGF antibody bevacizumab or an anti‐IL‐6 antibody, alone or in combination. We found that anti‐VEGF and anti‐IL‐6 antibodies inhibited the migration of breast cancer cells and that the combination had an higher inhibitory effect. We next evaluated the effects of recombinant VEGF and IL‐6 proteins on breast cancer cell growth and migration. IL‐6 and VEGF had not significant effects on the proliferation of breast carcinoma cells. In contrast, both VEGF and IL‐6 significantly increased the ability to migrate of MCF‐7, T47D and SK‐Br‐3 cells, with the combination showing a greater effect as compared with treatment with a single protein. The combination of VEGF and IL‐6 produced in breast cancer cells a more significant and more persistent activation of MAPK, AKT, and p38MAPK intracellular signaling pathways. These results suggest that MSC‐secreted IL‐6 and VEGF may act as paracrine factors to sustain breast cancer cell migration. J. Cell. Biochem. 113: 3363–3370, 2012. © 2012 Wiley Periodicals, Inc.     

A mechanistic modeling framework reveals the key principles underlying tumor metabolism

While aerobic glycolysis, or the Warburg effect, has for a long time been considered a hallmark of tumor metabolism, recent studies have revealed a far more complex picture. Tumor cells exhibit widespread metabolic heterogeneity, not only in their presentation of the Warburg effect but also in the nutrients and the metabolic pathways they are dependent on. Moreover, tumor cells can switch between different metabolic phenotypes in response to environmental cues and therapeutic interventions. A framework to analyze the observed metabolic heterogeneity and plasticity is, however, lacking. Using a mechanistic model that includes the key metabolic pathways active in tumor cells, we show that the inhibition of phosphofructokinase by excess ATP in the cytoplasm can drive a preference for aerobic glycolysis in fast-proliferating tumor cells. The differing rates of ATP utilization by tumor cells can therefore drive heterogeneity with respect to the presentation of the Warburg effect. Building upon this idea, we couple the metabolic phenotype of tumor cells to their migratory phenotype, and show that our model predictions are in agreement with previous experiments. Next, we report that the reliance of proliferating cells on different anaplerotic pathways depends on the relative availability of glucose and glutamine, and can further drive metabolic heterogeneity. Finally, using treatment of melanoma cells with a BRAF inhibitor as an example, we show that our model can be used to predict the metabolic and gene expression changes in cancer cells in response to drug treatment. By making predictions that are far more generalizable and interpretable as compared to previous tumor metabolism modeling approaches, our framework identifies key principles that govern tumor cell metabolism, and the reported heterogeneity and plasticity. These principles could be key to targeting the metabolic vulnerabilities of cancer.     

Nitrogen anabolism underlies the importance of glutaminolysis in proliferating cells

Glutaminolysis and the Warburg effect are the two most noticeable metabolic features of tumor cells, whereas their biological significance in cell proliferation remains elusive. A widely accepted current hypothesis is that tumor cells use glutamine as a preferred carbon source for energy and reducing power, which has been used to explain both glutaminolysis and the Warburg effect. Here we provide evidence to show that supplying nitrogen, not the carbon skeleton, underlies the major biological importance of glutaminolysis for proliferating cells. We show that alternative nitrogen supplying mechanisms rescue cell proliferation in glutamine-free media. Particularly, we show that ammonia is sufficient to maintain a long-term survival and proliferation of Hep3B in glutamine-free media. We also observed that nitrogen source restriction repressed carbon metabolic pathways, including glucose utilization. Based on these new observations and metabolic pathways well-established in published literature, we propose an alternative model that cellular demand for glutamate is a key molecule in nitrogen anabolism, which is the driving force of glutaminolysis in proliferating cells. Our model suggests that the Warburg effect may be a metabolic consequence secondary to the nitrogen anabolism.Key words: glutaminolysis, cancer, Warburg effect, transamination, glycolysis, hypoxia     

A second Warburg-like effect in cancer metabolism: The metabolic shift of glutamine-derived nitrogen

Carbon and nitrogen are essential elements for life. Glucose as a carbon source and glutamine as a nitrogen source are important nutrients for cell proliferation. About 100 years ago, it was discovered that cancer cells that have acquired unlimited proliferative capacity and undergone malignant evolution in their host manifest a cancer-specific remodeling of glucose metabolism (the Warburg effect). Only recently, however, was it shown that the metabolism of glutamine-derived nitrogen is substantially shifted from glutaminolysis to nucleotide biosynthesis during malignant progression of cancer—which might be referred to as a “second” Warburg effect. In this review, address the mechanism and relevance of this metabolic shift of glutamine-derived nitrogen in human cancer. We also examine the clinical potential of anticancer therapies that modulate the metabolic pathways of glutamine-derived nitrogen. This shift may be as important as the shift in carbon metabolism, which has long been known as the Warburg effect.     

Astragalus membranaceus lectin (AML) induces caspase-dependent apoptosis in human leukemia cells

Objectives: Recently, plant lectins have attracted great interest due to their various biological activities such as anti‐cancer, anti‐fungal and anti‐viral activities. We have reported earlier concerning anti‐proliferation of human cancer cell lines by a galactose‐binding lectin (AML), from a Chinese herb, Astragalus membranaceus. In the present study, detailed investigations into the mechanism of such anti‐proliferation properties have been carried out. Materials and methods: Mechanism of apoptosis initiation in K562 cells by AML was investigated by morphology, flow cytometry and western blot analysis. Results: AML induced apoptosis in a caspase‐dependent manner in the chronic myeloid leukemia cell line, K562. Furthermore, we observed that cytotoxicity and apoptosis of K562 cells induced by AML were completely abolished in presence of lactose or galactose. Conclusions: Our results suggest that AML could act as a potential anti‐cancer drug.     

Human umbilical cord Wharton's jelly stem cell (hWJSC) extracts inhibit cancer cell growth in vitro

Umbilical cord mesenchymal stem cells (MSCs) have been shown to inhibit breast cancer cell growth but it is not known whether this effect is specific to only breast cancer cells. We compared the effects of human Wharton's jelly stem cell (hWJSC) extracts [conditioned medium (hWJSC‐CM) and cell lysate (hWJSC‐CL)] on breast adenocarcinoma (MDA‐MB‐231), ovarian carcinoma (TOV‐112D), and osteosarcoma (MG‐63) cells. The cells were treated with either hWJSC‐CM (50%) or hWJSC‐CL (15 µg/ml) for 48–72 h and changes in cell morphology, proliferation, cycle, gene expression, migration, and cell death studied. All three cancer cell lines showed cell shrinkage, blebbing, and vacuolations with hWJSC‐CL and hWJSC‐CM compared to controls. MTT and BrdU assays showed inhibition of cell growth by 2–6% and 30–60%, while Transwell migration assay showed inhibition by 20–26% and 31–46% for hWJSC‐CM and hWJSC‐CL, respectively, for all three cancer cell lines. Cell cycle assays showed increases in sub‐G1 and G2/M phases for all three cancer cell lines suggestive of apoptosis and metaphase arrest. AnnexinV‐FITC and TUNEL positive cells seen in TOV‐112D and MDA‐MB‐231 suggested that inhibition was via apoptosis while the presence of anti‐BECLIN1 and anti‐LC3B antibodies seen with MG‐63 indicated autophagy. Upregulation of pro‐apoptotic BAX and downregulation of anti‐apoptotic BCL2 and SURVIVIN genes were observed in all three cancer cell lines and additionally the autophagy genes (ATG5, ATG7, and BECLIN1) were upregulated in MG‐63 cells. hWJSCs possess tumor inhibitory properties that are not specific to breast cancer cells alone and these effects are mediated via agents in its extracts. J. Cell. Biochem. 113: 2027–2039, 2012. © 2012 Wiley Periodicals, Inc.     

Loss of macrophage migration inhibitory factor impairs the growth properties of human HeLa cervical cancer cells

Objectives: This study aims to determine the role of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with cell proliferation and tumour growth in vivo. Materials and methods: Our team used RNA interference technology to knock down MIF expression in human HeLa cervical cancer cells and to establish a stable cell line lacking MIF function. Results: Our results showed that long‐term loss of MIF had little effect on cell morphology, but significantly inhibited their population growth and proliferation. The HeLa MIF‐knockdown cells retained normal apoptotic signalling pathways in response to TNF‐alpha treatment; however, they exhibited unique DNA profiles following doxorubicin treatment, suggesting that MIF may regulate a cell cycle checkpoint upon DNA damage. Our data also showed that knockdown of MIF expression in HeLa cells led to increased cell adhesion and therefore impaired their migratory capacity. More importantly, cells lacking MIF failed to either proliferate in soft agar or form tumours in vivo, when administered to nude mice. Conclusion: MIF plays a pivotal role in proliferation and tumourigenesis of human HeLa cervical carcinoma cells, and may represent a promising therapeutic target for cancer intervention.     

Melatonin Cytotoxicity Is Associated to Warburg Effect Inhibition in Ewing Sarcoma Cells

Melatonin kills or inhibits the proliferation of different cancer cell types, and this is associated with an increase or a decrease in reactive oxygen species, respectively. Intracellular oxidants originate mainly from oxidative metabolism, and cancer cells frequently show alterations in this metabolic pathway, such as the Warburg effect (aerobic glycolysis). Thus, we hypothesized that melatonin could also regulate differentially oxidative metabolism in cells where it is cytotoxic (Ewing sarcoma cells) and in cells where it inhibits proliferation (chondrosarcoma cells). Ewing sarcoma cells but not chondrosarcoma cells showed a metabolic profile consistent with aerobic glycolysis, i.e. increased glucose uptake, LDH activity, lactate production and HIF-1α activation. Melatonin reversed Ewing sarcoma metabolic profile and this effect was associated with its cytotoxicity. The differential regulation of metabolism by melatonin could explain why the hormone is harmless for a wide spectrum of normal and only a few tumoral cells, while it kills specific tumor cell types.     

Real-Time Motion Analysis Reveals Cell Directionality as an Indicator of Breast Cancer Progression

Cancer cells alter their migratory properties during tumor progression to invade surrounding tissues and metastasize to distant sites. However, it remains unclear how migratory behaviors differ between tumor cells of different malignancy and whether these migratory behaviors can be utilized to assess the malignant potential of tumor cells. Here, we analyzed the migratory behaviors of cell lines representing different stages of breast cancer progression using conventional migration assays or time-lapse imaging and particle image velocimetry (PIV) to capture migration dynamics. We find that the number of migrating cells in transwell assays, and the distance and speed of migration in unconstrained 2D assays, show no correlation with malignant potential. However, the directionality of cell motion during 2D migration nicely distinguishes benign and tumorigenic cell lines, with tumorigenic cell lines harboring less directed, more random motion. Furthermore, the migratory behaviors of epithelial sheets observed under basal conditions and in response to stimulation with epidermal growth factor (EGF) or lysophosphatitic acid (LPA) are distinct for each cell line with regard to cell speed, directionality, and spatiotemporal motion patterns. Surprisingly, treatment with LPA promotes a more cohesive, directional sheet movement in lung colony forming MCF10CA1a cells compared to basal conditions or EGF stimulation, implying that the LPA signaling pathway may alter the invasive potential of MCF10CA1a cells. Together, our findings identify cell directionality as a promising indicator for assessing the tumorigenic potential of breast cancer cell lines and show that LPA induces more cohesive motility in a subset of metastatic breast cancer cells.     

ASK-1 (apoptosis signal-regulating kinase 1) is a direct E2F target gene

S1P (sphingosine 1-phosphate) receptor expression and the effects of S1P on migration were studied in one papillary (NPA), two follicular (ML-1, WRO) and two anaplastic (FRO, ARO) thyroid cancer cell lines, as well as in human thyroid cells in primary culture. Additionally, the effects of S1P on proliferation, adhesion and calcium signalling were addressed in ML-1 and FRO cells. All cell types expressed multiple S1P receptors. S1P evoked intracellular calcium signalling in primary cultures, ML-1 cells and FRO cells. Neither proliferation nor migration was affected in primary cultures, whereas S1P partly inhibited proliferation in ML-1 and FRO cells. Low nanomolar concentrations of S1P inhibited migration in FRO, WRO and ARO cells, but stimulated ML-1 cell migration. Consistently, S1P1 and S1P3, which mediate migratory responses, were strongly expressed in ML-1 cells, and S1P2, which inhibits migration, was the dominating receptor in the other cell lines. The migratory effect in ML-1 cells was mediated by G(i) and phosphatidylinositol 3-kinase. Both S1P and the S1P1-specific agonist SEW-2871 induced Akt phosphorylation at Ser473. However, SEW-2871 failed to stimulate migration, whereas the S1P1/S1P3 antagonist VPC 23019 inhibited S1P-induced migration. The results suggest that aberrant S1P receptor expression may enhance thyroid cancer cell migration and thus contribute to the metastatic behaviour of some thyroid tumours.     

Modeling cancer metabolism on a genome scale

Cancer cells have fundamentally altered cellular metabolism that is associated with their tumorigenicity and malignancy. In addition to the widely studied Warburg effect, several new key metabolic alterations in cancer have been established over the last decade, leading to the recognition that altered tumor metabolism is one of the hallmarks of cancer. Deciphering the full scope and functional implications of the dysregulated metabolism in cancer requires both the advancement of a variety of omics measurements and the advancement of computational approaches for the analysis and contextualization of the accumulated data. Encouragingly, while the metabolic network is highly interconnected and complex, it is at the same time probably the best characterized cellular network. Following, this review discusses the challenges that genome‐scale modeling of cancer metabolism has been facing. We survey several recent studies demonstrating the first strides that have been done, testifying to the value of this approach in portraying a network‐level view of the cancer metabolism and in identifying novel drug targets and biomarkers. Finally, we outline a few new steps that may further advance this field.     

Differential role of proline-rich tyrosine kinase 2 and focal adhesion kinase in determining glioblastoma migration and proliferation

The propensity of malignant gliomas to invade surrounding brain tissue contributes to poor clinical outcome. Integrin-mediated adhesion to extracellular matrix regulates the migration and proliferation of many cell types, but its role in glioma progression is undefined. We investigated the role of the cytoplasmic tyrosine kinases FAK and Pyk2, potential integrin effectors, in the phenotypic determination of four different human glioblastoma cell lines. While FAK expression was similar between the four cell lines, increased FAK activity correlated with high proliferation and low migratory rates. In contrast, Pyk2 activity was significantly increased in migratory cell lines and depressed in proliferative cell lines. Overexpression of Pyk2 stimulated migration, whereas FAK overexpression inhibited cell migration and stimulated cellular proliferation. These data suggest that FAK and Pyk2 function as important signaling effectors in gliomas and indicate that their differential regulation may be determining factors in the temporal development of proliferative or migrational phenotypes.     

Glucosamine inhibits IL‐1β‐mediated IL‐8 production in prostate cancer cells by MAPK attenuation

Inflammation is a complex process involving cytokine production to regulate host defense cascades. In contrast to the therapeutic significance of acute inflammation, a pathogenic impact of chronic inflammation on cancer development has been proposed. Upregulation of inflammatory cytokines, such as IL‐1β and IL‐8, has been noted in prostate cancer patients and IL‐8 has been shown to promote prostate cancer cell proliferation and migration; however, it is not clear whether IL‐1β regulates IL‐8 expression in prostate cancer cells. Glucosamine is widely regarded as an anti‐inflammatory agent and thus we hypothesized that if IL‐1β activated IL‐8 production in prostate cancer cells, then glucosamine ought to blunt such an effect. Three prostate cancer cell lines, DU‐145, PC‐3, and LNCaP, were used to evaluate the effects of IL‐1β and glucosamine on IL‐8 expression using ELISA and RT‐PCR analyses. IL‐1β elevated IL‐8 mRNA expression and subsequent IL‐8 secretion. Glucosamine significantly inhibited IL‐1β‐induced IL‐8 secretion. IL‐8 appeared to induce LNCaP cell proliferation by MTT assay; involvement of IL‐8 in IL‐1β‐dependent PC‐3 cell migration was demonstrated by wound‐healing and transwell migration assays. Inhibitors of MAPKs and NFκB were used to pinpoint MAPKs but not NFκB being involved in IL‐1β‐mediated IL‐8 production. IL‐1β‐provoked phosphorylation of all MAPKs was notably suppressed by glucosamine. We suggest that IL‐1β can activate the MAPK pathways resulting in an induction of IL‐8 production, which promotes prostate cancer cell proliferation and migration. In this context, glucosamine appears to inhibit IL‐1β‐mediated activation of MAPKs and therefore reduces IL‐8 production; this, in turn, attenuates cell proliferation/migration. J. Cell. Biochem. 108: 489–498, 2009. © 2009 Wiley‐Liss, Inc.     

Mitochondrial Dysfunction and Permeability Transition in Osteosarcoma Cells Showing the Warburg Effect

Metabolic reprogramming in cancer is manifested by persistent aerobic glycolysis and suppression of mitochondrial function and is known as the Warburg effect. The Warburg effect contributes to cancer progression and is considered to be a promising therapeutic target. Understanding the mechanisms used by cancer cells to suppress their mitochondria may lead to development of new approaches to reverse metabolic reprogramming. We have evaluated mitochondrial function and morphology in poorly respiring LM7 and 143B osteosarcoma (OS) cell lines showing the Warburg effect in comparison with actively respiring Saos2 and HOS OS cells and noncancerous osteoblastic hFOB cells. In LM7 and 143B cells, we detected markers of the mitochondrial permeability transition (MPT), such as mitochondrial swelling, depolarization, and membrane permeabilization. In addition, we detected mitochondrial swelling in human OS xenografts in mice and archival human OS specimens using electron microscopy. The MPT inhibitor sanglifehrin A reversed MPT markers and increased respiration in LM7 and 143B cells. Our data suggest that the MPT may play a role in suppression of mitochondrial function, contributing to the Warburg effect in cancer.     

Phenethyl isothiocyanate potentiates anti‐tumour effect of doxorubicin through Akt‐dependent pathway

The present study aims to investigate the in vivo and in vitro anti‐tumour properties of phenethyl isothiocyanate (PEITC) alone and in combination with doxorubicin (Dox). The anti‐tumour activity was evaluated in vitro by MTT assay using cultured human breast cancer cell line (MCF‐7) and human hepatoma cell line (HepG‐2) cell lines. In vivo, Ehrlich solid tumour model was used. Tumour volume, weight and antioxidant parameters were determined. Immunohistochemistry analysis for active (cleaved) caspase‐3 was also performed. We tested the effect of PEITC treatment on pAkt/Akt ratio, NF‐κB p65 DNA binding activity and caspase‐9 enzyme activity in both MCF‐7 and HepG‐2 cell lines. Effect of PEITC treatment on cell migration was assessed by wound healing assay. PEITC and/or Dox treatment significantly inhibited solid tumour volume and tumour weight when compared with control mice. PEITC treatment significantly reduced oxidative stress caused by Dox treatment as indicated by significant increase in total antioxidant capacity and decrease in malondialdehyde level. Microscopic examination of tumour tissues showed a significant increase in active (cleaved) caspase‐3 expression in PEITC and/or Dox treated groups. PEITC showed a dose‐dependent inhibition of MCF‐7 and HepG‐2 cellular viability. PEITC inhibited Akt and NF‐κB activation and increased caspase‐9 activity in a dose‐dependent manner. PEITC treatment effectively inhibited both MCF‐7 and HepG‐2 cell migration. We can conclude that PEITC acts via multiple molecular targets to elicit anti‐carcinogenic activity. PEITC/Dox combination therapy might be a potential novel strategy, which may benefit patients with breast and liver cancers. Copyright © 2015 John Wiley & Sons, Ltd.