The cancer cell’s “power plants” as promising therapeutic targets: An overview

This introductory article to the review series entitled “The Cancer Cell’s Power Plants as Promising Therapeutic Targets” is written while more than 20 million people suffer from cancer. It summarizes strategies to destroy or prevent cancers by targeting their energy production factories, i.e., “power plants.” All nucleated animal/human cells have two types of power plants, i.e., systems that make the “high energy” compound ATP from ADP and P _i . One type is “glycolysis,” the other the “mitochondria.” In contrast to most normal cells where the mitochondria are the major ATP producers (>90%) in fueling growth, human cancers detected via Positron Emission Tomography (PET) rely on both types of power plants. In such cancers, glycolysis may contribute nearly half the ATP even in the presence of oxygen (“Warburg effect”). Based solely on cell energetics, this presents a challenge to identify curative agents that destroy only cancer cells as they must destroy both of their power plants causing “necrotic cell death” and leave normal cells alone. One such agent, 3-bromopyruvate (3-BrPA), a lactic acid analog, has been shown to inhibit both glycolytic and mitochondrial ATP production in rapidly growing cancers (Ko et al., Cancer Letts., 173, 83–91, 2001), leave normal cells alone, and eradicate advanced cancers (19 of 19) in a rodent model (Ko et al., Biochem. Biophys. Res. Commun., 324, 269–275, 2004). A second approach is to induce only cancer cells to undergo “apoptotic cell death.” Here, mitochondria release cell death inducing factors (e.g., cytochrome c). In a third approach, cancer cells are induced to die by both apoptotic and necrotic events. In summary, much effort is being focused on identifying agents that induce “necrotic,” “apoptotic” or apoptotic plus necrotic cell death only in cancer cells. Regardless how death is inflicted, every cancer cell must die, be it fast or slow.     

New insights into autophagic cell death in the gypsy moth Lymantria dispar: a proteomic approach

Autophagy is an evolutionary ancient process based on the activity of genes conserved from yeast to metazoan taxa. Whereas its role as a mechanism to provide energy during cell starvation is commonly accepted, debate continues about the occurrence of autophagy as a means specifically activated to achieve cell death. The IPLB-LdFB insect cell line, derived from the larval fat body of the lepidoptera Lymantria dispar, represents a suitable model to address this question, as both autophagic and apoptotic cell death can be induced by various stimuli. Using morphological and functional approaches, we have observed that the culture medium conditioned by IPLB-LdFB cells committed to death by the ATPase inhibitor oligomycin A stimulates autophagic cell death in untreated IPLB-LdFB cells. Moreover, proteomic analysis of the conditioned media suggests that, in IPLB-LdFB cells, oligomycin A promotes a shift towards lipid metabolism, increases oxidative stress and specifically directs the cells towards autophagic activity. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by an F.A.R. grant from the University of Modena and Reggio Emilia (D.M. and E.O.) and by an “Experimental approaches to the study of evolution” grant from the Department of Animal Biology of the University of Modena and Reggio Emilia (D.M.).     

The metabolism of proline,a stress substrate,modulates carcinogenic pathways

The resurgence of interest in tumor metabolism has led investigators to emphasize the metabolism of proline as a “stress substrate” and to suggest this pathway as a potential anti-tumor target. Proline oxidase, a.k.a. proline dehydrogenase (POX/PRODH), catalyzes the first step in proline degradation and uses proline to generate ATP for survival or reactive oxygen species for programmed cell death. POX/PRODH is induced by p53 under genotoxic stress and initiates apoptosis by both mitochondrial and death receptor pathways. Furthermore, POX/PRODH is induced by PPARγ and its pharmacologic ligands, the thiazolidinediones. The anti-tumor effects of PPARγ may be critically dependent on POX/PRODH. In addition, it is upregulated by nutrient stress through the mTOR pathway to maintain ATP levels. We propose that proline is made available as a stress substrate by the degradation of collagen in the microenvironmental extracellular matrix by matrix metalloproteinases. In a manner analogous to autophagy, this proline-dependent process for bioenergetics from collagen in extracellular matrix can be designated “ecophagy”.     

Immunohistochemical characterisation of differentiated CAD cells: expression of peptides and chromogranins

The CNS-derived cell line, CAD cell line, when grown in a protein free medium (PFM), differentiates to neuron-like cells with very long processes. It was previously studied biochemically and found to express TH activity, some neurospecific proteins, but no glial proteins. We have now further studied the CAD cells and focused on the expression of various neuropeptides, GAP-43 and GFAP. All peptides studied were present, including TH, but also GFAP, in contrast to earlier studies. A different kind of processes, short, slender and distributed like a “fringe” around cell body and along processes was observed, NESP55 but not other chromogranins was present in these “fringes”, GAP43 showed some degree of overlapping with NESP55. The results show that even after differentiation in PFM, the CAD cells express a palette of neuropeptides and chromogranins, catecholaminergic markers as well as the glia-specific GFAP. Our efforts to induce exocytosis/endocytosis from the peptide granules by high K⁺ were, however, unsuccessful. Due to long processes, the CAD cells may represent a good model for studying intracellular transport, and, since the cells express both neuronal and glial characteristics, it may be useful for investigating the influence of different trophic/growth factors on the expression of various neuronal characteristics.     

Autophagy–physiology and pathophysiology

“Autophagy” is a highly conserved pathway for degradation, by which wasted intracellular macromolecules are delivered to lysosomes, where they are degraded into biologically active monomers such as amino acids that are subsequently re-used to maintain cellular metabolic turnover and homeostasis. Recent genetic studies have shown that mice lacking an autophagy-related gene (Atg5 or Atg7) cannot survive longer than 12 h after birth because of nutrient shortage. Moreover, tissue-specific impairment of autophagy in central nervous system tissue causes massive loss of neurons, resulting in neurodegeneration, while impaired autophagy in liver tissue causes accumulation of wasted organelles, leading to hepatomegaly. Although autophagy generally prevents cell death, our recent study using conditional Atg7-deficient mice in CNS tissue has demonstrated the presence of autophagic neuron death in the hippocampus after neonatal hypoxic/ischemic brain injury. Thus, recent genetic studies have shown that autophagy is involved in various cellular functions. In this review, we introduce physiological and pathophysiological roles of autophagy.     

Raloxifene Induces Autophagy-Dependent Cell Death in Breast Cancer Cells via the Activation of AMP-Activated Protein Kinase

Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells.Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death.Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.     

An ultrasoft X-ray multi-microbeam irradiation system for studies of DNA damage responses by fixed- and live-cell fluorescence microscopy

Localized induction of DNA damage is a valuable tool for studying cellular DNA damage responses. In recent decades, methods have been developed to generate DNA damage using radiation of various types, including photons and charged particles. Here we describe a simple ultrasoft X-ray multi-microbeam system for high dose-rate, localized induction of DNA strand breaks in cells at spatially and geometrically adjustable sites. Our system can be combined with fixed- and live-cell microscopy to study responses of cells to DNA damage. This article has been submitted as a contribution to the Festschrift entitled “Uncovering cellular sub-structures by light microscopy” in honour of Professor Cremer’s 65th birthday. C. van Oven and P. M. Krawczyk contributed equally to this work.     

Mitochondrial matters of the heart: a plethora of regulatory modes to maintain function for a long lifetime

The human/animal heart, comprised of cells called “myocytes” is an incredible organ that to remain beating must be fueled constantly via the hydrolysis of adenosine tri-phosphate (ATP). Deriving most of its ATP from mitochondrial oxidative phosphorylation (ox phos), and a smaller amount from “glycolysis”, i.e., glucose conversion to pyruvate or lactate, the heart helps in the delivery of oxygen (via hemoglobin) to every organ/tissue in our body. Then, the empty (deoxy) hemoglobin returns to load more oxygen and the journey to tissues is repeated 24 h a day, year after year, until “death do us part”. To support this essential “pumping” process the heart must work constantly, i.e., 70–80 years (life expectancy in the U.S.). This is a remarkable feat when compared with one of our most costly people-made technologies, i.e., automobiles (cars). In the past century, it was rare to see the family car survive more than 10–15 years unless it had been subjected to motor replacement surgery. Most were laid to rest at a much earlier age. Now, in this new millennium should a brilliant car manufacturer succeed in constructing a car engine as efficient as the human heart, each family member requiring a car would need only one per life time. With this in mind, one of the major future “matters of the heart” is to keep it pumping, not only for the current 70–80 year life span but much longer. To do this depends on, among other matters, the two processes noted above, i.e., oxidative phosphorylation and glycolysis. The former is strictly a mitochondrial process that works only in the presence of oxygen whereas glycolysis, dependent on mitochondrial bound hexokinase 2 (MB-HK-2), works either in the presence or absence of oxygen. In addition, the MB-HK 2 is anti-apoptotic and helps with other factors to retard cell death. Current estimates reveal that the human heart of an individual living 70–80 years will have undergone 2.5–3.0 billion beats, a feat that is energetically feasible only due to the heart cells’ (cardiomyocytes) large population of mitochondria with bound HK-2.     

Characterization of the KG1a cell line for use in a cell migration based screening assay

High-throughput screening has become a popular method used to identify new “leads” for potentially therapeutic compounds. Further screening of these lead compounds is typically done with secondary assays which may utilize living, functioning cells as screening tools. A problem (or benefit) with these cell-based assays is that living cells are very sensitive to their environment. We have been interested in the process of stem cell migration and how it relates to the cellular therapy of bone marrow transplantation. In this study we describe a secondary, cell-based assay for screening the effects of variousin-vitro conditions on Immature Hematopoietic Cell (IHC) migration. Our results have revealed many subtle factors, such as the cell's adhesive characteristics, or the effect of a culture's growth phase, that need to be accounted for in a screening protocol. Finally, we show that exponentially growing KG1a cells (a human IHC cell line) were 10 times more motile than those in the lag or stationary phases. These data strongly suggest that KG1a cells secrete a chemokinetic factor during the exponential growth phase of a culture.     

Lead toxicity induces autophagy to protect against cell death through mTORC1 pathway in cardiofibroblasts

Heavy metals, such as lead (Pb²⁺), are usually accumulated in human bodies and impair human''s health. Lead is a metal with many recognized adverse health side effects and yet the molecular processes underlying lead toxicity are still poorly understood. In the present study, we proposed to investigate the effects of lead toxicity in cultured cardiofibroblasts. After lead treatment, cultured cardiofibroblasts showed severe endoplasmic reticulum (ER) stress. However, the lead-treated cardiofibroblasts were not dramatically apoptotic. Further, we found that these cells determined to undergo autophagy through inhibiting mammalian target of rapamycin complex 1 (mTORC1) pathway. Moreover, inhibition of autophagy by 3-methyladenine (3-MA) may dramatically enhance lead toxicity in cardiofibroblasts and cause cell death. Our data establish that lead toxicity induces cell stress in cardiofibroblasts and protective autophagy is activated by inhibition of mTORC1 pathway. These findings describe a mechanism by which lead toxicity may promote the autophagy of cardiofibroblasts cells, which protects cells from cell stress. Our findings provide evidence that autophagy may help cells to survive under ER stress conditions in cardiofibroblasts and may set up an effective therapeutic strategy for heavy metal toxicity.     

Question 9: Prospects for the Construction of Artificial Cells or Protocells

The construction of artificial cells or protocells that are a simplified version of contemporary cells will have implications for both the understanding of the origins of cellular Life and the design of “cell-like” chemical factories. In this short communication, we discuss the progress and remaining issues related to the construction of protocells from metabolic products. We further outline the de novo design of a simple chemical system that mimics the functional properties of a living cell without being composed of molecules of biological origin, thereby addressing issues related to Life’s origins. Presented at: International School of Complexity—4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Oligomycin A and the IPLB-LdFB insect cell line: actin and mitochondrial responses

Oligomycin A, an inhibitor of mitochondrial ATP synthase, provokes simultaneous and different responses in IPLB-LdFB insect cell line. The oligomycin A treatment causes mitochondrial loss, increase in reactive oxygen species (ROS), destabilization/reorganization of the actin microfilaments and, finally, autophagic cell death. We speculate that oligomycin A affects the mitochondria and that the impairment of these organelles leads to the generation of ROS in quantities that exceed the antioxidant capacity of the cell. This in turn would lead to a feedback loop of increased mitochondrial impairment, amplification of ROS production and the removal of damaged organelles through autophagy.     

Scanning electron microscopy ofMycoplasma pneumoniae on the membrane of individual ciliated tracheal cells

Summary Cell monolayer cultures were prepared from hamster tracheal explants by a collagenase exposure and subsequent incubation in Waymouth’s MAB 87/3 medium. The epithelial outgrowth occurred on glass cover slips. Cilia on the monolayers continued to beat normally after the “parent” explant was removed. Monolayer cultures infected withMycoplasma pneumoniae had significant amounts of attachment. A morphological analysis of the attachment was conducted with scanning electron microscopy. Clusters, cocci, and filaments ofM. pneumoniae all attached to the epithelial cells, but the filaments were especially common. Mycoplasmas were seen in association with both ciliated and nonciliated cell membranes. On ciliated cells, mycoplasmas were on the ciliary strands and on the cell membrane. When located immediately adjacent to or in between cilia, mycoplasmas were oriented vertically with the constricted attachment tip oriented down toward the host cell membrane. When located more than a micron away from the ciliary fibers, mycoplasmas lay horizontally along the epithelial cell membrane. The photographic data suggest that clusters or “sperules” of mycoplasmas may liberate individual mycoplasmas that attach to the cell membrane. It appears that the receptor sites forM. pneumoniae are rather uniformly distributed along the ciliated cell membrane, and are not restricted to the interciliary areas. Electron microscopy was done with the cooperation of Dr. R. Macleod and the staff of the Center for Electron Microscopy at the University of Illinois. Critical editorial review was provided by C. Dayton. This investigation was supported in part by grants to M. G. G. from the National Institute of Allergy and Infectious Diseases (AI 12559) and the National Heart, Lung, and Blood Institute (HL 23806), Bethesda, Maryland.     

Recent ostracods (Crustacea,Ostracoda) found in lowland springs of the provinces of Piacenza and Parma (Northern Italy)

The “fontanili” are artificial aquatic ecosystems, typical of the lowland plains of Northern Italy, exploiting natural resurgences of deep groundwater. These habitats are characterized by low variation in hydrologic, hydrochemical and thermal conditions throughout the year. Proper management is required to prevent the spring clogging by biomass accumulation. In spite of their importance as refugia for endangered species, many springs were completely abandoned in the last years and several of them will disappear. We report the results of a study carried out in 2001 on 31 springs of the provinces of Piacenza and Parma, distributed in seven areas defined on hydrological and geological considerations. Physical and chemical variables and parameters of waters were measured and ostracod samples were collected. Most of the springs showed high nitrate concentration, due to a diffuse pollution of agricultural origin. Twelve ostracod species in four families were identified. Ostracod valves were analysed by scanning electron microscopy. Cypria ophtalmica was found in all the springs; other relatively common species were Cyclocypris laevis,Notodromas persica, and Prionocypris zenkeri.The maximum number of species per site was four. Cypridopsis vidua,P. zenkeri, and N. persicashowed a very localized distribution in the study area. The ostracod fauna of the “fontanili” was compared to other species assemblages found in spring habitats and to the available information on recent freshwater ostracods reported for Italy.     

AMPK mediates a pro-survival autophagy downstream of PARP-1 activation in response to DNA alkylating agents

In this study we aim to elucidate the signaling pathway and biological function of autophagy induced by MNNG, a commonly used DNA alkylating agent. We first observed that MNNG is able to induce necrotic cell death and autophagy in Bax?/? Bak?/? double knockout MEFs. We analyzed the critical role of PARP-1 activation and ATP depletion in MNNG-mediated cell death and autophagy via AMPK activation and mTOR suppression. We provide evidence that suppression of AMPK blocks MNNG-induced autophagy and enhances cell death, suggesting the pro-survival function of autophagy in MNNG-treated cells. Taken together, data from this study reveal a novel mechanism in controlling MNNG-mediated autophagy via AMPK activation downstream of PARP-1 activation and ATP depletion.     

Subpopulation of nestin positive glial precursor cells occur in primary adult human brain cultures

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein considered to be the best astroglial marker. However, the predominant cell population in adult human brain tissue cultures does not express GFAP; these cells have been termed “glia-like” cells. The basic question about histological origin of adult human brain cultures remains unanswered. Some authors showed that “glia-like” cells in adult human brain cultures might be of non-glial origin. We examined primary explant tissue cultures derived from 70 adult human brain biopsies. Within first 5–10 days approximately 5–10% of the small explants became attached. Outgrowing cells were mostly flat cells. These cells formed confluent layer over 3–6 weeks in culture. At confluence the cultures contained 2–5% of microglial cells, 0.1% GFAP-positive astrocytes, less than 0.01% oligodendrocytes and 95–98% GFAP-negative “glia-like” cells. This population of flat “glia-like” cells was positively stained for vimentin, fibronectin, and 20–30% of these cells stained for nestin. Our findings revealed that 1 mM dibutyryl-cAMP addition, in serum free conditions, induced a reversible stellation in 5-10% of the flat “glia-like” cells but did not induce the expression of GFAP or nestin in morphologically changed stellate cells. These results demonstrate that “glia-like” cells in primary adult human brain cultures constitute heterogeneous cell populations albeit with similar morphological features. Two distinct subpopulations have been shown: (i) the one immunostained for nestin; and (ii) the other reactive for dibutyryl-cAMP treatment.     

The role of the mitochondria in mediating cytotoxicity of anti-cancer therapies

Optimal cytotoxic anticancer therapy, at the cellular level, requires effective and selective induction of cell death to achieve a net reduction of biomass of malignant tissues. Standard cytotoxic chemotherapeutics have been developed based on the observations that mitotically active cancer cells are more susceptible than quiescent normal cells to chromosomal, microtubular or metabolic poisons. More recent development of molecularly targeted drugs for cancer focuses on exploiting biological differentials between normal and transformed cells for selective eradication of cancers. The common thread of “standard” and “novel” cytotoxic drugs is their ability to activate the apoptosis-inducing machinery mediated by mitochondria, also known as the intrinsic death signaling cascade. The aim of this article is to provide an overview of the role of the mitochondria, an energy-generating organelle essential for life, in mediating death when properly activated by cytotoxic stresses.     

Soluble HLA/peptide monomers cross-linked with co-stimulatory antibodies onto a streptavidin core molecule efficiently stimulate antigen-specific T cell responses

Soluble MHC–peptide complexes, commonly referred to as tetramers, have been shown to induce strong cross-linking of TCR and CD8, resulting in a vigorous activation followed by a rapid non-apoptotic CD8⁺ T cell death. This has limited tetramer use for antigen-specific T cells isolation and cloning, as sorted tetramer positive cells were shown to possess compromised functional integrity. Here we show that the cross-linking of a secondary co-stimulatory signal into oligomeric MHC:peptide complexes prevents such cell death, and in contrast strongly stimulates antigen-specific T cell responses. Such soluble antigen-presenting complexes (sAPCs) containing MHC:peptide complexes linked to either anti-CD27 or anti-CD28 antibodies were capable of priming and expanding HLA-A*0201 restricted CMV specific T cells and also of generating functional HLA-A*0301 restricted BCR/ABL-specific T cell responses. These sAPCs constitute an encouraging alternative method for generating antigen-specific T cells that could be applied to a variety of antigens. A. I. Dodi and P. J. Travers contributed equally to this work. This paper is an original contribution from the meeting which took place on 28 and 29 May 2008 in Nottingham, UK, celebrating the contribution of Prof. I. A. “Tony” Dodi (^†29.1.2008) to the EU project “Network for the identification and validation of antigens and biomarkers in cancer and their application in clinical tumour immunology (ENACT)”.