Diverse memory paradigms in Drosophila reveal diverse neural mechanisms.

In this review, we aggregated the different types of learning and memory paradigms developed in adult Drosophila and attempted to assess the similarities and differences in the neural mechanisms supporting diverse types of memory. The simplest association memory assays are conditioning paradigms (olfactory, visual, and gustatory). A great deal of work has been done on these memories, revealing hundreds of genes and neural circuits supporting this memory. Variations of conditioning assays (reversal learning, trace conditioning, latent inhibition, and extinction) also reveal interesting memory mechanisms, whereas mechanisms supporting spatial memory (thermal maze, orientation memory, and heat box) and the conditioned suppression of innate behaviors (phototaxis, negative geotaxis, anemotaxis, and locomotion) remain largely unexplored. In recent years, there has been an increased interest in multisensory and multicomponent memories (context-dependent and cross-modal memory) and higher-order memory (sensory preconditioning and second-order conditioning). Some of this work has revealed how the intricate mushroom body (MB) neural circuitry can support more complex memories. Finally, the most complex memories are arguably those involving social memory: courtship conditioning and social learning (mate-copying and egg-laying behaviors). Currently, very little is known about the mechanisms supporting social memories. Overall, the MBs are important for association memories of multiple sensory modalities and multisensory integration, whereas the central complex is important for place, orientation, and navigation memories. Interestingly, several different types of memory appear to use similar or variants of the olfactory conditioning neural circuitry, which are repurposed in different ways.

Attenuation of photosynthesis in nanosilver-treated Arabidopsis thaliana is inherently linked to the particulate nature of silver.

Silver nanoparticles (AgNPs) are known to affect the physiology and morphology of plants in various ways, but the exact mechanism by which they interact with plant cells remains to be elucidated. An unresolved question of silver nanotoxicology is whether the interaction is triggered by the physical features of the particles, or by silver ions leached from their surface. In this study, we germinated and grew Arabidopsis thaliana seedlings in synthetic medium supplemented with sub-morbid concentrations (4 µg/mL) of AgNPs and silver nitrate (AgNO3). This treatment led to in planta accumulation of 106 µg/g and 97 µg/g of silver in the AgNO3- and AgNP-exposed seedlings, respectively. Despite the statistically indistinguishable silver accumulation, RNA sequencing data demonstrated distinct changes in the transcriptome of the AgNP-exposed, but not in the AgNO3-exposed plants. AgNP exposure induced changes in the expression of genes involved in immune response, cell wall organization, photosynthesis and cellular defense against reactive oxygen species. AgNO3 exposure, on the other hand, caused the differential expression of only two genes, neither of which belonged to any AgNP-enriched gene ontology categories. Moreover, AgNP exposure led to a 39% reduction (p < 0.001) in total chlorophyll concentration relative to untreated plants which was associated with a 56.9% and 56.2% drop (p < 0.05) in carbon assimilation rate at ambient and saturating light, respectively. Stomatal conductance was not significantly affected by AgNP exposure, and limitations to carbon assimilation, as determined through analysis of light and carbon dioxide (A/Ci) curves, were attributed to rates of electron transport, maximum carboxylation rates and triose phosphate use. AgNO3-exposure, on the other hand, did not lead to significant reduction either in chlorophyll concentration or in carbon assimilation rate. Given these data, we propose that the impact of AgNPs cannot be simply attributed to the presence of the metal in plants, but is innate to the particulate nature of nanosilver.

Active forgetting and neuropsychiatric diseases.

Recent and pioneering animal research has revealed the brain utilizes a variety of molecular, cellular, and network-level mechanisms used to forget memories in a process referred to as "active forgetting". Active forgetting increases behavioral flexibility and removes irrelevant information. Individuals with impaired active forgetting mechanisms can experience intrusive memories, distressing thoughts, and unwanted impulses that occur in neuropsychiatric diseases. The current evidence indicates that active forgetting mechanisms degrade, or mask, molecular and cellular memory traces created in synaptic connections of "engram cells" that are specific for a given memory. Combined molecular genetic/behavioral studies using Drosophila have uncovered a complex system of cellular active-forgetting pathways within engram cells that is regulated by dopamine neurons and involves dopamine-nitric oxide co-transmission and reception, endoplasmic reticulum Ca2+ signaling, and cytoskeletal remodeling machinery regulated by small GTPases. Some of these molecular cellular mechanisms have already been found to be conserved in mammals. Interestingly, some pathways independently regulate forgetting of distinct memory types and temporal phases, suggesting a multi-layering organization of forgetting systems. In mammals, active forgetting also involves modulation of memory trace synaptic strength by altering AMPA receptor trafficking. Furthermore, active-forgetting employs network level mechanisms wherein non-engram neurons, newly born-engram neurons, and glial cells regulate engram synapses in a state and experience dependent manner. Remarkably, there is evidence for potential coordination between the network and cellular level forgetting mechanisms. Finally, subjects with several neuropsychiatric diseases have been tested and shown to be impaired in active forgetting. Insights obtained from research on active forgetting in animal models will continue to enrich our understanding of the brain dysfunctions that occur in neuropsychiatric diseases.

Two Fifth-Generation Fighter Pilots Discovered with Latent Autoimmune Diabetes.

BACKGROUND: Fighter pilots undergo extensive medical screening but may still miss rare diseases like latent autoimmune diabetes in adults (LADA). LADA patients have circulating autoantibodies directed against pancreatic beta cell antigens and present with frank diabetes late in life which may elude conventional military flight screening.CASE REPORT: Two fifth-generation fighter pilots, a 38-yr-old man (patient 1) and a 27-yr-old man (patient 2), with no significant past medical histories developed symptoms of fatigue, weight loss, episodic polyuria, and arthralgia. Patient 1's symptoms were initially thought to have been caused by COVID-19, but he subsequently tested negative for viral infection. Lab work instead showed elevated TSH, HgbA1C 11.4%, positive GAD-65, anti-TPO, and anti-islet cell antibodies. Patient 2 developed symptoms following a military deployment and a 72-h diarrheal illness. Due to flight status, patient 2 did not seek expert medical attention for several months, but lab work found HgbA1C of 10.4%, positive GAD-66, and ZnT8 antibodies. Both patients were started on insulin therapy. Patient 1 was also started on levothyroxine for hypothyroidism and retired from flying duties. Patient 2 eventually transitioned to metformin without insulin and returned to flying duties with an aeromedical waiver.DISCUSSION: Our patients maintained peak physical fitness throughout their selection and aviation careers, which likely delayed their clinical presentation. Current USAF flight rules prohibit insulin use with flying fighter aircraft. Early antibody screening during pilot selection may be a cost-effective means of diagnosis as traditional screening techniques are unlikely to detect LADA.Zhang JX, Berry J, Kim NM, Gray JJ, Fotheringham S, Sauerwein TJ. Two fifth-generation fighter pilots discovered with latent autoimmune diabetes. Aerosp Med Hum Perform. 2022; 93(2):106-110.

Dopamine-based mechanism for transient forgetting.

Active forgetting is an essential component of the memory management system of the brain1. Forgetting can be permanent, in which prior memory is lost completely, or transient, in which memory exists in a temporary state of impaired retrieval. Temporary blocks on memory seem to be universal, and can disrupt an individual's plans, social interactions and ability to make rapid, flexible and appropriate choices. However, the neurobiological mechanisms that cause transient forgetting are unknown. Here we identify a single dopamine neuron in Drosophila that mediates the memory suppression that results in transient forgetting. Artificially activating this neuron did not abolish the expression of long-term memory. Instead, it briefly suppressed memory retrieval, with the memory becoming accessible again over time. The dopamine neuron modulates memory retrieval by stimulating a unique dopamine receptor that is expressed in a restricted physical compartment of the axons of mushroom body neurons. This mechanism for transient forgetting is triggered by the presentation of interfering stimuli immediately before retrieval.

Rac1 Impairs Forgetting-Induced Cellular Plasticity in Mushroom Body Output Neurons.

Active memory forgetting is a well-regulated biological process thought to be adaptive and to allow proper cognitive functions. Recent efforts have elucidated several molecular players involved in the regulation of olfactory forgetting in Drosophila, including the small G protein Rac1, the dopamine receptor DAMB, and the scaffold protein Scribble. Similarly, we recently reported that dopaminergic neurons mediate both learning- and forgetting-induced plasticity in the mushroom body output neuron MBON-γ2α'1. Two open questions remain: how does forgetting affect plasticity in other, highly plastic, mushroom body compartments and how do genes that regulate forgetting affect this cellular plasticity? Here, we show that forgetting reverses short-term synaptic depression induced by aversive conditioning in the highly plastic mushroom body output neuron MBON-γ1pedc>α/ß. In addition, our results indicate that genetic tampering with normal forgetting by inhibition of small G protein Rac1 impairs restoration of depressed odor responses to learned odor by intrinsic forgetting through time passing and forgetting induced acutely by shock stimulation after conditioning or reversal learning. These data further indicate that some forms of forgetting truly erase physiological changes generated by memory encoding.

Stromalin Constrains Memory Acquisition by Developmentally Limiting Synaptic Vesicle Pool Size.

Stromalin, a cohesin complex protein, was recently identified as a novel memory suppressor gene, but its mechanism remained unknown. Here, we show that Stromalin functions as a negative regulator of synaptic vesicle (SV) pool size in Drosophila neurons. Stromalin knockdown in dopamine neurons during a critical developmental period enhances learning and increases SV pool size without altering the number of dopamine neurons, their axons, or synapses. The developmental effect of Stromalin knockdown persists into adulthood, leading to strengthened synaptic connections and enhanced olfactory memory acquisition in adult flies. Correcting the SV content in dopamine neuron axon terminals by impairing anterograde SV trafficking motor protein Unc104/KIF1A rescues the enhanced-learning phenotype in Stromalin knockdown flies. Our results identify a new mechanism for memory suppression and reveal that the size of the SV pool is controlled genetically and independent from other aspects of neuron structure and function through Stromalin.

Dopamine Neurons Mediate Learning and Forgetting through Bidirectional Modulation of a Memory Trace.

It remains unclear how memory engrams are altered by experience, such as new learning, to cause forgetting. Here, we report that short-term aversive memory in Drosophila is encoded by and retrieved from the mushroom body output neuron MBOn-γ2α'1. Pairing an odor with aversive electric shock creates a robust depression in the calcium response of MBOn-γ2α'1 and increases avoidance to the paired odor. Electric shock after learning, which activates the cognate dopamine neuron DAn-γ2α'1, restores the response properties of MBOn-γ2α'1 and causes behavioral forgetting. Conditioning with a second odor restores the responses of MBOn-γ2α'1 to a previously learned odor while depressing responses to the newly learned odor, showing that learning and forgetting can occur simultaneously. Moreover, optogenetic activation of DAn-γ2α'1 is sufficient for the bidirectional modulation of MBOn-γ2α'1 response properties. Thus, a single DAn can drive both learning and forgetting by bidirectionally modulating a cellular memory trace.

Nonalcoholic fatty liver disease as a sentinel marker for the development of diabetes mellitus in non-obese subjects.

BACKGROUND: Non alcoholic fatty liver disease (NAFLD) is associated with substantial cardiometabolic morbidity. AIMS: We evaluated the long-term extrahepatic complications of NAFLD and sought to evaluate NAFLD in non-obese subjects. METHODS: A total of 2920 participants were retrospectively selected from a health check-up center in 2000, and followed through to December 2010. NAFLD was diagnosed using ultrasonography. Subjects were stratified according to body mass index, NAFLD, and metabolic syndrome. RESULTS: The prevalence of non-obese NAFLD subjects and metabolically unhealthy non-obese subjects was 14.4% and 8.7%, respectively. In the multivariate analysis, non-obese NAFLD subjects had a significantly higher risk for diabetes mellitus (DM; HR 2.69, 95% CI 1.72-4.20, P < 0.001); no increase was observed for hypertension or cardiovascular disease. Metabolically unhealthy non-obese subjects had a significantly higher risk for hypertension (HR 2.75, 95% CI 2.02-3.74, P < 0.001), DM (HR 5.72, 95% CI 3.68-8.89, P < 0.001), and cardiovascular disease (HR 2.93, 95% CI 1.53-5.63, P = 0.001). Subgroup analysis of non-obese subjects showed that NAFLD, without metabolic syndrome, conferred a higher risk for DM (HR 3.60, 95% CI 2.03-6.39, P < 0.001). CONCLUSION: Non-obese subjects with NAFLD are at a higher risk for DM independent of metabolic syndrome.

Dopamine Receptor DAMB Signals via Gq to Mediate Forgetting in Drosophila.

Prior studies have shown that aversive olfactory memory is acquired by dopamine acting on a specific receptor, dDA1, expressed by mushroom body neurons. Active forgetting is mediated by dopamine acting on another receptor, Damb, expressed by the same neurons. Surprisingly, prior studies have shown that both receptors stimulate cyclic AMP (cAMP) accumulation, presenting an enigma of how mushroom body neurons distinguish between acquisition and forgetting signals. Here, we surveyed the spectrum of G protein coupling of dDA1 and Damb, and we confirmed that both receptors can couple to Gs to stimulate cAMP synthesis. However, the Damb receptor uniquely activates Gq to mobilize Ca2+ signaling with greater efficiency and dopamine sensitivity. The knockdown of Gαq with RNAi in the mushroom bodies inhibits forgetting but has no effect on acquisition. Our findings identify a Damb/Gq-signaling pathway that stimulates forgetting and resolves the opposing effects of dopamine on acquisition and forgetting.

Explosive Decompression with Resultant Air Gas Embolism in a Fourth Generation Fighter at Ground Level.

BACKGROUND: Arterial gas embolism (AGE) is a rare condition in the flying community most often only ever seen in flight while operating at high altitude or incidents involving hypobaric chambers. This article describes a severe case of AGE that occurred in a pilot of a fourth generation fighter aircraft at ground level. The environmental control system (ECS) malfunctioned, causing an overpressurized cockpit and a subsequent explosive decompression when the pilot opened the canopy to egress. CASE REPORT: The ECS onboard fourth generation fighter aircraft is composed of many computer-controlled subsystems. When these components fail, the system can potentially overpressurize the cockpit. Combined with opening the canopy without prior venting, this overpressurization can lead to a situation akin to a diver surfacing too quickly. A pilot experienced this scenario and subsequently developed symptoms of arterial gas embolization-one form of decompression illness (DCI). We reviewed the design of the environmental control system and recommend that the cockpit must be slowly depressurized to decrease risk of injury from rapid decompression. DISCUSSION: Literature review showed three similar cases of ground-based overpressurization causing AGE symptoms, although these cases were maintenance personnel intentionally testing aircraft cabin integrity and not associated with aircraft intending flight.7 The lessons learned from this case can be used to identify and hopefully prevent severe DCI from ground level cockpit overpressurization and to further general understanding of aircraft ECS. Zhang JX, Berry JR, Beckstrand DP. Explosive decompression with resultant air gas embolism in a fourth generation fighter at ground level. Aerosp Med Hum Perform. 2016; 87(11):963-967.

Sleep Facilitates Memory by Blocking Dopamine Neuron-Mediated Forgetting.

Early studies from psychology suggest that sleep facilitates memory retention by stopping ongoing retroactive interference caused by mental activity or external sensory stimuli. Neuroscience research with animal models, on the other hand, suggests that sleep facilitates retention by enhancing memory consolidation. Recently, in Drosophila, the ongoing activity of specific dopamine neurons was shown to regulate the forgetting of olfactory memories. Here, we show this ongoing dopaminergic activity is modulated with behavioral state, increasing robustly with locomotor activity and decreasing with rest. Increasing sleep-drive, with either the sleep-promoting agent Gaboxadol or by genetic stimulation of the neural circuit for sleep, decreases ongoing dopaminergic activity, while enhancing memory retention. Conversely, increasing arousal stimulates ongoing dopaminergic activity and accelerates dopaminergic-based forgetting. Therefore, forgetting is regulated by the behavioral state modulation of dopaminergic-based plasticity. Our findings integrate psychological and neuroscience research on sleep and forgetting.

Active forgetting of olfactory memories in Drosophila.

Failure to remember, or forgetting, is a phenomenon familiar to everyone and despite more than a century of scientific inquiry, why we forget what we once knew remains unclear. If the brain marshals significant resources to form and store memories, why is it that these memories become lost? In the last century, psychological studies have divided forgetting into decay theory, in which memory simply dissipates with time, and interference theory, in which additional learning or mental activity hinders memory by reducing its stability or retrieval (for review, Dewar et al., 2007; Wixted, 2004). Importantly, these psychological models of forgetting posit that forgetting is a passive property of the brain and thus a failure of the brain to retain memories. However, recent neuroscience research on olfactory memory in Drosophila has offered evidence for an alternative conclusion that forgetting is an "active" process, with specific, biologically regulated mechanisms that remove existing memories (Berry et al., 2012; Shuai et al., 2010). Similar to the bidirectional regulation of cell number by mitosis and apoptosis, protein concentration by translation and lysosomal or proteomal degradation, and protein phosphate modification by kinases and phosphatases, biologically regulated memory formation and removal would be yet another example in biological systems where distinct and separate pathways regulate the creation and destruction of biological substrates.

System-like consolidation of olfactory memories in Drosophila.

System consolidation, as opposed to cellular consolidation, is defined as the relatively slow process of reorganizing the brain circuits that maintain long-term memory. This concept is founded in part on observations made in mammals that recently formed memories become progressively independent of brain regions initially involved in their acquisition and retrieval and dependent on other brain regions for their long-term storage. Here we present evidence that olfactory appetitive and aversive memories in Drosophila evolve using a system-like consolidation process. We show that all three classes of mushroom body neurons (MBNs) are involved in the retrieval of short- and intermediate-term memory. With the passage of time, memory retrieval becomes independent of α'/ß' and γ MBNs, and long-term memory becomes completely dependent on α/ß MBNs. This shift in neuronal dependency for behavioral performance is paralleled by shifts in the activity of the relevant neurons during the retrieval of short-term versus long-term memories. Moreover, transient neuron inactivation experiments using flies trained to have both early and remote memories showed that the α'/ß' MBNs have a time-limited role in memory processing. These results argue that system consolidation is not a unique feature of the mammalian brain and memory systems, but rather a general and conserved feature of how different temporal memories are encoded from relatively simple to complex brains.

Dopamine is required for learning and forgetting in Drosophila.

Psychological studies in humans and behavioral studies of model organisms suggest that forgetting is a common and biologically regulated process, but the molecular, cellular, and circuit mechanisms underlying forgetting are poorly understood. Here we show that the bidirectional modulation of a small subset of dopamine neurons (DANs) after olfactory learning regulates the rate of forgetting of both punishing (aversive) and rewarding (appetitive) memories. Two of these DANs, MP1 and MV1, exhibit synchronized ongoing activity in the mushroom body neuropil in alive and awake flies before and after learning, as revealed by functional cellular imaging. Furthermore, while the mushroom-body-expressed dDA1 dopamine receptor is essential for the acquisition of memory, we show that the dopamine receptor DAMB, also highly expressed in mushroom body neurons, is required for forgetting. We propose a dual role for dopamine: memory acquisition through dDA1 signaling and forgetting through DAMB signaling in the mushroom body neurons.

Cyclooxygenase-2 induces genomic instability, BCL2 expression, doxorubicin resistance, and altered cancer-initiating cell phenotype in MCF7 breast cancer cells.

INTRODUCTION: Cyclooxygenase (COX2) expression in primary breast cancer correlates with a worse prognosis. We reported previously that COX2 expression in MCF10A breast epithelial cells of basal subtype induces genomic instability. To understand the role of COX2 in estrogen receptor-positive (non-basal) breast cancer, we transfected the MCF7 cell line with COX2 and analyzed its chromosomal profile, BCL2 protein expression, and resistance to doxorubicin. We also analyzed cell cultures grown as mammospheres to determine whether COX2 expression affects the cancer-initiating ("stem") cell phenotype in MCF7 cells. METHODS: MCF7 Tet On cells (obtained from Clontech) were stably transfected with pTRE2pur-COX2 or pTRE2pur-COX2-GFP to produce COX2 or COX2-GFP protein, respectively. BCL2 protein was detected by Western blotting. Sensitivity of cells to drug treatment was analyzed by MTT assay. Groups were compared using Chi-Square test. We analyzed the genomic instability phenotype by chromosome analysis of control and COX2 transfected metaphase-arrested MCF7 cells after Giemsa staining. We assessed the tumorigenic potential of cells grown as mammospheres with a clonogenic assay. RESULTS: Cytogenetic analysis of early passage COX2 transfected MCF7 cells demonstrated significant genomic instability as compared to parental MCF7 cells. COX2 overexpression was associated with a significant increase in chromosomal aberrations (chromatid breaks, chromosome fusions, C anaphase). COX2 transfected MCF7 cells produced a significantly higher level of the anti-apoptotic protein BCL2 than the parental cells. In a functional assay, we found that COX2 expression correlated with increased resistance to doxorubicin. In a complimentary approach to determine tumorigenic potential of cells, we found that COX2 increased the ability of MCF7 cells to grow as mammospheres in culture, which correlated with an increase in clonogenic efficiency. CONCLUSIONS: We found that COX2 expression in MCF7 breast cancer cells induced genomic instability, BCL2 expression, and doxorubicin resistance, thus making them significantly more tumorigenic. This data suggests that COX-2 may be an important target for breast cancer treatment.

Olfactory memory traces in Drosophila.

In Drosophila, the fruit fly, coincident exposure to an odor and an aversive electric shock can produce robust behavioral memory. This behavioral memory is thought to be regulated by cellular memory traces within the central nervous system of the fly. These molecular, physiological, or structural changes in neurons, induced by pairing odor and shock, regulate behavior by altering the neurons' response to the learned environment. Recently, novel in vivo functional imaging techniques have allowed researchers to observe cellular memory traces in intact animals. These investigations have revealed interesting temporal and spatial dynamics of cellular memory traces. First, a short-term cellular memory trace was discovered that exists in the antennal lobe, an early site of olfactory processing. This trace represents the recruitment of new synaptic activity into the odor representation and forms for only a short period of time just after training. Second, an intermediate-term cellular memory trace was found in the dorsal paired medial neuron, a neuron thought to play a role in stabilizing olfactory memories. Finally, a long-term protein synthesis-dependent cellular memory trace was discovered in the mushroom bodies, a structure long implicated in olfactory learning and memory. Therefore, it appears that aversive olfactory associations are encoded by multiple cellular memory traces that occur in different regions of the brain with different temporal domains.

Cyclooxygenase-2 expression induces genomic instability in MCF10A breast epithelial cells.

BACKGROUND: Cyclooxygenase-2 (COX-2) is induced in many breast cancers and COX-2 expression correlates with a worse outcome in the clinic. We hypothesized that the induction of genomic instability is a major mechanism through which COX-2 contributes to breast cancer progression. METHODS: We transfected a normal immortalized breast epithelial cell line of Basal B subtype, MCF10A, with the pSG5-COX-2 vector and established the stably transfected cell line MCF10A/COX-2. We analyzed the genomic instability phenotype by chromosomal analysis of metaphase-arrested MCF10A and MCF10A/COX-2 cells after Giemsa staining. Groups were compared using chi(2) tests. To investigate the DNA damage checkpoint signaling, we analyzed the phosphorylation status of CHK1 protein with a phospho-specific antibody. RESULTS: Cytogenetic analysis of early passage transfected cells showed that COX-2 expression increased genomic instability compared with the MCF10A cells transfected with a luciferase vector alone. COX-2 overexpression was associated with a significant increase in chromosomal aberrations (fusions, breaks, and tetraploidy). There was a statistically significant increase in the number of polyploid cells in the COX-2 transfected cells versus the control (P=0.004). We also found that an inhibitory CHK1 phosphorylation at Ser-280 was dramatically increased upon COX-2 overexpression in MCF10A cells, thus explaining the mechanism of inactivation of an important cell cycle checkpoint. Further analysis of the MCF10A/COX-2 cells showed that these cells have acquired a premalignant phenotype characterized by a morphological transformation, a resistance to anoikis, a reduced requirement of epidermal growth factor for growth in culture, but their inability to establish tumors in a nude mouse model of malignancy. CONCLUSION: We found that COX-2 expression in MCF10A breast epithelial cells confers a premalignant phenotype that includes enhanced genomic instability and altered cell-cycle regulation.

Involvement of IL-8 in COX-2-mediated bone metastases from breast cancer.

BACKGROUND: Cyclooxygenase-2 (COX-2) overexpression by a primary tumor correlates with poor prognosis in breast cancer, including early spread to bone. Interleukin-8 (IL-8) stimulates osteoclastogenesis and resorption of bone, and elevated IL-8 levels predict early metastatic spread of breast cancer. The purpose of this study was to test our hypothesis that tumors that overexpress COX-2 induce IL-8 production. MATERIALS AND METHODS: We cotransfected MCF-10A (nonmalignant breast epithelial) cells, as well as MDA-231 (highly metastatic human breast cancer) cell lines with a pSG5-COX-2 vector and pEF1a-Luc-IRES-Neo vector (luciferase reporter). COX-2 overexpression was confirmed by Western blot and PGE2 (a product of the COX-2 pathway) immunoassay. IL-8 production was measured by immunoassay. In vivo testing used a nude mouse model to measure COX-2 and IL-8 production from breast cancer cells that had metastasized to bone (bone-seeking clones (BSCs)). Long bone metastases were localized and quantified by luciferase imaging (Xenogen IVIS system) and X-ray. BSCs were isolated and cultured and then tested for the production of PGE2 and IL-8. RESULTS: COX-2 overexpression caused a 4- to 5-fold increase in IL-8 production in both MCF-10A and MDA-231 cells in vitro. In vivo, we observed that the MDA-231-BSC (metastatic cells isolated from bone metastases) produced significantly greater levels of both PGE2 and IL-8 compared to the parental MDA-231 cells (P < 0.01). In contrast to the results obtained with these estrogen receptor-negative cell lines, COX-2 expression failed to induce IL-8 in the MCF-7 estrogen receptor-positive breast cancer cell line. Treatment with the COX-2 inhibitor NS-398 at a low 1-mu[scap]M dose reduced the production of IL-8 in COX-2-transfected MDA-231 cells by 30%, thus confirming the involvement of COX-2 in IL-8 induction. CONCLUSION: COX-2 expression induced formation of PGE2 and IL-8 in breast cancer cells. Since PGE2 and IL-8 stimulate osteoclasts to resorb bone, COX-2 inhibition is a potential target for treatment to prevent bone metastases.

COX-2 induces IL-11 production in human breast cancer cells.

BACKGROUND: Cyclooxygenase-2 (COX-2) is overexpressed in 40% of human invasive breast cancers. Interleukin-11 (IL-11), a potent mediator of osteoclastogenesis, is involved in breast cancer metastasis to bone. Since breast cancers that overexpress COX-2 are associated with a higher rate of metastasis to bone, we hypothesized that COX-2 expression in tumor cells would induce IL-11. MATERIALS AND METHODS: We transfected MCF-7 (poorly metastatic) and MDA-231 (highly metastatic) human breast cancer cell lines with COX-2 expression vectors. COX-2 overexpression was confirmed by Western blot and PGE(2) immunoassay, and IL-11 production was measured by immunoassay. We also used a nude mouse model to study COX-2 and IL-11 production from breast cancer cells that metastasized to bone. The bone-seeking clones (BSC) were isolated and cultured from the long bone metastases. RESULTS: COX-2 transfection caused an approximately 5- to 6-fold increase in IL-11 production in both MCF-7 and MDA-231 cells. MDA-435S-COX2-BSC (cells isolated from bone metastasis) produced elevated levels of IL-11 and PGE2 (an important mediator of COX-2) as compared to the parental MDA-435S-COX2 cells. Furthermore, a treatment with low 1- to 2-microm concentration NS-398 or Celecoxib significantly reduced the production of IL-11 in COX-2-transfected MDA-231 cells, thus confirming the involvement of COX-2 in IL-11 induction. CONCLUSION: COX-2-mediated production of IL-11 in breast cancer cells may be vital to the development of osteolytic bone metastases in patients with breast cancer, and a COX-2 inhibitor may be useful in inhibiting this process.