Mariana Rodrigues

How does the metabolism of tumour cells differ from that of normal cells

Tumour cells thrive in environments that would be hostile to their normal cell counterparts. Survival depends on the selection of cell lines that harbour modifications of both, gene regulation that shifts the balance between the cell cycle and apoptosis and those that involve the plasticity of the metabolic machinery. With regards to metabolism, the selected phenotypes usually display enhanced anaerobic glycolysis even in the presence of oxygen, the so-called Warburg effect, and anabolic pathways that provide precursors for the synthesis of lipids, proteins and DNA. The review will discuss the original ideas of Otto Warburg and how they initially led to the notion that mitochondria of tumour cells were dysfunctional. Data will be presented to show that not only the organelles are viable and respiring, but that they are key players in tumorigenesis and metastasis. Likewise, interconnecting pathways that stand out in the tumour phenotype and that require intact mitochondria such as glutaminolysis will be addressed. Furthermore, comments will be made as to how the peculiarities of the biochemistry of tumour cells renders them amenable to new forms of treatment by highlighting possible targets for inhibitors. In this respect, a case study describing the effect of a metabolite analogue, the alkylating agent 3BP (3-bromopyruvate), on glycolytic enzyme targets will be presented.

Methyl Jasmonate: Putative Mechanisms of Action on Cancer Cells Cycle, Metabolism, and Apoptosis

Methyl jasmonate (MJ), an oxylipid that induces defense-related mechanisms in plants, has been shown to be active against cancer cells both in vitro and in vivo, without affecting normal cells. Here we review most of the described MJ activities in an attempt to get an integrated view and better understanding of its multifaceted modes of action. MJ (1) arrests cell cycle, inhibiting cell growth and proliferation, (2) causes cell death through the intrinsic/extrinsic proapoptotic, p53-independent apoptotic, and nonapoptotic (necrosis) pathways, (3) detaches hexokinase from the voltage-dependent anion channel, dissociating glycolytic and mitochondrial functions, decreasing the mitochondrial membrane potential, favoring cytochrome c release and ATP depletion, activating pro-apoptotic, and inactivating antiapoptotic proteins, (4) induces reactive oxygen species mediated responses, (5) stimulates MAPK-stress signaling and redifferentiation in leukemia cells, (6) inhibits overexpressed proinflammatory enzymes in cancer cells such as aldo-keto reductase 1 and 5-lipoxygenase, and (7) inhibits cell migration and shows antiangiogenic and antimetastatic activities. Finally, MJ may act as a chemosensitizer to some chemotherapics helping to overcome drug resistant. The complete lack of toxicity to normal cells and the rapidity by which MJ causes damage to cancer cells turn MJ into a promising anticancer agent that can be used alone or in combination with other agents.

Energy metabolism in H460 lung cancer cells: effects of histone deacetylase inhibitors.

Background Tumor cells are characterized by accelerated growth usually accompanied by up-regulated pathways that ultimately increase the rate of ATP production. These cells can suffer metabolic reprogramming, resulting in distinct bioenergetic phenotypes, generally enhancing glycolysis channeled to lactate production. In the present work we showed metabolic reprogramming by means of inhibitors of histone deacetylase (HDACis), sodium butyrate and trichostatin. This treatment was able to shift energy metabolism by activating mitochondrial systems such as the respiratory chain and oxidative phosphorylation that were largely repressed in the untreated controls. Methodology/Principal Findings Various cellular and biochemical parameters were evaluated in lung cancer H460 cells treated with the histone deacetylase inhibitors (HDACis), sodium butyrate (NaB) and trichostatin A (TSA). NaB and TSA reduced glycolytic flux, assayed by lactate release by H460 cells in a concentration dependent manner. NaB inhibited the expression of glucose transporter type 1 (GLUT 1), but substantially increased mitochondria bound hexokinase (HK) activity. NaB induced increase in HK activity was associated to isoform HK I and was accompanied by 1.5 fold increase in HK I mRNA expression and cognate protein biosynthesis. Lactate dehydrogenase (LDH) and pyruvate kinase (PYK) activities were unchanged by HDACis suggesting that the increase in the HK activity was not coupled to glycolytic flux. High resolution respirometry of H460 cells revealed NaB-dependent increased rates of oxygen consumption coupled to ATP synthesis. Metabolomic analysis showed that NaB altered the glycolytic metabolite profile of intact H460 cells. Concomitantly we detected an activation of the pentose phosphate pathway (PPP). The high O2 consumption in NaB-treated cells was shown to be unrelated to mitochondrial biogenesis since citrate synthase (CS) activity and the amount of mitochondrial DNA remained unchanged. Conclusion NaB and TSA induced an increase in mitochondrial function and oxidative metabolism in H460 lung tumor cells concomitant with a less proliferative cellular phenotype.

Enhanced OXPHOS, glutaminolysis and β-oxidation constitute the metastatic phenotype of melanoma cells

Tumours display different cell populations with distinct metabolic phenotypes. Thus, subpopulations can adjust to different environments, particularly with regard to oxygen and nutrient availability. Our results indicate that progression to metastasis requires mitochondrial function. Our research, centered on cell lines that display increasing degrees of malignancy, focused on metabolic events, especially those involving mitochondria, which could reveal which stages are mechanistically associated with metastasis. Melanocytes were subjected to several cycles of adhesion impairment, producing stable cell lines exhibiting phenotypes representing a progression from non-tumorigenic to metastatic cells. Metastatic cells (4C11+) released the highest amounts of lactate, part of which was derived from glutamine catabolism. The 4C11+ cells also displayed an increased oxidative metabolism, accompanied by enhanced rates of oxygen consumption coupled to ATP synthesis. Enhanced mitochondrial function could not be explained by an increase in mitochondrial content or mitochondrial biogenesis. Furthermore, 4C11+ cells had a higher ATP content, and increased succinate oxidation (complex II activity) and fatty acid oxidation. In addition, 4C11+ cells exhibited a 2-fold increase in mitochondrial membrane potential (ΔΨmit). Consistently, functional assays showed that the migration of cells depended on glutaminase activity. Metabolomic analysis revealed that 4C11+ cells could be grouped as a subpopulation with a profile that was quite distinct from the other cells investigated in the present study. The results presented here have centred on how the multiple metabolic inputs of tumour cells may converge to compose the so-called metastatic phenotype.

Reciprocal modulation of histone deacetylase inhibitors sodium butyrate and trichostatin A on the energy metabolism of breast cancer cells.

Tumor cells display different bioenergetic profiles when compared to normal cells. In the present work we showed metabolic reprogramming by means of inhibitors of histone deacetylase (HDACis), sodium butyrate and trichostatin A in breast cancer cells representing different stages of aggressiveness and metabolic profile. When testing the effect of NaB and TSA on viability of cells, it was shown that non‐tumorigenic MCF‐10A cells were less affected by increasing doses of the drugs than the tumorigenic, hormone dependent, tightly cohesive MCF‐7, T‐47D and the highly metastatic triple‐negative MDA‐MB 231 cells. T‐47D cells were the most sensitive to treatment with both, NaB and TSA. Experiments measuring anchorage‐ independent growth of tumor cells showed that MCF‐7, T‐47D, and MDA‐MB‐231 cells were equally sensitive to the treatment with NaB. The NaB induced an attenuation of glycolysis, reflected by a decrease in lactate release in MCF‐7 and T47D lines. Pyruvate kinase activity was significantly enhanced by NaB in MDA‐MB‐231 cells only. In contrast, the inhibitor enhanced lactate dehydrogenase activity specifically in T‐47 D cells. Glucose‐6‐phosphate dehydrogenase activity was shown to be differentially modulated by NaB in the cell lines investigated: the enzyme was inhibited in MCF‐7 cells, whereas in T‐47D and MDA‐MB‐231 cells, G6PDH was activated. NaB and TSA were able to significantly increase the oxygen consumption by MDA‐MB‐231 and T‐47D cells. Collectively the results show that epigenetic changes associated to acetylation of proteins in general affect the energy metabolism in all cancer cell lines and that mitochondria may occupy a central role in metastasis. J. Cell. Biochem. 116: 797–808, 2015.

Cell Cycle and Energy Metabolism in Tumor Cells: Strategies for Drug Therapy

Recent results obtained from research on the intermediary metabolism of tumor cells have uncovered the biochemical reprogramming that takes place upon malignant transformation. Many features have been highlighted that are currently being exploited for specific chemotherapy. Many more will become available shortly as a consequence of the recognition of potentially useful targets for treatment. General interest in this area can be gauged by the number of recent patents that have been deposited, or are in the process of application. Because the metabolic subversion that is a hallmark of cancer cells involves a disruption of its homeostasis, the regulatory pathways dealt with in this review were broadly divided into those that encompass the main stages of the cell cycle and its various regulatory mechanisms and those that involve the aerobic glycolysis typical of cancer cells. It becomes apparent that both, the cell cycle and the intermediary metabolism are interconnected and rely on reactions many of which are dependent on kinases and phosphatases. Kinases and phosphatases are responsive to cellular redox signaling and may have a key role in determining whether cells progress towards malignant transformation as a result of continuous oxidative stress. The results discussed here underline aspects of the signaling pathways that lend themselves to specific inhibition by natural and synthetic compounds. The mitochondria and its role in programmed cell death are briefly commented, but special emphasis is placed on biochemical regulation at the level of chromatin structure, particularly the reactions that involve acetylation and deacetylation of histones. Within this context, inhibitors that act on histone deacetylases are discussed as promising alternatives to available treatments.

Dedicated Pediatricians in Emergency Department: Shorter Waiting Times and Lower Costs

Background Dedicated pediatricians in emergency departments (EDs) may be beneficial, though no previous studies have assessed the related costs and benefits/harms. We aimed to evaluate the net benefits and costs of dedicated emergency pediatricians in a pediatric ED. Methods Cost-consequences analysis of visits to a pediatric ED of a tertiary hospital. Two pediatric ED Medical Teams (MT) were compared: MT-A (May–September 2012), with general pediatrics physicians only; and MT-B (May–September 2013), with emergency dedicated pediatricians. The main outcomes analyzed were relevant clinical outcomes, patient throughput time and costs. Results We included 8,694 children in MT-A and 9,417 in MT-B. Medication use in the ED increased from 42.3% of the children in MT-A to 49.6% in MT-B; diagnostic tests decreased from 24.2% in MT-A to 14.3% in MT-B. Hospitalization increased from 1.3% in MT-A to 3.0% in MT-B; however, there was no significant difference in diagnosis-related group relative weight of hospitalized children in MT-A and MT-B (MT-A, 0.979; MT-B, 1.075). No differences were observed in ED readmissions or in patients leaving without being seen by a physician. The patient throughput time was significantly shorter in MT-B, with faster times to first medical observation. Within the cost domains analyzed, the total expenditures per children observed in the ED were 16% lower in MT-B: 37.87 euros in MT-A; 31.97 euros in MT-B. Conclusion The presence of dedicated emergency pediatricians in a pediatric ED was associated with significantly lower waiting times in the ED, reduced costs, and similar clinical outcomes.

Performance of HIV Prevention of Mother-To-Child Transmission Programs in Sub-Saharan Africa: Longitudinal Assessment of 64 Nevirapine-Based Programs Implemented in 25 Countries, 2000-2011

Background To evaluate the performance and to identify predictive factors of performance in prevention of mother-to-child HIV transmission programs (PMTCT) in sub-Saharan African countries. Methods From 2000 to 2011, PMTCT programs included in the Viramune Donation Programme (VDP) were prospectively followed. Each institution included in the VDP provided data on program implementation, type of management institution, number of PMTCT sites, key programs outputs (HIV counseling and testing, NVP regimens received by mothers and newborns). Nevirapine Coverage Ratio (NCR), defined as the number of women who should have received nevirapine (observed HIV prevalence x number of women in antenatal care), was used to measure performance. Included programs were followed every six months through progress reports. Results A total of 64 programs in 25 sub-Saharan African countries were included. The mean program follow-up was 48.0 months (SD = 24.5); 20,084,490 women attended in antenatal clinics were included. The overall mean NCR was 0.52 (SD = 0.25), with an increase from 0.37 to 0.57 between the first and last progress reports (p<.0001); NCR increased by 3.26% per year-program. Between the first and the last report, the number of women counseled and tested increased from 64.3% to 86.0% (p<.0001), the number of women post-counseled from 87.5% to 91.3% (p = 0.08). After mixed linear regression analysis, type of responsible institution, number of women attended in ANC, and program initiation in 2005-2006 were significant predictive factors associated with the NCR. The effect of the time period increased from earlier to later periods. Conclusion A longitudinal assessment of large PMTCT programs shows that scaling-up of programs was increased in sub-Saharan African countries. The PMTCT coverage increased throughout the study period, especially after 2006. Performance may be better for programs with a small or medium number of women attended in ANC. Identification of factors that predict PMTCT program performance may help in the development and expansion of additional large PMTCT services in sub-Saharan Africa.

Characterization of inorganic phosphate transport in the triple-negative breast cancer cell line, MDA-MB-231

Background Recent studies demonstrate that interstitial inorganic phosphate is significantly elevated in the breast cancer microenvironment as compared to normal tissue. In addition it has been shown that breast cancer cells express high levels of the NaPi-IIb carrier (SLC34A2), suggesting that this carrier may play a role in breast cancer progression. However, the biochemical behavior of inorganic phosphate (Pi) transporter in this cancer type remains elusive. Methods In this work, we characterize the kinetic parameters of Pi transport in the aggressive human breast cancer cell line, MDA-MB-231, and correlated Pi transport with cell migration and adhesion. Results We determined the influence of sodium concentration, pH, metabolic inhibitors, as well as the affinity for inorganic phosphate in Pi transport. We observed that the inorganic phosphate is dependent on sodium transport (K0,5 value = 21.98 mM for NaCl). Furthermore, the transport is modulated by different pH values and increasing concentrations of Pi, following the Michaelis-Menten kinetics (K0,5 = 0.08 mM Pi). PFA, monensin, furosemide and ouabain inhibited Pi transport, cell migration and adhesion. Conclusions Taken together, these results showed that the uptake of Pi in MDA-MB-231 cells is modulated by sodium and by regulatory mechanisms of intracellular sodium gradient. General Significance: Pi transport might be regarded as a potential target for therapy against tumor progression.

Societal impact of dengue outbreaks: Stakeholder perceptions and related implications. A qualitative study in Brazil, 2015.

Background The growing burden of dengue in many countries worldwide and the difficulty of preventing outbreaks have increased the urgency to identify alternative public health management strategies and effective approaches to control and prevent dengue outbreaks. The objectives of this study were to understand the impact of dengue outbreak on different stakeholders in Brazil, to explore their perceptions of approaches used by governmental authorities to control and prevent dengue outbreaks and to define the challenges and implications of preventing future outbreaks. Methods In 2015, a qualitative study was conducted in two urban states in Brazil: São Paulo, which was experiencing an outbreak in 2015, and Rio de Janeiro, which experienced outbreaks in 2011 and 2012. Face-to-face interviews using a semi-structured questionnaire were conducted with nine different categories of stakeholders: health workers (physicians, nurses), hospital administrators, municipal government representatives, community members and leaders, school administrators, business leaders and vector control managers. Interviews were focused on the following areas: impact of the dengue outbreak, perceptions of control measures implemented by governmental authorities during outbreaks and challenges in preventing future dengue outbreaks. Results A total of 40 stakeholders were included in the study. Health workers and community members reported longer waiting times at hospitals due to the increased number of patients receiving care for dengue-related symptoms. Health workers and hospital administrators reported that there were no major interruptions in access to care. Overall financial impact of dengue outbreaks on households was greatest for low-income families. Despite prevention and control campaigns implemented between outbreak periods, various stakeholders reported that dengue prevention and control efforts performed by municipal authorities remained insufficient, suggesting that efforts should be reinforced and better coordinated by governmental authorities, particularly during outbreak periods. Conclusion The study shows that a dengue outbreak has a multisectorial impact in the medical, societal, economic and political sectors. The study provides useful insights and knowledge in different stakeholder populations that could guide local authorities and government officials in planning, designing and initiating public health programs. Research focused on a better understanding of how communities and political authorities respond to dengue outbreaks is a necessary component for designing and implementing plans to decrease the incidence and impact of dengue outbreaks in Brazil.