Metformin is synthetically lethal with glucose withdrawal in cancer cells

Comment on: Menendez JA, et al. Cell Cycle 2012; 11: 2782-92.In a recent issue of Cell Cycle, Menendez and colleagues proposed a novel concept, that metformin is synthetically lethal with glucose withdrawal in cancer cells.¹ Historically, synthetic lethality has focused on how tumor cells are responsive to certain agents that only harbor specific constitutive epigenetic or genetic lesions.² More recent data from several groups have uncovered that altered tumor microenvironment could be used to confer synthetic lethality to specific drugs, defined as “contextual synthetic lethality,” that is microenvironment-mediated. For example, hypoxia-induced HR (homologous repair) defect has been shown to be synthetically lethal to PARP inhibition, while PARP inhibition, per se, did not alter HR inhibition or function, thus providing a prime example of “contextual synthetic lethality.”³ In this report, Menendez et al. have elegantly connected the glucose-deprived tumor microenvironment in primary tumors as a synthetic lethal partner to metformin. Metformin is a FDA-approved drug to treat diabetic patients that is gaining momentum as a repurposing drug for cancer treatment.⁴ Using several different breast cancer cells with and without oncogenic activation, the authors have shown that the glucose-rich conditions of the in vitro experiments dictates the use of very high concentrations of metformin, which are not applicable to glucose-starved in vivo conditions. While other reports have alluded to the effect of glucose withdrawal in killing genetically compromised cells to therapeutic effect of metformin in vitro,⁵ Menendez et al have provided a logical explanation for the use of very high concentrations of metformin to achieve anticancer effects in vitro in the high glucose-rich environment used in these experiments, which are clinically not applicable in vivo in patients.Based on these findings, it can be envisaged that in the tumor microenvironment, where the cancer cells are under extreme nutritional and hypoxic stress (a niche for cancer stem cells), metformin treatment could favor synthetic lethality and hence effectively can attenuate tumor growth. The tumor microenvironment thus enables the bioenergetic switch in favor of glycolysis and dependence on glucose and glutamine as a rapid source of nutrition. While the authors’ data clearly depicts how metformin eliminates the tolerance of the breast cancer cells to fluctuations in glucose concentrations, it is important to understand how the availability of other dominant sources of energy, such as glutamine, might participate in this scenario. It is plausible that subtype of breast cancers, i.e., basal vs luminal, might depend on different energy sources, albeit to a different extent.⁶ This is important, because tumor cells often acquire metabolic adaptability toward available preferred energy source to adapt well to nutritional stress via autophagy and altered metabolism.⁷ Along these lines, the authors rationalize the therapeutic targeting of the cancer stem cells by metformin through its synthetic lethal activity to the hyperglycotic phenotype often seen in CSC to sustain their stemness.⁸ Further characterization of how metformin treatment alters the metabolic nodes in cancer stem cells and/or p53-null cells would explain the underpinning mechanisms for increased susceptibility of these indolent and aggressive cancer cells toward metformin.It is well documented that metformin, by inhibiting complex I of respiratory chain in mitochondria (ETCI), induces a decrease in the ATP levels, and that glucose depletion also decreases ATP levels, albeit to varying levels. Therefore, it is possible that simultaneous targeting of both pathways (glycolytic pathway and OXPHOS) caused ATP depletion below a critical threshold, resulting in cell death. This concept is supported by the elegant study⁹ highlighting the effectiveness of combination of glycolysis inhibition by 2-DG and metformin in several preclinical models exhibiting anti-tumor effects, including MB-MDA231 used in this study.Since recent studies indicate that inhibiting glucose uptake with small-molecule inhibitors led to a decline in cylcin E2 and p-RB levels,¹⁰ it is a possibility that cell cycle inhibitor levels are also regulated under glucose withdrawal conditions, sensitizing cells to cytotoxic effects of metformin in breast cancer cells.Considering data from several studies, a view that metformin treatment has pleotropic effects on several signaling pathways under glucose-free conditions seems a practical possibility. Overall, this work offers several new insights into glucose-dependent mechanisms underpinning the mode of action of metformin as a viable therapeutic strategy.     

Tuning genetic clocks employing DNA binding sites

Periodic oscillations play a key role in cell physiology from the cell cycle to circadian clocks. The interplay of positive and negative feedback loops among genes and proteins is ubiquitous in these networks. Often, delays in a negative feedback loop and/or degradation rates are a crucial mechanism to obtain sustained oscillations. How does nature control delays and kinetic rates in feedback networks? Known mechanisms include proper selection of the number of steps composing a feedback loop and alteration of protease activity, respectively. Here, we show that a remarkably simple means to control both delays and effective kinetic rates is the employment of DNA binding sites. We illustrate this design principle on a widely studied activator-repressor clock motif, which is ubiquitous in natural systems. By suitably employing DNA target sites for the activator and/or the repressor, one can switch the clock “on” and “off” and precisely tune its period to a desired value. Our study reveals a design principle to engineer dynamic behavior in biomolecular networks, which may be largely exploited by natural systems and employed for the rational design of synthetic circuits.     

Basic fibroblast growth factor accelerates matrix degradation via a neuro-endocrine pathway in human adult articular chondrocytes

Pain-related neuropeptides released from synovial fibroblasts, such as substance P, have been implicated in joint destruction. Substance P-induced inflammatory processes are mediated via signaling through a G-protein-coupled receptor, that is, neurokinin-1 tachykinin receptor (NK(1)-R). We determined the pathophysiological link between substance P and its receptor in human adult articular cartilage homeostasis. We further examined if catabolic growth factors such as basic fibroblast growth factor (bFGF or FGF-2) or IL-1beta accelerate matrix degradation via a neural pathway upregulation of substance P and NK(1)-R. We show here that substance P stimulates the production of cartilage-degrading enzymes, such as matrix metalloproteinase-13 (MMP-13), and suppresses proteoglycan deposition in human adult articular chondrocytes via NK(1)-R. Furthermore, we have demonstrated that substance P negates proteoglycan stimulation promoted by bone morphogenetic protein-7, suggesting the dual role of substance P as both a pro-catabolic and anti-anabolic mediator of cartilage homeostasis. We report that bFGF-mediated stimulation of substance P and its receptor NK(1)-R is, in part, through an IL-1beta-dependent pathway.     

Immortalized feeders for the scale-up of human embryonic stem cells in feeder and feeder-free conditions

Human embryonic stem cells (hESC) are pluripotent cells that proliferate indefinitely in culture, whilst retaining their capacity for differentiation into different cell types. However, hESC cultures require culture in direct contact with feeder cells or conditioned medium (CM) from feeder cells. The most common source of feeders has been primary mouse embryonic fibroblast (MEF). In this study, we immortalized a primary MEF line with the E6 and E7 genes from HPV16. The immortal line, DeltaE-MEF, was able to proliferate beyond 7-9 passages and has an extended lifespan beyond 70 passages. When tested for its ability to support hESC growth, it was found that hESC continue to maintain the undifferentiated morphology for >40 passages both in co-culture with DeltaE-MEF and in feeder-free cultures supplemented with CM from DeltaE-MEF. The cultures also continue to express the pluripotent markers, Oct-4, SSEA-4, Tra-1-60, Tra-1-81, alkaline phosphatase and maintain a normal karyotype. In addition, these hESC formed teratomas when injected into SCID mice. Lastly, we demonstrated the feasibility of scaling-up significant quantities of undifferentiated hESC (>10(8) cells) using DeltaE-MEF in cell factories. The results from this study suggest that immortalized feeders can provide a consistent and reproducible source of feeders for hESC expansion and research.     

Chitinase production by Beauveria felinaRD 101: optimization of parameters under solid substrate fermentation conditions

Major parameters affecting the production of chitinase by Beauveria felinaRD 101 under solid substrate fermentation conditions have been optimized. Wheat bran moistened with 100 MS-HCl medium adjusted to pH 5.0, inoculated with 1 × 10¹⁰ conidia g^–1 initial dry bran and incubated at 28 °C for 6 days produced maximum chitinase activity of 6.34 U g^–1 initial dry substrate.     

What signals do herbivore-induced plant volatiles provide conspecific herbivores?

Herbivore-induced plant volatiles (HIPVs) have been opined as ‘indirect or direct defenses’ of plants and are extensively studied. In contrast, HIPVs may also indicate that plant defenses have been overcome by herbivores infesting the plant; however, studies on this aspect have so far received little attention. Using the interaction of Capsicum annum (Bell pepper) with its pest Scirtothrips dorsalis (Chilli thrips) as a model system, we studied the role of HIPVs in this selected insect–plant interaction. Multiple-choice olfactometer assays with headspace volatiles collected from different growth stages of un-infested C. annum plants represented by pre-flowering (PF), flowering (FL) and fruiting stages (FR) proved FR volatiles to be highly attractive to S. dorsalis. Further, FR plants were infested with S. dorsalis adults and HIPVs released by infested plants were collected and subjected to multiple-choice olfactometer bioassays. Thrips were significantly attracted to HIPVs than to headspace volatiles of un-infested FR plants or thrips body odour. Coupled GC-EAG with S. dorsalis and HIPVs or FR plant volatile revealed specific compounds that elicited an EAG response. Individual EAG-active compounds were less attractive to thrips, however, synthetic blends of EAG-active compounds at the ratio similar to headspace samples were found to be highly attractive. However, when given a choice between synthetic blends of HIPVs and FR, thrips were significantly attracted to synthetic blend of HIPVs. Our study provides empirical data on signals HIPVs may provide to conspecific herbivores and suggests that the role of HIPVs, mostly generalized as defense, may vary based on the interaction and must be studied closely to understand their ecological functions.     

A pilot study of an anti-endotoxin Ig-enriched bovine colostrum to prevent experimental sepsis

Despite the dramatic increase in antimicrobial resistance, there is a dearth of antibiotics in development and few pharmaceutical companies working in the field. Further, any new antibiotics are likely to have a short shelf life. Ab-based interventions offer alternatives that are not likely to be circumvented by the widely prevalent antibiotic resistance genes. Bovine colostrum (BC)—the first milk after parturition, rich in nutrients and immune components—promotes gut integrity and modulates the gut microbiome. We developed a hyperimmune BC (HBC) enriched in Abs to a highly conserved LOS core region of Gram-negative bacteria by immunizing pregnant cows with a vaccine comprised of detoxified LOS from Escherichia coli O111 Rc (J5) mutant non-covalently complexed to group B meningococcal outer membrane protein (J5dLOS/OMP). This vaccine generated robust levels of anti-J5 LOS Ab in the colostrum. When given orally to neutropenic rats challenged orally with Pseudomonas aeruginosa, administration of HBC improved survival compared to non-immune rats, while both BC preparations improved survival compared to PBS controls. Elevated circulating endotoxin levels correlated with mortality. HBC and to a lesser extent non-immune BC reduced bacterial burden from the liver, lung, and spleen. We conclude that HBC and to a lesser extent BC may be effective supplements that improve outcome from lethal gut-derived disseminated infection and may reduce transmission of Gram-negative bacilli from the gastrointestinal tract.