Nutrient-deprived Conditions Research Articles

Abstract LB-135: Stanniocalcin 2 attenuates tumor cell proliferation but suppresses apoptosis in nutrient-deprived conditions.

Abstract High levels of stanniocalcin2 (STC2), a member of the STC secreted glycoprotein family, correlates with tumor progression and poor prognosis. It has been reported that STC2 is over-expressed in response to various stresses, including hypoxia, endoplasmic reticulum stress, oxidative stress and irradiation. Glutamine (Gln) and glucose (Glc) are important nutrients for tumor cells, playing important roles as nitrogen and carbon sources to meet the bioenergetic, biosynthetic and reductive needs. Defective blood supply in solid tumors may result in nutrient insufficiency, including Gln and Glc insufficiency. It remains unknown how tumor cells respond to Gln- and Glc- insufficient microenvironment and regulate their viability and proliferation. Using gene profiling analysis with microarrays, we have identified STC2 as one of the most up-regulated genes in response to either Gln- or Glc- deprivation. We confirm that Gln-deprivation stimulates STC2 expression at both mRNA and protein levels in various human tumor cells by quantitative reverse transcription-real-time PCR and Western blot, respectively. Furthermore, we find that Gln-insufficiency activates both nuclear factor-κB (NF-κB) and ATF4 signaling pathways, which synergistically up-regulates STC2. Finally, we demonstrate that up-regulation of STC2 attenuates tumor cell proliferation but reduces apoptosis in Gln-insufficient conditions. These findings suggest a novel model that STC2 reprograms the priority of tumor cells from rapid proliferation to survival under stress conditions, indicating that STC2 may be a novel target for cancer therapy. Citation Format: Shuo Qie, Lifeng Tian, Chenguang Wang, Nianli Sang. Stanniocalcin 2 attenuates tumor cell proliferation but suppresses apoptosis in nutrient-deprived conditions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-135. doi:10.1158/1538-7445.AM2013-LB-135

Abstract 1892: Integrated energetics and flux analysis reveals differential metabolic reprogramming in highly and less invasive cancer cells.

Abstract Cancer cell metabolism was investigated using systems biology tools such as metabolic flux analysis (MFA) to reveal intracellular cancer cell metabolic flux distribution. To obtain a complete role of nutrient-induced deprivation in metabolic reprogramming of cancer cells, we need to use metabolic flux analysis to determine unknown intracellular fluxes. MFA provides a framework for the estimation of intracellular fluxes and energy balance analysis (EBA) ensures the thermodynamic feasibility of the computed optimal metabolic fluxes. MFA uses stoichiometric and mass balance constraints to compute the intracellular fluxes. Here, we have developed an integrated energy balance and flux balance analysis framework. Our framework allows estimation of intracellular fluxes and reveals specific adaptations and dysregulation of metabolic pathways in ovarian cancer cells as a result of the nutrient (glucose and glutamine) deprivation conditions. Glutamine and glucose are known to be important for cancer cell survival, growth and progression. Thus, there is a strong motivation to study metabolic alternations caused by glucose and glutamine deprivation and uncover links between various fuel sources for highly and less invasive cancer cells. Our findings indicate that NAD+/NADH ratio regulates glutamine's direct effect on lactate production irrespective of the bioenergetic substrate requirement for different kinds of cancer cells. Our findings, using quantification of metabolites pertaining to central carbon and nitrogen metabolism provided clues as to which pathways relevant to glycolysis, the pentose phosphate pathway, the TCA, urea cycles and lipid cycles, amino acid and nucleotide metabolisms may be altered by glucose and glutamine in cancer cells. This resulted in identification of dominant factors which control cancer metabolism. We believe that development of our oncosecretomics based metabolic strategy provides an unprecedented opportunity for the development of nutritional supplementation that can deregulate the adaptations and alterations induced by various nutrients in cancer metabolism. Citation Format: Abhinav Achreja, Lifeng Yang, Hongyun Zhao, Juan C. Marini, Deepak Nagrath. Integrated energetics and flux analysis reveals differential metabolic reprogramming in highly and less invasive cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1892. doi:10.1158/1538-7445.AM2013-1892 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

IRE-1 Dependent Expression of Phosphoribosyl Pyrophosphate Synthetase Genes in U87 Glioma Cells: Effect of Glucose or Glutamine Deprivation

Nutrient deprivation conditions are capable to induce the endoplasmic reticulum stress and are responsible for regulation of the expression of numerous growth factors which control the cell proliferation and angiogenesis in malignant tumors, including glioma. The ERN1 mediated endoplasmic reticulum stress response-signalling pathway is tightly linked to the proliferation because the blockade of ERN1 function suppresses the tumor growth via specific changes in the cell transcriptome. Activation of the pentose phosphate pathway is an important factor of enhanced cell proliferation and tumor growthbecause it controls the synthesis of nucleotides and respectively nucleic acids. We studied the effect of blockade the ERN1 signaling enzyme function in glioma cells on the expression of phosphoribosyl pyrophosphate synthetase (PRPS), an important enzyme of pentose phosphate pathway, as well as its dependence of glucose and glutamine deprivation. The expression of different genes of PRPS enzyme in glioma cells with complete suppression of ERN1 enzyme activity was measured by qPCR. For glucose and glutamine deprivation conditions the cells were exposurein the glucose or glutamine free medium for 16 hrs. The expression of PRPS1 and PRPS2 genes as well as phosphoribosyl pyrophosphate synthetase associated protein 1(PRPSAP1) is significantly increased in U87 glioma cells with suppressed function of ERN1 gene, but the expression PRPSAP2 gene is decreased. It was also shown that glucose or glutamine deprivation leads to suppression of PRPS1 and PRPS2 gene expressions in U87 glioma cells; however, the blockade of ERN1 function in glioma cells modifies the effect of glutamine deprivation. Moreover, the expression of PRPSAP1 and PRPSAP2 genes was resistant to glucose deprivation condition in control glioma cells, but increased in glioma cells with ERN1 loss of function. Results of this investigation demonstrated that the blockade of ERN1 signaling enzyme function affects the expression of genes encoding PRPS enzyme subunits in different ways and that dysregulation of the expression of important regulatory subunits of PRPS (PRPSAP1 and PRPSAP2) possibly changes the activity of this enzyme and participates in suppression of glioma growth via pentose phosphate pathway.

Ferulic Acid Induces Mammalian Target of Rapamycin Inactivation in Cultured Mammalian Cells

Ferulic acid (FA), a naturally occurring polyphenol abundant in vegetables and rice bran, is known to possess a potent antioxidant activity, thereby protecting cells from oxidative stress. In the present study, we show that in addition to its known anti-oxidant activity, ferulic acid exerts substantial inhibitory activity on cellular mammalian target of rapamycin (mTor)-signaling pathways. In HeLa cells and mouse primary hepatocytes cultured with conventional nutrient-rich media, ferulic acid (1 mM) elicited dephosphorylation of S6 kinase and its substrate ribosomal S6. The dephosphorylating activity of ferulic acid was almost comparable to that of rapamycin, an established mTor inhibitor (TORC1). We next investigated the effect of ferulic acid on autophagy, a major cellular degradative process, which significantly contributes to the maintenance of cell homeostasis. Using a conventional green fluorescent protein-microtubule-associated protein IA/IB light chain 3 (GFP-LC3) dot assay to evaluate autophagy flux, we showed that ferulic acid caused a significant increase in GFP-LC3 dots under serum-rich conditions in HeLa cells. The enhancement of autophagic flux by ferulic acid was almost equivalent to that of rapamycin. Furthermore, ferulic acid significantly enhanced autophagic degradation of (14)C-leucine-labeled long-lived proteins of cultured mouse hepatocytes under nutrient-rich conditions, but not nutrient-deprived conditions. These results indicate that ferulic acid is almost the equivalent of rapamycin in the ability to inhibit mTor (TORC1), which makes it a potent activator of basal autophagy.

Nematode-Trapping Fungi Eavesdrop on Nematode Pheromones

The recognition of molecular patterns associated with specific pathogens or food sources is fundamental to ecology and plays a major role in the evolution of predator-prey relationships. Recent studies showed that nematodes produce an evolutionarily highly conserved family of small molecules, the ascarosides, which serve essential functions in regulating nematode development and behavior. Here, we show that nematophagous fungi, natural predators of soil-dwelling nematodes, can detect and respond to ascarosides. Nematophagous fungi use specialized trapping devices to catch and consume nematodes, and previous studies demonstrated that most fungal species do not produce traps constitutively but rather initiate trap formation in response to their prey. We found that ascarosides, which are constitutively secreted by many species of soil-dwelling nematodes, represent a conserved molecular pattern used by nematophagous fungi to detect prey and trigger trap formation. Ascaroside-induced morphogenesis is conserved in several closely related species of nematophagous fungi and occurs only under nutrient-deprived conditions. Our results demonstrate that microbial predators eavesdrop on chemical communication among their metazoan prey to regulate morphogenesis, providing a striking example of predator-prey coevolution. We anticipate that these findings will have broader implications for understanding other interkingdom interactions involving nematodes, which are found in almost any ecological niche on Earth.

The Saccharomyces cerevisiae protein Stm1p facilitates ribosome preservation during quiescence

Once cells exhaust nutrients from their environment, they enter an alternative resting state known as quiescence, whereby proliferation ceases and essential nutrients are obtained through internal stores and through the catabolism of existing macromolecules and organelles. One example of this is ribophagy, the degradation of ribosomes through the process of autophagy. However, some ribosomes need to be preserved for an anticipated recovery from nutrient deprivation. We found that the ribosome-associated protein Stm1p greatly increases the quantity of 80S ribosomes present in quiescent yeast cells and that these ribosomes facilitate increased protein synthesis rates once nutrients are restored. These findings suggest that Stm1p can act as a ribosome preservation factor under conditions of nutrient deprivation and restoration.

Aurora kinase A inhibition-induced autophagy triggers drug resistance in breast cancer cells

We have previously shown that elevated expression of mitotic kinase aurora kinase A (AURKA) in cancer cells promotes the development of metastatic phenotypes and is associated clinically with adverse prognosis. Here, we first revealed a clinically positive correlation between AURKA and autophagy-associated protein SQSTM1 in breast cancer and further demonstrated that AURKA regulated SQSTM1 through autophagy. Indeed, depletion by siRNA or chemical inhibition of AURKA by the small molecule VX-680 increased both the level of microtubule-associated protein 1 light chain 3-II (LC3-II) and the number of autophagosomes, along with decreased SQSTM1. Conversely, overexpression of AURKA inhibited autophagy, as assessed by decreased LC3-II and increased SQSTM1 either upon nutrient deprivation or normal conditions. In addition, phosphorylated forms of both RPS6KB1 and mechanistic target of rapamycin (MTOR) were elevated by overexpression of AURKA whereas they were suppressed by depletion or inhibition of AURKA. Moreover, inhibition of MTOR by PP242, an inhibitor of MTOR complex1/2, abrogated the changes in both LC3-II and SQSTM1 in AURKA-overexpressing BT-549 cells, suggesting that AURKA-suppressed autophagy might be associated with MTOR activation. Lastly, repression of autophagy by depletion of either LC3 or ATG5, sensitized breast cancer cells to VX-680-induced apoptosis. Similar findings were observed in cells treated with the autophagy inhibitors chloroquine (CQ) and bafilomycin A1 (BAF). Our data thus revealed a novel role of AURKA as a negative regulator of autophagy, showing that AURKA inhibition induced autophagy, which may represent a novel mechanism of drug resistance in apoptosis-aimed therapy for breast cancer.

Synthesis and antitumor evaluation of arctigenin derivatives based on antiausterity strategy

A series of new (−)-arctigenin derivatives with variably modified O-alkyl groups were synthesized and their preferential cytotoxicity was evaluated against human pancreatic cancer cell line PANC-1 under nutrient-deprived conditions. The results showed that monoethoxy derivative 4i (PC50, 0.49 μM), diethoxy derivative 4h (PC50, 0.66 μM), and triethoxy derivative 4m (PC50, 0.78 μM) showed the preferential cytotoxicities under nutrient-deprived conditions, which were identical to or more potent than (−)-arctigenin (1) (PC50, 0.80 μM). Among them, we selected the triethoxy derivative 4m and examined its in vivo antitumor activity using a mouse xenograft model. Triethoxy derivative 4m exhibited also in vivo antitumor activity with the potency identical to or slightly more than (−)-arctigenin (1). These results would suggest that a modification of (−)-arctigenin structure could lead to a new drug based on the antiausterity strategy.

Involvement of annexin A8 in the properties of pancreatic cancer

Although Annexin A8 (ANXA8), a member of a superfamily of calcium and phospholipid binding proteins, is physiologically expressed in a tissue-specific manner, recent microarray studies reported that ANXA8 was also ectopically expressed in pancreatic cancers. We investigated the molecular mechanism of expression of ANXA8 in cancer cells and its functional role in pancreatic cancer cells. ANXA8 was diversely expressed in human cancer cell lines. Expression was enhanced by treatment with 5-aza-dC and butyrate, and correlated with methylation status at CpG in the promoter-exon 1 region. Inhibition of ANXA8 using siRNA in BxPC-3 cells which express ANXA8 at a high level elevated caspase-3 and -7 activities. In in vitro invasion assay, inhibition of ANXA8 using siRNA in BxPC-3 reduced the numbers of migrating cells, and down-regulated HIF-1α mRNA transcription. Overexpression of ANXA8 increased the number of viable cells and BrdU incorporation in PANC-1 cells, which express ANXA8 at a low level. Expression of ANXA8 was induced under conditions of nutrient deprivation, and overexpression of ANXA8 showed resistance against serum starvation in PANC-1 cells. In a promoter assay, co-transfection with the expression vector of ANXA8 and the vector of a reporter gene containing the promoter of HIF-1α enhanced HIF-1α promoter activity. In contrast, this effect of ANXA8 was inhibited by administration of BAPTA-AM, an intracellular Ca²⁺ chelator. These results suggest that ectopic ANXA8 expression in cancer cells might involve an epigenetic mechanism. ANXA8 might play an important role in calcium fluctuation-mediated HIF-1α transcriptional activation and cell viability.

Alleviation of autophagy by knockdown of Beclin-1 enhances susceptibility of hippocampal neurons to proapoptotic signals induced by amino acid starvation

Autophagy has been described as a cellular response to stressful stimuli like starvation. One of its primary functions is to recycle amino acids from degraded proteins for cellular survival under nutrient deprived conditions. Autophagy is characterized by double membrane cytosolic vesicles called autophagosomes and prolonged autophagy is known to result in autophagic (Type II) cell death. Beclin-1 is involved in the regulation of autophagy in mammalian cells. This study examined the potential impact of knockdown of beclin-1 in an autophagic response in HT22 neurons challenged with amino acid starvation (AAS). AAS exposure induced light chain-3 (LC-3)-immunopositive and monodansylcadaverine (MDC) fluorescent dye-labeled autophagosome formation in cell bodies as early as 3 h post-AAS in wild type cells. Elevated levels of the autophagosome-targeting LC3-II were also observed following AAS. In addition, neuronal death induced by AAS in HT22-cells led to a moderate activation of caspase-3, a slight upregulation of AIF and did not alter the HtrA2 levels. Autophagy inhibition by a knockdown of beclin-1 significantly reduced AAS-induced LC3-II increase, reduced accumulation of autophagosomes, and potentiated AAS-mediated neuronal death. Collectively, this study shows that the both apoptotic and autophagic machineries are inducible in cultured hippocampal HT22 neurons subjected to AAS. Our data further show that attenuation of autophagy by a knockdown of beclin-1 enhanced neurons susceptibility to proapoptotic signals induced by AAS and underlines that autophagy is per se a protective than a deleterious mechanism.

Polysome Profiling in Liver Identifies Dynamic Regulation of Endoplasmic Reticulum Translatome by Obesity and Fasting

Obesity-associated metabolic complications are generally considered to emerge from abnormalities in carbohydrate and lipid metabolism, whereas the status of protein metabolism is not well studied. Here, we performed comparative polysome and associated transcriptional profiling analyses to study the dynamics and functional implications of endoplasmic reticulum (ER)–associated protein synthesis in the mouse liver under conditions of obesity and nutrient deprivation. We discovered that ER from livers of obese mice exhibits a general reduction in protein synthesis, and comprehensive analysis of polysome-bound transcripts revealed extensive down-regulation of protein synthesis machinery, mitochondrial components, and bile acid metabolism in the obese translatome. Nutrient availability also plays an important but distinct role in remodeling the hepatic ER translatome in lean and obese mice. Fasting in obese mice partially reversed the overall translatomic differences between lean and obese nonfasted controls, whereas fasting of the lean mice mimicked many of the translatomic changes induced by the development of obesity. The strongest examples of such regulations were the reduction in Cyp7b1 and Slco1a1, molecules involved in bile acid metabolism. Exogenous expression of either gene significantly lowered plasma glucose levels, improved hepatic steatosis, but also caused cholestasis, indicating the fine balance bile acids play in regulating metabolism and health. Together, our work defines dynamic regulation of the liver translatome by obesity and nutrient availability, and it identifies a novel role for bile acid metabolism in the pathogenesis of metabolic abnormalities associated with obesity.

Dysregulation of Autophagy in Murine Fibroblasts Resistant to HSV-1 Infection

The mouse L cell mutant, gro29, was selected for its ability to survive infection by herpes simplex virus type 1 (HSV-1). gro29 cells are fully susceptible to HSV-1 infection, however, they produce 2000-fold less infectious virus than parental L cells despite their capacity to synthesize late viral gene products and assemble virions. Because productive HSV-1 infection is presumed to result in the death of the host cell, we questioned how gro29 cells might survive infection. Using time-lapse video microscopy, we demonstrated that a fraction of infected gro29 cells survived infection and divided. Electron microscopy of infected gro29 cells, revealed large membranous vesicles that contained virions as well as cytoplasmic constituents. These structures were reminiscent of autophagosomes. Autophagy is an ancient cellular process that, under nutrient deprivation conditions, results in the degradation and catabolism of cytoplasmic components and organelles. We hypothesized that enhanced autophagy, and resultant degradation of virions, might explain the ability of gro29 to survive HSV-1 infection. Here we demonstrate that gro29 cells have enhanced basal autophagy as compared to parental L cells. Moreover, treatment of gro29 cells with 3-methyladenine, an inhibitor of autophagy, failed to prevent the formation of autophagosome-like organelles in gro29 cells indicating that autophagy was dysregulated in these cells. Additionally, we observed robust co-localization of the virion structural component, VP26, with the autophagosomal marker, GFP-LC3, in infected gro29 cells that was not seen in infected parental L cells. Collectively, these data support a model whereby gro29 cells prevent the release of infectious virus by directing intracellular virions to an autophagosome-like compartment. Importantly, induction of autophagy in parental L cells did not prevent HSV-1 production, indicating that the relationship between autophagy, virus replication, and survival of HSV-1 infection by gro29 cells is complex.

Role of TRIB3 in regulation of insulin sensitivity and nutrient metabolism during short-term fasting and nutrient excess

We have suggested previously that Tribbles homolog 3 (TRIB3), a negative regulator of Akt activity in insulin-sensitive tissues, could mediate glucose-induced insulin resistance in muscle under conditions of chronic hyperglycemia (Liu J, Wu X, Franklin JL, Messina JL, Hill HS, Moellering DR, Walton RG, Martin M, Garvey WT. Am J Physiol Endocrinol Metab 298: E565-E576, 2010). In the current study, we have assessed short-term physiological regulation of TRIB3 in skeletal muscle and adipose tissues by nutrient excess and fasting as well as TRIB3's ability to modulate glucose transport and mitochondrial oxidation. In Sprague-Dawley rats, we found that short-term fasting enhanced insulin sensitivity concomitantly with decrements in TRIB3 mRNA (66%, P < 0.05) and protein (81%, P < 0.05) in muscle and increments in TRIB3 mRNA (96%, P < 0.05) and protein (~10-fold, P < 0.05) in adipose tissue compared with nonfasted controls. On the other hand, rats fed a Western diet for 7 days became insulin resistant concomitantly with increments in TRIB3 mRNA (155%, P < 0.05) and protein (69%, P = 0.0567) in muscle and a decrease in the mRNA (76%, P < 0.05) and protein (70%, P < 0.05) in adipose. In glucose transport and mitochondria oxidation studies using skeletal muscle cells, we found that stable TRIB3 overexpression impaired insulin-stimulated glucose uptake without affecting basal glucose transport and increased both basal glucose oxidation and the maximal uncoupled oxygen consumption rate. With stable knockdown of TRIB3, basal and insulin-stimulated glucose transport rates were increased, whereas basal glucose oxidation and the maximal uncoupled oxygen consumption rate were decreased. In conclusion, TRIB3 impacts glucose uptake and oxidation oppositely in muscle and fat according to levels of nutrient availability. The above data for the first time implicate TRIB3 as a potent physiological regulator of insulin sensitivity and mitochondrial glucose oxidation under conditions of nutrient deprivation and excess.

MicroRNA-125b promotes apoptosis by regulating the expression of Mcl-1, Bcl-w and IL-6R

The microRNA miR-125b is multi-faceted, with the ability to function as a tumor suppressor or an oncogene, depending on the cellular context. To date, the pro-apoptotic role of miR-125b and its underlying mechanisms are unexplored. In this study, both gain- and loss-of-function experiments revealed that miR-125b expression not only induced spontaneous apoptosis in various cell lines derived from the liver, lung and colorectal cancers, but also sensitized cancer cells to diverse apoptotic stimuli, including nutrient starvation and chemotherapeutic treatment. Furthermore, downregulation of miR-125b was a frequent event in hepatocellular carcinoma (HCC) tissues, and the miR-125b level was positively associated with the rate of apoptosis in HCC tissues. Subsequent investigations identified Mcl-1, Bcl-w and interleukin (IL)-6R as direct targets of miR-125b. Restoration of miR-125b expression not only diminished the expression of Mcl-1 and Bcl-w directly but also indirectly reduced the Mcl-1 and Bcl-xL levels by attenuating IL-6/signal transducer and activator of transcription 3 signaling. Consistent with these findings, introduction of miR-125b reduced the mitochondrial membrane potential and promoted the cleavage of pro-caspase-3. These data indicate that miR-125b may promote apoptosis by suppressing the anti-apoptotic molecules of the Bcl-2 family and miR-125b downregulation may facilitate tumor development by conferring upon cells the capability to survive under conditions of nutrient deprivation and chemotherapeutic treatment. Our findings highlight the importance of miR-125b in the regulation of apoptosis and suggest miR-125b as an attractive target for anti-cancer therapy.

Glycolytic Inhibition Alters Anaplastic Thyroid Carcinoma Tumor Metabolism and Improves Response to Conventional Chemotherapy and Radiation

Anaplastic thyroid carcinoma (ATC) accounts for more than 50% of thyroid cancer mortality and is generally refractory to conventional treatment. On the basis of recent studies, we hypothesized that ATC metabolism can be targeted to improve response to chemoradiotherapy. Eight established and authenticated ATC cell lines were sequenced at 140 sites contained within 26 commonly mutated genes to identify novel potential therapeutic targets. Cellular proliferation, energy, and reducing potential stores were measured under conditions of specific nutrient deprivation. Tumor metabolism was evaluated using hyperpolarized (13)C MRI in a murine orthotopic xenograft model of ATC. Sensitivity to chemotherapeutic agents and radiation (XRT) was assayed using cytotoxicity assays. We identified mutations in BRAF, NRAS, and KIT but failed to identify generalized novel targets for therapeutic intervention. ATC cell lines exhibited a mesenchymal phenotype and generalized dependence on glucose for energy, reducing potential and survival. Glycolytic inhibition using 2-deoxyglucose (2-DG) sensitized ATC cells to conventional chemotherapy and external beam radiation. In vivo, 2-DG induced a transient, but significant reduction in ATC metabolic activity. Generalized dependence of ATC cells on glucose catabolism makes them susceptible to the sensitizing effects of 2-DG for radiation therapy and chemotherapy. Under in vivo conditions, 2-DG can inhibit ATC metabolism. However, the modest magnitude and transient nature of this effect suggest the need for antimetabolic agents with more favorable pharmacodynamics to achieve therapeutic effects.

ΦSP-3, a Salmonella-specific lytic phage capable of infecting its host under nutrient-deprived states

Salmonella is a robust pathogen capable of surviving under various hostile conditions. The ability of a Salmonella-specific lytic phage, ΦSP-3, to infect its host under various nutrient-deprived conditions was studied. This phage was isolated from the intestinal contents of chicken. The identity of ΦSP-3 was confirmed by sequence analysis whereby ΦSP-3 showed maximum similarity towards T5-like phages of family Siphoviridae. The genome size of ΦSP-3 was estimated to be 88.43 kb by pulsed-field gel electrophoresis (PFGE) analysis. ΦSP-3 was able to infect its host under stationary phase, multiple nutrient-starved states, carbon-starved and nitrogen-starved conditions. ΦSP-3 failed to multiply only under phosphate-starved condition. Host range studies revealed the genus specificity of ΦSP-3. A wide host range within the genus and the capability of infecting bacterial host cells in ideal as well as nutrient-deprived conditions makes ΦSP-3 a desirable candidate as a biocontrol agent.

Expression of chitinase genes of Metarhizium anisopliae isolates in lepidopteran pests and on synthetic media

Pathogenecity of the well characterized entomopathogenic fungus Metarhizium anisopliae used for biocontrol of a wide range of insect pests secretes hydrolytic enzymes that degrade the host cuticle. The chitinolytic activity of high and low virulent isolates of M. anisopliae was assayed on minimal medium (MM) + colloidal chitin and MM supplemented with insect cuticles. Ex- pression pattern of four chitinase genes (chitinase (chi), chi 1, chi 2, chi 3) was profiled during pathogenic stages of the entomopathogen under in vitro and in vivo conditions. Reverse-transcription polymerase chain reaction (RT-PCR) analysis confirmed that chitinase cDNAs were expressed during the germination of fungus under nutrient-deprived conditions. RT-PCR analysis performed for the four chitinase genes on the two insect hosts Spodoptera litura and Helicoverpa armigera at six developmental stages of the pathogen displayed up-regulation in S. litura at mycosed and conidiated condition while with H. armigera there was expression only after 48 h of incubation. Differential expression of chi, chi 1 and chi 2 genes in vitro (nitrogen rich and nitrogen limiting media) and in vivo (live insect hosts S. litura and H. armigera) implicate the role of substrate differences in pathogenesis.

Abstract 1133: Glucose deprivation-induced intracellular reactive oxygen species activates the PI3K-AKT axis

Abstract Glucose is the most efficient energy source and various cancer cells depend on glycolysis for their energy production. On the other hand, tumor microenvironment is often characterized by nutrient deprived conditions. Glucose concentration of cancer tissues was much lower than that of adjacent non-cancerous tissues in colorectal cancers and other solid tumors. To maintain the survival and proliferation of cancer cells, glucose sensing and adaptation to lean nutritional circumstance must be well organized. Glucose sensing machinery has been studied in yeast system. Snf3, which shares sequence similarity with glucose transporters, and other plasma membrane proteins of S. cerevisiae are able to sense the present of glucose. However, the molecular mechanism of glucose sensing in mammalian cells remains to be elucidated. We have reported that pancreatic cancer cells showed tolerance to glucose deprivation. In these cells, glucose deprivation rapidly induced AKT phosphorylation through PI3K activity. In addition, suppressed AKT expression reduced survival of pancreatic cancer-derived PANC-1 cells under nutrient deprived condition. We assumed that regulation of AKT is relevant to glucose sensing and we further investigated the underlying mechanisms. Glucose analogue 2-deoxyglucose (2-DG) is transported into cells and recognized by the yeast glucose sensing system whereas 2-DG is not phosphorylated by hexokinases in glycolytic system. 2-DG treatment, as well as glucose deprivation, led AKT phosphorylation in PANC-1 cells. Hence, glucose transport may not be involved in the sensing for the AKT phosphorylation but downstream metabolic events may play roles in this phenomenon. Variety of metabolic stresses reportedly induced reactive oxygen species. Glucose deprivation induced slight but significant intracellular hydrogen peroxide (H2O2) level in PANC-1 cells. An anti-oxidant reagent N-acetyl cysteine (NAC) reduced both glucose deprivation-induced H2O2 increase and AKT phosphorylation. Furthermore, low dose extracellular H2O2 treatment increased AKT phosphorylation and this effect was suppressed by PI3K inhibitor LY294002. These results strongly suggest that glucose deprivation-induced H2O2 mediated AKT phosphorylation. Src is involved in an alternative PI3K activating pathway, and Ossa, a scaffold protein as known as FAM120A, specifically activates Src-PI3K pathway under oxidative stress. Glucose deprivation-induced AKT phosphorylation was suppressed by a Src inhibitor PP2. Furthermore, reduction of Ossa expression specifically blocked the glucose deprivation-induced AKT phosphorylation in PANC-1 cells. Taken together, increased intracellular H2O2 evokes PI3K-AKT axis under glucose deprived condition and pivotal roles of Src and Ossa is clarified. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1133. doi:1538-7445.AM2012-1133

Abstract 784A: Profiling pancreatic cancer metabolism and tumor microenvironment for therapy

Abstract Pancreatic cancers have one of the highest fatality rates among all cancers and their response to therapies is highly variable. We have recently shown that inhibition of lactate dehydrogenase A (LDHA) and glutaminase (GLS) through small drug-like molecules causes pancreatic tumor regression. These studies indicate that targeting the metabolic reprogramming of cancer cells is a promising new approach to pancreatic cancer therapy. In order to translate these metabolic inhibitors to the clinic, our goal is to characterize and enable targeted selection of patients based on predicted metabolic response. Although genetic mutations may ultimately be the prime causes of early stage cancer, they can also lead to changes in cancer metabolism which enable cancer cells to proliferate and survive even under the hypoxic and nutrient-deprived conditions often encountered in the tumor microenvironment. The biochemical changes in cancer cells, which determine their behavior in terms of growth, survival and mobility, represent a functional readout of responses to external agents. We hypothesize that the metabolites of tumors that are sensitive to metabolic inhibitors are measurably different from those of resistant ones. We can therefore propose these metabolites as potential functional predictors of response. The proposed research will also study the metabolic reprogramming of cancer cells in the tumor microenvironment that can affect pancreatic cancer sensitivity to LDHA and GLS inhibition. The proposed experiments will use Metabolomics technology to trace the metabolic prototypes of pancreatic orthotopic xenografts to identify the metabolic signature of FX11 or BPTES-sensitive tumors as compared to resistant ones. For an analysis of the effects due to the tumor hypoxic microenvironment, we will use our innovative tool, the HypoxCR reporter system, which can simultaneously detect hypoxic cells and proliferating cells to define the effects of the pancreatic cancer tumor microenvironment on their sensitivity to metabolic inhibitors. This will be the first evaluation of the hypoxic tumor microenvironment in regards to how it affects the response of subfractions of pancreatic orthotopic xenograft tumor cells to metabolic inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 784A. doi:1538-7445.AM2012-784A

Damnacanthal from the Congolese Medicinal Plant Garcinia huillensis has a Potent Preferential Cytotoxicity against Human Pancreatic Cancer PANC‐1 Cells

Screening of eight Congolese medicinal plants showed that the CHCl(3) and MeOH extracts of Aframomum melegueta (PC(50) = 47.8 µg/mL and 13.8 µg/mL, respectively) and CHCl(3) extracts of Garcinia huillensis (PC(50) = 17.8 µg/mL) and Securidaca longepedunculata (PC(50) = 23.4 µg/mL) had preferential cytotoxicity against human pancreatic cancer PANC-1 cells under nutrient-deprived conditions. The active constituents of the CHCl(3) extract of G. huillensis were examined and 12 known anthraquinones were identified. Among them, damnacanthal (1) caused preferential necrotic cell death of PANC-1 and PSN-1 cells under nutrient-deprived and serum-sensitive conditions (PC(50) = 4.46 µm and 3.77 µm, respectively).