NOS2 Polymorphism in Aspect of Left and Right-Sided Colorectal Cancer.

Background: The NOS2 gene polymorphism rs2297518 is associated with an increased level of NO, which could contribute to colorectal cancer (CRC) development. We hypothesized that the potential influence of the NOS2 gene polymorphism on cancer development may vary between right-sided and left-sided colon cancers, and rectal cancers. The aim of this study was to determine the rs2297518 polymorphism influence on colorectal cancer development with regard to tumor localization. Methods: This case-control study included 199 patients with CRC and 120 controls. The qPCR endpoint genotyping was conducted using the TaqMan® genotyping assay. Results: This study revealed significant differences in tumor characteristic and in the minor alelle A frequency in the NOS2 genotype between colorectal cancers with different localizations. The mucinous adenocarcinoma was diagnosed significantly more often in right-sided cancers than in left-sided (30.6% vs. 10.9%, p = 0.009) and rectal cancers (30.6% vs. 7.1%, p = 0.0003). The minor allele A of the NOS2 genotype was observed more frequently in right-sided cancers than in left-sided cancers (44.9% vs. 23.1%, p = 0.0137) and more frequently in rectal cancers than in left-sided cancers (40.0% vs. 23.1%, p = 0.0285). Conclusions: In conclusion, the results support the hypothesis that the SNP rs2297518 of the NOS2 gene influences colorectal cancer development with regard to tumor localization.

Immunometabolism in type 2 diabetes mellitus: tissue-specific interactions.

The immune system is frequently described in the context of its protective function against infections and its role in the development of autoimmunity. For more than a decade, the interactions between the immune system and metabolic processes have been reported, in effect creating a new research field, termed immunometabolism. Accumulating evidence supports the hypothesis that the development of metabolic diseases may be linked to inflammation, and reflects, in some cases, the activation of immune responses. As such, immunometabolism is defined by 1) inflammation as a driver of disease development and/or 2) metabolic processes stimulating cellular differentiation of the immune components. In this review, the main factors capable of altering the immuno-metabolic communication leading to the development and establishment of obesity and diabetes are comprehensively presented. Tissue-specific immune responses suggested to impair metabolic processes are described, with an emphasis on the adipose tissue, gut, muscle, liver, and pancreas.

FK506-Binding Protein 2 Participates in Proinsulin Folding.

Apart from chaperoning, disulfide bond formation, and downstream processing, the molecular sequence of proinsulin folding is not completely understood. Proinsulin requires proline isomerization for correct folding. Since FK506-binding protein 2 (FKBP2) is an ER-resident proline isomerase, we hypothesized that FKBP2 contributes to proinsulin folding. We found that FKBP2 co-immunoprecipitated with proinsulin and its chaperone GRP94 and that inhibition of FKBP2 expression increased proinsulin turnover with reduced intracellular proinsulin and insulin levels. This phenotype was accompanied by an increased proinsulin secretion and the formation of proinsulin high-molecular-weight complexes, a sign of proinsulin misfolding. FKBP2 knockout in pancreatic ß-cells increased apoptosis without detectable up-regulation of ER stress response genes. Interestingly, FKBP2 mRNA was overexpressed in ß-cells from pancreatic islets of T2D patients. Based on molecular modeling and an in vitro enzymatic assay, we suggest that proline at position 28 of the proinsulin B-chain (P28) is the substrate of FKBP2's isomerization activity. We propose that this isomerization step catalyzed by FKBP2 is an essential sequence required for correct proinsulin folding.

Glutathione S-Transferase P1 Genetic Variant's Influence on the HbA1c Level in Type Two Diabetic Patients.

GST (glutathione S-transferases) are capable of influencing glucose homeostasis, probably through regulation of the response to oxidant stress. The aim of our study was to investigate the relationship between GSTP1 gene polymorphism and glycated hemoglobin (HbA1c) levels in type two diabetic (T2D) patients. A total of 307 T2D patients were included. Analysis of the GSTP1 gene polymorphism (rs1695) was conducted using the TaqMan qPCR method endpoint genotyping. HbA1c was determined using a COBAS 6000 autoanalyzer. A univariable linear regression and multivariable linear regression model were used to investigate the association between mean HbA1c level and GSTP1 gene polymorphism, age at T2D diagnosis, T2D duration, therapy with insulin, gender, BMI, smoking status. GSTP1 Val/Val genotype, age at T2D diagnosis, T2D duration and therapy with insulin were statistically significant contributors to HbA1c levels (p < 0.05). Multivariable regression analysis revealed that GSTP1 (Val/Val vs. Ile/Ile) was associated with higher HbA1c even after adjustment for variables that showed a statistically significant relationship with HbA1c in univariable analyses (p = 0.024). The results suggest that GSTP polymorphism may be one of the risk factors for higher HbA1c in T2D patients. Our study is limited by the relatively small sample size, cross-sectional design, and lack of inclusion of other oxidative stress-related genetic variants.

Defective Proinsulin Handling Modulates the MHC I Bound Peptidome and Activates the Inflammasome in ß-Cells.

How immune tolerance is lost to pancreatic ß-cell peptides triggering autoimmune type 1 diabetes is enigmatic. We have shown that loss of the proinsulin chaperone glucose-regulated protein (GRP) 94 from the endoplasmic reticulum (ER) leads to mishandling of proinsulin, ER stress, and activation of the immunoproteasome. We hypothesize that inadequate ER proinsulin folding capacity relative to biosynthetic need may lead to an altered ß-cell major histocompatibility complex (MHC) class-I bound peptidome and inflammasome activation, sensitizing ß-cells to immune attack. We used INS-1E cells with or without GRP94 knockout (KO), or in the presence or absence of GRP94 inhibitor PU-WS13 (GRP94i, 20 µM), or exposed to proinflammatory cytokines interleukin (IL)-1ß or interferon gamma (IFNγ) (15 pg/mL and 10 ng/mL, respectively) for 24 h. RT1.A (rat MHC I) expression was evaluated using flow cytometry. The total RT1.A-bound peptidome analysis was performed on cell lysates fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC), followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing protein (NLRP1), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and (pro) IL-1ß expression and secretion were investigated by Western blotting. GRP94 KO increased RT1.A expression in ß-cells, as did cytokine exposure compared to relevant controls. Immunopeptidome analysis showed increased RT1.A-bound peptide repertoire in GRP94 KO/i cells as well as in the cells exposed to cytokines. The GRP94 KO/cytokine exposure groups showed partial overlap in their peptide repertoire. Notably, proinsulin-derived peptide diversity increased among the total RT1.A peptidome in GRP94 KO/i along with cytokines exposure. NLRP1 expression was upregulated in GRP94 deficient cells along with decreased IκBα content while proIL-1ß cellular levels declined, coupled with increased secretion of mature IL-1ß. Our results suggest that limiting ß-cell proinsulin chaperoning enhances RT1.A expression alters the MHC-I peptidome including proinsulin peptides and activates inflammatory pathways, suggesting that stress associated with impeding proinsulin handling may sensitize ß-cells to immune-attack.

Peanut-Shaped Gold Nanoparticles with Shells of Ceragenin CSA-131 Display the Ability to Inhibit Ovarian Cancer Growth In Vitro and in a Tumor Xenograft Model.

Gold nanoparticles-assisted delivery of antineoplastics into cancerous cells is presented as an effective approach for overcoming the limitations of systemic chemotherapy. Although ceragenins show great potential as anti-cancer agents, in some tumors, effective inhibition of cancer cells proliferation requires application of ceragenins at doses within their hemolytic range. For the purpose of toxicity/efficiency ratio control, peanut-shaped gold nanoparticles (AuP NPs) were functionalized with a shell of ceragenin CSA-131 and the cytotoxicity of AuP@CSA-131 against ovarian cancer SKOV-3 cells and were then analyzed. In vivo efficiency of intravenously and intratumorally administered CSA-131 and AuP@CSA-131 was examined using a xenograft ovarian cancer model. Serum parameters were estimated using ELISA methods. Comparative analysis revealed that AuP@CSA-131 exerted stronger anti-cancer effects than free ceragenin, which was determined by enhanced ability to induce caspase-dependent apoptosis and autophagy processes via reactive oxygen species (ROS)-mediated pathways. In an animal study, AuP@CSA-131 was characterized by delayed clearance and prolonged blood circulation when compared with free ceragenin, as well as enhanced anti-tumor efficiency, particularly when applied intratumorally. Administration of CSA-131 and AuP@CSA-131 prevented the inflammatory response associated with cancer development. These results present the possibility of employing non-spherical gold nanoparticles as an effective nanoplatform for the delivery of antineoplastics for the treatment of ovarian malignancy.

MCPIP1 is a novel link between diabetogenic conditions and impaired insulin secretory capacity.

During diabetes development insulin production and glucose-stimulated insulin secretion (GSIS) are defective due to inflammation-related, yet not fully understood mechanisms. MCPIP1 (monocyte chemotactic protein-induced protein-1) is a strong regulator of inflammation, and acts predominantly as a specific RNase. The impact of MCPIP1 on insulin secretory capacity is unknown. We show that the expression of the ZC3H12A gene, which encodes MCPIP1, was induced by T1DM- and by T2DM-simulating conditions, with a stronger effect of cytokines. The number of MCPIP1-positive pancreatic islet-cells, including beta-cells, was significantly higher in diabetic compared to nondiabetic individuals. In the 3'UTR regions of mRNAs coding for Pdx1 (pancreatic and duodenal homeobox 1), FoxO1 (forkhead box protein O1), and of a novel regulator of insulin handling, Grp94 (glucose-regulated protein 94), MCPIP1-target structures were detected. Overexpression of the wild type MCPIP1wt, but not of the mutant MCPIP1D141N (lacking the RNase activity), decreased the expression of genes involved in insulin production and GSIS. Additionally INS1-E-MCPIP1wt cells exhibited a higher Ire1 (inositol-requiring enzyme 1) expression. MCPIP1wt overexpression blunted GSIS and glucose-mediated calcium influx with no deleterious effects on glucose uptake or glucokinase activity. We identify MCPIP1 as a new common link between diabetogenic conditions and beta-cell failure. MCPIP1 may serve as an interesting target for novel beta-cell protective approaches.

Enhancer of Zeste Homolog 2 (EZH2) Mediates Glucolipotoxicity-Induced Apoptosis in ß-Cells.

Selective inhibition of histone deacetylase 3 (HDAC3) prevents glucolipotoxicity-induced ß-cell dysfunction and apoptosis by alleviation of proapoptotic endoplasmic reticulum (ER) stress-signaling, but the precise molecular mechanisms of alleviation are unexplored. By unbiased microarray analysis of the ß-cell gene expression profile of insulin-producing cells exposed to glucolipotoxicity in the presence or absence of a selective HDAC3 inhibitor, we identified Enhancer of zeste homolog 2 (EZH2) as the sole target candidate. ß-Cells were protected against glucolipotoxicity-induced ER stress and apoptosis by EZH2 attenuation. Small molecule inhibitors of EZH2 histone methyltransferase activity rescued human islets from glucolipotoxicity-induced apoptosis. Moreover, EZH2 knockdown cells were protected against glucolipotoxicity-induced downregulation of the protective non-canonical Nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) pathway. We conclude that EZH2 deficiency protects from glucolipotoxicity-induced ER stress, apoptosis and downregulation of the non-canonical NFκB pathway, but not from insulin secretory dysfunction. The mechanism likely involves transcriptional regulation via EZH2 functioning as a methyltransferase and/or as a methylation-dependent transcription factor.

Glucose-Regulated Protein 94 (GRP94): A Novel Regulator of Insulin-Like Growth Factor Production.

Mammals have two insulin-like growth factors (IGF) that are key mediators of somatic growth, tissue differentiation, and cellular responses to stress. Thus, the mechanisms that regulate the bioavailability of IGFs are important in both normal and aberrant development. IGF-I levels are primarily controlled via the growth hormone-IGF axis, in response to nutritional status, and also reflect metabolic diseases and cancer. One mechanism that controls IGF bioavailablity is the binding of circulating IGF to a number of binding proteins that keep IGF in a stable, but receptor non-binding state. However, even before IGF is released from the cells that produce it, it undergoes an obligatory association with a ubiquitous chaperone protein, GRP94. This binding is required for secretion of a properly folded, mature IGF. This chapter reviews the known aspects of the interaction and highlights the specificity issues yet to be determined. The IGF-GRP94 interaction provides a potential novel mechanism of idiopathic short stature, involving the obligatory chaperone and not just IGF gene expression. It also provides a novel target for cancer treatment, as GRP94 activity can be either inhibited or enhanced.

The inducible ß5i proteasome subunit contributes to proinsulin degradation in GRP94-deficient ß-cells and is overexpressed in type 2 diabetes pancreatic islets.

Proinsulin is a misfolding-prone protein, and its efficient breakdown is critical when ß-cells are confronted with high-insulin biosynthetic demands, to prevent endoplasmic reticulum stress, a key trigger of secretory dysfunction and, if uncompensated, apoptosis. Proinsulin degradation is thought to be performed by the constitutively expressed standard proteasome, while the roles of other proteasomes are unknown. We recently demonstrated that deficiency of the proinsulin chaperone glucose-regulated protein 94 (GRP94) causes impaired proinsulin handling and defective insulin secretion associated with a compensated endoplasmic reticulum stress response. Taking advantage of this model of restricted folding capacity, we investigated the role of different proteasomes in proinsulin degradation, reasoning that insulin secretory dynamics require an inducible protein degradation system. We show that the expression of only one enzymatically active proteasome subunit, namely, the inducible ß5i-subunit, was increased in GRP94 CRISPR/Cas9 knockout (KO) cells. Additionally, the level of ß5i-containing intermediate proteasomes was significantly increased in these cells, as was ß5i-related chymotrypsin-like activity. Moreover, proinsulin levels were restored in GRP94 KO upon ß5i small interfering RNA-mediated knockdown. Finally, the fraction of ß-cells expressing the ß5i-subunit is increased in human islets from type 2 diabetes patients. We conclude that ß5i is an inducible proteasome subunit dedicated to the degradation of mishandled proinsulin.

The intermediate proteasome is constitutively expressed in pancreatic beta cells and upregulated by stimulatory, low concentrations of interleukin 1 ß.

A central and still open question regarding the pathogenesis of autoimmune diseases, such as type 1 diabetes, concerns the processes that underlie the generation of MHC-presented autoantigenic epitopes that become targets of autoimmune attack. Proteasomal degradation is a key step in processing of proteins for MHC class I presentation. Different types of proteasomes can be expressed in cells dictating the repertoire of peptides presented by the MHC class I complex. Of particular interest for type 1 diabetes is the proteasomal configuration of pancreatic ß cells, as this might facilitate autoantigen presentation by ß cells and thereby their T-cell mediated destruction. Here we investigated whether so-called inducible subunits of the proteasome are constitutively expressed in ß cells, regulated by inflammatory signals and participate in the formation of active intermediate or immuno-proteasomes. We show that inducible proteasomal subunits are constitutively expressed in human and rodent islets and an insulin-secreting cell-line. Moreover, the ß5i subunit is incorporated into active intermediate proteasomes that are bound to 19S or 11S regulatory particles. Finally, inducible subunit expression along with increase in total proteasome activities are further upregulated by low concentrations of IL-1ß stimulating proinsulin biosynthesis. These findings suggest that the ß cell proteasomal repertoire is more diverse than assumed previously and may be highly responsive to a local inflammatory islet environment.

In silico approach to predict pancreatic ß-cells classically secreted proteins.

Pancreatic ß-cells, residents of the islets of Langerhans, are the unique insulin-producers in the body. Their physiology is a topic of intensive studies aiming to understand the biology of insulin production and its role in diabetes pathology. However, investigations about these cells' subset of secreted proteins, the secretome, are surprisingly scarce and a list describing islet/ß-cell secretome upon glucose-stimulation is not yet available. In silico predictions of secretomes are an interesting approach that can be employed to forecast proteins likely to be secreted. In this context, using the rationale behind classical secretion of proteins through the secretory pathway, a Python tool capable of predicting classically secreted proteins was developed. This tool was applied to different available proteomic data (human and rodent islets, isolated ß-cells, ß-cell secretory granules, and ß-cells supernatant), filtering them in order to selectively list only classically secreted proteins. The method presented here can retrieve, organize, search and filter proteomic lists using UniProtKB as a central database. It provides analysis by overlaying different sets of information, filtering out potential contaminants and clustering the identified proteins into functional groups. A range of 70-92% of the original proteomes analyzed was reduced generating predicted secretomes. Islet and ß-cell signal peptide-containing proteins, and endoplasmic reticulum-resident proteins were identified and quantified. From the predicted secretomes, exemplary conservational patterns were inferred, as well as the signaling pathways enriched within them. Such a technique proves to be an effective approach to reduce the horizon of plausible targets for drug development or biomarkers identification.

High-precision measurements of δ2H, δ18O and δ17O in water with the aid of cavity ring-down laser spectroscopy.

A thorough evaluation of measurement uncertainty together with control of short-term and long-term precision of measurements should be a basis of any successful quality assurance/quality control (QA/QC) strategy aimed at maintaining a high quality of the analytical process. Here we present the results of a comprehensive assessment of the analytical performance of a Picarro L2140-i CRDS laser spectrometer analysing δ2H, δ18O and δ17O in water. The assessment is based on results obtained during 15 months of continuous operation of this instrument (February 2017 to May 2018). The short-term precision of measured and derived quantities was 0.11, 0.036, 0.028, 0.23 ‰ and 11 per meg, for δ2H, δ18O, δ17O, d-excess and Δ17O, respectively, and is comparable to the precision reported by the manufacturer. The long-term precision of the L2140-i, defined as standard uncertainty of the time series of 153 analyses of a laboratory standard conducted throughout 15 months, was roughly two times lower (0.24, 0.053, 0.038, 0.37 ‰ and 21 per meg, for δ2H, δ18O, δ17O, d-excess and Δ17O). In-depth assessment of the measurement uncertainty of a single analysis revealed that assigned uncertainty of the calibration standards is an important component of the uncertainty budget, especially in case of δ2H analysis.

Endoplasmic Reticulum Chaperone Glucose-Regulated Protein 94 Is Essential for Proinsulin Handling.

Although endoplasmic reticulum (ER) chaperone binding to mutant proinsulin has been reported, the role of protein chaperones in the handling of wild-type proinsulin is underinvestigated. Here, we have explored the importance of glucose-regulated protein 94 (GRP94), a prominent ER chaperone known to fold insulin-like growth factors, in proinsulin handling within ß-cells. We found that GRP94 coimmunoprecipitated with proinsulin and that inhibition of GRP94 function and/or expression reduced glucose-dependent insulin secretion, shortened proinsulin half-life, and lowered intracellular proinsulin and insulin levels. This phenotype was accompanied by post-ER proinsulin misprocessing and higher numbers of enlarged insulin granules that contained amorphic material with reduced immunogold staining for mature insulin. Insulin granule exocytosis was accelerated twofold, but the secreted insulin had diminished bioactivity. Moreover, GRP94 knockdown or knockout in ß-cells selectively activated protein kinase R-like endoplasmic reticulum kinase (PERK), without increasing apoptosis levels. Finally, GRP94 mRNA was overexpressed in islets from patients with type 2 diabetes. We conclude that GRP94 is a chaperone crucial for proinsulin handling and insulin secretion.

Unexpected profile of sphingolipid contents in blood and bone marrow plasma collected from patients diagnosed with acute myeloid leukemia.

BACKGROUND: Impaired apoptotic pathways in leukemic cells enable them to grow in an uncontrolled way. Moreover, aberrations in the apoptotic pathways are the main factor of leukemic cells drug resistance. METHODS: To assess the presence of potential abnormalities that might promote dysfunction of leukemic cells growth, HPLC system was used to determine sphingosine (SFO), sphinganine (SFA), sphingosine-1-phosphate (S1P) and ceramide (CER) concentration in the blood collected from patients diagnose with acute myeloblastic leukemia (AML; n = 49) and compare to values of control (healthily) group (n = 51). Additionally, in AML group concentration of SFO, SFA, S1P and CER was determined in bone marrow plasma and compared to respective values in blood plasma. The concentration of S1P and CER binding protein - plasma gelsolin (GSN) was also assessed in collected samples using immunoblotting assay. RESULTS: We observed that in AML patients the average SFO, SFA and CER concentration in blood plasma was significantly higher (p < 0.001) compare to control group, when blood plasma S1P concentration was significantly lower (p < 0.001). At the same time the CER/S1P ratio in AML patient (44.5 ± 19.4) was about 54% higher compare to control group (20.9 ± 13.1). Interestingly the average concentration of S1P in blood plasma (196 ± 13 pmol/ml) was higher compare to its concentration in plasma collected from bone marrow (154 ± 21 pmol/ml). CONCLUSIONS: We hypothesize that changes in profile of sphingolipids concentration and some of their binding protein partners such as GSN in extracellular environment of blood and bone marrow cells in leukemic patients can be targeted to develop new AML treatment method(s).

A Human Variant of Glucose-Regulated Protein 94 That Inefficiently Supports IGF Production.

IGFs are critical for normal intrauterine and childhood growth and sustaining health throughout life. We showed previously that the production of IGF-1 and IGF-2 requires interaction with the chaperone glucose-regulated protein 94 (GRP94) and that the amount of secreted IGFs is proportional to the GRP94 activity. Therefore, we tested the hypothesis that functional polymorphisms of human GRP94 affect IGF production and thereby human health. We describe a hypomorphic variant of human GRP94, P300L, whose heterozygous carriers have 9% lower circulating IGF-1 concentration. P300L was found first in a child with primary IGF deficiency and was later shown to be a noncommon single-nucleotide polymorphism with frequencies of 1%-4% in various populations. When tested in the grp94(-/-) cell-based complementation assay, P300L supported only approximately 58% of IGF secretion relative to wild-type GRP94. Furthermore, recombinant P300L showed impaired nucleotide binding activity. These in vitro data strongly support a causal relationship between the GRP94 variant and the decreased concentration of circulating IGF-1, as observed in human carriers of P300L. Thus, mutations in GRP94 that affect its IGF chaperone activity represent a novel causal genetic mechanism that limits IGF biosynthesis, quite a distinct mechanism from the known genes in the GH/IGF signaling network.

Utility of blood procalcitonin concentration in the management of cancer patients with infections.

Diagnosis of infections in cancer patients is usually problematic since differentiating between infection and fever of unknown origin is often a considerable clinical challenge. In general, increase concentration of blood procalcitonin (PCT) is associated with severe bacterial infection. PCT with an optimal cutoff level of 0.5 ng/mL seems to be the most helpful biochemical parameter in detecting severe infections, mainly bloodstream infection, in patients with hematological cancers. In all clinical situations, the elevated level of PCT should be carefully analyzed, always with a thorough physical examination and an appropriate microbiological assessment.

Malignant transformation of CD4+ T lymphocytes mediated by oncogenic kinase NPM/ALK recapitulates IL-2-induced cell signaling and gene expression reprogramming.

Anaplastic lymphoma kinase (ALK), physiologically expressed only by nervous system cells, displays a remarkable capacity to transform CD4(+) T lymphocytes and other types of nonneural cells. In this study, we report that activity of nucleophosmin (NPM)/ALK chimeric protein, the dominant form of ALK expressed in T cell lymphomas (TCLs), closely resembles cell activation induced by IL-2, the key cytokine supporting growth and survival of normal CD4(+) T lymphocytes. Direct comparison of gene expression by ALK(+) TCL cells treated with an ALK inhibitor and IL-2-dependent ALK(-) TCL cells stimulated with the cytokine revealed a very similar, albeit inverse, gene-regulation pattern. Depending on the analysis method, up to 67% of the affected genes were modulated in common by NPM/ALK and IL-2. Based on the gene expression patterns, Jak/STAT- and IL-2-signaling pathways topped the list of pathways identified as affected by both IL-2 and NPM/ALK. The expression dependence on NPM/ALK and IL-2 of the five selected genes-CD25 (IL-2Rα), Egr-1, Fosl-1, SOCS3, and Irf-4-was confirmed at the protein level. In both ALK(+) TCL and IL-2-stimulated ALK(-) TCL cells, CD25, SOCS3, and Irf-4 genes were activated predominantly by the STAT5 and STAT3 transcription factors, whereas transcription of Egr-1 and Fosl-1 was induced by the MEK-ERK pathway. Finally, we found that Egr-1, a protein not associated previously with either IL-2 or ALK, contributes to the cell proliferation. These findings indicate that NPM/ALK transforms the target CD4(+) T lymphocytes, at least in part, by using the pre-existing, IL-2-dependent signaling pathways.

Decreased lactate concentration and glycolytic enzyme expression reflect inhibition of mTOR signal transduction pathway in B-cell lymphoma.

The application of kinase inhibitors in cancer treatment is growing rapidly. However, methods for monitoring the effectiveness of the inhibitors are still poorly developed and currently rely mainly on the tracking of changes in the tumor volume, a rather late and relatively insensitive marker of the therapeutic response. In contrast, MRS can detect changes in cell metabolism and has the potential to provide early and patient-specific markers of drug activity. Using human B-cell lymphoma models and MRS, we have demonstrated that the inhibition of the mTOR signaling pathway can be detected in malignant cells in vitro and noninvasively in vivo by the measurement of lactate levels. An mTOR inhibitor, rapamycin, suppressed lactic acid production in lymphoma cell line cultures and also diminished steady-state lactate levels in xenotransplants. The inhibition was time dependent and was first detectable 8 h after drug administration in cell cultures. In xenotransplants, 2 days of rapamycin treatment produced significant changes in lactic acid concentration in the tumor measured in vivo, which were followed by tumor growth arrest and tumor volume regression. The rapamycin-induced changes in lactate production were strongly correlated with the inhibition of expression of hexokinase II, the key enzyme in the glycolytic pathway. These studies suggest that MRS or (18) F-fluorodeoxyglucose positron emission tomography (FDG PET) detection of changes in glucose metabolism may represent effective noninvasive methods for the monitoring of mTOR targeting therapy in lymphomas and other malignancies. Furthermore, the measurement of glucose metabolic inhibition by MRS or FDG PET imaging may also prove to be effective in monitoring the efficacy of other kinase inhibitors given that the rapamycin-sensitive mTOR lies downstream of many oncogenic signaling pathways.