Context-dependent roles for ubiquitous mitochondrial creatine kinase CKMT1 in breast cancer progression.

Metabolic reprogramming is a hallmark of cancer, enabling cancer cells to rapidly proliferate, invade, and metastasize. We show that creatine levels in metastatic breast cancer cell lines and secondary metastatic tumors are driven by the ubiquitous mitochondrial creatine kinase (CKMT1). We discover that, while CKMT1 is highly expressed in primary tumors and promotes cell viability, it is downregulated in metastasis. We further show that CKMT1 downregulation, as seen in breast cancer metastasis, drives up mitochondrial reactive oxygen species (ROS) levels. CKMT1 downregulation contributes to the migratory and invasive potential of cells by ROS-induced upregulation of adhesion and degradative factors, which can be reversed by antioxidant treatment. Our study thus reconciles conflicting evidence about the roles of metabolites in the creatine metabolic pathway in breast cancer progression and reveals that tight, context-dependent regulation of CKMT1 expression facilitates cell viability, cell migration, and cell invasion, which are hallmarks of metastatic spread.

Primary breast tumor induced extracellular matrix remodeling in premetastatic lungs.

The premetastatic niche hypothesis proposes an active priming of the metastatic site by factors secreted from the primary tumor prior to the arrival of the first cancer cells. We investigated several extracellular matrix (ECM) structural proteins, ECM degrading enzymes, and ECM processing proteins involved in the ECM remodeling of the premetastatic niche. Our in vitro model consisted of lung fibroblasts, which were exposed to factors secreted by nonmalignant breast epithelial cells, nonmetastatic breast cancer cells, or metastatic breast cancer cells. We assessed ECM remodeling in vivo in premetastatic lungs of female mice growing orthotopic primary breast tumor xenografts, as compared to lungs of control mice without tumors. Premetastatic lungs contained significantly upregulated Collagen (Col) Col4A5, matrix metalloproteinases (MMPs) MMP9 and MMP14, and decreased levels of MMP13 and lysyl oxidase (LOX) as compared to control lungs. These in vivo findings were consistent with several of our in vitro cell culture findings, which showed elevated Col14A1, Col4A5, glypican-1 (GPC1) and decreased Col5A1 and Col15A1 for ECM structural proteins, increased MMP2, MMP3, and MMP14 for ECM degrading enzymes, and decreased LOX, LOXL2, and prolyl 4-hydroxylase alpha-1 (P4HA1) for ECM processing proteins in lung fibroblasts conditioned with metastatic breast cancer cell media as compared to control. Taken together, our data show that premetastatic priming of lungs by primary breast tumors resulted in significant ECM remodeling which could facilitate metastasis by increasing interstitial fibrillar collagens and ECM stiffness (Col14A1), disruptions of basement membranes (Col4A5), and formation of leaky blood vessels (MMP2, MMP3, MMP9, and MMP14) to promote metastasis.

A multimodal pipeline using NMR spectroscopy and MALDI-TOF mass spectrometry imaging from the same tissue sample.

NMR spectroscopy and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) are both commonly used to detect large numbers of metabolites and lipids in metabolomic and lipidomic studies. We have demonstrated a new workflow, highlighting the benefits of both techniques to obtain metabolomic and lipidomic data, which has realized for the first time the combination of these two complementary and powerful technologies. NMR spectroscopy is frequently used to obtain quantitative metabolite information from cells and tissues. Lipid detection is also possible with NMR spectroscopy, with changes being visible across entire classes of molecules. Meanwhile, MALDI MSI provides relative measures of metabolite and lipid concentrations, mapping spatial information of many specific metabolite and lipid molecules across cells or tissues. We have used these two complementary techniques in combination to obtain metabolomic and lipidomic measurements from triple-negative human breast cancer cells and tumor xenograft models. We have emphasized critical experimental procedures that ensured the success of achieving NMR spectroscopy and MALDI MSI in a combined workflow from the same sample. Our data show that several phospholipid metabolite species were differentially distributed in viable and necrotic regions of breast tumor xenografts. This study emphasizes the power of combined NMR spectroscopy-MALDI imaging to advance metabolomic and lipidomic studies.

Unlabeled aspirin as an activatable theranostic MRI agent for breast cancer.

Rationale: Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is emerging as an alternative to gadolinium-based contrast MRI. We have evaluated the possibility of CEST MRI of orthotopic breast tumor xenografts with unlabeled aspirin's conversion to salicylic acid (SA) through various enzymatic activities, most notably inhibition of cyclooxygenase (COX)-1/-2 enzymes. Methods: We measured the COX-1/-2 expression in four breast cancer cell lines by Western Blot analysis and selected the highest and lowest expressing cell lines. We then performed CEST MRI following aspirin treatment to detect SA levels and ELISA to measure levels of downstream prostaglandin E2 (PGE2). We also injected aspirin into the tail vein of mice growing orthotopic tumor xenografts which expressed high and low COX-1/-2 and acquired SA CEST MR images of these tumor xenografts for up to 70 minutes. Tumors were then harvested to perform Western Blot and ELISA experiments to measure COX-1/-2 expression and PGE2 levels, respectively. Results: Western Blots determined that SUM159 cells contained significantly higher COX-1/-2 expression levels than MDA-MB-231 cells, in line with higher levels of downstream PGE2. SA CEST MRI yielded similar contrast at approximately 3% for both cell lines, independent of COX-1/-2 expression level. PGE2 levels decreased by about 50% following aspirin treatment. Results from our mouse study aligned with cultured cells, the overall SA CEST MRI contrast in both MDA-MB-231 and SUM159 tumor xenograft models was 5~8% at one hour post injection. PGE2 levels were ten times higher in SUM159 than MDA-MB-231 and decreased by 50%. The CEST contrast directly depended on the injected dose, with ~6%, ~3% and ~1.5% contrast observed following injection of 100 µL of 300 mM, 200 mM and 150 mM aspirin, respectively. Conclusions: Our data demonstrate the feasibility of using aspirin as a noninvasive activatable CEST MRI contrast agent for breast tumor detection.

Kinetic and thermodynamic characterization of novel alkaline lipase from halotolerant Bacillus gibsonii.

A halotolerant bacterial strain isolated and identified as Bacillus gibsonii was used for extracellular lipase production. The bacterial strain was able to grow up to 1200 mM salt concentration and showed maximum growth at 600 mM NaCl concentration. The present study includes production of extracellular lipase enzyme and characterization of partially purified lipase with respect to its kinetic and thermodynamic behaviour. Maximum lipase activity was observed at 60 °C under alkaline pH (9.0) condition. The kinetic parameters such as Vmax, Km and Kcat were calculated as 158.73 U/mL, 0.539 mM and 483.93 min-1 at 60 °C, respectively, suggested thermostable nature of the enzyme. The thermal inactivation energy [Ea(d)] was calculated as 66.98 kJ/mol. The values of Gibb's free energy (86.31 kJ/mol), enthalpy (64.26 kJ/mol) and entropy (- 66.21 × 10-3 kJ/mol/K) for the enzyme inactivation obtained at 60 °C corroborated the assumption that 60 °C was the optimum temperature. Further, the deactivation rate constant (kd) values calculated at 60 °C and 80 °C were found to be 0.0907 and 0.182 min-1, respectively, which suggested that enzyme was more stable at 60 °C and it was partly inactivated at 80 °C.

Focus on the glycerophosphocholine pathway in choline phospholipid metabolism of cancer.

Activated choline metabolism is a hallmark of carcinogenesis and tumor progression, which leads to elevated levels of phosphocholine and glycerophosphocholine in all types of cancer tested so far. Magnetic resonance spectroscopy applications have played a key role in detecting these elevated choline phospholipid metabolites. To date, the majority of cancer-related studies have focused on phosphocholine and the Kennedy pathway, which constitutes the biosynthesis pathway for membrane phosphatidylcholine. Fewer and more recent studies have reported on the importance of glycerophosphocholine in cancer. In this review article, we summarize the recent literature on glycerophosphocholine metabolism with respect to its cancer biology and its detection by magnetic resonance spectroscopy applications.

The insertion sequence of the N2A region of titin exists in an extended structure with helical characteristics.

The giant sarcomere protein titin is the third filament in muscle and is integral to maintaining sarcomere integrity as well as contributing to both active and passive tension. Titin is a multi-domain protein that contains regions of repeated structural elements. The N2A region sits at the boundary between the proximal Ig region of titin that is extended under low force and the PEVK region that is extended under high force. Multiple binding interactions have been associated with the N2A region and it has been proposed that this region acts as a mechanical stretch sensor. The focus of this work is a 117 amino acid portion of the N2A region (N2A-IS), which resides between the proximal Ig domains and the PEVK region. Our work has shown that the N2A-IS region is predicted to contain helical structure in the center while both termini are predicted to be disordered. Recombinantly expressed N2A-IS protein contains 13% α-helical structure, as measured via circular dichroism. Additional α-helical structure can be induced with 2,2,2-trifluoroethanol, suggesting that there is transient helical structure that might be stabilized in the context of the entire N2A region. The N2A-IS region does not exhibit any cooperativity in either thermal or chemical denaturation studies while size exclusion chromatography and Fluorescence Resonance Energy Transfer demonstrates that the N2A-IS region has an extended structure. Combined, these results lead to a model of the N2A-IS region having a helical core with extended N- and C-termini.

Gallstones: A Worldwide Multifaceted Disease and Its Correlations with Gallbladder Carcinoma.

BACKGROUND: Gallstones (GS) associated diseases are among the most recurrent and frequent diseases delineated in India and United Arab Emirates. Several reports suggest that the association of GS with gallbladder cancer (GBC) is very high in Northern part of India; however, its occurrence in UAE and Southern part of India is notably low. Therefore, in the present study, we aimed to perform compositional analysis of GS in three different geographical areas by Solid State Nuclear Magnetic Resonance and Fourier Transformed Infrared spectroscopy. METHODS: Natural abundance 13C cross polarization magic angle spinning Nuclear Magnetic Resonance and Fourier Transform Infrared spectroscopy is employed for the analysis of human gallstone. RESULTS: Cholesterol, bilirubin and calcium carbonate were present in variant concentrations in GS obtained from three different geographical regions. Cholesterol was present predominantly in gallstones from North India. Bilirubin was found to be a main constituent in gallstones pertaining to South India. Whereas GS from UAE showed both cholesterol and bilirubin as their major constituents. Calcium carbonate was found in varying concentrations in gallstones acquired from different regions. CONCLUSION: Variation in environmental condition and dietary habits may contribute and affect the GS formation. Alterations in bile composition influence the GB and augment the crystallization of cholesterol. Analysis of different geographical regions GS could be an important stride to understand the etiology of GS diseases.

Metabonomic study of host-phage interaction by nuclear magnetic resonance- and statistical total correlation spectroscopy-based analysis.

We present a method for the qualitative and quantitative study of transient metabolic flux of phage infection at the molecular level. The method is based on statistical total correlation spectroscopy (STOCSY) and partial least squares discriminant analysis (PLS-DA) applied to nuclear magnetic resonance (NMR) metabonomic data sets. An algorithm for this type of study is developed and demonstrated. The method has been implemented on (1)H NMR data sets of growth media in planktonic cultures of Pseudomonas aeruginosa infected with bacteriophage pf1. Transient metabolic flux of various important metabolites, identified by STOCSY and PLS-DA analysis applied to the NMR data set, are estimated at various stages of growth. The opportunistic and nosocomial pathogen P. aeruginosa is one of the best-studied model organism for bacterial biofilms. Complete information regarding metabolic connectivity of this system is not possible by conventional spectroscopic approach. Our study presents temporal comparative (1)H NMR metabonomic analyses of filamentous phage pf1 infection in planktonic cultures of P. aeruginosa K strain (PAK). We exemplify here the potential of STOCSY and PLS-DA tools to gain mechanistic insight into subtle changes and to determine the transient flux associated with metabolites following metabolic perturbations resulting from phage infection. Our study has given new avenues in correlating existing postgenomic data with current metabonomic results in P. aeruginosa biofilms research.

Complete 13C NMR chemical shifts assignment for cholesterol crystals by combined CP-MAS spectral editing and ab initio GIPAW calculations with dispersion forces.

We report here the first fully ab initio determination of (13)C NMR spectra for several crystal structures of cholesterol, observed in various biomaterials. We combine Gauge-Including Projector Augmented Waves (GIPAW) calculations at relaxed structures, fully including dispersion forces, with Magic Angle Spinning Solid State NMR experiments and spectral editing to achieve a detailed interpretation of the complex NMR spectra of cholesterol crystals. By introducing an environment-dependent secondary referencing scheme in our calculations, not only do we reproduce the characteristic spectral features of the different crystalline polymorphs, thus clearly discriminating among them, but also closely represent the spectrum in the region of several highly overlapping peaks. This, in combination with spectral editing, allows us to provide a complete peak assignment for monohydrate (ChM) and low-temperature anhydrous (ChAl) crystal polymorphs. Our results show that the synergy between ab initio calculations and refined experimental techniques can be exploited for an accurate and efficient NMR crystallography of complex systems of great interest for biomaterial studies. The method is general in nature and can be applied for studies of various complex biomaterials.

Lipid profiling of cancerous and benign gallbladder tissues by 1H NMR spectroscopy.

Qualitative and quantitative (1) H NMR analysis of lipid extracts of gallbladder tissue in chronic cholecystitis (CC, benign) (n = 14), xanthogranulomatous cholecystitis (XGC, intermediate) (n = 9) and gallbladder cancer (GBC, malignant) (n = 8) was carried out to understand the mechanisms involved in the transformation of benign gallbladder tissue to intermediate and malignant tissue. The results revealed alterations in various tissue lipid components in gallbladder in CC, XGC and GBC. The difference in the nature of lipid components in benign and malignant disease may aid in the identification of the biological pathways involved in the etiopathogenesis of GBC. This is the first study on lipid profiling of gallbladder tissue by (1) H NMR spectroscopy, and has possible implications for the development of future diagnostic approaches.

Solid state (13)C NMR analysis of human gallstones from cancer and benign gall bladder diseases.

Natural abundance (13)C cross polarized (CP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) analysis of human gall bladder stones collected from patients suffering from malignant and benign gall bladder disease was carried out which revealed different polymorphs of cholesterol in these stones. All gall bladder stones in present study had cholesterol as their main constituent. (13)C CP-MAS NMR analysis revealed three forms of cholesterol molecules in these stones, which are anhydrous form, monohydrate crystalline with amorphous form and monohydrate crystalline form. Our study revealed that stones collected from patients associated with chronic cholecystitis (CC) disease have mostly different polymorph of cholesterol than stones collected from patients associated with gall bladder cancer (GBC). Such study will be helpful in understanding the mechanism of formation of gallstones which are associated with different gall bladder diseases. This is the first study by solid state NMR revealing different crystal polymorphism of cholesterol in human gallstones, extending the applicability of (13)C CP-MAS NMR technique for the routine study of gallstones.