Targeting pancreatic cancer with combinatorial treatment of CPI-613 and inhibitors of lactate metabolism.

Pancreatic cancer is the fourth leading cause of cancer death, with a 5-year survival rate of 10%. A stagnant high mortality rate over the last decades highlights the need for innovative therapeutic approaches. Pancreatic tumors pursue an altered metabolism in order to maintain energy generation under low nutrient influx and hypoxic conditions. Targeting these metabolic strategies might therefore be a reasonable therapeutic approach for pancreatic cancer. One promising agent is CPI- 613, a potent inhibitor of two enzymes of the tricarboxylic acid cycle. The present study evaluated the anti-cancerous efficacy of CPI-613 in combination with galloflavin, a lactate dehydrogenase inhibitor or with alpha-cyano-4-hydroxycinnamic acid, an inhibitor of monocarboxylate transporters. The efficacy of both combination therapies was tested in vitro on one human and two murine pancreatic cancer cell lines and in vivo in an orthotopic pancreatic cancer model. Tumor progression was evaluated by MRI and 18F-FDG PET-CT. Both combinatorial treatments demonstrated in vitro a significant inhibition of pancreatic cancer cell proliferation and induction of cell death. In contrast to the in vitro results, both combination therapies did not significantly reduce tumor growth in vivo. The in vitro results suggest that a combined inhibition of different metabolic pathways might be a promising approach for cancer therapy. However, the in vivo experiments indicate that applying a higher dosage or using other drugs targeting these metabolic pathways might be more promising.

MicroRNAs as systemic biomarkers to assess distress in animal models for gastrointestinal diseases.

Severity assessment of animal experiments is mainly conducted by using subjective parameters. A widely applicable biomarker to assess animal distress could contribute to an objective severity assessment in different animal models. Here, the distress of three murine animal models for gastrointestinal diseases was assessed by multiple behavioral and physiological parameters. To identify possible new biomarkers for distress 750 highly conserved microRNAs were measured in the blood plasma of mice before and after the induction of pancreatitis. Deregulated miRNA candidates were identified and further quantified in additional animal models for pancreatic cancer and cholestasis. MiR-375 and miR-203 were upregulated during pancreatitis and down regulated during cholestasis, whereas miR-132 was upregulated in all models. Correlation between miR-132 and plasma corticosterone concentrations resulted in the highest correlation coefficient, when compared to the analysis of miR-375, miR-203 and miR-30b. These results indicate that miR-132 might function as a general biomarker for distress, whereas the other miRNAs were altered in a disease specific manner. In conclusion, plasma miRNA profiling may help to better characterize the level of distress in mouse models for gastrointestinal diseases.

Galloflavin Plus Metformin Treatment Impairs Pancreatic Cancer Cells.

BACKGROUND/AIM: In this study, we evaluated the effect of galloflavin, an inhibitor of lactate dehydrogenase, in combination with metformin, an anti-diabetic drug and inhibitor of oxidative phosphorylation, on pancreatic ductal adenocarcinoma cells. MATERIALS AND METHODS: We explored the effect of galloflavin and metformin on proliferation and cell death of murine 6606PDA and human MIA PaCa-2 cells. RESULTS: We observed that monotherapies of galloflavin and metformin both inhibit proliferation and induce cancer cell death. Moreover, the combination of both agents increased these effects on pancreatic ductal adenocarcinoma cells. The inhibition of proliferation by this combination therapy can be detected under hypoxic and normoxic conditions, leading to the assumption that this therapy might impair insufficiently supplied solid tumors as well as small clusters of cancer cells, e.g. after metastatic dissemination. CONCLUSION: Galloflavin, especially in combination with metformin, has a strong anti-cancerous effect on pancreatic ductal adenocarcinoma cells.

Benefits of non-invasive methods compared to telemetry for distress analysis in a murine model of pancreatic cancer.

Prospective severity assessment is legally required in many countries to ensure high-quality research along with high welfare standards for laboratory animals. Mice and rats, the most common laboratory species, are prey animals that usually suppress signs of pain and suffering. Therefore, highly sensitive readout parameters are necessary to adequately quantify distress. The present study compared the performance of different non-invasive methods in determining animal distress, such as measuring body weight, distress score, faecal corticosterone metabolites, burrowing, and nesting behaviour, with continuous monitoring of heart rate, body temperature and activity by telemetry. The distress caused by two surgical interventions was compared and the burden caused by tumour growth was described. Transmitter implantation caused higher distress than laparotomy plus carcinoma cell injection into the pancreas. Surprisingly, no significant increase in distress was observed during tumour growth. The receiver operating characteristic curve analysis revealed that some non-invasive distress-parameters, i.e., distress-score and burrowing activity, exhibited slightly better performance to quantify distress than the most suitable parameters measured by telemetry. Due to the high burden caused by the implantation of the telemetric device, the use of non-invasive methods to assess distress in laboratory animals after surgical interventions should be favoured in future studies.

A novel multi-parametric analysis of non-invasive methods to assess animal distress during chronic pancreatitis.

Ethical responsibility, legal requirements and the need to improve the quality of research create a growing interest in the welfare of laboratory animals. Judging the welfare of animals requires readout parameters, which are valid and sensitive as well as specific to assess distress after different interventions. In the present study, we evaluated the sensitivity and specificity of different non-invasive parameters (body weight change, faecal corticosterone metabolites concentration, burrowing and nesting activity) by receiver operating characteristic curves and judged the merit of a multi-parametric analysis by logistic regression. Chronic pancreatitis as well as laparotomy caused significant changes in all parameters. However, the accuracy of these parameters was different between the two animal models. In both animal models, the multi-parametric analysis relying on all the readout parameters had the highest accuracy when predicting distress. This multi-parametric analysis revealed that C57BL/6 mice during the course of chronic pancreatitis often experienced less distress than mice after laparotomy. Interestingly these data also suggest that distress does not steadily increase during chronic pancreatitis. In conclusion, combining these non-invasive methods for severity assessment represents a reliable approach to evaluate animal distress in models such as chronic pancreatitis.

Blocking Autophagy in Cancer-Associated Fibroblasts Supports Chemotherapy of Pancreatic Cancer Cells.

In this study we evaluated the interaction of pancreatic cancer cells, cancer-associated fibroblasts, and distinct drugs such as α-cyano-4-hydroxycinnamate, metformin, and gemcitabine. We observed that α-cyano-4-hydroxycinnamate as monotherapy or in combination with metformin could significantly induce collagen I deposition within the stromal reaction. Subsequently, we demonstrated that cancer-associated fibroblasts impaired the anti-proliferation efficacy of α-cyano-4-hydroxycinnamate, metformin and gemcitabine. Interestingly, inhibition of autophagy in these fibroblasts can augment the anti-proliferation effect of these chemotherapeutics in vitro and can reduce the tumor weight in a syngeneic pancreatic cancer model. These results suggest that inhibiting autophagy in cancer-associated fibroblasts may contribute to strategies targeting cancer.

Structural adaptability of zinc binding sites: different structures in partially, fully, and heavy-metal loaded states.

The present study demonstrates that both the nature (Zn(II), Cd(II) or Hg(II)) and supply of metal ions determine whether zinc fingers fold into the well-known, fully loaded structures or alternatively populate a variety of structural states under substoichiometric conditions. Metal-bridged species are observed by perturbed angular correlation (PAC), EXAFS, UV spectroscopy, and stopped-flow kinetics. Transitions between structural states as adaptive reactions to changed metal-ion supply might represent intelligent system changes in zinc homeostasis, trafficking and signalling, and reflect features of heavy-metal toxicity at the molecular level. Because the zinc fingers exist in structural states that are different from the metal-free and fully loaded species, the prevailing view on metal-mediated molecular regulation in terms of "on and off control" might be oversimplified.

Zinc ion-induced domain organization in metallo-beta-lactamases: a flexible "zinc arm" for rapid metal ion transfer?

The reversible unfolding of metallo-beta-lactamase from Chryseobacterium meningosepticum (BlaB) by guanidinium hydrochloride is best described by a three-state model including folded, intermediate, and unfolded states. The transformation of the folded apoenzyme into the intermediate state requires only very low denaturant concentrations, in contrast to the Zn2-enzyme. Similarly, circular dichroism spectra of both BlaB and metallo-beta-lactamase from Bacillus cereus 569/H/9 (BcII) display distinct differences between metal-free and Zn2-enzymes, indicating that the zinc ions affect the folding of the proteins, giving a larger alpha-helix content. To identify the regions of the protein involved in this zinc ion-induced change, a hydrogen deuterium exchange study with matrix-assisted laser desorption ionization tandem time of flight mass spectrometry on metal-free and Zn1- and Zn2-BcII was carried out. The region spanning the metal binding metallo-beta-lactamases (MBL) superfamily consensus sequence His-X-His-X-Asp motif and the loop connecting the N- and C-terminal domains of the protein undergoes a zinc ion-dependent structural change between intrinsically disordered and ordered states. The inherent flexibility even appears to allow for the formation of metal ion-bridged protein-protein complexes which may account for both electrospray ionization-mass spectroscopy results obtained upon variation of the zinc/protein ratio and stoichiometry-dependent variations of 199mHg-perturbed angular correlation of gamma-rays spectroscopic data. We suggest that this flexible "zinc arm" motif, present in all the MBL subclasses, is disordered in metal-free MBLs and may be involved in metal ion acquisition from zinc-carrying molecules different from MBL in an "activation on demand" regulation of enzyme activity.

[Legal implications of the limitations of competition in healthcare systems]. / Juristische Implikationen der Grenzen des Wettbewerbs im Gesundheitswesen.

Limitations of competition in our healthcare system follow from both the natural results of competition and the natural requirements for working competition. The legal implications of the limitations of competition in our healthcare system are twofold: they comprise the right and the duty of the government and the legislator to ensure fair and functional regulation.

Studies on ternary metallo-beta lactamase-inhibitor complexes using electrospray ionization mass spectrometry.

Metallo-beta-lactamases (MBLs) are targets for medicinal chemistry as they mediate bacterial resistance to beta-lactam antibiotics. Electrospray-ionization mass spectrometry (ESI-MS) was used to study the inhibition by a set of mercaptocarboxylates of two representative MBLs with different optimal metal stoichiometries for catalysis. BcII is a dizinc MBL (Class B1), whilst the CphA MBL (Class B2) exhibits highest activity with a single zinc ion in the active site. Experimental parameters for the detection of the metallo-enzyme and the metallo-enzyme-inhibitor complexes were evaluated and optimized. Following investigations on the stoichiometry of metal binding, the affinity of the inhibitors was investigated by measuring the relative abundance of the complex compared to the metalloprotein. The results for the BcII enzyme were in general agreement with solution assays and demonstrated that the inhibitors bind to the dizinc form of the BcII enzyme. The results for the CphA(ZnII) complex unexpectedly revealed an increased affinity for the binding of a second metal ion in the presence of thiomandelic acid. The results demonstrate that direct ESI-MS analysis of enzyme:inhibitor complexes is a viable method for screening inhibitors and for the rapid assay of the enzyme:metal:inhibitor ratios.

Studies on ternary metallo-ß lactamase-inhibitor complexes using electrospray ionization mass spectrometry.

Metallo-ß-lactamases (MBLs) are targets for medicinal chemistry as they mediate bacterial resistance to ß-lactam antibiotics. Electrospray-ionization mass spectrometry (ESI-MS) was used to study the inhibition by a set of mercaptocarboxylates of two representative MBLs with different optimal metal stoichiometries for catalysis. BcII is a dizinc MBL (Class B1), whilst the CphA MBL (Class B2) exhibits highest activity with a single zinc ion in the active site. Experimental parameters for the detection of the metallo-enzyme and the metallo-enzyme-inhibitor complexes were evaluated and optimized. Following investigations on the stoichiometry of metal binding, the affinity of the inhibitors was investigated by measuring the relative abundance of the complex compared to the metalloprotein. The results for the BcII enzyme were in general agreement with solution assays and demonstrated that the inhibitors bind to the dizinc form of the BcII enzyme. The results for the CphA(ZnII) complex unexpectedly revealed an increased affinity for the binding of a second metal ion in the presence of thiomandelic acid. The results demonstrate that direct ESI-MS analysis of enzyme:inhibitor complexes is a viable method for screening inhibitors and for the rapid assay of the enzyme:metal:inhibitor ratios.

On the competition for available zinc.

Extended x-ray absorption fine structure (EXAFS) spectroscopy was combined with thermodynamic and kinetic approaches to investigate zinc binding to a zinc finger (C2H2) and a tetrathiolate (C4) peptide. Both peptides represent structural zinc sites of proteins and rapidly bind a single zinc ion with picomolar dissociation constants. In competition with EDTA the transfer of peptide-bound zinc ions proved to be 6 orders of magnitude faster than predicted for a dissociation-association mechanism thus requiring ligand exchange mechanisms via peptide-zinc-EDTA complexes. EXAFS spectra of C2H2 showed the expected Cys2His2-ligand geometry when fully loaded with zinc. For a 2-fold excess of peptide, however, the existence of zinc-bridged peptide-peptide complexes with dominating sulfur coordination could be clearly shown. Whereas zinc binding kinetics of C2H2 appeared as a simple second order process, the suggested mechanism for C4 comprises a zinc-bridged Zn-(C4)2 species as well as a Zn-C4 species with less than 4 metal-bound thiolates, which is supported by EXAFS results. A rapid equilibrium of bound and unbound states of individual ligands might explain the kinetic instability of zinc-peptide complexes, which enables fast ligand exchange during the encounter of occupied and unoccupied acceptor sites. Depending on relative concentrations and stabilities, this results in a rapid transfer of zinc ions in the virtual absence of free zinc ions, as seen for the zinc transfer to EDTA, or in the formation of zinc-bridged complexes, as seen for both peptides with excess of peptides over available zinc.

Coordination geometries of metal ions in d- or l-captopril-inhibited metallo-beta-lactamases.

d- and l-captopril are competitive inhibitors of metallo-beta-lactamases. For the enzymes from Bacillus cereus (BcII) and Aeromonas hydrophila (CphA), we found that the mononuclear enzymes are the favored targets for inhibition. By combining results from extended x-ray absorption fine structure, perturbed angular correlation of gamma-rays spectroscopy, and a study of metal ion binding, we derived that for Cd(II)1-BcII, the thiolate sulfur of d-captopril binds to the metal ion located at the site defined by three histidine ligand residues. This is also the case for the inhibited Co(II)1 and Co(II)2 enzymes as observed by UV-visible spectroscopy. Although the single metal ion in Cd(II)1-BcII is distributed between both available binding sites in both the uninhibited and the inhibited enzyme, Cd(II)1-CphA shows only one defined ligand geometry with the thiolate sulfur coordinating to the metal ion in the site composed of 1 Cys, 1 His, and 1 Asp. CphA shows a strong preference for d-captopril, which is also reflected in a very rigid structure of the complex as determined by perturbed angular correlation spectroscopy. For BcII and CphA, which are representatives of the metallo-beta-lactamase subclasses B1 and B2, we find two different inhibitor binding modes.

Substrate-activated zinc binding of metallo-beta -lactamases: physiological importance of mononuclear enzymes.

We have investigated the influence of substrate binding on the zinc ion affinity of representatives from the three metallo-beta-lactamase subclasses, B1 (BcII from Bacillus cereus and BlaB from Chryseobacterium meningosepticum), B2 (CphA from Aeromonas hydrophila), and B3 (L1 from Stenotrophomonas maltophilia). By competition experiments with metal-free apoenzymes and chromophoric zinc chelators or EDTA, we determined the dissociation constants in the absence and presence of substrates. For the formation of the monozinc enzymes we determined constants of 1.8, 5.1, 0.007, and 2.6 nm in the absence and 13.6, 1.8, 1.2, and 5.7 pm in the presence of substrates for BcII, BlaB, CphA, and L1, respectively. A second zinc ion binds in the absence (presence) of substrates with considerably higher dissociation constants, namely 1.8 (0.8), 0.007 (0.025), 50 (1.9), and 0.006 (0.12) microm for BcII, BlaB, CphA, and L1, respectively. We have concluded that the apo form might be the prevailing state of most of the metallo-beta-lactamases under physiological conditions in the absence of substrates. Substrate availability induces a spontaneous self-activation due to a drastic decrease of the dissociation constants, resulting in the formation of active mononuclear enzymes already at picomolar free zinc ion concentrations. In the presence of substrates, the binuclear state of the enzymes only exists at unphysiologic high zinc concentrations and might be of no biological relevance. From the competition experiments with EDTA it is further concluded that the reactivation rate does not depend on the pool of free zinc ions but proceeds via the EDTA-Zn(II)-enzyme ternary complexes.