RP1 is a phosphorylation target of CK2 and is involved in cell adhesion

RP1 (synonym: MAPRE2, EB2) is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser(236) in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP(236) show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser(236) by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.

Cancer risk and use of protease inhibitor or nonnucleoside reverse transcriptase inhibitor-based combination antiretroviral therapy: the d: a: d study.

BACKGROUND The association between combination antiretroviral therapy (cART) and cancer risk, especially regimens containing protease inhibitors (PIs) or nonnucleoside reverse transcriptase inhibitors (NNRTIs), is unclear. METHODS Participants were followed from the latest of D:A:D study entry or January 1, 2004, until the earliest of a first cancer diagnosis, February 1, 2012, death, or 6 months after the last visit. Multivariable Poisson regression models assessed associations between cumulative (per year) use of either any cART or PI/NNRTI, and the incidence of any cancer, non-AIDS-defining cancers (NADC), AIDS-defining cancers (ADC), and the most frequently occurring ADC (Kaposi sarcoma, non-Hodgkin lymphoma) and NADC (lung, invasive anal, head/neck cancers, and Hodgkin lymphoma). RESULTS A total of 41,762 persons contributed 241,556 person-years (PY). A total of 1832 cancers were diagnosed [incidence rate: 0.76/100 PY (95% confidence interval: 0.72 to 0.79)], 718 ADC [0.30/100 PY (0.28-0.32)], and 1114 NADC [0.46/100 PY (0.43-0.49)]. Longer exposure to cART was associated with a lower ADC risk [adjusted rate ratio: 0.88/year (0.85-0.92)] but a higher NADC risk [1.02/year (1.00-1.03)]. Both PI and NNRTI use were associated with a lower ADC risk [PI: 0.96/year (0.92-1.00); NNRTI: 0.86/year (0.81-0.91)]. PI use was associated with a higher NADC risk [1.03/year (1.01-1.05)]. Although this was largely driven by an association with anal cancer [1.08/year (1.04-1.13)], the association remained after excluding anal cancers from the end point [1.02/year (1.01-1.04)]. No association was seen between NNRTI use and NADC [1.00/year (0.98-1.02)]. CONCLUSIONS Cumulative use of PIs may be associated with a higher risk of anal cancer and possibly other NADC. Further investigation of biological mechanisms is warranted.

[Immunosuppressive drugs - how they work, their side effects and interactions]

The central issue in organ transplantation remains suppression of allograft rejection. Immunosuppression can be achieved by depleting lymphocytes, diverting lymphocyte traffic, or blocking lymphocyte response pathways. Immunosuppressive drugs include small-molecule drugs, depleting and nondepleting protein drugs (polyclonal and monoclonal antibodies), fusion proteins, intravenous immune globulin, and glucocorticoids. Small-molecule immunosuppressive agents include calcineurin-inhibitors (cyclosporine, tacrolimus), Target-of-Rapamycin Inhibitors (Sirolimus, Everolimus), inhibitors of nucleotide synthesis and azathioprine. The review covers the mode of action of these drugs with a special focus on belatacept, a new promising fusion protein. Different immuo-suppressive strategies mean also different safety profiles. Common side effects include the consequences of diminished immuno- response, i.e. infections and cancer (mainly involving the skin). Toxic side effects of immunosuppressive drugs range in a wide spectrum that involves almost every organ. The major interest of this toxic effects is the cardiovascular tolerance (with large differences from drug to drug), that are discussed seperately. The calcineurin- and mTOR-inhibitors are both metabolized by the CYP450 3A4 enzyme, which is also involved in the metabolism of many other drugs. The review discusses the most important interactions that in- or decreases the through level of these drugs.

Antisense-mediated melanoma inhibitor of apoptosis protein downregulation sensitizes G361 melanoma cells to cisplatin

Malignant melanoma is an aggressive form of skin cancer that is highly resistant to conventional therapies. The melanoma inhibitor of apoptosis protein is a potent inhibitor of apoptosis and is overexpressed in melanoma cells, but undetectable in most normal tissues including melanocytes. We designed 20-mer phosphorothioate antisense oligonucleotides complementary to five putatively single-stranded sites on the melanoma inhibitor of apoptosis protein mRNA and investigated their ability to sensitize G361 melanoma cells to cisplatin. Inhibition of melanoma inhibitor of apoptosis protein mRNA and protein expression were measured by real-time polymerase chain reaction and immunoblotting. Cell viability and apoptosis were quantitated by colorimetric viability assays and by annexin V staining, respectively. Oligonucleotide M706 was identified as the most efficient antisense sequence which downregulated melanoma inhibitor of apoptosis protein mRNA and protein levels in G361 cells by 68 and 78%, respectively. The specificity of target downregulation was confirmed using scrambled sequence control oligonucleotides that only marginally decreased melanoma inhibitor of apoptosis protein expression. Whereas downregulation of melanoma inhibitor of apoptosis protein moderately inhibited cell growth by 26%, in combination with cisplatin, this resulted in a supra-additive effect with almost 57% reduction in G361 cell viability compared with cisplatin alone (17%) (P<0.05). Cell death was mainly due to apoptosis as demonstrated by a 3- to 4-fold increase in annexin V-positive cells and typical morphological changes compared with controls. In summary, we describe a new antisense oligonucleotide that efficiently downregulates melanoma inhibitor of apoptosis protein expression and sensitizes melanoma cells to cisplatin.

Effective treatment of advanced colorectal cancer by rapamycin and 5-FU/oxaliplatin monitored by TIMP-1

AIM: The mTOR-inhibitor rapamycin has shown antitumor activity in various tumors. Bedside observations have suggested that rapamycin may be effective as a treatment for colorectal carcinomatosis. METHODS: We established an orthotopic syngenic model by transplanting CT26 peritoneal tumors in Balb/C mice and an orthotopic xenograft model by transplanting SW620 peritoneal tumors in nu/nu mice. Expression levels of tissue inhibitor of matrix-metalloproteinases 1 (TIMP-1) in the tumor and serum was determined by enzyme-linked immunosorbent assay. RESULTS: Rapamycin significantly suppressed growth of syngenic and xenografted peritoneal tumors. The effect was similar with intraperitoneal or oral rapamycin administration. Tumor suppression was further enhanced when rapamycin was combined with 5-fluorouracil and/or oxaliplatin. The combination treatment showed no acute toxicity. TIMP-1 serum levels correlated well (CC = 0.75; P < 0.01) with rapamycin treatment. CONCLUSIONS: Rapamycin suppressed advanced stage colorectal cancer, even with oral administration. Combining rapamycin with current chemotherapy regimens significantly increased antitumor efficacy without apparent toxicity. The treatment efficacy correlated with serum TIMP-1 levels, suggesting its potential as a surrogate marker in future clinical trials.

The Fatty Acid Biosynthesis Enzyme FabI Plays a Key Role in the Development of Liver-Stage Malarial Parasites

The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.

The proteasome inhibitor MLN-273 blocks exoerythrocytic and erythrocytic development of Plasmodium parasites.

Protein degradation is regulated during the cell cycle of all eukaryotic cells and is mediated by the ubiquitin-proteasome pathway. Potent and specific peptide-derived inhibitors of the 20S proteasome have been developed recently as anti-cancer agents, based on their ability to induce apoptosis in rapidly dividing cells. Here, we tested a novel small molecule dipeptidyl boronic acid proteasome inhibitor, named MLN-273 on blood and liver stages of Plasmodium species, both of which undergo active replication, probably requiring extensive proteasome activity. The inhibitor blocked Plasmodium falciparum erythrocytic development at an early ring stage as well as P. berghei exoerythrocytic progression to schizonts. Importantly, neither uninfected erythrocytes nor hepatocytes were affected by the drug. MLN-273 caused an overall reduction in protein degradation in P. falciparum, as demonstrated by immunoblots using anti-ubiquitin antibodies to label ubiquitin-tagged protein conjugates. This led us to conclude that the target of the drug was the parasite proteasome. The fact that proteasome inhibitors are presently used as anti-cancer drugs in humans forms a solid basis for further development and makes them potentially attractive drugs also for malaria chemotherapy.

Bone morphogenetic protein-7 is a MYC target with prosurvival functions in childhood medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumor in children. It is known that overexpression and/or amplification of the MYC oncogene is associated with poor clinical outcome, but the molecular mechanisms and the MYC downstream effectors in MB remain still elusive. Besides contributing to elucidate how progression of MB takes place, most importantly, the identification of novel MYC-target genes will suggest novel candidates for targeted therapy in MB. A group of 209 MYC-responsive genes was obtained from a complementary DNA microarray analysis of a MB-derived cell line, following MYC overexpression and silencing. Among the MYC-responsive genes, we identified the members of the bone morphogenetic protein (BMP) signaling pathway, which have a crucial role during the development of the cerebellum. In particular, the gene BMP7 was identified as a direct target of MYC. A positive correlation between MYC and BMP7 expression was documented by analyzing two distinct sets of primary MB samples. Functional studies in vitro using a small-molecule inhibitor of the BMP/SMAD signaling pathway reproduced the effect of the small interfering RNA-mediated silencing of BMP7. Both approaches led to a block of proliferation in a panel of MB cells and to inhibition of SMAD phosphorylation. Altogether, our findings indicate that high MYC levels drive BMP7 overexpression, promoting cell survival in MB cells. This observation suggests the potential relevance of targeting the BMP/SMAD pathway as a novel therapeutic approach for the treatment of childhood MB.

Evidence that N-acetylcysteine inhibits TNF-alpha-induced cerebrovascular endothelin-1 upregulation via inhibition of mitogen- and stress-activated protein kinase

N-acetylcysteine (NAC) is neuroprotective in animal models of acute brain injury such as caused by bacterial meningitis. However, the mechanism(s) by which NAC exerts neuroprotection is unclear. Gene expression of endothelin-1 (ET-1), which contributes to cerebral blood flow decline in acute brain injury, is partially regulated by reactive oxygen species, and thus a potential target of NAC. We therefore examined the effect of NAC on tumor necrosis factor (TNF)-alpha-induced ET-1 production in cerebrovascular endothelial cells. NAC dose dependently inhibited TNF-alpha-induced preproET-1 mRNA upregulation and ET-1 protein secretion, while upregulation of inducible nitric oxide synthase (iNOS) was unaffected. Intriguingly, NAC had no effect on the initial activation (i.e., IkappaB degradation, nuclear p65 translocation, and Ser536 phosphorylation) of NF-kappaB by TNF-alpha. However, transient inhibition of NF-kappaB DNA binding suggested that NAC may inhibit ET-1 upregulation by inhibiting (a) parallel pathway(s) necessary for full transcriptional activation of NF-kappaB-mediated ET-1 gene expression. Similar to NAC, the MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and the protein kinase inhibitor H-89 selectively inhibited ET-1 upregulation without affecting nuclear p65 translocation, suggesting that NAC inhibits ET-1 upregulation via inhibition of mitogen- and stress-activated protein kinase (MSK). Supporting this notion, cotreatment with NAC inhibited the TNF-alpha-induced rise in MSK1 and MSK2 kinase activity, while siRNA knock-down experiments showed that MSK2 is the predominant isoform involved in TNF-alpha-induced ET-1 upregulation.

A novel open channel blocker of GABA-A receptors

GABA-A receptors are chloride ion channels composed of five subunits, mediating fast synaptic and tonic inhibition in the mammalian brain. 19 different subunit isoforms have been identified, with the major receptor type in mammalian adult brain consisting of α1, β2, and γ2 subunits. GABA-A receptors are the target of numerous sedating and anxiolytic drugs such as benzodiazepines. The currently known endogenous ligands are GABA, neurosteroids and the endocannabinoid 2- arachidonoyl glycerol (2-AG). The pharmacological properties of this chloride ion channel strictly depend on receptor subunit composition and arrangement. GABA-A receptors bind and are inhibited by epileptogenic agents such as picrotoxin, and cyclodiene insecticides such as dieldrin. We screened aromatic monovalent anions with five-fold symmetry for inhibition of GABA-A receptors. One of the anions, PCCPinhibited currents elicited by GABA with comparable potency as picrotoxin. This inhibition showed all characteristics of an open channel block. The GABA-A receptor ion channel is lined by residues from the M2 membrane-spanning segment. To identify important residues of the pore involved in the interaction with the blocking molecules PCCP-, a mutation scan was performed in combination with subsequent analysis of the expressed mutant proteins using electrophysiological techniques. In a second project we characterised a light-switchable modulator of GABA-A receptors based on propofol. It was my responsibility to investigate the switching kinetics in patch clamp experiments. After its discovery in 1980, propofol has become the most widely used intravenous general anaesthetic. It is commonly accepted that the anaesthesia induced by this unusually lipophilic drug mostly results from potentiation of GABA induced currents. While GABA-A receptors respond to a variety of ligands, they are normally not sensitive towards light. This light sensitivity could be indirectly achieved by using modulators that can be optically switched between an active and an inactive form. We tested an azobenzene derivative of propofol where an aryldiazene unit is directly coupled to the pharmacophore. This molecule was termed azopropofol (AP2). The effect of AP2 on Cl- currents was investigated with electrophysiological techniques using α1β2γ2 GABA-A receptors expressed in Xenopus oocytes and HEK-cells. In the third project we wanted to investigate the functional role of GABA-A receptors in the liver, and their possible involvement in cell proliferation. GABA-A receptors are also found in a wide range of peripheral tissues, including parts of the peripheral nervous system and non-neural tissues such as smooth muscle, the female reproductive system, liver and several cancer tissues. However their precise function in non neuronal or cancerous cells is still unknown. For this purpose we investigated expression, localization and function of the hepatocytes GABA-A receptors in model cell lines and healthy and cancerous hepatocytes.

PARP Inhibitor Resistance - What Is Beyond BRCA1 or BRCA2 Restoration?

Nearly 10 years ago the usefulness of poly(ADP-ribose) polymerase (PARP) inhibitors to kill BRCA1 or BRCA2-deficient cells was reported, and this finding has served as a prime example of the concept of synthetic lethality in the context of anticancer therapy. The clinical translation of this finding has undergone several ups and downs, however. Despite spectacular responses seen in some patients with BRCA-deficient breast or ovarian cancers, other patients did not show the expected benefit from PARP inhibitor therapy. Thus, like for all novel tailored anti-cancer drugs, upfront and secondary resistance remain major hurdles in the implementation of the initial preclinical finding. We know at least one clinically relevant mechanism of PARP inhibitor resistance: the reversion of BRCA function by secondary mutations. Nevertheless, it is also clear that this mechanism does not explain all cases of resistance. At the moment, we only have a poor understanding of BRCA reversion-independent resistance mechanisms. Preclinical data have pointed in several directions, e.g. increased drug efflux, reduced drug target levels, or alternative DNA repair. Here, we discuss these mechanisms with a focus on potential DNA repair adaptations.

The Effect of Everolimus Initiation and Calcineurin Inhibitor Elimination on Cardiac Allograft Vasculopathy in De Novo Recipients: One-Year Results of a Scandinavian Randomized Trial.

Early initiation of everolimus with calcineurin inhibitor therapy has been shown to reduce the progression of cardiac allograft vasculopathy (CAV) in de novo heart transplant recipients. The effect of de novo everolimus therapy and early total elimination of calcineurin inhibitor therapy has, however, not been investigated and is relevant given the morbidity and lack of efficacy of current protocols in preventing CAV. This 12-month multicenter Scandinavian trial randomized 115 de novo heart transplant recipients to everolimus with complete calcineurin inhibitor elimination 7-11 weeks after HTx or standard cyclosporine immunosuppression. Ninety-five (83%) patients had matched intravascular ultrasound examinations at baseline and 12 months. Mean (± SD) recipient age was 49.9 ± 13.1 years. The everolimus group (n = 47) demonstrated significantly reduced CAV progression as compared to the calcineurin inhibitor group (n = 48) (ΔMaximal Intimal Thickness 0.03 ± 0.06 and 0.08 ± 0.12 mm, ΔPercent Atheroma Volume 1.3 ± 2.3 and 4.2 ± 5.0%, ΔTotal Atheroma Volume 1.1 ± 19.2 mm(3) and 13.8 ± 28.0 mm(3) [all p-values ≤ 0.01]). Everolimus patients also had a significantly greater decline in levels of soluble tumor necrosis factor receptor-1 as compared to the calcineurin inhibitor group (p = 0.02). These preliminary results suggest that an everolimus-based CNI-free can potentially be considered in suitable de novo HTx recipients.

Treatment with the HIV protease inhibitor Nelfinavir triggers the unfolded protein response and may overcome proteasome inhibitor resistance of multiple myeloma in combination with bortezomib: a phase I trial (SAKK 65/08).

Downregulation of the unfolded protein response mediates proteasome inhibitor resistance in Multiple Myeloma.The Human Immunodeficieny Virus protease inhibitor nelfinavir activates the unfolded protein response in vitro. We determined dose limiting toxicity and recommended dose for phase II of nelfinavir in combination with the proteasome inhibitor bortezomib. 12 patients with advanced hematological malignancies were treated with nelfinavir (2500 - 5000 mg/d p.o., d 1-14, 3+3 dose escalation) and bortezomib (1.3 mg/m2, d 1, 4, 8, 11; 21 day cycles). A run in phase with nelfinavir monotherapy allowed pharmakokinetic/pharmakodynamic assessment of nelfinavir in the presence or absence of concomittant bortezomib. Endpoints included dose limiting toxicity, activation of the unfolded protein response, proteasome activity, toxicity and response to trial treatment. Nelfinavir 2 x 2500 mg was the recommended phase II dose identified. Nelfinavir alone significantly upregulated expression of proteins related to the unfolded protein response in peripheral blood mononuclear cells and inhibited proteasome activity. Of 10 evaluable patients in the dose escalation cohort, 3 achieved a partial response, 4 stable disease for ≥ 2 cycles, while 3 had progressive disease as best response. In an exploratory extension cohort with 6 relapsed, bortezomib-refractory, lenalidomide-resistant myeloma patients treated at the recommended phase II dose, 3 reached a partial response, 2 a minor response and one progressive disease. The combination of nelfinavir with bortezomib is safe and shows promising signals for activity in advanced, bortezomib-refractory MM. Induction of the unfolded protein response by nelfinavir may overcome the biological features of proteasome inhibitor resistance. (Trial registration NCT01164709).

Deposition and biokinetics of inhaled nanoparticles

Particle biokinetics is important in hazard identification and characterization of inhaled particles. Such studies intend to convert external to internal exposure or biologically effective dose, and may help to set limits in that way. Here we focus on the biokinetics of inhaled nanometer sized particles in comparison to micrometer sized ones.The presented approach ranges from inhaled particle deposition probability and retention in the respiratory tract to biokinetics and clearance of particles out of the respiratory tract. Particle transport into the blood circulation (translocation), towards secondary target organs and tissues (accumulation), and out of the body (clearance) is considered. The macroscopically assessed amount of particles in the respiratory tract and secondary target organs provides dose estimates for toxicological studies on the level of the whole organism. Complementary, microscopic analyses at the individual particle level provide detailed information about which cells and subcellular components are the target of inhaled particles. These studies contribute to shed light on mechanisms and modes of action eventually leading to adverse health effects by inhaled nanoparticles.We review current methods for macroscopic and microscopic analyses of particle deposition, retention and clearance. Existing macroscopic knowledge on particle biokinetics and microscopic views on particle organ interactions are discussed comparing nanometer and micrometer sized particles. We emphasize the importance for quantitative analyses and the use of particle doses derived from real world exposures.

Individual dosage of digoxin in patients with heart failure

After the publication of DIG trial, the therapeutic target of serum digoxin concentration (SDC) for the treatment of heart failure (HF) has been lowered (0.40-1.00 ng/ml). However, the majority of equations to calculate digoxin dosages were developed for higher SDCs. Recently, a new equation was validated in Asian population for low SDCs by Konishi et al., but results in Caucasians are unknown.