Activated protein kinase C (PKC) contributes to tumor survival and proliferation, provoking the development of inhibitory agents as potential cancer therapeutics. Immunotoxins are antibody-based recombinant proteins that employ antibody fragments for cancer targeting and bacterial toxins as the cytotoxic agent. Pseudomonas exotoxin-based immunotoxins act via the ADP-ribosylation of EF2 leading to the enzymatic inhibition of protein synthesis. Combining PKC inhibitors with the immunotoxin SS1P, targeted to surface mesothelin, was undertaken to explore possible therapeutic strategies. Enzastaurin but not two other PKC inhibitors combined with SS1P to produce synergistic cell death via apoptosis. Mechanistic insights of the synergistic killing centered on the complete loss of the prosurvival Bcl2 protein, Mcl-1, the loss of AKT and the activation of caspase 3/7. Synergy was most evident when cells exhibited resistance to the immunotoxin alone. Further, because PKC inhibition by itself was not sufficient to enhance SS1P action, enzastaurin must target other kinases that are involved in the immunotoxin pathway. Protein Kinase C (PKC) enzymes contribute to growth, survival and angiogenesis, all features that are frequently up-regulated in cancer Because most antibodies do not exhibit cell-killing activity in an unmodified form, they are frequently joined to toxic molecules to increase killing activity The immunotoxin, SS1P, is targeted to surface mesothelin which is up-regulated on a number of epithelial cancers including pancreatic, lung, ovarian and mesotheliomas To investigate new approaches for enhancing immunotoxin action, we reasoned that kinase inhibitors might be a particularly apt choice because they target survival pathways and because they do not require the expression of new gene products to be effective. We surveyed three inhibitors of PKC and report that enzastaurin exhibited immunotoxin enhancing action while the other two did not. Enhancement was noted with SS1P and to a lesser extent with a model immunotoxin targeting the transferrin receptor. However, there was no enhancement of other agents that inhibit protein synthesis such as diphtheria toxin and cycloheximide, suggesting a specific action on the PE-based immunotoxin pathway.Escherichia coli as described previously. Cycloheximide was purchased from Sigma.Enzastaurin was purchased from Selleck Chemicals LLC, dissolved in DMSO at 10 mmol/L and stored frozen at −80°C. Sotrastaurin was purchased from Axon Medchem, dissolved in DMSO at 10 mm/L and stored at −20°C. Go6976 was purchased from EMD Millipore, dissolved in DMSO at 5 mmol/L and stored at −20°C. SS1P and HB21-PE40 were produced recombinantly in The KB3-1 cells  were obtained from Dr Michael Gottesman, NCI and grown in DMEM plus 10% FBS 3H-leucine (2 µCi/ml) for 2 hrs in 96-well plates. Cells were collected on filter mats and samples counted using a Wallac Beta plate reader.CellTiter-Glo (Promega) and Caspase-Glo (Promega) measured cytotoxic activity and were used according to the directions supplied by the manufacturers. Routinely, cells were incubated for 48 hr prior to the addition of CellTiter-Glo reagent or overnight when measuring caspase activity. To stain treated cells, methylene blue (0.5% w/v) in 50% (v/v) methanol/water was added for ∼15 min. Treated cells were assayed for inhibition of protein synthesis by the addition of Immunotoxin-treated cells in the presence or absence of Enzastaurin, Sotrastaurin and Go6976 were washed with PBS, and then solubilized with RIPA buffer containing both protease and phosphatase inhibitors. Precast NU-PAGE 8–16% gels were used to separate cell lysates. Lysates transferred to nitrocellulose membranes were probed with rabbit anti-Mcl-1 , rabbit anti-Akt  anti-β-actin . The primary antibody was detected with goat anti-rabbit or anti-mouse HRP (Jackson Immunoresearch).Because combination therapies are frequently more effective in cancer treatment than single agents, we evaluated three PKC inhibitors in combination with SS1P, an immunotoxin targeted to surface mesothelin. Enzastaurin, sotrastaurin and Go6976 at 5 (not shown) and 10 uM were incubated with KB cells either alone or in combination with SS1P. After 48 hr, cytotoxicity was determined using the CellTiterGlo assay. By itself, enzastaurin showed only modest inhibitory activity at 10 uM. However, when added in combination, the toxicity of SS1P was greatly enhanced . At 50 nA matrix of enzastaurin and immunotoxin concentrations was arrayed to determine the fold-enhancement of combination treatments versus immunotoxin alone. Enzastaurin at 4 uM exhibited a small (∼2-fold) enhancement of SS1P activity toward KB cells  while thThe CellTiterGlo assay detects cellular ATP levels and reports on the energy status of the cell. To determine if combinations of SS1P and enzastaurin enhanced apoptosis we conducted similar combination experiments and assayed for caspase activation. For each cell line, immunotoxin-mediated apoptosis was enhanced in the presence of 10 uM enzastaurin. In the SS1P resistant lines, enhancement of apoptosis was 5–11-fold, for KLM1 and KB cells respectively . EnhanceTo determine if enzastaurin was a working at the level of protein translation, we incubated cells with three agents that inhibit protein synthesis. Cycloheximide reduced ATP levels at 1 and 5 ug/ml (only the 1 ug/ml concentration is shown) but there was no enhancement in the presence of either 2 or 16 uM enzastaurin . BecauseDesignating a combination treatment as ‘synergistic’ implies that both compounds enhance each other’s action. Immunotoxins inhibit protein synthesis leading to the loss of Mcl-1. Inhibition of PKC can lead to the inactivation of AKT resulting in the re-activation of GSK beta that results in the phosphorylation and degradation of Mcl-1. Thus Mcl-1 is a common, albeit indirect target of both compounds. To examine the roles of immunotoxin and enzastaurin in protein synthesis inhibition, we incubated KB cells with single agents or combinations and measured the incorporation of 3H-leucine into cellular proteins. SS1P at 100 ng/ml reduced protein synthesis by approximately 25% Further, in a time course we noted that substantial activation of caspase coincided with loss of Mcl-1. Little activation was noted when enzastaurin and SS1P were combined at 4 and 8 hrs. Only at 16 hrs did combinations of 500 ng/ml of SS1P and enzastaurin at 10 uM result in ∼ 8-fold increase in caspase and complete loss of Mcl-1 .In searching for a mechanism to explain the loss of Mcl-1 we considered that enzastaurin was interacting with the immunotoxin pathway to make it more effective, which subsequently resulted in the loss of Mcl-1. Our early data showing that other PKC inhibitors did not produce the same level of immunotoxin enhancement led to a comparison among these agents and a determination of cellular Mcl-1 levels. We noted that only enzastaurin and not sotrastaurin or Go6976, when combined with SS1P, resulted in a complete loss of Mcl-1. Thus, there was specificity to enzastaurin action. In searching for a particular action of enzastaurin in the loss of Mcl-1, we considered the upstream pathway and examined AKT levels. AKT is known to positively regulate Mcl-1 levels Using cytotoxicity assays that detect energy stores, mitochondrial ‘health’ and the activation of the apoptotic pathway we have shown synergistic action of SS1P and enzastaurin. In addition here we report the loss of growth on plastic culture dishes . Cells gInhibiting PKC is one of many potential strategies to improve cancer treatment outcomes Initially, we surveyed three PKC inhibitors as agents that could be combined with PE-based immunotoxins. Two of these, sotrastaurin and Go6976, exhibited some antagonism, whereby immunotoxin treatment rendered these inhibitors less potent  B and C.Clearly, the inhibition of PKC, by itself, does not cause an enhancement of immunotoxin action. We know this because sotrastaurin and Go6976 do not synergize with immunotoxin action. In fact there was evidence of antagonism - see Enhancement by compounds that are, by themselves, non-toxic constitutes de facto ‘synergy’. Therefore, for each of the three cell lines tested, KB, KLM1 and HAY, enzastaurin and SS1P exhibited synergistic cell killing. Killing included reductions in ATP levels, caspase activation and loss of attachment from culture dishes. Investigations of mechanism revealed several features. First, both agents in combination resulted in greater reductions in protein synthesis than immunotoxin alone. Further, enzastaurin but not sotrastaurin or Go6976 in combination with immunotoxins resulted in complete loss of the prosurvival protein Mcl-1. Loss of Mcl-1 is considered pivotal in many models of cell death and in some previous reports of immunotoxin action It is unlikely that concentrations of enzastaurin at 10–16 uM can be achieved in patients with mesothelin-positive tumors. However, as mentioned above, like many kinase inhibitors, enzastaurin is known to be a multi-kinase inhibitor Figure S1To determine the relative IC50 values for KB(A), KLM1(B) and HAY cells (C) the Cell Viability (Cell-Titer Glo) data were normalized where each concentration of enzastaurin was considered ‘100% of control’ and % inhibition for each concentration of immunotoxin was then calculated from the resulting graphs.(PPTX)Click here for additional data file.


LZAP  is a putative tumor suppressor that inhibits RelA, Chk1 and Chk2 and activates p53. LZAP is lost in a portion of human head and neck squamous cell carcinoma and experimental loss of LZAP expression is associated with enhanced invasion, xenograft tumor growth and angiogenesis. p38 MAPK can increase or decrease proliferation and cell death depending on cellular context. LZAP has no known enzymatic activity, implying that its biological functions are likely mediated by its protein-protein interactions. To gain further insight into LZAP activities, we searched for LZAP-associated proteins (LAPs). Here we show that the LZAP binds p38, alters p38 cellular localization, and inhibits basal and cytokine-stimulated p38 activity. Expression of LZAP inhibits p38 phosphorylation in a dose-dependent fashion while loss of LZAP enhances phosphorylation and activation with resultant phosphorylation of p38 downstream targets. Mechanistically, the ability of LZAP to alter p38 phosphorylation depended, at least partially, on the p38 phosphatase, Wip1. Expression of LZAP increased both LZAP and Wip1 binding to p38. Taken together, these data suggest that LZAP activity includes inhibition of p38 phosphorylation and activation. ARF in human and p19ARF in mice) and activate p53, both in the presence and absence of ARF, resulting in a G1 cell cycle arrest and inhibition of clonogenic growth LZAP  was originally identified as a binding partner of the Cdk5 activator p35 wild-type p53-induced phosphatase 1  p38MAPK belongs to a family of stress-activated MAPKs that responds to cellular stress and cytokines. Expression patterns suggest that p38α may be the primary p38 kinase in most cell types Here, we describe that the putative tumor suppressor LZAP bound and inhibited p38MAPK. Conversely, depletion of LZAP enhanced phosphorylation and activity of p38. LZAP did not alter p38 activating kinases (MKKs); however, LZAP increased association of p38 with Wip1 and LZAP dependent inhibition of p38 phosphorylation was at least partially dependent on Wip1. Given that LZAP inhibits p38 activity and that the role of p38 in cancers can vary from growth inhibitory to growth promoting, results presented here suggest that LZAP activities in tumors may be complex.Described LZAP activities include inhibition of cellular proliferation, inhibition of anchorage-independent growth, and enhancement of response to chemotherapeutic agents To confirm the predicted interaction between LZAP and p38, Myc-tagged LZAP was transiently expressed singly or with Flag-tagged p38 in mammalian U2OS cells before immunoprecipitation. When co-expressed, bands corresponding to Myc-tagged LZAP and untagged LZAP were detected in p38 immunoprecipitates . Consistent with our previous findings, LZAP expression increased nuclear localization of p38 as indicated by immunofluorescence  activate p38 through direct phosphorylation at thr180 and tyr182 p38-mediated phosphorylation of ATF2 activates ATF2 transcriptional activity in vivo and to directly dephosphorylate and inactivate p38 Loss of LZAP did not result in activation of upstream p38-activating kinases MKK3 or MKK6 , suggestTo determine if the Wip1 phosphatase was involved in LZAP's inhibition of p38, binding of Wip1 to p38 was determined following transient expression of p38 singly or with increasing expression of LZAP. Before p38 immunoprecipitation, cells were UV treated to increase p38 phosphorylation and expression of endogenous Wip1 Expression of LZAP resulted in increased association between p38 and its direct phosphatase, Wip1, suggesting that decreased phosphorylation and activity of p38 following LZAP expression may be mediated by Wip1. To explore this possibility, phosphorylation of p38 was compared following transient expression of LZAP with or without siRNA mediated inhibition of Wip1 expression. As expected, expression of LZAP resulted in decreased p38 phosphorylation . IncreasBased on loss of expression in human HNSCC and cellular effects associated with loss of LZAP such as enhanced invasion, anchorage independent growth, angiogenesis, and growth of xenograft tumors, LZAP has been identified as a putative tumor suppressor Here we report that LZAP bound to the stress activated protein kinase p38, altered p38 subcellular localization and inhibited p38 phosphorylation and activity. Mechanistically, our data suggest that LZAP inhibition of p38 phosphorylation depends on the Wip1 phosphatase, and that in the presence of LZAP, more Wip1 is associated with p38. Conceivably, LZAP may sequester p38 in the nucleus in a complex with Wip1 as a means of p38 inactivation. Alternatively, unphosphorylated nuclear p38 may have unknown activities or may be sequestered in the nucleus so that upon loss of LZAP, rapid activation of p38 could occur through phosphorylation by nuclear kinases.Interestingly, LZAP was found to bind p38 through two independent and non-overlapping domains, an amino-terminal region (αα 1-303), and a carboxy-terminal region (αα 329-506) of LZAP. Based on gel filtration, LZAP has been reported to exist in large molecular weight complexes and the pattern of independent regions of LZAP binding to its partners, as we observed, has been reported and may be critical for formation of large protein complexes containing LZAP The mechanism of LZAP activity toward the growing list of LZAP-associated proteins (LAPs) is not well understood. LZAP has no described enzymatic activity, suggesting that LZAP may exert its effects through association with other proteins. The finding that a portion of LZAP exists in large molecular weight complexes combined with a potential for LZAP to oligomerize lends credence to this argument and further suggests that LZAP may serve to bring together effector proteins. A large portion of LZAP interacting proteins are phosphorylated, and LZAP expression has been associated with decreased phosphorylation of these proteins. These observations led us to explore if LZAP had activity to inhibit p38 upstream kinases or to activate p38 phosphatases. We found that LZAP did not alter kinase activity, as measured by phosphorylation, of MKK3 or MKK6; however, LZAP was found to increase association of p38 with its direct phosphatase Wip1 . It is uDepending on cellular context, p38 can mediate opposing cellular responses as an inducer or inhibitor of proliferation and apoptosis GTGGACCGAGT5′-CAAGGTAT) The full coding sequence and truncation mutants of LZAP were subcloned into pcDNA3-Myc3 and pET-His expression vectors LZAP polyclonal rabbit antibody has been previously described 2, in growth media with 10% fetal bovine serum (FBS) . Cell lines were obtained from ATCC or collaborators and have been passed in the Yarbrough lab with biannual authentication of identity based on microsatellite analyses of 3 markers . Plasmids were transfected using FuGene6  according to the manufacturer's instructions. The total amount of transfected DNA in any single experiment was kept constant by adding control vector (pcDNA3). Small interfering RNA (siRNA) was transfected at 20 nM using Lipofectamine RNAiMAX . Control siRNA duplex (nontargeting #1) was purchased from Dharmacon . The LZAP siRNA-2 was previously described 5′-CAAGGTATGTGGACCGAGT (sense strand); the sequence of Wip1 siRNA is: 5′- GGCUUUCUCGCUUGUCACC dTdT Cell lines were maintained at 37°C with 5% COCells were lysed in 0.5%(v/v) Nonidet P40 lysis buffer Briefly, cells were fixed with paraformaldehyde permeabilized with Triton X-100, and blocked with BSA. Target proteins were visualized following incubation with primary antibodies followed by fluorophore secondary antibodies and visualization as described 2, and 40 mg/ml digitonin). The lysate was incubated on ice for 5 min and centrifuged , and the supernatant was designated as soluble cytosolic fraction. The pellet was washed with the same buffer before adding RIPA lysis buffer , incubated on ice for 5 min and centrifuged , to obtain the nuclear fraction. Whole cell lysates were prepared using RIPA buffer, as described Cells were scraped in cytosolic lysis buffer  as described Table S1LZAPpredicted motifs from Scansite. LZAP protein coding sequence was inputed for motif scan, and chose high stringency criteria to look for all possible binding motifs.(TIF)Click here for additional data file.


PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways are thought to be the central transducers of oncogenic signals in solid malignancies, and there has been a lot of enthusiasm for developing inhibitors of these pathways for cancer therapy. Some preclinical models have suggested that combining inhibitors of both parallel pathways may be more efficacious, but it remains unknown whether dual inhibition with high enough concentrations of the drugs to achieve meaningful target inhibition is tolerable in a clinical setting. Furthermore, the predictive factors for dual inhibition are unknown.Non-small cell lung cancer (NSCLC) cell lines (n=12) with the most frequent oncogenic backgrounds  were exposed to PI3K inhibitors  or MEK inhibitor (CI-1040) alone or in combination and analysed with an MTS growth/cytotoxicity assay and statistically by combination index analysis. The activity of the intracellular signaling pathways in response to the inhibitor treatments was analysed with a western blot using phospho-specific antibodies to AKT, ERK1/2, S6, and 4E-BPI. For the differential dosing schedule experiments, additional breast and colon cancer cell lines known to be sensitive to dual inhibition were included.Two of the 12 NSCLC cell lines tested, H3122 (ALK translocated) and H1437 (triple-negative), showed increased cytotoxicity upon dual MEK and PI3K inhibition. Furthermore, MDA-MB231 (breast) and HCT116 (colon), showed increased cytotoxicity upon dual inhibition, as in previous studies. Activation of parallel pathways in the dual inhibition-sensitive lines was also noted in response to single inhibitor treatment. Otherwise, no significant differences in downstream intracellular pathway activity (S6 and 4E-BPI) were noted between PI3K alone and dual inhibition other than the increased cytotoxicity of the latter. In the alternative dosing schedules two out of the four dual inhibition-sensitive cell lines showed similar cytotoxicity to continuous PI3K and short (15min) MEK inhibition treatment.Therapy with a dual PI3K and MEK inhibitor combination is more efficient than either inhibitor alone in some NSCLC cell lines. Responses to dual inhibition were not associated with any specific oncogenic genotype and no other predictive factors for dual inhibition were noted. The maximal effect of the dual PI3K and MEK inhibition can be achieved with alternative dosing schedules which are potentially more tolerable clinically. Ras, Raf or PI3KCA and inactivation of certain tumor suppressors such as PTEN lead to constitutive activation of these pathways , Promega; Madison, WI) supplemented with phenazine methosulfate  according to the manufacturer’s guidelines. The absorbances were read on a plate reader  at a wavelength of 488nm. The data were displayed graphically using GraphPad Prism , with the absorbance in the non-treated wells as the reference value (100%). The combination index (CI) was calculated using Calcusyn software , and a 3.3:1 ratio of the PI3K inhibitors to the MEK inhibitor was used in the CI analysis. CI values at ED50 are presented.The cells were plated onto 6-well plates and treated with the drugs 24-48h later for 6 or 72 h, after which they were lysed in RIPA buffer . Protein concentrations were measured using the Bio-Rad Protein Assay  and the concentrations in individual samples were equalized before adding 3x Laemmli buffer to a final concentration of 1x. Equal amounts of protein were run on 7.5% SDS-PAGE gels, transferred to PVDF membranes, probed with the antibodies and developed using the ECL chemiluminescence system  for detection on radiographic films, which were scanned to an electronic format. All the antibodies used were from Cell Signaling Technologies : pAKT (S473), AKT, pERK (T202/Y204), ERK, pS6 (Thr389), S6, p4E-BP1 (Thr37/46), 4E-BP1, cleaved PARP. Anti-rabbit HRP conjugated antibody was used as a secondary antibody.http://www.cellsignal.com/products/7982.html).The PathScan analysis was carried out with the PathScan® RTK Signaling Antibody Array kit  according to the manufacturer’s guidelines. In brief, cells were plated on plates of diameter 6 cm and drugged the following day for 24 h. Whole cell lysates were collected, protein concentrations were determined using the Bio-Rad Protein Assay  and the protein concentrations were equalized. The lysates were applied to nitrocellulose membranes and incubated over night, washed, exposed to the secondary antibodies, developed with ECL and imaged with a Fujifilm LAS-3000 Luminescent Image analyzer and the ImageReader LAS-3000 program. The array target map can be found through the manufacturer’s homepage (K-Ras mutant), HCC827 (EGFR mutant) and H3122 (EML4-ALK translocated), representing the three most frequent oncogenic genotypes of the disease, to establish concentration frames for the target inhibition. In the Western blots ZSTK474 at a 3.3μM concentration induced complete downregulation of pAKT, an immediate downstream target of PI3K, while PI-103 induced a similar inhibition at concentrations of 1 to 3.3 μM  and PI-103 (PI3K and mTOR inhibitor) and CI-1040 (MEK inhibitor). We first addressed the effects of these inhibitors alone in the NSCLC lines A549 , EGFR (n=3), ALK (n=3), or triple-negative (n=3) oncogenic genotypes. Analogously to the cell lines in the preliminary experiments, all the cell lines tested here showed a major reduction in cell growth in response to the PI3K inhibitors alone, with no significant differences between ZSTK474 or PI-103  and a triple-negative negative line (H1437) showed synergistic responses to dual inhibition  with the 3 Figure . The res2 with th3 Figure . There wn Figure , Table 1n Figure . AnalogoKRas mutant colorectal line, were exposed to single inhibitors or dual inhibition and analyzed with the MTS assay. As in the previous work, both the cell lines showed synergistic responses to dual inhibition , breast cancer (MDA-MB231) and colon cancer (HCT116) lines, which showing major synergy upon dual inhibition, were further studied for cell signaling in response to the inhibitors. All the cell lines downregulated pAKT and its downstream target pS6 completely in response to 6h of treatment with the PI3K inhibitor ZSTK474 or PI-103 (3.3 μM) Figure . DownregWhen the PI3K and MEK inhibitors were administered simultaneously the inhibition of the targets was similar to that seen with single inhibitor treatment Figure . Dual inFurther analysis of the dual inhibition of the central RTKs and signaling nodes was carried out with the PathScan Antibody Array, which investigates the phosphorylation status of 28 RTKs and 11 signaling nodes concurrently. Attention was focused on the dual inhibition-sensitive H1437 and MDA-MB231 lines. A low level of RTK activation was noted in untreated cells of both cell lines, H1437 showing some activity with c-MET Figure , while iin vitro models, administration of both drugs at doses inducing major downregulation of the target for long periods of time may be too toxic in a clinical setting. We therefore set out to investigate concurrent administration of PI3K and MEK inhibitors to cell lines sensitive to dual inhibition with alternative dosing schedules. The MTS assays showed that for maximal reduction in the number of living cells in all the lines, dual inhibition needed to be administered for longer periods of time. The therapy was significantly more effective when it was administered throughout the 72 h experiment as compared with 15 min, 4 h or 24 h periods , while similar cytotoxicity was seen with 72 h inhibition of PI3K and MEK concurrently  while the other inhibitor was administered concurrently for 15 min, 4 h or 24 h at the beginning of the drug exposure. The results varied significantly between the cell lines tested. In the H1437 and MDA-MB231 lines concurrent inhibition of PI3K and MEK for 15 min with continued PI3K inhibition for 72 h achieved similar cytotoxicity to concurrent inhibition for 72 h Figure . ConversWe next investigated alternative dosing of the dual inhibition of cell signaling. The dual inhibition-sensitive lines were exposed to the PI3K inhibitors and MEK inhibitor concurrently for 15 min, after which treatment was continued with a single inhibitor for the remainder of the 6 h period. pAKT downregulation was complete or nearly complete when the cells were treated for only 15 min and with PI3K inhibitors for 6 h Figure , while cWe went on further to analyze whether the alternative dosing could also result in apoptosis in the H3122 cell line, the only line identified as inducing apoptosis in response to dual inhibition. When the cells was treated for 15 min with dual inhibition and treatment with either the PI3K inhibitors or the MEK inhibitor was continued for 48 h, marked PARP cleavage was seen in all the treatments Figure . FurtherRaf mutant melanoma, where both RAF and MEK inhibitors have high clinical activity. There is preclinical evidence that combining the inhibitors of both pathways provides more efficient cancer therapy [The PI3K-AKT and RAS-RAF-MEK-ERK signaling pathways are thought to be the central mediators of oncogenic signals in solid malignancies. Multiple inhibitors targeting PI3K, AKT, RAF and MEK are under development for cancer therapy, but early-phase clinical trials suggest that the single agent efficiency of such inhibitors seems to be limited, except in the case of the  therapy ,4,6,7, aKRas mutant colorectal cancers have shown additive cytotoxicity or reversal of resistance when MEK inhibitors have been combined with inhibitors of the PI3K-AKT-mTOR pathway [KRas, EGFR and ALK wild-type cell H1437 is of a rare oncogenic genotype, a MEK1 mutant, and has previously been identified as being sensitive to MEK inhibitor treatment alone [in vivo, it is likely to make the selection of patients for such treatment difficult, since no predictive biomarkers of feedback activation exist.We investigated here the dual pharmacological inhibition of PI3K and MEK in NSCLC cell line models with specific oncogenic genotypes. All the cell lines tested were highly responsive to single-agent PI3K inhibitors, showing a strong correlation with maximal target inhibition. This suggests that the PI3K-AKT pathway has a central role in transmitting oncogenic signals from various upstream sources, and therefore the responses to pathway inhibition are not limited to any specific cancer genotype. Furthermore, the data suggest a central role for pathway activation in the proliferation of carcinomas. The cytotoxicity of PI3K inhibitors seemed to be comparable when a PI3K ZSTK474) or PI3K/mTOR (PI-103) inhibitors alone were used, suggesting that only PI3K inhibition matters for cytotoxicity, as administration of the MEK inhibitor seemed to have limited activity or none at all in the models tested. Two out of the twelve cell lines tested showed significantly increased cytotoxicity in response to the concurrent administration of PI3K and MEK inhibitors. Analogously to previous studies, the activity of dual inhibition was not associated with any specific oncogenic genotype, since ALK translocation-positive (H3122) and triple-negative (H1437) cell lines were the most responsive ones . In MEK 74 or PI3nt alone . Based oALK translocated cell line, showed apoptosis in response to short concurrent administration of the drugs even though longer concurrent administration led to maximal cytotoxicity. Interestingly, short courses of ALK inhibition (15 min) induced comparable cytotoxicity to long administration of either an ALK inhibitor or a dual inhibitor combination, even though the ALK inhibitor is reversible in its mode of action and some recovery of the target inhibition is known to occur within 6h. In the light of our in vitro data, one could hypothesize that even a short course of dual inhibitor administration could have similar clinical effects with better tolerability. Analogously, a recent work has shown that intermittent administration of concurrent PI3K and MEK inhibition can induce robust growth inhibition in cancer cell lines [in vitro models, however, and correlation with the in vivo situation is not always a straightforward matter.Even though dual inhibition of PI3K-AKT and MEK has been identified as an effective cancer therapy in preclinical models, it questionable whether this therapy is tolerable in a clinical setting concentrations high enough to achieve sufficient target inhibition. Early-phase clinical trials are in progress to test different doses and dosing schedules, but the optimal administration for maximal efficiency and tolerability remains to be elucidated. In the light of recent data from the ASCO 2012 Annual Meeting, PI3K and MEK inhibitor combination treatments are now being tested in concurrent and intermittent schedules ,23. The ll lines . Better The interconnectivity of the PI3K-AKT-mTOR and RAS-RAF-MEK-ERK pathways makes the idea of their concurrent dual inhibition an appealing one. The present cell signaling experiments also showed high interconnectivity of these two pathways, since in many instances inhibition of one pathway resulted in concurrent feedback activation of the other. Furthermore, another MEK inhibition-induced feedback mechanism was identified in the MDA-MB231 cell line which led to the activation of 4E-BP1 independently of PI3K-AKT. Previous studies have suggested that the PI3K-AKT-mTOR and RAS-RAF-MEK-ERK pathway signals converge at 4E-BP1, and that its inhibition may be a major determinant of the efficiency of dual inhibition . ConversThe most important findings to emerge from this investigation of the concurrent dual inhibition of PI3K and MEK for cancer therapy purposes are the fact that alternative dosing schedules result in comparable cytotoxicity to that achieved with continuous treatment schedules, and that the responses to dual inhibition can be achieved in multiple cancer genotypes. The present preclinical data may offer new leads for clinical progress towards more efficient and tolerable cancer therapies.The authors’ declare no competing interests.JPK conceived the study design and was coordinated the work. JPK and EJ carried out the laboratory experiments, participated in the gathering, analysis and interpretation of the data, and drafted, read and approved the final version of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/612/prepub


Backgound: Hexokinase II binds to mitochondria and promotes cell survival.Results: Akt phosphorylates HK-II but not the threonine 473 mutant. The phosphomimetic T473D mutant decreases its dissociation from mitochondria induced by G-6P and increases cell viability against stress.Conclusion: Akt phosphorylates HK-II at Thr-473, resulting in increased mitochondrial HK-II and cell protection.Significance: The Akt-HK-II signaling nexus is important in cell survival. Hexokinase II (HK-II) is an enzyme that catalyzes the first step in glycolysis and localizes not only in the cytosol but also at mitochondria. Akt, activated by insulin-like growth factor 1 (IGF-1) treatment in neonatal rat ventricular myocytes, translocates to mitochondria and increases mitochondrial HK-II binding. Expression of an HK-II-dissociating peptide diminished IGF-1-induced increases in mitochondrial HK-II as well as protection against hydrogen peroxide treatment, suggesting an important role of mitochondrial HK-II in IGF-1/Akt-mediated protection. We hypothesized, on the basis of an Akt phosphorylation consensus sequence present in HK-II, that Thr-473 is the target of Akt kinase activity. Indeed, recombinant kinase-active Akt robustly phosphorylates WT HK-II, but not Thr-473 mutants. Phosphomimetic (T473D)HK-II, but not non-phosphorylatable (T473A)HK-II, constitutively increased mitochondrial binding compared with WT HK-II and concomitantly confers greater protection against hydrogen peroxide. Glucose 6-phosphate (G-6P), a product of the catalytic activity of HK-II, is well known to dissociate HK-II from mitochondria. Addition of G-6P to isolated mitochondria dose-dependently dissociates WT HK-II, and this response is inhibited significantly in mitochondria isolated from cardiomyocytes expressing T473D HK-II. Pretreatment with IGF-1 also inhibits G-6P-induced overexpressed or endogenous HK-II dissociation, and this response was blocked by Akt inhibition. These results show that Akt phosphorylation of HK-II at Thr-473 is responsible for the Akt-mediated increase in HK-II binding to mitochondria. This increase is, at least in part, due to the decreased sensitivity to G-6P-induced dissociation. Thus, phosphorylation-mediated regulation of mitochondrial HK-II would be a critical component of the protective effect of Akt. Mitochondria are critical for energy generation and are also recognized as gatekeepers in the control of cell survival. Loss of mitochondrial integrity leads to necrotic or apoptotic cell death –4. CardiHexokinase (HK) catalyzes phosphorylation of glucose, producing glucose 6-phosphate (G-6P). Hexokinase II (HK-II) is a predominant isoform in insulin-sensitive tissues such as heart, skeletal muscle, and adipose tissues and is known to be up-regulated in many types of tumors, whereas hexokinase I (HK-I) is the main isoform in the brain but is also expressed ubiquitously , 10. EarThere is increasing evidence that mitochondrial HKs protect mitochondria against insults –25. For XXRS/T) conserved in mouse, rat, and human, implying that HK-II is phosphorylated by Akt  supplemented with 15% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). Cells were serum-starved for 24 h prior to the experiment. After serum starvation, cells were infected with adenoviruses and cultured for 24 h.Neonatal rat ventricular myocytes (NRVMs) were isolated from 1- to 2-day-old Sprague-Dawley rat pups using a kit (Worthington). Myocytes were plated at a density of 3.5 × 10BC054472) was obtained (Open Biosystems), amplified by PCR, and inserted into pENTR/D-TOPO (Life Sciences) for further manipulation. The Thr-473 single-point mutations and N-terminal deletion were carried out using the QuikChange site-directed mutagenesis method (Agilent Technologies). To produce 15NG, the N-terminal bases comprising the first 15 amino acids (MIASHMIACLFTELN) of HK-II were fused onto the N-terminal of GFP in pENTR. A linker sequence (GGSGG) separated the two molecules. The resulting constructs were inserted, using Gateway cloning, into the pAd/CMV/V5 DEST plasmid (Life Sciences) and were transfected into HEK293A cells for adenovirus production. These constructs are illustrated in Mouse HK-II cDNA ), and shaken at 600 rpm for 2 min at 4 °C. CHAPS was added to 0.1% final concentration and shaken for 5 min at 4 °C. 110 μl of buffer C ) was added and spun down at 700 × g for 10 min to remove cell debris and nuclei. The supernatant was transferred and spun at 12,000 × g for 15 min at 4 °C. The supernatant was again spun down at 20,000 × g for 3 min, and the resultant supernatant was saved as the cytosolic fraction. The pellet was washed in buffer D ) and saved as the mitochondrial fraction. For Western blotting, the isolated mitochondria were lysed in radioimmune precipitation assay buffer composed of 150 mm NaCl, 50 mm Tris (pH 7.4), 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.2 mm EDTA and supplemented with 200 μm Na3VO4, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm PNPP, and 1 mm PMSF.Cardiomyocytes were washed twice with ice-cold PBS, collected in ice-cold PBS, spun down at 900 × m KCl, 20 mm MOPS, 10 mm Tris·HCl (pH 7.4) and 0.2 mm EDTA. G-6P was added to mitochondria at various concentrations and incubated for 15 min at room temperature. The mitochondrial suspension was spun down at 20,000 × g for 3 min, the supernatant was transferred, and the pellet was resuspended in the buffer.The mitochondrial pellet was resuspended in a buffer containing 120 mg for 7 min, and then the supernatants were saved as whole cell lysates. Protein concentration was measured by Micro BCA protein assay kit (Thermo Scientific). Samples were then mixed with LDS sample buffer and reducing agent (Invitrogen), heated at 75 °C for 10 min, and then equal amounts of protein (10–30 μg) were loaded onto SDS-PAGE (Invitrogen NuPage system). Proteins were transferred to PVDF membranes (Millipore). Membranes were blocked with 5% milk in TBS-Tween for 1 h at room temperature and probed with primary antibody at 4 °C overnight at 1:1000 dilution in 5% BSA/TBS-Tween. All Western blotting antibodies used in this study were from Cell Signaling Technology. Blots were washed with TBS-Tween  and incubated with secondary antibodies (1:2000∼1:5000 dilution) in 5% milk/TBS-Tween for 1 h.Cardiomyocytes were washed three times with ice-cold PBS and harvested in radioimmune precipitation assay buffer. Samples were nutated at 4 °C for 10 min, spun down at 20,000 × in vitro phosphorylation experiment, immunocomplexes were washed with ice-cold radioimmune precipitation assay buffer and resuspended in kinase reaction buffer containing 20 mm Hepes, 2 mm DTT, and 5 mm MgCl2 with or without 200 μm ATP and recombinant Akt (Millipore). Incubations were carried out at 30 °C for 20 min, spun down, resuspended in 2× LDS sample buffer, boiled for 5 min, and subjected to Western blotting.HK-II was immunoprecipitated from whole cell lysate as described previously . Brieflym TMRE (Life Technology) for 30 min. Cells were washed twice with DPBS containing 1 mm CaCl2 and 1 mm MgCl2. Fluorescence was visualized using an Olympus LV1000 confocal microscope.NRVMs were grown on glass-bottom 3.5-cm dishes and infected with adenoviruses. 24 h later, cells were loaded with 50 nm Calcein Blue AM (eBioscience) for 30 min at room temperature, washed twice with DPBS containing 1 mm CaCl2 and 1 mm MgCl2, and fluorescence was detected using an Infinite M200PRO microplate reader (TECAN).Cell viability was measured using Calcein Blue AM. Briefly, cells were loaded with 4 μt test. Experiments with more than two groups were compared by analysis of variance followed by the Tukey post-hoc test. p < 0.05 was considered statistically significant.Results are reported as mean ± S.E. Comparisons of two groups were accomplished using unpaired Student's m IGF-1 for 30 min, and the mitochondrial and cytosolic fractions were isolated and subjected to Western blotting (A). In response to IGF-1 treatment, phosphorylated and total Akt were increased in the mitochondrial fraction, which was blocked by pretreatment with Akt inhibitor V (triciribine). Mitochondrial HK-II was also increased by IGF-1, and this response was abolished by Akt inhibition. These results support our previous observations using leukemia inhibitory factor that Akt translocates to mitochondria upon activation and leads to increased mitochondrial HK-II binding in cardiomyocytes.NRVMs were treated with 10 nblotting A. In resB). Using cells infected at a low MOI, we characterized the intracellular distribution of 15NG versus untagged GFP together with TMRE to detect active mitochondria. As shown in Fig. 2C, GFP fluorescence in cells infected with Ad-15NG was colocalized with TMRE fluorescence . However, cells infected with Ad-GFP showed diffuse fluorescence throughout the cell, demonstrating preferential localization of 15NG to mitochondria. Cell viability was not affected by the expression of 15NG at this level of expression . Thus, at a low level of expression, the dissociating peptide is unlikely to affect mitochondrial integrity and, thus, cell survival.The HK-II N terminus peptide attached to a cell-permeable sequence (antennapedia or TAT) has been used as a method of dissociating HK-II from mitochondria to determine the role of mitochondrial HK-II , 23, 34.Fig. 2E). IGF-1 treatment increased total Akt, phospho-Akt, and HK-II in the mitochondrial fraction. 15NG expression diminished the increase in HK-II in the mitochondrial fraction but did not affect total and phospho-Akt levels. To determine whether the increase in HK-II binding to mitochondria contributes to IGF-1-mediated cell protection, cardiomyocytes were treated with 100 μm hydrogen peroxide for 4 h, and cell viability was measured (F). IGF-1 treatment increased cell viability against hydrogen peroxide in cardiomyocytes, and this protection was blocked by inhibition of Akt, suggesting a critical role of Akt in IGF-1-mediated protection. This IGF-1/Akt-induced protection was greatly attenuated in cells expressing 15NG. These results support the notion that increased mitochondrial HK-II contributes to Akt-mediated protection against oxidative stress in cardiomyocytes.To determine whether the expression of 15NG inhibits the IGF-1-induced increase in HK-II binding to mitochondria, cells were infected with GFP (control) or 15NG adenovirus at 50 MOI, stimulated with IGF-1, and then the mitochondrial fractions were isolated E. IGF-1 measured F. IGF-1 TD) and non-phosphorylatable T473A mutants (TA) (A). GFP (control), WT, T473D, and T473A were expressed adenovirally in cells, and HK-II was immunoprecipitated. The immunoprecipitates were then subjected to in vitro phosphorylation using recombinant kinase-active Akt  in the presence or absence of ATP. Phosphorylation of HK-II was assessed by PAS antibody (phosphorylated Akt consensus sequence antibody) or P-Thr antibody. Recombinant active Akt catalyzed a robust ATP-dependent phosphorylation of WT HK-II but did not increase phosphorylation of Thr-473 mutants over control (endogenous HK-II) levels, providing direct evidence that Thr-473 in HK-II is phosphorylated by Akt. To determine whether Akt, activated by IGF-1, phosphorylates endogenous HK-II, cells were treated with IGF-1 with or without Akt inhibitor V. HK-II was then immunoprecipitated and blotted using PAS or P-Thr antibody (B). IGF-1 treatment increased PAS or P-Thr signals, which were blocked by Akt inhibitor V, confirming that HK-II is phosphorylated by Akt.To test the hypothesis that Thr-473 in the Akt consensus sequence found in HK-II is the phosphorylation site for Akt, we generated the phosphomimetic T473D (nts (TA) A. GFP (cantibody B. IGF-1 A). Overexpression of WT, T473D, or T473A increased the amount of HK-II in the mitochondrial fraction. Slight increases were observed in the cytosolic fractions. T473D showed a significantly larger increase in the mitochondrial fraction compared with WT or T473A (A and B). The effect of IGF-1 treatment was examined. Similar to Fig. 2D, IGF-1 increased endogenous or overexpressed WT HK-II in the mitochondrial fraction, and IGF-1 treatment did not change mitochondrial HK-II in T473D HK-II expressing cells. Nor did it increase the mitochondrial HK-II in T473A-expressing cells over control (GFP) cells, corresponding to endogenous HK-II phosphorylation and association.We then evaluated the mitochondrial binding of WT, T473D, and T473A HK-II A. Overexor T473A , A and BC. The WT showed a slight but significant increase in HK-II mitochondrial binding over the control. Remarkably, the T473D mutant showed a robust increase in mitochondrial HK-II, whereas the T473A mutant did not increase the ratio over the control. These results suggest that phosphorylation of Thr-473 sensitizes HK-II to associate with mitochondria. IGF-1 treatment increased the ratio in GFP- and WT-expressing cells. In cells expressing T473D, the ratio was not increased by IGF-1 treatment. The ratio in T473A-expressing cells was increased by IGF-1, but this response was similar to control cells treated with IGF-1.Although the MOI of each adenovirus was chosen carefully to achieve equivalent protein expression, higher expression can result in more mitochondrial HK-II. To normalize the difference in the protein expression levels of each construct, the ratio of mitochondrial HK-II to total HK-II  is quantified in Fig. 4D). Western blotting demonstrated that mitochondrial HK-II was not increased in cells expressing ΔN HK-II despite high overexpression and that IGF-1 was ineffective in promoting mitochondrial binding of ΔN HK-II (data not shown), confirming that the N terminus is essential and sufficient for HK-II binding to mitochondria.The N terminus of HK-II has been considered to mediate its binding to mitochondria. The N terminus deletion mutation (ΔN HK-II), GFP, WT, or T473D were overexpressed in NRVMs, and the cells were fractionated D. WesterTo determine whether phosphorylation-dependent increases in mitochondrial HK-II binding support cell survival in cardiomyocytes, cells were infected with WT, T473D, T473A, and ΔN HK-II, treated with hydrogen peroxide, and then cell viability was assessed . WT HK-IWe next investigated the mechanism by which phosphorylation at Thr-473 by Akt may increase the amount of HK-II at mitochondria.A). As expected, G-6P dose-dependently dissociated WT HK-II from mitochondria up to ∼80% at 3 mm. Remarkably, this response was decreased significantly, and the dose-dependent curve was shifted rightward, in mitochondria isolated from cells expressing T473D HK-II (B). On the other hand, the T473A mutant did not inhibit G-6P-dependent dissociation of HK-II from mitochondria (A and B). The level of COXIV in the pellet was unchanged, nor was it detected in the supernatant, suggesting that HK-II release was not due to disruption of mitochondria. Similar dose-dependent curves were obtained in mitochondria supplemented with succinate and rotenone (data not shown).It has been established that G-6P can dissociate HK-II from mitochondria, and we hypothesized that phosphorylation of HK-II counteracts the dissociating effect of G-6P. To test the hypothesis, mitochondria were isolated from cells expressing WT or T473D HK-II, incubated with G-6P for 15 min, and the supernatant (dissociated HK-II) and pellet (mitochondria-bound HK-II) were subjected to Western blotting A. As exp3D HK-II B. On thechondria , A and Bm G-6P for 15 min (C). G-6P-induced HK-II release to the supernatant was inhibited significantly by IGF-1 treatment and was reversed by pretreatment with an Akt inhibitor, suggesting Akt mediated inhibition of G-6P-dependent HK-II dissociation. The effect of G-6P on the dissociation of endogenous HK-II was also tested (D). After 30 min of IGF-1 treatment in the presence or absence of Akt inhibitor V, mitochondria were isolated, and the effect of 0.3 mm G-6P was examined. G-6P-induced dissociation was inhibited significantly in cells treated with IGF-1, and this inhibitory effect was abolished by inhibition of Akt.To further determine the effect of Akt-mediated phosphorylation of HK-II on G-6P-dependent dissociation, cells overexpressing WT HK-II were treated with IGF-1 in the presence or absence of an Akt inhibitor, and mitochondria were isolated and treated with 0.3 mr 15 min C. G-6P-io tested D. After Although it has been demonstrated that an increased level of HK-II at mitochondria is protective and is increased by protective interventions but decreased under stress, it has not been fully determined which molecular signals regulate the level of HK-II at mitochondria. Here we demonstrate, for the first time, that Thr-473 in HK-II is phosphorylated by Akt and that this phosphorylation leads to increases in mitochondrial HK-II binding through inhibition of G-6P-dependent dissociation, conferring resistance to oxidative stress .Overexpression of WT HK-II increases mitochondrial HK-II and confers protection against hydrogen peroxide, which is enhanced significantly in HK-II T473D-expressing cells, whereas ΔN HK-II, lacking the ability to bind to mitochondria, does not confer protection. Conversely, mitochondrial HK-II dissociation peptide 15NG) inhibits the IGF-1-mediated increase in mitochondrial HK-II and cellular protection. These results strongly support the protective effect of an increase in mitochondrial HK-II against stress NG inhibi. AlthougThe mechanisms by which HK-II prevent opening of the mPTP are not fully understood. Previous studies have suggested a link between HK-II, a negative regulator of the mPTP, and cyclophilin D, a positive regulator , 41, 42.It has been demonstrated that Akt, upon its activation at the plasma membrane, translocates to different subcellular compartments, including the nucleus and mitochondria , 43, 44.We confirmed that Akt activation induced by IGF-1 phosphorylates and increases mitochondrial binding of HK-II  and 4. Um, which is in agreement with G-6P concentration increases of ∼0.17 to ∼1.3 mm in perfused working heart (50 of G-6P versus HK-II (A and B) strongly suggests that phosphorylation of Thr-473 decreases the sensitivity of HK-II to G-6P-induced mitochondrial dissociation and, thereby, increases mitochondrial HK-II. Importantly, Akt activation stimulated by IGF-1 also inhibits exogenous WT and endogenous HK-II dissociation induced by G-6P. To our knowledge, this is the first presentation that the inhibitory effect of G-6P on mitochondrial HK-II binding is negatively regulated by posttranscriptional modification. There was no difference in G-6P-induced inhibition of the catalytic activity of HK-II in WT, T473D, and T473A.It has been well established that G-6P induces dissociation of HK-II from mitochondria , 49–51. ng heart  and withus HK-II , 54. Ourus HK-II , A and BG-6P is known to be accumulated in the heart during ischemia about 10-fold , 56, andThe Pederson laboratory , 27, 58 In conclusion, we demonstrate that Akt phosphorylates HK-II at Thr-473, inducing increases in HK-II binding to mitochondria and conferring resistance to cytotoxic insult. This is attributable, at least in part, to decreased sensitivity of phosphorylated HK-II to G-6P-induced dissociation. We suggest that this regulation of mitochondrial HK-II binding by Akt-mediated phosphorylation plays a crucial role in cell survival.


The Na+/K+-ATPase is also implicated in regulating tight junctions and cardiotonic steroid (CTS)-induced signal transduction via SRC. We investigated the expression of SRC family kinase (SFK) members, Src and Yes, during preimplantation development and determined whether SFK activity is required for blastocyst formation. Embryos were collected following super-ovulation of CD1 or MF1 female mice. RT-PCR was used to detect SFK mRNAs encoding Src and Yes throughout preimplantation development. SRC and YES protein were localized throughout preimplantation development. Treatment of mouse morulae with the SFK inhibitors PP2 and SU6656 for 18 hours resulted in a reversible blockade of progression to the blastocyst stage. Blastocysts treated with 10−3 M ouabain for 2 or 10 minutes and immediately immunostained for phosphorylation at SRC tyr418 displayed reduced phosphorylation while in contrast blastocysts treated with 10−4 M displayed increased tyr418 fluorescence. SFK inhibition increased and SFK activation reduced trophectoderm tight junction permeability in blastocysts. The results demonstrate that SFKs are expressed during preimplantation development and that SFK activity is required for blastocyst formation and is an important mediator of trophectoderm tight junction permeability.The Na Blastocyst formation is a prerequisite for the initiation of pregnancy, however, the majority of mammalian preimplantation embryos fail to complete this developmental interval and implant +/K+-ATPase that creates a trans-trophectodermal ion gradient Blastocyst formation is regulated by the combined actions of ion transporters, water channels, and intercellular junctions +/K+-ATPase to modulate the ion transport function of the pump +/K+-ATPase function Ouabain is a cardiotonic steroid that is primarily known as a plant-derived chemical that specifically binds to the Na+/K+-ATPase ion transport, research applied primarily to cell lines has indicated that ouabain binding to the cell also regulates SRC pathway signalling +/K+-ATPase receptor regulates cellular function via activation of SRC and its downstream mechanisms In addition to regulating NaSrc family kinase members, Src and Yes, during preimplantation development. We establish concentrations of ouabain that both activate and inhibit SFK activation at the blastocyst stage. Furthermore, we demonstrate that SFK activity is necessary for blastocyst formation, and more specifically, regulates trophectoderm tight junction function. We therefore conclude that the developing blastocyst has the capacity to respond to ouabain by activating SFKs and that this process is an important mediator of tight junction function, and thus overall blastocyst formation.In this study we present evidence for the expression of Src and Yes mRNAs were consistently detected, using RT-PCR applied to three preimplantation development series  experimental replicates, throughout mouse preimplantation development , that are known to block the ion transport functions of the Na+/K+-ATPase in the mouse −4 M) that do not. After a 2 minute ouabain treatment, blastocysts exhibited consistent variations in SRC tyr418 phosphorylation  control, KSOMaa, 10−3 M ouabain, and 10−4 M ouabain treatment groups, respectively, were 10, 24, 32, 32. These results demonstrate the ability of different concentrations of ouabain and varying treatment times to induce disparate changes in SRC phosphorylation fluorescence in blastocysts. A high ouabain concentration, that blocks progression to the blastocyst stage, elicited reduced phosphorylation fluorescence levels on tyr418, indicating reduced SRC activity. A lower ouabain concentration that does not reduce development to the blastocyst stage elevated tyr418 phosphorylation fluorescence levels, indicating increased SRC activity.Following a 10 minute ouabain treatment, a greater difference in SRC tyr418 fluorescence in blastocysts was observed between treatment groups. Blastocysts cultured in 10controls . In turnNext we investigated the role of SFK signaling in regulating preimplantation development and more specifically on the regulation of trophectoderm tight junction permeability. Morulae treated with PP2 displayed a significant reduction in blastocyst formation after an 18 hour treatment at 20 µM, 30 µM and 50 µM, when compared to both control groups, P≤0.05 . Of the Embryos were randomly selected and removed from each treatment group  following the initial 18 hour culture period and were fixed instead of being placed into the recovery KSOMaa culture experiment. Application of immunofluorescence methods for phosphorylation on tyr418 of SRC revealed a concentration dependent loss of tyr418 phosphorylation of SRC after treatment in PP2, when compared to embryos in the KSOMaa or DMSO+KSOMaa . This ouA second SFK inhibitor, SU6656, was used to test for consistency of outcomes after SFK inhibition. Morulae cultured in SU6656 exhibited a significant reduction in their ability to cavitate after an 18 hour treatment period, P≤0.05 . Seventy−4 M ouabain decreased tight junction permeability to 11±3%  that increases SFK phosphorylation decreases trophectoderm tight junction permeability. In total our results demonstrate that SFK activity facilitates blastocyst formation and that SFKs are a potent mediator of trophectoderm tight junction function in the blastocyst.Our outcomes demonstrate that mRNAs encoding SFK family members SRC and YES are detectable during preimplantation development including blastocyst stage mouse embryos. Ouabain treatment has concentration dependent effects on SFK activation; higher concentrations known to block NaSrc and Yes, along with Fyn are ubiquitously expressed Fgr is present in B cells and myeloid cells, as are Lyn, Lck, Blk and Hck, which are also present in T cells and brain tissue Src, Yes and Fyn would be expected to be present during early mouse development. We restricted our analysis to include only Src and Yes as we were only able to obtain reliable antisera for these two members of the SFK family. The detection of mRNAs for Src and Yes during preimplantation development and most importantly within blastocysts implies a role for these SFK family members in regulating early development and allowed for experiments aimed at determining which kinases could be tethered to the Na+/K+-ATPase pump and activated in the signaling pathway regulating tight junctions in the preimplantation embryo. However, our results do not validate that only Src, and Yes have a role to play in blastocyst formation as it is quite possible proteins from other family members may be present in the blastocyst and contribute to the outcomes reported in our study. It will be important in the future to obtain effective antisera for these additional family members to characterize their localization patterns during preimplantation development.We first investigated the presence of SFK mRNAs during preimplantaiton development to provide a foundation for our studies. Our main focus was to determine if Src and Yes mRNAs were present prior to and following blastocyst formation. There are eight known SFK family members, Immunofluorescence revealed SRC and YES protein localization patterns also during preimplantation development and most importantly at the morula and blastocyst stages. SRC fluorescence was more intense when compared to YES, however our results do not precisely measure the abundance of these proteins. Immunofluoresence is a sensitive method that is very effective at revealing protein localization patterns. While several studies have developed ways to quantify immunofluoresence signals it is challenging to standardize conditions sufficiently to allow for a precise quantitiation of target proteins levels to occur. Because of this limitation we did not attempt to precisely measure variations in immunofluorescence signals in this study. We have confined ourselves to simply reporting consistent localization patterns and in providing qualitative assessment of consistent and obvious changes in fluorescence intensity following ouabain treatment. This is a limitation of our study but providing quantitative fold differences in fluorescence intensity may be misleading as its precise relationship to protein abundance or phosphorylation cannot be assured.+/K+-ATPase signalling in other cell systems such as cardiac myocytes, and speculation exists that the other SFKs could be a part of their own signalling pathway specific to different tissues +/K+-ATPase in the trophectoderm +/K+-ATPase pump in a signalling complex regulating tight junctions. Although it is possible that all SFKs interact with the Na+/K+-ATPase because of their similar structure, it would likely be in a tissue specific manner due to the difference in signals and protein distribution among cell types +/K+-ATPase responses.SRC has been linked to ouabain induced Na+/K+-ATPase α1-subunit, while lower concentrations will activate signalling pathways transduced through SRC +/K+-ATPase activity in cardiac myocytes, by way of activating SRC/EGFR, PI3K and ROS production +/K+-ATPase α1 knockout studies have even demonstrated increased basal SRC activity, and the loss of ouabain induction of SRC signalling  cells or pre-treating cells with PP2, caused ouabain to lose its ability to activate ERK Many studies report that high concentrations of ouabain will block the ion transport function of the rodent Na+/K+-ATPase pump. Although these outcomes were consistent and novel, the results did not convincingly exhibit a maximal effect following 2 minutes. Following 10 minutes of ouabain treatment, obvious differences between the level of fluorescence of tyr418 was observed between treatment groups. Blastocysts treated with 10−3 M ouabain exhibited a reduction in tyr418 phosphorylation fluorescence while those treated in 10−4 M ouabain demonstrated an increase in tyr418 phosphorylation fluorescence. Of greater significance, the percentage of embryos exhibiting higher tyr418 phosphorylation fluorescence doubled in the 10 minute group when compared to blastocysts treated for 2 min. The 10 minute treatment also resulted in higher intensity fluorescence when compared to the 2 min group, suggesting a time dependent aspect of ouabain stimulated SRC activity. This outcome has revealed a novel mode of SRC regulation in the blastocyst. This, together with the localization of SRC protein to the basolateral domain of embryonic cells, and the existing literature showing ouabain eliciting its effects through the Na+/K+-ATPase, implicates the pump in this signalling pathway in the mouse blastocyst. An important limitation of our study is that we were not able to precisely measure these changes in tyr418 phosphorylation by employing methods such as Western blotting. The primary reason for this was attempts to perform this analysis using samples derived from several 100 blastocysts per lane failed in detecting either total SRC, Yes or phosphorylated SRC. This is not completely surprising as the application of Western blotting methods to quantify protein changes during preimplantation stage embryos often fail even with the application to samples derived from 100's of early embryos due to low overall protein abundance in such samples. While we do not report specific fold changes in fluorescence the observations we do report were consistent and while qualitative in nature due reflect obvious changes in fluorescence intensity representative of tyr418 phosphorylation. We have validated the specificity of the antisera used in this study by conducting tissue and cell sample Western blots  is associated with disrupted tight junction associated protein (ZO-1 and occluden) distribution in treated blastocysts. In addition, it was reported by Violette +/K+-ATPase β1-subunit results in a loss of blastocyst formation, also associated with a disrupted trophectoderm tight junction associated protein distribution +/K+-ATPase β1-subunit may also be subject to SFK regulation. These will represent the subject of our future experiments. In addition, the ouabain-Na+/K+-ATPase-SRC complex is often associated with activation of ERK1/2, and now that SRC is implicated in interacting with ZO-1, regulation of trophectoderm tight junctions through this signalling cascade is very likely.This effect was also observed using 40 kDa FITC-detxtran, which showed no statistical significance between the EGTA and PP2 treated groups. This outcome establishes that SFK inhibition is just as effective at disrupting tight junction function as EGTA treatment. Remarkably, 10+/K+-ATPase pump as a signal transducer, rather than an ion transporter responsible for maintaining osmotic gradients. A number of novel findings pertaining to blastocyst development have been uncovered. Firstly, Src and Yes mRNAs and protein are detectable at the blastocyst stage. Secondly, 10−3 M ouabain reduces SFK activity, whereas 10−4 M ouabain increases tyr418 phosphorylation. Localizing SRC in the basolateral domain of blastocyst cells, and identifying that SFK activity is regulated by CTS, strongly suggests the involvement of the Na+/K+-ATPase in transducing this signal. Thirdly, SFK activity is required for blastocyst development. Lastly, SFKs regulates trophectoderm tight junction function in the blastocyst. Considering that little is known of how tight junctions could be regulated in the blastocyst, or of the signaling cascade initiated by CTS binding to the Na+/K+-ATPase in embryos, this study has increased our understanding of the mechanisms controlling blastocyst formation. It would seem to be reasonable to propose that the Na+/K+-ATPase is present in caveolae of the trophectoderm, creating a signalosome capable of transducing SFK signals following ouabain activation of the Na/K-ATPase. This research therefore addresses the basic mechanisms controlling early mammalian development and has uncovered new principles for understanding how blastocyst formation is regulated.In conclusion, this study has investigated the unconventional function of the Na2 in air atmosphere at 37°C for the time outlined by the experimental design. All animal care and embryo collection methods were conducted using approved protocols from the Canadian Council of Animal Care and the University of Western Ontario Animal Care and Veterinary Services. The approved University of Western Ontario animal care protocol number for these studies is 2010-021Watson.Female CD1  or MF1 mice  were superovulated by intraperitoneal (IP) injection of 5 IU pregnant mare's serum gonadotropin  followed 48 hours later by 5 IU human chorionic gonadotropin . Superovulated female mice were mated with CD1 males . Successful mating was indicated by the presence of a vaginal plug the following morning (day 1). Time post-hCG was used to measure the developmental age of the embryos. Preimplantation mouse embryos were collected at 18 hours (1-cell), 48 hours (2-cell), 55 hours (4-cell), 66 hours (8-cell), 77 hours (morula) and 90 hours (blastocyst) post hCG. All embryo stages were flushed from the reproductive tract using M2 medium . Embryos were washed several times in M2 medium with HEPES or KSOMaa and collected in pools to be 1) frozen at −80°C until their use in RNA extraction; 2) fixed for indirect immunofluorescence; or 3) cultured in EmbryoMax® KSOMaa (potassium simplex optimized medium with amino acids) Liquid Mouse Embryo Media  under mineral oil and maintained in culture under 5% COTotal RNA was extracted from pools of 20 or 40 preimplantation stage mouse embryos, stored at −80°C in M2 medium with HEPES immediately after collection, using the PicoPure® RNA Isolation Kit . Samples were treated with DNAse I  to remove any possible DNA contamination.Total RNA was reverse transcribed (RT) using the suggested protocol of Sensiscript RT . Each sample was diluted to a concentration of one embryo equivalent per microliter. PCR reactions were carried out in a volume of 50 µL consisting of 5 µL 10× PCR buffer, 2 µL 50 mM magnesium chloride, 1 µL 10 mM dNTP , 1 µM of each appropriate PCR primer, 38.8 µL HyPure™ water , 0.2 µL Platimum® Taq DNA Polymerase  and 1 µL cDNA for the embryonic stage being tested. Amplification reactions were performed using a Techne Touchgene Gradient DNA thermal cycler . PCR reactions were repeated a minimum of three times using cDNA prepared from embryos at each indicated stage, isolated from a minimum of three developmental series . Positive (lung tissue cDNA) and negative control (no cDNA template) samples were included for each primer set in each experiment. Lung RNA was extracted by the Trizol Reagent® protocol suggested by the manufacturer . The identity of each PCR product was confirmed by sequence analysis which resulted in a 100% sequence identity for both the Src and Yes PCR products derived from embryo and control samples .Src, and Yes, based on available mouse nucleotide sequences in GenBank.Primer pairs for PCR were designed and synthesized  for Rabbit polyclonal antiserum raised against the phosphorylated tyrosine residue 418 of human SRC  was utilized at a dilution of 1∶100 to localize SRC phosphorylation in mouse embryos. Rabbit polyclonal antisera against total human SRC  and human YES  were used at dilutions of 1∶200 to localize the proteins in mouse embryos. Western blotting utilizing standard SDS-polyacrylamide Gel Electrophoresis (PAGE) and chemi-luminescence detection methods was applied to verify the specificity of the SRC and YES antisera employed in the study .Mouse preimplantation stage embryos were collected from the reproductive tracts of superovulated female mice as described above. Embryo pools were fixed in 2% paraformaldehyde in PBS for 20 minutes at room temperature. Fixed embryos were incubated in embryo blocking buffer  for 1 hour followed by one wash in 1× PBS for 20 minutes at 37°C. Embryos were incubated with primary antisera diluted in antibody dilution buffer (ADB)  for 1 hour at 37°C or overnight at 4°C. Embryos were then washed 3× in ADB for 30 minutes at 37°C, then incubated with FITC-conjugated secondary antibody  at a 1∶200 dilution in ADB for 1 hour at 37°C or overnight at 4°C. To stain nuclear DNA, the first 30 minute wash in ADB included 4′,6-diamindino-2-phenylindole dihydrochloride (DAPI) , diluted to 1∶2000 from 1 mg/mL stock solution. Two additional washes in 400 µL ADB of 30 minutes each at 37°C were applied. Fully processed embryos were mounted onto glass slides in a drop of Vectashield  under Vaseline-elevated glass coverslips and sealed with nail polish. Immunofluorescence imaging employed an Olympus Fluoview 1000 laser scanning confocal microscope. For all immunofluorescence protocols, embryos serving as negative controls were incubated in ADB rather than primary antibody.2 in air atmosphere for 1 hour. Blastocysts were flushed from mouse uteri, washed thoroughly through M2 medium with HEPES, then KSOMaa and were incubated in 20 µL KSOMaa drops for 1 hour, after which they were placed in KSOMaa/ouabain  for 2 or 10 minutes. Immediately following ouabain treatment, blastocysts were fixed in 2% paraformaldehyde for 20 minutes at room temperature and were then immunostained for phosphorylation of tyr418 of SRC.Culture drops of KSOMaa were equilibrated at 37°C and 5% CO2 in air atmosphere. Morulae were flushed from uteri, washed thoroughly through M2 medium with HEPES, then KSOMaa and placed into 20 µL culture drops of KSOMaa, or KSOMaa plus DMSO, or KSOMaa plus PP2. Morulae were cultured for 18 hours at which time progression to the blastocyst stage was assessed. SU6656 at concentrations of 1 µM, 5 µM, 10 µM were also used in the same experimental design described above.Culture drops of KSOMaa, DMSO (vehicle control for PP2), and PP2  were incubated for 1 hour at 37°C and 5% CO−4 M ouabain or KSOMaa/20 µM PP2 for 3 hours. After the 3 hour time point, blastocysts were transferred to 4 or 40 kDa FITC-dextran in KSOMaa for 30 minutes. Following this incubation, blastocysts were immediately washed in three 50 µL wash drops of KSOMaa and placed in a fourth clean KSOMaa drop for immediate visualization using a Zeiss fluorescent compound microscope equipped with epi-fluorescent optics.To investigate the effects of SRC inhibition on tight junction permeability, blastocysts were flushed from uteri, were washed through KSOMaa and placed into 15 µL culture drops of KSOMaa, KSOMaa/2 mM EGTA (ethylene glycol Bis- (β-aminoethyl ether) N,N,N,N -tetraacetic acid), KSOMaa/10Statistical analysis of data was performed using SigmaStat®, version 3.5, software. The results from culture in ouabain, PP2 and SU6656 are all presented as the mean ± standard error from three independent experimental replicates. For testing significant differences between culture groups, data were subjected to one-way analysis of variance (ANOVA). Data analysis for ouabain and PP2 trials passed normality tests and were followed by the Holm-Sidak post hoc test. Data for the SU6656 and tight junction permeability experiments were evaluated by applying an ANOVA on ranks. As a post-hoc test, the Student-Neuman-Keul method was employed. For all data analysis, P≤0.05 was considered statistically significant.Figure S1A,B. Supplementary Data – Src and Yes antiserum western blotting in control tissues. SRC and YES antibodies were tested on liver, pancreas, mammary and cancerous acinar cell line control protein extracts . The SRC antiserum resulted in the detection of a single band of expected molecular weight (60 kDa) in liver and AR42J (A). SRC protein was not detected in mammary protein samples but was detected in both total pancreas tissue samples (A). The doublet detected in pancreatic tissue is suggestive of SRC phophorylation in this tissue (A). Western blot conducted using the YES antiserum also validated the specificity of the antiserum in the two pancreas protein tissue samples by producing bands of expected size in each sample (B). Yes protein was undetectable in liver, mammary gland tissue or AR42J cells (B).(TIF)Click here for additional data file.


The response of the postnatal heart to growth and stress stimuli includes activation of a network of signal transduction cascades, including the stress activated protein kinases such as p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK) and the extracellular signal-regulated kinase (ERK1/2) pathways. In response to increased workload, the mitogen-activated protein kinase kinase (MAPKK) MEK1 has been shown to be active. Studies embarking on mitogen-activated protein kinase (MAPK) signaling cascades in the heart have indicated peroxisome-proliferators activated-receptors (PPARs) as downstream effectors that can be regulated by this signaling cascade. Despite the importance of PPARα in controlling cardiac metabolism, little is known about the relationship between MAPK signaling and cardiac PPARα signaling.Using co-immunoprecipitation and immunofluorescence approaches we show a complex formation of PPARα with MEK1 and not with ERK1/2. Binding of PPARα to MEK1 is mediated via a LXXLL motif and results in translocation from the nucleus towards the cytoplasm, hereby disabling the transcriptional activity of PPARα. Mice subjected to voluntary running-wheel exercise showed increased cardiac MEK1 activation and complex formation with PPARα, subsequently resulting in reduced PPARα activity. Inhibition of MEK1, using U0126, blunted this effect.Here we show that activation of the MEK1-ERK1/2 pathway leads to specific inhibition of PPARα transcriptional activity. Furthermore we show that this inhibitory effect is mediated by MEK1, and not by its downstream effector kinase ERK1/2, through a mechanism involving direct binding to PPARα and subsequent stimulation of PPARα export from the nucleus. Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor which is believed to act as a sensor of fatty acids (FA) and FA derivatives to enable the cell to adapt to environmental changes through regulation of a large number of processes such as inflammation, differentiation and metabolism During the development of cardiac hypertrophy, myocardial fatty acid oxidation (FAO) rates decrease and glucose utilization increases 2-terminal kinase (JNK) and the extracellular signal-regulated kinase (ERK1/2) pathways The response of the postnatal heart to growth and stress stimuli includes activation of a network of signal transduction cascades, including the stress activated protein kinases such as p38 mitogen-activated protein kinase (MAPK), c-Jun NHcpt1a promoter harboring a functional PPRE. Co-transfection of a PPARα expression vector and stimulation with Wy-14643 as synthethic ligand (2 hr) resulted in increased luciferase activity . Next, we repeated the experiment using PPARα-GFPΔLxxLL and analyzed subcellular localization in the presence of absence of MEK1 and Wy-14643. In this case, Wy-14643 induced nuclear localization of PPARα-GFPΔLxxLL was not affected by ectopic MEK1 expression . QuantifIn conclusion, the combined experiments demonstrate that the ERK1/2 selective MAPKK MEK1 interacts with the LXXLL binding pocket of PPARα and forces translocation out of the nucleus to the cytosol as a novel mechanism whereby MEK1 signaling inhibits the transcriptional activity of PPARα.In vivo, MEK1 is activated in hearts of mice subjected to exercise training in vivo, we chose voluntary running-wheel exercise as a model to stimulate physiological cardiac hypertrophy and activate cardiac MEK1 in mice. After 4 weeks of voluntary wheel exercise, mice demonstrated a substantial cardiac growth response as evidenced by increased HW/BW ratios , carboxylesterase 3 (ces3), carnitine o-palmitoyltransferase 1(cpt1a), and acyl-coenzyme a dehydrogenase (acadvl), while exercise in the presence of U0126 fully relieved the inhibition of these PPARα target genes , protein kinase A (PKA), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). MAPK signaling pathways have been reported to be very important in the regulation of cellular differentiation, proliferation and stress responsiveness. Consisting of three major branches of sequentially signaling pathways, the MEK1 signaling pathway, which culminates in ERK1/2 activation, is hypothesized to regulate the growth and adaptation of the heart to both physiological and pathological stimuli in vivo with no detectable threonine or tyrosine activity Functional verification of the transcriptional activity of PPARα, after activation of the MEK1 pathway, resulted in a dramatic decrease of PPARα ability to activate a mCPT reporter. Although it was previously shown that activated ERK1/2 is able to phosphorylate PPARα An important factor that participates in the regulation of PPAR as well as MAPK signaling is their subcellular localization Unfortunately, little is known about the regulation of the intracellular distribution of PPARs, though cytosolic localization of PPARs has been reported as well as their bindings ability to the cytosolic/membrane proteins such as HSP90 cpt1a, acadv1, hadhsc), but also binding proteins that are involved in cellular transport of FA (cd36). This effect was less apparent for the regulation of genes involved the glycolysis/gluconeogenesis, where glucose transporter GLUT3 was up regulated but other components of the glycolysis were downregulated. Nonetheless, this expression profile rendered cardiomyocytes more resistant to energy deprivation following deoxyglucose exposure In this context one could assume that MEK1, via the inhibition of PPARα, has a role in the regulation of metabolic processes in the heart. Indeed, gene profiling studies executed on cardiomyocytes overexpressing a constitutively active form of MEK1 (MEK-EE) demonstrated a significant decrease of genes coding for proteins involved in FA metabolism Gene expression studies performed with RNA isolated from ventricles of mice subjected to transverse aortic banding indicated a downregulation of PPARα expression and several of its target genes In contrast, mice subjected to increased workload by voluntary running-wheel exercise showed no significant differences in cardiac PPARα levels compared to untrained mice. Using voluntary running-wheel exercise as a functional in vivo model to stimulate cardiac MEK1 activation, we show that MEK1 signaling inhibits the transcriptional activity of PPARα. Interestingly, MEK1 transgenic mouse lines showed a mild concentric hypertrophy with thicker septum and left ventricular posterior wall with very few signs of histopathology or interstitial fibrosis This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University Medical Center Utrecht, Utrecht, the Netherlands (Permit Number: 2009.II.03.024). Male C57BL6/J mice were subjected to voluntary cage wheel exercise as described Low passage HEK293 and NKL-TAg cells 4 cells/well). After 24 hours, transient transfections were performed as described Transfections were performed in 24-well plates  linked to firefly luciferase (mCPT1-luc) was described previously The reporter plasmid containing the Immunoprecipitation assays were performed as described previously One microgram of total RNA was used as template for Superscript reverse transcriptase II (Promega). For real time-PCR, a BioRad iCycler (Biorad) and SYBR Green was used in combination with specific primer sets designed to detect transcripts (primer sequences available upon request).2, 10 mM KCl, supplemented with 2 mM DTT, 0.4 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 2 µg/ml pepstatin, and 1 mM Na3VO4), resuspended in buffer A, and incubated on ice for 10 min. Nuclei were pelleted at 3000 rpm for 10 min and resuspended in buffer C  glycerol, 1.5 mM MgCl2) supplemented with 2 mM DTT, 0.4 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 2 µg/ml pepstatin, and 1 mM Na3VO4. Nuclear proteins were extracted by stirring at 4°C for 30 min. After centrifugation at 13,500 rpm for 30 min, the supernatant was dialyzed against buffer Z-100  glycerol, 2 mM DTT, 0.4 mM PMSF, 1 mM Na3VO4, 100 mM KCl) at 4°C. The dialysate was clarified by centrifugation at 13,500 rpm for 30 min at 4°C and designated as crude nuclear extract.Cells were washed with ice-cold PBS, scraped into 5 ml of PBS and pelleted by centrifugation at 1500 rpm for 10 min at 4°C. Cell pellets were washed with buffer A  and secondary monoclonal anti-mouse Texas-Red  antibodies were diluted using 1% BSA in TBS and incubations were carried out at room temperature for 1 hour. Cells were washed 3 times with PBS for 5 minutes, mounted with coverslips in Vectashield mounting medium H-1000 , and analyzed by immunofluorescence microscopy using a Zeiss LSM 510 META instrument Results are presented as means ± SEM. Statistical analyses were performed using INSTAT 3.0 software  and Student’s t-test or ANOVA followed by Tukey’s post-test when appropriate. Statistical significance was accepted at a P value <0.05.


Rasagiline possesses no amphetamine-like properties, by contrast with the related compound selegiline . Although the exact distribution of MAO isoforms in different neurons and tissues is not known, dopamine behaves largely as a MAO-A substrate in vivo, but following loss of dopaminergic axonal varicosities from the striatum, metabolism by glial MAO-B becomes increasingly important. Following subchronic administration to normal rats, rasagiline increases levels of dopamine in striatal microdialysate, possibly by the build-up of β-phenylethylamine, which is an excellent substrate for MAO-B, and is an effective inhibitor of the plasma membrane dopamine transporter (DAT). Both of these mechanisms may participate in the anti-Parkinsonian effect of rasagiline in humans. Rasagiline possesses neuroprotective properties in a variety of primary neuronal preparations and neuron-like cell lines, which is not due to MAO inhibition. Recent clinical studies have also demonstrated possible neuroprotective properties of the drug in human Parkinsonian patients, as shown by a reduced rate of decline of symptoms over time.Rasagiline (Azilect) is a highly selective and potent propargylamine inhibitor of monoamine oxidase (MAO) type B. Like other similar propargylamine inhibitors, rasagiline binds covalently to the N5 nitrogen of the flavin residue of MAO, resulting in irreversible inactivation of the enzyme. Therapeutic doses of the drug which inhibit brain MAO-B by 95% or more cause minimal inhibition of MAO-A, and do not potentiate the pressor or other pharmacological effects of tyramine. Metabolic conversion of the compound  The most important and debilitating symptoms of Parkinson’s disease are those resulting from dopamine (DA) depletion in the nigro-striatal pathway, although the disease process starts at an earlier stage, and is marked by symptoms such as reduced olfactory sensitivity, autonomic dysfunction, and affective disorder.Rasagiline exerts its anti-Parkinsonian effect by inhibiting the oxidative breakdown of DA in the striatum, and can be used both as monotherapy in the early stages of the disease, or as an adjunct to L-dopa, in the advanced stages.The enzyme MAO is responsible for the oxidative deamination of a wide range of biogenic and xenobiotic amines, including DA, noradrenaline, adrenaline, tyramine, serotonin, β-phenylethylamine, N-methylhistamine, benzylamine, and methoxy metabolites of the parent amines, such as metanephrine and normetanephrineThe first clinical use of MAO inhibitors was in the treatment of depressive disease, an effect mediated by inhibition of the degradation of noradrenaline and 5-HT, and consequent increased levels of these amines at their receptors. In the Parkinsonian patient, in whom DA levels are reduced, inhibition of DA oxidative metabolism can also be effective in returning neurotransmitter levels towards normal; however, non-selective inhibition of MAO can cause dangerous increases in amine levels, especially in conjunction with a monoamine precursor such as L-dopa or indirectly acting amine such as tyramine. Following the introduction of the selective inhibitors clorgyline and selegiline, together with biochemical experiments which succeeded in separating different isoforms of the enzyme, MAO was shown to exist in two isoforms known as MAO-A and MAO-B, which show different selectivities for substrates and inhibitorsThe enzyme MAO is widely distributed in the body’s tissues, with a high degree of expression in the gastro-intestinal tract and liver, as well as neuronal tissue, and also in lung, heart, placenta, and nearly all other organs. For the current discussion, however, the most important aspect of the selective distribution of MAO-A and MAO-B is their selective expression in neurons and cells of the nervous system. Within neurons, MAO enzyme molecules are synthesized in the perikaryon, and inserted into the mitochondrial outer membrane. Mitochondria are transported to the axon terminals by axonal transport. Little is known about the way in which MAO subtypes are trafficked along the axons, but this process may well be important in determining the subtype expressed in axonal varicosities. The selective occurrence of MAO subtypes in neuronal and glial tissue is an important factor in understanding how subtype-selective MAO inhibitors may affect synaptic neurotransmitter levels.in situ hybridization and immunohistochemistry, MAO-A has been localized to noradrenergic perikarya of locus coeruleus, while MAO-B was the predominant subtype expressed in serotonergic cell bodies of the raphe nucleus and in glial cells.in vivo.24Sympathetic denervation studies showed that MAO-A is the predominant subtype in sympathetic post-ganglionic neurons.The breakdown of monamines by MAO can be described by the equation:The first selective MAO-B inhibitor to be described was selegiline, which was synthesized in 1965 by Knoll and Magyar, based on methamphetamine with the addition of a propargyl group .25 Follo26in vitro and in vivo pharmacological tests, selegiline was shown not to potentiate the actions of tyramine, while at the same time potentiating those of β-phenylethylamine.27In a large number of Selegiline was introduced into clinical medicine for treatment of Parkinson’s disease by Birkmayer and associates.in vivo and in vitro, but is significantly more potent than selegiline, both in rat and man. Both inhibitors will inhibit the A form of the enzyme at higher doses.Rasagiline is a close chemical relative of selegiline, but possesses the important distinction that its major metabolite is 1-aminoindan, which does not possess amphetamine-like activity, and does not possess appreciable affinity for any of the catecholaminergic or serotonergic receptor groups.The propargyl derivative inhibitors are irreversible site-directed inhibitors, which form covalent linkage with the N5 nitrogen of flavin, a component of the enzyme active site. When used clinically, the drugs are administered at a low daily dose, which inhibits a small fraction of the enzyme at each administration. The degree of enzyme inhibition thereby increases with successive doses of the inhibitor. The aim is to use a daily dose at which nearly complete inhibition of the enzyme occurs after about 10 days, so that subsequent drug administration maintains the extensive inhibition of the enzyme by inhibiting newly synthesized enzyme.Rasagiline is mainly metabolized by the hepatic cytochrome P450 enzyme 1A2, with production of 1-aminoindan as the major metabolite.38in vitro.1-adrenoceptor agonists. By combining biochemical determination of tissue MAO activities with pharmacological response to tyramine and noradrenaline, we were able to show that tyramine potentiation occurred following 80% or more inhibition of MAO-A, but not of MAO-B.40The advent of rasagiline enabled confirmation of the hypothesis that tyramine potentiation results from inhibition of MAO-A but not MAO-B. This point was extensively studied by us in pharmacological experiments using the rat vas deferens preparation At the whole animal level, cheese effect can theoretically result from a decrease in breakdown of orally administered tyramine in intestinal tract and liver, tissues which both express large amounts of both subtypes of the MAO enzyme. Our work with the isolated tissue preparation, however, showed that an important part of the cheese effect is potentiation of tyramine’s ability to release noradrenaline at the level of the neuron. Selective inhibition of MAO within the sympathetic neuron could be working in two ways: either by increasing the level of tyramine within the neuron, or by increasing the cytoplasmatic level of noradrenaline available for release. The latter would appear to be the most significant mechanism. Release of noradrenaline by tyramine is non-exocytotic. As shown by Trendelenburg and associates,During my sabbatical studies at NIH, Bethesda, Maryland, in the laboratories of Drs I. Kopin, D. Goldstein, and K. Bankiewycz, I used the micro-dialysis technique to study the metabolism of DA in rat striatum which had been depleted of dopaminergic innervation by local application of the neurotoxin 6-hydroxydopamine to the substantia nigra. These studies showed that MAO-A is the dominant enzyme subtype in the metabolism of DA in rat striatum, both in intact striatum, and following loss of dopaminergic input.–10 M) below those required for MAO inhibition (1 X 10–8M).As described above, selegiline was found to possess neuroprotective effect in isolated neuronal preparations. Neuroprotection was observed at concentrations below those required for MAO inhibition, and other MAO inhibitors did not consistently produce neuroprotection. We observed neuroprotection by rasagiline both in dopaminergic and non-dopaminergic rat embryonic mesencephalic neurons.The intracellular mechanism of action of rasagiline’s anti-apoptotic effect has been extensively investigated by Youdim and co-workers.At the whole animal level, rasagiline has been observed to reverse MPTP-induced reduction of tyrosine hydroxylase-positive neurons in the substantia nigra in mice as well as the neurological deficit caused by the MPTP administration.Clinical anti-Parkinsonian effect of rasagiline was described in two major clinical trials. The first (TEMPO)Rasagiline is a selective MAO-B inhibitor which is devoid of the amphetamine-like actions of its predecessor, selegiline. The drug has two distinct actions: selective irreversible inhibition of MAO-B, and neuroprotective effect not dependent on MAO inhibition. When used in patients, the possibility exists that its action to retard disease progression is linked to MAO inhibition, since activation of DA receptors can induce release of a variety of neurotrophic factors, but reduced disease progression could also be an expression of the neuroprotective action seen in laboratory experiments. Further understanding of the mechanism of clinical action in retardation of disease progression will require development of better techniques to understand the consequences of inhibition of MAO-B in the human brain.