Co-delivery of paclitaxel and cetuximab by nanodiamond enhances mitotic catastrophe and tumor inhibition.

The poor intracellular uptake and non-specific binding of anticancer drugs into cancer cells are the bottlenecks in cancer therapy. Nanocarrier platforms provide the opportunities to improve the drug efficacy. Here we show a carbon-based nanomaterial nanodiamond (ND) that carried paclitaxel (PTX), a microtubule inhibitor, and cetuximab (Cet), a specific monoclonal antibody against epidermal growth factor receptor (EGFR), inducing mitotic catastrophe and tumor inhibition in human colorectal cancer (CRC). ND-PTX blocked the mitotic progression, chromosomal separation, and induced apoptosis in the CRC cells; however, NDs did not induce these effects. Conjugation of ND-PTX with Cet (ND-PTX-Cet) was specifically binding to the EGFR-positive CRC cells and enhanced the mitotic catastrophe and apoptosis induction. Besides, ND-PTX-Cet markedly decreased tumor size in the xenograft EGFR-expressed human CRC tumors of nude mice. Moreover, ND-PTX-Cet induced the mitotic marker protein phospho-histone 3 (Ser10) and apoptotic protein active-caspase 3 for mitotic catastrophe and apoptosis. Taken together, this study demonstrated that the co-delivery of PTX and Cet by ND enhanced the effects of mitotic catastrophe and apoptosis in vitro and in vivo, which may be applied in the human CRC therapy.

Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7.

ZAC, an encoding gene mapped at chromosome 6q24-q25 within PSORS1, was previously found over-expressed in the lower compartment of the hyperplastic epidermis in psoriatic lesions. Cytokines produced in the inflammatory dermatoses may drive AP-1 transcription factor to induce responsive gene expressions. We demonstrated that mZac1 can enhance AP-1-responsive S100A7 expression of which the encoding gene was located in PSORS4 with HaCaT keratinocytes. However, the mZac1-enhanced AP-1 transcriptional activity was suppressed by curcumin, indicating the anti-inflammatory property of this botanical agent and is exhibited by blocking the AP-1-mediated cross-talk between PSORS1 and PSORS4. Two putative AP-1-binding sites were found and demonstrated to be functionally important in the regulation of S100A7 promoter activity. Moreover, we found curcumin reduced the DNA-binding activity of AP-1 to the recognition element located in the S100A7 promoter. The S100A7 expression was found to be upregulated in the lesioned epidermis of atopic dermatitis and psoriasis, which is where this keratinocyte-derived chemoattractant engaged in the pro-inflammatory feedback loop. Understanding the regulatory mechanism of S100A7 expression will be helpful to develop therapeutic strategies for chronic inflammatory dermatoses via blocking the reciprocal stimuli between the inflammatory cells and keratinocytes.

Systematic Biological Filter Design with a Desired I/O Filtering Response Based on Promoter-RBS Libraries.

In this study, robust biological filters with an external control to match a desired input/output (I/O) filtering response are engineered based on the well-characterized promoter-RBS libraries and a cascade gene circuit topology. In the field of synthetic biology, the biological filter system serves as a powerful detector or sensor to sense different molecular signals and produces a specific output response only if the concentration of the input molecular signal is higher or lower than a specified threshold. The proposed systematic design method of robust biological filters is summarized into three steps. Firstly, several well-characterized promoter-RBS libraries are established for biological filter design by identifying and collecting the quantitative and qualitative characteristics of their promoter-RBS components via nonlinear parameter estimation method. Then, the topology of synthetic biological filter is decomposed into three cascade gene regulatory modules, and an appropriate promoter-RBS library is selected for each module to achieve the desired I/O specification of a biological filter. Finally, based on the proposed systematic method, a robust externally tunable biological filter is engineered by searching the promoter-RBS component libraries and a control inducer concentration library to achieve the optimal reference match for the specified I/O filtering response.

The inhibitory mechanism by curcumin on the Zac1-enhanced cyclin D1 expression in human keratinocytes.

BACKGROUND: ZAC, a zinc finger protein regulating cell cycle arrest and apoptosis, mRNA was found highly expressed in the hyper-proliferative epidermal compartment of psoriatic skin. On the other hand, curcumin has been tried for treatment of psoriasis partly due to its anti-proliferative property. OBJECTIVES: Since cyclin D1 is a positive regulator for cell-cycle progression and its expression can be inhibited by curcumin, we would like to test whether the expression of cyclin D1 can be affected by Zac1. The cross-talk between curcumin and Zac1 upon the regulation of cyclin D1 expression will also be explored in the HaCaT cell line. METHODS: Cyclin D1 promoter luciferase reporter was used to measure the transcriptional activity of Zac1 in the absence or presence of curcumin treatment for HaCaT cells. Likewise, RT-PCR, western blotting and flow cytometry were employed to evaluate the expression of Zac1, cyclin D1 and other negative regulators of S phase entry in cell-cycle. RESULTS: Zac1 enhances the expression of cyclin D1, but curcumin decreases both the expression of Zac1 and cyclin D1. Interestingly, Zac1-induced cyclin D1 promoter activity is abolished by curcumin. Supportively, curcumin indeed exhibits an inhibitory effect to prevent cultured keratinocytes from entry into S phase in the cell cycle. CONCLUSIONS: These findings revealed that Zac1 modulates not only cell differentiation and apoptosis but also cell proliferation. The experimental results implied that curcumin may inhibit the expression of ZAC, consequently down-regulate the cyclin D1 expression and decelerate cell-cycle progression of psoriatic keratinocytes.

Systematic design methodology for robust genetic transistors based on I/O specifications via promoter-RBS libraries.

BACKGROUND: Synthetic genetic transistors are vital for signal amplification and switching in genetic circuits. However, it is still problematic to efficiently select the adequate promoters, Ribosome Binding Sides (RBSs) and inducer concentrations to construct a genetic transistor with the desired linear amplification or switching in the Input/Output (I/O) characteristics for practical applications. RESULTS: Three kinds of promoter-RBS libraries, i.e., a constitutive promoter-RBS library, a repressor-regulated promoter-RBS library and an activator-regulated promoter-RBS library, are constructed for systematic genetic circuit design using the identified kinetic strengths of their promoter-RBS components.According to the dynamic model of genetic transistors, a design methodology for genetic transistors via a Genetic Algorithm (GA)-based searching algorithm is developed to search for a set of promoter-RBS components and adequate concentrations of inducers to achieve the prescribed I/O characteristics of a genetic transistor. Furthermore, according to design specifications for different types of genetic transistors, a look-up table is built for genetic transistor design, from which we could easily select an adequate set of promoter-RBS components and adequate concentrations of external inducers for a specific genetic transistor. CONCLUSION: This systematic design method will reduce the time spent using trial-and-error methods in the experimental procedure for a genetic transistor with a desired I/O characteristic. We demonstrate the applicability of our design methodology to genetic transistors that have desirable linear amplification or switching by employing promoter-RBS library searching.

Regulation of presynaptic neurotransmission by macroautophagy.

mTOR is a regulator of cell growth and survival, protein synthesis-dependent synaptic plasticity, and autophagic degradation of cellular components. When triggered by mTOR inactivation, macroautophagy degrades long-lived proteins and organelles via sequestration into autophagic vacuoles. mTOR further regulates synaptic plasticity, and neurodegeneration occurs when macroautophagy is deficient. It is nevertheless unknown whether macroautophagy modulates presynaptic function. We find that the mTOR inhibitor rapamycin induces formation of autophagic vacuoles in prejunctional dopaminergic axons with associated decreased axonal profile volumes, synaptic vesicle numbers, and evoked dopamine release. Evoked dopamine secretion was enhanced and recovery was accelerated in transgenic mice in which macroautophagy deficiency was restricted to dopaminergic neurons; rapamycin failed to decrease evoked dopamine release in the striatum of these mice. Macroautophagy that follows mTOR inhibition in presynaptic terminals, therefore, rapidly alters presynaptic structure and neurotransmission.

Age and α-synuclein expression interact to reveal a dependence of dopaminergic axons on endogenous Akt/PKB signaling.

The mechanisms underlying the chronic neurodegeneration that occurs in Parkinson's disease (PD) are unknown. One emerging hypothesis is that neural systems deteriorate and eventually degenerate due to a primary failure of either extrinsic neurotrophic support or the intrinsic cellular pathways that mediate such support. One of the cellular pathways that have been often identified in mediating neurotrophic effects is that of PI3K/Akt signaling. In addition, recent observations have suggested a primary failure of PI3K/Akt signaling in animal models and in PD patients. Therefore, to explore the possible role of endogenous Akt signaling in maintaining the viability and functionality of substantia nigra (SN) dopamine neurons, one of the principal systems affected in PD, we have used an adeno-associated viral vector to transduce them with a dominant negative (DN) form of Akt, the pleckstrin homology (PH) domain alone (DN(PH)-Akt). In addition, we have examined the effect of DN(PH)-Akt in murine models of two risk factors for human PD: advanced age and increased expression of α-synuclein. We find that transduction of these neurons in normal adult mice has no effect on any aspect of their morphology at 4 or 7weeks. However, in both aged mice and in transgenic mice with increased expression of human α-synuclein we observe decreased phenotypic expression of the catecholamine synthetic enzyme tyrosine hydroxylase (TH) in dopaminergic axons and terminals in the striatum. In aged transgenic α-synuclein over-expressing mice this reduction was 2-fold as great. We conclude that the two principal risk factors for human PD, advanced age and increased expression of α-synuclein, reveal a dependence of dopaminergic neurons on endogenous Akt signaling for maintenance of axonal phenotype.

Akt suppresses retrograde degeneration of dopaminergic axons by inhibition of macroautophagy.

Axon degeneration is a hallmark of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Such degeneration is not a passive event but rather an active process mediated by mechanisms that are distinct from the canonical pathways of programmed cell death that mediate destruction of the cell soma. Little is known of the diverse mechanisms involved, particularly those of retrograde axon degeneration. We have previously observed in living animal models of degeneration in the nigrostriatal projection that a constitutively active form of the kinase, myristoylated Akt (Myr-Akt), demonstrates an ability to suppress programmed cell death and preserve the soma of dopamine neurons. Here, we show in both neurotoxin and physical injury (axotomy) models that Myr-Akt is also able to preserve dopaminergic axons due to suppression of acute retrograde axon degeneration. This cellular phenotype is associated with increased mammalian target of rapamycin (mTor) activity and can be recapitulated by a constitutively active form of the small GTPase Rheb, an upstream activator of mTor. Axon degeneration in these models is accompanied by the occurrence of macroautophagy, which is suppressed by Myr-Akt. Conditional deletion of the essential autophagy mediator Atg7 in adult mice also achieves striking axon protection in these acute models of retrograde degeneration. The protection afforded by both Myr-Akt and Atg7 deletion is robust and lasting, because it is still observed as protection of both axons and dopaminergic striatal innervation weeks after injury. We conclude that acute retrograde axon degeneration is regulated by Akt/Rheb/mTor signaling pathways.

Clinical progression in Parkinson disease and the neurobiology of axons.

Despite tremendous growth in recent years in our knowledge of the molecular basis of Parkinson disease (PD) and the molecular pathways of cell injury and death, we remain without therapies that forestall disease progression. Although there are many possible explanations for this lack of success, one is that experimental therapeutics to date have not adequately focused on an important component of the disease process, that of axon degeneration. It remains unknown what neuronal compartment, either the soma or the axon, is involved at disease onset, although some have proposed that it is the axons and their terminals that take the initial brunt of injury. Nevertheless, this concept has not been formally incorporated into many of the current theories of disease pathogenesis, and it has not achieved a wide consensus. More importantly, in view of growing evidence that the molecular mechanisms of axon degeneration are separate and distinct from the canonical pathways of programmed cell death that mediate soma destruction, the possibility of early involvement of axons in PD has not been adequately emphasized as a rationale to explore the neurobiology of axons for novel therapeutic targets. We propose that ongoing degeneration of axons, not cell bodies, is the primary determinant of clinically apparent progression of disease, and that future experimental therapeutics intended to forestall disease progression will benefit from a new focus on the distinct mechanisms of axon degeneration.

The Wld(S) mutation delays anterograde, but not retrograde, axonal degeneration of the dopaminergic nigro-striatal pathway in vivo.

For many neurodegenerative disorders, such as Parkinson's disease, there is evidence that the disease first affects axons and terminals of neurons that are selectively vulnerable. This would suggest that it may be possible to forestall progression by targeting the cellular mechanisms of axon degeneration. While it is now clear that these mechanisms are distinct from the pathways of programmed cell death, they are less well known. Compelling evidence of the distinctiveness of these mechanisms has derived from studies of the Wld(S) mutation, which confers resistance to axon degeneration. Little is known about how this mutation affects degeneration in dopaminergic axons, those that are affected in Parkinson's disease. We have characterized the Wld(S) phenotype in these axons in four models of injury: two that utilize the neurotoxin 6-hydroxydopamine or axotomy to induce anterograde degeneration, and two that use these methods to induce retrograde degeneration. For both 6-hydroxydopamine and axotomy, Wld(S) provides protection from anterograde, but not retrograde degeneration. This protection is observed as preserved immunostaining for tyrosine hydroxylase in axons and striatum, and by structural integrity visualized by GFP in tyrosine hydroxylase-GFP mice. Therefore, Wld(S) offers axon protection, but it reveals fundamentally different processes underlying antero- and retrograde degeneration in this system.

Regulation of the postnatal development of dopamine neurons of the substantia nigra in vivo by Akt/protein kinase B.

Following mitosis, specification and migration during embryogenesis, dopamine neurons of the mesencephalon undergo a postnatal naturally occurring cell death event that determines their final adult number, and a period of axonal growth that determines pattern and extent of target contacts. While a number of neurotrophic factors have been suggested to regulate these developmental events, little is known, especially in vivo, of the cell signaling pathways that mediate these effects. We have examined the possible role of Akt/Protein Kinase B by transduction of these neurons in vivo with adeno-associated viral vectors to express either a constitutively active or a dominant negative form of Akt/protein kinase B. We find that Akt regulates multiple features of the postnatal development of these neurons, including the magnitude of the apoptotic developmental cell death event, neuron size, and the extent of target innervation of the striatum. Given the diversity and magnitude of its effects, the regulation of the development of these neurons by Akt may have implications for the many psychiatric and neurologic diseases in which these neurons may play a role.

JNK2 and JNK3 combined are essential for apoptosis in dopamine neurons of the substantia nigra, but are not required for axon degeneration.

Activation of c-jun N-terminal kinase (JNK) by the mitogen-activated protein kinase cascade has been shown to play an important role in the death of dopamine neurons of the substantia nigra, one of the principal neuronal populations affected in Parkinson's disease. However, it has remained unknown whether the JNK2 and JNK3 isoforms, either singly or in combination, are essential for apoptotic death, and, if so, the mechanisms involved. In addition, it has been unclear whether they play a role in axonal degeneration of these neurons in disease models. To address these issues we have examined the effect of single and double jnk2 and jnk3 null mutations on apoptosis in a highly destructive neurotoxin model, that induced by intrastriatal 6-hydroxydopamine. We find that homozygous jnk2/3 double null mutations result in a complete abrogation of apoptosis and a prolonged survival of the entire population of dopamine neurons. In spite of this complete protection at the cell soma level, there was no protection of axons. These studies provide a striking demonstration of the distinctiveness of the mechanisms that mediate cell soma and axon degeneration, and they illustrate the need to identify and target pathways of axon degeneration in the development of neuroprotective therapeutics.

Rescue of p53 blockage by the A(2A) adenosine receptor via a novel interacting protein, translin-associated protein X.

Blockage of the p53 tumor suppressor has been found to impair nerve growth factor (NGF)-induced neurite outgrowth in PC-12 cells. We report herein that such impairment could be rescued by stimulation of the A(2A) adenosine receptor (A(2A)-R), a G protein-coupled receptor implicated in neuronal plasticity. The A(2A)-R-mediated rescue occurred in the presence of protein kinase C (PKC) inhibitors or protein kinase A (PKA) inhibitors and in a PKA-deficient PC-12 variant. Thus, neither PKA nor PKC was involved. In contrast, expression of a truncated A(2A)-R mutant harboring the seventh transmembrane domain and its C terminus reduced the rescue effect of A(2A)-R. Using the cytoplasmic tail of the A(2A)-R as bait, a novel-A(2A)-R-interacting protein [translin-associated protein X (TRAX)] was identified in a yeast two-hybrid screen. The authenticity of this interaction was verified by pull-down experiments, coimmunoprecipitation, and colocalization of these two molecules in the brain. It is noteworthy that reduction of TRAX using an antisense construct suppressed the rescue effect of A(2A)-R, whereas overexpression of TRAX alone caused the same rescue effect as did A(2A)-R activation. Results of [(3)H]thymidine and bromodeoxyuridine incorporation suggested that A(2A)-R stimulation inhibited cell proliferation in a TRAX-dependent manner. Because the antimitotic activity is crucial for NGF function, the A(2A)-R might exert its rescue effect through a TRAX-mediated antiproliferative signal. This antimitotic activity of the A(2A)-R also enables a mitogenic factor (epidermal growth factor) to induce neurite outgrowth. We demonstrate that the A(2A)-R modulates the differentiation ability of trophic factors through a novel interacting protein, TRAX.

Essential role of cAMP-response element-binding protein activation by A2A adenosine receptors in rescuing the nerve growth factor-induced neurite outgrowth impaired by blockage of the MAPK cascade.

We found in the present study that stimulation of the A(2A) adenosine receptor (A(2A)-R) using an A(2A)-selective agonist (CGS21680) rescued the blockage of nerve growth factor (NGF)-induced neurite outgrowth when the NGF-evoked MAPK cascade was suppressed by an MEK inhibitor (PD98059) or by a dominant-negative MAPK mutant (dnMAPK). This action of A(2A)-R (designated as the A(2A)-rescue effect) can be blocked by two inhibitors of protein kinase A (PKA) and was absent in a PKA-deficient PC12 variant. Activation of the cAMP/PKA pathway by forskolin exerted the same effect as that by A(2A)-R stimulation. PKA, thus, appears to mediate the A(2A)-rescue effect. Results from cAMP-response element-binding protein (CREB) phosphorylation at serine 133, trans-reporting assays, and overexpression of two dominant-negative CREB mutants revealed that A(2A)-R stimulation led to activation of CREB in a PKA-dependent manner and subsequently reversed the damage of NGF-evoked neurite outgrowth by PD98059 or dnMAPK. Expression of an active mutant of CREB readily rescued the NGF-induced neurite outgrowth impaired by dnMAPK, further strengthening the importance of CREB in the NGF-mediated neurite outgrowth process. Moreover, simultaneous activation of the A(2A)-R/PKA/CREB-mediated and the phosphatidylinositol 3-kinase pathways caused neurite outgrowth that was not suppressed by a selective inhibitor of TrkA, indicating that transactivation of TrkA was not involved. Collectively, CREB functions in conjunction with the phosphatidylinositol 3-kinase pathway to mediate the neurite outgrowth process in PC12 cells.