Ahmad R. Bassiouny

Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage

The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein, plays a central role in the repair of oxidative base damage via the DNA base excision repair (BER) pathway. The mammalian AP-endonuclease (APE1) overexpression is often observed in tumor cells, and confers resistance to various anticancer drugs; its downregulation sensitizes tumor cells to those agents via induction of apoptosis. Here we show that wild type (WT) but not mutant p53 negatively regulates APE1 expression. Time-dependent decrease was observed in APE1 mRNA and protein levels in the human colorectal cancer line HCT116 p53(+/+), but not in the isogenic p53 null mutant after treatment with camptothecin, a DNA topoisomerase I inhibitor. Furthermore, ectopic expression of WTp53 in the p53 null cells significantly reduced both endogenous APE1 and APE1 promoter-dependent luciferase expression in a dose-dependent fashion. Chromatin immunoprecipitation assays revealed that endogenous p53 is bound to the APE1 promoter region that includes a Sp1 site. We show here that WTp53 interferes with Sp1 binding to the APE1 promoter, which provides a mechanism for the downregulation of APE1. Taken together, our results demonstrate that WTp53 is a negative regulator of APE1 expression, so that repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of apoptosis in response to DNA damage.

Biosorption of lead and cadmium using marine algae

The use of algae (Ulva fasciata, green and Sargassum sp., brown) to reduce lead and cadmium levels from mono-metal solutions was investigated. The brown algae showed higher efficiency for the accumulation of lead (∼1.5 times) and cadmium (∼2 times) than green algae. The optimum pH value is found to be between 4 and 5.5. Regarding biomass concentration, an increase in metals percentage removal and a decrease in metal uptake capacity coincided with the increase in biomass concentration. All light metals (Ca, Mg and Na) showed a suppressive effect on biosorption capacity. The enhancement of biosorption in the case of NaOH was obvious. The biosorption process (65–90%) occurred within 3 min. Experimental data were in high agreement with the pseudo-second-order kinetic model and Freundlich model for lead and cadmium biosorption using different biosorbents. In the desorption study, 0.2 mol⋅L−1 HCl recorded the best concentration for the elution of metals from the biomass. The biosorption capacity decreased over the four operational cycles for both lead and cadmium. Infrared analysis showed that amino, hydroxyl and carboxyl functional groups provide the major biosorption sites for metal binding. Use of the above-mentioned algae for cheap metal absorbance is considered as one water treatment criterion.

Valproic acid potentiates curcumin-mediated neuroprotection in lipopolysaccharide induced rats.

The etiology of neuroinflammation is complex and comprises multifactorial, involving both genetic and environmental factors during which diverse genetic and epigenetic modulations are implicated. Curcumin (Cur) and valproic acid (VPA), histone deacetylase 1 inhibitor, have neuroprotective effects. The present study was designed with an aim to investigate the ability of co-treatment of both compounds (Cur or VPA, 200 mg/kg) for 4 weeks to augment neuroprotection and enhance brain recovery from intra-peritoneal injection of (250 μg/kg) lipopolysaccharide-stimulated neuroinflammatory condition on rat brain cortex. Cortex activation and the effects of combined treatment and production of proinflammatory mediators, cyclooxygenase-2 (COX-2), APE1, and nitric oxide/inducible nitric oxide synthase (iNOS) were investigated. Neuroinflammation development was assessed by histological analyses and by investigating associated indices [β-secretase (BACE1), amyloid protein precursor (APP), presenilin (PSEN-1), and PSEN-2)]. Furthermore we measured the expression profile of lethal-7 (let-7) miRNAs members a, b, c, e, and f in all groups, a highly abundant regulator of gene expression in the CNS. Protein and mRNA levels of neuroinflammation markers COX-2, BACE1, APP, and iNOS were also attenuated by combined therapy. On the other hand, assessment of the indicated five let-7 members, showed distinct expression profile pattern in the different groups. Let-7 a, b, and c disappeared in the induced group, an effect that was partially suppressed by co-addition of either Cur or VPA. These data suggest that the combined treatment induced significantly the expression of the five members when compared to rats treated with Cur or VPA only as well as to self-recovery group, which indicates a possible benefit from the synergistic effect of Cur-VPA combination as therapeutic agents for neuroinflammation and its associated disorders. The mechanism elucidated here highlights the particular drug-induced expression profile of let-7 family as new targets for future pharmacological development.

Inhibition of NF-ĸB, Bcl-2 and COX-2 Gene Expression by an Extract of Eruca sativa Seeds during Rat Mammary Gland Carcinogenesis

The effect of Eruca sativa seed extract (SE) on nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2) and B-cell lymphoma-2 (Bcl-2) gene expression levels was investigated in rat mammary gland carcinogenesis induced by 7,12 dimethylbenz(α)anthracene (DMBA). DMBA increased NF-κB, COX-2 and Bcl-2 gene expression levels and lipid peroxidation (LP), while, decreased glutathione-S-transferase (GST) and superoxide dismutase (SOD) activities and total antioxidant concentration (TAC) compared to the control group. After DMBA administration, SE treatment reduced NF-κB, COX-2 and Bcl-2 gene expression levels and LP. Hence, SE treatment reduced inflammation and cell proliferation, while increasing apoptosis, GST and SOD activities and TAC. Analysis revealed that SE has high concentrations of total flavonoids, triterpenoids, alkaloids and polyphenolic compounds such as gallic, chlorogenic, caffeic, 3,4-dicaffeoyl quinic, 3,5-dicaffeoyl quinic, tannic, cinnamic acids, catechin and phloridzin. These findings indicate that SE may be considered a promising natural product from cruciferous vegetables against breast cancer, especially given its high antioxidant properties.

Combination between Taxol-Encapsulated Liposomes and Eruca sativa Seed Extract Suppresses Mammary Tumors in Female Rats Induced by 7,12 Dimethylbenz(α)anthracene

Taxol (paclitaxel) is a powerful anti-cancer drug widely used against several types of malignant tumors. Because Taxol may exert several side effects, a variety of formulations have been developed. One of these features liposomes, regarded as one of the most promising drug carriers, biocompatible and best able to reduce drug toxicity without changing efficacy against tumor cells. Eruca sativa seed extract (SE) is considered a promising natural product from cruciferous vegetables against breast cancer, increasing chemotherapeutic and eliminating harmful side effects. The effects of Taxol-encapsulated liposomes (T) alone and in combination between Eruca sativa seed extract on nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2) and B-cell lymphoma-2 (Bcl-2) gene expression levels were investigated in rat mammary gland carcinogenesis induced by 7,12 dimethylbenz(α) anthracene (DMBA) using qRT-PCR. The results showed that DMBA increased NF-κB, COX-2 and Bcl-2 gene expression levels and lipid peroxidation (LP), while decreasing glutathione-S-transferase (GST) and superoxide dismutase (SOD) activities and total antioxidant concentration (TAC) compared to the control group. T and T-SE treatment reduced NF-κB, COX-2 and Bcl-2 gene expression levels and LP. Hence, T and T-SE treatment appeared to reduce inflammation and cell proliferation, while increasing apoptosis, GST and SOD activities and TAC.

Neuroprotective role of curcumin and resveratrol combination against AlCl3 induced Alzheimer?s disease in rats

I monocyte-derived effector cells are speculated to play a major pathologic role in the pathogenesis of numerous inflammatory neurological diseases. However, no treatment option currently exists that is capable of specifically modulating these cells. We show that intravenously infused 500 nm diameter negatively-charged, biodegradable immune-modifying-nanoparticles (IMPs) formulated from the FDA-approved biopolymer, poly(lactic-co-glycolic) acid (PLGA), are specifically taken up by inflammatory monocytes, in an opsonin-independent fashion, via the macrophage receptor with collagenous structure (MARCO). Subsequently, these monocytes no longer trafficked to sites of inflammation, rather, IMP infusion caused their sequestration in the spleen (through apoptotic cell clearance mechanisms) and ultimately caspase 3-mediated apoptosis. Administration of IMPs immediately following disease initiation in mouse models of relapsing experimental autoimmune encephalomyelitis (R-EAE), acute spinal cord injury, epilepsy and lethal West Nile virus encephalitis, markedly reduced monocyte accumulation at CNS inflammatory foci, significantly reduced disease symptoms and promoted tissue repair. Together these data highlight the intricate interplay between scavenger receptors, the spleen and inflammatory monocyte function; indicate a major pathologic role for blood-borne inflammatory monocytes rather than CNS-resident microglia in a variety of infectious and non-infectious neuroinflammatory diseases; and support the translation of IMPs for therapeutic use in a range of neuroinflammatory diseases caused or potentiated by inflammatory monocytes.Multiple sclerosis is an autoimmune demyelinating disease that affects more that 2.5 million people worldwide. Biochemical and in vitro data put forward many therapeutic candidates that have to be verified in vivo models of MS. Experimental autoimmune encephalomyelitis (EAE) is a widely utilized model that replicates many aspects of MS. The traditionally used assessment of behavioural outcomes is performed by two independent observers in the blind to experimental setup manner. Such way of assessment is person dependent and allow only evaluate a small number of parameters. Also, the scores often are different among different laboratories. Here we propose to utilize 40 parameters behavioural assessment during development and progression of EAE. We used 24hr/day recording of animals before and three weeks after induction of EAE. The films were analysed using Clever Sys software. We found dramatic differences in behavioural outcomes even within the first week of the EAE induction. First, we noted a significant reduction of total distance walked per day. Mice with EAE walked two-fold less after 1-week post-EAE and four-fold less after two weeks of EAE. Hanging behaviour significantly declined (up to 30 fold) in mice within two weeks of EAE. On the other hand, grooming behaviour significantly increased in the first week of EAE. Detailed analysis of mouse activity revealed that mice are most active between 20h and 24h and least active during 11h and 15hr. In conclusion, we demonstrate that multiple parameter automated analysis of behavioural outcomes may help to validate therapeutic targets and give insights into the mechanisms of the neuroinflammation during MS.We have investigated microglia polarization in the framework of microglia interactions with primary brain tumors. In a series of in vitro experiments, we have characterized the influence of glioma-soluble factors on microglial function, comparing the effects of media harvested under basal conditions with those of media obtained after inducing a pro-inflammatory activation state in glioma cells. Microglia exposed to basal glioma-derived factors (a condition resembling the early stage of pathology), shows increased M2b polarization status and up-regulation of IL-10 only. At variance, when exposed to activated glioma-derived factors (a condition mimicking the late stage of pathology), microglia presents as a mixture of polarization phenotypes (M1 and M2a/b), with up-regulation of iNOS, arginase and IL-10. In this paradigm, the inhibition of mTOR shifts polarization of glioma-activated microglial cells towards the M1 phenotype, thus preventing the induction of a M2 status that would promote tumor growth. Investigations are currently underway on 54 surgical specimens of glioblastoma multiforme to confirm the influence of brain tumors on microglia polarization. An apparently unrelated line of research in our lab was addressed to investigate the effects of antiretroviral drugs exposure on microglia cultures, seeking for putative mechanisms of neurotoxicity. We found that certain NNRTIs and PIs increased NO production thorough a mechanism independent from iNOS induction. Rather, these agents increased the availability of the iNOS substrate L-arginine by blocking arginase, a wellestablished marker of M2 polarization. Thus, the investigation of microglia polarization markers turns out to be a common background linking studies on the most different patho-physiological conditions involving the CNS.P disease (PD), the second most common neurodegenerative disorder affecting, more severely, the aged population is a complicating disease in terms of diagnosis and management. Many investigations have identified biomarkers for accelerating improved diagnosis and tracking of the disease. However, many of them require sound technical expertise and expensive modes of assessment in addition to the problem of the inadequate/lack of accessibility of the brain tissue of PD patients. In view of such scenario we did investigate biochemical and molecular analyses of blood samples from PD patients and found potential candidates which could be validated as peripheral markers for implication in risk assessment, diagnosis and also in assessing the course of the treatment. Understanding the robust link of oxidative stress and neural cell damage in the neurodegenerative process, we assessed the mRNA expression of reportedly highly vulnerable water channel, aquaporin (AQP4) and Phosphatidyl ethnolamine binding protein (PBP), brain derived neurotrophic factor (BDNF) and tropomyosin receptor factor (NTRK2) in the blood samples of PD and non-PD. The study included 140 PD patients and 70 healthy controls. RNA isolation was carried out using blood samples of the subjects recruited in the study and used for qRT-PCR analysis of AQP4, Tyroine hydroxylase as well as PBP. The data were significantly evaluated with SPSS/10 software. Hypothesis testing method was performed and p values of less than 0.05 were considered to indicate statistical significance. The mRNA expressions of AQP4 and TH were found to be reduced whereas that of PBP was found to be elevated when compared with those of healthy control samples. Further, NMR and MS analyses of the lipid content of the samples had surprisingly revealed a new candidate molecules exclusively present in the PD population. The selected PD candidate genes in the study are anticipated to provide valuable resources for developing and understanding of the molecular mechanism associated with PD and for discovering potential diagnostic/therapeutic/pathological markers for PD, as it is a widely accepted fact that peripheral changes could be considered as marker of brain changes in PD patients.C is the first subcomponent of the complement classical pathway. Classically, it is known to bind to immune complexes containing IgG and IgM and trigger the complement cascade leading up to the generation of membrane attack complex and C3a and C5a fragments that cause cellular infiltration. In addition, C1q is also known as a charge pattern recognition molecule which is involved in the clearance of non-self and altered self. It has become increasingly evident that C1q (and other complement proteins) can be made locally by microglia, neurons and atrocytes within the central nervous system. Thus, C1q can bind beta amyloid aggregates and precipitate neuroinflammation and neurodegeneration in Alzheimer’s Disease (AD) via classical pathway as well as microglia activation. It turns out that developmental expression of C1q is essential for synaptic prunning and C1q deficiency can have aberrant neuropathogical consequences. Therefore, under pathological conditions, regulating C1q expression and its target binding can have therapeutic benefits. We have identified two recombinant inhibitors of the classical pathway that interfere at the C1q function level. We have also immunologically characterised a natural model of AD, a Chilean rodent called Octodon degus, and found a direct link between complement activation (via C1q upregulation) and burrowing behaviour.A there is currently no effective treatment for Alzheimer’s disease (AD), significant advances have been made in suppressing its progress, using conventional antibodies that block aggregation of the abnormally folded proteins. The major limitation in targeting plaques and aggregates associated with Alzheimer in the CNS is the blood-brain barrier (BBB), which restricts the access of biologics and drugs to the brain parenchyma. To that end, we used anti-oligomer PRIOC-antibody-secreting mesenchymal stem cells (MSCs) modified by Zinc Finger Nuclease (ZFN) technology. Tg2576 and htau mice models were used to assess the therapeutic efficacy of a sustained local delivery of PRIOC mAbs in situ. This led to substantial reduction of Aβ plaques and aggregate formation/reduction and neuropathology and improvement of cognitive deficits in these animals. Our preliminary results indicate that this strategy might be useful in reducing Aβ burden and neuropathology recognized in Alzheimer patients.S is the leading cause of disability in adults and an important risk factor for bone fracture. Alternatively, stroke is one of the most devastating complications of bone fracture. In the United States, approximately 70,000 stroke victims suffer from bone fracture within the first year after their stroke. Reported date showed that about 0.2% to 4.1% patients suffer from stroke after hip surgery. However, the impact of bone fracture on stroke recovery has not been fully studies. We have studied the influence of bone fracture shortly before or after ischemic stroke on stroke recovery in a mouse model. We found that bone fracture increases alarmins and pro-inflammatory cytokines in the blood and increase stroke related injury as well as functional deficits through augmenting the neuroinflammatory response. Mice with stroke and bone fracture have more severe functional deficits, larger infarct sizes, and more CD68+ macrophages in the peri-infarct region than mice that have stroke only. Bone fracture also increases oxidative stress in the injury and promotes pro-inflammatory polarization (M1) of macrophage. Antiinflammatory therapies reduce the negative impact of bone fracture on stroke recovery.

Synergistic effect of curcumin on temozolomide inhibition of cancer stem cell-like properties and reduced chemoresistance of Glioblastoma C6

K questions in adult stem cell biology revolve around origin, tissue home and physiological role of adult stem cell populations. Mesenchymal stem cells (MSC) have remained elusive with regard to their in vivo physiology. Recent observations suggest that almost all adult tissues contain mesenchymal-like progenitors in a perivascular niche. Those cells can differentiate into mesodermal cell types and may even be endowed with tissue specific differentiation capacities. We have isolated and characterized a previously unrecognized progenitor cell population in the adult human brain. This cell population exhibits characteristics of mesenchymal stem cells (CD105, CD90, CD73, CD29) but in its native state does not express hematopoetic (CD34, CD45), endothelial (CD31), microglial (CD14, CD11b), glial and neuronal progenitor markers (GFAP, O4). We demonstrate at a clonal level, that the progenitors have true multilineage potential not only towards a mesodermal but also neuroectodermal phenotype and can differentiate into neurons. Thus, the vasculature in the adult human brain contains progenitor cells with multilineage capacity that may represent a reservoir that can be exploited in attempts to repair the damaged or diseased brain.T cell-environment communication is a dynamic procedure. Therefore, when biomaterials are developed for regulating cell behaviour, the functionality of biomaterials needs to be regulated in a dynamic manner to satisfy dynamic cell requirements. To this end, we have developed a new generation of adaptive hydrogels that can mimic the functions of extracellular matrices (ECMs). ECMs have complicated, dynamic functions. The artificial ECMs play three critical roles when interacting with cells. First, the hydrogel networks are used as the fundamental structural support to provide cells with a biocompatible environment to survive. Second, multiple growth factors can be sequentially released at desired time points. Third, the interactions between cell receptors and aptamers tethered to the hydrogel networks can be switched on and off. Therefore, the artificial ECMs can dynamically provide cells with biophysical and biochemical cues. In this presentation, we will discuss the synthesis of aptamerfunctionalized hydrogels, the sequential release of multiple growth factors from artificial ECMs, and the dynamic regulation of cell-aptamer interactions in the artificial ECMs. It is believed that this research will open a new avenue of developing adaptive biomaterials for tissue regeneration.I optimal conditions, damaged bone heals by a precisely regulated multi-stage process that takes a few weeks. Although the physiological mechanisms that govern the repair of different types of bone are somewhat variable, they all exhibit a highly anabolic repair phase of finite duration and capacity. If the extent of trauma is too great, or if healing potential is diminished by bone disease, the bone simply does not heal. This is a significant health concern given a current fracture non-union rate of 10%, a spinal fusion failure rate of up to 40%, at least 10 million osteoporosis sufferers in the United States, and about half of all cancer sufferers face destructive metastases to bone. Here, we discuss the lessons our group have learned from the presumptive progenitors of osteoblasts, the bone marrow mesenchymal stem cell (MSC). This work has led to the development of novel cytotherapeutic preparations and biologically complex matrices for unprecedented levels of stem cell retention and bone repair in experimental models. The study of malignant bone disease is also discussed, and how our work on MSCs has led to the characterization of bone degradation mechanisms caused by malignancy. Novel methods for the reversal of bone damage during malignancy are introduced.C therapy with small RNA agents against multiple viral targets is critical to efficient inhibition of viral production. We have previously validated this approach by expressing multiple anti-HIV RNAs from independent Pol III promoters within a single gene therapy construct, however, the high transcription rate of Pol III promoters often leads to toxicity as a consequence of saturating the endogenous RISC machinery. An alternative approach takes advantage of an endogenous polycistronic miRNA cluster driven by a Pol II promoter which has been engineered as a multiplexing platform. We tested combinations of different classes of therapeutic anti-HIV RNAs expressed within the context of an intronic MCM7 platform replacing the endogenous miRNAs with siRNAs targeting HIV-1 tat and rev messages, a nucleolar-localizing RNA ribozyme that targets the conserved U5 region of HIV-1 transcripts for degradation, or nucleolar TAR and RBE RNA decoys. We demonstrate the versatility of the MCM7 platform for expression and efficient processing of all RNA elements. Three of the combinatorial constructs tested potently suppressed viral replication during a one-month HIV challenge, with greater than five-logs of inhibition compared to unprotected HIV-1 infected CEM T-cells. One of the most effective constructs contains an anti-HIV siRNA combined with a nucleolar-localizing U5 ribozyme and nucleolar-localizing TAR RNA decoy. To the best of our knowledge, this is the first successful demonstration of functional combinations of different types of small inhibitory RNAs expressed from a single intron transcriptional unit.