Ling-Sun Jen

Targeting specific cell signaling transduction pathways by dietary and medicinal phytochemicals in cancer chemoprevention

Natural phytochemicals derived from dietary sources or medicinal plants have gained significant recognition in the potential management of several human clinical conditions. Much research has also been geared towards the evaluation of plant extracts as effective prophylactic agents since they can act on specific and/or multiple molecular and cellular targets. Plants have been an abundant source of highly effective phytochemicals which offer great potential in the fight against cancer by inhibiting the process of carcinogenesis through the upregulation of cytoprotective genes that encode for carcinogen detoxifying enzymes and antioxidant enzymes. The mechanistic insight into chemoprevention further includes induction of cell cycle arrest and apoptosis or inhibition of signal transduction pathways mainly the mitogen-activated protein kinases (MAPK), protein kinases C (PKC), phosphoinositide 3-kinase (PI3K), glycogen synthase kinase (GSK) which lead to abnormal cyclooxygenase-2 (COX-2), activator protein-1 (AP-1), nuclear factor-kappaB (NF-κB) and c-myc expression. Effectiveness of chemopreventive agents reflects their ability to counteract certain upstream signals that leads to genotoxic damage, redox imbalances and other forms of cellular stress. Targeting malfunctioning molecules along the disrupted signal transduction pathway in cancer represent a rational strategy in chemoprevention. NF-κB and AP-1 provide mechanistic links between inflammation and cancer, and moreover regulate tumor angiogenesis and invasiveness, indicating that signaling pathways that mediate their activation provide attractive targets for new chemotherapeutic approaches. Thus cell signaling cascades and their interacting factors have become important targets of chemoprevention and phenolic phytochemicals and plant extracts seem to be promising in this endeavor.

Amyloid-Beta Peptide Is Toxic to Neurons In Vivo via Indirect Mechanisms

We have studied the neurotoxicity of amyloid-beta (Abeta) after a single unilateral intravitreal injection. Within the retina apoptotic cells were seen throughout the photoreceptor layer and the inner nuclear layer but not in the ganglion cell layer at 48 h after injection of Abeta(1-42) compared to vehicle control and control peptide. At 5 months, there was a significant reduction in total cell numbers in the ganglion cell layer in Nissl stained retinas. There was glial cell dysfunction with upregulation of glial fibrillary acidic protein and a reduction in the expression of Müller cell associated proteins in the injected retinas. These results suggest an indirect cytotoxic effect of Abeta on retinal neurons and an important role for dysfunction of Müller glia in mediating Abeta neurotoxicity.

Ergothioneine treatment protects neurons against N-methyl-d-aspartate excitotoxicity in an in vivo rat retinal model

Injection of the glutamate agonist N-methyl-D-aspartate into the vitreous body of the rat eye resulted in a number of morphological changes in the retina. Most apparent was a dramatic reduction in the density and sizes of neurons accompanied by a decrease in amyloid precursor protein and glial fibrillary acidic protein immunoreactivity. Cell counts revealed that 81% of ganglion cells and 43% of non-ganglion cells were lost as a result of the treatment. However, in animals treated with the antioxidant ergothioneine, these figures dropped to 44 and 31%, respectively. Thus, ergothioneine appears to be neuroprotective in this system and the data suggest that antioxidants may provide a useful means of modulating glutamate-based toxicity.

Amyloid beta peptide causes chronic glial cell activation and neuro-degeneration after intravitreal injection.

We have previously demonstrated that amyloid beta (Aβ) peptide is acutely toxic to retinal neurones in vivo and that this toxicity is mediated by an indirect mechanism. We have now extended these studies to look at the chronic effect of intravitreal injection of Aβ peptides on retinal ganglion cells (RGC), the projection neurones of the retina and the glial cell response. 5 months after injection of Aβ1−42 or Aβ42−1 there was no significant reduction in RGC densities but there was a significant reduction in the retinal surface area after both peptides. Phosphate‐buffered saline (PBS) injection had no effect on retinal size or RGC density. There was a pronounced reduction in the number of large RGCs with a concomitant significant increase in medium and small RGCs. There was no change in cell sizes 5 months after injection with PBS. At 5 months after injection of both peptides, there was marked activation of Muller glial cells and microglia. There was also expression of the major histocompatibility complex (MHC) class II molecule on some of the microglial cells but we saw no evidence of T‐cell infiltration into the injected retinas. In order to elucidate potential toxic mechanisms, we have looked at levels of glutamine synthetase and nitric oxide synthase. As early as 2 days after injection we noted that activation of Muller glia was associated with a decrease in glutamine synthetase immuno‐reactivity but there was no detectable expression of inducible nitric oxide synthase in any retinal cells. These results suggest that chronic activation of glial cells induced by Aβ peptides may result in chronic atrophy of projection neurones in the rat retina.

The antioxidant cocktail effective microorganism X (EM-X) inhibits oxidant-induced interleukin-8 release and the peroxidation of phospholipids in vitro

The antioxidant beverage EM-X is derived from the ferment of unpolished rice, papaya, and sea-weeds with effective microorganisms. Oxidative stress enhances the expression of proinflammatory genes, causing the release of the chemokine interleukin-8 (IL-8), which mediates a multitude of inflammatory events. Human alveolar epithelial cells (A549) were treated with H(2)O(2) (100 microM) or TNF-alpha (10ng/ml) alone or with the addition of EM-X (100 microl/ml), incubated for 20h, and the release of IL-8, measured using ELISA. EM-X inhibited the release of IL-8 at the transcriptional level in A549 cells. EM-X also decreased the iron/ascorbate dependent peroxidation of ox-brain phospholipids in a concentration dependent manner. A TEAC value of 0.10+/-0.05mM was obtained for EM-X, indicating antioxidant potential. We suggest that the anti-inflammatory and antioxidant properties of EM-X are dependent on the flavonoid contents of the beverage.

Free Radicals, Antioxidants and Diabetes: Embryopathy, Retinopathy, Neuropathy, Nephropathy and Cardiovascular Complications

The metabolic disturbances in the insulin-dependent diabetes mellitus or type 1 and noninsulin-dependent diabetes mellitus or type 2 are associated with a number of complications including cardiovascular diseases, nephropathy, neuropathy, retinopathy leading to blindness and embryopathy or congenital malformations. Maternal diabetes is associated with a high incidence of congenital malformations and fetal abortions. Heart and kidney anomalies, along with central nervous system defects are frequent manifestations of a maternal diabetic environment. Glycation products from excess glucose can chemically modify DNA causing mutations and complex DNA rearrangements. Therefore, DNA damage in fetal tissues as a result of maternal diabetes may reflect a level of genomic injury sufficient to affect embryonic development. The formation of advanced glycation end products can accelerate vascular occlusion by quenching the vasodilating agent nitric oxide. Interaction with high-affinity receptors located on monocytes and macrophages can enhance the production of free radicals and reactive oxygen/nitrogen species, the secretion of tumor necrosis factor-α, interleukin-1 and insulin-like growth factor 1 which can proliferate endothelial, mesangial and smooth muscle cells and hence contribute significantly to the pathogenesis of cardiovascular complications. Retinopathy is characterized by increased vascular permeability, by vascular closure, together with the growth of new blood vessels on the retina and posterior surface of the vitreous. Reactive oxygen species are involved in decreased retinal blood flow, increased vascular permeability and disruption of blood-retinal barrier. The involvement of oxidative stress in the pathology of diabetes from its associated cardiovascular dysfunctions, nephropathy, neuropathy, retinopathy (leading to blindness) and embryopathy or congenital malformations, suggests that potential management of diabetes could benefit from use of dietary biofactors in medicinal and food plants. There is therefore a requirement for research to focus on the molecular mechanisms of action of extracts and/or the biofactors flavonoids, proanthocyanidins, alkaloids, etc. derived from these plants.

Bioefficacy of Mauritian Endemic Medicinal Plants: Assessment of Their Phenolic Contents and Antioxidant Potential

Abstract The role of free radicals in the etiology and development of a wide range of clinical disorders has continued to fuel the suggestion that phenolic antioxidants can offer a realistic promise to reduce the incidence of a number of pathologies involving oxidative stress. In this study, the total phenol, flavonoid, and proanthocyanidin contents of the Mauritian medicinal plants Crinum mauritianum. Lodd. (Asteraceae), Gaertnera psychotroides. DC (Rubiaceae), Psidia terebinthina. A.J. Scott (Asteraceae), and Tylophora coriacea. Marais. (Monimiaceae) were assessed and contrasted with their antioxidant potential. The antioxidant propensity was evaluated by the ability of the extracts to scavenge hypochlorous acid and hydroxyl radical and the ABTS⋅ + radical including their abilities to inhibit microsomal lipid peroxidation. The endemic plants Badula multiflora. A. DC. (Myrsinaceae), Croton vaughanii. L. (Euphorbiaceae), Erythroxylum macrocarpum. Lam. (Erythroxylaceae), Ochna mauritiana. Lam. (Ochnaceae), Tambourissa cordifolia. Lorence. (Monimiaceae), and Turraea rigida. Vent. (Meliaceae) were similarly investigated. Badula multiflora. and Erythoxylum macrocarpum. showed highest antioxidant activity in the TEAC and FRAP assay. Badula multiflora., Ochna mauritiana., and Gaertnera psychotroides. were very potent scavengers of hypochlorous acid and inhibited microsomal lipid peroxidation induced by 2,2′-azobis.(2-amidinopropane) hydrochloride (AAPH), suggesting that the inhibition was intrinsically linked to peroxyl radical scavenging. The antioxidant activity of Gaertnera psychotroides., Tylophora coriacea., Psidia terebinthina., and Crinum mauritianum. may account for the therapeutic effects of these extracts, in particular, in conditions characterized by inflammation and oxidative mechanisms. While these polyphenolic-rich endemics are good sources of natural prophylactic antioxidants, there is an urgent need for sustainable conservation programs for their protection.

Differential effects of interleukin-1β and S100B on amyloid precursor protein in rat retinal neurons

Purpose: Interleukin-1β (IL-1β) and S100B calcium binding protein B (S100B) have been implicated in the pathogenesis of Alzheimer’s disease. Both are present in and around senile plaques and have been shown to increase levels of amyloid precursor protein (APP) mRNA in vitro. However, it is not known how either of these substances affects APP in vivo. Methods: We have studied the effects of IL-1β and S100B on the expression and processing of APP using a retinal-vitreal model. We have also investigated the effect of amyloid beta peptide (Aβ) on APP in the same system and the regulation of S100B production by Aβ and IL-1β from retinal glial cells. Results: Retinal ganglion cells constitutively express APP. However, after intravitreal injection of IL-1β or Aβ there was a marked reduction in APP levels as detected by Western blotting and IL-1β produced a decrease in APP immunoreactivity (IR). Nissl staining showed that the integrity of the injected retinas was unchanged after injection. Two days after S100B injection, there was a small reduction in APP-IR but this was accompanied by the appearance of some intensely stained large ganglion cells and there was some up-regulation in APP holoprotein levels on Western blot. Seven days post-S100B injection, these large, highly stained cells had increased in number throughout the retina. Injection of Aβ and IL-1β also caused an increase in S100B production within the retinal Müller glial cells. Conclusion: These results support the hypothesis that S100B (a glial-derived neurotrophic factor) and IL-1β (a pro-inflammatory cytokine) can modulate the expression and processing of APP in vivo and so may contribute to the progression of Alzheimer’s disease.

Developmental regulation and possible alternative cleavage of presenilin 1 in the rat retina.

Presenilin 1 (PS1) is a multitransmembrane protein well known for being mutated in most cases of familial Alzheimers disease. Although its pathological effect is clear, its biological functions are not yet fully understood, but it appears to be involved in development and apoptosis. To investigate the role of PS1 in developmental processes we have studied the expression and proteolytic processing of this protein in the developing rat retina. PS1 appears to be developmentally regulated in the retina, and the pattern of PS1 immunoreactivity is consistent with a role in retinal lamination and pattern formation. Interestingly, no correlation was observed between PS1-positive cells and cells undergoing programmed cell death, suggesting that PS1 does not play a role in apoptosis occurring during this period. Moreover, we observed a change in the pattern of PS1 proteolytic fragments suggestive of a novel alternative cleavage site in the PS1 molecule.

Acute and chronic effects of intravitreally injected β-amyloid on the neurotransmitter system in the retina

The potential cytotoxic effect of aggregated Abeta(1-42) to neurons that express classical neurotransmitters, including acetylcholine, gamma-amino butyric acid, catecholamines and serotonin was assessed. The cholinergic system has been the central focus of the therapeutic drug strategies in amyloid-depositing pathologies such as Alzheimers disease. Aggregated Abeta(1-42) has a multisystem cytotoxic effect causing non-specific reduction in immunoreactivity, dysfunction, or loss of retinal nerve cells. The extent of this was investigated using immunocytochemistry, TUNEL staining for apoptosis, and measurement of cell density as well as retinal surface area. There was a differential acute and/or chronic effect of Abeta on choline acetyltransferase, gamma-aminobutyric acid and 5-tryptamine hydroxylase systems, observed with the increasing time course of 6h to 5 months, and a bilateral/systemic effect. In contrast, the overall pattern of catecholaminergic system, as revealed by tyrosine hydroxylase immunoreactivity of the retina, appears to have remained relatively unaffected by Abeta (however this may reflect neuronal loss due to reduction in the retinal surface). This is the first in vivo evidence in a CNS model to show that not only all major neurotransmitter systems are differentially affected by Abeta aggregates but the effect may vary from one transmitter system to another under the same experimental conditions in situ and in a dose- and time-dependent manner.