Stephen Hsu

Green tea polyphenols reduce autoimmune symptoms in a murine model for human Sjogren's syndrome and protect human salivary acinar cells from TNF-a-induced cytotoxicity

Sjogrens syndrome (SS) is a relatively common autoimmune disorder. A key feature of SS is lymphocytic infiltration of the salivary and lacrimal glands, associated with the destruction of secretory functions of these glands. Current treatment of SS targets the symptoms but is unable to reduce or prevent the damage to the glands. We reported previously that the major green tea polyphenol (GTP) epigallocatechin-3-gallate (EGCG) inhibits autoantigen expression in normal human keratinocytes and immortalized normal human salivary acinar cells (Hsu et al. 2005). However, it is not known whether GTPs have this effect in vivo, if they can reduce lymphocytic infiltration, or protect salivary acinar cells from tumor necrosis factor-α (TNF-α)-induced cytotoxicity. Here, we demonstrate that in the NOD mouse, a model for human SS, oral administration of green tea extract reduced the serum total autoantibody levels and the autoimmune-induced lymphocytic infiltration of the submandibular glands. Further, we show that EGCG protected normal human salivary acinar cells from TNF-α-induced cytotoxicity. This protection was associated with specific phosphorylation of p38 MAPK, and inhibitors of the p38 MAPK pathway blocked the protective effect. In conclusion, GTPs may provide a degree of protection against autoimmune-induced tissue damage in SS, mediated in part through activation of MAPK elements.

Effects of oral consumption of the green tea polyphenol EGCG in a murine model for human Sjogren's syndrome, an autoimmune disease.

SIGNIFICANCE Protection of glandular cells from autoimmune-induced damage would be of significant clinical benefit to Sjogrens syndrome (SS) patients. Epigallocatechin-3-gallate (EGCG) possesses anti-apoptotic, anti-inflammatory, and autoantigen-inhibitory properties. AIMS To investigate if EGCG protects against certain autoimmune-induced pathological changes in the salivary glands of the non-obese diabetic (NOD) mouse model for SS. MAIN METHODS Animals were provided with either water or water containing 0.2% EGCG. At the age of 8, 16 and 22 weeks, submandibular salivary gland tissue and serum samples were collected for pathological and serological analysis. KEY FINDINGS Significant lymphocyte infiltration was observed in the salivary glands of the water-fed group at the age of 16 weeks, while the EGCG group showed reduced lymphocyte infiltration. By 22 weeks of age, water-fed animals demonstrated elevated levels of apoptotic activity within the lymphocytic infiltrates, and high levels of serum total anti-nuclear antibody, compared to EGCG-fed animals. Remarkably, proliferating cell nuclear antigen (PCNA) and Ki-67 levels in the salivary glands of water-fed NOD mice were significantly elevated in comparison to BALB/c control mice; in contrast, PCNA and Ki-67 levels in EGCG-fed NOD animals were similar to BALB/c mice. These results indicate that EGCG protects the NOD mouse submandibular glands from autoimmune-induced inflammation, and reduces serum autoantibody levels. Abnormal proliferation, rather than apoptosis, appears to be a characteristic of the NOD mouse gland that is normalized by EGCG. The evidence suggests that EGCG could be useful in delaying or managing SS-like autoimmune disorders.

Green tea polyphenol induces caspase 14 in epidermal keratinocytes via MAPK pathways and reduces psoriasiform lesions in the flaky skin mouse model

Abstract:  Psoriasiform lesions are characterized by hyperproliferation and aberrant differentiation of epidermal keratinocytes, accompanied by inflammation, leading to a disrupted skin barrier with an abnormal stratum corneum. The expression and proteolytic processing of caspase 14, a member of the caspase family which is associated with epithelial cell differentiation, planned cell death, and barrier formation, is altered in psoriatic epidermis. We recently reported that human psoriatic tissues lack normal expression of caspase 14 [J Dermatol Sci37 (2005) 61], and caspase 14 is induced by EGCG, a green tea polyphenol (GTP), in exponentially growing normal human epidermal keratinocytes (NHEK) [J Pharmacol Exp Ther315 (2005) 805]. This suggests that GTPs may have beneficial effects on psoriasiform lesions. The current study aimed to determine whether MAPK pathways are required for GTP‐induced caspase 14 expression in NHEK and if GTPs can modulate the expression of pathological markers in the psoriasiform lesions that develop in the flaky skin mouse. The results indicate that the p38 and JNK MAPK pathways are required for EGCG‐induced expression of caspase 14 in NHEK. Importantly, topical application of 0.5% GTPs significantly reduced the symptoms of epidermal pathology in the flaky skin mice, associated with efficient caspase 14 processing and reduction in proliferating cell nuclear antigen levels. This suggests that GTP‐activated pathways may be potential targets for novel therapeutic approaches to the treatment of some psoriasiform skin disorders.

Inhibition of Herpes Simplex Virus type 1 with the modified green tea polyphenol palmitoyl-epigallocatechin gallate

Green tea polyphenol epigallocatechin gallate (EGCG) is a strong antioxidant that has previously been shown to reduce the number of plaques in HIV-infected cultured cells. Modified EGCG, palmitoyl-EGCG (p-EGCG), is of interest as a topical antiviral agent for herpes simplex virus (HSV-1) infections. This study evaluated the effect of p-EGCG on HSV-infected Vero cells. Results of cell viability and cell proliferation assays indicate that p-EGCG is not toxic to cultured Vero cells and show that modification of the green tea polyphenol epigallocatechin gallate (EGCG) with palmitate increases the effectiveness of EGCG as an antiviral agent. Furthermore, p-EGCG is a more potent inhibitor of herpes simplex virus 1 (HSV-1) than EGCG and can be topically applied to skin, one of the primary tissues infected by HSV. Viral binding assay, plaque forming assay, PCR, real-time PCR, and fluorescence microscopy were used to demonstrate that p-EGCG concentrations of 50 μM and higher block the production of infectious HSV-1 particles. p-EGCG was found to inhibit HSV-1 adsorption to Vero cells. Thus, p-EGCG may provide a novel treatment for HSV-1 infections.

A Mechanism-Based In Vitro Anticancer Drug Screening Approach for Phenolic Phytochemicals

Plant-derived phenolic compounds, including polyphenols (e.g., tannins), flavonoids, and phenolic acids, have been under investigation for their anticancer therapeutic and chemoprevention properties. Recently, certain mechanisms underlying the differential effects of green tea polyphenols (GTPPs) on tumor versus normal cells have been determined. These suggest that GTPPs may simultaneously activate multiple pathways. However, existing screening methods are insufficient for the identification of agents that possess both a cytotoxic effect on tumor cells and a protective effect on normal cells. The current study describes the establishment of an in vitro survival/apoptosis testing system based on detecting these mechanisms by a double-fluorescence method. This system is able to screen potential chemopreventive or therapeutic agents from (but not limited to) plant-derived compounds based on the pathways differentially activated by the agents. Tumor cell death and normal cell survival are detected simultaneously, in a device that co-cultures normal human cells adjacent to human tumor cells.

Ni(II) ions dysregulate cytokine secretion from human monocytes

Nickel-containing alloys are used in dentistry because of their low cost, but poor corrosion behavior increases their risk of causing adverse biological responses. Intraorally, nickel-containing alloys accumulate bacterial plaque that triggers periodontal inflammation via toxins such as lipopolysaccharide (LPS). Recent evidence suggests that in monocytes, Ni(II) amplifies LPS-induced secretion of several cytokines that mediate periodontal destruction. Thus, we investigated the effects of Ni(II), with or without LPS, on the secretion of a broader array of cytokines from monocytes. We then measured monocytic expression of two proteins, Nrf2 and thioredoxin-1 (Trx1), that influence the regulation of cytokine secretion. Cytokine arrays were used to measure the effects of 0-50 microM Ni(II) on cytokine secretion from human THP1 monocytes, with or without LPS activation. Immunoblots were used to estimate Nrf2 and Trx1 levels. Our results indicate that both Ni(II) alone and Ni(II) with LPS have broad-based effects on cytokine secretion. Ni(II) increased Nrf2 levels by threefold, and LPS amplified the effects of Ni(II) by 10-fold. Trx1 levels did not change under any condition tested. Our results suggest that Ni(II)-induced changes in cytokine secretion by monocytes are diverse and may be influenced by Nrf2 but are not likely influenced by changes in whole-cell Trx1 levels.

Protective effect of green tea polyphenols on tributyltin-induced oxidative damage detected by in vivo and in vitro models

The current study investigated the protective effects of green tea polyphenols (GTPP) on TBT‐induced oxidative damage. The results showed that reactive oxygen species (ROS) production and malondialdehyde content of the liver in mice exposed to TBT were reduced in the GTPP‐treated group compared to the untreated group. The intracellular ROS level was elevated in TBT‐treated human FL cells in a time‐dependent manner. Comet assay data demonstrated that the number of cells with damaged DNA in untreated mice was found to be significantly higher compared to GTPP‐treated mice. Damage to the nuclei and mitochondria observed in TBT‐treated mice were alleviated in mice treated with both TBT and GTPP. The results represent the first observation that GTPP were effective in reducing TBT‐induced oxidative damage both in vivo and in vitro. The possible protective mechanism may be due to the powerful ability of GTPP to scavenge ROS and prevent DNA breaks. We conclude that GTPP could be an effective agent or food supplement to reduce the cytotoxicity of TBT.

Transforming growth factor β1 dysregulation in a human oral carcinoma tumour progression model

Abstract. A human oral tumour progression model was established that consists of normal epithelial cells and three cell lines representing stages from dysplastic to metastatic cells. To investigate the impact of exogenous transforming growth factor‐β1 on this model system, we analysed the responsiveness of those cells to transforming growth factor‐β1 and explored the potential mechanism underlying the transforming growth factor‐β1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor‐β1, although to different degrees. Transforming growth factor‐β1 induced the expression of cyclin‐dependent kinase inhibitors p15INK4B, p21WAF1/CIP1 and p27KIP1. In contrast, transforming growth factor‐β1 was found to stimulate the invasive potential of one cell type that represents the most advanced stage of tumour phenotype, suggesting that the impact of transforming growth factor‐β1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression.

Epigallocatechin-3-gallate modulates anti-oxidant defense enzyme expression in murine submandibular and pancreatic exocrine gland cells and human HSG cells

Abstract Sjogren’s syndrome (SS) and type-1 diabetes are prevalent autoimmune diseases in the USA. We reported previously that epigallocatechin-3-gallate (EGCG) prevented and delayed the onset of autoimmune disease in non-obese diabetic (NOD) mice, a model for both SS and type-1 diabetes. EGCG also normalized the levels of proteins related to DNA repair and anti-oxidant activity in NOD.B10.Sn-H2 mice, a model for primary SS, prior to disease onset. The current study examined the effect of EGCG on the expression of anti-oxidant enzymes in the submandibular salivary gland and the pancreas of NOD mice and cultured human salivary gland acinar cells. NOD mice consuming 0.2% EGCG daily dissolved in water showed higher protein levels of peroxiredoxin 6 (PRDX6), a major anti-oxidant defense protein, and catalase, while the untreated NOD mice exhibited significantly lowered levels of PRDX6. Similarly, pancreas samples from water-fed NOD mice were depleted in PRDX6 and superoxide dismutase, while EGCG-fed mice showed high levels of these anti-oxidant enzymes. In cultured HSG cells EGCG increased PRDX6 levels significantly, and this was inhibited by p38 and JNK inhibitors, suggesting that the EGCG-mediated increase in protective anti-oxidant capacity is regulated in part through mitogen-activated protein kinase pathway signaling. This mechanism may explain the higher levels of PRDX6 found in EGCG-fed NOD mice. These preclinical observations warrant future preclinical and clinical studies to determine whether EGCG or green tea polyphenols could be used in novel preventive and therapeutic approaches against autoimmune diseases and salivary dysfunction involving oxidative stress.

Dentin Sialophosphoprotein (DSPP) Gene-Silencing Inhibits Key Tumorigenic Activities in Human Oral Cancer Cell Line, OSC2

Background We determined recently that dentin sialophosphoprotein (DSPP), a member of the SIBLING (Small integrin-binding ligand N-linked glycoproteins) family of phosphoglycoproteins, is highly upregulated in human oral squamous cell carcinomas (OSCCs) where upregulation is associated with tumor aggressiveness. To investigate the effects of DSPP-silencing on the tumorigenic profiles of the oral cancer cell line, OSC2, short-hairpin RNA (shRNA) interference was employed to silence DSPP in OSC2 cells. Methodology/Principal Findings Multiple regions of DSPP transcript were targeted for shRNA interference using hDSP-shRNA lentiviral particles designed to silence DSPP gene expression. Control shRNA plasmid encoding a scrambled sequence incapable of degrading any known cellular mRNA was used for negative control. Following puromycin selection of stable lines of DSSP-silenced OSC2 cells, phenotypic hallmarks of oral carcinogenesis were assayed by western blot and RT-PCR analyses, MTT (cell-viability), colony-formation, modified Boyden-Chamber (migration and invasion), and flow cytometry (cell-cycle and apoptosis) analyses. DSPP-silenced OSC2 cells showed altered cell morphology, reduced viability, decreased colony-formation ability, decreased migration and invasion, G0/G1 cell-cycle arrest, and increased tumor cell sensitivity to cisplatin-induced apoptosis. Furthermore, MMP-2, MMP-3, MMP-9, VEGF, Ki-67, p53, and EGFR were down-regulated. There was a direct correlation between the degree of DSPP-silencing and MMP suppression, as indicated by least squares regression: MMP-2 {(y = 0.850x, p<0.001) (y = 1.156x, p<0.001)}, MMP-3 {(y = 0.994x, p<0.001) (y = 1.324x, p = 0.004)}, and MMP-9 {(y = 1.248x, p = 0.005, y = 0.809, p = 0.013)}. Conclusions/Significance DSPP-silencing in OSC2 cell decreased salient hallmarks of oral tumorigenesis and provides the first functional evidence of a potential key role for DSPP in oral cancer biology. The down-regulation of MMP-2, MMP-3, MMP-9, p53 and VEGF in DSPP-silenced OSC2 cells provides a significant functional/molecular framework for deciphering the mechanisms of DSPP activities in oral cancer biology.