CYP2E1 deficit mediates cholic acid-induced malignant growth in hepatocellular carcinoma cells.

BACKGROUND: Increased level of serum cholic acid (CA) is often accompanied with decreased CYP2E1 expression in hepatocellular carcinoma (HCC) patients. However, the roles of CA and CYP2E1 in hepatocarcinogenesis have not been elucidated. This study aimed to investigate the roles and the underlying mechanisms of CYP2E1 and CA in HCC cell growth. METHODS: The proteomic analysis of liver tumors from DEN-induced male SD rats with CA administration was used to reveal the changes of protein expression in the CA treated group. The growth of CA-treated HCC cells was examined by colony formation assays. Autophagic flux was assessed with immunofluorescence and confocal microscopy. Western blot analysis was used to examine the expression of CYP2E1, mTOR, AKT, p62, and LC3II/I. A xenograft tumor model in nude mice was used to examine the role of CYP2E1 in CA-induced hepatocellular carcinogenesis. The samples from HCC patients were used to evaluate the clinical value of CYP2E1 expression. RESULTS: CA treatment significantly increased the growth of HCC cells and promoted xenograft tumors accompanied by a decrease of CYP2E1 expression. Further studies revealed that both in vitro and in vivo, upregulated CYP2E1 expression inhibited the growth of HCC cells, blocked autophagic flux, decreased AKT phosphorylation, and increased mTOR phosphorylation. CYP2E1 was involved in CA-activated autophagy through the AKT/mTOR signaling. Finally, decreased CYP2E1 expression was observed in the tumor tissues of HCC patients and its expression level in tumors was negatively correlated with the serum level of total bile acids (TBA) and gamma-glutamyltransferase (GGT). CONCLUSIONS: CYP2E1 downregulation contributes to CA-induced HCC development presumably through autophagy regulation. Thus, CYP2E1 may serve as a potential target for HCC drug development.

Bisphenol P and bisphenol M promote triple-negative breast cancer metastasis through activation of AKT pathways.

Bisphenol P (BPP) and bisphenol M (BPM) are increasing in our living environment as analogues of bisphenol A (BPA), but little is known about their biological effect. In this study, we investigated the effects of low to medium dose exposure of BPP and BPM on triple negative breast cancer (TNBC). We found that BPP and BPM exposure didn't affect proliferation of TNBC cell lines MDA-MB-231 and 4 T1, but significantly promoted cells migration and invasion. The effect of BPP and BPM on promoting TNBC metastasis was further confirmed in mouse models. Low concentrations of BPP and BPM significantly increased the expression of epithelial-mesenchymal transition (EMT) marker and related proteins such as N-cadherin, MMP-9, MMP-2 and Snail, and also enhanced phosphorylation of AKT both in vitro and in vivo. When PI3K inhibitor wortmannin was applied to specifically inhibit phosphorylation of AKT, the expression of target genes markedly decreased, and the TNBC metastasis induced by low-concentration BPP and BPM were reversed. In conclusion, these results showed that PI3K/AKT signaling regulate BPP/BPM-induced metastasis of TNBC by triggering EMT. This study provides insights into the effects and the potential mechanisms of BPP and BPM on TNBC, raising concerns about the risk of using these two bisphenols as the alternative of BPA.

Autophagy blockage and lysosomal dysfunction are involved in diallyl sulfide-induced inhibition of malignant growth in hepatocellular carcinoma cells.

Diallyl sulfide (DAS), as a major component of garlic extracts, has been shown to inhibit growth of hepatocellular carcinoma cells (HCC), but the underlying mechanism is still elusive. In this study, we aimed to explore the involvement of autophagy in DAS-induced growth inhibition of HepG2 and Huh7 hepatocellular carcinoma cells. We studied growth of DAS-treated HepG2 and Huh7 cells using the MTS and clonogenic assays. Autophagic flux was examined by immunofluorescence and confocal microscopy. The expression levels of autophagy-related proteins AMPK, mTOR, p62, LC3-II, LAMP1, and cathepsin D in the HepG2 and Huh7 cells treated with DAS as well as the tumors formed by HepG2 cells in the nude mice in the presence or absence of DAS were examined using western blotting and immunohistochemistry analysis. We found that DAS treatment induced activation of AMPK/mTOR, and accumulation of LC3-II and p62 both in vivo and in vitro. DAS inhibited autophagic flux through blocking the fusion of autophagosomes with lysosomes. Furthermore, DAS induced an increase in lysosomal pH and inhibition of Cathepsin D maturation. Co-treatment with an autophagy inhibitor (Chloroquine, CQ) further enhanced the growth inhibitory activity of DAS in HCC cells. Thus, our findings indicate that autophagy is involved in DAS-mediated growth inhibition of HCC cells both in vitro and in vivo.

Downregulation of estrogen receptor-α36 expression attenuates metastasis of hepatocellular carcinoma cells.

This study aimed to examine the role of estrogen receptor (ER)-α36 in the metastasis of hepatocellular carcinoma (HCC) and in the epithelial-mesenchymal transition (EMT). HCC HepG2 and Huh7 cells with the knocked-down level of ER-α36 expression were established. Cell growth and migration of the HepG2 and Huh7 cell variants were studied using MTS, transwell, and wound-healing assays, and the metastatic abilities of HepG2 cell variants were examined using a tail-vein injection model in nude mice. Levels of EMT markers, Src phosphorylation in HepG2 and Huh7 cell variants, and tumors formed by HepG2 cell variants in the nude mice were examined using Western blot and immunohistochemistry. We found that the growth and metastatic abilities of HepG2 and Huh7 cells with the knocked-down level of ER-α36 expression (HepG2/Si36 and Huh7/Si36) were significantly reduced, with increased levels of cytokeratin and E-Cadherin expression, and decreased levels of Vimentin, Snail, Slug and the Src phosphorylation, compared to the HCC cells transfected with an empty vector (HepG2/Vector and Huh7/Vector). We also found ER-α36 knockdown suppressed the lung metastasis of HepG2 cells with the involvement of EMT and the Src pathway in vivo. The Src inhibitor PP2 suppressed the growth and migration of HepG2/Vector and Huh7/Vector cells with decreased Vimentin, Snail, and Slug and increased cytokeratin and E-Cadherin expressions, but failed to induce the migration and the EMT markers in HepG2/Si36 and Huh7/Si36 cells. ER-α36 is involved in the metastasis of HCC cells through the regulation of EMT and the Src signaling pathway.

Estrogen receptor-α36 is involved in diallyl sulfide-induced inhibition of malignant growth of HepG2 and Huh7 hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a highly malignant disease that currently lacks effective treatment. Epidemiological studies have suggested the preventive role of raw garlic intake in different tumors, such as HCC. Although diallyl sulfide (DAS), the main component of garlic extracts, has been reported to inhibit the growth of HCC cells, the underlying mechanism remains elusive. This study aimed to investigate the inhibitory effect of DAS on the growth of HepG2 and Huh7 hepatocellular carcinoma cells and its underlying mechanism. HepG2 and Huh7 cells were treated with DAS and nude mice were intrahepatically injected with human HCC HepG2 cells and maintained with or without DAS administration for 28 days. MTS and clonogenic assays revealed that DAS inhibited the growth and clonogenicity of HepG2 and Huh7 hepatocellular carcinoma cells. Furthermore, DAS inhibited the growth of xenograft tumors accompanied by a decreased rate of pathological karyomitosis as observed by H&E staining. The expression levels of estrogen receptor-α36 (ER-α36) and epidermal growth factor receptor (EGFR) in HepG2 and Huh7 cells and in xenograft tumors derived from HepG2 cells after DAS treatment were detected by immunohistochemistry and western blotting. We found that DAS disrupted the positive regulatory loop between ER-α36 and EGFR, and decreased the phosphorylation of AKT at Ser 473 both in vivo and in vitro. DAS also induced cell apoptosis, as evidenced by Hoechst and TUNEL staining. Western blotting revealed activation of caspase3, increased BAX and decreased Bcl-2 expression. However, the ER-α36 expression knockdown attenuated DAS-induced ERK and AKT phosphorylation in HCC cells. DAS was also able to inhibit ER-α36-mediated activation of the MAPK/ERK signaling induced by estrogen. Thus, our results indicate that ER-α36 signaling is involved in DAS-induced inhibition of HCC cell growth both in vitro and in vivo.

Primary hepatic mucosa-associated lymphoid tissue lymphoma: case report and literature review.

BACKGROUND: The prevalence of primary hepatic mucosa-associated lymphoid tissue (MALT) lymphomas is extremely low. Here, we describe a case of this disease misdiagnosed as hepatocellular carcinoma (HCC) and review relevant literature to prevent future misdiagnoses. CASE PRESENTATION: a 58-year-old woman complained about abdominal pain for more than four months. About two months prior, she came to our hospital with elevated levels of HBV DNA and positive HBsAg and HBcAb. After two months of entecavir treatment, HBV DNA decreased to a normal level. She returned to the hospital with worsened abdominal pain for over a month. Magnetic resonance imaging and systemic positron emission tomography-computed tomography identified two nodes in the liver, and she was diagnosed with HCC. The patient then underwent a laparoscopic hepatectomy. Microscopic examination showed a diffuse infiltrate of small-to-medium-sized lymphocytes and lymphoepithelial lesions. Immunohistochemical staining showed that most of the lymphoid cells were strongly positive for CD20, CD79a, BCL2, IgM and weakly positive for IgD, while negative for CD3, CD10, BCL6, MUM1, CD43, CD5, cyclin D1, CD23, CD30, and PD1. The Ki-67 index of lymphoid cells was 5%. Further pathologic analysis confirmed the diagnosis of primary hepatic MALT lymphoma. The patient received antiviral treatment and recovered well with no sign of relapse for 17 months. CONCLUSIONS: Primary hepatic MALT lymphoma is an uncommon disease that is difficult to diagnose and has no widely accepted treatment. Surgical resection is a good choice for both diagnosis and local therapy, and strict follow-up of the patient is essential.

Estrogen receptor-α36-mediated rapid estrogen signaling regulates 78 kDa glucose-regulated protein expression in gastric carcinoma cells.

To determine whether estrogen receptor-α36 (ER-α36) -mediated rapid estrogen signaling is associated with 78 kDa glucose-regulated protein (GRP78) expression in gastric cancer, 86 samples of gastric tumor tissue with corresponding normal and tumor-adjacent tissues were used to examine expression patterns of GRP78 and ER-α36. Immunohistochemistry demonstrated that 55/86 (63.95%) patients with gastric carcinoma, and western blot analysis revealed that GRP78 was upregulated in 15/20 (75%) of tumor specimens. GRP78 expression was positively associated with ER-α36 expression, the male sex and lymph node metastasis (P<0.05). Estrogen treatment increased GRP78 and ER-α36 expression, as well as GSK-3ß phosphorylation in established gastric cancer SGC-7901 cells. The steady-state level of GRP78 protein expression and the level of phosphorylated GSK-3ß at Ser9 were decreased in SGC-7901 cells with ER-α36 knockdown. Forced expression of ER-α36 in SGC-7901 cells, however, led to an increase in GRP78 expression and GSK-3ß phosphorylation. It may therefore be concluded that ER-α36-mediated rapid estrogen signaling positively regulates GRP78 expression, presumably via the GSK-3ß pathway, which may be associated with gastric carcinogenesis.

GRP78 positively regulates estrogen­stimulated cell growth mediated by ER­α36 in gastric cancer cells.

Estrogen receptor (ER)­α36, a novel isoform of ER, primarily mediates non­classical estrogen signaling. It has been reported that ER­α36­mediated growth stimulating signals are involved in the malignancy of gastric tumor cells. However, the mechanism underlying the regulation of ER­α36 function in development of gastric cancer remains to be elucidated. The present study investigated the role of 78 kDa glucose­regulated protein (GRP78) in the regulation of ER­α36 expression and signaling during the growth of gastric tumor cells. It was demonstrated that GRP78 expression was detectable in gastric cancer tumor tissues, and was positively­correlated with tumor stage, lymphatic metastasis and ER­α36 expression (P<0.05). An increased growth rate, and increased expression of ER­α36 and the cell cycle regulator cyclin D1 was detected in cells with GRP78 overexpression (SGC­High78 cells). SGC­High78 cells are more sensitive to estrogen compared with SGC­Control cells. Therefore, the results of the present study demonstrated that GRP78 positively regulated ER­α36 expression and signaling with cell growth in gastric cancer, which is involved in gastric carcinogenesis.

Effects of 17ß-estradiol and tamoxifen on gastric cancer cell proliferation and apoptosis and ER-α36 expression.

The present study aimed to investigate the effects of 17ß-estradiol and tamoxifen, an agonist and inhibitor of the estrogen receptor (ER), respectively, on the proliferation and apoptosis of gastric cancer cells, as well as the messenger (m)RNA expression levels of ER-α36. Nested reverse transcription-polymerase chain reaction (RT-PCR) confirmed that ER-α36 was expressed in the BGC823, MKN45 and SGC7901 human gastric cancer cell lines. Subsequently, the BGC823 cell line was stimulated with various concentrations of 17ß-estradiol or tamoxifen for 24 or 48 h, and the proliferation, apoptosis and mRNA expression levels of ER-α36 were determined by water-soluble tetrazolium (WST)-1 assay, flow cytometry and RT-quantitative PCR, respectively. The activity of BGC823 cells was significantly increased following treatment with 10-12 mol/l 17ß-estradiol for 24 h (P=0.013), as compared with the control, and reached a peak at 48 h (P=0.002). Notably, the activity of BGC823 cells was decreased with increasing concentrations of 17ß-estradiol, although it remained higher compared with that of the control. In the tamoxifen-treated groups, the cell activity decreased as the drug concentration increased. The apoptosis rate was markedly reduced in the 17ß-estradiol group after 24 h (10-12 mol/l, P=0.013; 10-11 mol/l, P=0.023; and 10-10 mol/l, P=0.017) and after 48 h (10-12 mol/l, P=0.002; 10-11 mol/l, P=0.011; and 10-10 mol/l, P=0.033), whereas the rate of apoptosis increased as the tamoxifen concentration increased (24 h: 5×10-6 mol/l, P=0.002; and 10-5 mol/l, P=0.001; and 48 h: 5×10-6 mol/l, P=0.014 and 10-5 mol/l, P=0.0021), as compared with the control group. The mRNA expression levels of ER-α36 were significantly increased after 24 h of treatment with 10-12 mol/l (P=0.024), 10-11 mol/l (P=0.0113) and 10-10 mol/l (P=0.0037) 17ß-estradiol compared with the control group when the concentration of 17ß-estradiol was low, and the same was observed after 48 h of treatment 10-12 mol/l (P=0.0164), 10-11 mol/l (P=0.0342) and 10-10 mol/l (P=0.0198) 17ß-estradiol. The mRNA expression levels of ER-α36 were significantly decreased with increasing concentrations of tamoxifen after 24 h (5×10-6 mol/l, P=0.0233; and 10-5 mol/l, P=0.007) and after 48 h (5×10-6 mol/l, P=0.001; and 10-5 mol/l, P=0.0153). In addition, the ability of tamoxifen to inhibit the growth of gastric cancer cells was concentration-dependent. The results of the present study suggested that gastric cancer cells were sensitive to the effects of 17ß-estradiol and tamoxifen, and that tamoxifen is able to induce gastric cancer cell apoptosis. The expression levels of ER-α36 were upregulated, and the growth of gastric cancer cells was increased, following treatment with 17ß-estradiol, thus suggesting that gastric cancer tumors are stimulated by estrogen.

Fas expression is downregulated in gastric cancer.

The aim of the present study was to investigate Fas expression in tumor samples from patients with gastric cancer, in order to determine the involvement of the Fas signaling pathway. The protein expression levels of Fas, caspase-8, caspase­3 and poly (adenosine diphosphate­ribose) polymerase 1 (PARP1) were examined in gastric cancer specimens and their associations with clinical pathological parameters were analyzed with immunohistochemical staining and western blot analysis. The mRNA expression was quantified with quantitative PCR and apoptosis was examined with a FACScan flow cytometer. The results demonstrated that the downregulation of Fas expression was correlated with less histological differentiation, gender (male), and increased lymph node and distant metastases (P<0.05). In the AGS established gastric cancer cell line, upregulation of the Fas signaling pathway promoted the apoptosis of gastric cancer cells by upregulating the expression of caspase­8 and caspase­3, and downregulating the expression of PARP1. The present study demonstrated that Fas was associated with gastric cancer and promoted the apoptosis of gastric cancer cells via caspase­8, caspase­3 and PARP1. These results suggested that caspase­8, caspase­3 and PARP1 may be triggers of gastric cancer, and upregulation of caspase­8 and caspase­3 expression, or inhibition of PARP1 expression may improve the therapeutic outcome in patients with gastric cancer.

Characterization of sphere­forming cells with stem­like properties from the gastric cancer cell lines MKN45 and SGC7901.

Traditionally, it was presumed that gastric cancer was derived from tumor cells with stem­like properties. In the present study, stem­like cells from the gastric cancer cell lines MKN45 and SGC7901 were enriched by growing them as spheres in a defined serum­free medium. Following enrichment for stem­like cells, cluster of differentiation (CD)24 and CD44 were applied as candidate stem cell markers to examine the expression profile. It was revealed that the sphere­derived cells contained a higher proportion of cells expressing the stem cell surface markers CD24 and CD44 when compared with the parental cells. It was also identified that the expression of cytokeratin 18 in sphere­derived cells was decreased and the expression of vimentin and aldehyde dehydrogenase 1 (ALDH1) was increased compared with the parental cells. This finding supports the existence of a population of tumor sphere­forming cells with stem cell properties in the MKN45 and SGC7901 cell lines. Furthermore, the stem cell population was enriched in cells expressing CD24, CD44, vimentin and ALDH1 cell surface markers. These results support the existence of gastric cancer stem cells and provide an alternative approach to the diagnosis and treatment of gastric cancer.

Involvement of the Akt signaling pathway in ER-α36/GRP94-mediated signaling in gastric cancer.

Glucose-regulated protein 94 (GRP94) has been implicated in the promotion of tumor proliferation and metastasis. Previous studies have found that GRP94 is involved in the malignant growth of gastric carcinoma cells through estrogen receptor-α36 (ER-α36)-mediated estrogen signaling, but the underlying mechanism remains unclear. In the present study, we examined the expression levels of GRP94 and ER-α36 in tumor specimens from gastric cancer patients by immunohistochemistry, and found that both GRP94 and ER-α36 were highly expressed in the cytoplasms of gastric carcinoma cells. Furthermore, treatment with 17ß-estradiol at a concentration of 10-12 M for 24 h increased the expression levels of GRP94 and ER-α36, and the phosphorylation levels of Akt at the Ser473 site (Ser473-Akt). In established SGC7901 gastric cancer cells with knockdown of ER-α36 expression, the levels of GRP94 and Ser473-Akt expression were significantly reduced. Thus, the Akt signaling pathway is a potentially important signaling pathway in ER-α36-GRP94-mediated gastric carcinogenesis.

Biphasic ER-α36-mediated estrogen signaling regulates growth of gastric cancer cells.

To examine the expression patterns of ER-α36 and Cyclin D1 in human gastric cancer tissues and to investigate the effects of ER-α36-mediated estrogen signaling on the growth of gastric cancer cells, 117 samples of formalin-fixed and paraffin-embedded gastric cancer tumor tissues and 40 fresh gastric cancer tumor tissues were analyzed with immunohistochemistry assay and western blot analysis. ER-α36 expression was well correlated with gender (male:female ratio 2.88:1, P=0.01), invasion to serosa (P=0.01) as well as Cyclin D1 expression (P<0.01). The effects of different concentrations of estrogen on the growth of different gastric cancer cells and normal gastric cells as well as gastric cancer SGC7901 cells with different levels of ER-α36 expression were examined. SGC7901 cells with high levels of ER-α36 expression exhibited estrogen hypersensitivity, high growth rate and high levels of Cyclin D1 expression while SGC7901 cells with knocked-down levels of ER-α36 expression were insensitive to estrogen stimulation, grew slowly and expressed less Cyclin D1. Our results indicate that ER-α36 mediates biphasic estrogen signaling in the growth of gastric cancer cells.

Estrogen protects SGC7901 cells from endoplasmic reticulum stress-induced apoptosis by the Akt pathway.

Several previous studies have demonstrated that estrogen may protect cancer cells from endoplasmic reticulum stress-induced apoptosis. However, the molecular mechanisms involved are not fully understood. In the present study, human gastric adenocarcinoma SGC7901 cells were treated with tunicamycin (TM) to induce endoplasmic reticulum stress. This was demonstrated by increased glucose-regulated protein 78 expression and enhanced phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase. Endoplasmic reticulum stress induced caspase-3-mediated apoptosis with the inhibition of Akt; the latter of which was measured by the activity-dependent phosphorylation at Ser473 of Akt. Simultaneous treatment of 10-9 M 17ß-estradiol (E2) with TM may protect SGC7901 cells from endoplasmic reticulum stress-induced apoptosis by counteracting the inhibitory effect of TM on Akt, causing an increase in the phosphorylation of Ser473-Akt. It was concluded that low concentrations of E2 may counteract endoplasmic reticulum stress-induced inactivation of Akt to block caspase-3-mediated apoptosis.

Involvement of ER-α36 in the malignant growth of gastric carcinoma cells is associated with GRP94 overexpression.

AIMS: This study aimed to examine the involvement of glucose-regulated protein 94 (GRP94) in oestrogen receptor-α36 (ER-α36)-mediated oestrogen signalling in gastric cancer development. METHODS AND RESULTS: A total of 130 formalin-fixed and paraffin-embedded gastric tumour samples with corresponding normal gastric and tumour-adjacent tissues were used. High levels of GRP94 expression (2+ or 3+) were observed in 109 of 130 gastric carcinomas (83.85%) by immunohistochemistry, and in 13 of 18 tumour specimens (72.22%) with Western blot analysis. GRP94 expression was correlated positively with gender, tumour stage, lymph node metastasis and ER-α36 expression (P < 0.05). Oestrogen treatment up-regulated both GRP94 and ER-α36 expression in gastric cancer SGC7901 cells. In addition, steady state levels of GRP94 protein were decreased in established gastric cancer SGC7901 cells with knocked-down levels of ER-α36 expression and in xenograft tumours formed by these cells. Forced expression of recombinant ER-α36 in SGC7901 cells, however, up-regulated the levels of GRP94 expression. CONCLUSIONS: Glucose-regulated protein 94 is a downstream effector of ER-α36-mediated oestrogen signalling, and may be involved in ER-α36 function during gastric carcinogenesis.

The antagonistic effect between STAT1 and Survivin and its clinical significance in gastric cancer.

In previous studies, we observed that STAT1 and Survivin correlated negatively with gastric cancer tissues, and that the functions of the IFN-γ-STAT1 pathway and Survivin in gastric cancer are the same as those reported for other types of cancer. In this study, the SGC7901 gastric cancer cell line and 83 gastric cancer specimens were used to confirm the relationship between STAT1 and Survivin, as well as the clinical significance of this relationship in gastric cancer. IFN-γ and STAT1 and Survivin antisense oligonucleotides (ASONs) were used to knock down the expression in SGC7901 cells. The protein expression of STAT1 and Survivin was tested by immunocytochemical and image analysis methods. A gastric cancer tissue microarray was prepared and tested by immunohistochemical methods. Data were analyzed by the Spearman's rank correlation analysis, the χ(2) test and Cox's multivariate regression analysis. Upon knockdown of IFN-γ, STAT1 and Survivin expression by ASON in the SGC7901 cell line, an antagonistic effect was observed between STAT1 and Survivin. In gastric cancer tissues, STAT1 showed a negative correlation with depth of invasion (p<0.05) in gastric cancer tissues exhibiting a negative Survivin protein expression. Furthermore, in tissues exhibiting a negative STAT1 protein expression, Survivin correlated negatively with N stage (p<0.05). Pathological and molecular markers were used to conduct Cox's multivariate regression analysis, and depth of invasion and N stage were found to be prognostic factors (p<0.05). On the other hand, in tissues exhibiting a negative Survivin protein expression, Cox's multivariate regression analysis revealed that the differentiation type and STAT1 protein expression were prognostic factors (p<0.05). There is an antagonistic effect between STAT1 and Survivin in gastric cancer, and this antagonistic effect is of clinical significance in gastric cancer.

LiCl attenuates thapsigargin-induced tau hyperphosphorylation by inhibiting GSK-3ß in vivo and in vitro.

Abnormal hyperphosphorylation of microtubule-associated protein tau is involved in the pathogenesis of several neurodegenerative diseases, including Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress is indicated to play an important role in neurodegeneration and activation of glycogen synthase kinase-3ß (GSK-3ß), an integral kinase in tau phosphorylation. To explore the effect of ER stress on tau phosphorylation, we treated cultured cells (HEK293 and SH-SY5Y cells) and rat brain with thapsigargin, an ER stress inducer. We found that the phosphorylation level of tau was significantly increased after thapsigargin treatment. By using a cell-free reconstitution system, we also observed that co-culture of the thapsigargin-treated ER fraction from HEK293/wt (without tau) with cytoplasm prepared from HEK293/tau induced an increased tau phosphorylation. Concurrently, activation of GSK-3ß as evidenced by an increased phospho-GSK-3ß at Tyr-216 and decreased phospho-GSK-3ß at Ser-9 both in vitro and in vivo was detected. Application of lithium chloride, a GSK-3ß inhibitor, could efficiently attenuate the thapsigargin-induced tau hyperphosphorylation with suppressed activation of GSK-3ß in cell cultures and rat brains. Our data provide further evidence supporting the role of ER stress in tau hyperphosphorylation and the protective role of lithium.

Constant illumination induces Alzheimer-like damages with endoplasmic reticulum involvement and the protection of melatonin.

Most patients with Alzheimer's disease (AD) present decreased levels of melatonin, a day-night rhythm-related hormone. To investigate the role of melatonin deficiency in AD, we used constant illumination to interrupt melatonin metabolism and measured some of the AD-like alterations in rats. Concomitant with decreased serum melatonin, the rats developed spatial memory deficits, tau hyperphosphorylation at multiple sites, activation of glycogen synthase kinase-3 and protein kinase A, as well as suppression of protein phosphatase-1. Prominent oxidative damage and organelle lesions, demonstrated by increased expression of endoplasmic reticulum (ER) stress-related proteins including BiP/GRP78 and CHOP/GADD153, decreased number of rough ER and free ribosome, thinner synapses, and increased superoxide dismutase and monoamine oxidase were also observed in the light exposed rats. Simultaneous supplement of melatonin partially arrested the behavioral and molecular impairments. It is suggested that melatonin deficiency may be an upstream effector responsible for the AD-like behavioral and molecular pathologies with ER stress-involved mechanisms.