Parecoxib and 5-Fluorouracil Synergistically Inhibit EMT and Subsequent Metastasis in Colorectal Cancer by Targeting PI3K/Akt/NF-κB Signaling.

Colorectal cancer is one of the most common causes of cancer mortality worldwide, and innovative drugs for the treatment of colorectal cancer are continually being developed. 5-Fluorouracil (5-FU) is a common clinical chemotherapeutic drug. Acquired resistance to 5-FU is a clinical challenge in colorectal cancer treatment. Parecoxib is a selective COX-2-specific inhibitor that was demonstrated to inhibit metastasis in colorectal cancers in our previous study. This study aimed to investigate the synergistic antimetastatic activities of parecoxib to 5-FU in human colorectal cancer cells and determine the underlying mechanisms. Parecoxib and 5-FU synergistically suppressed metastasis in colorectal cancer cells. Treatment with the parecoxib/5-FU combination induced an increase in E-cadherin and decrease in ß-catenin expression. The parecoxib/5-FU combination inhibited MMP-9 activity, and the NF-κB pathway was suppressed as well. Mechanistic analysis denoted that the parecoxib/5-FU combination hindered the essential molecules of the PI3K/Akt route to obstruct metastatic colorectal cancer. Furthermore, the parecoxib/5-FU combination could inhibit reactive oxygen species. Our work showed the antimetastatic capacity of the parecoxib/5-FU combination for treating colorectal cancers via the targeting of the PI3K/Akt/NF-κB pathway.

Parecoxib expresses anti-metastasis effect through inhibition of epithelial-mesenchymal transition and the Wnt/ß-catenin signaling pathway in human colon cancer DLD-1 cell line.

Colorectal cancer is the third leading cause of cancer death in Taiwan. Current treatments involve combination of surgical resection, radiation, and chemotherapy. These treatments have demonstrated to increased five-year survival of a patient with colorectal cancer. However, metastasis is a major capability of cancer cells that causes poor prognosis, recurrence, and even death. Epidemiological and clinical studies have suggested the use of non-steroidal anti-inflammatory drugs (NSAIDs) as an effective class of compounds to prevent colon cancer. Parecoxib is an NSAID and the only parenterally administered selective cyclooxygenase (COX)-2 inhibitor. In this study, we evaluated whether parecoxib inhibits the metastasis of DLD-1 human colon cancer cells, a COX-2 null cell line, and the underlying mechanism. Cell migration of the DLD-1 cells was significantly inhibited by parecoxib treatment as shown by the Transwell migration assay. This enhanced anti-migration effect was correlated with the attenuated phosphorylation of Akt, expression of vimentin (a mesenchymal marker), and ß-catenin, and corresponded with the upregulated GSK3ß and E-cadherin (an epithelial marker). These findings suggested that parecoxib could inhibit the epithelial-mesenchymal transition (EMT) and metastasis in human colon cancer cells by downregulating ß-catenin. Thus, parecoxib could provide a novel prospective strategy for a combination treatment with chemotherapeutic drugs against metastasis of human colon cancer.

Anti-fibrotic effect of rosmarinic acid on inhibition of pterygium epithelial cells.

AIM: To investigate the anti-fibrosis effect of rosmarinic acid (RA) in pterygium epithelial cells (PECs) to determine if RA is a potent agent for treating pterygium. METHODS: The PECs (1×104 cells/mL) were treated with 100 µmol/L of RA for 1, 3 and 6h. After RA treatment, the cell viability was determined by staining with acridine orange/DAPI and analysis via a NucleoCounter NC-3000. The protein expression levels of type I collagen, transforming growth factor beta-1 (TGF-ß1), TGF-ß type II receptor (TGF-ßRII), p-Smad1/5, p-Smad2, p-Smad3, and Smad4 of the cell lysates were measured by Western blot analysis. RESULTS: The cell viability of PECs was significantly decreased after RA treatment (P<0.01). As the result, RA reduced the protein expression of type I collagen and TGF-ß1 of PECs. Additionally, RA also inhibited TGF-ß1/Smad signaling by decreasing the protein expressions of TGF-ßRII, p-Smad1/5, p-Smad2, p-Smad3, and Smad4. CONCLUSION: This study demonstrate that RA could inhibit fibrosis of PECs by down-regulating type I collagen expression and TGF-ß1/Smad signaling. Therefore, RA is a potent therapeutic agent for the treatment of pterygium.

Nephroprotective effect of electrolyzed reduced water against cisplatin-induced kidney toxicity and oxidative damage in mice.

BACKGROUND: Cisplatin is a potent chemotherapeutic drug for cancer therapy, but it has serious side effects in clinical treatment, particularly nephrotoxicity. The purpose of this study was to evaluate the protective effect of electrolyzed reduced water (ERW) on renal injury caused by cisplatin. METHODS: Animals were divided into four groups as follows: normal control group, cisplatin control group, ERW control group and ERW + cisplatin group. Each group comprised 10 animals, which were orally treated with normal saline or ERW daily companion by administration of one dose of cisplatin for 28 days. Animals in the cisplatin group received an intraperitoneal single-dose injection of cisplatin (20 mg/kg body weight) as a single i.p. dose on the 25th day of the experiment. We determined the hydration state in urine and the level of serum markers of kidney function, the levels of glutathione (GSH) and thiobarbituric acid-reactive substances (TBARS) levels and the activities of glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT) and superoxidase dismutase (SOD) in kidney and histopathological changes. RESULTS: After administration of ERW, the reduced urinary osmolality was increased and elevated Na+, K+, Mg2+ and Ca2+ levels in urine were significantly decreased in cisplatin-induced renal injury mice. Besides, the results demonstrated that significantly decreased elevated serum levels of creatinine and blood urea nitrogen (BUN) and the levels of TBARS in the kidneys that were induced by cisplatin. Moreover, ERW treatment was also found to markedly increase (p < 0.05) the activities of GPx, GR, CAT and SOD, and to increase GSH content in the kidneys. Histopathology showed that ERW protects against cisplatin-induced renal injury to both the proximal and distal tubules. CONCLUSION: ERW exhibits potent nephroprotective effects on cisplatin-induced kidney damage in mice, likely due to both the increase in antioxidant-defense system activity and the inhibition of lipid peroxidation.

Propyl Gallate Exerts an Antimigration Effect on Temozolomide-Treated Malignant Glioma Cells through Inhibition of ROS and the NF-κB Pathway.

In this study, we demonstrated that temozolomide (TMZ) and propyl gallate (PG) combination enhanced the inhibition of migration in human U87MG glioma cells. PG inhibited the TMZ-induced reactive oxygen species (ROS) generation. The mitochondrial complex III and NADPH oxidase are two critical sites that can be considered to regulate antimigration in TMZ-treated U87MG cells. PG can enhance the antimigration effect of TMZ through suppression of metalloproteinase-2 and metalloproteinase-9 activities, ROS generation, and the NF-κB pathway and possibly provide a novel prospective strategy for treating malignant glioma.

Anti-oxidant and anti-inflammatory effects of hydrogen-rich water alleviate ethanol-induced fatty liver in mice.

AIM: To investigate the effects of hydrogen-rich water (HRW) treatment on prevention of ethanol (EtOH)-induced early fatty liver in mice. METHODS: In vitro reduction of hydrogen peroxide by HRW was determined with a chemiluminescence system. Female mice were randomly divided into five groups: control, EtOH, EtOH + silymarin, EtOH + HRW and EtOH + silymarin + HRW. Each group was fed a Lieber-DeCarli liquid diet containing EtOH or isocaloric maltose dextrin (control diet). Silymarin was used as a positive control to compare HRW efficacy against chronic EtOH-induced hepatotoxicity. HRW was freshly prepared and given at a dosage of 1.2 mL/mouse trice daily. Blood and liver tissue were collected after chronic-binge liquid-diet feeding for 12 wk. RESULTS: The in vitro study showed that HRW directly scavenged hydrogen peroxide. The in vivo study showed that HRW increased expression of acyl ghrelin, which was correlated with food intake. HRW treatment significantly reduced EtOH-induced increases in serum alanine aminotransferase, aspartate aminotransferase, triglycerol and total cholesterol levels, hepatic lipid accumulation and inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6. HRW attenuated malondialdehyde level, restored glutathione depletion and increased superoxide dismutase, glutathione peroxidase and catalase activities in the liver. Moreover, HRW reduced TNF-α and IL-6 levels but increased IL-10 and IL-22 levels. CONCLUSION: HRW protects against chronic EtOH-induced liver injury, possibly by inducing acyl ghrelin to suppress the pro-inflammatory cytokines TNF-α and IL-6 and induce IL-10 and IL-22, thus activating antioxidant enzymes against oxidative stress.

ß-mangostin suppresses human hepatocellular carcinoma cell invasion through inhibition of MMP-2 and MMP-9 expression and activating the ERK and JNK pathways.

ß-mangostin is a dietary xanthone that has been reported to have the anticancer properties in some human cancer cell types. However, the antimetastatic effect and molecular mechanism of ß-mangostin action in human hepatocellular carcinoma (HCC) cells remain unknown. In this study, we found that ß-mangostin did not induce cytotoxicity in human HCC cells (SK-Hep-1, Huh-7 and HA22T/VGH cells). ß-mangostin could inhibit migration and invasion of human HCC cells. Meanwhile, ß-mangostin significantly decreased the protein activities and expression of matrix metalloproteinase (MMP)-2 and MMP-9 via increasing the activation of MEK1/2, ERK1/2, MEK4 and JNK1/2 signaling pathways. Furthermore, using specific inhibitor for ERK1/2 (PD98059) and JNK1/2 (JNKII) significantly restored the expression of MMP-2/-9 and invasion by ß-mangostin treatment in Huh-7 cells. In addition, ß-mangostin effectively restored the protein levels and transcription activity of MMP-2 and MMP-9 in siERK or siJNK-transfected Huh-7 cells, concomitantly with promotion on cell migration and invasion. Taken together, these findings are the first to demonstrate the antimetastatic activity of ß-mangostin against human HCC cells, which may act as a promising therapeutic agent for the treatment of HCC.

Inhibitory effects of rosmarinic acid on pterygium epithelial cells through redox imbalance and induction of extrinsic and intrinsic apoptosis.

Pterygium is a common tumor-like ocular disease, which may be related to exposure to chronic ultraviolet (UV) radiation. Although the standard treatment for pterygium is surgical intervention, the recurrence rate of pterygium is high when no effective inhibitory drug is used after surgery. Rosmarinic acid (RA) is a polyphenol antioxidant with many biological activities, including anti-UV and anti-tumor properties. This study aimed to examine the inhibitory effects of RA on pterygium epithelial cells (PECs). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay was used to examine the cell cytotoxicity of PECs after RA treatment. A fluorescent probe, DCFH-DA (2',7'-dichlorofluorescin diacetate), was stained with PECs to measure intracellular reactive oxygen species (ROS) levels. Antioxidant activity assays were used to measure the levels of superoxide dismutase (SOD) and catalase (CAT) in PECs. Western blot analysis was used to determine the protein expression of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), quinone acceptor oxidoreductase 1 (NQO1), and apoptosis-associated proteins. RA significantly reduced the cell viability of the PECs. Treatment with RA remarkably increased the Nrf2 protein expression levels in the nucleus, HO-1 and NQO1 protein expression levels, and the activities of SOD and CAT. As a result, intracellular ROS levels in PECs were decreased. Additionally, the induction of extrinsic apoptosis on PECs by RA was associated with increasing expressions levels of Fas, Fas-associated protein with death domain (FADD), tumor necrosis factor-alpha (TNF-α), and caspase 8 protein. Moreover, the induction of intrinsic apoptotic cell death in PECs was confirmed through upregulation of cytochrome c, Bax, caspase 9, and caspase 3 and downregulation of Bcl-2 and pro-caspase 3. Our study demonstrated that RA could inhibit the viability of PECs through regulation of extrinsic and intrinsic apoptosis pathways. Therefore, RA may have potential as a therapeutic medication for pterygium.

The Chinese medicine JC-001 enhances the chemosensitivity of Lewis lung tumors to cisplatin by modulating the immune response.

BACKGROUND: JC-001 is a Chinese medicine that can modulate the immunity in Hepa 1-6 tumor-bearing mice, and we questioned whether JC-001 can serve as efficient adjuvant chemotherapy. We aimed to identify a novel approach for enhancing cis-diamminedichloroplatinum (II) (CDDP)-based chemotherapy by immunomodulation. METHODS: The anti-tumor activity in vitro was determined based on foci formation and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A LLC1 tumor xenograft model was used to analyze the activity of tumor rejection in vivo. The tumors were analyzed through hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) staining and cytokine arrays. RESULTS: JC-001 suppressed foci formation and reduced the viability of Lewis lung carcinoma (LLC1) cells in vitro. JC-001 suppressed LLC1 tumor growth in immunodeficient BALB/c nude mice and in immunocompetent C57BL/6 mice to an even greater extent. Furthermore, JC-001 up-regulated interferon-γ expression in the tumor microenvironment, enhanced the Th1 response in tumor-bearing mice, and increased the chemosensitivity of LLC1 tumors to CDDP chemotherapy. The results of our study suggest that JC-001 is associated with low cytotoxicity and can significantly suppress tumor growth by enhancing the Th1 response. CONCLUSION: JC-001 is a Chinese medicine with potential clinical applications in CDDP-based chemotherapeutic regimens.

Antitumor Activity of the Chinese Medicine JC-001 Is Mediated by Immunomodulation in a Murine Model of Hepatocellular Carcinoma.

JC-001 is a Chinese medicine that has been used to treat liver disease; however, its significance in cancer treatment has not been characterized. In this study, we used an immunocompetent tumor model to characterize the antitumor activity of JC-001. A total of 48 Hepa 1-6 tumor-bearing C57BL/6 mice were randomly grouped into 4 groups and treated with H2O or JC-001 via oral administration. After hepatoma cell lines, including HepG2, Hep3B, SK-Hep-1, and Hepa 1-6, underwent 96 hours of JC-001 treatment, a low cytotoxic effect was observed. In contrast, no direct cytotoxic effect of JC-001 on a normal human liver cell line, THLE-3, was observed under the same incubation conditions. Using a murine tumor model, we found that tumor growth could be inhibited by JC-001 in C57BL/6 mice but not in immunodeficient mice. Histopathological analysis of tumors from C57BL/6 mice revealed immune cell infiltration in tumors from the JC-001-treated group, as observed by hematoxylin and eosin staining; in addition, Ki67, hypoxia-inducible factor-1-α, and high mobility group box 1 expression levels were suppressed in the tumors. Both the coculture assay and murine spleen mRNA quantitative PCR analyses demonstrated that JC-001 could suppress Th17 immunity. Our data suggest that JC-001 is a Chinese medicine with low cytotoxicity that can significantly suppress tumor growth by immune regulation. This herbal remedy has great potential for future clinical application in hepatoma therapy.

Novel Function of Isoamylamine Improves Survival in Endotoxemic Mice by Ameliorating Coagulopathy and Attenuating MMP-9 Expression Through p-ERK/p-p38 Signaling at Early Stage.

When a host suffers endotoxemic shock or septic shock, it results in many symptoms including disseminated intravascular coagulation (DIC). Septic shock (SS) causes coagulation time to decrease and then gradually increase, finally becoming prolonged and giving rise to DIC. Isoamylamine (IA) is one of the main components of grape products and can improve the survival rate of endotoxin lipopolysaccharide (LPS)-induced endotoxemic shock. The aim of this study was to elucidate if IA ameliorates coagulopathy in the early phase of LPS-induced damage. We studied the effects of IA on the coagulation system of extrinsic (prothrombin time [PT]) and intrinsic (activated partial thromboplastin time [aPTT]) pathways. PT and aPTT were tested in plasma drawn from mice following intraperitoneal (IP) injection of 1 mL of 1,000 ppm IA after LPS administration. Shortened PT was ameliorated by 1,000 ppm IA 1 h after LPS administration, but there was no effect on aPTT. In conclusion, IA 1,000 ppm partially intervenes in the early phase of LPS-induced damage, shortening plasma PT 1 h after LPS treatment in mice. Furthermore, we found 1,000 ppm IA also could attenuate MMP-9 expression through p-ERK/p-p38 signaling in mice hepatocyte extracts. This study focused on the effects of IA on blood coagulation function and inflammatory proteins. In the current situation of absence of effective treatment for SS, IA can increase survival rate and may offer another choice of patient avoiding causing death during endotoxemic shock.

Synergistic antioxidant activity of resveratrol with genistein in high-glucose treated Madin-Darby canine kidney epithelial cells.

Resveratrol (Re), a stilbenoid, is associated with a potential benefit in controlling certain biomarkers in type II diabetes. Genistein (Ge), a phytoestrogen, may act as an antioxidant and thus may diminish damaging effects of free radicals in tissues. In the present study, a potential synergistic antioxidant effect of an Re/Ge combination on high-glucose (HG) incubation in Madin-Darby canine kidney (MDCK) epithelial cells was evaluated. Compared with the treatment of Re or Ge alone, the Re/Ge combination synergistically decreased intracellular reactive oxygen species (ROS) and hydroxyl radicals in MDCK cells. This synergistic antioxidant effect correlated with the inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression and an increase in γ-glutamylcysteine synthetase expression. In addition, mitochondrial complex I, NADPH oxidase, xanthine oxidase and lipoxygenase contributed towards ROS overproduction when the MDCK cells were incubated with HG. In conclusion, the Re/Ge combination synergistically enhanced the antioxidant effect in HG-incubated kidney cells, possibly through an enhanced antioxidant regulation mechanism. The Re/Ge combination may be a potential benefit against oxidative stress in diabetes mellitus.

miR-302 Attenuates Amyloid-ß-Induced Neurotoxicity through Activation of Akt Signaling.

Deficiency of insulin signaling has been linked to diabetes and ageing-related neurodegenerative diseases such as Alzheimer's disease (AD). In this regard, brains exhibit defective insulin receptor substrate-1 (IRS-1) and hence result in alteration of insulin signaling in progression of AD, the most common cause of dementia. Consequently, dysregulation of insulin signaling plays an important role in amyloid-ß (Aß)-induced neurotoxicity. As the derivation of induced pluripotent stem cells (iPSC) involves cell reprogramming, it may provide a means for regaining the control of ageing-associated dysfunction and neurodegeneration via affecting insulin-related signaling. To this, we found that an embryonic stem cell (ESC)-specific microRNA, miR-302, silences phosphatase and tensin homolog (PTEN) to activate Akt signaling, which subsequently stimulates nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) elevation and hence inhibits Aß-induced neurotoxicity. miR-302 is predominantly expressed in iPSCs and is known to regulate several important biological processes of anti-oxidative stress, anti-apoptosis, and anti-aging through activating Akt signaling. In addition, we also found that miR-302-mediated Akt signaling further stimulates Nanog expression to suppress Aß-induced p-Ser307 IRS-1 expression and thus enhances tyrosine phosphorylation and p-Ser 473-Akt/p-Ser 9-GSK3ß formation. Furthermore, our in vivo studies revealed that the mRNA expression levels of both Nanog and miR-302-encoding LARP7 genes were significantly reduced in AD patients' blood cells, providing a novel diagnosis marker for AD. Taken together, our findings demonstrated that miR-302 is able to inhibit Aß-induced cytotoxicity via activating Akt signaling to upregulate Nrf2 and Nanog expressions, leading to a marked restoration of insulin signaling in AD neurons.

Folate Deficiency Triggered Apoptosis of Synoviocytes: Role of Overproduction of Reactive Oxygen Species Generated via NADPH Oxidase/Mitochondrial Complex II and Calcium Perturbation.

Despite a plethora of literature has documented that osteoarthritis (OA) is veritably associated with oxidative stress-mediated chondrocyte death and matrix degradation, yet the possible involvement of synoviocyte abnormality as causative factor of OA has not been thoroughly investigated. For this reason, we conduct the current studies to insight into how synoviocytes could respond to an episode of folate-deprived (FD) condition. First, when HIG-82 synoviocytes were cultivated under FD condition, a time-dependent growth impediment was observed and the demise of these cells was demonstrated to be apoptotic in nature mediated through FD-evoked overproduction of reactive oxygen species (ROS) and drastically released of cytosolic calcium (Ca2+) concentrations. Next, we uncovered that FD-evoked ROS overproduction could only be strongly suppressed by either mitochondrial complex II inhibitors (TTFA and carboxin) or NADPH oxidase (NOX) inhibitors (AEBSF and apocynin), but not by mitochondrial complex I inhibitor (rotenone) and mitochondrial complex III inhibitor (antimycin A). Interestingly, this selective inhibition of FD-evoked ROS by mitochondrial complex II and NOX inhibitors was found to correlate excellently with the suppression of cytosolic Ca2+ release and reduced the magnitude of the apoptotic TUNEL-positive cells. Taken together, we present the first evidence here that FD-triggered ROS overproduction in synoviocytes is originated from mitochondrial complex II and NOX. Both elevated ROS in tandem with cytosolic Ca2+ overload serve as final arbitrators for apoptotic lethality of synoviocytes cultivated under FD condition. Thus, folate supplementation may be beneficial to patients with OA.

Environmentally relevant concentration of arsenic trioxide and humic acid promoted tumor progression of human cervical cancer cells: In vivo and in vitro studies.

In a previous study, treatment at higher concentrations of arsenic trioxide or co-exposure to arsenic trioxide and humic acid was found to be inhibited cell growth of cervical cancer cells (SiHa cells) by reactive oxygen species generation. However, treatment at lower concentrations slightly increased cell viability. Here, we investigate the enhancement of progression effects of environmentally relevant concentration of humic acid and arsenic trioxide in SiHa cell lines in vitro and in vivo by measuring cell proliferation, migration, invasion, and the carcinogenesis-related protein (MMP-2, MMP-9, and VEGF-A) expressions. SiHa cells treated with low concentrations of humic acid and arsenic trioxide alone or in co-exposure significantly increased reactive oxygen species, glutathione levels, cell proliferation, scratch wound-healing activities, migration abilities, and MMP-2 expression as compared to the untreated control. In vivo the tumor volume of either single drug (humic acid or arsenic trioxide) or combined drug-treated group was significantly larger than that of the control for an additional 45 days after tumor cell injection on the back of NOD/SCID mice. Levels of MMP-2, MMP-9, and VEGF-A, also significantly increased compared to the control. Histopathologic effects of all tumor cells appeared round in cell shape with high mitosis, focal hyperkeratosis and epidermal hyperplasia in the skin, and some tumor growth in the muscle were observed. Our results may indicate that exposure to low concentrations of arsenic trioxide and humic acid is associated with the progression of cervical cancer. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1121-1132, 2016.

The Anthropometric Measurement of Schober's Test in Normal Taiwanese Population.

The measurement of lower back mobility is essential in the assessment of lower back pain including ankylosing spondylitis. Original Schober's test (OST) and modified Schober's test (MST) are popularly conducted in daily rheumatology and orthopedics clinical practices. To our knowledge, this report is the only anthropometric reference study in a normal oriental population. The OST declined with age from 5.0 cm in the youngest (20-30 years old) to 3.1 cm in the aged (70-80 years old) male subjects and from 3.6 cm to 2.4 cm in the female subjects. The male OST was significantly more than the female OST. There was a good correlation between OST and MST in each of the three age groups of both sexes.

Hydrogen-rich water attenuates amyloid ß-induced cytotoxicity through upregulation of Sirt1-FoxO3a by stimulation of AMP-activated protein kinase in SK-N-MC cells.

Amyloid ß (Aß) peptides are identified in cause of neurodegenerative diseases such as Alzheimer's disease (AD). Previous evidence suggests Aß-induced neurotoxicity is linked to the stimulation of reactive oxygen species (ROS) production. The accumulation of Aß-induced ROS leads to increased mitochondrial dysfunction and triggers apoptotic cell death. This suggests antioxidant therapies may be beneficial for preventing ROS-related diseases such as AD. Recently, hydrogen-rich water (HRW) has been proven effective in treating oxidative stress-induced disorders because of its ROS-scavenging abilities. However, the precise molecular mechanisms whereby HRW prevents neuronal death are still unclear. In the present study, we evaluated the putative pathways by which HRW protects against Aß-induced cytotoxicity. Our results indicated that HRW directly counteracts oxidative damage by neutralizing excessive ROS, leading to the alleviation of Aß-induced cell death. In addition, HRW also stimulated AMP-activated protein kinase (AMPK) in a sirtuin 1 (Sirt1)-dependent pathway, which upregulates forkhead box protein O3a (FoxO3a) downstream antioxidant response and diminishes Aß-induced mitochondrial potential loss and oxidative stress. Taken together, our findings suggest that HRW may have potential therapeutic value to inhibit Aß-induced neurotoxicity.

Humic Acid Increases Amyloid ß-Induced Cytotoxicity by Induction of ER Stress in Human SK-N-MC Neuronal Cells.

Humic acid (HA) is a possible etiological factor associated with for several vascular diseases. It is known that vascular risk factors can directly increase the susceptibility to Alzheimer's disease (AD), which is a neurodegenerative disorder due to accumulation of amyloid ß (Aß) peptide in the brain. However, the role that HA contributes to Aß-induced cytotoxicity has not been demonstrated. In the present study, we demonstrate that HA exhibits a synergistic effect enhancing Aß-induced cytotoxicity in cultured human SK-N-MC neuronal cells. Furthermore, this deterioration was mediated through the activation of endoplasmic reticulum (ER) stress by stimulating PERK and eIF2α phosphorylation. We also observed HA and Aß-induced cytotoxicity is associated with mitochondrial dysfunction caused by down-regulation of the Sirt1/PGC1α pathway, while in contrast, treating the cells with the ER stress inhibitor Salubrinal, or over-expression of Sirt1 significantly reduced loss of cell viability by HA and Aß. Our findings suggest a new mechanism by which HA can deteriorate Aß-induced cytotoxicity through modulation of ER stress, which may provide significant insights into the pathogenesis of AD co-occurring with vascular injury.

Dunaliella salina exhibits an antileukemic immunity in a mouse model of WEHI-3 leukemia cells.

Dunaliella salina has been shown to have antioxidant property and induce apoptotic cell death of human cancer cells in vitro. However, there is no information available on D. salina showing an antileukemia effect or immunomodulatory activity in vivo. This study applied D. salina to syngeneic leukemia-implanted mice (BALB/c and WEHI-3) to investigate its immunological and antileukemia properties. Oral administration of D. salina (184.5, 369, and 922.5 mg/kg) inhibited spleen metastasis and prolonged the survival in BALB/c mice that had received an intravenous injection of WEHI-3 cells. The results revealed that D. salina had reduced spleen enlargement in murine leukemia. It had also increased the population and proliferation of T-cells (CD3) and B-cells (CD19) following Con A/LPS treatment on flow cytometry and MTT assay, respectively. Furthermore, D. salina increased the phagocytosis of macrophages and enhanced the cytotoxicity of natural killer cells on flow cytometry and LDH assay. Moreover, D. salina enhanced the levels of interferon-γ and interleukin 2 (IL-2) but reduced the levels of IL-4 and IL-10 in leukemic mice. In conclusion, these results demonstrated that the application of D. salina had beneficial effects on WEHI-3 leukemic mice by prolonging survival via modulating the immune responses.

Induction of macrophage cell-cycle arrest and apoptosis by humic acid.

Humic acid (HA) in well water is associated with Blackfoot disease and various cancers. Previously, we reported that acute humic acid exposure (25-200 µg/mL for 24 hr) induces inflammation in RAW264.7 macrophages. In this study, we observed that prolonged (72 hr) HA exposure (25-200 µg/mL) induces cell-cycle arrest and apoptosis in cultured RAW264.7 cells. We also observed that exposing macrophages to HA arrests cells in the G2 /M phase of the cell cycle by reducing cyclin A/B1 , Cdc2, and Cdc25C levels. Treating macrophages with HA triggers a sequence of events characteristic of apoptotic cell death including loss of cell viability, morphological changes, internucleosomal DNA fragmentation, sub-G1 accumulation. Molecular markers of apoptosis associated with mitochondrial dysfunction were similarly observed, including cytochrome c release, caspase-3 or caspase-9 activation, and Bcl-2/Bax dysregulation. In addition to the mitochondrial pathway, HA-induced apoptosis may also be mediated through the death receptor and ER stress pathways, as evidence by induction of Fas, caspase-8, caspase-4, and caspase-12 activity. HA also upregulates p53 expression and causes DNA damage as assessed by the comet assay. These findings yield new insight into the mechanisms by which HA exposure may trigger atherosclerosis through modulation of the macrophage-mediated immune system.