Combined Effects of Fludarabine and Interferon Alpha on Autophagy Regulation Define the Phase of Cell Survival and Promotes Responses in LLC-MK2 and K562 Cells.

Autophagy is a known mechanism of cells under internal stress that regulates cellular function via internal protein recycling and the cleaning up of debris, leading to healthy live cells. However, the stimulation of autophagy by external factors such as chemical compounds or viral infection mostly tends to induce apoptosis/cell death. This study hypothesizes that manipulation of the autophagy mechanism to the pro-cell survival and/or decreased pro-viral niche can be a strategy for effective antiviral and anticancer treatment. Cells susceptible to viral infection, namely LLC-MK2, normal monkey epithelium, and K562, human immune-related lymphocyte, which is also a cancer cell line, were treated with fludarabine nucleoside analog (Fdb), interferon alpha (IFN-α), and a combination of Fdb and IFN-α, and then were evaluated for signs of adaptive autophagy and STAT1 antiviral signaling by Western blotting and immunolabeling assays. The results showed that the low concentration of Fdb was able to activate an autophagy response in both cell types, as demonstrated by the intense immunostaining of LC3B foci in the autophagosomes of living cells. Treatment with IFN-α (10 U/mL) showed no alteration in the initiator of mTOR autophagy but dramatically increased the intracellular STAT1 signaling molecules in both cell types. Although in the combined Fdb and IFN-α treatment, both LLC-MK2 and K562 cells showed only slight changes in the autophagy-responsive proteins p-mTOR and LC3B, an adaptive autophagy event was clearly shown in the autophagosome of the LLC-MK2 cell, suggesting the survival phase of the normal cell. The combined effect of Fdb and IFN-α treatment on the antiviral response was identified by the level of activation of the STAT1 antiviral marker. Significantly, the adaptive autophagy mediated by Fdb was able to suppress the IFN-α-mediated pSTAT1 signaling in both cell types to a level that is appropriate for cellular function. It is concluded that the administration of an appropriate dose of Fdb and IFN-α in combination is beneficial for the treatment of some types of cancer and viral infection.

Characterization of recombinant dengue-2 virus derived from a single nucleotide substitution in the 5' noncoding region.

Variants of wild-type dengue serotype 2 (DEN-2) virus containing nucleotide substitutions at positions 14, 15, or 57 in the 5' NCR were constructed by PCR-mediated site-directed mutagenesis. All three viruses containing a single point substitution demonstrated attenuation phenotype as evidenced by decreases replication and plaque size in cell culture assay. All three variants were less neurovirulent in newborn mice compared to the wild type. The mutants were immunogenic in adult mice immunogenicity and maintained stable replication characteristics following passage in mice. The variant viruses were competent for replication in Aedes aegypi mosquito vector, albeit at lower levels of infection and dissemination in the mosquito than the wild-type Den-2 16681 virus. Although all of the viruses, including the wild type, were found transmissible in mosquito life cycles, they were found subsequentially decreased in efficiency of infection, transmission, and dissemination rates along the mosquito generations and all of them remained genetically stable.

A rice bran phytochemical, cyanidin 3-glucoside, inhibits the progression of PC3 prostate cancer cell.

Prostate cancer is one of the high incidences and the most invasive cancer that is also highly resistant to chemotherapy. Currently, several natural products have been considering using as the supplements for anti-cancer therapy. This study aims to identify the potential active anti-cancer ingredients in the bran extracts of the native Thai rice (Luempua cultivar). Rice bran fraction enriched in anthocyanins was successively isolated and processed until the major purified compound obtained. The sub-fractions and the purified, rice bran, cyanidin 3-glucoside (RBC3G), were studied for biological effects (cell viability, migration, and invasion assays) on human prostatic cancer (PC3) cells using immunohistochemical-staining and immuno-blotting approaches. The sub-fractions and the purified RBC3G inhibited epithelial mesenchymal transition (EMT) characteristics of PC3 cells by blocking the expression of several cytoskeletal associate proteins in a concentration dependent manner, leading to decreasing of the cancer cell motility. RBC3G reduced the expression of Smad/Snail signaling molecules but enhanced the expression of cell surface protein, E-cadherin, leading to a delay tumor transformation. The RBC3G also inhibited matrix metalloproteinase-9 and nuclear factor-kappa B expression levels and the enzymes activity in PC3 cells, leading to a slow cell migration/invasion process. The results suggested that RBC3G blunt and/or delay the progressive cancer cell behaviors by inhibit EMT through Smad signaling pathway(s) mediating Snail/E-cadherin expression.

Anti-Tumor and Immune Enhancing Activities of Rice Bran Gramisterol on Acute Myelogenous Leukemia.

BACKGROUND: Acute myelogenous leukemia (AML) is a cancer of the blood that most commonly affects human adults. The specific cause of AML is unclear, but it induces abnormality of white blood cells that grow rapidly and accumulate in bone marrow interfering with the production and functions of the normal blood cells. AML patients face poor prognosis and low quality of life during chemotherapy or transplantation of hematopoietic stem cells due to the progressive impairment of their immune system. The goal of this study is to find natural products that have the potential to delay growth or eliminate the abnormal leukemic cells but cause less harmful effect to the body's immune system. METHODS AND FINDINGS: The unsaponified fraction of Riceberry rice bran (RBDS) and the main pure compound, gramisterol, were studied for cytotoxicity and biological activities in WEHI-3 cells and in the leukemic mouse model induced by transplantation of WEHI-3 cells intraperitoneally. In the in vitro assay, RBDS and gramisterol exerted sub-G1 phase cell cycle arrest with a potent induction of apoptosis. Both of them effectively decreased cell cycle controlling proteins (cyclin D1 and cyclin E), suppressed cellular DNA synthesis and mitotic division, and reduced anti-apoptosis Bcl-2 protein, but increased apoptotic proteins (p53 and Bax) and activated caspase-3 enzyme in the intrinsic cell death stimulation pathway. In leukemic mice, daily feeding of RBDS significantly increased the amount of immune function-related cells including CD3+, CD19+, and CD11b+, and elevated the serum levels of IFN-γ, TNF-α, IL-2, and IL-12ß cytokines, but suppressed IL-10 level. At the tumor sites, CD11b+ cells were polarized and became active phagocytotic cells. Treatment of mice normal immune cells with gramisterol alone or a combination of gramisterol with cytokines released from RBDS-treated leukemic mice splenocytes culture synergistically increased pSTAT1 transcriptional factor that up-regulated the genes controlling cell survival and function. Phosphorylation of STAT1 was absent in WEHI-3. Instead, similar treatments significantly decreased pSTAT3 signaling that regulates transcription of genes controlling tumor growth and proliferation. CONCLUSIONS: Rice bran gramisterol possesses a promising anti-cancer effect against a tumor of white blood cells and induces the production of anti-cancer immune-related cytokines. Gramisterol induces cell cycle arrest and apoptosis via suppression of pSTAT3 signaling control of tumor cells' growth and progression. Gramisterol increased IFN-γ production and prevented the dysfunctional immune system of leukemic mice by enhancing pSTAT1 transcription signal controlling proliferation and functions of hematopoietic cells in the spleen. Together with IFN-γ, gramisterol efficiently facilitates leukemic mice immune system modulation leading to improvement of the AML condition. Administration of RBDS containing gramisterol potentiates immune recovery of leukemic mice and extends their survival. This finding encourages the medicinal application of rice bran gramisterol as a palliative treatment or an alternative agent for future drug development against AML.

Focal adhesion kinase and Src phosphorylations in HGF-induced proliferation and invasion of human cholangiocarcinoma cell line, HuCCA-1.

AIM: To study the role of focal adhesion kinase (FAK) and its association with Src in hepatocyte growth factor (HGF)-induced cell signaling in cholangiocarcinoma progression. METHODS: Previously isolated HuCCA-1 cells were re-characterized by immunofluorescent staining and reverse transcriptase-polymerase chain reaction assay for the expression of cytokeratin 19, HGF and c-Met mRNA. Cultured HuCCA-1 cells were treated with HGF and determined for cell proliferation and invasion effects by MTT and invasion assays. Western blotting, immunoprecipitation, and co-immunoprecipitation were also performed to study the phosphorylation and interaction of FAK and Src. A novel Src inhibitor (AZM555130) was applied in cultures to investigate the effects on FAK phosphorylation inhibition and on cell proliferation and invasion. RESULTS: HGF enhanced HuCCA-1 cell proliferation and invasion by mediating FAK and Src phosphorylations. FAK-Src interaction occurred in a time-dependent manner that Src was proved to be an upstream signaling molecule to FAK. The inhibitor to Src decreased FAK phosphorylation level in correlation with the reduction of cell proliferation and invasion. CONCLUSION: FAK plays a significant role in signaling pathway of HGF-responsive cell line derived from cholangiocarcinoma. Autophosphorylated Src, induced by HGF, mediates Src kinase activation, which subsequently phosphorylates its substrate, FAK, and signals to cell proliferation and invasion.

Receptor-binding cancer antigen expressed on SiSo cells can be detected in metastatic lymph nodes from gastrointestinal cancers.

AIM: To investigate the expression of receptor-binding cancer antigen expressed on SiSo cells (RCAS1) in metastatic lymph nodes from gastrointestinal cancer. METHODS: Metastatic lymph nodes from gastrointestinal cancer were detected for RCAS1 by immunohistochemical staining and mRNA in situ hybridization. RESULTS: A total of 102 metastatic lymph nodes from bile duct, gastric, colon and pancreatic cancer were investigated for RCAS1 expression. The immunoreactivity of RCAS1 was identified in 100% of metastatic lymph nodes. Both local and distant metastatic lymph nodes showed RCAS1 expression. On the contrary, specimens of non-cancerous lymph nodes were negative for RCAS1. The result of mRNA in situ hybridization was also confirmed by the finding of immunohistochemical staining. RCAS1 mRNA was detected in all tumor cells that metastasized to lymph nodes. CONCLUSION: All metastatic lymph nodes express RCAS1 in tumor cells at both mRNA levels, and RCAS1 that should be used as a complementary factor for identification of metastatic lymph nodes from gastrointestinal cancers.

Different Responses in MMP/TIMP Expression of U937 and HepG2 Cells to Dengue Virus Infection.

Disease severities following dengue virus (DV) infection are the result of increased vascular permeability leading to hypovolemic shock. Matrix metalloproteinases (MMPs) are believed to play a key role in promoting such severities. A previous study reported that supernatants of DV-infected dendritic cells (DCs), which contained high levels of MMP-2 and MMP-9, induced vascular leakage in a mouse model. In the present study, we investigated whether hepatocytes (HepG2) and monocytes (U937) could be additional sources of MMPs during DV infection. HepG2 and U937 cells were exposed to DV serotype 2 strain 16681. The secretion of MMP-2 and MMP-9 was detected using gelatin zymography. We found that DV infection in the HepG2 cells promoted MMP-2 production while that in the U937 cells promoted MMP-9 production. Semi-quantitative RT-PCR results also confirmed that DV infection in the HepG2 cells up-regulated the expression of MMP-2 mRNA, whereas that in the U937 cells enhanced the expression of MMP-9 mRNA. We monitored the expression of endogenous TIMP-1 and TIMP-2. DV infection induced TIMP-1 expression in the U937 cells. However, lower expression of TIMP-2 was observed in the infected HepG2 cells. We believed that following DV infection, monocytes and hepatocytes can act as MMP-9 and MMP-2 producers, respectively. Their responses could be attributed to the disturbance of TIMP expression by DV in different cell types.

Structures of phytosterols and triterpenoids with potential anti-cancer activity in bran of black non-glutinous rice.

Structures of some bioactive phytochemicals in bran extract of the black rice cv. Riceberry that had demonstrated anti-cancer activity in leukemic cell line were investigated. After saponification with potassium hydroxide, separation of the unsaponified fraction by reversed-phase high performance liquid chromatography (HPLC) resulted in four sub-fractions that had a certain degree of anti-proliferation against a mouse leukemic cell line (WEHI-3 cell), this being IC50 at 24 h ranging between 2.80-467.11 µg/mL. Further purification of the bioactive substances contained in these four sub-fractions was performed by normal-phase HPLC. Structural characterization by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) resulted in, overall, the structures of seven phytosterols and four triterpenoids. Four phytosterols, 24-methylene-ergosta-5-en-3ß-ol, 24-methylene-ergosta-7-en-3ß-ol, fucosterol, and gramisterol, along with three triterpenoids, cycloeucalenol, lupenone, and lupeol, were found in the two sub-fractions that showed strong anti-leukemic cell proliferation (IC50 = 2.80 and 32.89 µg/mL). The other sterols and triterpenoids were campesterol, stigmasterol, ß-sitosterol and 24-methylenecycloartanol. Together with the data from in vitro biological analysis, we suggest that gramisterol is a significant anti-cancer lead compound in Riceberry bran extract.

No expression of human leukocyte antigen G (HLA-G) in colorectal cancer cells.

Although there is a specific antitumor immune response in the body, colorectal cancer cells progressively develop. This fact indicated that the cancer cells could have a variety of mechanisms to evade or escape the immune system. HLA-G is identified to inhibit the recognition of NK-cell in various kinds of cancers. This study investigated the expression of HLA-G in colorectal cancer. Eighty five specimens of colorectal cancer, carcinoma in situ and adenomatous polyp were examined by immunohistochemistry and RT-PCR for the detection of human leukocyte antigen (HLA)-G The expression of HLA-G was not found in all colorectal specimens (85/85) both protein level and transcription level, suggesting that the expression of HLA-G is not a possible immune evasion mechanism of colorectal cancer cell.

Melatonin attenuates cisplatin-induced HepG2 cell death via the regulation of mTOR and ERCC1 expressions.

AIM: To elucidate the effects of melatonin on cisplatin-induced hepatocellular carcinoma (HepG2) cell death and to identify potential cross-talk pathways. METHODS: Hepatocellular carcinoma HepG2 cells were treated with melatonin and/or cisplatin for 24 to 48 h. Cell viability and the 50% cytotoxic concentration (CC50) were calculated by MTT assays. The effects and intracellular events induced by the selected concentrations of melatonin (1 mmol/L) and cisplatin (20 µmol/L) were investigated. Cell death and survival detection were primarily evaluated using a fluorescence microscope to assess 4',6 diamideno-2-phenylindol DNA staining and acridine orange lysosome staining and then further analyzed with immunocytochemistry using an anti-LC3 antibody. The potential molecular responses mediated by melatonin against cisplatin after the combined treatment were investigated by reverse transcription-polymerase chains reaction and Western blot analyses of the genes and proteins associated with cell survival and death. A cell cycle analysis was performed using a flow cytometry assay. RESULTS: Melatonin had a concentration-dependent effect on HepG2 cell viability. At 1 mmol/L, melatonin significantly increased the cell viability percentage and decreased reactive oxygen species production due to cisplatin. Melatonin reduced cisplatin-induced cell death, decreasing phosphorylated p53 apoptotic protein, cleaved caspase 3 and Bax levels but increasing anti-apoptotic Bcl-2 gene and protein expression. When combined with cisplatin, melatonin induced S phase (DNA synthesis) cell cycle arrest and promoted autophagic events in HepG2 cells. Melatonin also had a concentration-dependent effect on Beclin-1 and its autophagic regulator mammalian target of rapamycin (mTOR) as well as the DNA excision repair cross complementary 1 (ERCC1) protein. The expression levels of these proteins were altered in HepG2 cells during cisplatin or melatonin treatment alone. In the combination treatment, melatonin reversed the effects of cisplatin by suppressing the over-expression of mTOR and ERCC 1 and enhancing the expression levels of Beclin-1 and microtubule-associated protein-light chain3-II, leading to intracellular autophagosome progression. CONCLUSION: Melatonin attenuated cisplatin-induced cell death in HepG2 cells via a counter-balance between the roles of apoptotic- and autophagy-related proteins.

Long-term supplementation of high pigmented rice bran oil (Oryza sativa L.) on amelioration of oxidative stress and histological changes in streptozotocin-induced diabetic rats fed a high fat diet; Riceberry bran oil.

Diabetes is a serious health problem. Searching for alternative natural antioxidants is considered important strategy to manage diabetes. This study evaluated the effect of Riceberry bran oil (RBBO) supplementation on oxidative stress and organ histology in streptozotocin-induced diabetic rats fed a high fat (HF) diet. Adult male Sprague-Dawley rats with hyperglycemia were divided into four groups: DM group fed a HF diet alone; DMRL group fed a HF diet and 5% RBBO; DMRM group fed a HF diet and 7.5% RBBO; DMRH group fed a HF diet and 15% RBBO. Normal rats were used as normal control and were divided into NC and NR group fed a normal diet containing either 5% corn oil or 5% RBBO, respectively. After 12 weeks, RBBO significantly decreased malondialdehyde and restored superoxide dismutase, catalase, glutathione peroxidase, coenzyme Q(10) and ORAC levels in diabetic rats. RBBO additionally improved the regenerative changes of the pancreas, kidneys, heart and liver. These findings indicate that pigmented RBBO could provide beneficial effect on diabetes by decreasing oxidative stress and recovering organ histology.

Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication.

Insertion of green fluorescent protein (GFP) encoding-gene into virus genes has provided a valuable tool for flavivirus research. This study aimed to develop dengue virus (DENV) replicons expressing GFP reporter that would provide a fast in vitro system to analyze functional roles of specific DENV sequences in viral replication. Two classes of recombinant replicon constructs were generated; one was a RNA-launched replicon with a GFP gene directly inserted into a full-length DENV genome (FL-DENV/GFP), and the other consisted of 4 types of DNA-launched DENV subgenomic replicons with GFP replacement at various structural genes (Δ-DENV/GFP). The FL-DENV/GFP resulted in GFP expression in transfected cells with no viable DENV being recovered from the transfection. The Δ-DENV/GFP constructs with partial structural gene deletion (ΔC-, ΔCprM/M-, ΔprM/M-, or ΔE-) expressed bright and long lasting GFP. The GFP expression intensity in living cells correlated well with the level of RNA replication. Various mutations in the 5'noncoding region of DENV-2 previously shown to be important genetic determinants for virus replication and mouse virulence were incorporated into the 5 different replicon constructs. Characterizations of 29 mutants demonstrated that these replicons can provide a useful platform for a quick and powerful in vitro system to analyze genetic determinants of DENV replication. These constructs can also be useful for development of vectors expressing foreign genes for various researches.

Evaluation of genotoxic and anti-mutagenic properties of cleistanthin A and cleistanthoside A tetraacetate.

Cleistanthin A (CleinA) and cleistanthoside A (CleisA) isolated from plant Phyllanthus taxodiifolius Beille have previously shown potent anticancer effects. To promote their medicinal benefits, CleisA was modified to cleistanthoside A tetraacetate (CleisTA) and evaluated for genotoxic and anti-mutagenic properties in comparison with CleinA. Both compounds showed no significant mutagenic activity to S. typhimulium bacteria and no cytotoxic effect to normal mammalian cells. The non genotoxic effect of CleinA was further confirmed by un-alteration of cytokinesis-block proliferation index (CBPI) and micronucleus (MN) frequency assays in Chinese hamster lung fibroblast (V79) cells, and of CleisTA was confirmed by un-changes of human peripheral blood lymphocytes (HPBL) chromosomal structure assay. Moreover, the metabolic form of CleinA efficiently demonstrated cytostasis effect to V79 cell and prevented mutagen induced Salmonella TA98 and TA100 reversion, whereas both metabolic and non-metabolic forms of CleisTA reduced HPBL mitotic index (%M.I) in a concentration-dependent relationship. The results support CleinA and CleisTA as the new lead compounds for anti-cancer drug development.

Pharmacological activity of Kaempferia parviflora extract against human bile duct cancer cell lines.

A crude ethanol extract of Kaemperia parviflora Wall. Ex Baker and a purified compound, 5,7,4-trimethoxyflavone (KP.8.10), were evaluated for pharmacological effects on human cholangiocarcinoma cell lines (HuCCA-1 and RMCCA-1). The cells were incubated with various concentrations of extract for various time periods and metabolic activity (MTT assay) was assessed for cell viability. The results showed a dose-dependent effect of both crude ethanol extract and the pure compound. CC50s for the crude extract on HuCCA-1 and RMCCA-1 cells were 46.1 microg/ml and 62.0 microg/ml, respectively. Values for the pure compound could not be determined because of solubility problems. Interestingly, K. parviflora ethanol extract and KP.8.10 at low concentrations (10-20 microg/ml and 2.5-5 microg/ml, respectively) markedly reduced rhHGF-induced invasion by HuCCA-1 and RMCCA-1 cells across matrix-coated transwell plates. Higher concentrations of K. parviflora ethanol extract (60 and 80 microg/ml) and KP.8.10 (20 microg /ml) dramatically changed the cellular morphology and caused death in both cell types. KP.8.10 further exhibited progressive action via caspase-3 mitochondrial enzyme activation, enhancing cellular toxicity in a time-dose dependent fashion. Therefore, 5,7,4-trimethoxyflavone appeared to be a bioactive component of K. parviflora extract capable of exerting anti-cancer action. The results suggested a benefit of this edible plant in prevention and treatment of cholangiocarcinoma.

Estrogen-related fecundity reduction of Lymnaea ollula following Fasciola gigantica infection.

Infection by trematode parasites generally affects life history traits of their intermediate hosts. Reduction in life expectancy and reproductive capacity have previously been documented in Fasciola gigantica-infected Lymnaea ollula, but the influence of the endocrine system on this specific host­parasite interaction has not been previously examined. In the present study, we observed survival, growth pattern, and reproductive output of L. ollula following exposure to F. gigantica. Both the survival and the growth pattern of infected snails were similar to those of the non-infected control group. However, a significant difference was apparent in fecundity, as infected snails consistently showed lower levels of egg and embryo production throughout a 7-wk observation period. Semi-quantitative RT-PCR also revealed down-regulation of estrogen receptor expression in infected snails during the first 4 wk of infection. Nonetheless, the inhibition of estrogen signaling was transient, as they regained expression in the later phase of infection. It is, therefore, suggested that other hormones of the complex endocrine system may be involved in the reduced fecundity of L. ollula following F. gigantica infection.

Substitution or deletion mutations between nt 54 and 70 in the 5' non-coding region of dengue type 2 virus produce variable effects on virus viability.

A C57U nucleotide mutation in a predicted RNA stem structure (nt 11-16/56-61) of the 5' non-coding region (5'NCR) of dengue 2 (DEN-2) 16681 virus is partially attenuating, but unstable during serial passage of certain candidate DEN-2 PDK-53-based vaccine viruses containing this mutation. Here, 11 different mutations (one or more point substitution and/or deletion) between nt 54 and 70 in the 5'NCR of the pD2/IC-30P-A (16681) infectious clone are described. Four mutants were infectious. Three mutants with single point substitutions replicated well in cell culture and exhibited variable neurovirulence in mice. Constructs containing multiple substitutions or any deletions failed to produce infectious viruses. Unexpectedly, a double C57U+G58C mutant replicated as efficiently as D2/IC-30P-A virus, and was more neurovirulent for newborn ICR mice. Thus, despite its predicted additional disruption of the RNA stem structure, the engineered contiguous secondary G58C mutation caused reversion of the partially attenuated phenotype caused by the 5'NCR-C57U mutation.

Dengue-virus-infected dendritic cells trigger vascular leakage through metalloproteinase overproduction.

Dengue virus (DV) is an important re-emerging arthropod-borne virus of global significance. The defining characteristic of DV infection-associated pathology is haemorrhagic fever, which often leads to a fatal shock-like syndrome (DHF/DSS) owing to an increase in vascular endothelial permeability. Here, we show, in a viral dose-dependent manner, that DV-infected immature dendritic cells overproduce soluble gelatinolytic matrix metalloproteinase (MMP)-9-and to a lesser extent MMP-2-which enhances endothelial permeability, but which are reduced by specific inhibitors and a neutralizing anti-MMP-9 antibody. This permeability was associated with a loss of expression of the platelet endothelial adhesion molecule 1 (PECAM-1) and vascular endothelium (VE)-cadherin cell adhesion molecules and redistribution of F-actin fibres. These in vitro observations were confirmed in an in vivo vascular-leakage mouse model. These results provide a molecular basis for DHF/DSS that could be a basis for a general model of haemorrhagic fever-inducing viruses, and identify a new therapeutic approach for the treatment of viral-induced vascular leakage by specifically targeting gelatinolytic metalloproteases.