A Novel Multidrug Combination Mitigates Rat Liver Steatosis Through Activating AMPK Pathway During Normothermic Machine Perfusion.

BACKGROUND: Hepatic steatosis is now the leading cause of liver discards in deceased donors. Previous studies [Yarmush formula (Y) defatting] have successfully reduced the fat content by treating rat steatotic livers on extracorporeal normothermic machine perfusion (NMP) with a multidrug combination including the GW compounds that were linked to an increased risk of carcinogenesis. METHODS: We developed a novel multidrug combination by replacing the GW compounds with 2 polyphenols, epigallocatechin-3-gallate (E) and resveratrol (R). Sixteen rat livers were placed on NMP and assigned to control, Y defatting, Y + E + R defatting, or Y'-GW + E + R defatting groups (Y'-GW = 90% dose-reduced Y defatting, n = 4/group). RESULTS: All livers in defatting groups had significant decreases in hepatic triglyceride content at the end of the experiment. However, livers treated with our novel Y'-GW + E + R combination had evidence of increased metabolism and less hepatocyte damage and carcinogenic potential. Our Y'-GW + E + R combination had increased phosphorylation of AMP-activated protein kinase (P = 0.019) and acetyl-CoA carboxylase (P = 0.023) compared with control; these were not increased in Y + E + R group and actually decreased in the Y group. Furthermore, the Y'-GW + E + R group had less evidence of carcinogenic potential with no increase in AKT phosphorylation compared with control (P = 0.089); the Y (P = 0.031) and Y + E + R (P = 0.035) groups had striking increases in AKT phosphorylation. Finally, our Y'-GW + E + R showed less evidence of hepatocyte damage with significantly lower perfusate alanine aminotransferase (P = 0.007) and aspartate aminotransferase (P = 0.014) levels. CONCLUSIONS: We have developed a novel multidrug combination demonstrating promising defatting efficacy via activation of the AMP-activated protein kinase pathway with an optimized safety profile and reduced hepatotoxicity during ex vivo NMP.

Disrupting cholesterol esterification by bitter melon suppresses triple-negative breast cancer cell growth.

Triple negative breast cancer (TNBC) is aggressive with a worse prognosis. We have recently shown that bitter melon extract (BME) treatment was more effective in inhibition of TNBC tumor growth in mouse models as compared to ER positive breast tumor growth. Aberrant dysregulation of lipid metabolism is associated with breast cancer progression, however, anti-cancer mechanism of BME linking lipid metabolism in breast cancer growth remains unexplored. Here, we observed that accumulation of esterified cholesterol was reduced in BME treated TNBC cell lines as compared to control cells. We next evaluated expression levels of acyl-CoA: cholesterol acyltransferase 1 (ACAT-1) in TNBC cells treated with BME. Our results demonstrated that BME treatment inhibited ACAT-1 expression in TNBC cells. Subsequently, we found that sterol regulatory element-binding proteins-1 and -2, and FASN was significantly reduced in BME treated TNBC cell lines. Low-density lipoprotein receptor was also downregulated in BME treated TNBC cells as compared to control cells. We further demonstrated that BME feeding reduced tumor growth in TNBC mammospheres implanted into NSG mice, and inhibits ACAT-1 expression. To our knowledge, this is the first report demonstrating BME suppresses TNBC cell growth through ACAT-1 inhibition, and have potential for additional therapeutic regimen against human breast cancer.

Shikonin Induces Apoptotic Cell Death via Regulation of p53 and Nrf2 in AGS Human Stomach Carcinoma Cells.

Shikonin, which derives from Lithospermum erythrorhizon, has been traditionally used against a variety of diseases, including cancer, in Eastern Asia. Here we determined that shikonin inhibits proliferation of gastric cancer cells by inducing apoptosis. Shikonin's biological activity was validated by observing cell viability, caspase 3 activity, reactive oxygen species (ROS) generation, and apoptotic marker expressions in AGS stomach cancer cells. The concentration range of shikonin was 35-250 nM with the incubation time of 6 h. Protein levels of Nrf2 and p53 were evaluated by western blotting and confirmed by real-time PCR. Our results revealed that shikonin induced the generation of ROS as well as caspase 3-dependent apoptosis. c-Jun-N-terminal kinases (JNK) activity was significantly elevated in shikonin-treated cells, thereby linking JNK to apoptosis. Furthermore, our results revealed that shikonin induced p53 expression but repressed Nrf2 expression. Moreover, our results suggested that there may be a co-regulation between p53 and Nrf2, in which transfection with siNrf2 induced the p53 expression. We demonstrated for the first time that shikonin activated cell apoptosis in AGS cells via caspase 3- and JNK-dependent pathways, as well as through the p53-Nrf2 mediated signal pathway. Our study validates in partly the contribution of shikonin as a new therapeutic approaches/ agent for cancer chemotherapy.

Chemopreventive Properties of Genipin on AGS Cell Line via Induction of JNK/Nrf2/ARE Signaling Pathway.

Roles of dietary phytochemicals in cancer chemoprevention via induction of nuclear factor-erythroid-2-related factor 2 (Nrf2)-mediated antioxidant enzymes have been well established in a number of studies. In this study, FACS analysis was used to reveal that the intracellular reactive oxygen species level decreased at 0-25 µM of genipin treatment. Furthermore, immunofluorescence analysis and Western blotting were used to demonstrate that genipin treatment resulted in the upregulation and nuclear translocation of Nrf2, as well as upregulation of gastrointestinal glutathione peroxidase. Finally, we found that C-Jun-NH2-kinase (JNK) was also dose-dependently activated, where depleting JNK by using a biochemical inhibitor indicated that JNK was upstream of Nrf2. Interestingly, the antioxidant effects were limited to the treatment in the lower dosage of genipin, where higher dosage of genipin treatment resulted in the increased reactive oxygen species level and cytotoxicity. Thus, this study demonstrates for the first time that lower dosage of genipin results in the induction of JNK/Nrf2/ARE signaling pathway and protection from cell death.

Induction of apoptosis by genipin inhibits cell proliferation in AGS human gastric cancer cells via Egr1/p21 signaling pathway.

Natural compounds are becoming important candidates in cancer therapy due to their cytotoxic effects on cancer cells by inducing various types of programmed cell deaths. In this study, we investigated whether genipin induces programmed cell deaths and mediates in Egr1/p21 signaling pathways in gastric cancer cells. Effects of genipin in AGS cancer cell lines were observed via evaluation of cell viability, ROS generation, cell cycle arrest, and protein and RNA levels of p21, Egr1, as well as apoptotic marker genes. The cell viability of AGS cells reduced by genipin treatment via induction of the caspase 3-dependent apoptosis. Cell cycle arrest was observed at the G2/M phase along with induction of p21 and p21-dependent cyclins. As an upstream mediator of p21, the transcription factor early growth response-1 (Egr1) upregulated p21 through nuclear translocation and binding to the p21 promoter site. Silencing Egr1 expression inhibited the expression of p21 and downstream molecules involved in apoptosis. We demonstrated that genipin treatment in AGS human gastric cancer cell line induces apoptosis via p53-independent Egr1/p21 signaling pathway in a dose-dependent manner.

Shikonin induces cell cycle arrest in human gastric cancer (AGS) by early growth response 1 (Egr1)-mediated p21 gene expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Lithospermum erythrorhizon, a naphthoquinone compound derived from a shikonin, has long been used as traditional Chinese medicine for treatment of various diseases, including cancer. To evaluate the cytotoxic effects of shikonin on AGS gastric cancer cells via induction of cell cycle arrest. MATERIALS AND METHODS: We observed the effects of 12.5-100 ng/mL dosage of shikonin treatment on AGS cancer cell line with the incubation time of 6h. Cytotoxic effects were assessed by measuring the changes in the intracellular ROS, appearance of senescence phenotype, cell cycle progression, CDK and cyclins expression levels upon shikonin treatment. We also examined upon the activation of Egr1-mediated p21 expression, by siRNA transfection, Luciferase assay, and ChIP assay. RESULTS: In this study, we found that shikonin inhibits cell proliferation by arresting cell cycle progression at the G2/M phase via modulation of p21 in AGS cells. Also, our results revealed that the p21 gene was transactivated by early growth response1 (Egr1) in response to the shikonin treatment. Transient Egr1 expression enhanced shikonin-induced p21 promoter activity, whereas the suppression of Egr1 expression by small interfering RNA attenuated the ability of shikonin to induce p21 promoter activity. CONCLUSION: Our results suggested that the anti-proliferative activity of shikonin was due to its ability to induce cell cycle arrest via Egr1-p21 signaling pathway. Thus, the work stated here validates the traditional use of shikonin in the treatment of cancer.

Hantaan virus surveillance targeting small mammals at Dagmar North Training Area, Gyeonggi Province, Republic of Korea, 2001-2005.

In response to a hemorrhagic fever with renal syndrome case in November 2000, a seasonal rodent-borne disease surveillance program was initiated at Dagmar North Training Area (DNTA), Gyeonggi Province, Republic of Korea. From April 2001-December 2005, 1,848 small mammals were captured. Apodemus agrarius accounted for 92.5%, followed by Mus musculus (3.6%), Crocidura lasiura (2.1%), and Microtus fortis (1.1%). Three species of rodents were found to be antibody-positive (Ab+) for Hantaan virus (HTNV): A. agrarius (22.3%), M. musculus (9.1%), and M. fortis (5.0%). Ab+ rates for A. agrarius increased with increasing weight (age), except for those weighing <10 g. The peak HTNV transmission period in Korea coincided with the peak reproductive potential of A. agrarius during the fall (August/September) surveys. HTNV strains from DNTA were distinct from HTNV strains from the People's Republic of China. From these studies, more accurate risk assessments can be developed to better protect personnel from rodent-borne diseases.

Comparison of innate immune responses to pathogenic and putative non-pathogenic hantaviruses in vitro.

Hantaviruses are human pathogens that cause hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. The mechanisms accounting for the differences in virulence between pathogenic and non-pathogenic hantaviruses are not well known. We have examined the pathogenesis of different hantavirus groups by comparing the innate immune responses induced in the host cell following infection by pathogenic (Sin Nombre, Hantaan, and Seoul virus) and putative non-pathogenic (Prospect Hill, Tula, and Thottapalayam virus) hantaviruses. Pathogenic hantaviruses were found to replicate more efficiently in interferon-competent A549 cells than putative non-pathogenic hantaviruses. The former also suppressed the expression of the interferon-ß and myxovirus resistance protein genes, while the transcription level of both genes increased rapidly within 24 h post-infection in the latter. In addition, the induction level of interferon correlated with the activation level of interferon regulatory factor-3. Taken together, these results suggest that the observed differences are correlated with viral pathogenesis and further indicate that pathogenic and putative non-pathogenic hantaviruses differ in terms of early interferon induction via activation of the interferon regulatory factor-3 in infected host cells.

Small-molecule probe using dual signals to monitor leucine aminopeptidase activity.

Leucine aminopeptidases (LAPs) are widely distributed in organisms from bacteria to humans, and play crucial roles in cell maintenance and cell growth. Thus, assays for LAP are necessary for measuring its activity and inhibitor potency. In this Letter, we report a small-molecule probe which exhibits colorimetric and fluorogenic changes according to LAP activity.

Combined omics analysis identifies transmembrane 4 L6 family member 1 as a surface protein marker specific to human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are promising for cell therapy and regenerative medicine; but their lack of specific markers renders the cell culture at potential contamination risk with other cell types, in particular, fibroblasts. In this study, we mapped 2 differential transcriptome data of MSCs compared, one to mononuclear cells and the other to fibroblasts, onto the membrane proteome data, the analysis of which led to an identification of transmembrane 4 L6 family member 1 (TM4SF1) as a surface protein marker candidate that could discriminate MSCs simultaneously from blood cells and fibroblasts. Our analyses confirmed that TM4SF1 was abundantly expressed on MSCs but neither on other blood/tissue cells nor on fibroblasts. TM4SF1 immunoselection from bone marrow and adipose tissues yielded homogeneous cell populations that were highly similar to MSCs, in terms of morphology, immunophenotype, and differentiation potential. These findings indicate that TM4SF1 can serve as a surface protein marker which singly identifies MSCs from diverse cell sources, in particular, fibroblast-rich connective tissues.

Serological surveillance of scrub typhus, murine typhus, and leptospirosis in small mammals captured at Twin Bridges Training Area, Gyeonggi Province, Republic of Korea, 2005-2007.

Soldiers from the Republic of Korea and the United States conduct armistice military operations at Twin Bridges Training Area (TBTA) located near the demilitarized zone (DMZ) and are exposed to zoonotic disease pathogens that small mammals and their potentially disease-carrying ectoparasites transmit. TBTA is a 36 km2 rural training site with small villages and various forms of agriculture along its boundary. At TBTA, rodents, insectivores, and their ectoparasites are commonly found in association with unmanaged habitats of various densities of tall grasses, herbaceous plants, shrubs, briars, and crawling vegetation. Rodents and insectivores were collected during the winter (November-December 2005 and December 2006) and early spring (March 2007), and serologically tested for the presence of scrub typhus, murine typhus, and leptospirosis antibodies. Of the six species of small mammals collected, Apodemus agrarius, the common striped field mouse and known reservoir of scrub typhus, was the most frequently collected (96.1%), followed by Crocidura lasiura (2.5%), Micromys minutus (0.5%), Myodes regulus (0.5%), Mus musculus (0.3%), and Rattus rattus (0.1%). A. agrarius (56.1%), M. musculus (66.7%), M. minutus (25%), and R. rattus (100%) were positive for scrub typhus antibodies. Only A. agrarius (14.7%) and C. lasiura (4.5%) were positive for murine typhus antibodies, whereas only A. agrarius (1.5%) was seropositive for leptospirosis. Seroprevalence rates of scrub typhus and murine typhus based on weight and sex of A. agrarius are presented.

Serological surveillance of scrub typhus, murine typhus, and leptospirosis in small mammals captured at firing points 10 and 60, Gyeonggi province, Republic of Korea, 2001-2005.

Soldiers from the Republic of Korea and the United States conducting peacetime military operations at various training sites and multiple range complexes located near the demilitarized zone separating North and South Korea are exposed to rodents and their potentially disease-carrying ectoparasites. These diseases include scrub typhus, murine typhus, and leptospirosis. Many of the training sites are rural or semi-rural, surrounded or co-located with various forms of agriculture, and are infested with rodents and insectivores (as well as their ectoparasites), which are commonly found in association with unmanaged tall grasses, scrub, and crawling vegetation habitats. For 5 years, rodents and insectivores were collected seasonally (spring, summer, fall, and winter) at firing points 10 and 60 near the demilitarized zone and serologically tested for the presence of scrub typhus, murine typhus, and leptospirosis antibodies. Of the nine species of small mammals collected, Apodemus agrarius, the common striped field mouse and known reservoir of scrub typhus, was the most frequently collected (90.6%). Only four of the nine species captured, A. agrarius (60.9%), Micromys minutus (100%), Mus musculus (55.6%), and Rattus norvegicus (46.7%), were positive for scrub typhus. Of all the small mammals captured, only A. agrarius was positive for murine typhus (0.3%) and leptospirosis (1.3%). Seasonal and annual prevalence rates based on weight and sex are presented.

Purification and characterization of a biosurfactant produced by Pseudomonas sp. G11 by asymmetrical flow field-flow fractionation (AsFlFFF).

Three hundred and thirty two bacterial colonies were isolated from soil contaminated by an oil spill. All the bacteria were cultured in a liquid medium individually, and the surface tensions of the media were compared. The bacterium whose culture medium had the lowest surface tension was identified as Pseudomonas sp. G11. A biosurfactant was produced by cultivation of the Pseudomonas sp. G11 in the LB media. For extraction of the biosurfactant, two solvent systems were used (n-hexane and a 2:1 (v/v) mixture of chloroform/MeOH), and the results were compared. Various experimental conditions (solvent composition, flow rate, etc.) were tested to optimize the analysis of the biosurfactant by asymmetrical flow field-flow fractionation (AsFlFFF). The biosurfactant was successfully separated from the culture medium by AsFlFFF when pure water was used as the carrier. From the retention data, the hydrodynamic diameter (dH) and molecular weight (M) of the biosurfactant were determined by AsFlFFF. The molecular weight was determined by using pullulans as the calibration standards. The dH and M were 49 nm and 2.3 x 10(5) Da when extracted with n-hexane, and 39 nm and 1.13 x 10(5) Da when extracted with the 2:1 mixture of chloroform/MeOH, respectively.