Label-free quantitative proteomics reveals aberrant expression levels of LRG, C9, FN, A1AT and AGP1 in the plasma of patients with colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the major causes of cancer-related death worldwide. Although commercial biomarkers of CRC are currently available, they are still lacking in terms of sensitivity and specificity; thus, searching for reliable blood-based biomarkers are important for the primary screening of CRC. METHODS: Plasma samples of patients with non-metastatic (NM) and metastatic (M) CRC and healthy controls were fractionated using MARS-14 immunoaffinity chromatography. The flow-through and elute fractions representing low- and high-abundant proteins, respectively, were analyzed by label-free quantitative proteomics mass spectrometry. The functional analysis of the proteins with greater than 1.5-fold differential expression level between the CRC and the healthy control groups were analyzed for their biological processes and molecular functions. In addition, the levels of plasma proteins showing large alterations in CRC patients were confirmed by immunoblotting using two independent cohorts. Moreover, receiver operating characteristic (ROC) curve analysis was performed for individual and combinations of biomarker candidates so as to evaluate the diagnostic performance of biomarker candidates. RESULTS: From 163 refined identifications, five proteins were up-regulated and two proteins were down-regulated in NM-CRC while eight proteins were up-regulated and three proteins were down-regulated in M-CRC, respectively. Altered plasma proteins in NM-CRC were mainly involved in complement activation, while those in M-CRC were clustered in acute-phase response, complement activation, and inflammatory response. Results from the study- and validation-cohorts indicate that the levels of leucine-rich alpha-2-glycoprotein-1(LRG), complement component C9 (C9), alpha-1-acid glycoprotein 1 (AGP1), and alpha-1-antitrypsin (A1AT) were statistically increased, while fibronectin (FN) level was statistically decreased in CRC patients compared to healthy controls, with most alterations found in a metastatic stage-dependent manner. ROC analysis revealed that FN exhibited the best diagnostic performance to discriminate CRC patients and healthy controls while AGP1 showed the best discrimination between the disease stages in both cohorts. The combined biomarker candidates, FN + A1AT + AGP1, exhibited perfect discriminatory power to discriminate between the CRC population and healthy controls whereas LRG + A1AT + AGP1 was likely to be the best panel to discriminate the metastatic stages in both cohorts. CONCLUSIONS: This study identified and quantified distinct plasma proteome profiles of CRC patients. Selected CRC biomarker candidates including FN, LRG, C9, A1AT, and AGP1 may be further applied for screening larger cohorts including disease groups from other types of cancer or other diseases.

Protein A/G-based immunochromatographic test for serodiagnosis of pythiosis in human and animal subjects from Asia and Americas.

Pythiosis is a life-threatening infectious disease of both humans and animals living in Asia, Americas, Africa, and parts of Australia and New Zealand. The etiologic pathogen is the fungus-like organism Pythium insidiosum The disease has high mortality and morbidity rates. Use of antifungal drugs are ineffective against P. insidiosum, leaving radical surgery the main treatment option. Prompt treatment leads to better prognosis of affected individuals, and could be achieved by early and accurate diagnosis. Since pythiosis has been increasingly reported worldwide, there is a need for a rapid, user-friendly, and efficient test that facilitates the diagnosis of the disease. This study aims to develop an immunochromatographic test (ICT), using the bacterial protein A/G, to detect anti-P. insidiosum IgGs in humans and animals, and compare its diagnostic performance with the established ELISA. Eighty-five serum samples from 28 patients, 24 dogs, 12 horses, 12 rabbits, and 9 cattle with pythiosis, and 143 serum samples from 80 human and 63 animal subjects in a healthy condition, with thalassemia, or with other fungal infections, were recruited for assay evaluation. Detection specificities of ELISA and ICT were 100.0%. While the detection sensitivity of ELISA was 98.8%, that of ICT was 90.6%. Most pythiosis sera, that were falsely read negative by ICT, were weakly positive by ELISA. In conclusion, a protein A/G-based ICT is a rapid, user-friendly, and efficient assay for serodiagnosis of pythiosis in humans and animals. Compared to ELISA, ICT has an equivalent detection specificity and a slightly lower detection sensitivity.

p­STAT3 influences doxorubicin and etoposide resistance of A549 cells grown in an in vitro 3D culture model.

Tumor microenvironment undoubtedly has a significant impact on therapeutic responses. Abundant evidence suggests that the 3D in vitro culture holds great promise for drug discovery and development by bridging the gap between conventional 2D culture and animal models. The present study described 3D basement membrane culture of A549 cells, which mimics the complex 3D arrangement of tumors in vivo and elucidates the underlying mechanisms of microenvironmental influences on cellular functions and therapeutic efficacy. A549 cells cultured in 3D undergo G0/G1 phase arrest and decreased migratory and invasive capacity, indicating dormant cell characteristics. Hypoxia, apoptosis and stemness were demonstrated in the A549 cells in 3D architecture compared with the 2D­cultured counterparts. More importantly, cells in the 3D environment exhibited increased resistance to different classes of anticancer agents. Western blotting revealed changes in the levels of key cancer­associated pathways, phosphorylated (p)­STAT3, p­ERK, and p­Akt, in response to 3D culture compared with 2D monolayer culture. Notably, mechanistic analysis using specific inhibitors showed that the STAT3 inhibitor overcomes the 3D culture­induced doxorubicin and etoposide resistance. These results implicated an important role of p­STAT3 in conferring chemoresistance in 3D­cultured A549 cells, as well as the use of STAT3 inhibitor as a potential chemosensitizer to improve drug sensitivity. Thus, 3D culture systems, that more closely resemble in vivo tumor biology, may be more effective models in searching for novel chemotherapeutic agents and therapeutic targets for cancer treatment.

JAK2/STAT3­mediated dose­dependent cytostatic and cytotoxic effects of sesquiterpene lactones from Gymnanthemum extensum on A549 human lung carcinoma cells.

Due to drug resistance and disease recurrence, lung cancer remains one of the primary cancer­related causes of death in both men and women worldwide. In addition, lung cancer is clinically silent and thus most patients are at an advanced stage at the time of diagnosis. The limited efficiency of current conventional chemotherapies necessitates the search for novel effective anticancer agents. The present study demonstrated the anti­proliferative effect and apoptosis­inducing activity of three sesquiterpene lactones isolated from Gymnanthemum extensum, vernodalin (VDa), vernolepin (VLe) and vernolide (VLi), on A549 human lung cancer cells. Treatment with sub­cytotoxic doses (cell viability remaining >75%) of VDa, VLe and VLi, arrested progression of the A549 cell cycle at the G0/G1 phase, while cytotoxic doses of the three compounds induced G2/M phase arrest and apoptosis. Mechanistic studies revealed that VDa, VLe and VLi may exert their anti­tumor activity through the JAK2/STAT3 pathway. Molecular docking analysis confirmed that VDa, VLe and VLi formed hydrogen bonds with the FERM domain of JAK2 protein. Overall, the present study highlighted the potential therapeutic value of VDa, VLe and VLi to be further developed as anticancer agents for the treatment of lung cancer.

Effects of topical exposure to a mixture of chlorpyrifos, cypermethrin and captan on the hematological and immunological systems in male Wistar rats.

Most Thai orchid farmers heavily used pesticide mixtures, and were shown to have various hematologic/immunologic alterations. The present study investigated the effect of exposure of male Wistar rats to a mixture of three pesticides (chlorpyrifos, cypermethrin and captan) that are most often used by the farmers. Three groups of 10 rats were dermally exposed to three different doses (high, middle and low) for 28 consecutive days. The rats showed significant changes in body, liver, kidneys and adrenals weights. Significant changes were observed in various biological parameters including hematotoxicity (increased leukocyte and platelet counts, percent neutrophil, decreased RBC count, percent lymphocyte and eosinophil), hepatotoxicity (increased serum AST, decreased serum ALP, cholesterol, triglyceride, serum protein and albumin), and immunotoxicity (decrease in numbers of NK cells, decrease splenic proliferative response to LPS, and increase in serum IgG). These results confirm the potential health danger of exposure to these pesticide mixtures in orchid farmers.

Effect of pesticide exposure on immunological, hematological and biochemical parameters in thai orchid farmers- a cross-sectional study.

Various studies have found that many Thai orchid farmers used excessive amounts of pesticides without proper protective gear, but no toxicological study has been made. This cross-sectional study aimed to evaluate the immunological, hematological and biochemical statuses of these farmers. Sixty four orchid farmers and 60 controls were studied. Plasma cholinesterase activity, the percentage and absolute number of B lymphocytes (CD19+) were significantly lower in the farmers group (3966.32±1165.48 U/L, 11.61±4.09% and 312.26±164.83 cells/mm3, respectively) as compared to those of controls (5048.85±1139.40 U/L, 14.32±4.23%, 420.34±195.18 cells/mm3, respectively). There was a statistically significant higher level of serum IgE among the orchid farmers (0.031±0.011 mg/dL vs. 0.018±0.007 mg/dL) but not IgG, IgA and IgM, levels. Serum lysozyme level, lymphocyte proliferative responses to mitogens, hematological parameters and kidney function test, were not significantly different between the two groups. The liver function profiles showed significantly lower levels of albumin and serum protein in the farmer group. Thus frequent pesticide exposure resulted in subtle changes of some biological parameters. These changes, though may not be clinically significant, strongly indicated that caution in handing pesticides by these farmers is warranted.