The high FKBP1A expression in WBCs as a potential screening biomarker for pancreatic cancer.

Given the limitation of current routine approaches for pancreatic cancer screening and detection, the mortality rate of pancreatic cancer cases is still critical. The development of blood-based molecular biomarkers for pancreatic cancer screening and early detection which provide less-invasive, high-sensitivity, and cost-effective, is urgently needed. The goal of this study is to identify and validate the potential molecular biomarkers in white blood cells (WBCs) of pancreatic cancer patients. Gene expression profiles of pancreatic cancer patients from NCBI GEO database were analyzed by CU-DREAM. Then, mRNA expression levels of three candidate genes were determined by quantitative RT-PCR in WBCs of pancreatic cancer patients (N = 27) and healthy controls (N = 51). ROC analysis was performed to assess the performance of each candidate gene. A total of 29 upregulated genes were identified and three selected genes were performed gene expression analysis. Our results revealed high mRNA expression levels in WBCs of pancreatic cancer patients in all selected genes, including FKBP1A (p < 0.0001), PLD1 (p < 0.0001), and PSMA4 (p = 0.0002). Among candidate genes, FKBP1A mRNA expression level was remarkably increased in the pancreatic cancer samples and also in the early stage (p < 0.0001). Moreover, FKBP1A showed the greatest performance to discriminate patients with pancreatic cancer from healthy individuals than other genes with the 88.9% sensitivity, 84.3% specificity, and 90.1% accuracy. Our findings demonstrated that the alteration of FKBP1A gene in WBCs serves as a novel valuable biomarker for patients with pancreatic cancer. Detection of FKBP1A mRNA expression level in circulating WBCs, providing high-sensitive, less-invasive, and cost-effective, is simple and feasible for routine clinical setting that can be applied for pancreatic cancer screening and early detection.

New Phenolic Glycosides from Coelogyne fuscescens Lindl. var. brunnea and Their Cytotoxicity against Human Breast Cancer Cells.

The phytochemical investigation of the whole plants of Coelogyne fuscescens Lindl. var. brunnea led to the discovery of three new phenolic glycosides, i.e., coelofusides A-C (1-3) and 12 known compounds (4-15). For the first time, we reported the nuclear magnetic resonance (NMR) data of 4-O-(6'-O-glucosyl-4″-hydroxybenzoyl)-4-hydroxybenzyl alcohol (4) in this study. The identification of the structures of newly discovered compounds was done through the analysis of their spectroscopic data [NMR, mass spectrometry, ultraviolet, Fourier transform infrared, optical rotation, and circular dichroism (CD)]. In comparison to anticancer drugs (i.e., etoposide and carboplatin), we evaluated anticancer potential of the isolated compounds on two different breast cancer cell lines, namely, T47D and MDA-MB-231. Human fibroblast HaCaT cells were used as the control cells. After a 48 h incubation, flavidin (8), coelonin (10), 3,4-dihydroxybenzaldehyde (11), and oxoflavidin (12) showed significant cytotoxic effects against breast cancer cells. Among them, oxoflavidin (12) exhibited the most potent cytotoxicity on MDA-MB-231 with an IC50 value of 26.26 ± 4.33 µM. In the nuclear staining assay, oxoflavidin induced apoptosis after 48 h in both T47D and MDA-MB-231 cells in a dose-dependent manner. Furthermore, oxoflavidin upregulated the expression of apoptotic genes, such as p53, Bax, poly(ADP-ribose) polymerase, caspase-3, and caspase-9 genes while significantly decreasing antiapoptotic protein (Bcl-2) expression levels.

Changes in immune cell subtypes during ageing.

BACKGROUND: The immune system comprises many different types of cells, each with different functions and properties during immune defence. The numbers and types of immune cells in the circulation is highly dynamic and regulated by infections, ageing and certain types of cancers. It is recognised that immune function decreases during ageing, but the biological age at which these functional changes occur is variable, and how ageing affects the different sub-types of lymphocytes, monocytes and NK cells in the circulation is not fully defined. METHODS: In this study, we recruited 24 healthy volunteers over the age range of 23y to 89y and measured the numbers of different subclasses of circulating cells by immuno-phenotyping and flow cytometry. RESULTS: We show increased monocyte:lymphocyte ratios in a > 50y cohort and most T cell subsets were decreased, except for CD4+ cells, which were increased in this cohort. In addition, there was NK cell expansion and increased HLA-DR+ T cells, but decreased numbers of classical monocytes and increased numbers of CD4+ monocytes in this >50y cohort. CONCLUSIONS: These data indicate that healthy ageing is associated with changes in both the major cell groups but also individual subclasses of cells, and these are likely to result from continuous immune challenge and impaired development.

Breast Cancer Sera Changes in Alu Element Methylation Predict Metastatic Disease Progression.

BACKGROUND/AIM: During metastatic disease development, the cancer-immune system crosstalk induces epigenetic modifications to immune cells, impairing their functions. Recently, Alu elements methylation changes were widely studied in terms of early cancer detection. This study aimed to demonstrate in vitro Alu element methylation changes in peripheral immune cells in a metastatic setting and examine their prognostic values in metastatic breast cancer. MATERIALS AND METHODS: Sera from sixteen metastatic cancer patients and sixteen healthy participants were obtained and used to culture normal peripheral immune cells. After 48 h of incubation, the percentage and pattern of Alu element methylation were examined for clinical relevance. RESULTS: We found that the Alu element hypomethylation was affected by age in the cancer group. Intriguingly, a decrease in Alu element methylation was found in patients with early progressive disease. Moreover, an increase in unmethylated cytosine (mCuC) loci was related to the poorer prognosis group. Accordingly, the decrease in Alu element methylation and the increase in mCuC loci pattern in peripheral immune cells correlated with poorer prognosis and early progression in metastatic breast cancer. CONCLUSION: Alu element hypomethylation in immune cells and their increased mCuC foci were related to the early progression of breast cancer. These warrant the use of Alu element methylation changes for diagnostic and therapeutic purposes in breast cancer.

Upregulation of p16INK4A in Peripheral White Blood Cells as a Novel Screening Marker for Colorectal Carcinoma.

OBJECTIVE: Screening of colorectal cancer (CRC) is important for the early detection. CRC is relating to aging and immuno-senescence. One such senescent marker is p16INK4A expression in immune cells. The objective of the study is to investigate the protein expression of p16INK4A in peripheral white blood cells as a screening marker for colorectal cancer. METHODS: A case-control studies were conducted. Cases were patients with colorectal cancer and controls were matched with cases based on age and sex. Peripheral blood was collected from patients and controls and the protein p16INK4A was measured with immunofluorescent techniques. The p16INK4A levels from cases and controls were evaluated using ROC analysis to be used as a screening marker in CRC patients. Mean fluorescent intensity of p16INK4A of cases and controls were analyzed in CD45+, CD3+ or CD14+ cells. The p16INK4A levels of cases were also correlated with clinical data. RESULT: Statistically significant increased expression of p16INK4A levels were found in cases compared to controls. p16INK4A in peripheral immune cells had 78% sensitivity and 71% specificity which can possibly be used as a diagnosis tool for colorectal cancer. P16INK4A-positive cell percentage and mean florescent intensity were significantly higher in CD45+ cells, CD3 positive cells and CD14 positive cells. No significant correlation was observed with the clinical data and p16INK4A level of CRC patients. CONCLUSION: The significant increase of p16 INK4A expression level in peripheral immune cells represents potential for use as a CRC screening marker.

The roles of HMGB1-produced DNA gaps in DNA protection and aging biomarker reversal.

The endogenous DNA damage triggering an aging progression in the elderly is prevented in the youth, probably by naturally occurring DNA gaps. Decreased DNA gaps are found during chronological aging in yeast. So we named the gaps "Youth-DNA-GAPs." The gaps are hidden by histone deacetylation to prevent DNA break response and were also reduced in cells lacking either the high-mobility group box (HMGB) or the NAD-dependent histone deacetylase, SIR2. A reduction in DNA gaps results in shearing DNA strands and decreasing cell viability. Here, we show the roles of DNA gaps in genomic stability and aging prevention in mammals. The number of Youth-DNA-GAPs were low in senescent cells, two aging rat models, and the elderly. Box A domain of HMGB1 acts as molecular scissors in producing DNA gaps. Increased gaps consolidated DNA durability, leading to DNA protection and improved aging features in senescent cells and two aging rat models similar to those of young organisms. Like the naturally occurring Youth-DNA-GAPs, Box A-produced DNA gaps avoided DNA double-strand break response by histone deacetylation and SIRT1, a Sir2 homolog. In conclusion, Youth-DNA-GAPs are a biomarker determining the DNA aging stage (young/old). Box A-produced DNA gaps ultimately reverse aging features. Therefore, DNA gap formation is a potential strategy to monitor and treat aging-associated diseases.

The expression of FLNA and CLU in PBMCs as a novel screening marker for hepatocellular carcinoma.

Early detection improves survival and increases curative probability in hepatocellular carcinoma (HCC). Peripheral blood mononuclear cells (PBMCs) can provide an inexpensive, less-invasive and highly accurate method. The objective of this study is to find the potential marker for HCC screening, utilizing gene expression of the PBMCs. Data from the NCBI GEO database of gene expression in HCC patients and healthy donor's PBMCs was collected. As a result, GSE 49515 and GSE 58208 were found. Using both, a statistical significance test was conducted in each gene expression of each data set which resulted in 187 genes. We randomized three selected genes (FLNA, CAP1, and CLU) from the significant p-value group (p-values < 0.001). Then, a total of 76 healthy donors, 153 HCC, 20 hepatic fibrosis, 20 non-alcoholic fatty liver were collected. Quantitative RT-PCR (qRT-PCR) was performed in cDNA from all blood samples from the qRT-PCR, The Cycle threshold (Ct) value of FLNA, CLU, CAP1 of HCC group (28.47 ± 4.43, 28.01 ± 3.75, 29.64 ± 3.90) were lower than healthy group (34.23 ± 3.54, 32.90 ± 4.15, 32.18 ± 5.02) (p-values < 0.0001). The accuracy, sensitivity and specificity of these genes as a screening tool were: FLNA (80.8%, 88.0%, 65.8%), CLU (63.4%, 93.3%, 31.3%), CAP1 (67.2%, 83.3%, 39.1%). The tests were performed in two and three gene combinations. Results demonstrated high accuracy of 86.2%, sensitivity of 85% and specificity of 88.4% in the FLNA and CLU combination. Furthermore, after analyzed using hepatic fibrosis and non-alcoholic fatty liver as a control, the FLNA and CLU combination is shown to have accuracy of 76.9%, sensitivity of 77.6% and specificity of 75%. Also, we founded that our gene combination performs better than the current gold standard for HCC screening. We concluded that FLNA and CLU combination have high potential for being HCC novel markers. Combined with current tumor markers, further research of the gene's expression might help identify more potential markers and improve diagnosis methods.

Quantitative STAU2 measurement in lymphocytes for breast cancer risk assessment.

Although mammograms play a key role in early breast cancer detection, the test is not applicable to all women, for example, women under the age of 40. The development of a noninvasive blood test with high sensitivity and accessibility will improve the effectiveness of breast cancer screening programmes. Secretory factors released from cancer cells can induce the expression of certain genes in a large number of white blood cells (WBCs). Therefore, cancer-dependent proteins in WBCs can be used as tumour markers with high sensitivity. Five proteins (LMAN1, AZI2, STAU2, MMP9 and PLOD1) from a systemic analysis of a variety of array data of breast cancer patients were subjected to immunofluorescence staining to evaluate the presence of fixed WBCs on 96-well plates from 363 healthy females and 358 female breast cancer patients. The results revealed that the average fluorescence intensity of anti-STAU2 and the percentage of STAU2-positive T and B lymphocytes in breast cancer patients (110.50 ± 23.38 and 61.87 ± 12.44, respectively) were significantly increased compared with those in healthy females (56.47 ± 32.03 and 33.02 ± 18.10, respectively) (p = 3.56 × 10-71, odds ratio = 24.59, 95% CI = 16.64-36.34). The effect of secreted molecules from breast cancer cells was proven by the increase in STAU2 intensity in PBMCs cocultured with MCF-7 and T47D cells at 48 h (p = 0.0289). The test demonstrated 98.32%, 82.96%, and 48.32% sensitivity and 56.47%, 83.47%, and 98.62% specificity in correlation with the percentage of STAU2-positive cells at 40, 53.34 and 63.38, respectively. We also demonstrated how to use the STAU2 test for the assessment of risk in women under the age of 40. STAU2 is a novel breast cancer marker that can be assessed by quantitative immunofluorescence staining of fixed WBCs that are transportable at room temperature via mail, representing a useful risk assessment tool for women without access to mammograms.

Tumor-induced DNA methylation in the white blood cells of patients with colorectal cancer.

The secretions of cancer cells alter epigenetic regulation in cancer stromal cells. The present study investigated the methylation changes in white blood cells (WBCs) caused by the secretions of colorectal cancer (CRC) cells. Changes in the DNA methylation of peripheral blood mononuclear cells (PBMCs) from normal individuals co-cultured with CRC cells were estimated using a methylation microarray. These changes were then compared against the DNA methylation changes and mRNA levels observed in the WBCs of patients with CRC. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) and matrix metalloproteinase 9 (MMP9) were selected to assess the DNA methylation of the WBCs from CRC patients using real-time methylation-specific PCR. The majority of the genes analyzed presented high levels of mRNA in the WBCs of the patients with CRC and DNA methylation in the co-cultured PBMCs. Intragenic methylation revealed the strongest association (P=8.52×10-21). For validation, MMP9 and PLOD1 were selected and used to test WBCs from 32 patients with CRC and 57 normal controls. The intragenic MMP9 methylation was commonly found (P<0.0001) with high sensitivity (90.63%) and high specificity (96.49%), and a positive predictive value of 93.33% and a negative predictive value of 93.22%. PLOD1 methylation was revealed to have lower sensitivity (30.00%) but higher specificity (97.92%). In addition to circulating WBCs, MMP9 protein expression was observed in infiltrating WBCs and the metastatic lymph nodes of patients with CRC. In conclusion, CRC cells secrete factors that induce genome wide DNA methylation changes in the WBCs of patients with CRC. These changes, including intragenic MMP9 methylation in WBCs, are promising CRC biomarkers to be tested in future CRC screening studies.

Breast cancer upregulating genes in stromal cells by LINE-1 hypermethylation and micrometastatic detection.

UNLABELLED: Changes in the methylation level of genes containing LINE-1 alter host gene regulation. AIM: This study demonstrates that paracrine signaling of breast cancer influences the epigenetic regulation of stromal cells. METHODS: We proved in vitro and in vivo breast cancer promoted LINE-1 methylation exists exclusively in female stromal cells. RESULTS: Genes containing LINE-1 of breast cancer stromal cells were upregulated. Furthermore, one of the genes, MUC-1, was demonstrated to have expression in plasma cells from the lymph nodes of patients with lymph node metastasis or micrometastasis. CONCLUSION: Breast cancer sends a paracrine signal to stroma cells causing LINE-1 epigenetic regulation. Moreover, the regulated genes in stroma cells are potential biomarkers for detecting breast cancer micrometastasis.

Alu methylation in serum from patients with nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a common cancer in Southern China and Southeast Asia. Alu elements are among the most prevalent repetitive sequences and constitute 11% of the human genome. Although Alu methylation has been evaluated in many types of cancer, few studies have examined the levels of this modification in serum from NPC patients. OBJECTIVE: To compare the Alu methylation levels and patterns between serum from NPC patients and normal controls. MATERIALS AND METHODS: Sera from 50 NPC patients and 140 controls were examined. Quantitative combined bisulfite restriction analysis-Alu (qCOBRA-Alu) was applied to measure Alu methylation levels and characterize Alu methylation patterns. Amplified products were classified into four patterns according to the methylation status of 2 CpG sites: hypermethylated (methylation at both loci), partially methylated (methylation of either of the two loci), and hypomethylated (unmethylated at both loci). RESULTS: A comparison of normal control sera with NPC sera revealed that the latter presented a significantly lower methylation level (p=0.0002) and a significantly higher percentage of hypomethylated loci (p=0.0002). The sensitivity of the higher percentage of Alu hypomethyted loci for distinguishing NPC patients from normal controls was 96%. CONCLUSIONS: Alu elements in the circulating DNA of NPC patients are hypomethylated. Moreover, Alu hypomethylated loci may represent a potential biomarker for NPC screening.

Alu hypomethylation in smoke-exposed epithelia and oral squamous carcinoma.

BACKGROUND: Alu elements are one of the most common repetitive sequences that now constitute more than 10% of the human genome and potential targets for epigenetic alterations. Correspondingly, methylation of these elements can result in a genome-wide event that may have an impact in cancer. However, studies investigating the genome-wide status of Alu methylation in cancer remain limited. OBJECTIVES: Oral squamous cell carcinoma (OSCC) presents with high incidence in South-East Asia and thus the aim of this study was to evaluate the Alu methylation status in OSCCs and explore with the possibility of using this information for diagnostic screening. We evaluated Alu methylation status in a) normal oral mucosa compared to OSCC; b) peripheral blood mononuclear cells (PBMCs) of normal controls comparing to oral cancer patients; c) among oral epithelium of normal controls, smokers and oral cancer patients. MATERIALS AND METHODS: Alu methylation was detected by combined bisulfite restriction analysis (COBRA) at 2 CpG sites. The amplified products were classified into three patterns; hypermethylation ((m)C(m)C), partial methylation (uC(m)C+(m)C(u)C), and hypomethylation ((u)C(u)C). RESULTS: The results demonstrate that the %(m)C(m)C value is suitable for differentiating normal and cancer in oral tissues (p=0.0002), but is not significantly observe in PBMCs. In addition, a stepwise decrease in this value was observed in the oral epithelium from normal, light smoker, heavy smoker, low stage and high stage OSCC (p=0.0003). Furthermore, receiver operating characteristic (ROC) curve analyses demonstrated the potential of combined %mC or %(m)C(m)C values as markers for oral cancer detection with sensitivity and specificity of 86.7% and 56.7%, respectively. CONCLUSIONS: Alu hypomethylation is likely to be associated with multistep oral carcinogenesis, and might be developed as a screening tool for oral cancer detection.

Synthesis and theoretical study of molecularly imprinted nanospheres for recognition of tocopherols.

Molecular imprinting is a technology that facilitates the production of artificial receptors toward compounds of interest. The molecularly imprinted polymers act as artificial antibodies, artificial receptors, or artificial enzymes with the added benefit over their biological counterparts of being highly durable. In this study, we prepared molecularly imprinted polymers for the purpose of binding specifically to tocopherol (vitamin E) and its derivative, tocopherol acetate. Binding of the imprinted polymers to the template was found to be two times greater than that of the control, non-imprinted polymers, when using only 10 mg of polymers. Optimization of the rebinding solvent indicated that ethanol-water at a molar ratio of 6:4 (v/v) was the best solvent system as it enhanced the rebinding performance of the imprinted polymers toward both tocopherol and tocopherol acetate with a binding capacity of approximately 2 mg/g of polymer. Furthermore, imprinted nanospheres against tocopherol was successfully prepared by precipitation polymerization with ethanol-water at a molar ratio of 8:2 (v/v) as the optimal rebinding solvent. Computer simulation was also performed to provide mechanistic insights on the binding mode of template-monomer complexes. Such polymers show high potential for industrial and medical applications, particularly for selective separation of tocopherol and derivatives.