Detection of annexin A autoantibodies in sera from colorectal cancer patients.

BACKGROUND: Annexins (ANXAs) belong to a superfamily of closely related calcium and membrane-binding proteins and are overexpressed in some carcinomas. The overexpression of ANXAs presumably induces an autoantibody response, but this has not been demonstrated for sera from colorectal cancer (CRC) patients. STUDY: We examined serum samples from 220 CRC patients and 216 healthy volunteers to evaluate the ANXA autoantibody response in patients by using an enzyme-linked immunosorbent assay with recombinant ANXA A4 as the antigen. RESULTS: The sensitivity of the anti-ANXA response from CRC patient sera was about 85% and the specificity was about 61.6%. When a cut-off value of 5.0 ng/mL was chosen for carcinoembryonic antigen (CEA) in the same sera samples, the sensitivity and specificity values were 43.2% and 85.2%, respectively. Combined detection using ANXA autoantibodies and CEA produced better sensitivity (71.8%) and specificity (79.2%) compared with CEA sensitivity (43.2%) and anti-ANXA specificity (61.6%). The area under a receiver operating characteristic curve was 0.794 for ANXA autoantibodies, 0.666 for CEA, and 0.84 for both markers together. Importantly, in comparison to CEA (27.5% seropositivity), ANXA autoantibody showed a remarkable change (81.3% seropositivity) at the early stage of CRC. CONCLUSIONS: Measurement of ANXA autoantibody levels may provide an alternative detection indicator for CRC, particularly among early-stage patients.

Identification of phospholipid scramblase 1 as a biomarker and determination of its prognostic value for colorectal cancer.

The purpose of this study was to examine the expression of phospholipid scramblase 1 (PLSCR1) in tumor tissues and plasma specimens of patients with colorectal cancer (CRC), as well as analyze its association with clinical parameters. The expression levels of PLSCR1 protein in 104 matched CRC and adjacent normal tissue sections and 50 pairs of CRC tissue blocks were determined by use of immunohistochemical and Western blot analyses, respectively. To evaluate the diagnostic potential of PLSCR1, the plasma levels of PLSCR1 were investigated in 111 additional subjects (59 CRC patients and 52 healthy controls) by Western blot. PLSCR1 was overexpressed in malignant adenocarcinoma tissues compared with normal colorectal mucosa (P < 0.001). In addition, the plasma level of PLSCR1 was not only significantly elevated in CRC patients compared with healthy individuals (P < 0.001), but it was also substantially increased in early stage CRC (P < 0.001). Importantly, the overall sensitivity and specificity of PLSCR1 for CRC detection were 80% and 59.6%, respectively. The area under the ROC curve of PLSCR1 for CRC diagnosis is 0.75, which increases to 0.8 if combined with the measurement of carcinoembryonic antigen. Univariate analysis with the Cox regression model revealed that elevated PLSCR1 expression indicated a poor prognosis for CRC. This study showed that PLSCR1 protein levels were significantly elevated in both the cancer tissue and plasma of CRC patients. Moreover, the plasma levels of PLSCR1 were significantly elevated in patients with early stage CRC compared with healthy individuals, suggesting that PLSCR1 might be used as a noninvasive serological diagnostic and prognostic biomarker for CRC.

Comparison of membrane fraction proteomic profiles of normal and cancerous human colorectal tissues with gel-assisted digestion and iTRAQ labeling mass spectrometry.

The aim of this study was to uncover the membrane protein profile differences between colorectal carcinoma and neighboring normal mucosa from colorectal cancer patients. Information from cellular membrane proteomes can be used not only to study the roles of membrane proteins in fundamental biological processes, but also to discover novel targets for improving the management of colorectal cancer patients. We used solvent extraction and a gel-assisted digestion method, together with isobaric tags with related and absolute quantitation (iTRAQ) reagents to label tumoral and adjacent normal tissues in a pairwise manner (n = 8). For high-throughput quantification, these digested labeled peptides were combined and simultaneously analyzed using LC-MS/MS. Using the shotgun approach, we identified a total of 438 distinct proteins from membrane fractions of all eight patients. After comparing protein expression between cancerous and corresponding normal tissue, we identified 34 upregulated and eight downregulated proteins with expression changes greater than twofold (Student's t-test, P < 0.05). Among these, the overexpression of well-established biomarkers such as carcinoembryonic antigens (CEACAM5, CEACAM6), as well as claudin-3, HLA class I histocompatibility antigen A-1, tapasin and mitochondrial solute carrier family 25A4 were confirmed by western blotting. We conclude that gel-assisted digestion and iTRAQ labeling MS is a potential approach for uncovering and comparing membrane protein profiles of tissue samples that has the potential to identify novel biomarkers.

Multiple serological biomarkers for colorectal cancer detection.

The aim of this study was to initiate a survey of human autoantibody responses to a panel of select colorectal tumor-associated antigens identified by previous serological analysis of a cDNA expression library and to subsequently identify multiple serological biomarkers for the detection of colorectal cancer. For screening of autoantibodies against colorectal tumor-associated antigens, sera from 94 colorectal cancer patients and 54 normal controls were analyzed by enzyme-linked immunosorbent assay using recombinant rCCCAP, rHDAC5, rP53, rNMDAR and rNY-CO-16 proteins as coating antigens. Seropositivity among colorectal cancer patients to the 5 individual coating antigens varied from 18.1% to 35.1%. Seropositivity to any of the 5 coating antigens was 58.5% and combining this analysis with evaluation of serum carcinoembryonic antigen (> or =5 ng/ml) significantly increased the seropositivity to 77.6%. Seropositivity of early-stage (Dukes' Stages A and B) colorectal cancer patients to CEA was 21.9%, and seropositivity to any of the 5 colorectal cancer-associated antigens was 53.7%, and the combination of these 2 measurements resulted in a higher diagnostic capacity (65.9%) than either marker alone. In conclusion, these results collectively indicated that combined detection of serum autoantibody profiles against our panel of colorectal tumor-associated antigens and the analysis of carcinoembryonic antigen provides a promising diagnostic biomarker for colorectal cancer, particularly among early-stage patients.

(-)-Anonaine induces apoptosis through Bax- and caspase-dependent pathways in human cervical cancer (HeLa) cells.

(-)-Anonaine has been shown to have some anticancer activities, but the mechanisms of (-)-anonaine inducing cell death of human cancer cells is not fully understood. We investigated the mechanisms of apoptosis induced by (-)-anonaine in human HeLa cancer cells. Treatment with (-)-anonaine induces dose-dependent DNA damage that is correlated with increased intracellular nitric oxide, reactive oxygen species, glutathione depletion, disruptive mitochondrial transmembrane potential, activation of caspase 3, 7, 8, and 9, and poly ADP ribose polymerase cleavage. Our data indicate that (-)-anonaine up-regulated the expression of Bax and p53 proteins in HeLa cancer cells. The apoptosis and expression of Bax induced by (-)-anonaine could be inhibited when the HeLa cells were pretreated with Boc-Asp(OMe)-fmk, which is a broad caspases inhibitor. There was no obvious DNA damage in the (-)-anonaine-treated Madin-Darby canine kidney and Vero cell lines. Both Madin-Darby canine kidney and Vero cell lines are kidney epithelial cellular morphology. These results suggest that (-)-anonaine might be considered a potent compound for chemotherapy against cervical cancer or a health food supplement for cancer chemoprevention.

The anticancer effect of protein-extract from Bidens alba in human colorectal carcinoma SW480 cells via the reactive oxidative species- and glutathione depletion-dependent apoptosis.

Bidens alba has been used for healing cuts, injuries, swellings, hypertension, jaundice, and diabetes in some countries. However, the effect of B. alba on human cancer remains poorly understood. The goal of this study was to investigate whether B. alba protein-extract could have an anticancer property against human colorectal cancer. The human colorectal cancer SW 480 cells treated with the protein-extract of B. alba would cause marked DNA damages and apoptosis-related cellular morphologies. Treatment with 225 microg/ml B. alba protein-extract also led to the SW480 cells to produce readily intracellular reactive oxygen species (ROS) after 1h of treatment and last to 24 h. The intracellular glutathione (GSH) depletion occurred after 12-24h of treatment. The treatment of the protein-extract would also caused mitochondrial transmembrane potential (DeltaPsi(m)) to decrease and cytosolic cytochrome c to increase. The caspase 3/7 activities were activated from 3 to 6 h after the treatment. The percentages of apoptosis induced by the protein-extract of B. alba decreased 26.4%, 10.1%, and 29.4% when the SW 480 cells were pretreated with Vitamin C, N-acetylcysteine, and Boc-Asp(OMe)-fmk, respectively. Taken together, we demonstrated for the first time that the protein-extract of B. alba could induce apoptosis that was related to the ROS production and GSH depletion in human colorectal cancer. The protein-extract of B. alba might have therapeutic value against the human colorectal cancer.