Risk factor assessment to predict the likelihood of a diagnosis of metastasis for indeterminate hepatic lesions found at computed tomography in patients with rectal cancer.

AIM: To assess the significant factors on rectal magnetic resonance imaging (MRI) to predict the likelihood of a diagnosis of metastasis for indeterminate hepatic lesions found at computed tomography (CT) in patients with rectal cancer. MATERIALS AND METHODS: A total of 207 patients with rectal cancer who underwent preoperative contrast-enhanced abdominopelvic CT, and rectal and liver MRI were included. Univariate analysis and multivariate logistic regression were used to evaluate the determining factors for the significance of indeterminate hepatic lesions on CT in patients with rectal cancer. RESULTS: Hepatic metastases were diagnosed in 29 (20.9%) of 139 patients who had indeterminate hepatic lesions on preoperative CT obtained for rectal cancer. On univariate analysis, carcinoembryonic antigen level, N stage, mesorectal fascia (MRF) invasion, diameter of superior haemorrhoidal vein, and mesorectal vascular lesion (MVL) grade on rectal MRI (p<0.05) were associated with the possibility of metastasis for indeterminate hepatic lesions on CT. On multivariate analysis, MVL grade and MRF invasion on rectal MRI were independent factors associated with the possibility of metastasis for indeterminate hepatic lesions on CT (p<0.0005 and p=0.0066, respectively). CONCLUSION: MVL grade and MRF invasion on rectal MRI are independent factors for estimating hepatic metastasis among indeterminate hepatic lesions on CT in patients with rectal cancer.

Epigenetic modification of Nrf2 in 5-fluorouracil-resistant colon cancer cells: involvement of TET-dependent DNA demethylation.

5-Fluorouracil (5-FU) is a widely used anticancer drug for the treatment of colorectal cancer (CRC). However, resistance to 5-FU often prevents the success of chemotherapy. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcriptional regulator and a possible target to overcome 5-FU resistance. The present study examined epigenetic changes associated with Nrf2 induction in a human CRC cell line (SNUC5) resistant to 5-FU (SNUC5/5-FUR). Nrf2 expression, nuclear translocation, and binding to promoter were higher in SNUC5/5-FUR cells than in SNUC5 cells. The activated Nrf2 in SNUC5/5-FUR cells led to an increase in the protein expression and activity of heme oxygenase-1 (HO-1), an Nrf2-regulated gene. SNUC5/5-FUR cells produced a larger amount of reactive oxygen species (ROS) than SNUC5 cells. The siRNA- or shRNA-mediated knockdown of Nrf2 or HO-1 significantly suppressed cancer cell viability and tumor growth in vitro and in vivo, resulting in enhanced 5-FU sensitivity. Methylation-specific (MS) or real-time quantitative MS-PCR data showed hypomethylation of the Nrf2 promoter CpG islands in SNUC5/5-FUR cells compared with SNUC5 cells. Expression of the DNA demethylase ten-eleven translocation (TET) was upregulated in SNUC5/5-FUR cells. ROS generated by 5-FU upregulated TET1 expression and function, whereas antioxidant had the opposite effect. These results suggested that the mechanism underlying the acquisition of 5-FU resistance in CRC involves the upregulation of Nrf2 and HO-1 expression via epigenetic modifications of DNA demethylation.

A ginseng metabolite, compound K, induces autophagy and apoptosis via generation of reactive oxygen species and activation of JNK in human colon cancer cells.

Compound K (20-O-(ß-D-glucopyranosyl)-20(S)-protopanaxadiol) is an active metabolite of ginsenosides and induces apoptosis in various types of cancer cells. This study investigated the role of autophagy in compound K-induced cell death of human HCT-116 colon cancer cells. Compound K activated an autophagy pathway characterized by the accumulation of vesicles, the increased positive acridine orange-stained cells, the accumulation of LC3-II, and the elevation of autophagic flux. Whereas blockade of compound K-induced autophagy by 3-methyladenein and bafilomycin A1 significantly increased cell viability. In addition, compound K augmented the time-dependent expression of the autophagy-related proteins Atg5, Atg6, and Atg7. However, knockdown of Atg5, Atg6, and Atg7 markedly inhibited the detrimental impact of compound K on LC3-II accumulation and cell vitality. Compound K-provoked autophagy was also linked to the generation of intracellular reactive oxygen species (ROS); both of these processes were mitigated by the pre-treatment of cells with the antioxidant N-acetylcysteine. Moreover, compound K activated the c-Jun NH2-terminal kinase (JNK) signaling pathway, whereas downregulation of JNK by its specific inhibitor SP600125 or by small interfering RNA against JNK attenuated autophagy-mediated cell death in response to compound K. Compound K also provoked apoptosis, as evidenced by an increased number of apoptotic bodies and sub-G1 hypodiploid cells, enhanced activation of caspase-3 and caspase-9, and modulation of Bcl-2 and Bcl-2-associated X protein expression. Notably, compound K-stimulated autophagy as well as apoptosis was induced by disrupting the interaction between Atg6 and Bcl-2. Taken together, these results indicate that the induction of autophagy and apoptosis by compound K is mediated through ROS generation and JNK activation in human colon cancer cells.

Ginseng total saponin enhances the phagocytic capacity of canine peripheral blood phagocytes in vitro.

The clinical and pharmacological activities of ginseng are known to modulate immune function, metabolic processes and neuro-endocrine system activities. Ginseng saponins are the principle active ingredients in the formation of immune stimulating complexes. The objective of this study was to evaluate the in vitro effect of ginseng total saponin (GTS) on the phagocytic capacity of canine peripheral blood phagocytes. GTS itself did not cause any direct effect on the phagocytic capacity of peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMN) but not peripheral blood monocytes. However, the phagocytic capacity of PMN and monocytes, but not PBMC, was enhanced by the culture supernatant from PBMC treated with GTS. The phagocytic capacity of PMN and monocytes was also increased by treatment with recombinant canine (rc) tumor necrosis factor (TNF)-alpha. The ability of the culture supernatant from GTS-treated PBMC to stimulate the phagocytic capacity of phagocytes was inhibited by addition of anti-rc TNF-alpha polyclonal antibody (pAb) prior to the culture. The amount of TNF-alpha in the culture supernatant from PBMC was shown to increase upon treatment of GTS as compared with that of vehicle-treated PBMC culture supernatant. These results suggest that GTS has an immunoenhancing effect on the phagocytic capacity of canine peripheral blood phagocytes, which is mainly mediated by TNF-alpha released from GTS-stimulated PBMC.

A feasibility study of multispectral image analysis of skin tumors.

To develop a noninvasive, early-detection method for skin cancers, a feasibility study of multispectral image analysis was investigated. The three most frequently occurring skin cancer types, ten basal-cell carcinomas (BCCs), ten squamous-cell carcinomas (SCCs) and five malignant melanomas (MMs) were studied, along with ten normal moles. Images were acquired by a charge-coupled device camera using eight narrow-band filters ranging from 450 nm to 800 nm, at 50-nm intervals. To extract main features of these tumors, principal components analysis (PCA) was performed, because it projects the multidimensional (here, eight-dimensional) data in the direction of maximum data variance. Then, the primary PCA components for red, green, and blue subset images were analyzed in terms of hue-saturation-intensity (HSI). By hue distributions, the BCCs and SCCs were differentiated from the MMs and normal moles. Texture information was used to further classify tumor types after the HSI analysis. The texture analysis, performed using a spatial gray-level co-occurrence matrix (SGCM), could separate MMs from normal moles. The BCCs and SCCs were further studied by Fisher's linear discriminant analysis. Distribution was described as a Gaussian mixture model. By this classification procedure, seven BCCs, eight SCCs, five MMs, and ten NMs were correctly classified. Three BCCs and two SCCs were unseparable. Thus, multispectral skin cancer image analysis has potential to diagnose skin cancers.

Preliminary study of real-time fiber optic based protein C biosensor.

Deficiency of protein C (PC), one of the human body's key anticoagulants, can lead to massive thrombotic complications. There is a diagnostic need to perform real-time assays, in order to quickly identify and treat this disease. An immuno-optical biosensor for the diagnosing of PC deficiencies and monitoring of PC concentrations is being developed for this purpose. Monoclonal antibody against PC (anti-PC) is immobilized on the surface of a tapered quartz fiber that is enclosed in a glass tube (capacity approximately 200 microL). Following sample injection, PC within a sample binds to the anti-PC in a highly specific reaction. The system is then probed with a fluorophore-tagged secondary antibody against PC. Excitation light is applied through the fiber, and the fluorescence intensity is correlated with the PC concentration in the sample. This study presents (1) a feasibility, direct binding assay, (2) a comparison of methods to immobilize anti-PC upon the fiber (direct immobilization vs an avidin-biotin bridge), and (3) effectiveness of an elution step to regenerate the fiber. PC-deficient patients typically have a concentration range less than 2.5 microg/mL. It was found that the sensor could detect PC levels down to 0.1 microg/mL in pure buffer with minimal optimization. Avidin-biotin immobilization of the primary antibody produced enhanced signals, up to 470% of the original intensities. Preliminary fiber regeneration tests achieved nearly a 50% increase in fiber lifetime with the use of a CaCl(2) elution step. Ultimately, further development may lead to automation and the use of the system as a multi-blood factor analyzer.

Preliminary study of biosensor optimization for the detection of protein C.

Methods to develop an immuno-optical biosensor for the detection and monitoring of Protein C (PC) concentrations are described. A tapered quartz fiber is enclosed in a glass tube (capacity approximately 300 microliters) and monoclonal antibody against PC (anti-PC) is immobilized on the surface of this fiber. PC within a sample, when injected into the chamber, will bind to the anti-PC in a specific reaction. The system is then probed with a fluorophore tagged secondary antibody against PC, also binding to PC in a specific reaction. Excitation light is applied through the fiber, and the amount of fluorescence is correlated with the PC concentration in the sample. This study offers encouraging results for the detection of PC deficiency in real-time.

Localization of a fluorescent object in highly scattering media via frequency response analysis of near infrared-time resolved spectroscopy spectra.

Frequency response analysis via pulse testing for engineering systems and near infrared (NIR) time resolved spectroscopy (TRS) for biological system characterization involve identical principles: the system of interest is disturbed by an input pulse and the output response is observed. Since a sharp pulse, such as the Dirac delta function, contains the information of multimodulation frequencies (theoretically from 0 to infinity in frequency) a narrow pulse TRS input can produce a wide range frequency response for identifying any system of interest. Currently used NIR-TRS spectral analyses either fit the spectra with a known theoretical solution or use photon mean time-of-flight. Transforming the system time domain representation to the frequency domain generates five system parameters that can be valuable for process identification utility: steady state gain, time constant, system order, and magnitude ratio and phase shift over a wide frequency range. Optical contrast agents or fluorescent agents can be used to enhance the capability of optical instruments in detecting biological heterogeneities. In this article, magnitude ratio, phase shift, and other system parameters derived from the transfer function of systems with both a fluorescent absorber and a regular absorber are correlated with the position of the absorber. This technique is important in that ultimately it can be used to enhance optical medical imaging.

Application of near-IR time-resolved spectroscopy and frequency response analysis for deep vein thrombosis detection.

Frequency response analysis is applied to analyze NIR-TRS spectra in a tissue model with a simulated thrombus. The value changes in parameters obtained from frequency response analysis are correlated with heterogeneity position in three dimensions. The goal of this research is to noninvasively localize deep vein thrombosis in the human leg through the use of this novel combination.

Feasibility study of a single- and multiple-source near-infrared phase-modulation device for characterizing biologic systems.

Near-infrared (NIR) spectroscopy is an inexpensive and noninvasive optical method to characterize biologic and physiologic systems. Measurements of oxygen saturation in various organs, cytochrome oxidase concentration, and glucose concentration in tissue are examples of NIR spectroscopy applications. This instrument also has potential for diagnostic imaging by comparing the optical properties of a given point in tissue with those of the surrounding areas, e.g., tumor localization, hematoma detection, or deep vein thrombosis detection. Currently, NIR spectroscopy is available in three modalities: time-resolved spectroscopy (TRS), continuous-wave spectroscopy (CWS), and a phase-modulation device. This paper describes a single-source phase-modulation device and a multiple-source (in- and anti-phased) device and their application to system identification. The phase and intensity information for homogeneous systems is correlated with system and instrumental parameters. Phase and intensity changes resulting from the presence of one and two absorbers are illustrated. The sensitivity of in- and anti-phased phase-modulation devices (phased arrays) is demonstrated. The use of amplitude-modulated phased array for heterogeneity detection is described.

Chromatographic process identification for protein C purification using frequency response analysis.

Frequency response analysis is applied for the analysis of liquid chromatography output of protein separation. Reduced data from simple chromatograms suggest that various Bode plot parameters, magnitude ratios, phase shift, the steady state gain, break frequency, and system order in the frequency domain, can be used to gain phenomenological insights on the system. Such an approach is advantageous because the validity of the model can be checked for two plots, the magnitude ratio vs. frequency and the phase shift vs. frequency, as compared to a single plot in the time domain. This approach also provides a useful empirical-tool which can be quantifiably used for process validation and scale-up, especially for immunoaffinity and immobilized metal affinity chromatographic systems used for protein C purification.

Separation of recombinant human protein C from transgenic animal milk using immobilized metal affinity chromatography.

Protein C is an important serine protease due to its ability to proteolytically cleave activated Factors V and VIII. Excess coagulation and blood agglutination can lead to plugged capillaries, thereby reducing oxygen transport to interstitial tissues. To treat patients with hereditary and acquired protein C deficiency would require a greater amount of Protein C than that available from human plasma. However, the potential demand for this protein could be met by the production of human protein C from transgenic animal mammary glands. Thus, research into inexpensive, efficient methods to purify proteins from transgenic animal milk will be a critical area of study for the large scale production of protein C. Immobilized metal affinity chromatography (IMAC) is a novel method for the purification of protein C. A proposed method of purification is to take advantage of protein C's strong metal ion binding characteristics with IMAC to assist in the separation from transgenic animal milk. The separation procedure is benchmarked against current systems in use by the American Red Cross for purification of Protein C from transgenic porcine milk. Common problems in developing separation schemes for new therapeutics are the initial availability of the product (protein), and time-to-market concerns. Extensive experimental tests for scaleable purification schemes are often cost and time prohibitive. In order to optimize an IMAC protocol with minimal waste of time and resources, total quality management tools have been adopted. Initial experiments were designed to choose buffer conditions, eluents, immobilized valence metals, and flow rates using Taguchi experimental design, which is a total quality management (TQM) tool. One of the values of Taguchi methods lies in the use of Latin orthogonal sets. Through the use of the orthogonal sets, the total number of experiments may be reduced, shortening the focus time on optimal conditions.

Reusable, real-time, immuno-optical protein C biosensor.

A Protein C (PC) biosensor can be used to diagnose PC deficiency, to monitor the PC level in the blood of PC deficient patients, and to measure the PC concentration in other PC-containing samples, such as PC producing animal cell culture broth or transgenic animal milk. A fully functional biosensor requires extremely high sensitivity and specificity, and real-time measurement. To satisfy these requirements, it is proposed to develop an immuno-optical fiber biosensor that utilizes PC-specific biomolecules (PC probes) tagged with fluorophores. The method involves immobilizing monoclonal antibody against PC (anti-PC) on the surface of an optical fiber. When PC in a sample is adsorbed to the anti-PC on the fiber, it can be reached with the fluorophore tagged PC-probe. The intensity of light transported through the optical fiber, therefore, can be correlated with the concentration of PC in the sample. The sensor will be designed so it can be reused, following a simple elution step, thus reducing diagnostic expense. The preliminary study shows encouraging future for the real-time optical PC biosensor.