Background: Boron neutron capture therapy (BNCT) selectively kills tumor cells while sparing adjacent normal cells. Boric acid (BA)-mediated BNCT showed therapeutic efficacy in treating hepatocellular carcinoma (HCC) in vivo. However, DNA damage and corresponding responses induced by BA-mediated BNCT remained unclear. This study aimed to investigate whether BA-mediated BNCT induced DNA double-strand breaks (DSBs) and to explore DNA damage responses in vitro. Materials and Methods: Huh7 Human HCC cells were treated with BA and irradiated with neutrons during BA-BNCT. Cell survival and DNA DSBs were examined by clonogenic assay and expression of phosphorylated H2A histone family member X (γH2AX), respectively. The DNA damage response was explored by determining the expression levels of DNA repair- and apoptosis-associated proteins and conducting a cell-cycle analysis. Results: DNA DSBs induced by BA-mediated BNCT were primarily repaired through the homologous recombination pathway. BA-mediated BNCT induced G2/M arrest and apoptosis in HCC. Conclusion: Our findings may enable the identification of radiosensitizers or adjuvant drugs for potentiating the therapeutic effectiveness of BA-mediated BNCT for HCC.

BACKGROUNDFeasibility and efficacy of boric acid (BA)-mediated boron neutron capture therapy (BNCT) was first demonstrated by eliminating hepatocellular carcinoma (HCC) in a rat model. Furthermore, selective uptake of BA by liver tumor cells was shown in a rabbit model. To gain further insight, this study aimed to investigate the mechanisms of transportation and selective uptake of BA in HepG2 liver tumor cells.MATERIALS AND METHODSTransportation of BA in HepG2 cells was analyzed by time-course assays and by analyzing the rate of diffusion versus the concentration of BA. The effect of different tumor conditions on BA uptake was studied by treating HepG2 cells with 25 μg 10B/ml BA under different concentrations of glucose, at different pH and in the presence of water-soluble cholesterol.RESULTSHepG2 cells mainly uptake BA by simple diffusion. Cell membrane permeability may also contribute to tumor-specific uptake of BA.CONCLUSIONThe selective uptake of BA was achieved primarily by diffusion, while other factors, such as low pH and increased membrane fluidity, which are hallmarks of HCC, might further enhance BA uptake.

Lipopolysaccharide (LPS, endotoxin) is the major component of Gram-negative bacterial outer surface membrane. LPS released from bacteria into bloodstream during infection may cause serious unwanted stimulation of host's immune system and lead to septic shock of the patient. Recently, we have developed a strategy to increase salt resistance and LPS neutralization of short antimicrobial peptides by adding β-naphthylalanine end-tags to their termini. Herein, correlations between membrane immersion depth, orientation, and antiendotoxin activities of the antimicrobial peptides S1 and S1-Nal-Nal have been investigated via solution structure, paramagnetic resonance enhancement, and saturation transfer difference NMR studies. Unlike the parent peptide S1, S1-Nal-Nal rotated its two terminal β-naphthylalanine residues into the hydrophobic lipid A motif of LPS micelles. The LPS-induced inflammation may then be prohibited by the blocked lipid A motif.

We describe a strategy to boost anticancer activity and reduce normal cell toxicity of short antimicrobial peptides by adding positive charge amino acids and non-nature bulky amino acid β-naphthylalanine residues to their termini. Among the designed peptides, K4R2-Nal2-S1 displayed better salt resistance and less toxicity to hRBCs and human fibroblast than Nal2-S1 and K6-Nal2-S1. Fluorescence microscopic studies indicated that the FITC-labeled K4R2-Nal2-S1 preferentially binds cancer cells and causes apoptotic cell death. Moreover, a significant inhibition in human lung tumor growth was observed in the xenograft mice treated with K4R2-Nal2-S1. Our strategy provides new opportunities in the development of highly effective and selective antimicrobial and anticancer peptide-based therapeutics.

A microfluidic immunoassay system was developed for the study of the enhancement of protein binding reaction. The system mainly consisted of a thermopneumatic actuator and a reaction chamber. Reagent was pre-installed in the on-chip reservoir and manipulated by the actuator. Such design could eliminate the external tubing connections in order to reduce the waste of reagent and improve the portability. The on-chip actuator could manipulate the reagent bi-directionally to induce vortexes in the chamber. Enhancement of protein binding reaction was demonstrated by the protein model pair, i.e., mouse IgG and anti-mouse IgG. By such bi-directional fluid motion, more binding opportunities between suspended protein and its surface-immobilized counterpart were generated to improve the performance of immunoassay. It showed that an 83.74 % enhancement of the binding reaction was achieved, compared with the static situation. As a whole, the proposed microfluidic system is highly integrated and can enhance the protein binding efficiency using such novel design. The developed system can be easily extended to multi-reagents immunoassay protocols and provides a useful platform for point-of-care applications.

Blood coagulation is an extremely complicated and dynamic physiological process. Monitoring of blood coagulation is essential to predict the risk of hemorrhage and thrombosis during cardiac surgical procedures. In this study, a high throughput microfluidic chip has been developed for the investigation of the blood coagulation process under temperature and hematocrit variations. Electrical impedance of the whole blood was continuously recorded by on-chip electrodes in contact with the blood sample during coagulation. Analysis of the impedance change of the blood was conducted to investigate the characteristics of blood coagulation process and the starting time of blood coagulation was defined. The study of blood coagulation time under temperature and hematocrit variations was shown a good agreement with results in the previous clinical reports. The electrical impedance measurement for the definition of blood coagulation process provides a fast and easy measurement technique. The microfluidic chip was shown to be a sensitive and promising device for monitoring blood coagulation process even in a variety of conditions. It is found valuable for the development of point-of-care coagulation testing devices that utilizes whole blood sample in microliter quantity.

(Cancer Sci 2010; 101: 624–630)Gastroesophageal reflux has recently been implicated as a causative factor in upper aerodigestive tract carcinogenesis. Esophageal squamous cell carcinomas (ESCCs) have developed in duodenal‐content reflux animals without any known carcinogen present. We established a cell line, designated ESCC‐DR, from a thoracic metastatic tumor in a reflux animal. To gain insight into the genomic alterations associated with duodenal content reflux‐induced carcinogenesis, we first performed comparative genomic hybridization using an Agilent rat 244K array in ESCC‐DR and identified many chromosomal gains and losses. Of the many genes identified, we detected an interesting ezrin amplicon that has been recently reported in human ESCC. Ezrin, which cross‐links the cytoskeleton and plasma membrane, is involved in the growth and metastatic potential of cancer cells. Overexpression of ezrin protein in ESCC‐DR was confirmed by Western blotting. We also compared ezrin protein expression levels and patterns in hyperplastic, dysplastic, ESCC, and metastatic sites developed in two distinct reflux models using immunohistochemistry. Immunohistochemical staining of ezrin revealed overexpression in the nucleus, and the cytoplasm as well as plasma membrane of ESCC cells. Phosphorylated ERM (ezrin, radixin, moesin) was expressed at the leading edge, or invasive front, of larger metastatic sites. Taken together, duodenal reflux has a great potential for initiating malignancy, and thus likely plays a role in development of ESCC. Ezrin probably influences the growth and invasiveness of ESCC cells, and phosphorylation is only required in metastatic behavior of tumor cells at the leading edge and invasive front.

The endogenous DNA adducts O6‐carboxymethyl‐deoxyguanosine (O6‐CM‐dG) and 3‐ethanesulfonic acid‐deoxycytidine (3‐ESA‐dC) are produced from N‐nitroso bile acid conjugates, such as N‐nitrosoglycocholic acid (NO‐GCA) and N‐nitrosotaurocholic acid (NO‐TCA), respectively. Formation of these DNA adducts in vivo was here analyzed by 32P‐postlabeling in the glandular stomach of rats subjected to duodenal content reflux surgery. In this model, all duodenal contents, including bile acid conjugates, flow back from the jejunum into the gastric corpus. The levels of O6‐CM‐dG found at 4 and 8 weeks after surgery were 40.9 ± 9.4 and 56.3 ± 3.2 per 108 nucleotides, respectively, whereas the sham operation groups had values of 5.8 ± 2.3 and 5.9 ± 0.5 per 108 nucleotides. Moreover, adduct spots corresponding to 3‐ESA‐dC were detected in both duodenal reflux and sham operation groups and levels in the duodenal reflux groups were around four‐fold elevated at 11.2 ± 1.0 and 8.9 ± 1.0 per 108 nucleotides after 4 and 8 weeks, respectively. When the duodenal reflux animals were treated with a nitrite trapping agent, thiazolidine‐ 4‐carboxylic acid (thioproline, TPRO), the levels of O6‐CM‐dG and 3‐ESA‐dC were reduced to the same levels as in the sham operation animals. These observations suggest that NO‐TCA and NO‐GCA are formed by nitrosation of glycocholic acid and taurocholic acid, respectively, and these nitroso compounds produce DNA adducts in the glandular stomach of rats subjected to duodenal content reflux surgery. (Cancer Sci 2008; 99: 1741–1746)

Background/aims In the duodenal content reflux model of rats, we noted an elevation of serum bile acid and swelling of the thyroid gland. This study was designed to elucidate whether bile acids (BAs) also enhance thyroid function. Methods In varying lengths of period after esophago-jejunostomy without gastrectomy, which causes duodenal content reflux, rats were sacrificed and blood samples were taken from the heart for analyses of BAs and triiodothyronine (T3), thyroxine (T4), free T3 (fT3), free T4 (fT4), and thyroid-stimulating hormone (TSH) in the serum. Results Macroscopically, at 10 and 30 weeks after operation, thyroid glands in the reflux model showed a symmetric enlargement because of the presence of diffuse hypertrophy of the thyroid follicular epithelium. At both time points, no significant differences were detected in T3, T4, fT3, and fT4 levels between the reflux model and the control group, whereas, at 10 weeks after operation, the animals with the reflux showed significantly lower serum TSH levels and greater thyroid weight than those in the control group. An inverse correlation between serum BAs and TSH levels was noted in the reflux model but not in the control group. Microscopically, thyroid follicles were greater in size and number, with paler colloids in the reflux model than the control group. Conclusions The present results suggest that high serum BAs cause hyperplasia of the thyroid follicles and the reduction of TSH. The effects of BAs on thyroid hormones, thus, include the induction of overall hyperthyroidism. Therefore, the strict monitoring of serum TSH levels is of vital importance if BAs are used for the treatment of obesity.

The dietary components responsible for the development of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) remain unclear. Wistar rats were divided into four groups based on their diet: a low soybean‐oil diet, a low cow‐fat diet, a high soybean‐oil diet, and a high cow‐fat diet. First, we evaluated the bile acid composition of the bile juice in each group without operation, using high‐performance liquid chromatography. Because only high cow‐fat intake induced changes in the composition of bile acids in bile juice, we then selected animals fed with a low soybean‐oil diet and those with a high cow‐fat diet to carry out esophago‐jejunostomy for reflux of the duodenal contents, and compared sequential morphological changes between these groups up to 30 weeks after surgery. At 30 weeks after surgery, the reflux animals in the high cow‐fat group showed a significantly higher incidence of BE and Barrett's dysplasia than those in the low soybean‐oil group, and the incidence of EAC in the high cow‐fat group was also slightly higher than that in the low soybean‐oil group. High dietary animal fat changed the bile‐acid composition and increased the concentration of taurine conjugates in the bile juice. These increased bile acids promoted the development of BE and Barrett's dysplasia leading to EAC. (Cancer Sci 2007; 98: 1683–1688)

Squamous cell carcinoma (SCC) of the human esophagus has a multifactorial etiology involving several environmental and/or genetic factors. Recently, gastroesophageal reflux has been implicated as a causative factor in upper aerodigestive tract carcinogenesis. The development of esophageal squamous cell carcinoma (ESCC) in a duodenal contents reflux model without any known carcinogen present has been reported previously. In this study, the duodenal contents reflux model without gastrectomy was used. At 60 weeks post-operatively, all surviving animals had malignant lesions as follows: ESCC (40%), esophageal adenocarcinoma (EAC) (20%) and adenosquamous carcinoma (40%). In one subject, a well-differentiated ESCC was detected with thoracic dissemination and metastases in lymph nodes. A novel cell line, designated ESCC-DR, was established from the thoracic metastatic tumor at the 60th post-operative week. These cells were transplanted into nude mice, and the developed nodules represented a well differentiated ESCC, resembling that of the parent site. Duodenal contents reflux has a great potential for malignant initiation and plays a role in developing not only EAC but also ESCC.

OBJECTIVES:The main factors considered responsible for the onset of laryngeal cancer are tobacco smoking and alcohol abuse. Recently, gastroesophageal reflux has also been implicated as a causative factor in several laryngeal disorders, including laryngeal cancer. However, the significance of gastroesophageal reflux in laryngeal cancer is controversial.METHODS:We investigated the histologic features of the esophagus and larynx in a rat model of reflux of the duodenal contents. Cell proliferation was also evaluated in laryngeal samples by detection of Ki67 antigen.RESULTS:In this reflux model, laryngitis with infiltration of inflammatory cells and proliferation of small mucous glands was evident from 10 weeks after operation, and basal cell hyperplasia around the epiglottis and hyperplastic changes in the larynx were detected at 30 weeks. No dysplastic or malignant lesions were detected in the laryngeal samples within the duration of the experiment, in spite of detection of malignancy in 31.3% of lesions in esophageal samples at 30 weeks. The Ki67 index at each week was significantly higher than that of the control animals.CONCLUSIONS:Previous studies have shown smoking and alcohol abuse to have refluxogenic effects. Reflux of duodenal contents causes laryngitis. Reflux does not appear to be an independent risk factor for laryngeal carcinogenesis, but it may enhance the acknowledged etiologic risk factors, namely, smoking and alcohol abuse, by promoting cell proliferation.