Transport of ribavirin in human myelogenous leukemia cell line K562.

The anticancer effect of ribavirin, a purine nucleoside analogue, has been studied using cultured cancer cells such as the human myelogenous leukemia cell line K562. In order to exert its pharmacological effect, ribavirin has to enter cancer cells. However, there is little information concerning the transport mechanism of ribavirin into K562 cells. In this study, therefore, we examined the uptake mechanism of ribavirin in K562 cells. The uptake of ribavirin in K562 cells was time- and temperature-dependent, and was saturable with a Km value of 1.5 mM. Ribavirin uptake was inhibited by nucleosides such as adenosine and uridine, and by inhibitors of equilibrative nucleoside transporter 1 (ENT1) such as S-(4-nitrobenzyl)-6-thioinosine and dipyridamole in a concentration-dependent manner. In addition, the expression of ENT1 mRNA in K562 cells was confirmed by real-time PCR. On the other hand, Na+-dependence of ribavirin uptake was not observed, suggesting the involvement of ENT1, but not Na+-dependent concentrative nucleoside transporters, in ribavirin uptake in K562 cells. Treatment of K562 cells with sodium butyrate induced erythroid differentiation, but ribavirin uptake activity and sensitivity of the uptake to various inhibitors were not different between native and differentiated K562 cells. These results suggest that ribavirin uptake into K562 cells is mainly mediated by ENT1, which may have a pivotal role in anticancer effect of ribavirin.

Efficacy of programmed intermittent bolus epidural analgesia in thoracic surgery: a randomized controlled trial.

BACKGROUND: Continuous epidural infusion (CEI) has some disadvantages, such as increased local anesthetic consumption and limited area of anesthetic distribution. Programmed intermittent bolus (PIB) is a technique of epidural anesthesia in which boluses of local anesthetic are automatically injected into the epidural space. The usefulness of PIB in thoracic surgery remains unclear. In this study, we aimed to compare the efficacies of PIB epidural analgesia and CEI in patients undergoing thoracic surgery. METHODS: This randomized prospective study was approved by the Institutional Review Board. The study included 42 patients, who were divided into CEI (n = 21) and PIB groups (n = 21). In the CEI group, patients received continuous infusion of the local anesthetic at a rate of 5.1 mL/90 min. In the PIB group, a pump delivered the local anesthetic at a dose of 5.1 mL every 90 min. The primary endpoints were the frequency of patient-controlled analgesia (PCA) and the total dose of local anesthetic until 36 h following surgery. Student's t-test, the chi-square test, and the Mann-Whitney U test were used for statistical analyses. RESULTS: The mean number of PCA administrations and total amount of local anesthetic were not significantly different between the two groups up to 24 h following surgery. However, the mean number of PCA administrations and total amount of local anesthetic at 24-36 h after surgery were significantly lower in the PIB group than in the CEI group (median [lower-upper quartiles]: 0 [0-2.5] vs. 2 [0.5-5], P = 0.018 and 41 [41-48.5] vs. 47 [43-56], P = 0.035, respectively). Hypotension was significantly more frequent in the PIB group than in the CEI group at 0-12 h and 12-24 h (3.3% vs. 0.5%, P = 0.018 and 7.9% vs. 0%, P = 0.017, respectively). CONCLUSION: PIB can reduce local anesthetic consumption in thoracic surgery. However, it might result in adverse events, such as hypotension. TRIAL REGISTRATION: This randomized prospective study was approved by the Institutional Review Board (IRB No. 15-9-06) of the Fukuoka University Hospital, Fukuoka, Japan, and was registered in the clinical trials database UMIN ( ID 000019904 ) on 24 November 2015. Written informed consent was obtained from all patients.

Functional expression of breast cancer resistance protein and cholesterol effect in human erythrocyte membranes.

In human erythrocyte membranes, various influx and efflux transporters are functionally expressed. However, their transport characteristics and modulation under disease states are not fully understood. In this study, we first examined the expression and detailed transport characteristics of breast cancer resistance protein (BCRP), an efflux ABC transporter, using inside-out membrane vesicles (IOVs) prepared from human erythrocytes, and then studied the effect of membrane cholesterol on BCRP function. The expression of BCRP was confirmed by western blotting; most of them being homodimers. The uptake of lucifer yellow (LY), a fluorescent BCRP substrate, into IOVs was time-, temperature-, and ATP-dependent, and the concentration of ATP which induced half-maximal stimulation of LY uptake was calculated to be 0.39 mM. The uptake of LY by IOVs was saturable with a Km value of 166 µM, and was inhibited by various BCRP inhibitors and substrates, such as fumitremorgin C and mitoxantrone. When membrane cholesterol content was increased by treating IOVs with cholesteryl hemisuccinate, LY uptake decreased with increasing cholesterol content. These results suggest that transport activity of BCRP in human erythrocyte membranes may be suppressed under disease states, such as hypercholesterolemia, that increase membrane cholesterol content.

Vascular endothelial growth factors C and D and lymphangiogenesis at the early stage of esophageal squamous cell carcinoma progression.

We conducted a detailed study of lymphangiogenesis and subsequent lymph node metastasis in early-stage esophageal squamous cell carcinoma (ESCC) using immunostaining for D2-40 and vascular endothelial growth factor (VEGF)-C and D. The study materials included 13 samples of normal squamous epithelium, 6 samples of low-grade intraepithelial neoplasia (LGIN), and 60 samples of superficial ESCC (M1 and M2 cancer 24; M3 or deeper cancer 36). We assessed lymphatic vessel density (LVD) using D2-40 and immunoreactivity for VEGF-C and D in relation to histological type, lymphatic invasion, and lymph node metastasis. LVD in M1 and M2 lesions and M3 or deeper lesions was significantly higher than in normal squamous epithelium (P < 0.001). High expression of VEGF-C and D was observed in M1 and M2 cancer and in M3 or deeper cancer, but not in normal squamous epithelium or LGIN. LVD in VEGF-C- and D-positive cases was significantly higher than in negative cases (P < 0.001). In M3 or deeper cancer, the correlation between VEGF-C or D status and lymphatic invasion or lymph node metastasis was not significant. LVD in cases with positive lymphatic invasion and those with lymph node metastasis was significantly higher than in cases lacking either (P = 0.02 and 0.03, respectively). ESCC cells produce VEGF-C and D from the very early stage of progression. VEGF-C and D activate lymphangiogenesis, and this increase of lymphatic vessels leads to lymphatic invasion and subsequent lymph node metastasis.

Endocytoscopic observation of various esophageal lesions at ×600: can nuclear abnormality be recognized?

Endocytoscopy (ECS) is a novel endoscopic technique that allows detailed diagnostic examination of the gastrointestinal tract at the cellular level. We previously reported that use of ECS at ×380 magnification (GIF-Y0002) allowed a pathologist to diagnose esophageal squamous cell carcinoma (ESCC) with high sensitivity (94.9%) but considerably low specificity (46.7%) because this low magnification did not reveal information about nuclear abnormality. In the present study, we used the same magnifying endoscope to observe various esophageal lesions, but employed digital 1.6-fold magnification to achieve an effective magnification of ×600, and evaluated whether this improved the diagnostic accuracy in distinguishing neoplastic from non-neoplastic lesions.We examined the morphology of surface cells using vital staining with toluidine blue and compared the histological features of 40 cases, including 19 case of ESCC and 21 non-neoplastic esophageal lesions (18 cases of esophagitis, 1 case of glycogenic acanthosis, 1 case of leiomyoma, and 1 case of normal squamous epithelium). One endoscopist classified the lesions using the type classification, and we consulted one pathologist for judgment of the ECS images as 'neoplastic', 'borderline', or 'non-neoplastic'. At ×600 magnification, the pathologist confirmed that nuclear abnormality became evident, in addition to the information about nuclear density provided by observation at ×380. The overall sensitivity and specificity with which the endoscopist was able to predict neoplastic lesions using the type classification was 100% (19/19) and 90.5% (19/21), respectively, in comparison with values of 94.7% (18/19 cases) and 76.2% (16/21), respectively, for the pathologist using a magnification of ×600. The pathologist diagnosed two non-neoplastic lesions and one case of ESCC showing an apparent increase of nuclear density with weak nuclear abnormality as 'borderline'. Among the 21 non-cancerous lesions, two cases of esophagitis that were misdiagnosed by the endoscopist were also misinterpreted as 'neoplastic' by the pathologist. We have shown, by consultation with a pathologist, that an ECS magnification of ×600 (on a 19-inch monitor) is adequate for recognition of nuclear abnormality. We consider that it is feasible to diagnose esophageal neoplasms on the basis of ECS images, and that biopsy histology can be omitted if a combination of increased nuclear density and nuclear abnormality is observed.

The etiologic role of human papillomavirus in penile cancers: a study in Vietnam.

BACKGROUND: We investigated the aetiologic role of human papillomavirus (HPV) in 120 penile squamous cell carcinomas (PSCCs) from Vietnam. METHODS: Human papillomavirus DNA was detected by PCR using SPF10 primers and a primer set targeting HPV-16 E6. The INNO-LiPA HPV genotyping kit was used to determine genotype. Human papillomavirus-16 viral load and physical status were determined by real-time PCR. P16(INK4A) protein expression was investigated by immunohistochemistry. RESULTS: Human papillomavirus DNA was detected in 27 of 120 (23%) PSCCs. The most frequently detected genotype was HPV-16 (24 of 27 cases, 89%). In 16 of 18 (89%) HPV-16-positive cases, the HPV DNA was considered to be integrated into the host genome. The geometric mean of the HPV-16 viral load was 0.4 copies per cell. P16(INK4A) overexpression was significantly related to PSCCs infected with high-risk HPV (P=0.018) and HPV-16 copy numbers (P<0.001). CONCLUSION: Human papillomavirus-16 DNA integration and p16(INK4A) overexpression in high-risk HPV detected PSCCs suggested an aetiologic role of high-risk HPV in the development of PSCCs.

MUC1 enhances hypoxia-driven angiogenesis through the regulation of multiple proangiogenic factors.

Pancreatic cancer is one of the most lethal malignancies due to its aggressive growth and rapid development of distant metastases. In this context, mucin 1 (MUC1) overexpression and hypoxia are frequently observed events. However, their functional relationship remains largely unknown. This study provides evidence that MUC1 is overexpressed by hypoxia and contributes to hypoxia-driven angiogenesis. Using the conditioned medium obtained from hypoxia-stressed AsPC1 cells treated with MUC1 siRNAs, we demonstrated that MUC1 enhanced the endothelial tube formation, proliferation and migration ability, which induced by hypoxia-conditioned medium (HCM). In addition, MUC1 was significantly induced by hypoxia, especially in the pancreatic cancer cells derived from metastatic tumors (AsPC1, HPAF2 or Capan1), and MUC1-cytoplasmic tail (MUC1-CT) accumulated in the nucleus under hypoxia. As noted in a previous report, MUC1-CT was recruited to genomic regions upstream of the connective tissue growth factor (CTGF) accompanied with ß-catenin and p53, resulting in the hypoxic induction of CTGF. Moreover, hypoxia-induced MUC1 partially regulated two other hypoxia-inducible proangiogenic factors including vascular endothelial growth factor-A and platelet-derived growth factor-B. The neutralization assay revealed that endothelial tube formation induced by HCM was clearly suppressed by antibodies against these three factors, suggesting the importance of these factors in hypoxia-driven angiogenesis. In summary, this is the first report demonstrating a pivotal role of MUC1 in controlling the hypoxia-driven angiogenesis through the regulation of multiple proangiogenic factors in pancreatic cancer. Our findings provide the novel insights into the understanding of complex interactions between pancreatic cancer cells and tumor microenvironments.

Promoter CpG methylation in cancer cells contributes to the regulation of MUC4.

Mucin 4 (MUC4) is a high molecular weight transmembrane mucin that is overexpressed in many carcinomas and is a risk factor associated with a poor prognosis. In this study, we show that the DNA methylation pattern is intimately correlated with MUC4 expression in breast, lung, pancreas and colon cancer cell lines. We mapped the DNA methylation status of 94 CpG sites from -3622 to +29 using MassARRAY analysis that utilises base-specific cleavage of nucleic acids. MUC4-negative cancer cell lines and those with low MUC4 expression (eg, A427) were highly methylated near the transcriptional start site, whereas MUC4-positive cell lines (eg, NCI-H292) had low methylation levels. Moreover, 5-aza-2'-deoxycytidine and trichostatin A treatment of MUC4-negative cells or those with low MUC4 expression caused elevation of MUC4 mRNA. Our results suggest that DNA methylation in the 5' flanking region play an important role in MUC4 gene expression in carcinomas of various organs. An understanding of epigenetic changes in MUC4 may contribute to the diagnosis of carcinogenic risk and prediction of outcome in patients with cancer.

Human papillomavirus detected in female breast carcinomas in Japan.

To investigate the aetiological role of human papillomavirus (HPV) in breast cancer, we examined the presence, genotype, viral load, and physical status of HPV in 124 Japanese female patients with breast carcinoma. Human papillomavirus presence was examined by PCR using SPF10 primers, and primer sets targeting the E6 region of HPV-16, -18, and -33. The INNO-LiPA HPV genotyping kit was used to determine genotype. Human papillomavirus DNA was detected in 26 (21%) breast carcinomas. The most frequently detected HPV genotype was HPV-16 (92%), followed by HPV-6 (46%), HPV-18 (12%), and HPV-33 (4%). In 11 normal epithelium specimens adjacent to 11 HPV-16-positive carcinomas, 7 were HPV-16-positive. However, none of the normal breast tissue specimens adjacent to HPV-negative breast carcinomas were HPV-positive. The real-time PCR analysis suggested the presence of integrated form of viral DNA in all HPV-16-positive samples, and estimated viral load was low with a geometric mean of 5.4 copies per 10(4) cells. In conclusion, although HPV DNA was detected in 26 (21%) breast carcinomas and, in all HPV-16-positive cases, the HPV genome was considered integrated into the host genome, their low viral loads suggest it is unlikely that integrated HPV is aetiologically involved in the development of Japanese breast carcinomas that we examined.

Subtraction 3D CT angiography with the orbital synchronized helical scan technique for the evaluation of postoperative cerebral aneurysms treated with cobalt-alloy clips.

BACKGROUND AND PURPOSE: CT angiography (CTA) has been used for the evaluation of intracranial aneurysms and recently has been applied to assess postoperative aneurysms treated with titanium-alloy clips. We investigated the clinical usefulness of subtraction CTA by using the orbital synchronized helical scan technique (OSHST) for evaluating intracranial aneurysms surgically treated with cobalt-alloy clips. MATERIALS AND METHODS: We scanned an agar gel phantom with a cobalt-alloy clip mounted in the center by using subtraction CT with and without OSHST. Eighteen patients (20 aneurysms) who underwent surgery with cobalt-alloy clips were postoperatively evaluated with subtraction CTA with OSHST, and the results were compared with those from digital subtraction angiography. Two neuroradiologists independently evaluated the 3D CTA images and source images with and without subtraction for the presence of residual flow in the aneurysm and stenotic change in parent or neighboring arteries. RESULTS: For the phantom study, significantly fewer artifacts from clips were noted on images obtained by using subtraction CT with OSHST than on those obtained without OSHST. For the clinical study, subtraction CTA with OSHST also showed fewer clip artifacts than did conventional CTA. Image quality was poor, and we were unable to diagnose residual neck for 5% (1/20) with subtraction CTA with OSHST and 75% (15/20) with conventional CTA. For evaluation of adjacent vessels, image quality was poor for none (0/20) with subtraction CTA with OSHST and for 55% (11/20) with conventional CTA. For subtraction CTA with OSHST, sensitivity in detecting residual neck was 1.0, and specificity was 0.94. For conventional CTA, sensitivity and specificity were both 0.25. CONCLUSIONS: OSHST is a useful technique for subtracting cobalt-alloy clips, and subtraction CTA with OSHST is available for evaluating aneurysms after clipping with cobalt-alloy clips.

Human papillomavirus-16 is integrated in lung carcinomas: a study in Chile.

The human papillomavirus (HPV) was detected in 20 (29%) out of 69 lung carcinomas (LCs) in Chile, by PCR and Southern blot, and was more frequently detected in squamous cell carcinoma (SQC) than in adenocarcinomas (46 vs 9%, P=0.001). HPV-16, positive in 11 cases, was the most frequently detected HPV genotype determined by DNA sequencing. HPV-16 E2/E6 ratio, estimated from real-time PCR analysis, was much lower than the unity, suggesting that at least a partial HPV-16 genome was integrated in all but one HPV-16-positive SQCs. The remaining one case was suspected to have only episomal HPV-16. Although the viral load was low in most of the LCs, a case showed the HPV-16 copy number as high as 8479 per nanogram DNA, which was even a few times higher than the minimum viral load of seven cervical carcinomas (observed viral load: 3356-609 392 per nanogram DNA). The expression of the HPV-16/18 E6 protein was found in only two HPV-16-positive SQCs (13%) but not in the case with the highest viral load. Although the viral load was in general very low and HPV E6 expression is none or weak, further studies seem warranted to examine aetiological involvement of high-risk HPV in lung carcinogenesis.

Insights into infant neuroblastomas based on an analysis of neuroblastomas detected by mass screening at 6 months of age in Japan.

BACKGROUND/PURPOSE: Mass screening (MS) for neuroblastoma (NB) at 6 months of age in Japan was discontinued in 2004. We have previously reported that the majority of NB detected by MS showed a good prognosis, with only a few cases demonstrating an unfavorable outcome (J Pediatr Surg 2002, Cancer 2001). This study aims to provide insights into infant NB by assessing the details of the clinical courses in patients treated with a standard regimen and the biological features of such cases using highly sensitive methods at one institution in Japan. METHODS: In 76 NB detected through MS treated at Kyushu University Hospital, the clinical features and MYCN amplification, 1p deletion, 17q gain, the expression level of TRKA using FISH and the quantitative PCR were analyzed. RESULTS: Of these 76 persons with NB treated at one institution, 97 % are still alive, while 2 cases died from other diseases. Three patients experienced a recurrence after complete remission (CR), and 2 patients demonstrated refractory disease since the initial diagnosis. Two of the 3 NB patients with recurrence have demonstrated a 2nd CR, while one case still has multiple active diseases. Regarding the findings of highly sensitive biological analyses, 5/74 (7 %) showed MYCN amplification, 2/24 (8 %) cases had a 1p deletion, 3/33 (9 %) cases had a 17q gain, 5/50 (10 %) cases had diploidy, 1/25 (4 %) cases had a low expression of TRKA, and 2/76 (3 %) cases had an unfavorable histology. Of the 76 NB, 13 tumors (17 %) had one or more unfavorable factors (UF). Of the 5 refractory NB, 1 case had 3 UF, 1 case had 2 UF, 1 case had 1 UF, and 2 cases had no UF. As a result, 60 % of the refractory NB had one or more UF. CONCLUSIONS: Of the NB detected by MS at one institution in Japan, 17 % had one or more unfavorable factors (UF) and might have a higher risk of recurrence than the patients with no UF, although the unfavorable biology of several refractory cases is still unclear even after highly sensitive analyses. At least one-fifth of the NB cases detected by MS are anticipated cases. In infantile neuroblastomas, it may therefore be most important to analyze biologically prognostic factors using highly sensitive methods followed by immediate surgical intervention. Since the MS program has been discontinued in Japan, it will be necessary in future to assess the mortality and characteristics of NB detected clinically.

Association between the HER2 expression and histological differentiation in Wilms tumor.

Human epidermal growth factor receptors (HER) play a critical role in the branching morphogenesis of renal tubules. In the current study, we analyzed the expression of HER2 in Wilms tumor and assessed the role of this gene in the tumorgenesis of Wilms tumor. During the period from 1960 to 2005, 40 patients with Wilms tumor were treated in our department. Twenty-four of those patients (except those with clear cell sarcoma of the kidney and malignant rhabdoid tumor of the kidney) were collected and assessed. The histological component of each Wilms tumor was divided into three categories (epithelial, blastemal, and mesenchymal) and the extent of HER2 protein expression was analyzed immunohistochemically. The normal kidney tissue accompanied with 12 cases of Wilms tumor was also examined. In the normal kidney, HER2 showed a strong immunoreactivity in the cell membranes of the collecting tubules and in the endothelial cells. Of 24 cases, 15 cases showed an epithelial component, while 24 cases had a blastemal component and 21 cases had a mesenchymal component, respectively. Among the 15 specimens with epithelial cell differentiation, eight (53.3%) showed HER2 immunoreactive epithelial cells. HER2 immunoreactive blastemal cells were present in 11 (45.8%) of 24 specimens with blastemal cells. On the other hand, only 3 (14.3%) of 21 specimens containing mesenchymal cells showed HER2 immunoreactivity. These results suggest that the extent of HER2 expression is associated with epithelial differentiation in Wilms tumor. These histological findings may therefore help to explain the development of Wilms tumor from the standpoint of histological differentiation.

Reliability of fingertip skin-surface temperature and its related thermal measures as indices of peripheral perfusion in the clinical setting of the operating theatre.

During the perioperative period, evaluation of digital blood flow would be useful in early detection of decreased circulating volume, thermoregulatory responses or anaphylactoid reactions, and assessment of the effects of vasoactive agents. This study was designed to assess the reliability of fingertip temperature, core-fingertip temperature gradients and fingertip-forearm temperature gradients as indices of fingertip blood flow in the clinical setting of the operating theatre. In 22 adult patients undergoing abdominal surgery with general anaesthesia, fingertip skin-surface temperature, forearm skin-surface temperature, and nasopharyngeal temperature were measured every five minutes during the surgery. Fingertip skin-surface blood flow was simultaneously estimated using laser Doppler flowmetry. These measurements were made in the same upper limb with an IV catheter (+ IV group, n=11) or without an IV catheter (-IV group, n=11). Fingertip blood flow, transformed to a logarithmic scale, significantly correlated with any of the three thermal measures in both the groups. Their rank order as an index of fingertip blood flow in the -IV group was forearm-fingertip temperature gradient (r=-0.86) > fingertip temperature (r=0.83) > nasopharyngeal-fingertip temperature gradient (r=-0.82), while that in the +IV group was nasopharyngeal-fingertip temperature gradient (r=-0.77) > fingertip temperature (r=0.71) > forearm-fingertip temperature gradient (r=-0.66). The relation of fingertip blood flow to each thermal measure in the -IV/group was stronger (P<0.05) than that in the +IV group. In the clinical setting of the operating theatre, using the upper limb without IV catheters, fingertip skin-surface temperature, nasopharyngeal-fingertip temperature gradients, and forearm-fingertip temperature gradients are almost equally reliable measures of fingertip skin-surface blood flow.

Overexpression of cyclooxygenase-2 in high-grade human transitional cell carcinoma of the upper urinary tract.

OBJECTIVE: To examine cyclooxygenase (COX)-2 expression (a key enzyme in the synthesis of prostaglandins, and involved in carcinogenesis of human epithelial tumours) in human transitional cell carcinomas (TCCs) of the renal pelvis and ureter, and to determine whether COX-2 expression correlates with the clinicopathological characteristics of the disease. MATERIALS AND METHODS: Specimens from 144 patients with TCC of the upper urinary tract who had undergone nephroureterectomy were analysed immunohistochemically, and 23 were also analysed by immunoblotting. RESULTS: Immunoblot analysis showed COX-2 immunoreactivity in 17 (74%) of 23 tumours, but not in normal transitional epithelium. COX-2 was localized to the cytoplasm of cancer cells and expressed in 108 (75%) of 144 tumours, as assessed by immunohistochemical analysis. COX-2 expression correlated with tumour grade (P < 0.008), being detected in one of nine grade 1, 77 (79%) of 97 grade 2 and 30 (79%) of 38 grade 3 tumours. Other variables including tumour stage were not associated with COX-2 expression. CONCLUSIONS: We show for the first time that COX-2 is frequently expressed in TCC of the upper urinary tract and is associated with the degree of tumour cell differentiation, indicating that COX-2 may be involved in TCC carcinogenesis at an early and/or late stage, and could be a useful target for chemoprevention of this type of cancer.