No-touch pancreatectomy for invasive ductal carcinoma of the pancreas.

BACKGROUND: Pancreatectomy is the only effective treatment for cancers of the pancreas. Surgeons usually grasp tumors during pancreatectomy; however, this procedure may increase the risk of squeezing and shedding of the cancer cells into the portal vein, retroperitoneum, and/or peritoneal cavity. In an effort to overcome these problems, we have developed surgical techniques for no-touch pancreatectomy. METHODS: From April 2008 through September 2013, 52 patients have been operated on no-touch pancreatectomy for invasive ductal carcinoma of the pancreas by a single operator (M.H.). Among them, 40 received pancreatoduodenectomy (PD), and 12 did distal pancreatectomy (DP). Twenty two cases (42%) required SMV-PV resection. This is a study to see if pancreatectomy can be technically done using a no-touch surgical technique without deteriorating the post-operative prognosis. During the procedure, the pancreatic tumor is neither grasped nor squeezed by the surgeon. Furthermore, for improved dissection of the retroperitoneal tissue (leftward and posterior margins for PD and rightward and posterior margins for DP), we use a hanging and clamping maneuver and dissection behind Gerota fascia. RESULTS: Overall 2- and 5-year survival rates were 64 and 42% with mean follow-up periods of 34.4 months (range: 6-68 months). Recurrence free 2- and 5-year survival rates were 49 and 31%, respectively. The 5-year survival rates of patients with JPS-stage III and those with JPS-stage IV were 57 and 20%, respectively. The 5-year survival rates of patients with UICC-stage IIA and those with UICC- stage IIB were 49 and 39%, respectively. Patients with UICC-stage III or IV did not survive for more than 2 years. CONCLUSIONS: No-touch pancreatectomy has many theoretic advantages that merit further investigation in future randomized controlled trials.

Novel heat shock response mechanism mediated by the initiation nucleotide of transcription.

Among SigA-dependent promoters in Bacillus subtilis, we compared the nucleotide sequences of heat shock responding and non-responding promoters. Chimeric promoter experiments revealed that the heat shock response could be ascribed to the initiation nucleotide (iNTP) of the transcription. Our in vivo reporter assay results indicated that a full response was achieved using GTP, a reduced response was observed using ATP, and no additional expression was observed using UTP or CTP. We then investigated the in vitro transcription assay in more detail. Enhanced transcription that was dependent upon the iNTP was observed when heat treatment was administered during the pre-initiation period. We next analyzed the efficiency of open complex formation using potassium permanganate footprinting, and our results revealed an increase in the ratio of open complex formation at elevated temperatures. Based on this, we suggest that the overall intensification of transcription at high temperatures was derived from the high efficiency of open complex formation together with the high affinity of RNA polymerase (RNAP) for the initiation nucleotide GTP. To determine if this mechanism observed in B. subtilis RNAP is common among bacterial species, we performed similar experiments using Escherichia coli RNAP. Our results indicated that E. coli RNAP also exhibited both temperature- and iNTP-dependent enhancement of transcription. Although the temperature ranges and the ratios of enhancement are somewhat different, the overall heat shock response mechanism mediated by the iNTP of transcription appears to be conserved among bacterial RNAP.

Roles of Autophagy and Pancreatic Secretory Trypsin Inhibitor in Trypsinogen Activation in Acute Pancreatitis.

The focus of the review is on roles of autophagy and pancreatic secretory trypsin inhibitor (PSTI), an endogenous trypsin inhibitor, in trypsinogen activation in acute pancreatitis. Acute pancreatitis is a disease in which tissues in and around the pancreas are autodigested by pancreatic digestive enzymes. This reaction is triggered by the intrapancreatic activation of trypsinogen. Autophagy causes trypsinogen and cathepsin B, a trypsinogen activator, to colocalize within the autolysosomes. Consequently, if the resultant trypsin activity exceeds the inhibitory activity of PSTI, the pancreatic digestive enzymes are activated, and they cause autodigestion of the acinar cells. Thus, autophagy and PSTI play important roles in the development and suppression of acute pancreatitis, respectively.

Induction of interleukin-8 (CXCL-8) by tumor necrosis factor-alpha and leukemia inhibitory factor in pancreatic carcinoma cells: Impact of CXCL-8 as an autocrine growth factor.

Pancreatic carcinoma is one of the most lethal of the gastrointestinal malignant tumors. Chronic inflammation leads to cancer development and progression. Interleukin-8 (CXCL-8) is a CXC chemokine, which plays an important role in neutrophil chemotaxis and activation. We previously reported that CXCL-8 was produced by a variety of human carcinoma cells and tissues, and that CXCL-8 promoted proliferation in pancreatic carcinoma cells (SUIT-2). In the present study, we analyzed whether various cytokines affect cell proliferation by CXCL-8 expression in pancreas carcinoma cells. All examined pancreatic carcinoma cells expressed CXCL-8 and TNFRII mRNA constitutively in RPMI-1640 medium without FBS. TNF-alpha, LIF, IL-1beta, IL-6, IL-8, or IFN-beta enhanced the expression of CXCL-8 mRNA, but IL-10 did not in Hs-700T cells. Actinomycin D suppressed and cycloheximide augmented CXCL-8 mRNA which was induced by TNF-alpha or not. The half-life of CXCL-8 mRNA was 36.5 min by TNF-alpha and 35.2 min by no stimulation. In our previous study, LIF promoted cell growth in Hs-700T cells. LIF induced CXCL-8 mRNA in a dose- and time-dependent manner. Addition of recombinant CXCL-8 did not induce cell growth of Hs-700T. Anti-CXCL-8 IgG significantly suppressed cell growth. CXCL-8 would act as an autocrine growth factor in Hs-700T cells, which expressed CXCL-8 mRNA highly without stimulation. Curcumin (diferuloylmethane), NF-kappaB inhibitor, suppressed cell proliferation in Hs-700T cells. These results suggest that CXCL-8 plays a pivotal role in progression of pancreatic cancer, and its expression is influenced by inflammatory cytokines in pancreatic tumor microenvironment.

Leukemia inhibitory factor functions as a growth factor in pancreas carcinoma cells: Involvement of regulation of LIF and its receptor expression.

Leukemia inhibitory factor (LIF) is a pleiotrophic cytokine, which plays an important role in inducing cancer cachexia. We have previously reported that LIF promotes cell proliferation in some human carcinoma cells through c-fos, jun-B and cyclin-E expression. In the present study, we analyzed the regulation of LIF and its receptor (LIFR) expression in pancreatic carcinoma cells. Seven pancreatic carcinoma cells expressed constitutively LIF and its heterodimer receptor (LIFR and gp130) mRNA in RPMI-1640 medium without FBS. The amount of LIF immunoreactive protein was 132.5+/-52 pg/10(6) cells in culture supernatants without FBS. Pro-inflammatory cytokines, such as TNF-alpha, IL-1beta, IL-6, IL-8, or LIF, enhanced the expression of LIF mRNA in Hs-700T and Hs-766T cells. Addition of LIF significantly induced cell proliferation of Hs700T in 13 days LIF dose-dependently. However, anti-LIF IgG failed to suppress cell proliferation in Hs-700T cells. LIF acted as a paracrine growth factor in Hs-700T cells, which expressed low amount of LIF without stimuli. Cellular signal transductions by LIF was down-regulated by inhibitors of protein kinase C (PKC), protein tyrosine kinase (PTK), and Ca/Calmodulin. LIF induced phosphorylation of STAT3. Moreover, exogenous LIF upregulated the expression of LIFR mRNA. Antisense LIFR oligonucleotide significantly suppressed cell growth in the presence of LIF in Hs-700T cells. These results suggest that cytokine network might alter the expression and responsiveness to LIF in tumor microenvironment.

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) blocks the chemotaxis of neutrophils by inhibiting signal transduction through IL-8 receptors.

We investigated the impact of curcumin on neutrophils. Chemotactic activity via human recombinant IL-8 (hrIL-8) was significantly inhibited by curcumin. Curcumin reduced calcium ion flow induced by internalization of the IL-8 receptor. We analyzed flow cytometry to evaluate the status of the IL-8 receptor after curcumin treatment. The change in the distribution of receptors intracellularly and on the cell surface suggested that curcumin may affect the receptor trafficking pathway intracellulary. Rab11 is a low molecular weight G protein associated with the CXCR recycling pathway. Following curcumin treatment, immunoprecipitation studies showed that the IL-8 receptor was associated with larger amounts of active Rab11 than that in control cells. These data suggest that curcumin induces the stacking of the Rab11 vesicle complex with CXCR1 and CXCR2 in the endocytic pathway. The mechanism for antiinflammatory response by curcumin may involve unique regulation of the Rab11 trafficking molecule in recycling of IL-8 receptors.

Combined radical retropubic prostatectomy and abdominoperineal excision of the rectum for locally invasive rectal cancer as a less invasive surgery: report of a case.

The optimal therapy for carcinoma of the rectum with invasion of the prostate gland has not been established. For a patient who has rectal carcinoma invading into the prostate and seminal vesicles and not invading into any other pelvic viscera, we performed combined radical retropubic prostatectomy and abdominoperineal excision of the rectum with reconstruction of the urinary tract by anastomosis of the ureter to the bladder. En bloc excision yielded negative surgical margins. After the operation, the patient had an infection of the abdominal wound and leakage of the anastomosis of the urethra to the bladder. These complications were treated conservatively and improved without becoming critical. The patient now has satisfactory postoperative function of voiding. This technique obviates the need for urinary diversion or urinary reconstruction such as the neobladder in the case of total pelvic exenteration. We consider this procedure is of benefit for improving the quality of life of patients with rectal cancer invading into the prostate.

Signal of proteinase-activated receptor-2 contributes to highly malignant potential of human pancreatic cancer by up-regulation of interleukin-8 release.

Proteinase-activated receptor-2 (PAR-2) is expressed in various tissues, including cancer lesions. However, the functional consequences of PAR-2 expression in cancer cells, especially in pancreatic cancer cells, are poorly understood. To clarify the biological significance of PAR-2 signaling in pancreatic cancer, we examined the production of growth factors and cytokines, such as IL-6, IL-8, bFGF, TGF-beta1, and VEGF, by specific ELISAs. Two human pancreatic cancer cell lines, SUIT2 and MiaPaCa2, which have been shown to express PAR-2, were stimulated by trypsin and PAR-2 activating peptide (PAR-2AP: SLIGKV-NH2). After 24 h, the culture supernatants were collected and specific ELISAs were performed. Although no significant changes were observed in the release of IL-6, bFGF, TGF-beta1, or VEGF, that of IL-8 was significantly up-regulated by PAR-2 agonists in a dose-dependent manner. In addition, IL-8 receptor expression was found in pancreatic cancer cells and fibroblasts. These results suggest that the PAR-2 signal up-regulates IL-8 release from pancreatic cancer cells. This up-regulated IL-8 has an effect on the pancreatic cancer cells in an autocrine manner and on the fibroblasts in a paracrine manner. Thus, this signal might contribute to tumor progression and characteristic fibrosis in pancreatic cancer.

Isolation of a novel human gene, APCDD1, as a direct target of the beta-Catenin/T-cell factor 4 complex with probable involvement in colorectal carcinogenesis.

To clarify the molecular mechanisms of human carcinogenesis associated with abnormal beta-catenin/T-cell factor (Tcf) signaling, we have been using cDNA microarrays to search for genes whose expression is significantly altered after introduction of wild-type APC into SW480 colon cancer cells. These experiments identified a novel human gene, termed APCDD1, that was down-regulated in the cancer cells by exogenous wild-type APC; its expression was also reduced in response to transduction of AXIN1. Moreover, we documented elevated expression of APCDD1 in 18 of 27 primary colon cancer tissues compared with corresponding noncancerous mucosae. A reporter gene assay using the 5'-flanking region of APCDD1 indicated that transfection of beta-catenin together with wild-type Tcf4 into HeLa cells increased the reporter activity through two putative Tcf/lymphoid enhancer factor-binding motifs upstream of the transcription start site, indicating that APCDD1 is one of the direct targets of this transcription complex. Exogenous APCDD1 promoted growth of colon cancer cells both in vitro and in vivo, whereas transfection with antisense S-oligodeoxynucleotides decreased cell/tumor growth. These data suggest that APCDD1 is directly regulated by the beta-catenin/Tcf complex and that its elevated expression is likely to contribute to colorectal tumorigenesis.

[A 6-year-survival case of esophageal adenosquamous cancer with liver metastases cured by multidisciplinary therapy].

A 65-year-old man was diagnosed as esophageal cancer with multiple liver metastases (S2 10 mm, S7 10 mm, S8 15 mm). The preoperative diagnosis was stage IV (T 3 N 3 M 1 Pl 0), and he was operated palliatively by esophagocardiofundectomy and intrathoracic anastomosis for oral food intake. The postoperative histological diagnosis was adenosquamous carcinoma. One month after the operation he was administered orally UFT-E (300 mg/day) and PSK (3g/day). He was also treated by hepatic arterial infusion therapy with CDDP (10 mg/week). After 180 mg of CDDP, liver metastases were evaluated for PR. This therapy was discontinued after 410 mg of CDDP by vomiting and hypotension. 16 months after, DOC (20 mg/week) was given by arterial infusion and CR of liver metastases was achieved 18 months after. Then he was given 840 mg of DOC and oral administration of UFT-E and PSK was performed for about 5 years. He was free from the recurrence of cancer as an outpatient and had a good QOL. We think that esophageal cancer with liver metastasis should be aggressively treated surgically so as to allow oral food intake, and liver metastasis should be treated with chemotherapy because postoperative hepatic arterial infusion therapy is effective and provides a good QOL.

Correlation of translocation of tight junction protein Zonula occludens-1 and activation of epidermal growth factor receptor in the regulation of invasion of pancreatic cancer cells.

Mitogen-activated protein kinase signal transduction pathway was isolated as a potential factor related to cancer cell dissociation in dissociated (PC-1.0 and AsPC-1) and non-dissociated (PC-1 and Capan-2) pancreatic cancer cells in our previous works. On the other hand, changes of structure and function of tight junction (TJ) are reported to be correlated with carcinogenesis and tumor development. In this study, the translocation of TJ protein Zonula occludens-1 (ZO-1) and the activation of epidermal growth factor receptor (EGFR) were examined to demonstrate the involvement and correlation of TJ protein translocation and EGFR activation in the cell dissociation and subsequent invasion of pancreatic cancer. Immunocytochemistry, fluorescence intensity analysis and in vitro invasion assay were performed in dissociated and non-dissociated pancreatic cancer cells. The obvious translocation of cell-cell junction localized ZO-1 protein to the cytoplasm and nucleus, simultaneous activation of EGFR, as well as the dissociation of cell colonies of non-dissociated pancreatic cancer cells were induced by dissociation factor treatment. However, EGFR inhibitor, AG1478, treatment significantly induced the redistribution of ZO-1 protein to the sites of cell-cell junction and the cell aggregation, as well as simultaneous suppression of EGFR activation in both the dissociated and the non-dissociated pancreatic cancer cells. In addition, AG1478 treatment markedly enhanced the in vitro invasion of non-dissociated pancreatic cancer cells. Translocation of TJ protein ZO-1 is closely involved in the induction of invasion through EGFR activation in pancreatic cancer cells.

Involvement of matrix metalloproteinase-7 in invasion-metastasis through induction of cell dissociation in pancreatic cancer.

Epidermal growth factor receptor (EGFR) mediated mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway was isolated as invasion-metastasis related factor in pancreatic cancer in our previous studies. Matrix metalloproteinase-7 (MMP-7) and tight junction (TJ) proteins are indicated to be involved in cancer invasion-metastasis. To clarify the underlying mechanism of involvement of MMP-7 in cancer invasion, western blotting, invasion assay and immunohistochemistry were performed in dissociated (PC-1.0 and AsPC-1) and non-dissociated (PC-1 and Capan-2) pancreatic cancer cells, as well as pancreatic cancer tissues. Intracellular MMP-7 protein presented as pre-proenzyme and its expression was decreased by AG1478 (EGFR inhibitor) or U0126 (MEK inhibitor) treatment in pancreatic cancer cells. Activated MMP-7 protein was only detected in the medium of PC-1.0 and AsPC-1 cells, but not detected in the medium of PC-1 and Capan-2 cells. Moreover, MMP-7 treatment significant induced the dissociation of cell colonies in PC-1 and Capan-2 cells. Synchronously, TJ structure was apparently disrupted and translocation of TJ proteins to cytoplasm or extracellular medium was induced in PC-1 and Capan-2 cells. Furthermore, MMP-7 treatment markedly increased the in vitro invasion of PC-1 and Capan-2 cells. In addition, MMP-7 expression at the invasive front was obviously stronger than that at the center of pancreatic cancer tissues. Activation of MMP-7 protein is closely involved in disruption of TJ structure and consequent induction of cell dissociation as well as invasion in pancreatic cancer. EGFR mediated MEK/ERK signaling pathway is implied to be involved in regulation of MMP-7 expression in pancreatic cancer cells.

Clinical significance of MHC class II-associated invariant chain expression in human gastric carcinoma.

MHC class II antigens serve as restricted elements for cell presenting antigens to CD4+ helper T cells. CD4+ T cells and CD8+ cytotoxic T cells, which are tumor-infiltrating lymphocytes (TILs), and play a major role in the survey and attack against tumor cells in primary lesions. Invariant chain (Ii) has several functions in MHC class II-restricted antigen presentation. In addition, Ii is found to be closely involved in the regulation of anti-tumor immunity in several tumor types. However, the significance of Ii expression in tumor cells is not fully illustrated. Immunohistochemical staining of Ii expression was performed in 58 cases of human gastric carcinoma specimens. The prognostic analysis of patients with gastric carcinoma was also performed. A total of 67.2% (39/58) gastric carcinomas were found to be Ii-positive, whereas only 20.7% (12/58) showed positive immunoreactivity with anti-MHC class II determinants. Furthermore, Ii expression showed significant correlation with the differentiation of gastric carcinoma (p<0.05). Ii expression also showed an inverse correlation with the frequency of TILs around carcinoma tissues, as well as with the prognosis of gastric carcinoma (p<0.01). Ii expression is closely correlated with anti-tumor immunity in human gastric carcinoma. Therefore, Ii may serve as an independent clinical marker for poor biological behavior and prognostic analysis in patients with gastric carcinoma.

Mechanisms of peritoneal metastasis after operation for non-serosa-invasive gastric carcinoma: an ultrarapid detection system for intraperitoneal free cancer cells and a prophylactic strategy for peritoneal metastasis.

PURPOSE: This aims of this study are to establish an ultra-rapid quantitative reverse transcription-PCR (RT-PCR) protocol that enables the diagnosis of i.p. cancer spread during operation, to reveal the mechanisms of peritoneal metastasis from non-serosa-invasive gastric carcinoma, and to evaluate the effect of the extensive intraoperative peritoneal lavage (EIPL) using the ultra-rapid quantitative RT-PCR as a prophylactic strategy for peritoneal metastasis. EXPERIMENTAL DESIGN: Peritoneal lavage samples from 63 patients with non-serosa-invasive gastric carcinoma were obtained at laparotomy and immediately after lymph node dissection. To identify the free cancer cells in the samples, carcinoembryonic antigen- and cytokeratin 20-specific RT-PCRs were performed using the LightCycler method in combination with an automated mRNA extractor. In addition, EIPL was performed in five cases with serosa-invasive gastric carcinoma, and its efficacy was evaluated by the ultra-rapid quantitative RT-PCR protocol. RESULTS: The method enabled us to complete the detection of cancer cells within approximately 70 min. Both the carcinoembryonic antigen and cytokeratin 20 mRNA in i.p. lavages after lymph node dissection were identified in three (14.3%), four (26.7%), and six (46.2%) patients with submucosal, muscularis propria, and subserosal tumors, respectively. Lymph node metastasis was the independent predictor of the existence of i.p. free cancer cells. The ultra-rapid quantitative RT-PCR demonstrated that EIPL reduced free cancer cells from 3.8 x 10(5) +/- 1.4 x 10(5) cells to 2.8 +/- 1.5 cells/100 ml lavage after six to eight washes, and they disappeared after seventh to ninth wash. CONCLUSIONS: The present study proved that lymph node dissection opened lymphatic channels and spread viable cancer cells into the peritoneal cavity. It is suggested that the combination of the novel detection system with the intraoperative therapy of EIPL can be a useful prophylactic strategy for peritoneal metastasis from gastric carcinoma.

Mouse homologue of a novel human oncofetal antigen, glypican-3, evokes T-cell-mediated tumor rejection without autoimmune reactions in mice.

PURPOSE AND EXPERIMENTAL DESIGN: We recently identified glypican-3 (GPC3) overexpressed specifically in human hepatocellular carcinoma, as based on cDNA microarray analysis of 23,040 genes, and we reported that GPC3 is a novel tumor marker for human hepatocellular carcinoma and melanoma. GPC3, expressed in almost all hepatocellular carcinomas and melanomas, but not in normal tissues except for placenta or fetal liver, is a candidate of ideal tumor antigen for immunotherapy. In this study, we attempted to identify a mouse GPC3 epitope for CTLs in BALB/c mice, and for this, we set up a preclinical study to investigate the usefulness of GPC3 as a target for cancer immunotherapy in vivo. RESULTS: We identified a mouse GPC3-derived and Kd- restricted CTL epitope peptide in BALB/c mice. Inoculation of this GPC3 peptide-specific CTL into s.c. Colon26 cancer cells transfected with mouse GPC3 gene (C26/GPC3) led to rejection of the tumor in vivo, and i.v. inoculation of these CTLs into sublethally irradiated mice markedly inhibited growth of an established s.c. tumor. Inoculation of bone marrow-derived dendritic cells pulsed with this peptide prevented the growth of s.c. and splenic C26/GPC3 accompanied with massive infiltration of CD8+ T cells into tumors. Evidence of autoimmune reactions was never observed in surviving mice that had rejected tumor cell challenges. CONCLUSIONS: We found the novel oncofetal protein GPC3 to be highly immunogenic in mice and elicited effective antitumor immunity with no evidence of autoimmunity. GPC3 is useful not only for diagnosis of hepatocellular carcinoma and melanoma but also for possible immunotherapy or prevention of these tumors.

Pancreatectomy using the no-touch isolation technique followed by extensive intraoperative peritoneal lavage to prevent cancer cell dissemination: a pilot study.

CONTEXT: In pancreatic cancer, even for patients who have undergone curative resection (R0), survival analysis has revealed a poor survival rate due to cancer recurrence. Because the operation itself might have caused the dissemination of these cancer cells, the no-touch isolation technique and extensive intraoperative peritoneal lavage may be a potential operative procedure for improving the outcome. PATIENTS: Eight patients treated by the no-touch isolation technique were compared with 10 patients treated using conventional techniques. MAIN OUTCOME MEASURES: Cancer cell detection rates in the portal venous blood, frequency of recurrence, and survival rate. We also analyzed the lymphatic fluid squeezed from the resected cancerous pancreatic tissue. RESULTS: In 5 out of 10 cases (50%) in the conventional procedure group, CEA mRNA was identified in the portal blood after tumor manipulation, while only 1 out of 8 cases (13%) in the no-touch isolation technique group was positive for portal CEA mRNA. All lymphatic fluid samples squeezed from the resected cancerous pancreatic tissue were positive (8/8) for CEA mRNA. The recurrence rate was 90% (9/10) in the conventional procedure group, and 38% (3/8) in the no-touch isolation technique group (P=0.043). In the conventional procedure group, hepatic metastasis, local recurrence, peritoneal dissemination, and extraabdominal recurrence were identified in 6 (60%), 4 (40%), 4 (40%), and 2 patients (20%), respectively. On the other hand, among the no-touch isolation technique group, recurrence was identified in 1 (13%), 1 (13%), 0 (0%), and 1 patient (13%), respectively. There was no peritoneal dissemination along with the decreased hepatic recurrence rate. Mean (+/-SEM) survival time was 21.2+/-5.8 months for the conventional procedure group and 41.5+/-5.6 months for the no-touch isolation technique group (P=0.018). The 3-year survival rate was 12.5+/-11.5% for the conventional procedure group and 75.0+/-21.7% for the no-touch isolation technique group. CONCLUSION: This study presented the potential of cancer dissemination during the intraoperative manipulation of tumors and its contribution to cancer recurrence, as well as the significance of the no-touch isolation technique and extensive intraoperative peritoneal lavage for pancreatic cancer surgery.

[Utility of convex echo probe in laparoscopic radio frequency ablation therapy for hepatocellular carcinoma].

We have examined the utility of the convex echo probe, which has the fine gutter of a puncture needle in laparoscopic radio frequency ablation therapy. When we use a flexible linear echo probe in RFA treatment, we have to puncture tumor with the hand piece in free hand. But it is difficult to treat in the case of HCC which is located in S1 and the lower area of S5 and S6 because we have a narrow space where colon, duodenum and netz are close for safe and exact puncturing of the tumor. We used a convex echo probe in RFA to the above mentioned area of the liver. We punctured with the hand piece exactly and easily without preliminary puncturing of the tumor. So we can perform RFA treatment successfully and safely by a choice of an appropriate echo probe.

Genetic pathways of 'de novo' colorectal carcinomas with reference to fetal-type glycogen phosphorylase positive foci.

'De novo' carcinogenesis has been advocated besides 'adenoma carcinoma sequence' as another dominant pathway leading to the colorectal carcinoma. Our previous study demonstrated that brain (fetal)-type glycogen phosphorylase (BGP) positive foci in the transitional mucosa (BGP foci) have frequent p53 mutations and that the distribution of BGP foci has a close relationship with the location of 'de novo' carcinoma. The aims of the present study were to investigate further genetic alterations in the BGP foci and to clarify the mechanism of 'de novo' carcinogenesis. Twenty-eight colorectal carcinomas with invasion into submucosa or superficial muscularis propria without any adenoma component expressing immunoreactive p53 protein were selected from 168 resected specimens. Investigations of the p53, K-ras and APC mutations was performed in the BGP foci, BGP negative colorectal mucosa and 'de novo' carcinoma using PCR-SSCP and DNA squencing. In all 28 cases, immunoreactive BGP was positive in the carcinomas and the BGP foci were observed sporadically in the mucosa adjacent to the carcinoma. No K-ras mutation was observed in either carcinoma or BGP foci in any of the cases. Mutations of p53 and APC were 14 (50.0%) and 9 (32.1%) in 'de novo' carcinomas, and 11 (39.3%) and 1 (3.6%) in BGP foci, respectively. Both p53 and APC mutations were detected in 8 and 1, p53 mutation alone in 6 and 10, APC mutation alone in 1 and 0 out of 28 carcinomas and BGP positive foci, respectively. These results suggest that the BGP foci may play a very important role in the 'de novo' colorectal carcinogenesis from the frequent genetic alterations of p53, and that there may be two major pathways, i.e., the p53-APC pathway and the p53 alone pathway, from the chain of genetic alterations between BGP foci and 'de novo' carcinoma.

Relationship between activation of epidermal growth factor receptor and cell dissociation in pancreatic cancer.

In our previous investigations, mitogen-activated protein kinase kinase 2 (MEK2)/extracellular signal-regulated kinase 2 (ERK2) signaling pathway was found to be correlated with the cell dissociation induced by dissociation factor (DF) in pancreatic cancer cells. In this study, the expressions of epidermal growth factor receptor (EGFR), phosphorylated EGFR (p-EGFR), and its downstream kinases MEK1/2 and ERK1/2, were analyzed to clarify the regulatory mechanism of cell dissociation in pancreatic cancer cells. Two hamster (PC-1.0 and PC-1) and two human (AsPC-1 and Capan-2) pancreatic cancer cell lines were used. Immunocytochemical study was performed using anti-EGFR, p-EGFR, phosphorylated MEK1/2 (p-MEK1/2), and phosphorylated ERK1/2 (p-ERK1/2) antibodies. DF-treatment markedly induced the expressions of EGFR, p-EGFR, p-MEK1/2, p-ERK1/2, as well as the dissociation of cell colonies in PC-1 and Capan-2 cells. In contrast, AG1478 (an EGFR inhibitor) treatment significantly induced the cell aggregation in PC-1.0 and AsPC-1 cells which usually grew as single cells, but strongly suppressed the expressions of EGFR, p-EGFR, p-MEK1/2, and p-ERK1/2. These observations demonstrate that activation of EGFR is closely involved in cell dissociation in pancreatic cancer through activating MEK/ERK signaling pathway.

Arrangement of expression and distribution of tight junction protein claudin-1 in cell dissociation of pancreatic cancer cells.

Mitogen-activated protein kinase kinase 2 (MEK2) was isolated previously as a potential factor related to cancer cell dissociation in highly (PC-1.0) and weakly (PC-1) invasive pancreatic cancer cells. On the other hand, changes of structure and function of tight junction (TJ) are reported to be correlated with carcinogenesis and tumor development. In this study, immunocytochemistry and Western blot analysis were performed in pancreatic cancer cells using anti-claudin-1, MEK2 and phosphorylated MEK1/2 (p-MEK1/2) antibodies to reveal the correlation between TJ and cancer cell dissociation, as well as the involvement of MEK2 in regulation of TJ in cell dissociation of pancreatic cancer. After incubation with conditioned medium of PC-1.0 cells, plasma membrane distribution of claudin-1 was obviously disrupted, and expressions of MEK2 and p-MEK1/2, as well as dissociation of cell colonies, were significantly induced in PC-1 and CAPAN-2 cells. However, U0126 (a MEK1/2 inhibitor) treatment apparently induced the plasma membrane distribution of claudin-1 and aggregation of single cells in PC-1.0 and AsPC-1 cells, synchronously seriously suppressed MEK2 and p-MEK1/2 expression. Arrangement of expression and distribution of claudin-1 is closely related to cell dissociation status in pancreatic cancer cells through MEK2 activation.