Targeting Pyruvate Kinase M2 and Lactate Dehydrogenase A Is an Effective Combination Strategy for the Treatment of Pancreatic Cancer.

Reprogrammed glucose metabolism is one of the hallmarks of cancer, and increased expression of key glycolytic enzymes, such as pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA), has been associated with poor prognosis in various malignancies. Targeting these enzymes could attenuate aerobic glycolysis and inhibit tumor proliferation. We investigated whether the PKM2 activator, TEPP-46, and the LDHA inhibitor, FX-11, can be combined to inhibit in vitro and in vivo tumor growth in preclinical models of pancreatic cancer. We assessed PKM2 and LDHA expression, enzyme activity, and cell proliferation rate after treatment with TEPP-46, FX-11, or a combination of both. Efficacy was validated in vivo by evaluating tumor growth, PK and LDHA activity in plasma and tumors, and PKM2, LDHA, and Ki-67 expression in tumor tissues following treatment. Dual therapy synergistically inhibited pancreatic cancer cell proliferation and significantly delayed tumor growth in vivo without apparent toxicity. Treatment with TEPP-46 and FX-11 resulted in increased PK and reduced LDHA enzyme activity in plasma and tumor tissues and decreased PKM2 and LDHA expression in tumors, which was reflected by a decrease in tumor volume and proliferation. The targeting of glycolytic enzymes such as PKM2 and LDHA represents a promising therapeutic approach for the treatment of pancreatic cancer.

Numerical and Experimental Study of Mechanisms Involved in Boiling Histotripsy.

The aim of boiling histotripsy is to mechanically fractionate tissue as an alternative to thermal ablation for therapeutic applications. In general, the shape of a lesion produced by boiling histotripsy is tadpole like, consisting of a head and a tail. Although many studies have demonstrated the efficacy of boiling histotripsy for fractionating solid tumors, the exact mechanisms underpinning this phenomenon are not yet well understood, particularly the interaction of a boiling vapor bubble with incoming incident shockwaves. To investigate the mechanisms involved in boiling histotripsy, a high-speed camera with a passive cavitation detection system was used to observe the dynamics of bubbles produced in optically transparent tissue-mimicking gel phantoms exposed to the field of a 2.0-MHz high-intensity focused ultrasound (HIFU) transducer. We observed that boiling bubbles were generated in a localized heated region and cavitation clouds were subsequently induced ahead of the expanding bubble. This process was repeated with HIFU pulses and eventually resulted in a tadpole-shaped lesion. A simplified numerical model describing the scattering of the incident ultrasound wave by a vapor bubble was developed to help interpret the experimental observations. Together with the numerical results, these observations suggest that the overall size of a lesion induced by boiling histotripsy is dependent on the sizes of (i) the heated region at the HIFU focus and (ii) the backscattered acoustic field by the original vapor bubble.

Vps33b is crucial for structural and functional hepatocyte polarity.

BACKGROUND & AIMS: In the normal liver, hepatocytes form a uniquely polarised cell layer that enables movement of solutes from sinusoidal blood to canalicular bile. Whilst several cholestatic liver diseases with defects of hepatocyte polarity have been identified, the molecular mechanisms of pathogenesis are not well defined. One example is arthrogryposis, renal dysfunction and cholestasis syndrome, which in most patients is caused by VPS33B mutations. VPS33B is a protein involved in membrane trafficking that interacts with RAB11A at recycling endosomes. To understand the pathways that regulate hepatocyte polarity better, we investigated VPS33B deficiency using a novel mouse model with a liver-specific Vps33b deletion. METHODS: To assess functional polarity, plasma and bile samples were collected from Vps33b liver knockout (Vps33bfl/fl-AlfpCre) and control (Vps33bfl/fl) mice; bile components or injected substrates were quantitated by mass spectrometry or fluorometry. For structural analysis, livers underwent light and transmission electron microscopy. Apical membrane and tight junction protein localisation was assessed by immunostaining. Adeno-associated virus vectors were used for in vivo gene rescue experiments. RESULTS: Like patients, Vps33bfl/fl-AlfpCre mice showed mislocalisation of ATP-binding cassette proteins that are specifically trafficked to the apical membrane via Rab11a-positive recycling endosomes. This was associated with retention of bile components in blood. Loss of functional tight junction integrity and depletion of apical microvilli were seen in knockout animals. Gene transfer partially rescued these defects. CONCLUSIONS: Vps33b has a key role in establishing structural and functional aspects of hepatocyte polarity and may be a target for gene replacement therapy. LAY SUMMARY: Hepatocytes are liver cells with tops and bottoms; that is, they are polarised. At their bottoms they absorb substances from blood. They then, at their tops, secrete these substances and their metabolites into bile. When polarity is lost, this directional flow of substances from blood to bile is disrupted and liver disease follows. In this study, using a new mouse model with a liver-specific mutation of Vps33b, the mouse version of a gene that is mutated in most patients with arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome, we investigated how the Vps33b gene product contributes to establishing hepatocyte polarity. We identified in these mice abnormalities similar to those in children with ARC syndrome. Gene transfer could partly reverse the mouse abnormalities. Our work contributes to the understanding of VPS33B disease and hepatocyte polarity in general, and may point towards gene transfer mediated treatment of ARC liver disease.

A Novel Approach to Ultrasound-Mediated Tissue Decellularization and Intra-Hepatic Cell Delivery in Rats.

Liver transplantation is the mainstay of treatment for end stage liver diseases, including metabolic and congenital liver diseases. The number of suitable donor organs is, however, limited, and a whole-liver transplant requires complex surgery. Cell therapy, such as intra-portal hepatocytes transplantation, has been considered as a bridging therapy to liver transplantation but has shown a mixed clinical outcome with limited success, including low level of engraftment of transplanted hepatocytes. Here, we report a novel cell delivery technique in a rat model by creating a cavity inside the liver parenchyma by non-invasive high intensity focused ultrasound histotripsy. Our in vivo experimental results together with histologic observations show that direct injection of cells inside the cavity can facilitate successful uptake, proliferation and integration of the transplanted hepatocytes in the recipient liver. We were able to restore the plasma albumin level to 50% of the normal level in Nagase analbuminemic rats (serum albumin level of the Nagase rats was initially nil) by cell therapy after high intensity focused ultrasound-mediated histotripsy. We believe that this novel technique would enable the delivery of a large number of cells into the liver to restore liver function, particularly as a treatment for metabolic liver diseases. This novel method of intra-hepatic hepatocyte transplantation might be an invaluable tool for cell therapy in the future.

Pyruvate Kinase M2 and Lactate Dehydrogenase A Are Overexpressed in Pancreatic Cancer and Correlate with Poor Outcome.

Pancreatic cancer has a 5-year survival rate of less than 4%. Despite advances in diagnostic technology, pancreatic cancer continues to be diagnosed at a late and incurable stage. Accurate biomarkers for early diagnosis and to predict treatment response are urgently needed. Since alteration of glucose metabolism is one of the hallmarks of cancer cells, we proposed that pyruvate kinase type M2 (M2PK) and lactate dehydrogenase A (LDHA) enzymes could represent novel diagnostic markers and potential therapeutic targets in pancreatic cancer. In 266 tissue sections from normal pancreas, pancreatic cystic neoplasms, pancreatic intraepithelial neoplasia (PanIN) and cancer, we evaluated the expression of PKM2, LDHA, Ki-67 and CD8+ by immunohistochemistry and correlated these markers with clinicopathological characteristics and patient survival. PKM2 and LDHA expression was also assessed by Western blot in 10 human pancreatic cancer cell lines. PKM2 expression increased progressively from cyst through PanIN to cancer, whereas LDHA was overexpressed throughout the carcinogenic process. All but one cell line showed high expression of both proteins. Patients with strong PKM2 and LDHA expression had significantly worse survival than those with weak PKM2 and/or LDHA expression (7.0 months vs. 27.9 months, respectively, p = 0.003, log rank test). The expression of both PKM2 and LDHA correlated directly with Ki-67 expression, and inversely with intratumoral CD8+ cell count. PKM2 was significantly overexpressed in poorly differentiated tumours and both PKM2 and LDHA were overexpressed in larger tumours. Multivariable analysis showed that combined expression of PKM2 and LDHA was an independent poor prognostic marker for survival. In conclusion, our results demonstrate a high expression pattern of two major glycolytic enzymes during pancreatic carcinogenesis, with increased expression in aggressive tumours and a significant adverse effect on survival.

A novel rat model of liver regeneration: possible role of cytokine induced neutrophil chemoattractant-1 in augmented liver regeneration.

BACKGROUND: Liver resection is the mainstay of treatment for most of the liver tumors. Liver has a unique capability to restore the lost volume following resection, however, most of the primary tumors grow in a liver with preexisting parenchymal diseases and secondary tumors often present in multiple liver lobes precluding a safe curative resection. Two-stage hepatectomy and portal vein ligation (PVL) are used to achieve a safer future remnant liver volume (FRLV), however, these procedures take several weeks to achieve adequate FRLV. A recently introduced faster alternative two-stage hepatectomy, also know as associated liver partitioning and portal vein ligation for staged hepatectomy (ALPPS), produces a desirable FRLV in days. METHODS: To have an insight into the mechanism of ALPPS associated liver regeneration, we reproduced a rat model of ALPPS and compared the results with the PVL group. RESULTS: Our results convincingly showed an advantage of the ALPPS procedure over PVL group in terms of early regeneration, however, in 1-week time the amount of regeneration was comparable. An early regeneration in the ALPPS group coincided with an early entry of hepatocytes into the cell proliferation phase, a significant increase in portal pressure and increase in hepatic enzymes in the ALPPS group compared with the PVL group. According to the protein array evaluation of 29 cytokines/chemokines, cytokine induced neutrophil chemoattractant-1 had the highest expression whereas IL-6 had the highest fold (>6 vs PVL group) expression at the early phase of regeneration in the ALPPS group. CONCLUSIONS: This unique rat model of ALPPS would help to improve our understanding about the liver generation process and also will help in further refinement of the ALPPS procedure for the clinical benefit.

Role of toll-like receptor 4 in mediating multiorgan dysfunction in mice with acetaminophen induced acute liver failure.

Strategies for the prevention of multiorgan dysfunction (MOD) in acetaminophen (APAP)-induced acute liver failure (ALF) are an unmet need. Our study tested the hypothesis that sterile inflammation induced by APAP in a mouse model would activate toll-like receptor 4 (TLR4) in the liver and extrahepatic organs and lead to the progression of ALF and MOD and that the administration of the novel TLR4 antagonist STM28 (a peptide formed of 17 amino-acids) would prevent liver injury and associated MOD. ALF and, subsequently, MOD were induced in TLR4-knockout (KO) mice (B6.B10ScN-Tlr4 (lpsdel) /JthJ) and wild-type (WT) mice (C57BL/6) with APAP (500 mg/kg). A second set of experiments was conducted to evaluate the effects of a pretreatment with a novel TLR4 antagonist, STM28, on APAP-induced MOD in CD1 mice. Animals were sacrificed at the coma stage, and plasma, peripheral blood cells, liver, kidneys, and brain were collected. Biochemistry values and cytokines were measured. Liver and kidneys were studied histologically and were stained for TLR4 and activated Kupffer cells, and the expression of nuclear factor kappa B-p65 was quantified with western blotting. Brain water was measured in the frontal cortex. After APAP administration, TLR4-KO (NFkBp65) mice were relatively protected from liver necrosis and end-organ dysfunction and had significantly better survival than WT controls (P < 0.01). STM28 attenuated liver injury and necrosis, reduced creatinine levels, and delayed the time to a coma significantly. The increases in cytokines in the plasma and liver, including TLR4 expression and the activation of Kupffer cells, after APAP administration were reduced significantly in the STM28-treated animals. The increased number of circulating myeloid cells was reduced significantly after STM28 treatment. In conclusion, these data provide evidence for an important role of the TLR4 antagonist in the prevention of the progression of APAP-induced ALF and MOD.

Importance of Connexin-43 based gap junction in cirrhosis and acute-on-chronic liver failure.

BACKGROUND & AIMS: In cirrhosis, superimposed inflammation often culminates in acute-on-chronic liver failure (ACLF) but the mechanism underlying this increased sensitivity is not clear. Cx43 is a ubiquitous gap junction protein that allows transmission of signals between cells at a much higher rate than the constitutively expressed gap junctions. The aims of the study were to test the hypothesis that inflammation drives the increased expression of hepatic Cx43 and to determine its role by Cx43 inhibition. METHODS: Four weeks after bile-duct ligation (BDL) or sham operation, rats were treated with an anti-TNF antibody, or saline; with or without LPS (1mg/kg); given 3h prior to termination. Biochemistry and cytokines were measured in the plasma and hepatic protein expression (NFkB, TNFα, iNOS, 4HNE, Cx26, 32, and 43) and confocal microscopy (Cx26, 32, and 43) were performed. The effect of a Cx43-specific inhibitory peptide was studied in a mouse BDL model. RESULTS: BDL animals administered LPS developed typical features of ACLF but animals administered infliximab were relatively protected. Cx26/32 expression was significantly decreased in BDL animals while Cx43 was significantly increased and increased further following LPS. Infliximab treatment prevented this increase. However, inhibiting Cx43 in BDL mice produced detrimental effects with markedly greater hepatocellular necrosis. CONCLUSIONS: The results of this study show for the first time an increased expression of hepatic Cx43 in cirrhosis and ACLF, which was related to the severity of inflammation. This increased Cx43 expression is likely to be an adaptive protective response of the liver to allow better cell-to-cell communication.

Pyruvate kinase M2 is a novel diagnostic marker and predicts tumor progression in human biliary tract cancer.

BACKGROUND: The early diagnosis of biliary tract cancer (BTC) remains challenging, and there are few effective therapies. This study investigated whether the M2 isotype of pyruvate kinase (M2-PK), which serves as the key regulator of cellular energy metabolism in proliferating cells, could play a role in the diagnosis and therapy of BTC. METHODS: Plasma and bile M2-PK concentrations were measured by enzyme-linked immunosorbent assay in 88 patients with BTC, 79 with benign biliary diseases, and 17 healthy controls. M2-PK expression was assayed in a BTC tissue array by immunohistochemistry. The role of M2-PK in tumor growth, invasion, and angiogenesis was evaluated in BTC cell lines by retrovirus-mediated M2-PK transfection and short hairpin RNA silencing techniques. RESULTS: Sensitivity (90.3%) and specificity (84.3%) of bile M2-PK for malignancy were significantly higher than those for plasma M2-PK and serum carbohydrate antigen 19-9. M2-PK expression was specific for cancer cells and correlated with microvessel density. M2-PK positivity was a significant independent prognostic factor by multivariable analysis. Transfection of M2-PK in a negatively expressed cell line (HuCCT-1 cells) increased cell invasion, whereas silencing in an M2-PK-positive cell line (TFK cells) decreased tumor nodule formation and cellular invasion. A significant increase in endothelial tube formation was noted when supernatants from M2-PK-transfected cells were added to an in vitro angiogenesis assay, whereas supernatants from silenced cells negated endothelial tube formation. CONCLUSIONS: Bile M2-PK is a novel tumor marker for BTC and correlates with tumor aggressiveness and poor outcome. Short hairpin RNA-mediated inhibition of M2-PK indicates the potential of M2-PK as a therapeutic target.

Dynamic tracking of stem cells in an acute liver failure model.

AIM: To investigate a dual labeling technique, which would enable real-time monitoring of transplanted embryonic stem cell (ESC) kinetics, as well as long-term tracking. METHODS: Liver damage was induced in C57/BL6 male mice (n = 40) by acetaminophen (APAP) 300 mg/kg administered intraperitoneally. Green fluorescence protein (GFP) positive C57/BL6 mouse ESCs were stained with the near-infrared fluorescent lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) immediately before transplantation into the spleen. Each of the animals in the cell therapy group (n = 20) received 5 × 10(6) ESCs 4 h following treatment with APAP. The control group (n = 20) received the vehicle only. The distribution and dynamics of the cells were monitored in real-time with the IVIS Lumina-2 at 30 min post transplantation, then at 3, 12, 24, 48 and 72 h, and after one and 2 wk. Immunohistochemical examination of liver tissue was used to identify expression of GFP and albumin. Plasma alanine aminotransferase (ALT) was measured as an indication of liver damage. RESULTS: DiR-stained ESCs were easily tracked with the IVIS using the indocyanine green filter due to its high background passband with minimal background autofluorescence. The transplanted cells were confined inside the spleen at 30 min post-transplantation, gradually moved into the splenic vein, and were detectable in parts of the liver at the 3 h time-point. Within 24 h of transplantation, homing of almost 90% of cells was confirmed in the liver. On day three, however, the DiR signal started to fade out, and ex vivo IVIS imaging of different organs allowed signal detection at time-points when the signal could not be detected by in vivo imaging, and confirmed that the highest photon emission was in the liver (P < 0.0001). At 2 wk, the DiRsignal was no longer detectable in vivo; however, immunohistochemistry analysis of constitutively-expressed GFP was used to provide an insight into the distribution of the cells. GFP +ve cells were detected in tissue sections resembling hepatocytes and were dispersed throughout the hepatic parenchyma, with the presence of a larger number of GFP +ve cells incorporated within the sinusoidal endothelial lining. Very faint albumin expression was detected in the transplanted GFP +ve cells at 72 h; however at 2 wk, few cells that were positive for GFP were also strongly positive for albumin. There was a significant improvement in serum levels of ALT, albumin and bilirubin in both groups at 2 wk when compared with the 72 h time-point. In the cell therapy group, serum ALT was significantly (P = 0.016) lower and albumin (P = 0.009) was significantly higher when compared with the control group at the 2 wk time-point; however there was no difference in mortality between the two groups. CONCLUSION: Dual labeling is an easy to use and cheap method for longitudinal monitoring of distribution, survival and engraftment of transplanted cells, and could be used for cell therapy models.

Role of peroxisome proliferator-activated receptor-gamma (PPARgamma) during liver regeneration in rats.

BACKGROUND AND AIM: Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear receptor superfamily, is widely expressed in adipocytes and other tissues, including the liver. Several reports have shown that PPARgamma activation induced cell-cycle arrest and apoptosis in tumor cells. We investigated the role of the PPARgamma/ligand system and the effect of the PPARgamma agonist during liver regeneration. METHODS: Expression of PPARgamma and serum levels of 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) by enzyme immunoassay were evaluated in rats following partial hepatectomy (PH group). Further, the effect of the PPARgamma agonist, pioglitazone, on liver regeneration (PH + PGZ group) was evaluated by proliferating cell nuclear antigen labeling index, relative liver weight, and expression of cell-cycle regulators. RESULTS: The number of PPARgamma-stained hepatocytes decreased at 24 h (PH, 15.8 +/- 2.2%; sham, 35.5 +/- 2.4%; P < 0.001) and increased in the late phase of liver regeneration compared to the sham-operated group (P < 0.001 at 48-120 h). The peaks of serum 15d-PGJ2 (627.0 +/- 91.1 pg/ml) and PPARgamma expression (90.6 +/- 3.1%) coincided in the late phase of liver regeneration. Also, oral administration of pioglitazone inhibited hepatocyte proliferation, in terms of the proliferating cell nuclear antigen (PCNA) labeling index and p27 expression during the late phase of liver regeneration, and caused a transient reduction in liver mass when compared to the PH group. CONCLUSIONS: These results indicate that the PPARgamma/ligand system may be one of the key negative regulators of hepatocyte proliferation and may be responsible for the inhibition of liver growth in the late phase of liver regeneration.

Expression of metastin and a G-protein-coupled receptor (AXOR12) in epithelial ovarian cancer.

BACKGROUND: Metastin, a product of the KiSS-1 gene, is a ligand for a G-protein-coupled receptor (AXOR12) and is a strong suppressant of metastasis. The aim of this study was to evaluate whether metastin and AXOR12 gene expressions affect prognosis of patients with epithelial ovarian cancer. METHODS: The expression levels of metastin, AXOR12 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression were analysed by the real-time quantitative reverse transcription-polymerase chain reaction in 76 epithelial ovarian cancer surgical specimens. Their expression (metastin/GAPDH and AXOR12/GAPDH ratios) was correlated with the clinical findings. Furthermore, cellular distribution of metastin and AXOR12 mRNA was examined by in situ hybridisation on tissue sections. RESULTS: The median and range of mRNA expression for metastin and AXOR12 were 0.047 and 0.01-13.57, and 4.00 and 0.011-135.13, respectively. Patients were dichotomised into two groups having low and high expressions by using the median value as the cutoff. A good agreement was noticed between metastin and AXOR12 gene expression levels (kappa coefficient; 0.74). The presence of residual tumour following resection was negatively associated with metastin (P=0.0084) and AXOR12 (P=0.0148) gene expressions indicating an association of low expression of these genes in more aggressive, and advanced tumours. By univariate Cox regression analysis, the prognosis of the patients with low AXOR12 gene expression was significantly worse than those with high AXOR12 gene expression (P=0.030). The combination of metastin and AXOR12 gene expression level was also significantly associated with the prognosis (P=0.049). Transcripts for both metastin and AXOR12 were detected in the epithelial ovarian carcinoma cells. CONCLUSIONS: These results present a new insight into the understanding of the biological behaviour of epithelial ovarian cancer. Metastin/AXOR12 signalling may suppress the invasive phenotype of epithelial ovarian cancer.

Appraisal of a revised lymph node classification system for esophageal squamous cell cancer.

BACKGROUND: Node-positive patients with esophageal carcinoma constitute a heterogeneous population with a variable prognosis, which the current staging system insufficiently addresses. To that end, 863 patients with a curative resection for esophageal squamous cell carcinoma were analyzed to evaluate a useful and simple nodal classification system. METHODS: Along with standard conventional clinicopathologic factors, data for metastatic lymph node (MLN) number, metastatic to examined LN ratio (MLN ratio), and MLN size were evaluated. The greatest microscopic dimension of the metastatic tumor inside the largest MLN (MLN size) was measured on histopathologic slides. Patients with MLNs were classified into n1 (< 9 mm) and n2 (> or = 9 mm) groups, according to size of MLNs (n-stage). RESULTS: The paratracheal LNs most frequently contained the largest MLN and among them the right recurrent laryngeal LNs were the most common site (81.8%). Patients were stratified into significant groups by all the nodal criteria. In multivariable analysis, MLN size n-stage and MLN ratio N-stage were the best independent predictors for disease-free and overall survival, respectively. In the disease-free survival, MLN ratio N-stage subcategories were divided into prognostic groups according to the n-stage. A combined nodal staging strategy combining the n-stage and N-stage had the strongest prognostic value and was used for the tumor-node-metastasis classification with distinct separation of patients into prognostic groups. CONCLUSIONS: Results of this study indicate that the MLN size may serve as an accurate metric to classify node-positive patients and a combination of the MLN ratio and size may have synergism in classifying node-positive patients into prognostically homogenous groups.

Differential expression of RUNX genes in human esophageal squamous cell carcinoma: downregulation of RUNX3 worsens patient prognosis.

BACKGROUND: The RUNX proteins are a family of transcriptional factors that have essential functions during embryogenesis and development, whereas deregulation in expression of RUNXs is often linked to tumor formation. To date, there has been no study describing the precise expression, prognostic impact and methylation status of RUNXs in esophageal squamous cell carcinoma. METHODS: Resected specimens from 61 patients with esophageal SCC were used to identify the expression of RUNX1, RUNX2 and RUNX3 by real-time RT-PCR. Localization of mRNA was done by in situ hybridization, expression of Smad4 was evaluated by immunohistochemistry, and the methylation status of RUNX3 was analyzed by methylation-specific PCR. RESULTS: RUNX3 had a significant impact on patient prognosis with worse survival in the RUNX3-negative group. In early tumors (T1/T2), the prevalence of lymph vessel invasion and the number of metastatic lymph nodes were significantly higher in RUNX3-negative tumors. Furthermore, RUNX3 became a strong prognostic factor only in Smad4-positive tumors. Also, the methylation status of the RUNX3 promoter had a significant correlation with the loss of RUNX3 expression. CONCLUSION: Downregulation of RUNX3 may play a role in disease progression of esophageal SCC, and hypermethylation of the promoter region might be one of the crucial pathways to silence RUNX3 gene.

Prognosis of patients with gastric carcinoma treated in remote island hospitals in Japan: a multivariable analysis.

BACKGROUND: Treating gastric cancer in remote island hospitals remains a major clinical challenge. Factors affecting prognosis of patients treated in general hospitals are still at large. We sought to determine the characteristics of gastric cancer in the Amamiooshima (Amami) archipelago of Japan and also evaluated the independent prognostic factors by the Cox regression analysis. MATERIALS AND METHODS: A total of 125 patients treated in four sister hospitals in the Amami were analyzed. RESULTS: The median age of patients with resection was 74 years and almost 85% patients had diffuse type of cancer. The 5-year overall survival was 34% for all patients and 58% for those who had a resection. Among the several clinicopathological factors, operation method (distal vs. total gastrectomy), splenectomy, lymphatic and venous invasion, T-stage, metastatic lymph node (MLN) size n-stage and UICC N-stage had significant impact on survival. Only MLN size and intraoperative blood loss had independent effect on survival by multivariable analysis. CONCLUSION: Improved perioperative care may yield a reasonable patient survival in elderly patients with gastric carcinoma treated in remote hospitals. Restricting amount of intraoperative blood loss may further improve the patient prognosis and MLN size may serve as a new metric to stage gastric cancers.

Surgical treatment of superficial esophageal cancer.

OBJECTIVE: The worldwide incidence of superficial esophageal cancer (SEC) is increasing. The aim of this study is to review the systematic surgical outcomes of esophagectomy for SEC. DATA SOURCES: Only manuscripts written in English and written between 1980 and 2003 were selected from MEDLINE. The keywords consisting of superficial esophageal cancer, early esophageal cancer, and early stage or superficial stage or stage I in esophageal cancer were searched. STUDY SELECTION: There were no exclusion criteria for published information relevant to the topics. The most representative articles were selected when there were several articles from the same institution. Case reports were excluded. DATA EXTRACTIONS: Thirty-two manuscripts were finally collected from MEDLINE and eight articles were also added from reference lists of the pertinent literatures. In evaluating the statistical analysis of the complications of the reported literature, collective method was used. DATA SYNTHESIS: The collected information was organized. CONCLUSIONS: The conclusions drawn from those articles showed that the overall prevalence of SEC accounted around 10% and increased to 25% in the 2000s. The overall incidence of lymph node metastasis of SEC was about 25% and its incidences in mucosal and submucosal cancer were 5 and 35%, respectively. The percentage of the cases of squamous cell carcinoma (SCC) vs adenocarcinoma (AC) widely varied depending on the geographic locations reported; most SCC cases were from the Asian countries and most AC cases were from the European countries. Clinical significance of multimodal treatment for SEC has dramatically developed in the recent era and could provide various potential therapeutic options for SEC. These concepts make it possible to individualize surgical management of SEC as part of various multimodal treatments. The operative approaches for SEC varied from minimally invasive thoracoscopic esophagectomy, limited transabdominal distal esophagectomy, conventional transthoracic esophagectomy, transhiatal esophagectomy without thoracotomy, en bloc esophagectomy, and to extended esophagectomy with a complete three-field lymph node dissection. A 5-year overall survival rate of SEC after esophagectomy was good (46 to 83%) to excellent (71 and 100%) for mucosal SEC, but far from satisfactory (33 and 78%) for submucosal SEC. Early diagnosis, development of multimodal treatment, standardization of the surgical procedure including routine lymph node dissection, and improved perioperative management of patients have led to a better survival for patients with SEC.

Tetracycline analogues (doxycycline and COL-3) induce caspase-dependent and -independent apoptosis in human colon cancer cells.

Tetracycline analogues (TCNAs) possess cytotoxic activities as well as matrix metalloproteinase (MMP) inhibitory properties. Previously, we demonstrated that doxycycline (DOXY) could induce apoptosis in human HT29 colon cancer cells. In present study, the molecular apoptotic mechanisms induced by two kinds of TCNAs, designated as DOXY and COL-3 (chemically modified tetracycline-3; 6-demethyl, 6-deoxy, 4-dedimethylamino tetracycline), were evaluated in cultured HT29 cells. Both TCNAs inhibited the proliferation of 6 different colorectal cancer cell lines in a dose-dependent manner. Especially, COL-3 had a stronger effect on cancer cells than DOXY. Apoptotic changes were actually observed by 10 mug/ml COL-3 and 20 mug/ml DOXY in a time-dependent manner. COL-3 produced the increase in cytosolic cytochrome c and the loss of mitochondrial membrane potential after 3 hr treatment, and thereafter activated caspases. In case of DOXY, these changes were observed after 24 hr. Bax translocation was not a prerequisite for cytochrome c releasing in COL-3 treatment. Pretreated pancaspase inhibitor (Z-VAD-FMK) reduced COL-3 and DOXY mediated apoptosis up to 81.3 and 35.3%, as compared with nontreated cells, respectively. These data indicated that TCNAs could induce mitochondria-mediated apoptosis through both caspase-dependent and -independent pathway. In fact, endonuclease G and apoptosis-inducing factor were released into cytosol after the treatment of TCNAs, which indicated that caspase-independent apoptotic pathway is also one of the key mechanisms for the treatment of TCNAs. Taken together, we believe that TCNAs could have strong potentials for clinical application in treating colorectal cancers and improve cancer chemotherapy.

Expression of hypoxia inducible factor-1alpha during liver regeneration induced by partial hepatectomy in rats.

BACKGROUND/AIMS: It has been well established that hypoxia inducible factor-1alpha (HIF-1alpha) transcribes essential factors in cell preservation, including angiogenesis, during hypoxia. However, the transition of HIF-1alpha expression during liver regeneration remains unknown. In this study, a role of HIF-1alpha was experimentally elucidated in relation to sinusoidal endothelial reconstruction during liver regeneration. METHODS: Expression of HIF-1alpha was evaluated in the regenerating liver following 70% hepatectomy in rats. Expressions of nuclear HIF-1alpha, vascular endothelial growth factor (VEGF) and fms-like tyrosine kinase-1 (flt-1) were measured by Western blot and liver blood flow by a laser Doppler. Sinusoidal endothelial cell area (SECA) and HIF-1alpha were localized by immunohistochemistry. HIF-1alpha mRNA was measured by reverse transcription-polymerase chain reaction. RESULT: Liver blood flow and SECA were lowest 36 and 72 h following hepatectomy, respectively. Nuclear HIF-1alpha peaked at 24 h and continuously increased 72-120 h following hepatectomy. This transition was fully supported by histological HIF-1alpha expression and HIF-1alpha mRNA up-regulation. VEGF and flt-1 peaked at 120 and 12 h, respectively. CONCLUSIONS: A significant evaluation in HIF-1alpha expression was revealed in regenerating rat livers. HIF-1alpha expression was preceded by VEGF and flt-1 expression and thus may be related to sinusoidal endothelial reconstruction.

Expression of trefoil factor family members correlates with patient prognosis and neoangiogenesis.

PURPOSE: Trefoil factor family (TFF) peptides are thought to contribute to epithelial protection and restitution by virtue of their protease-resistant nature and their strong affinity for mucins. However, they are often overexpressed in tumors, where they seem to be negative prognostic factors, possibly contributing to tumor spread, although the precise mechanisms have not been defined. EXPERIMENTAL DESIGN: Tissue sections from 111 patients with curatively resected advanced gastric carcinoma were immunohistochemically stained for TFF2, ITF (TFF3), and CD34. Microvessel density was expressed as number and area of microvessels. Results were correlated with clinicopathological characteristics and patient survival. RESULTS: Forty-nine (44.1%) and 41 (36.9%) tumors were immunohistochemically positive for TFF3 and TFF2, respectively. Among the various clinicopathologic variables, overexpression of TFF3 had a significant correlation with patient age only. In addition, a significantly higher prevalence of positive TFF2 staining was detected in large, diffuse tumors and in tumors with lymph node metastasis. The number of microvessels had a significant correlation with both TFF3 and TFF2 staining, whereas the area of microvessels had a significant correlation only with TFF3 staining. Both TFF3 and TFF2 were independent predictors of a worse disease-free survival. TFF3 had a gender-specific negative survival advantage, with a 91.3% disease-free survival in female patients with TFF3-negative advanced gastric carcinoma. CONCLUSIONS: Induction of increased tumor vascularity might be one of the mechanisms by which TFFs confer metastatic phenotype and frequent disease recurrence in gastric carcinomas. Female patients with TFF3-negative advanced gastric carcinoma have comparable survival as that reported for patients with early gastric carcinoma.

Role of tumor-associated macrophages (TAM) in advanced gastric carcinoma: the impact on FasL-mediated counterattack.

BACKGROUND: Exactly what role does tumor-derived Fas ligand (FasL) play in cancer: maintaining the immune privilege site or inducing a pro-inflammatory effect? One possible hypothesis is that tumor-associated macrophages (TAM) act as the mediator that enables apoptosis of anti-tumor immune cells without FasL-related inflammation. We have evaluated the tumor FasL expression and TAM along the tumor margin and/or in cancer stroma, and their impact on the infiltration of immune-competent cells into the tumor nest. PATIENTS AND METHODS: Tissue specimens from consecutive 84 advanced gastric carcinoma patients, who had undergone a curative resection, were evaluated for TAM (CD68+ cells), tumor FasL expression and immune status (CD8 + T cells). RESULTS: A high number of TAM significantly correlated with lymph node metastasis, intestinal type tumor and FasL expression. Although TAM had a tendency for an inverse correlation with the number of CD8+ T cells within the tumor nest (nest CD8) (p=0.0592), there was no correlation between FasL expression and nest CD8 (p=0.2158). This inverse association was found to be stronger in cases with both FasL-positive and high TAM tumors than in others (p=0.0139). The combination parameter of FasL-positive and high TAM became an independent prognostic factor in Cox's multivariate analysis, along with the pT status, nest CD8 and tumor cell apoptosis. CONCLUSION: We suggest that TAM works harmoniously with tumor-derived FasL and serves as a barrier against the infiltration of CD8+ T cells into the cancer nest.