The effect of p53 gene expression on the inhibition of cell proliferation by paclitaxel.

BACKGROUND/AIMS: We evaluated the relationship between p53 status and paclitaxel (PTX)-induced inhibition of the growth of human stomach cancer cells. MATERIALS AND METHODS: We made use of two human stomach cancer cell lines, MKN45 and MKN28. Growth inhibition in response to PTX was evaluated by MTT method. We used flow cytometry to monitor the cell cycle and western blot analysis to evaluate the expression of signaling molecules. RESULTS: PTX inhibited the proliferation of both stomach cancer cell lines in a dose-dependent manner. However, PTX cytotoxicity was stronger in MKN28 cells. Flow cytometric analysis showed that 1 muM PTX enhanced the percentage of MKN 45 cells in the sub-G1 phase of the cell cycle, whereas it increased the percentage of MKN 28 cells arrested at G2/M phase. 1 muM PTX was found to increase cyclin B1 production in MKN28 cells, but not in MKN 45 cells. In contrast, PTX-treatment led to an increase in the cleaved form of caspase-3 in MKN45, but not MKN28 cells. An inhibitor of p53, pifithrin-alpha, antagonized the expression of the cleaved form of caspase-3 in MKN45 cells. CONCLUSION: Both p53 status and cyclin-B1 expression might be useful for predicting the therapeutic response of stomach cancer to PTX.

Extracellular signal-regulated kinase phosphorylation due to menadione-induced arylation mediates growth inhibition of pancreas cancer cells.

BACKGROUND: Cytotoxicity of Vitamin K3 (VK3) is indicated to have the same mechanism with oxidative stress (H(2)O(2)). In the present study, we analyzed the differences and/or similarities in the cellular responses to oxidative stress and VK3 to clarify the mechanism of growth inhibition. METHODS: Cell viability was determined by a test method with 3-[4, 5-dimethyl-thiazol]-2, 5-dephenyl tetrazolium bromide (MTT). Expressions of cellular proteins were evaluated by Western blot analysis. RESULTS: The IC50 was calculated to be 47.3 +/- 4.1 microM for VK3 and 2.2 +/- 1.2 microM for H(2)O(2). By Western blot analysis, VK3 or H(2)O(2) was shown to induce rapid phosphorylation of extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinases (JNKs). H(2)O(2)-induced phosphorylation of ERK and JNK was almost complete inhibited by more than 100-muM genistein. VK3-induced JNK phosphorylation was blocked by 100-microM genistein, but ERK phosphorylation was not inhibited completely even if 400-microM genistein was used. H(2)O(2)-induced inhibition of cell proliferation was completely blocked by 400-microM genistein, but the VK3 effect was reduced 72.8 +/- 5.4% by the same concentration of genistein. H(2)O(2)-induced JNK phosphorylation and ERK phosphorylation were inhibited by staurosporine, protein kinase C (PKC) inhibitor. VK3-induced JNK phosphorylation was also blocked, but ERK phosphorylation was not affected. Staurosporine had no effect on VK3- or H(2)O(2)-induced growth inhibition. Treatment with a non-thiol antioxidant agent, catalase, completely abrogated H(2)O(2)-induced JNK and ERK phosphorylation, but a thiol antioxidant, L: -cystein, had no effect on phosphorylation of them. The VK3-induced JNK phosphorylation was inhibited by catalase, but not L: -cystein. But ERK phosphorylation was not inhibited by catalase and was abrogated completely by the thiol antioxidant. Catalase, but not L: -cystein, blocked H(2)O(2)-induced growth inhibition, and L: -cystein, but not catalase, blocked VK3-induced effects on cell proliferation completely. CONCLUSION: VK3-induced ERK phosphorylation occurs by a different mechanism from oxidative stress, and it might have an important role to induce growth inhibition.

The utility of vitamin K3 (menadione) against pancreatic cancer.

BACKGROUND: To evaluate the efficacy of vitamin K3 (VK3) against pancreatic cancer, the molecular mechanism of VK3 or gemcitabine (GEM)-induced inhibition of proliferation was characterized. MATERIALS AND METHODS: The cell viability was determined using the 3-[4,5-dimethylthiazol]-2,5-diphenyl tetrazolium bromide (MTT) test method. The expressions of cellular proteins were evaluated by Western blot analysis. For morphological studies of the in vivo transplanted cancer cells, the tissues were stained with hematoxylin and eosin. RESULTS: The IC50 of VK3 for pancreatic cancer cells was calculated for 42.1 +/- 3.5 microM. Western blot analysis showed that VK3 induced rapid phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) 30 minutes after application. ERK but not JNK phosphorylation was maintained for at least 12 hours. Activation of apoptosis by VK3, as shown by molecular weight shifts of the pro-activated 32-kDa form of caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage of the 112-kDa form, was found. Treatment with the thiol antioxidant, L-cysteine (>0.2 mM), completely abrogated the VK3-induced phosphorylation of ERK, but not the JNK, and inhibition of proliferation. A caspase-3 inhibitor antagonized caspase-3 activation, but had no inhibitory effect on the proliferative activity of VK3. GEM at concentrations >0.1 microg/ml was found to inhibit cell proliferation after 24 hours. GEM also induced phosphorylation of JNK, activation of caspase-3 and accumulation of cyclin B1. Local application of VK3 was found to induce extensive tumor tissue necrosis, but slight hematemesis without necrosis was observed 48 hours after GEM injection. In Western blot, ERK but not JNK phosphorylation, was clearly detected in response to VK3 injection into the tumor tissue. CONCLUSION: The action of VK3 may lead to a favorable outcome against pancreatic cancer, and the detection of ERK phosphorylation in the tissue is important for predicting this effect.

Growth inhibition of unresectable tumors induced by hepatic cryoablation: report of two cases.

The efficacy of percutaneous cryosurgery (PCS) as a treatment strategy for unresected liver tumor was evaluated in two cases. The first patient was a 64-year-old man who was found to have multiple liver tumors after undergoing gastrectomy for gastric cancer (T3, N0, M0, Stage II). Two PCS treatments under local anesthesia decreased the size of both the treated and untreated tumors. The second patient was a 61-year-old man in whom multiple liver tumors were discovered after hepatectomy for metastases of a duodenal gastrointestinal stromal tumor, which also had been treated surgically. A third surgery was performed for mass reduction. The patient showed stable improvement after surgery, and PCS combined with administration of polysaccharide-Kureha was selected to treat the unresectable tumors. PCS was performed once a week with an overnight hospital stay. After nine PCS treatment, the remarkable reduction in the size and number of liver tumors was observed, even among non-treated tumors. The patient remains in good condition without tumors 21 months after treatment.

Expression of vascular endothelial growth factor receptors is closely related to the histological grade of hepatocellular carcinoma.

Angiogenesis is important for tumor growth, and is regulated by angiogenetic factors such as vascular endothelial growth factor (VEGF). In the present study, we investigated whether or not expression of VEGF receptors (VEGFRs) is related to the proliferation of tumor cells in hepatocellular carcinoma (HCC). We simultaneously stained proliferation marker Ki-67 antigen and either VEGFR1 (Flt-1) or VEGFR2 (Flk-1) on paraffin-embedded tissue sections from 50 cases of surgically resected human HCC. Based on the staining pattern of VEGFRs, we classified the cases into 4 categories; receptor double-negative, Flt-1 single-positive, Flk-1 single-positive, receptor double-positive. Interestingly, the Ki-67 index was significantly lower in receptor double-negative cases in comparison to that in either Flt-1 single-positive or Flk-1 single-positive cases (P = 0.0491, P = 0.0196, respectively). Moreover, the index was also significantly lower in receptor double-positive cases in comparison to either Flt-1 single-positive or Flk-1 single-positive cases (P = 0.0026, P < 0.0001, respectively). We further investigated 35 cases showing a Ki67 index > 10% to determine the expression of VEGFRs on Ki-67 antigen-positive proliferating cells. Surprisingly, the histological grade of HCC and the expression pattern of VEGFRs showed a characteristic relation; the well-differentiated HCC cases were all distributed in the Flk-1-positive group (7/7), moderately differentiated HCC cases were distributed in either the Flt-1 or Flk-1 single-positive group (20/21), and poorly differentiated HCC cases were predominantly distributed in either the receptor double-negative or double-positive group (6/7). These findings suggest that the expression pattern of VEGFRs influences the histological differentiation of HCC.

A modified reconstruction method to prevent critical complications after pancreatoduodenectomy.

BACKGROUND/AIMS: A new method of reconstructing the pancreatic stump after pancreatoduodenectomy (PD) is necessary to improve the postoperative mortality rate. Thus, we modified the pancreatoenteric procedure to reduce anastomotic leakage from the pancreatic stump after PD, and we conducted a study to evaluate the usefulness of the new procedure on the basis of patients' postoperative condition. METHODOLOGY: We compared the postoperative condition of 21 patients who underwent PD with the new separated loop (SL) reconstruction (6 men, 11 women; mean age, 67.7+/-7.2 years) to that of 31 patients (12 men, 19 women; mean age, 66.8+/-10.3 years) who underwent PD with pancreatogastrostomy (PG). In the SL reconstruction procedure, the proximal jejunum is brought up behind the colon, and an end-to-side choledochojejunostomy is made with a single layer of interrupted sutures. Approximately 20cm of the jejunum is fitted with a fixed stomach tube for postoperative enteral feeding, and the cut proximal jejunum is positioned next to the pancreatic stump. A pancreatic tube is inserted into the lumen of the pancreatic duct and fixed without closing the pancreatic duct. Pancreatojejunostomy is achieved as an end-to-end anastomosis with the pancreatic stump telescoping into the proximal jejunum. Approximately 20cm of the jejunum is anastomosed side-to-end to the stomach, and end-to-side jejunojejunostomy is made to complete a Y-type reconstruction. Each patient's postoperative condition was also assessed on the basis of serum albumin (ALB), cholinesterase and total cholesterol (T-CHO) levels on postoperative days (PODs) 14 and 28. RESULTS: A high level of amylase in drainage fluid was noted in two (6.5%) and delayed gastric emptying in four (12.9%) of the patients in the PG group. There were no complications in the SL group. Postoperative levels of ALB on POD 14 and T-CHO on POD 28 were significantly higher than in the PG group. CONCLUSIONS: The SL method is safe and does not induce complications after PD. Our results indicate that this method may provide a favored outcome.

Challenge for a better combination with basic evidence.

5-Fluorouracil (5-FU) has been the most widely accepted and studied chemotherapeutic agent, and many combination chemotherapeutic regimens have been reported. However, until recently, a standard regimen for metastatic gastric cancer had not been established. The combination of S-1 and cisplatin is a good candidate as a standard first-line regimen for metastatic gastric cancer. On the other hand, interest in biochemical modulation has become wide spread recently. The low level of dihydropyrimidine denhydrogenase (DPD), thymidylate synthase (TS) activities, and a high level of orotate phosphoribosyl-transferase (OPRT) activity enhance the antitumor effect of 5-FU and S-1. Docetaxel is one of the agents that modulate these enzyme expressions and activities. Moreover, the response rate of combination therapy of docetaxel and S-1 for metastatic gastric cancer was 56.3% and median survival time was 14.3 months in a phase II study, showing it to be a good candidate for a new standard regimen for gastric cancer. A phase III collaborative study, START (S-1 and Taxotere for advanced gastric cancer randomized phase III trial), is now under way in Japan and Korea.

Serum cytokine levels in response to hepatic cryoablation.

BACKGROUND AND OBJECTIVES: Cryogenic treatment sometimes stimulates the immune system by releasing intracellular antigens. We evaluated anti-tumor immune response by analyzing alterations in serum cytokine levels. METHODS: Percutaneous cryosurgery was performed in 13 patients with unresectable tumors. Serum levels of interleukin (IL) -4, -6, and -10, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma were measured by enzyme-linked immunosorbent assay (ELISA). The Th1/Th2 ratio was estimated from the IFN-gamma/IL-4 ratio. RESULTS: Levels of serum factors in the immune reaction (IR) group, in which tumor necrosis was identified not only in the treated area but also away from the treated area, were compared with those in the local effect (LE) group. Serum amyloid A (SAA), C-reactive protein (CRP), and IL-6 levels were increased in both groups after three treatments. The serum IL-10 level tended to increase with the number of treatments. Pretreatment IL-10 levels in the LE group were significantly greater than those in the IR group, and the maximum value in the LE group (59.5 +/- 13.2 pg/ml) was greater than that in the IR group (47.0 +/- 15.0 pg/ml). The TNF-alpha level was increased in the IR group. Pretreatment TNF-alpha levels and maximum levels in response to treatment were significantly greater in the IR group than in the LE group (P = 0.0313). The Th1/Th2 ratio was increased in the IR group, and the maximum ratio was significantly greater in the IR group than in the LE group. CONCLUSION: It might be possible to evaluate the appearance of immune responses to cryosurgery by monitoring serum cytokine levels.

Clinicopathological features of hepatocellular carcinoma evaluated by vascular endothelial growth factor expression.

AIM: To evaluate the significance of the expression of vascular endothelial growth factor (VEGF), its correlation with clinicopathological variables were studied in the tissue of hepatocellular carcinoma (HCC) and surrounding liver. METHODS: In 56 samples (tumor and non-tumor liver tissue) collected from 28 patients, VEGF expression was examined by immunohistochemistry and western blot analysis. RESULTS: The value of VEGF expression by western blotting was correlated with immunohistochemical staining grade. In tumor tissue, the value of VEGF expression correlated with tumor size (P = 0.034), á-fetoprotein (P = 0.036) and protein induced by vitamin K absence-II by simple regression, and histological grade (P = 0.0132) by the unpaired t-test. The level of VEGF expression in non-tumor liver was found to correlate with the value of serum albumin (P = 0.008), cholinesterase (P = 0.012) and prothrombin activity (P = 0.046). The frequency of simple nodular type in gross appearance decreased in cases with high tumor/non-tumor (T/N) ratio (P = 0.022), and the degree of portal vein invasion progressed with an increase in the T/N ratio (P = 0.008). The T/N ratio was significantly higher in early recurrence cases (P = 0.0081). CONCLUSION: This study on the expression of VEGF might be useful to estimate the liver condition and the clinicopathological features of HCC.

Vascular endothelial growth factor protects hepatoma cells against oxidative stress-induced cell death.

BACKGROUND: The aim of the present study was to examine coordination of the vascular endothelial growth factor (VEGF) and VEGF receptor (Flk-1) system and to study control of VEGF expression by oxidative stress, which is considered a model for chronic liver disease. METHODS: Cell viability was determined by test method with 3-[4, 5-dimethylthiazol-2-yl]-2, 5-dephenyl tetrazolium bromide (MTT). Expressions of cellular proteins were evaluated by western blot analysis. RESULTS: The c-Met tyrosine phosphorylation in PLC/PRF/5 hepatoma cells was increased by treatment with 20 ng/mL hepatocyte growth factor (HGF), and extracellular signal-regulated kinase (ERK) was also activated. Although Flk-1 was phosphorylated in response to VEGF (>50 ng/mL), phosphorylated ERK was not detected at these concentrations. A total of 5.0 and 10 micromol/L hydrogen peroxide (H(2)O(2)) caused cell death in a dose-dependent manner after 24 h. On western blot analysis at 1 h with H(2)O(2), rapid phosphorylation of both ERK1/2 and c-Jun NH(2)-terminal kinase (JNK) was observed. In the first 6 h, H(2)O(2) induced cell death for 58.4 +/- 6.8%, whereas the presence of 100 ng/mL VEGF improved the survival rate to 77.2 +/- 4.2%. The VEGF significantly decreased H(2)O(2)-induced cell death after 12 h, whereas HGF (20 ng/mL) did not have a similar effect. When cells were incubated with 5 micromol/L H(2)O(2), expression of VEGF protein was detected. Furthermore, H(2)O(2)-induced phosphorylation of ERK and JNK was also reduced by VEGF (100 ng/mL). In contrast, HGF did not induce phosphorylation of ERK and JNK. CONCLUSION: Hepatoma cells might be able to survive under continuous oxidative stress through expression of VEGF.

Identification of an immune tolerance reaction in response to pretreatment with frozen pancreatic tissue in islet cell transplantation in rats.

OBJECTIVES: Whereas transplantation of insulin-secreting pancreatic islets may provide long-term control of glucose metabolism in patients with diabetes mellitus, transplant rejection remains a problem. In this study, we tried pretreatment with frozen pancreatic tissue in a rat model of islet cell transplantation to determine whether induction of immune tolerance is feasible. METHODS: Isolated islet cells from Wistar rats were transplanted into the spleen of recipient rats that were sensitized and rats that were not sensitized with frozen pancreatic tissue. Spleens were analyzed histologically and then examined immunohistochemically for expression of insulin, a pancreas-specific gene. With the use of cDNA primers for proinsulin, RT-PCR was performed to detect islet cells in the spleen. RESULTS: Although islet cells were present in spleens at posttransplantation day (PTD) 1, islet cell clusters in recipients without pretreatment were markedly destroyed on PTD 14 on histologic examination. In contrast, islet cell clusters in recipients sensitized with frozen pancreatic tissue appeared similar to those at PTD 1 even at PTD 14. Proinsulin gene expression was found to be specific to pancreatic tissue. In recipients sensitized with frozen pancreatic tissue, proinsulin gene expression was identified in recipient spleens, even at PTD 14, whereas it was undetected in the absence of pretreatment. In recipients transplanted with islet cells and treated simultaneously with frozen pancreatic tissue, proinsulin gene expression was completely eliminated. The immunohistologic study also showed the presence of insulin-producing islet cells in the spleens of rats sensitized with frozen pancreatic tissue. CONCLUSIONS: Inhibition of the immune reaction in transplant rejection may be mediated by pretreatment with frozen donor tissue.