Toshimasa Tsujinaka

Synthesis of a new cell penetrating calpain inhibitor (calpeptin)

N-terminal of Leu-norleucinal or Leu-methioninal was modified to obtain a cell penetrative peptide inhibitor against calpain. Benzyloxycarbonyl (Z) derivatives had less active against papain than phenylbutyryl derivatives and leupeptin. Z-Leu-nLeu-H (calpeptin) was more sensitive to calpain I than Z-Leu-Met-H and leupeptin. Calpeptin was most potent among synthesized inhibitors in terms of preventing the Ca2+-ionophore induced degradation of actin binding protein and P235 in intact platelets. After 30 min incubation with intact platelets, calpeptin completely abolished calpain activity in platelets but no effect was observed in case of leupeptin. Calpeptin also inhibited 20K phosphorylation in platelets stimulated by thrombin, ionomycin or collagen. Thus calpeptin was found to be a useful cell-penetrative calpain inhibitor.

Genetic polymorphisms of drug‐metabolizing enzymes and susceptibility to head‐and‐neck squamous‐cell carcinoma

We have investigated the association between the polymorphisms of drug‐metabolizing enzymes and susceptibility to head‐and‐neck squamous‐cell carcinoma (HNSCC). PCR‐based analysis was performed on 145 Japanese patients and 164 healthy Japanese controls to determine genotypes of polymorphisms in CYP1A1, CYP2E1, GSTM1, GSTP1, and NAT2. Patients and controls were compared by multivariate analysis. The CYP1A1 Val/Val genotype was seen more frequently in patients than in controls [odds ratio (OR) 4.1, p = 0.038). The frequency of the slow plus intermediate NAT2 genotypes was also higher in patients (OR 2.0, p = 0.039). When we analyzed the distributions of the genotypes in 69 laryngeal and 45 pharyngeal cancer patients, laryngeal cancer patients had a higher frequency of NAT2 slow or intermediate genotype (OR 2.7, p = 0.011) and GSTP1 AA genotype (OR 2.4, p = 0.047) than controls. Pharyngeal cancer patients had a higher frequency of the CYP1A1 Val/Val genotype than controls (OR 5.7, p = 0.034), suggesting that different organs may be responsive to different chemicals from the environment. Furthermore, 23 patients who developed multiple cancers (HNSCC plus other) were compared with 115 patients with HNSCC alone. There was no significant difference in the polymorphisms between the 2 groups, though excessive alcohol consumption (more than 50 g/day of ethanol) appeared to be a risk factor for multiple cancers (p = 0.053). Int. J. Cancer 80:685–688, 1999.

Anti-interleukin-6 receptor antibody prevents muscle atrophy in colon-26 adenocarcinoma-bearing mice with modulation of lysosomal and ATP-ubiquitin-dependent proteolytic pathways

Progression of skeletal muscle atrophy is one of the characteristic features in cancer patients. Interleukin‐6 (IL‐6) has been reported to be responsible for the loss of lean body mass during cancer cachexia in colon‐26 adenocarcinoma (C‐26)‐bearing mice. This study was carried out to elucidate the intracellular proteolytic pathways operating in skeletal muscle in C‐26‐bearing mice, and to examine the effect of anti IL‐6 receptor antibody on muscle atrophy. On day 17 after tumor inoculation, the gastrocnemius muscle weight of C‐26‐bearing mice had significantly decreased to 69% of that of the pair‐fed control mice. This weight loss occurred in association with increases in the mRNA levels of cathepsins B and L, poly‐ubiquitin (Ub) and the subunits of proteasomes in the muscles. Furthermore, enzymatic activity of cathepsin B+L in the muscles also increased to 119% of the control. The administration of antimurine IL‐6 receptor antibody to C‐26‐bearing mice reduced the weight loss of the gastrocnemius muscles to 84% of that of the control mice, whose enzymatic activity of cathepsin B+L and mRNA levels of cathepsin L and poly‐Ub were significantly suppressed compared with those of the C‐26‐bearing mice. Our data indicate that both the lysosomal cathepsin pathway and the ATP‐dependent proteolytic pathway might be involved in the muscle atrophy of C‐26‐bearing mice. The results also suggest that anti IL‐6 receptor antibody could be a potential therapeutic agent against muscle atrophy in cancer cachexia by inhibiting these proteolytic systems.

Gastrectomy plus chemotherapy versus chemotherapy alone for advanced gastric cancer with a single non-curable factor (REGATTA): a phase 3, randomised controlled trial

BACKGROUND Chemotherapy is the standard of care for incurable advanced gastric cancer. Whether the addition of gastrectomy to chemotherapy improves survival for patients with advanced gastric cancer with a single non-curable factor remains controversial. We aimed to investigate the superiority of gastrectomy followed by chemotherapy versus chemotherapy alone with respect to overall survival in these patients. METHODS We did an open-label, randomised, phase 3 trial at 44 centres or hospitals in Japan, South Korea, and Singapore. Patients aged 20-75 years with advanced gastric cancer with a single non-curable factor confined to either the liver (H1), peritoneum (P1), or para-aortic lymph nodes (16a1/b2) were randomly assigned (1:1) in each country to chemotherapy alone or gastrectomy followed by chemotherapy by a minimisation method with biased-coin assignment to balance the groups according to institution, clinical nodal status, and non-curable factor. Patients, treating physicians, and individuals who assessed outcomes and analysed data were not masked to treatment assignment. Chemotherapy consisted of oral S-1 80 mg/m(2) per day on days 1-21 and cisplatin 60 mg/m(2) on day 8 of every 5-week cycle. Gastrectomy was restricted to D1 lymphadenectomy without any resection of metastatic lesions. The primary endpoint was overall survival, analysed by intention to treat. This study is registered with UMIN-CTR, number UMIN000001012. FINDINGS Between Feb 4, 2008, and Sept 17, 2013, 175 patients were randomly assigned to chemotherapy alone (86 patients) or gastrectomy followed by chemotherapy (89 patients). After the first interim analysis on Sept 14, 2013, the predictive probability of overall survival being significantly higher in the gastrectomy plus chemotherapy group than in the chemotherapy alone group at the final analysis was only 13·2%, so the study was closed on the basis of futility. Overall survival at 2 years for all randomly assigned patients was 31·7% (95% CI 21·7-42·2) for patients assigned to chemotherapy alone compared with 25·1% (16·2-34·9) for those assigned to gastrectomy plus chemotherapy. Median overall survival was 16·6 months (95% CI 13·7-19·8) for patients assigned to chemotherapy alone and 14·3 months (11·8-16·3) for those assigned to gastrectomy plus chemotherapy (hazard ratio 1·09, 95% CI 0·78-1·52; one-sided p=0·70). The incidence of the following grade 3 or 4 chemotherapy-associated adverse events was higher in patients assigned to gastrectomy plus chemotherapy than in those assigned to chemotherapy alone: leucopenia (14 patients [18%] vs two [3%]), anorexia (22 [29%] vs nine [12%]), nausea (11 [15%] vs four [5%]), and hyponatraemia (seven [9%] vs four [5%]). One treatment-related death occurred in a patient assigned to chemotherapy alone (sudden cardiopulmonary arrest of unknown cause during the second cycle of chemotherapy) and one occurred in a patient assigned to chemotherapy plus gastrectomy (rapid growth of peritoneal metastasis after discharge 12 days after surgery). INTERPRETATION Since gastrectomy followed by chemotherapy did not show any survival benefit compared with chemotherapy alone in advanced gastric cancer with a single non-curable factor, gastrectomy cannot be justified for treatment of patients with these tumours. FUNDING The Ministry of Health, Labour and Welfare of Japan and the Korean Gastric Cancer Association.

Prognostic significance of heat shock proteins 27 and 70 in patients with squamous cell carcinoma of the esophagus.

Heat shock proteins (HSPs) first were defined as proteins induced by heat shock and other environmental and pathophysiologic stresses and are implicated in protein‐protein interactions such as folding, translocation, and prevention of inappropriate protein aggregation. Many of their functions suggest that they play important roles in cancer.

Influence of Overweight on Surgical Complications for Gastric Cancer: Results From a Randomized Control Trial Comparing D2 and Extended Para-aortic D3 Lymphadenectomy (JCOG9501)

BackgroundThe impact of overweight on the outcome of gastrectomy with lymphadenectomy is controversial, and data from a well-controlled, randomized study are needed to identify a possible relationship.MethodsWe used data from 523 patients registered for a prospective randomized trial comparing D2 and extended para-aortic D3 lymphadenectomy to compare the effects of body mass index (BMI) and the extent of lymphadenectomy for the development of general or major surgical complications (anastomotic leakage, abdominal abscess, and pancreatic fistula).ResultsSeventy-seven patients were classified as overweight with BMI ≥ 25, and 38 and 39 of these patients underwent a D2 or D3 lymphadenectomy, respectively. Among the 446 patients classified as nonoverweight with BMI < 25, 225 received D2 and 221 received D3 lymphadenectomy. Surgical complications, operation time, and blood loss were statistically significantly associated with BMI, and logistic regression analysis revealed that overweight directly affected the occurrence of surgical complications even after considering operation time and blood loss as intermediate factors instead of outcome variables. Among patients undergoing D2 lymphadenectomy, being overweight increased the risk for surgical complications and blood loss, whereas overweight was associated with only blood loss and operation time among patients receiving D3 lymphadenectomy.ConclusionsOverweight increased the risk of surgical complications in patients undergoing gastrectomy both directly and indirectly through operation time and blood loss. The impact of overweight on surgical complications was more evident in patients undergoing a D2 dissection.

CYP1A1 CYP2E1 and GSTM1 polymorphisms are not associated with susceptibility to squamous-cell carcinoma of the esophagus

We investigated the genetic polymorphisms of CYPIAI, CYP2EI and GSTMI in Japanese esophageal cancer patients (n = 53) with a histological diagnosis of squamous‐cell carcinoma, to determine whether susceptibility to esophageal cancer is associated with these polymorphisms. There were no significant differences in the frequency distribution of any one of the 3 polymorphisms between esophageal cancer patients and 132 healthy Japanese controls. The genotype distributions in tobacco smokers or alcohol drinkers were also quite similar for male patients and male controls. The age at onset of esophageal cancer was also similar for patients with any genotype of the 3 polymorphisms. We conclude that the 3 polymorphisms are unlikely to be associated with esophageal cancer susceptibility. Int. J. Cancer 71:192–195, 1997.

E‐cadherin gene variants in gastric cancer families whose probands are diagnosed with diffuse gastric cancer

To identify germline E‐cadherin mutations responsible for the predisposition to diffuse gastric cancer (DGC) among the Japanese, we screened 17 patients with familial aggregation of gastric cancer by sequencing analysis. All the patients were diagnosed with DGC and had at least 1 sibling with gastric cancer. Two novel E‐cadherin gene variants were detected. One was detected in 1 patient only and associated with an amino acid substitution (Val/Met) at codon 832 in the region essential for binding to β‐catenin. The M832 variant was detected not only in the proband but also in 2 other gastric cancer patients in the family. Immunohistochemical analysis of gastric cancer tissue from the proband revealed that E‐cadherin expression was markedly reduced and β‐catenin expression was also reduced in cancer cells. However, no significant difference in the activity of β‐catenin binding was detected between the M832 variant and V832 wild‐type E‐cadherin in immunofluorescence and immunoprecipitation/Western blot analyses. The other was detected in 5 patients and was located in the splice donor site (IVS1+6T/C); however, RT‐PCR analysis indicated that the IVS+6C variant did not cause an aberrant splicing. Thus, the M832 variant could be a germline mutation causative of familial aggregation of DGC, although further functional studies are needed to understand the pathogenic significance of this variant.

Prospective randomized trial of preoperative enteral immunonutrition followed by elective total gastrectomy for gastric cancer.

Perioperative enteral immunonutrition is thought to reduce postoperative morbidity in patients undergoing major gastrointestinal surgery. This study assessed the clinical effects of preoperative enteral immunonutrition in well nourished patients with gastric cancer undergoing total gastrectomy.

Appraisal of Treatment Strategy by Staging Laparoscopy for Locally Advanced Gastric Cancer

Abstract More accurate preoperative staging is necessary to determine the treatment strategy for locally advanced gastric cancer. Thirty-two patients with T3 or T4 gastric cancer expected to undergo curative resection based on conventional examinations underwent staging laparoscopy. The disease stages determined were compared with those obtained by conventional methods. The discrepancy rate of disease staging was 16 of 32 (50.0%), with down-staging in 5 of 32 (15.6%) and up-staging in 11 of 32 (34.4%). Of the 32 patients, 13 (40.6%) were found to have unsuspected peritoneal dissemination. The positive predictive value for peritoneal metastasis by staging laparoscopy was 100%, whereas the negative predictive value was 89% (17/19). The accuracy rate was 94%. After laparoscopy, 15 of the 32 (46.9%) were diagnosed as candidates for curative resection. Of these 15 patients who underwent surgery, 13 (86.7%) underwent curative resection (1 R0 and 12 R1); the remaining two underwent R2 resection because of peritoneal metastasis that was undetected by staging laparoscopy. Patients with tumors judged noncurable by laparoscopy (n= 11) received neoadjuvant chemotherapy. In 7 of the 11 cases, salvage surgery was done (one R0, three R1, three R2 resections). A second staging laparoscopy was performed in four cases to determine the indication for salvage surgery. Three of the four were judged to be curable and underwent curative resection. Staging laparoscopy is an effective tool for detecting unsuspected peritoneal metastasis, and it can increase the curative resection rate and decrease unnecessary laparotomy for advanced gastric cancer. Second-look laparoscopy enables accurate assessment of the chemotherapeutic response, which can help in decisions about salvage surgery.