COTI-2, A Novel Thiosemicarbazone Derivative, Exhibits Antitumor Activity in HNSCC through p53-dependent and -independent Mechanisms.

PURPOSE: TP53 mutations are highly prevalent in head and neck squamous cell carcinoma (HNSCC) and associated with increased resistance to conventional treatment primarily consisting of chemotherapy and radiation. Restoration of wild-type p53 function in TP53-mutant cancer cells represents an attractive therapeutic approach and has been explored in recent years. In this study, the efficacy of a putative p53 reactivator called COTI-2 was evaluated in HNSCC cell lines with different TP53 status.Experimental Design: Clonogenic survival assays and an orthotopic mouse model of oral cancer were used to examine in vitro and in vivo sensitivity of HNSCC cell lines with either wild-type, null, or mutant TP53 to COTI-2 alone, and in combination with cisplatin and/or radiation. Western blotting, cell cycle, live-cell imaging, RNA sequencing, reverse-phase protein array, chromatin immunoprecipitation, and apoptosis analyses were performed to dissect molecular mechanisms. RESULTS: COTI-2 decreased clonogenic survival of HNSCC cells and potentiated response to cisplatin and/or radiation in vitro and in vivo irrespective of TP53 status. Mechanistically, COTI-2 normalized wild-type p53 target gene expression and restored DNA-binding properties to the p53-mutant protein in HNSCC. In addition, COTI-2 induced DNA damage and replication stress responses leading to apoptosis and/or senescence. Furthermore, COTI-2 lead to activation of AMPK and inhibition of the mTOR pathways in vitro in HNSCC cells. CONCLUSIONS: COTI-2 inhibits tumor growth in vitro and in vivo in HNSCC likely through p53-dependent and p53-independent mechanisms. Combination of COTI-2 with cisplatin or radiation may be highly relevant in treating patients with HNSCC harboring TP53 mutations.

Gemcitabine alone or in combination with cisplatin in patients with biliary tract cancer: a comparative multicentre study in Japan.

BACKGROUND: A British randomised study of gemcitabine plus cisplatin (GC) combination showed promising results in biliary tract cancer (BTC) patients. In our study, we evaluated the efficacy and safety of this combination compared with gemcitabine alone (G) in Japanese BTC patients. METHODS: Overall, 84 advanced BTC patients were randomised to either cisplatin 25 mg m(-2) plus gemcitabine 1000 mg m(-2) on days 1, 8 of a 21-day cycle (GC-arm), or single-agent gemcitabine 1000 mg m(-2) on days 1, 8 and 15 of a 28-day cycle (G-arm). Treatments were repeated for at least 12 weeks until disease progression or unacceptable toxicity occurred, up to a maximum of 48 weeks. RESULTS: A total of 83 patients were included in the analysis. For the GC and G-arms, respectively, the 1-year survival rate was 39.0 vs 31.0%, median survival time 11.2 vs 7.7 months, median progression-free survival time 5.8 vs 3.7 months and overall response rate 19.5 vs 11.9%. The most common grade 3 or 4 toxicities (GC-arm/G-arm) were neutropenia (56.1%/38.1%), thrombocytopenia (39.0%/7.1%), leukopenia (29.3%/19.0%), haemoglobin decrease (36.6%/16.7%) and gamma-GTP increase (29.3%/35.7%). CONCLUSIONS: Gemcitabine plus cisplatin combination therapy was found to be effective and well tolerated, suggesting that it could also be a standard regimen for Japanese patients.

Clonally expanded T lymphocytes from atomic bomb survivors in vitro show no evidence of cytogenetic instability.

Abstract Genomic instability has been suggested as a mechanism by which exposure to ionizing radiation can lead to cancer in exposed humans. However, the data from human cells needed to support or refute this idea are limited. In our previous study on clonal lymphocyte populations carrying stable-type aberrations derived from A-bomb survivors, we found no increase in the frequency of sporadic additional aberrations among the clonal cell populations compared with the spontaneous frequency in vivo. That work has been extended by using multicolor FISH (mFISH) to quantify the various kinds of chromosome aberrations known to be indicative of genomic instability in cloned T lymphocytes after they were expanded in culture for 25 population doublings. The blood T cells used were obtained from each of two high-dose-exposed survivors (>1 Gy) and two control subjects, and a total of 66 clonal populations (36 from exposed and 30 from control individuals) were established. For each clone, 100 metaphases were examined. In the case of exposed lymphocytes, a total of 39 additional de novo stable, exchange-type aberrations [translocation (t) + derivative chromosome (der)] were found among 3600 cells (1.1%); the corresponding value in the control group was 0.6% (17/3000). Although the ratio (39/3600) obtained from the exposed cases was greater than that of the controls (17/3000), the difference was not statistically significant (P = 0.101). A similar lack of statistical difference was found for the total of all structural chromosome alterations including t, der, dicentrics, duplications, deletions and fragments (P = 0.142). Thus there was no clear evidence suggesting the presence of chromosome instabilities among the clonally expanded lymphocytes in vitro from A-bomb survivors.

Changes of ROS during a two-day ultra-marathon race.

To assess oxidative stress (OS) induced by endurance exercise, concentrations of serum reactive oxygen species (ROS) were determined in 70 Japanese male amateur runners completing a two-day ultra-marathon race. Serum ROS levels were analyzed at three time points: before the race (baseline), after the 1st day race (mid-race), and after the 2nd day race (goal) (post-race). The means (SE) of ROS were 151.4(3.7) (U. CARR.), 168.7(4.4), and 156.8(4.4), respectively. Significant positive trends were noted between age and serum ROS concentrations at the three race points (p<0.05 for all). After adjusting for age, BMI and average monthly running distance, the baseline serum ROS concentrations were positively associated with completion times of the first-day race, in particular (p<0.05), suggesting that the concentrations may predict physical performance. The ROS production increased at mid-race (p<0.05), but the levels returned to baseline levels at post-race, indicating that an antioxidant defense system may develop post-race to reduce OS.

Modulation of connexin 43 in rotenone-induced model of Parkinson's disease.

Gap junctional communication plays an important role in various models of brain pathology, but the changes of gap junctions in Parkinsonism are still not understood. In this study, we show that a major gap junctional protein, connexin43 (Cx43), in astrocytes is enhanced both in a rat Parkinson's disease (PD) model induced with rotenone, a widely used pesticide that inhibits mitochondrial complex I, and in vitro in cultured astrocytes stimulated with rotenone. Enhancement of Cx43 protein levels in rotenone-treated cultured astrocytes occurred in parallel with an increase in gap junctional intercellular communication, but was not accompanied with an increase in Cx43 mRNA levels. Furthermore, the rotenone-induced increase of Cx43 protein levels both in vitro and in vivo was associated with increased levels of phosphorylated Cx43, which is required for gap junctional intercellular communication. In our rat PD model, phosphorylated Cx43 was selectively enhanced in the basal ganglia regions, which contain DA neurons or their terminal areas. The increase of Cx43 levels was lower in the substantia nigra pars compacta and the striatum than in the substantia nigra pars reticulata and the globus pallidus. Our findings indicate that modulation of Cx43 protein, and consequently gap junctional cellular communication, in astrocytes may play an important role in PD pathology.

Cytogenetic differences in breast cancer samples between German and Japanese patients.

BACKGROUND: Japanese and German breast cancer cases differ substantially in the frequency of egfr amplification. AIMS: To unravel further the cytogenetic differences between Japanese and German breast cancer cases. METHODS: Forty one Japanese breast cancer cases were evaluated by means of comparative genomic hybridisation (CGH). The results were compared with the CGH results from 161 German breast cancer cases. RESULTS: The mean number of genetic alterations/case was significantly higher in German premenopausal patients with breast cancer than in their Japanese counterparts. Japanese breast cancer cases revealed a higher number of chromosome 17p losses. Losses of 8p were associated with oestrogen receptor (ER) negativity in Japanese patients with breast cancer, whereas in the German patients gains of 3q and 6q were associated with the lack of ER expression. CONCLUSIONS: The interethnic differences of invasive breast cancer are reflected by cytogenetic aberrations, which are also associated with the differential expression of the ER.

Acute brain injury in hypoglycaemia-induced hemiplegia.

BACKGROUND: The development of hemiplegia as a result of hypoglycaemia was first described in 1928. However, the mechanism remains unclear. CASE REPORT: We report a case of a 58-year-old male with diabetes, who developed left hemiplegia during a severe hypoglycaemic event. Results Diffusion-weighted magnetic resonance imaging detected an increased signal intensity in the pons, indicating that the patient's hemiplegia resulted from acute brain injury. CONCLUSIONS: This report provides evidence that acute brain injury may be a cause of the neurological deficit.

Personality types, lifestyle, and sensitivity to mental stress in association with NK activity.

We conducted a cross-sectional study among 302 healthy Japanese male workers to make a mechanistic approach to the association between personality types and cancer; two types of personality, the emotionally unstable-introvert and the emotionally stable-extravert, were compared with each other in lifestyle, mental stress status, and biological markers such as plasma levels of neurotransmitters and NK activity of peripheral lymphocytes. We first found that emotionally unstable-introverts have a more unhealthy lifestyle associated with low NK activity than among stable-extraverts, along with higher sensitivity to mental stress (also known to suppress NK activity) than stable-extraverts. Second, emotionally unstable-introverts were found to have in fact decreased NK activity along with higher plasma levels of noradrenaline, when compared with stable-extraverts. Our results thus demonstrate that emotionally unstable-introverts have a decreased capacity of immunological host defense against cancer, which is possibly due to two factors, unhealthy lifestyle and high sensitivity to mental stress.

Elevated levels of connective tissue growth factor, WISP-1, and CYR61 in primary breast cancers associated with more advanced features.

To gain insight into the role of the CCN genes in human breast carcinomas, we quantified connective tissue growth factor (CTGF), WISP-1, CYR61, and human NOV (NOVH) mRNA expression levels in samples from 44 primary breast tumors and seven normal breasts using quantitative real-time PCR assay. Overexpression of CTGF, WISP-1, CYR61, and NOVH was found in 55 (24 of 44), 46 (20 of 44), 39 (17 of 44), and 11% (5 of 44) primary breast tumors, respectively. Statistical univariate analysis was performed to explore the links between expression of the CCN genes and clinical and pathological parameters. Interestingly, significant associations were found between CTGF expression versus stage, tumor size, lymph node status, and age at diagnosis; WISP-1 mRNA levels versus stage, tumor size, lymph node, and HER-2/neu overexpression; and CYR61 expression with stage, tumor size, lymph node, age, and estrogen receptor expression. In contrast to CTGF, WISP-1, and CYR61, no significant correlation was found between NOVH expression and any of the clinical and pathological factors. Furthermore, multivariate classification tree model analysis showed that stage and lymph node involvement were important for predicting CTGF expression in breast cancers; the stage, age, and HER-2/neu status were key factors for WISP-1 expression; and the stage, age, and estrogen receptor were valuable predictors for CYR61 expression. In summary, these results suggest that CTGF, WISP-1, and CYR61 may play a role in the progression of breast cancer and might serve as a valuable tool for monitoring tumor status of breast cancer patients.

Natural killer activity and its changes among participants in a smoking cessation intervention program--a prospective pilot study of 6 months' duration.

We examined the effect of smoking cessation on natural killer (NK) activity of peripheral blood lymphocytes in terms of a prospective study of 27 Japanese subjects who participated in a smoking cessation intervention program. This program was delivered by means of group-counseling offering 7 sessions of about 2 hours over 6 months to help smokers to discontinue the habit. Thirteen subjects ceased smoking (quitters), while 14 continued to smoke (cigarette smokers). NK activity before the intervention was correlated positively with age (correlation coefficient=0.46, P<0.05). NK activity remained almost constant among quitters, comparing the activity before and after the intervention, while it decreased among cigarette smokers although it was not statistically significant. In the subgroup analysis, NK activity increased among those aged less than 65 years, or urine cotinine levels over 800 ng/ml before the intervention, especially among quitters, but there were no statistical significances. Multiple regression analysis showed changes in NK activity were correlated significantly only with age (standard regression coefficient=-0.44, P<0.05). These findings suggest that smoking cessation intervention programs might have been more effective for younger than elder subjects in consideration of NK activity.

Cancer prevention with green tea and monitoring by a new biomarker, hnRNP B1.

The study of green tea polyphenols as a cancer preventative is approaching a new era, with significant results accumulating rapidly. This paper briefly reviews four topics related to mechanisms of action of tea polyphenols: (I) identification of the genes commonly affected by EGCG, as demonstrated by Clontech's Atlas cDNA Expression Array; (II) the significance of heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1) as a new biomarker for early detection of lung cancer, and inhibition of its expression by EGCG; (III) the synergistic or additive effects of EGCG with the cancer preventive agents, sulindac and tamoxifen, on induction of apoptosis in PC-9 cells and on inhibition of intestinal tumor development in multiple intestinal neoplasia (Min) mice; (IV) the results of a 10 year prospective cohort study demonstrating the effectiveness of daily consumption of green tea in preventing cancer, and a prototype study for developing green tea beverage as cancer preventive.

Breast cancer. Cyr61 is overexpressed, estrogen-inducible, and associated with more advanced disease.

To identify genes involved in breast cancer, polymerase chain reaction-selected cDNA subtraction was utilized to construct a breast cancer-subtracted library. Differential screening of the library isolated the growth factor-inducible immediate-early gene Cyr61, a secreted, cysteine-rich, heparin binding protein that promotes endothelial cell adhesion, migration, and neovascularization. Northern analysis revealed that Cyr61 was expressed highly in the invasive breast cancer cell lines MDA-MB-231, T47D, and MDA-MB-157; very low levels were found in the less tumorigenic MCF-7 and BT-20 breast cancer cells and barely detectable amounts were expressed in the normal breast cells, MCF-12A. Univariate analysis showed a significant or borderline significant association between Cyr61 expression and stage, tumor size, lymph node positivity, age, and estrogen receptor levels. Interestingly, expression of Cyr61 mRNA increased 8- to 12-fold in MCF-12A and 3- to 5-fold in MCF-7 cells after 24- and 48-h exposure to estrogen, respectively. Induction of Cyr61 mRNA was blocked by tamoxifen and ICI182,780, inhibitors of the estrogen receptor. Stable expression of Cyr61 cDNA under the regulation of a constitutive promoter in MCF-7 cells enhanced anchorage-independent cell growth in soft agar and significantly increased tumorigenicity and vascularization of these tumors in nude mice. Moreover, overexpression of Cyr61 in MCF-12A normal breast cells induced their tumor formation and vascularization in nude mice. In summary, these results suggest that Cyr61 may play a role in the progression of breast cancer and may be involved in estrogen-mediated tumor development.

Molecular significance of excess body weight in postmenopausal breast cancer patients, in relation to expression of insulin-like growth factor I receptor and insulin-like growth factor II genes.

A number of epidemiological and clinical studies have revealed that excess body weight increases the risk of postmenopausal breast cancer and also adversely affects subsequent malignant progression. To elucidate the molecular mechanisms underlying these observations, we examined mRNA expression of various genes in normal (non-cancerous) mammary gland and cancer tissue of Japanese patients with primary breast cancer, in association with their body mass index (BMI). On the basis of analysis of 106 breast cancer patients, we found that mRNA expression of insulin-like growth factor I receptor (IGF-IR) and insulin-like growth factor II (IGF-II) in the normal mammary gland showed a significant and positive association with increased BMI among postmenopausal patients. Furthermore, the positive association of increased BMI with IGF-IR mRNA expression was also found in postmenopausal breast cancer tissue, while this association was not observed among premenopausal patients. In addition, increased mRNA expression of cyclin D1 and bcl-2 was observed in association with increased mRNA levels of IGF-IR among the patients regardless of menopausal status. These findings suggest that the molecular consequence of the increased BMI is the increased expression of IGF-II and IGF-IR, resulting in development of postmenopausal breast cancer and its progression mediated through modulation of the cell cycle and apoptosis.

Antibodies to tissue transglutaminase: comparison of ELISA and immunoprecipitation assay in the presence and in the absence of calcium ions.

Endomysial antibodies are characteristic of coeliac disease and tissue transglutaminase (tTG) has been identified as a major component of the endomysial antigen. tTG autoantibodies were measured in sera from patients with coeliac disease using ELISA based on guinea pig tTG and immunoprecipitation assay (IPA) based on 35S-labelled human tTG produced in an in vitro transcription/translation system. In addition, the effect of calcium ions on the interaction between tTG autoantibodies in the two assays was studied. Under standard (i.e. Ca2+-free) conditions, 36/39 (92%) coeliac sera were positive for IgA tTG antibodies by ELISA and 34/39 (87%) sera were positive by IPA. Comparison of ELISA and IPA results showed three sera positive by ELISA but negative by IPA and one serum which was positive by IPA but negative by ELISA. Bland and Altman analysis of the correlation between the ELISA and IPA showed that the results for 37 out of 39 samples were in the agreement. The results by ELISA carried out without and with Ca2+ were in good agreement (r=0.99; n=39). IPA using Ca2+ containing buffer detected fewer samples compared to IPA using standard assay buffer however the results of the two assays also showed a good agreement (r=0.93; n=39). Our studies confirm that antibodies to tTG are good markers of coeliac disease and indicate that the autoantibody binding sites on tTG are formed in a way which is essentially independent of Ca2+.

A new function of green tea: prevention of lifestyle-related diseases.

In the normal human life span, there occur lifestyle-related diseases that may be preventable with nontoxic agents. This paper deals with the preventive activity of green tea in some lifestyle-related diseases. Green tea is one of the most practical cancer preventives, as we have shown in various in vitro and in vivo experiments, along with epidemiological studies. Among various biological effects of green tea, we have focused on its inhibitory effect on TNF-alpha gene expression mediated through inhibition of NF-kappaB and AP-1 activation. Based on our recent results with TNF-alpha-deficient mice, TNF-alpha is an endogenous tumor promoter. TNF-alpha is also known to be a central mediator in chronic inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. We therefore hypothesized that green tea might be a preventive agent for chronic inflammatory diseases. To test this hypothesis, TNF-alpha transgenic mice, which overexpress TNF-alpha only in the lungs, were examined. The TNF-alpha transgenic mouse is an animal model of human idiopathic pulmonary fibrosis which also frequently develops lung cancer. Expressions of TNF-alpha and IL-6 were inhibited in the lungs of these mice after treatment with green tea in drinking water for 4 months. In addition, judging from the results of a prospective cohort study in Saitama Prefecture, Japan, green tea helps to prevent cardiovascular disease. In this study, a decreased relative risk of death from cardiovascular disease was found for people consuming over 10 cups of green tea a day, and green tea also had life-prolonging effects on cumulative survival. These data suggest that green tea has preventive effects on both chronic inflammatory diseases and lifestyle-related diseases (including cardiovascular disease and cancer), resulting in prolongation of life span.

Natural cytotoxic activity of peripheral-blood lymphocytes and cancer incidence: an 11-year follow-up study of a general population.

BACKGROUND: One of the most critical questions in immunosurveillance is whether differences between individuals with regards to natural immunological host defence can predict future development of cancer. Although this question has so far remained open, there are clear indications of significant roles of several naturally cytotoxic lymphocytes in preventing the development of cancer. We began a prospective cohort study among a Japanese general population in 1986, using various immunological and biochemical markers. METHODS: Natural cytotoxic activity of peripheral-blood mononuclear cells was assessed by isotope-release assay in 3625 residents of a Japanese population mostly older than 40 years of age, between 1986 and 1990. Immunological and biochemical markers were also measured, and participants were given a questionnaire on lifestyle. We did an 11-year follow-up survey of the cohort members looking at cancer incidence and death from all causes, and analysed the association between cytotoxic activity of peripheral-blood lymphocytes assessed at baseline and cancer incidence found in the subsequent follow-up. FINDINGS: 154 cancer cases were used in the analysis. When we categorised the cytotoxic activity of peripheral-blood lymphocytes by tertiles, age-adjusted relative risk of cancer incidence (all sites) was 0.72 (95% CI 0.45-1.16) for men with high cytotoxic activity, and 0.62 (0.38-1.03) for men with medium cytotoxic activity, taking the risk of those with low cytotoxic activity as reference. For women with high cytotoxic activity relative risk was 0.52 (0.28-0.95), and for those with medium cytotoxic activity 0.56 (0.31-1.01). For both sexes with high and medium cytotoxic activity risk was 0.63 (0.43-0.92) and 0.59 (0.40-0.87), respectively. INTERPRETATION: Our results indicate that medium and high cytotoxic activity of peripheral-blood lymphocytes is associated with reduced cancer risk, whereas low activity is associated with increased cancer risk suggesting a role for natural immunological host defence mechanisms against cancer.