Inhibition of mTOR by temsirolimus contributes to prolonged survival of mice with pleural dissemination of non-small-cell lung cancer cells

Temsirolimus (CCI-779), a recently synthesized analogue of rapamycin, specifically inhibits mTOR and has been approved for clinical use in renal cell carcinoma. Recent reports have indicated the growth inhibitory effect of temsirolimus in some cancers including non-small-cell lung carcinoma (NSCLC). In this study, we aimed to explore the potential therapeutic use of temsirolimus as a treatment for NSCLC. Using cultured NSCLC cells (A549, H1299, and H358), we determined the effect of temsirolimus on cell proliferation and its antitumor effects on subcutaneous tumors, as well as its contribution to the survival of mice having pleural dissemination of cancer cells, mimicking advanced NSCLC. Temsirolimus suppressed proliferation of NSCLC cells in a dose-dependent manner, with an IC(50) of <1 nM. Western blot analysis revealed that temsirolimus treatment specifically inhibited the phosphorylation of mTOR and its downstream effectors in 1 h, accompanied by an increased cell population in the G(0) /G(1) phase, but according to flow cytometry, the cell population did not increase in the sub-G(0) phase. When NSCLC subcutaneous tumor-bearing mice were treated with temsirolimus, tumor volume was significantly reduced (tumor volume on day 35: vehicle vs temsirolimus = 1239 vs 698 cm(3) ; P < 0.05). Furthermore, prolonged survival was observed in pleural disseminated tumor-bearing mice with temsirolimus treatment (median survival: vehicle vs temsirolimus = 53.5 vs 72.5 days; P < 0.05). These results suggest that temsirolimus could be useful for NSCLC treatment, due to its antiproliferative effect, and could be a potential treatment for advanced NSCLC, giving prolonged survival.     

Evaluation of changes of cell-surface thiols as a new biomarker for in vitro sensitization test

In order to find a novel biomarker for a simple assay to predict skin sensitization, we evaluated cell-surface thiols as a biomarker reflecting intracellular signaling in THP-1 cells (human monocytic cell line). First, we found that a decrease of cell-surface thiols on hapten-treated THP-1 cells was induced in parallel with phosphorylation of p38 MAPK. Next, we confirmed that 2-mercaptoethanol in the culture medium and the differentiation state of THP-1 cells did not affect the changes of cell-surface thiols by hapten. Changes of cell-surface thiols on THP-1 cells were detected after 2 h treatment with most allergens (e.g., DNCB, NiSO₄), as well as some non-allergens (e.g., Tween80, benzalkonium chloride), though other non-allergens (e.g., SDS, glycerol) had no effect. When either a significant decrease or increase of cell-surface thiols (more than 15% in each case) was used as a criterion, the results using 36 allergens and 16 non-allergens were in good accordance with those of in vivo assays. Finally, we confirmed that ATP, which is released as a consequence of cytotoxicity, did not affect the changes of cell-surface thiols. Our results suggest that changes of cell-surface thiols may be useful for an in vitro sensitization assay, which we designate as the SH test.     

Efficacy of preoperative radiotherapy to portal vein tumor thrombus in the main trunk or first branch in patients with hepatocellular carcinoma

Background The prognosis of hepatocellular carcinoma (HCC) with portal vein tumor thrombus (PVTT) in the main trunk or the first branch is very poor. Methods Radiotherapy (RT) to PVTT was followed by hepatectomy within 2 weeks. The dose used was 30–36 Gy, in 10–12 fractions, for 15–20 days. The efficacy of preoperative RT to PVTT in the main trunk or first branch was evaluated by comparing results in patients who underwent hepatectomy (group R; n = 15) with preoperative RT and those without preoperative RT (group N; n = 28). Results The 1-, 3-, and 5-year survival rates in group R were 86.2%, 43.5%, and 34.8%, respectively, while these values in group N were 39.0%, 13.1%, and 13.1%, respectively. The survival curve of group R was significantly better than that of group N (P = 0.0359). In group R, five (83.3%) of six patients whose tumor thrombus was completely necrosed (based on pathological examination) and whose follow-up period was over 2 years survived for more than 2 years. Female sex (P = 0.0066), multiple tumors (P = 0.0369), and absence of preoperative RT (P = 0.0359) were ranked as significant factors for a poor prognosis by univariate analysis. Multivariate analysis revealed absence of preoperative RT and female sex to be significant factors for a poor prognosis. Conclusion Preoperative RT to PVTT in the main trunk or first branch improved the prognosis of patients with HCC with PVTT, and could be a promising new modality in the treatment of these patients.     

IGFBP-3 regulates esophageal tumor growth through IGF-dependent and independent mechanisms

Insulin-like growth factor binding protein (IGFBP)-3 exerts either proapoptotic or growth stimulatory effects depending upon the cellular context. IGFBP-3 is overexpressed frequently in esophageal cancer. Yet, the role of IGFBP-3 in esophageal tumor biology remains elusive. To delineate the functional consequences of IGFBP-3 overexpression, we stably transduced Ha-Ras(V12)-transformed human esophageal cells with either wild-type or mutant IGFBP-3, the latter incapable of binding Insulin-like growth factor (IGFs) as a result of substitution of amino-terminal Ile56, Leu80, and Leu81 residues with Glycine residues. Wild-type, but not mutant, IGFBP-3 prevented IGF-1 from activating the IGF-1 receptor and AKT, and suppressed anchorage-independent cell growth. When xenografted in nude mice, in vivo bioluminescence imaging demonstrated that wild-type, but not mutant IGFBP-3, abrogated tumor formation by the Ras-transformed cells with concurrent induction of apoptosis, implying a prosurvival effect of IGF in cancer cell adaptation to the microenvironment. Moreover, there was more aggressive tumor growth by mutant IGFBP-3 overexpressing cells than control cell tumors, without detectable caspase-3 cleavage in tumor tissues, indicating an IGF-independent growth stimulatory effect of mutant IGFBP-3. In aggregate, these data suggest that IGFBP-3 contributes to esophageal tumor development and progression through IGF-dependent and independent mechanisms.     

Over-expression of 14-3-3sigma in budding colorectal cancer cells modulates cell migration in the presence of tenascin-C

Epigenetic silencing of the 14-3-3sigma gene by CpG hypermethylation has been reported in many kinds of cancers, but has been considered inapplicable in the colorectal variety. The expression of 14-3-3sigma in colorectal cancer is located primarily in the invasive area. The interaction between tumor cells and the extracellular matrix (ECM) is involved in tumor invasion. In the current study, we investigated the correlation between 14-3-3sigma expression and the ECM, focusing especially on the presence of tenascin-C (TNC) at the invasive area of colorectal cancers. Correlations between the immunohistochemical expression of 14-3-3sigma and TNC, as well as other clinicopathological factors, were evaluated in 123 colorectal carcinoma tissues. 14-3-3sigma expression was frequently observed in budding tumor cells in the invasive area and expression was significantly correlated with budding formation (p=0.001), pTNM classification (p=0.001) and stromal TNC expression (p=0.004). Using colorectal cancer cell lines and ECMs, the up-regulation of 14-3-3sigma mRNA levels was investigated by semi-quantitative RT-PCR. TNC surrounding the tumor cells increased 14-3-3sigma mRNA expression 1.8- to 2.2-fold in HCT116 cells. The effect of 14-3-3sigma over-expression on tumor cell migration was investigated using an agarose-cell droplet migration assay. Over-expression of 14-3-3sigma up-regulated HCT116 cell migration on TNC (p<0.001). We concluded that the expression of 14-3-3sigma in the invasive area modulates tumor cell migration in certain types of colorectal cancer and thus facilitates tumor progression.     

Development of integrated backscatter intravascular ultrasound for tissue characterization of coronary plaques

Tissue characterization of plaques of coronary arteries is important to clarify the process of acute coronary syndrome and prevent it. The purpose of this study is to develop an online integrated backscatter intravascular ultrasound (IB-IVUS) system and validate the diagnostic accuracy for the characterization of coronary plaques. A personal computer equipped with custom software was connected to an IVUS imaging system. Images were acquired from 242 segments of 46 coronary arteries from 25 cadavers obtained at autopsy. In the training study, a total of 724 regions-of-interests on color-coded maps were compared with histologic images. In the validation study, a total of 192 cross-sections of coronary arteries were evaluated. Receiver operating characteristic curve analysis showed that the cut-off points of -49 dB (area under curve = 0.98) and -29 dB (area under curve = 0.99) were the most reliable predictors of lipid pools, fibrosis and calcification. In the validation study, the analysis using IB values classified fibrous, lipid-rich and fibrocalcific plaque components with a high accuracy of 93%, 90% and 96%, respectively. The overall agreement between histologic and IB-IVUS diagnoses (n = 175) was high (Cohen's kappa = 0.81). The IB-IVUS system provides high diagnostic accuracy for analysis of tissue characteristics of coronary plaques.     

Comparison of gelatin particle agglutination and hemagglutination inhibition tests for measles seroepidemiology studies

Summary.  The prevalence of measles antibody in Japan was surveyed with a newly developed gelatin particle agglutination (PA) test, and the results compared with those of the hemagglutination inhibition (HI) test. The two age-distribution curves of the PA antibody-positive rates at ≥1:8 and ≥1:32 were almost the same in all the age groups, except the less-than-1-year-old group for which the rate at ≥1:8 was higher than that at ≥1:32 (p<0.05, χ² test). In the vaccinated children, all groups older-than-1-year of age had antibody-positive levels of 96% or more. In contrast, in the unvaccinated children, there was a sharp increase in antibody-positive rates between the 1- and 4-year-old groups, indicative that about 80% of the children were infected by wild measles virus at these ages. A significant number of PA antibody-positive specimens were antibody-negative (<1:8) by HI. The percentage of specimens in this category, PA (+) but HI (−), was greatest in infants less than one year old, and least in young children, but it increased with age to 97% of the HI (−) specimens from adults of more than 20 years of age. The PA test therefore detected some measles antibodies that HI could not. This test is simple and useful for making serosurveys in both developed and developing countries. Received January 27, 1997 Accepted June 16, 1997     

Intracellular calcium increasing at the beginning of reperfusion assists the early recovery of myocardial contractility after diltiazem cardioplegia

Objectives: We investigated the ability of diltiazem to prevent myocardial injury by assessing heart function and intracellular calcium concentrations before and after ischemia-reperfusion. Method: Isolated rat hearts underwent cardioplegia using the Langendorff perfusion model and were subjected to normothermic global ischemia for 60 minutes. The recovery rates for the heart function (heart rate, coronary flow, left ventricular systolic pressure) after reperfusion were monitored, and the intracellular Ca concentration was measured during ischemia and during the following reperfusion. Experimental groups were divided into three groups according to the diltiazem concentration used in the cardioplegic solution (potassium 20 mmol/l in Ringer's solution): (1) Group A: diltiazem 2.5 mg/l; (2) Group B: diltiazem 5 mg/l; and (3) Group C: no diltiazem. Results: Intracellular calcium concentration increased in all 3 groups during ischemia, but was significantly lower in Group B compared to either Group A or Group C. The heart function was significantly higher for Group A than for Group B or Group C. The hearts in Group B displayed markedly poor recovery in contractility and in heart rate. Conclusions: Generally, a decrease in intracellular Ca concentration improves the heart function during ischemia and after reperfusion. However, this study showed that some increase in intracellular Ca at the beginning of reperfusion assisted the contractility of rat heart.