Jun-ichi Ogawa

ABCA3 is a lamellar body membrane protein in human lung alveolar type II cells1

The ABCA3 gene, of the ABCA subclass of ATP‐binding cassette (ABC) transporters, is expressed exclusively in lung. We report here the cloning, molecular characterization, and distribution of human ABCA3 in the lung. Immunoblot analysis using the specific antibody reveals a 150‐kDa protein in the crude membrane fraction of human lung. Immunohistochemical analyses of alveoli show that ABCA3 is expressed only in the type II cells expressing surfactant protein A. At the ultrastructural level, ABCA3 immunoreactivity was detected mostly at the limiting membrane of the lamellar bodies. Since members of the ABCA transporter family are known to be involved in transmembrane transport of endogenous lipids, our findings suggest that ABCA3 plays an important role in the formation of pulmonary surfactant in type II cells.

Tumor-Derived TGFβ-1 Induces Dendritic Cell Apoptosis in the Sentinel Lymph Node

Lymphatic flux from a primary tumor initially flows into a tumor-draining lymph node (LN), the so-called sentinel LN (SLN). Carried by the lymph fluid are a variety of mediators produced by the tumor that can influence immune responses within the SLN, making it a good model with which to investigate tumor-related immunology. For instance, dendritic cell (DC) numbers are reduced in SLNs from melanoma and breast cancer patients. In the present study, we investigated the mechanism by which DC numbers were reduced within SLNs from patients with non-small cell lung cancer. We found that the incidence of apoptosis among DCs was higher in SLNs than in non-SLNs, as were levels of TGFβ-1. In contrast, levels of TGFβ-1 mRNA did not differ between SLNs and non-SLNs, but were 30 times higher in tumors than in either LN type. In vitro, incubation for 2 days with TGFβ-1 induced apoptosis among both cultured DCs and DCs acutely isolated from normal thoracic LNs, effects that were blocked by the TGFβ-1 inhibitor DAN/Fc chimera. Taken together, these results suggest that tumor-derived TGFβ-1 induces immunosuppression within SLNs before the movement of tumor cells into the SLNs, thereby facilitating metastasis within those nodes.

Endothelial Rho and Rho kinase regulate neutrophil migration via endothelial myosin light chain phosphorylation.

The transendothelial migration of neutrophils is a critical step in acute inflammation, which we previously showed to be regulated by endothelial myosin light chain (MLC) kinase. Recent studies suggest that Rho and Rho kinase are also key mediators of MLC phosphorylation, but their roles in neutrophil migration have not been investigated. In the present study, a transwell chamber migration assay system incorporating endothelial monolayer was used to examine the numbers of migrating neutrophils, endothelial F‐actin and myosin II rearrangement, and endothelial MLC phosphorylation at selected times during the neutrophil migration in vitro. The results showed that pretreating endothelial cells with C3 (Rho inhibitor) or Y‐27632 (Rho kinase inhibitor) significantly diminished neutrophil migration, actin polymerization, myosin II filament formation, and MLC phosphorylation normally associated with the migration. These data suggest that endothelial Rho and Rho kinase regulate transendothelial neutrophil migration by modulating the cytoskeletal events that mediate such migration.

Expression of histone deacetylase 1 correlates with a poor prognosis in patients with adenocarcinoma of the lung.

UNLABELLED Histone deacetylase (HDAC) inhibitors (HDACis) are now attracting attention as promising therapeutic agents for the treatment of cancer. However, the relation between HDAC1 expression and the clinicopathological characteristics of non-small cell lung cancer (NSCLC) is not well understood. We therefore investigated the relationship between HDAC1 expression by tumors and the clinicopathological characteristics of patients with adenocarcinoma of the lung. METHODS We used semi-quantitative real time reverse transcription polymerase chain reaction to assess expression of HDAC1 mRNA in tumor samples from 93 patients with adenocarcinoma of the lung. We then correlated HDAC1 mRNA levels with known clinicopathological factors. RESULTS We found that the 5-year disease-free survival rate among patients strongly expressing HDAC1 was significantly poorer than among those expressing lower levels (P=0.005 by log-rank test). Multivariate Cox proportional hazard analyses revealed that being male (hazard ratio, 3.56; 95% CI, 1.58-8.42; P=0.002), nodal metastasis (hazard ratio, 7.98; 95% CI, 2.99-22.1; P<.001) and HDAC1 (hazard ratio, 2.17; 95% CI, 1.04-4.84; P=0.039) were all independent factors affecting 5-year disease-free survival. CONCLUSION Stronger HDAC1 expression by tumor cells is an independent predictor of a poor prognosis in patients with adenocarcinoma of the lung.

Hand-Sewn Cervical Anastomosis Versus Stapled Intrathoracic Anastomosis After Esophagectomy for Middle or Lower Thoracic Esophageal Cancer: A Prospective Randomized Controlled Study

PurposeThe type of anastomosis and its outcome can affect postoperative morbidity, mortality, and quality of life after esophagectomy. We compared the outcomes of cervical hand-sewn anastomosis (CHS) and intrathoracic stapled anastomosis (ITS) performed after esophagectomy and gastric reconstruction.MethodsThirty-two patients with middle or lower thoracic esophageal cancer were prospectively randomized to undergo CHS (n = 18) or ITS (n = 14) after esophagectomy. We compared clinical data, postoperative symptoms, and long-term survival in the two groups.ResultsThe rates of anastomotic leak and stricture in the CHS and ITS groups were 16.7% versus 7.1% and 0% versus 14.2%, respectively, which do not represent significant differences. The respective rates of recurrent laryngeal nerve palsy were 38.8% versus 7.1% (P < 0.05), and proximal esophageal resection was 15 mm longer (P < 0.05) in the CHS group. There were no significant differences in symptoms 6 months after surgery, or in the overall 5-year survival rates (72.2% and 85.7%, respectively).ConclusionsThe two methods of anastomosis yielded similar anastomotic outcomes. Although the incidence of recurrent laryngeal nerve injury was higher after CHS, and proximal esophageal resection was longer, this had little impact on postoperative symptoms and long-term survival.

Connexin26-mediated gap junctional communication reverses the malignant phenotype of MCF-7 breast cancer cells

A growing body of evidence indicates that the gap junction (GJ) plays a pivotal role in tumor suppression by exerting cell‐cell communication. It has, however, been reported that expression of connexin26 (Cx26) protein is induced in human ductal carcinomas of the breast and that its amount increases in proportion to the grade of malignancy. We thus examined the effects of over‐expressed Cx26 on growth characteristics in GJ‐deficient human MCF‐7 breast cancer cells that maintain the phenotype of early‐stage cancers. MCF‐7 cells were transfected with Cx26 cDNA, and several clones of stable transformants exhibiting a high level of cell‐cell communication were established. When they were examined in terms of various growth characteristics in vitro, the proliferation rate and the saturation density were drastically reduced in Cx26‐transfected clones compared with the mock‐transfectant. The anchorage‐independent growth capacity was also decreased by 50–75% after transfection of Cx26. Furthermore, the cell migration toward growth factors and cell invasion into Matrigel in a Boyden chamber were suppressed to 5–10% and 20–60%, respectively, of the control in Cx26‐transfected clones. When implanted into the mammary fat pads of nude mice in the presence of an excess of 17β‐estradiol, Cx26‐transfected clones tended to show slower tumor growth than the mock‐transfectant, although the difference was not statistically significant. Our results strongly suggest that the induction of Cx26 protein observed in human breast cancers, reported previously, may not be very relevant to the development of breast cancers, and that Cx26 can function as a tumor suppressor in breast cancer cells.

A novel method for sentinel lymph node mapping using magnetite in patients with non-small cell lung cancer.

OBJECTIVE The detection rate of sentinel lymph nodes in patients with non-small cell lung cancer using isosulfan blue dye is too low for clinical use. Although exposure to radioactivity is reportedly minimal, special procedures are nonetheless required when a radioactive isotope is used as a tracer. Therefore, to eliminate the need for a radioactive tracer and to obtain a better detection rate than is obtained with isosulfan blue dye, we have developed a novel method that employs magnetite as the tracer. The aim of the present study was to test the feasibility of this technique. METHODS The tracer employed was ferumoxides, a colloidal superparamagnetic iron oxide of nonstoichiometric magnetite. Thirty-eight non-small cell lung cancer patients participated in the study; each received 5 mL of ferumoxides, injected around the tumor intraoperatively. Fifteen minutes after injection, lung resection and lymph node dissection were carried out. The magnetic force within the lymph nodes was measured using a highly sensitive handheld magnetometer ex vivo. All lymph nodes were also subjected to conventional histological analysis. RESULTS The rate of detection of sentinel lymph nodes was 81.6% (31/38). The accuracy, sensitivity, and false-negative rates were 96.8% (30/31), 85.7% (6/7), and 14.3% (1/7), respectively. CONCLUSION Intraoperative sentinel lymph node mapping using ferumoxides and a highly sensitive magnetometer is a safe, accurate, and sensitive way to detect sentinel lymph nodes in non-small cell lung cancer patients.

Cisplatin Induces Fas Expression in Esophageal Cancer Cell Lines and Enhanced Cytotoxicity in Combination with LAK Cells

Objective: To establish a new therapeutic approach for the treatment of esophageal cancer, we investigated an alternative mechanism of immunotherapy for sensitizing target cells to effector cells. Methods: Six human esophageal cancer cell lines were used. The expression of Fas antigen on tumor cells was determined by flow cytometry. The cytotoxic effect of cis-dichlorodiammineplatinum (CDDP) and anti-Fas antibody was evaluated using an MTT assay. The cytotoxic activity of LAK cells was measured by a 51Cr release assay. Results: Five out of six esophageal cancer cell lines expressed Fas antigen at various levels (26.2–61.5%), and Fas expression increased after CDDP treatment. The antitumor effect of anti-Fas antibody on the esophageal cancer cell line and the antitumor effect of LAK cells activated by IL-2 were enhanced by pretreatment with CDDP. After concanamycin A treatment to specifically evaluate Fas-dependent cytotoxicity, LAK cells expressing Fas ligand killed only Fas-positive cells, but not Fas-negative cells. An anti-Fas neutralizing antibody inhibited this cytotoxicity. DNA fragmentation was shown in a cell line that was treated with CDDP and anti-Fas antibody, and also in the targeted esophageal cancer cell line cocultured with LAK cells. Conclusion: Our results suggest a potential clinical application of CDDP as a Fas inducer to make esophageal tumors susceptible to Fas antigen and LAK cytotoxicity.

Increased expression of myosin light chain kinase mRNA is related to metastasis in non-small cell lung cancer.

Background: The invasiveness of tumor cells depends in large part on their motility, which in turn depends on cytoskeletal function. A major cytoskeletal component involved in cell motility is myosin II, the classical form of myosin first identified in muscle but also expressed in nonmuscle cells. Myosin II is activated by myosin light chain kinase (MLCK), which phosphorylates it on its regulatory light chain. In this context, the contribution made by MLCK to tumor cell motility and invasiveness has been investigated extensively in vitro, but clinical evidence of such a contribution has been lacking. In the present study, therefore, we examined the role of MLCK in the metastasis of non-small cell lung cancer (NSCLC) in a clinical setting. Methods: We measured MLCK mRNA expression in tumor samples from 39 NSCLC patients using real-time semiquantitative reverse transcription polymerase chain reaction carried out in a LightCycler. We then correlated MLCK mRNA expression with known clinicopathological factors. Results: We found that levels of MLCK mRNA expression were higher in patients who showed disease recurrence and distant metastasis than in those who did not. Moreover, the 3-year disease-free survival rate among patients showing lower levels of MLCK mRNA expression [log10(MLCK/GAPDH) <1.4] was significantly greater than among those showing higher MLCK mRNA expression [log10(MLCK/GAPDH) ≧1.4] (87.5 vs. 50.0%; log-rank test, p = 0.021). Conclusion: These findings are the first clinical evidence that expression of MLCK is correlated with disease recurrence and distant metastasis in NSCLC.

Inhibition of heat shock protein 90 sensitizes melanoma cells to thermosensitive ferromagnetic particle-mediated hyperthermia with low Curie temperature

Heat shock protein (Hsp) 90 is a key regulator of a variety of oncogene products and cell‐signaling molecules, and the therapeutic benefit of its inhibition in combination with radiation or chemotherapy has been investigated. In addition, hyperthermia has been used for many years to treat various malignant tumors. We previously described a system in which hyperthermia was induced using thermosensitive ferromagnetic particles (FMP) with a Curie temperature (Tc = 43˚C) low enough to mediate automatic temperature control, and demonstrated its antitumor effect in a mouse melanoma model. In the present study, we examined the antitumor effects of combining a Hsp90 inhibitor (geldanamycin; GA) with FMP‐mediated hyperthermia. In cultured B16 melanoma cells, GA exerted an antitumor effect by increasing the cells’ susceptibility to hyperthermia and reducing expression of Akt. In an in vivo study, melanoma cells were subcutaneously injected into the backs of C57BL/6 mice. FMP were then injected into the resultant tumors, and the mice were divided into four groups: group I, no treatment (control); group II, one hyperthermia treatment; group III, GA alone; and group IV, GA with hyperthermia. When exposed to a magnetic field, the temperature of tissues containing FMP increased and stabilized at the Tc. In group IV, complete regression of tumors was observed in five of nine mice (56%), whereas no tumor regression was seen in groups I–III. Our findings suggest that inhibition of Hsp90 with hyperthermia increases its antitumor effect. Thus, the combination of FMP‐mediated, self‐regulating hyperthermia with Hsp90 inhibition has important implications for the treatment of cancer. (Cancer Sci 2009; 100: 558–564)