Significance of ZO-1, an Intercellular Adhesion Molecule, as a Prognostic Marker in Lung Adenocarcinoma.

In epithelial tissues, intercellular adhesion structures are formed between adjacent cells via intercellular adhesion factors, such as zonula occludens (ZO-1), to maintain the structure and function of tissues and organs, thereby contributing to homeostasis. Epithelial cells are polarized into apical and basal regions by tight junctions (TJs), a type of intercellular adhesion structure, and thus, their intracellular organelles are asymmetrically distributed. Normal epithelial cells maintain their cellular function by controlling cytoskeletal reorganization, motility, and division by maintaining asymmetry in their intracellular organelles. Among the features common to many cancer tissues are abnormalities in cell polarity and intercellular adhesion. Lung adenocarcinoma consists of a mixture of five different histologic types that can be distinguished in the same section: lepidic, papillary, acinar, micropapillary, and solid patterns. Therefore, it is often difficult to accurately assess histological images because the staining differs according to the histological types. In the present study, we evaluated ZO-1 staining based on histological features observed in a single section and examined its relationship to clinicopathological features. In non-tumor areas, ZO-1 was expressed on the plasma membrane and in the cytoplasm of normal alveolar epithelial cells. However, in tumor areas, ZO-1 staining was mainly localized in the cytoplasm and on the plasma membrane only in a few cells. ZO-1-negative cases tended to have poorer prognoses in all histological types, with a poorer prognosis in the solid pattern. These results suggest that ZO-1 expression in solid-pattern lung adenocarcinoma may be a useful prognostic marker.

Protective effect of ebselen on bleomycin-induced lung fibrosis: analysis of the molecular mechanism of lung fibrosis mediated by oxidized diacylglycerol.

The molecular mechanisms underlying the development of pulmonary fibrosis remain unknown, and effective treatments have not yet been developed. It has been shown that oxidative stress is involved in lung fibrosis. Oxidized diacylglycerol (DAG) produced by oxidative stress is thought to play an important role in lung fibrosis. This study assessed the effect of oxidized DAG in an animal model of pulmonary fibrosis induced by aspiration of bleomycin (BLM) into the lungs. The inhibitory effect of ebselen on pulmonary fibrosis was also investigated. In lung fibrotic tissue induced by BLM, an increase in lipid peroxides and collagen accumulation was observed. Moreover, the levels of oxidized DAG, which has strong protein kinase C (PKC) activation activity, were significantly increased over time following the administration of BLM. Western blotting showed that phosphorylation of PKCα and δ isoforms was increased by BLM. Oral administration of ebselen significantly suppressed the increase in oxidized DAG induced by BLM and improved lung fibrosis. PKCα and δ phosphorylation were also significantly inhibited. The mRNA expression of α-smooth muscle actin and collagen I (marker molecules for fibrosis), as well as the production of transforming growth factor-ß and tumor necrosis factor-α(a potentially important factor in the fibrotic process), were increased by BLM and significantly decreased by ebselen. The administration of BLM may induce lipid peroxidation in lung tissue, while the oxidized DAG produced by BLM may induce overactivation of PKCα and δ, resulting in the induction of lung fibrosis.

Possible Glutathione Peroxidase 4-Independent Reduction of Phosphatidylcholine Hydroperoxide: Its Relevance to Ferroptosis.

Ferroptosis is mainly caused by iron-mediated peroxidation of phospholipids and has recently attracted attention due to its involvement in various diseases. At the center of it is supposedly the inability of glutathione peroxidase 4 (GPX4) to reduce excess peroxidized phospholipids (e.g., phosphatidylcholine hydroperoxide (PCOOH)) that trigger ferroptosis. However, the involvement of enzymes other than GPX4 in ferroptosis is scarcely known. To elucidate this matter, we evaluated the uptake of PCOOH in a GPX4 knockout (KO) human hepatoma cell line HepG2 generated using CRISPR-Cas9. After confirming that GPX4 expression in the KO cells was below the detection limit, we cultured both wild-type (WT) and GPX4 KO HepG2 cells in a medium containing 50 µM PCOOH for 1-8 hours. By analyzing the level of PCOOH and its reduction product (phosphatidylcholine hydroxide, PCOH) in cells using liquid chromatography-tandem mass spectrometry, we detected the cellular uptake of PCOOH. On top of this, we detected a large amount of PCOH not only in WT HepG2 but also in GPX4 KO HepG2, thus indicating the notable involvement of enzymes other than GPX4 (e.g., other GPX family, glutathione S-transferase, thioredoxin, or peroxiredoxin) in reducing PCOOH. Further corroboration of these findings hopefully leads to the development of novel methods to prevent ferroptosis-related diseases by targeting enzymes other than GPX4.

Par3 and ZO-1 Membrane Clustering is an Indicator of Poor Prognosis in Lung Squamous Cell Carcinoma.

Epithelial cells form epithelial tissue structures by joining together via intercellular adhesion structures composed of intercellular adhesion factors such as zona occludins-1 (ZO-1). Epithelial cells are polarized at the apical and basal regions, and are bordered by intercellular adhesion structures called tight junctions; the organelles within epithelial cells are distributed asymmetrically. Maintenance of this asymmetry in normal epithelial cells is essential for normal cytoskeletal remodeling, movement, and cell division. The key factor regulating cell polarity is called partitioning-defective protein 3 (Par3). Abnormalities in cell polarity and intercellular adhesion are common features of many cancer tissues. Mutation and loss of cell polarity regulators contributes to the immortalization of normal cells and to the malignant transformation of cancer cells. In this study, we investigated the relationship between the subcellular localization of Par3 and ZO-1 and clinicopathological features of lung squamous cell carcinoma (lung SqCC). Both molecules were localized to the cell membrane in normal lung tissue, but the levels were lower at this location in pulmonary tumor tissue compared with normal lung tissue. Both Par3 and ZO-1 accumulated in clusters on the cell membrane (hereinafter, "foci"). Tumor size, recurrence rate, and mortality rate were significantly higher in patients with Par3 foci compared to those without Par3 foci. Rates of lymph node metastasis, recurrence, and mortality were significantly higher in patients with ZO-1 foci than in those without ZO-1 foci. The expression of Par3 and ZO-1 mRNA was not s ignificantly different in s amples from p atients with foci versus those without. These results strongly suggest that the presence of Par3 and ZO-1 foci on the membrane may be a useful prognostic marker for lung SqCC.

Inositol hexakisphosphate kinase 2 promotes cell death of anterior horn cells in the spinal cord of patients with amyotrophic lateral sclerosis.

We have previously reported that inositol hexakisphosphate kinase (InsP6K)2 mediates cell death. InsP6K2 is abundantly expressed in anterior horn cells of the mammalian spinal cord. We investigated the role of InsP6K2 in spinal cords of patients with amyotrophic lateral sclerosis (ALS). Autopsy specimens of lumbar spinal cords from ten patients with sporadic ALS and five non-neurological disease patients (NNDPs) were obtained. We performed quantitative real-time PCR, immunostaining, and western blotting for InsP6K1, InsP6K2, InsP6K3, protein kinase B (Akt), casein kinase 2 (CK2), and 90-kDa heat-shock protein (HSP90). In contrast to InsP6K1 and InsP6K3 mRNA expression, InsP6K2 levels in anterior horn cells of the spinal cord were significantly increased in ALS patients compared to NNDPs. In ALS patients, InsP6K2 translocated from the nucleus to the cytoplasm. However, we observed a decrease in HSP90, CK2, and Akt activity in ALS patients compared to NNDPs. A previous study reported that InsP6K2 activity is suppressed after binding to HSP90 and subsequent phosphorylation and degradation by CK2, thus decreasing InsP6K2 activity. However, InsP7, which is generated by InsP6K2, can compete with Akt for PH domain binding. Consequently, InsP7 can inhibit Akt phosphorylation. Our results suggest that InsP6K2 is activated in the spinal cord of patients with ALS and may play an important role in ALS by inducing cell death mechanisms via Akt, CK2, and HSP90 pathways.

Selenium and Glutathione-Depleted Rats as a Sensitive Animal Model to Predict Drug-Induced Liver Injury in Humans.

Drug-induced liver injury (DILI) is one of the most serious and frequent drug-related adverse events in humans. Selenium (Se) and glutathione (GSH) have a crucial role for the hepatoprotective effect against reactive metabolites or oxidative damage leading to DILI. The hepatoprotective capacity related to Se and GSH in rodents is considered to be superior compared to the capacity in humans. Therefore, we hypothesize that Se/GSH-depleted rats could be a sensitive animal model to predict DILI in humans. In this study, Se-deficiency is induced by feeding a Se-deficient diet and GSH-deficiency is induced by l-buthionine-S,R-sulfoxinine treatment via drinking water. The usefulness of this animal model is validated using flutamide, which is known to cause DILI in humans but not in intact rats. In the Se/GSH-depleted rats from the present study, decreases in glutathione peroxidase-1 protein expression and GSH levels and an increase in malondialdehyde levels in the liver are observed without any increase in plasma liver function parameters. Five-day repeated dosing of flutamide at 150 mg/kg causes hepatotoxicity in the Se/GSH-depleted rats but not in normal rats. In conclusion, Se/GSH-depleted rats are the most sensitive for detecting flutamide-induced hepatotoxicity in all the reported animal models.

Hyperadrenocorticism Caused by a Pituitary Ganglioglioma in a Dog.

An 11-year-old castrated male Chihuahua dog was presented with complaints of polyuria, polydipsia, abdominal enlargement, and alopecia. Hyperadrenocorticism was diagnosed on the basis of clinical signs, blood tests, adrenocorticotropin-stimulation test results, and an elevated serum adrenocorticotropin concentration. Contrast-enhanced magnetic resonance imaging showed that the pituitary gland was enlarged, compatible with a pituitary macroadenoma. Pituitary-dependent hyperadrenocorticism was suspected, and transsphenoidal hypophysectomy was thus performed for complete resection of the tumor. After surgery, the serum adrenocorticotropin concentration normalized and the hyperadrenocorticism resolved. Histological and immunocytochemical analyses revealed a benign tumor composed of mature neuronal cells and glial cells, suggestive of a ganglioglioma with immunolabeling for adrenocorticotropin. Careful analysis of the resected tumor revealed no pituitary adenoma tissue. The clinical and histopathologic findings indicated that the ganglioglioma was directly responsible for the hyperadrenocorticism. This is the first case of hyperadrenocorticism caused by a ganglioglioma in a dog.

Presence of human herpes virus 1-thymidine kinase in testis of azoospermic infertile herpes-infected patients.

Infection with human herpes virus 1 (HHV1) is a suspected cause of human male infertility. However, the correlation between HHV1 infection and infertility is still unclear. We have previously generated transgenic rats that ectopically express the HHV1 thymidine kinase gene (HHV1-TK) in post-meiotic spermatids and found they had aberrant spermatogenesis and infertility. Therefore, we hypothesized that human infertility might be caused by HHV1 infection. Here, we examined whether HHV1-TK is expressed in human testis by analyzing the presence of its transcript and protein. Specimens were collected by biopsy from 30 azoospermic infertile male patients. RT-PCR and immunohistochemistry showed that 23 patients were positive for HHV1-TK expression, while seven patients were negative. Thus, we demonstrated HHV1-TK expression, indicating HHV1 infection, in the testis of human azoospermic infertile males for the first time; our findings represent a great advancement toward the verification of our hypothesis that HHV1-TK expression might cause human infertility.

Localization of SOX2-positive stem/progenitor cells in the anterior lobe of the common marmoset (Callithrix jacchus) pituitary.

Studies on mouse and rat pituitaries reported that Sox2-expressing cells play roles as stem/progenitor cells in the adult pituitary gland. The presence of cells with stem cell-like properties in the pituitary adenoma and SOX2-positive cells has been demonstrated in the human pituitary. However, considering the difficulty in fully examining the stem/progenitor cell properties in the human pituitary, in the present study, we analyzed the SOX2-positive cells in the pituitary of the adult common marmoset (Callithrix jacchus), which is used as a non-human primate model. Immunohistochemistry demonstrated that localization pattern of SOX2-positive cells in the common marmoset pituitary was similar to that observed in the rodent pituitary, i.e., in the two types of niches (marginal cell layer and parenchymal-niche) and as scattered single cells in the parenchyma of the anterior lobe. Furthermore, most of the SOX2-positive cells express S100 and were located in the center or interior of LAMININ-positive micro-lobular structures. Collectively, the present study reveals properties of SOX2-positive cells in the common marmoset pituitary and suggests that the common marmoset proves to be a useful tool for analyzing pituitary stem/progenitor cells in a non-human primate model.

Determination of triacylglycerol oxidation mechanisms in canola oil using liquid chromatography-tandem mass spectrometry.

Triacylglycerol (TG), the main component of edible oil, is oxidized by thermal- or photo- oxidation to form TG hydroperoxide (TGOOH) as the primary oxidation product. Since TGOOH and its subsequent oxidation products cause not only the deterioration of oil quality but also various toxicities, preventing the oxidation of edible oils is essential. Therefore understanding oxidation mechanisms that cause the formation of TGOOH is necessary. Since isomeric information of lipid hydroperoxide provides insights about oil oxidation mechanisms, we focused on dioleoyl-(hydroperoxy octadecadienoyl)-TG (OO-HpODE-TG) isomers, which are the primary oxidation products of the most abundant TG molecular species (dioleoyl-linoleoyl-TG) in canola oil. To secure highly selective and sensitive analysis, authentic OO-HpODE-TG isomer references (i.e., hydroperoxide positional/geometrical isomers) were synthesized and analyzed with HPLC-MS/MS. With the use of the method, photo- or thermal- oxidized edible oils were analyzed. While dioleoyl-(10-hydroperoxy-8E,12Z-octadecadienoyl)-TG (OO-(10-HpODE)-TG) and dioleoyl-(12-hydroperoxy-9Z,13E-octadecadienoyl)-TG (OO-(12-HpODE)-TG) were characteristically detected in photo-oxidized oils, dioleoyl-(9-hydroperoxy-10E,12E-octadecadienoyl)-TG and dioleoyl-(13-hydroperoxy-9E,11E-octadecadienoyl)-TG were found to increase depending on temperature in thermal-oxidized oils. These results prove that our methods not only evaluate oil oxidation in levels that are unquantifiable with peroxide value, but also allows for the determination of oil oxidation mechanisms. From the analysis of marketed canola oils, photo-oxidized products (i.e., OO-(10-HpODE)-TG and OO-(12-HpODE)-TG) were characteristically accumulated compared to the oil analyzed immediately after production. The method described in this paper is valuable in the understanding of oil and food oxidation mechanisms, and may be applied to the development of preventive methods against food deterioration.

Liposome-Encapsulated Hemoglobin Accelerates Skin Wound Healing in Diabetic dB/dB Mice.

Since liposome-encapsulated hemoglobin with high O2 affinity (h-LEH, P50 O2  = 10 mm Hg) has been reported to accelerate skin wound healing in normal mice, it was tested in dB/dB mice with retarded wound healing, as seen in human diabetics. Two full-thickness dorsal wounds 6 mm in diameter encompassed by silicone stents were created in dB/dB mice. Two days later (day 2), the animals were randomly assigned to receive intravenous h-LEH (2 mL/kg, n = 7) or saline (2 mL/kg, n = 7). The same treatment was repeated 4 days after wounding (day 4), and the size of the skin lesions was analyzed by photography, surface perfusion was detected by Laser-Doppler imager, and plasma cytokines and chemokines were determined on days 0, 2, 4, and 7, when all animals were euthanized for morphological studies. The size of the ulcer compared to the skin defect or silicone stent became significantly reduced on days 4 and 7 in mice treated with h-LEH (47 ± 8% of original size), similar to the level in wild-type mice, compared to saline-treated dB/dB mice (68 ± 18%, P < 0.01). Mice treated with h-LEH had significantly attenuated inflammatory cytokines, increased surface perfusion, and increased Ki67 expression on day 7 in accordance with the ulcer size reduction, while there was no significant difference in chemokines, histological granulation, epithelial thickness, and granulocyte infiltration detected by immunohistochemical staining in the ulcer between the treatment groups. The results suggest that h-LEH (2 mL/kg) early after wounding may accelerate skin wound healing in dB/dB mice to levels equivalent to wild-type mice probably via mechanism(s) involving reduced hypoxia, increased surface perfusion, suppressed inflammation, accelerated in situ cell proliferation and protein synthesis.

Cartilage Oligomeric Matrix Protein Increases in Photodamaged Skin.

Cartilage oligomeric matrix protein (COMP) is a structural component of cartilage. Recent studies have described COMP as a pathogenic factor that promotes collagen deposition in fibrotic skin disorders such as scleroderma and keloid skin. Although collagen, a major dermis component, is thought to decrease in photoaged skin, recent reports have demonstrated the presence of tightly packed collagen fibrils with a structural resemblance to fibrosis in the papillary dermis of photoaged skin. Here we examined how photoaging damage relates to COMP expression and localization in photoaged skin. In situ hybridization revealed an increase in COMP-mRNA-positive cells with the progress of photoaging in preauricular skin (sun-exposed skin). The signal intensity of immunostaining for COMP increased with photoaging in not only the papillary dermis but also the reticular dermis affected by advancing solar elastosis. Immunoelectron microscopy detected the colocalization of COMP with both elastotic materials and collagen fibrils in photoaged skin. Ultraviolet light A irradiation of human dermal fibroblasts induced COMP expression at both the mRNA and protein levels. Ultraviolet light A-induced COMP expression was inhibited by an anti-transforming growth factor-ß antibody or SB431542, an activin receptor-like kinase 5 inhibitor. These results suggest that the transforming growth factor-ß-mediated upregulation of COMP expression may contribute to the modulation of dermal extracellular matrix in the photoaging process.

Reduced Expression of Hugl 1 Contributes to the Progression of Lung Squamous Cell Carcinoma.

Cell polarity and cell-cell adhesion play a critical role in the regulation of normal tissue architecture and function. Disruption of cell adhesion and cell polarity is often associated with neoplastic tumors. Loss of apical-basal polarity in epithelial cells is one of the hallmarks of aggressive and invasive cancers. Several polarity proteins including atypical protein kinase C (aPKC), Par 6, Par 3, and Lethal giant larvae (Lgl, the human homologues of which are called Hugl 1 and Hugl 2) are localized at the leading edge of migrating cells, and play critical roles during directional migration. Herein, we investigated the expression of aPKC, Par 6, Par 3, Hugl 1, and Hugl 2 in lung squamous cell carcinoma (SqCC). An inverse correlation was observed between the expression of Hugl 1 and lung SqCC progression. Results of immunohistochemistry and real-time RT-PCR analyses showed that reduced expression of Hugl 1 predicts poor survival in lung SqCC patients. The expression of Hugl 1 was inversely correlated with both overall survival rate and tumor stage. On the other hand, no associations were observed between the expressions of Hugl 2, Par 6, and Par 3 and lung SqCC progression. These findings indicate that the reduced expression of Hugl 1 could be considered as a poor prognostic factor in human lung cancers.

A Histopathological Study of Multi-hormone Producing Proliferative Lesions in Estrogen-induced Rat Pituitary Prolactinoma.

Rats with estrogen-induced prolactin-producing pituitary adenoma (E2-PRLoma) have been employed as an animal model of human PRL-producing pituitary adenoma in a large number of studies. Presently, we found that long-term administration of estrogen to SD rats resulted in the development of E2-PRLomas, some of which included multi-hormone producing nodules. We herein report results of histopathological analyses of these lesions. PRLoma models were created in female SD rats by 22 weeks or longer administration of a controlled-release preparation of estradiol at a dose of 10 mg/kg/2 weeks. Ten of the 11 PRLoma model rats had proliferative nodular lesions composed of large eosinophilic cells like gonadotrophs inside the PRLoma. These lesions were positive for PRL, TSHß, and α subunits and were negative for GH, LHß, ACTH, and S-100. Double immunostaining revealed that these large eosinophilic cells showed coexpression of PRL and TSHß, PRL and α subunits, and TSHß and α subunits. Those results clarified that long-term estrogen administration to female SD rats induced multi-hormone producing neoplastic pituitary nodules that expressed PRL, TSHß, and α subunits. We studied these neoplastic nodules obtained by laser microdissection to acquire findings similar to those of the immuno-histochemical analysis. We consider that this animal model is useful for pathogenesis analyses and therapeutic agent development concerning human multi-hormone producing pituitary adenomas.

Flavonoids enhance melanogenesis in human melanoma cells.

Flavonoids are pigmentary compounds existing widely in plants. We have reported that quercetin (3, 3', 4', 5, 7-pentahydroxylflavone), one of the typical flavonoids, strongly promotes melanogenesis by melanocytes. Meanwhile, there are 8000 or more flavonoids having a chemical structure different from each other in the natural world. Their distinctive chemical properties suggest that they may be different in melanogenic actions. In the present study, the melanogenic actions of 14 flavonoids were analyzed to correlate their chemical structures with melanogenic actions. To evaluate the effects of flavonoids on melanogenesis, the HMV II cell line derived from human malignant melanoma was used. Flavonols including quercetin, kaempferol, rhamnetin and fisetin, flavones including apigenin, luteolin and chrysin, and isoflavones including genestein showed melanogenesis-promoting actions but rutin, robinetin, myricetin, ipriflavone, epigalocatechin gallate (EGCg) and naringin did not. From analyses of the relationships between the chemical structures of flavonoids and their melanogenesis-promoting actions, it was inferred that a hydroxyl group bound to the phenyl group plays an important role in stimulating melanogenesis. From the above results, 8 flavonoids were identified as melanogenesis promoters. Also, correlations were established between the melanogenesis-promoting actions of flavonoids and their chemical structures.

Effects of liposome-encapsulated hemoglobin on gastric wound healing in the rat.

Liposome-encapsulated hemoglobin (LEH) may improve microcirculation and oxygen (O2 ) metabolism at a surgical wound to accelerate its healing. Ten mL/kg of LEH with high (h-LEH) or low O2 -affinity (l-LEH), homologous red blood cells (RBC), empty liposome or saline as a control was infused before a 10-mm incision and interrupted suture closure of the gastric wall in a total of 110 rats. Two and 4 days later, the stomach was excised for bursting pressure determination and histological sampling. The dose-response relationship was examined in 70 additional rats receiving progressively reduced doses of h-LEH. Hypoxia-inducible factor-1α (HIF-1α) was stained immunohistochemically in 54 other rats to examine its accumulation at the anastomotic sites. Bursting pressure of the surgical wound was significantly higher 2 days after surgery only in the h-LEH-treated rats (P < 0.05), but not at 4 days after surgery, when other rats showed increased bursting pressure to a nonsignificant level. Histological examination revealed less granulocyte infiltration, better granulation, and more macrophage infiltration in h-LEH-treated rats at 2 days, but no longer at 4 days postsurgery. Dose-response study revealed that 0.4 mL/kg of h-LEH (hemoglobin 24 mg/kg) was effective for elevating bursting pressure at 2 days. h-LEH-treated rats had significantly suppressed HIF-1α accumulation in the wound 6, 24, and 48 h after surgery as compared with control animals treated with homologous RBC or saline. In conclusion, the results suggest that h-LEH, but not l-LEH or homologous transfusion, may accelerate wound healing early after gastric incision and anastomosis in the rat. The mechanism(s) appears to be related to improved O2 supply, aerobic metabolism, and suppressed inflammation in the wound.

Defect of tropomyosin-related kinase B isotype expression in ovarian clear cell adenocarcinoma.

Tropomyosin-related kinase B (TrkB) is a functional signal molecule that correlates with cell survival and epithelial-mesenchymal transition (EMT), which is essential for the invasiveness of malignant cancer cells. While a truncated isoform of TrkB has a dominant negative effect, full-length TrkB with its tyrosine kinase domain is predicted to play a role in cancer progression. Because ovarian clear cell adenocarcinoma (CCA) shows worse prognosis compared to other cancer types, we investigated the correlation between TrkB isoforms and the progression of CCA. Ovarian adenocarcinoma and benign tumor samples were obtained from Tokai University Hospital and Juntendo University Hospital. These samples were examined for the TrkB expression of isotype-specific proteins and mRNAs by immunohistochemistry and domain-specific semi-quantitative reverse transcription polymerase chain reaction. While TrkB mRNA expression was detected in all of the ovarian tissues and TrkB protein expression was predominant in ovarian cancer tissues, the number of tissues expressing the tyrosine kinase-truncated isoforms (T-Shc or T1) decreased according to the clinical stage of CCA. Irregular isoforms were also observed in some CCA samples. The decrease in T-Shc and T1 were less obvious in mucinous adenocarcinoma and not observed in serous or endometrioid adenocarcinoma. Decreased expression of the truncated isoforms (T-Shc and T1) was associated with CCA progression. These results demonstrate that irregular expression of TrkB isoforms is a characteristic of CCA tissues. The unique TrkB expression profile may be useful for the diagnosis of CCA subtypes.

Stimulation of melanogenesis by nordihydroguaiaretic Acid in human melanoma cells.

Nordihydroguaiaretic acid (NDGA), a lignan found in vegetables, fruits and legumin, has been shown to possess antineoplastic, antiviral and antioxidant characteristics. In this study, we examined the effect of NDGA on melanogenesis in human melanoma cells (HMVII). In vitro, NDGA does not alter mushroom tyrosinase activity. However, in NDGA-treated HMVII cells, cellular tyrosinase activity increased in both a time- and dose-dependent manner. The concomitant increases in melanin content in NDGA-treated cells indicated an elevation of melanin synthesis by tyrosinase activation. In addition, after a 7-day incubation, melanin content in 20 µM NDGA-treated cells increased 5.02 fold. Tyrosinase protein also increased by treatment with NDGA. Nevertheless, tyrosinase mRNA was not altered in NDGA-treated cells. Our results suggest that NDGA can increase tyrosinase activity and de novo synthesis of melanin in human melanoma cells. We found that NDGA is a novel potent stimulator of melanogenesis in human melanoma cells.

Localization of aPKC lambda/iota and its interacting protein, Lgl2, is significantly associated with lung adenocarcinoma progression.

Atypical protein kinase C lambda/iota (aPKC λ/ι) is expressed in several human cancers; however, the correlation between aPKC λ/ι localization and cancer progression in human lung adenocarcinoma (LAC) remains to be clarified. We found that patients with a high level of aPKC λ/ι expression in LAC had significantly shorter overall survival than those with a low level of aPKC λ/ι expression. In addition, localization of aPKC λ/ι in the apical membrane or at the cell-cell contact was associated with both lymphatic invasion and metastasis. The intercellular adhesion molecule, E-cadherin, was decreased in LACs with highly expressed aPKC λ/ι at the invasion site of tumor cells. This result suggested that the expression levels of aPKC λ/ι and E-cadherin reflect the progression of LAC. On double-immunohistochemical analysis, aPKC λ/ι and Lgl2, a protein that interacts with aPKC λ/ι, were co-localized within LACs. Furthermore, we found that Lgl2 bound the aPKC λ/ι-Par6 complex in tumor tissue by immune-cosedimentation analysis. Apical membrane localization of Lgl2 was correlated with lymphatic invasion and lymph node metastasis. These results thus indicate that aPKC λ/ι expression is altered upon the progression of LAC. This is also the first evidence to show aPKC λ/ι overexpression in LAC and demonstrates that aPKC λ/ι localization at the apical membrane or cell-cell contact is associated with lymphatic invasion and metastasis of the tumor.

Over-expression of BACH2 is related to ongoing somatic hypermutation of the immunoglobulin heavy chain gene variable region of de novo diffuse large B-cell lymphoma.

The basic region-leucine zipper (bZip) factor BTB, CNC homology 2 (BACH2) is known to have important roles in class switch recombination and somatic hypermutation (SHM) of the immunoglobulin (Ig) gene. In this study, we investigated the relationship between the expression of BACH2 and the status of SHM of the Ig heavy chain gene variable region (IgHV) for SHM in diffuse large B-cell lymphoma (DLBCL). We examined 20 cases of DLBCL, 13 of which were germinal center B-cell (GCB) DLBCL and 7 were non-GCB DLBCL. Seven cases were negative, 6 were positive (cytoplasmic expression) and 7 were strongly positive (both nuclear and cytoplasmic expression) for BACH2. Confirmed mutation (CM) was identified in 8 cases and the CM index (number of confirmed mutations per 10 subclones) was distributed from 0 to 5. A CM index of 7 strongly positive (over-expression) cases with BACH2 were distributed from 0 to 5, and that of 7 negative and 6 positive cases were distributed from 0 to 1. Over-expression of BACH2 was statistically related to CM index (P = 0.008). In conclusion, over-expression of BACH2 is critical for ongoing SHM of IgHV in DLBCL, and our data suggest that BACH2 may play an essential role for SHM of the Ig gene in B-cell lymphoma.