ROS1 as a possible prognostic biomarker of cervical adenocarcinoma: An exploratory analysis with next-generation sequencing.

OBJECTIVES: Given the differences in clinical and biological characteristics between cervical adenocarcinoma and squamous cell carcinoma, this study aimed to conduct an exploratory analysis to examine the molecular characteristics of cervical adenocarcinoma in a Japanese population. METHODS: This study explored the simultaneous testing of multiple mutations targeting cervical adenocarcinoma using next-generation sequencing (NGS). The following genes were analyzed: BCAR4, CD274, PDCD1LG2, KRAS, ARID1A, PTEN, ALK, EGFR, ROS1, BRAF, PIK3CA, EP300, EBXW7, SHCBP1, TGFBR2, SMAD4, ERBB2, ERBB3, and KLF5. Tumor tissue and blood samples were obtained at the time of primary treatment. The NGS-based molecular profiles obtained from Tokai University (49 specimens) were compared with the registered data in The Cancer Genome Atlas (TCGA) database (133 specimens). RESULTS: The study cohort had higher rates of adenocarcinoma than the TCGA cohort (44.9% vs. 18.0%; P = 0.001). The adenocarcinomas in the study cohort had more alterations in ROS1, EGFR, EP300, SHCBP1, ALK, and PIK3CA than those in the TCGA cohort. Among them, ROS1 had the highest number of gene alterations (median, 7.00 ± 2.63). In the study cohort, patients with a high number of ROS1 alterations had a significantly higher recurrence rate (5-year recurrence rate, 48.8% vs. 14.6%; hazard ratio [HR], 4.32; 95% confidence interval [CI], 1.20-15.50; P = 0.014) and lower overall survival than those with low alterations (5-year survival rate, 70.7% vs. 93.1%; HR, 7.15; 95% CI, 1.08-58.22; P = 0.032). CONCLUSION: The current exploratory analysis suggests that ROS1 gene alteration may be a prognostic biomarker in cervical adenocarcinoma in Japanese patients.

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.

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.

Acute Effects of Transdermal Administration of Jojoba Oil on Lipid Metabolism in Mice.

Background and objectives: Aroma therapy is a complementary therapy using essential oils diluted with carrier oils. Jojoba oils have been widely used as carrier oils. However, limited information is available regarding their effects on blood biochemical parameters. This study aimed to investigate the effect of transdermal administration of jojoba oil on blood biochemical parameters in mice. Materials and Methods: Eight-week-old male hairless mice were randomly divided into naïve control and treatment groups. In the treatment group, mice were topically administered 4 µL of jojoba oil, per gram of body weight, on the dorsa 30 min before euthanasia. Thereafter, serum biochemical parameters were assayed, and gene expression was analyzed in various tissues via a real-time polymerase chain reaction. Results: Serum non-esterified fatty acid (NEFA) levels increased significantly 30 min after topical application of jojoba oil (p < 0.05). Atgl was significantly upregulated in the liver (p < 0.05), and Atgl upregulation in the liver was positively correlated with serum NEFA levels (r = 0.592, p < 0.05). Furthermore, a trend of decreasing fatty acid trafficking-related gene (FABPpm, FATP-1, FATP-3, and FATP-4) expression in the skin after topical application of jojoba oil (p = 0.067, 0.074, 0.076, and 0.082, respectively) was observed. Conclusions: Serum NEFA levels were elevated 30 min after transdermal administration of jojoba oil. The mechanisms of elevated serum NEFA levels might be related to both enhanced lipolysis in the liver and reduced fatty acid trafficking in the skin.

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.

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.

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.

Protein kinase D1 regulates cofilin-mediated F-actin reorganization and cell motility through slingshot.

Dynamic actin remodelling processes at the leading edge of migrating tumour cells are concerted events controlled by a fine-tuned temporal and spatial interplay of kinases and phosphatases. Actin severing is regulated by actin depolymerizing factor (ADF)/cofilin, which regulates stimulus-induced lamellipodia protrusion and directed cell motility. Cofilin is activated by dephosphorylation through phosphatases of the slingshot (SSH) family. SSH activity is strongly increased by its binding to filamentous actin (F-actin); however, other upstream regulators remain unknown. Here we show that in response to RhoA activation, protein kinase D1 (PKD1) phosphorylates the SSH enzyme SSH1L at a serine residue located in its actin-binding motif. This generates a 14-3-3-binding motif and blocks the localization of SSH1L to F-actin-rich structures in the lamellipodium by sequestering it in the cytoplasm. Consequently, expression of constitutively active PKD1 in invasive tumour cells enhanced the phosphorylation of cofilin and effectively blocked the formation of free actin-filament barbed ends and directed cell migration.

An up-to-date anti-cancer treatment strategy focusing on HIF-1alpha suppression: its application for refractory ovarian cancer.

Hypoxia inducible factor-1alpha (HIF-1alpha) predominantly determines the transcriptional activity of HIF-1, which induces the certain genetic expressions to participate in the proliferation and progression of the tumor. It is supposed that HIF-1alpha is also an extremely important factor in cancer treatment. Based on the results of our recent analyses using ovarian tumors, which indicated the close association of HIF-1alpha expression with the acquisition of malignancy and the characterization of histology, we further investigated the possibility of a new strategy of cancer therapy that targeted HIF-1alpha inhibition in the ovarian carcinoma. The cell line HUOCA-II, which originates from the refractory ovarian clear cell adenocarcinoma, was treated with rapamycin. The inhibitory effect of HIF-1alpha was analyzed by immunohistochemistry and western blotting. It was demonstrated that inhibition of HIF-1alpha and vascular endothelial growth factor (VEGF) expressions would lead to the down-regulation of tumor cell proliferation. Interestingly, there was little or no change in GLUT-1 expression by rapamycin administration. Thus, the inhibition of GLUT-1 may also be a key for the new strategy of cancer therapy as well as HIF-1alpha and VEGF.