Id-1 is not expressed in the luminal epithelial cells of mammary glands

BACKGROUND: The family of inhibitor of differentiation/DNA binding (Id) proteins is known to regulate development in several tissues. One member of this gene family, Id-1, has been implicated in mammary development and carcinogenesis. Mammary glands contain various cell types, among which the luminal epithelial cells are primarily targeted for proliferation, differentiation and carcinogenesis. Therefore, to assess the precise significance of Id-1 in mammary biology and carcinogenesis, we examined its cellular localization in vivo using immunohistochemistry. METHODS: Extracts of whole mammary glands from wild type and Id-1 null mutant mice, and tissue sections from paraffin-embedded mouse mammary glands from various developmental stages and normal human breast were subjected to immunoblot and immunohistochemical analyses, respectively. In both these procedures, an anti-Id-1 rabbit polyclonal antibody was used for detection of Id-1. RESULTS: In immunoblot analyses, using whole mammary gland extracts, Id-1 was detected. In immunohistochemical analyses, however, Id-1 was not detected in the luminal epithelial cells of mammary glands during any stage of development, but it was detected in vascular endothelial cells. CONCLUSION: Id-1 is not expressed in the luminal epithelial cells of mammary glands

N -Ethyl- N -Nitrosourea Induces Retinal Photoreceptor Damage in Adult Rats

Seven-week-old male Lewis rats received a single intraperitoneal injection of N-ethyl-N-nitrosourea (ENU) (100, 200, 400 or 600 mg/kg), and retinal damage was evaluated 7 days after the treatment. Sequential morphological features of the retina and retinal DNA damage, as determined by a TUNEL assay and phospho-histone H2A.X (γ-H2AX), were analyzed 3, 6, 12, 24 and 72 hr, 7 days, and/or 30 days after 400 mg/kg ENU treatment. Activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) was analyzed immunohistochemically by poly (ADP-ribose) (PAR) expression in response to DNA damage of the retina. All rats that received ≥ 400 mg/kg of ENU developed retinal degeneration characterized by the loss of photoreceptor cells in both the central and peripheral retina within 7 days. In the 400 mg/kg ENU-treated rats, TUNEL-positive signals were only located in the photoreceptor cells and peaked 24 hr after ENU treatment. The γ-H2AX signals in inner retinal cells appeared at 24 hr and peaked at 72 hr after ENU treatment, and the PAR signals selectively located in the photoreceptor cell nuclei appeared at 12 hr and peaked at 24 hr after ENU treatment. However, degeneration was restricted to photoreceptor cells, and no degenerative changes in inner retinal cells were seen at any time points. Retinal thickness and the photoreceptor cell ratio in the central and peripheral retina were significantly decreased, and the retinal damage ratio was significantly increased 7 days after ENU treatment. In conclusion, ENU induced retinal degeneration in adult rats that was characterized by photoreceptor cell apoptosis through PARP activity

Conjugated docosahexaenoic acid suppresses KPL-1 human breast cancer cell growth in vitro and in vivo: potential mechanisms of action

Introduction The present study was conducted to examine the effect of conjugated docosahexaenoic acid (CDHA) on cell growth, cell cycle progression, mode of cell death, and expression of cell cycle regulatory and/or apoptosis-related proteins in KPL-1 human breast cancer cell line. This effect of CDHA was compared with that of docosahexaenoic acid (DHA). Methods KPL-1 cell growth was assessed by colorimetric 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; cell cycle progression and mode of cell death were examined by flow cytometry; and levels of expression of p53, p21Cip1/Waf1, cyclin D1, Bax, and Bcl-2 proteins were examined by Western blotting analysis. In vivo tumor growth was examined by injecting KPL-1 cells subcutaneously into the area of the right thoracic mammary fat pad of female athymic mice fed a CDHA diet. Results CDHA inhibited KPL-1 cells more effectively than did DHA (50% inhibitory concentration for 72 hours: 97 μmol/l and 270 μmol/l, respectively). With both CDHA and DHA growth inhibition was due to apoptosis, as indicated by the appearance of a sub-G1 fraction. The apoptosis cascade involved downregulation of Bcl-2 protein; Bax expression was unchanged. Cell cycle progression was due to G0/G1 arrest, which involved increased expression of p53 and p21Cip1/Waf1, and decreased expression of cyclin D1. CDHA modulated cell cycle regulatory proteins and apoptosis-related proteins in a manner similar to that of parent DHA. In the athymic mouse system 1.0% dietary CDHA, but not 0.2%, significantly suppressed growth of KPL-1 tumor cells; CDHA tended to decrease regional lymph node metastasis in a dose dependent manner. Conclusion CDHA inhibited growth of KPL-1 human breast cancer cells in vitro more effectively than did DHA. The mechanisms of action involved modulation of apoptosis cascade and cell cycle progression. Dietary CDHA at 1.0% suppressed KPL-1 cell growth in the athymic mouse system.</p

MSLN gene silencing has an anti-malignant effect on cell lines overexpressing mesothelin deriving from malignant pleural mesothelioma

Genes involved in the carcinogenetic mechanisms underlying malignant pleural mesothelioma (MPM) are still poorly characterized. So far, mesothelin (MSLN) has aroused the most interest. It encodes for a membrane glycoprotein, frequently over-expressed in various malignancies such as MPM, and ovarian and pancreatic cancers. It has been proposed as a diagnostic and immunotherapeutic target with promising results. However, an alternative therapeutic approach seems to rise, whereby synthetic molecules, such as antisense oligonucleotides, could be used to inhibit MSLN activity. To date, such a gene-level inhibition has been attempted in two studies only, both on pancreatic and ovarian carcinoma cell lines, with the use of silencing RNA approaches. With regard to MPM, only one cell line (H2373) has been employed to study the effects of MSLN depletion. Indeed, the knowledge on the role of MSLN in MPM needs expanding. Accordingly, we investigated the expression of MSLN in a panel of three MPM cell lines, i.e., NCI-H28, Mero-14, and IstMes2; one non-MPM cell line was used as reference (Met5A). MSLN knock-down experiments on MSLN-overexpressing cells were also performed through silencing RNA (siRNA) to verify whether previous findings could be generalized to a different set of cell cultures. In agreement with previous studies, transient MSLN-silencing caused decreased proliferation rate and reduced invasive capacity and sphere formation in MSLN-overexpressing Mero-14 cells. Moreover, MSLN-siRNA combined with cisplatin, triggered a marked increase in apoptosis and a decrease in proliferation as compared to cells treated with each agent alone, thereby suggesting a sensitizing effect of siRNA towards cisplatin. In summary, our findings confirm that MSLN should be considered a key molecular target for novel gene-based targeted therapies of cancer

Exosomes Derived from Fisetin-Treated Keratinocytes Mediate Hair Growth Promotion

Enhanced telomerase reverse transcriptase (TERT) levels in dermal keratinocytes can serve as a novel target for hair growth promotion. Previously, we identified fisetin using a system for screening food components that can activate the TERT promoter in HaCaT cells (keratinocytes). In the present study, we aimed to clarify the molecular basis of fisetin-induced hair growth promotion in mice. To this end, the dorsal skin of mice was treated with fisetin, and hair growth was evaluated 12 days after treatment. Histochemical analyses of fisetin-treated skin samples and HaCaT cells were performed to observe the effects of fisetin. The results showed that fisetin activated HaCaT cells by regulating the expression of various genes related to epidermogenesis, cell proliferation, hair follicle regulation, and hair cycle regulation. In addition, fisetin induced the secretion of exosomes from HaCaT cells, which activated β-catenin and mitochondria in hair follicle stem cells (HFSCs) and induced their proliferation. Moreover, these results revealed the existence of exosomes as the molecular basis of keratinocyte-HFSC interaction and showed that fisetin, along with its effects on keratinocytes, caused exosome secretion, thereby activating HFSCs. This is the first study to show that keratinocyte-derived exosomes can activate HFSCs and consequently induce hair growth