For quantification, the samples were normalized against the expression of -actin-encoding mRNA using the CTmethod

For quantification, the samples were normalized against the expression of -actin-encoding mRNA using the CTmethod. MDA/LUC-shUGT8 cells. In addition, it was found that expression of UGT8 in MDA-MB-231 cells increased their resistance to apoptosis induced by doxorubicin in vitro. Therefore, these data suggest that accumulation of GalCer in tumor cells inhibits apoptosis, which would facilitates metastatic cells to survive in the hostile microenvironment of tumor in target organ. == Introduction == In 1874 Thudichum isolated from bovine brain the lipid fraction that was highly enriched in galactosylceramide (then cerebroside)[1], for which the final structure was established in 1952 by Carter and Greenwood[2]and its enzymatic synthesis was described by Morrel and Radin in 1969[3]. Since then, GalCer was primarily seen to be one of the major myelin stabilizing components[4]. This glycolipid, in addition to oligodendrocytes and Schwann cells, is also highly expressed in kidney and testis[5][6]. However, in contrast to many other glycosphingolipids, little is known about GalCer expression in human cancers except oligodendrogliomas and astrocytomas[7][8]. GalCer is synthesized by highly specific, reticulum-localized, glycosyltransferase UDP-ceramide:galactose galactosyltransferase (UGT8, EC 2.4.1.47)[9]. This enzyme is up-regulated in ER-negative breast cancer[10][11]and ovarian cancer as shown by microarray studies[12]. Using the same approach, UGT8 was listed as one of six genes predicting breast cancer lung metastases[13]. Recently, our studies with the use of immunohistochemistry and real-time PCR on the expression of UGT8 in breast cancer tissue specimens revealed significant increase in UGT8 expression in (1) metastatic vs. primary tumors, (2) tumors of malignancy grades G3 vs. G2 as well as G3 vs. G1 and (3) node-positive vs. node-negative tumors[14]. The predictive ability of increased expression of UGT8 was validated at the mRNA level in three independent cohorts of breast cancer patients. Therefore, our data suggested that UGT8 is a significant index of tumor aggressiveness and a potential marker for the prognostic evaluation of lung metastases in breast cancer. We also analyzed the presence of UGT8 and GalCer in breast cancer cell lines and found that cells with luminal epithelial-like phenotype did not express or weakly expressed UGT8 and GalCer, in contrast to malignant, mesenchymal-like cells forming metastases in nude mice[14]. GalCer is synthesized by transferring galactose to ceramide, which is SMIP004 the second messenger molecule involved in such basic cellular processes as induction of growth arrest, differentiation, senescence and apoptosis[15]. It is widely accepted that ceramide is part of specific signaling pathways related to cellular stress response and many stressors like cytokines, serum deprivation, heat shock, ionizing radiation, and chemotherapeutics generate enhanced ceramide production[16]. Among different ceramide activities, special attention was paid to the pro-apoptotic properties of this molecule[15],[17]as a potential target for cancer chemotherapy[18]. De novo synthesis is responsible for the accumulation of ceramide in receptor-dependent and receptor-independent induction of apoptosis in cancer cells by such chemotherapeutics as etoposides[19]or doxorubicin[20]. Using MCF-7 cells as a model, it was shown that ionizing radiation induces apoptosis of tumor cells by Rabbit Polyclonal to BLNK (phospho-Tyr84) activating acid sphingomyelinase[21]. The same enzyme as well as neutral sphingomyelinase are involved in death receptor-mediated apoptosis of breast cancer cells[22][23]. On the other hand, ceramide, synthesized de novo or/and generated from other compounds, can be converted to several metabolites as ceramide 1-phosphate[24], sphingosine/sphingosine 1-phosphate[25], sphingomyelin[26], and 1-O-acylcermide[27]. Ceramide can also be glycosylated to form glucosylceramide (GlcCer) or GalCer. It is now well established that tumor cells, in order to escape apoptosis induced by various chemiotherapeutics and mediated by the accumulation of ceramide, convert this active lipid molecule to GlcCer[15],[28][29]. In contrast, little attention has been paid to an alternative ceramide glycosylation pathway by the formation of GalCer. Interestingly, few years ago, it was proposed, however without any experimental evidence, that accumulation of GalCer in tumor cells could inhibit apoptosis which facilitates metastatic cells to survive in the hostile microenvironment of the target organ[30]. It was also documented that the tumor microenvironment by itself is the source of SMIP004 numerous cell stresses, such as hypoxia, acidosis, hyperglycemia, hyperosmotic pressure, SMIP004 high cell density, and free radicals which affect cancer cells[31]. The involvement of UGT8 and GalCer in cellular stress response was shown in normal kidney cells subjected to hiperosmotic or hipertermic stresses. Such conditions generated UGT8 overexpression and synthesis of large amounts of GalCer as a way to decrease the level of pro-apotptotic ceramide[32][33]..