O-GlcNAc transferase regulates p21 protein levels and cell proliferation through the FoxM1-Skp2 axis in a p53-independent manner.

The protein product of the CDKN1A gene, p21, has been extensively characterized as a negative regulator of the cell cycle. Nevertheless, it is clear that p21 has manifold complex and context-dependent roles that can be either tumor suppressive or oncogenic. Most well studied as a transcriptional target of the p53 tumor suppressor protein, there are other means by which p21 levels can be regulated. In this study, we show that pharmacological inhibition or siRNA-mediated reduction of O-GlcNAc transferase (OGT), the enzyme responsible for glycosylation of intracellular proteins, increases expression of p21 in both p53-dependent and p53-independent manners in nontransformed and cancer cells. In cells harboring WT p53, we demonstrate that inhibition of OGT leads to p53-mediated transactivation of CDKN1A, while in cells that do not express p53, inhibiting OGT leads to increased p21 protein stabilization. p21 is normally degraded by the ubiquitin-proteasome system following ubiquitination by, among others, the E3 ligase Skp-Cullin-F-box complex; however, in this case, we show that blocking OGT causes impairment of the Skp-Cullin-F-box ubiquitin complex as a result of disruption of the FoxM1 transcription factor-mediated induction of Skp2 expression. In either setting, we conclude that p21 levels induced by OGT inhibition correlate with cell cycle arrest and decreased cancer cell proliferation.

The roles and regulation of MDM2 and MDMX: it is not just about p53.

Most well studied as proteins that restrain the p53 tumor suppressor protein, MDM2 and MDMX have rich lives outside of their relationship to p53. There is much to learn about how these two proteins are regulated and how they can function in cells that lack p53. Regulation of MDM2 and MDMX, which takes place at the level of transcription, post-transcription, and protein modification, can be very intricate and is context-dependent. Equally complex are the myriad roles that these two proteins play in cells that lack wild-type p53; while many of these independent outcomes are consistent with oncogenic transformation, in some settings their functions could also be tumor suppressive. Since numerous small molecules that affect MDM2 and MDMX have been developed for therapeutic outcomes, most if not all designed to prevent their restraint of p53, it will be essential to understand how these diverse molecules might affect the p53-independent activities of MDM2 and MDMX.

Hexosamine Biosynthetic Pathway and Glycosylation Regulate Cell Migration in Melanoma Cells.

The Hexosamine Biosynthetic Pathway (HBP) is a branch of glycolysis responsible for the production of a key substrate for protein glycosylation, UDP-GlcNAc. Cancer cells present altered glucose metabolism and aberrant glycosylation, pointing to alterations on HBP. Recently it was demonstrated that HBP influences many aspects of tumor biology, including the development of metastasis. In this work we characterize HBP in melanoma cells and analyze its importance to cellular processes related to the metastatic phenotype. We demonstrate that an increase in HBP flux, as well as increased O-GlcNAcylation, leads to decreased cell motility and migration in melanoma cells. In addition, inhibition of N- and O-glycosylation glycosylation reduces cell migration. High HBP flux and inhibition of N-glycosylation decrease the activity of metalloproteases 2 and 9. Our data demonstrates that modulation of HBP and different types of glycosylation impact cell migration.

Hyperglycemia and aberrant O-GlcNAcylation: contributions to tumor progression.

A number of cancer types have shown an increased prevalence and a higher mortality rate in patients with hyperglycemic associated pathologies. Although the correlation between diabetes and cancer incidence has been increasingly reported, the underlying molecular mechanisms beyond this association are not yet fully understood. Recent studies have suggested that high glucose levels support tumor progression through multiple mechanisms that are hallmarks of cancer, including cell proliferation, resistance to apoptosis, increased cell migration and invasiveness, epigenetic regulation (hyperglycemic memory), resistance to chemotherapy and altered metabolism. Most of the above occur because hyperglycemia through hexosamine biosynthetic pathway leads to aberrant O-GlcNAcylation of many intracellular proteins that are involved in those mechanisms. Deregulated O-GlcNAcylation is emerging as a general feature of cancer. Despite strong evidence suggesting that aberrant O-GlcNAcylation is or may be involved in the acquisition of all cancer hallmarks, it remains out of the list of the next generation of emerging hallmarks. Here, we discuss some of the current understanding on how hyperglycemia affects cancer cell biology and how aberrant O-GlcNAcylation stands in this context.

Pomolic acid induces apoptosis and inhibits multidrug resistance protein MRP1 and migration in glioblastoma cells.

Glioblastoma (GBM), the most aggressive of primary brain tumors, determine short survival and poor quality of life. Therapies used for its treatment are not effective and chemotherapy failure is partially due to multidrug resistance (MDR) mechanisms present in the tumor cells. New therapeutic strategies are needed in order to improve survival in GBM. The present study investigated the activity of the pentacyclic triterpene pomolic acid (PA) in GBM. Pomolic acid decreased the viability and induced apoptosis of GBM cells as demonstrated by DNA fragmentation. It also induced uncoupling of mitochondria membrane potential and activation of caspase-3 and -9. Pomolic acid-induced apoptosis is dependent on reactive oxygen species (ROS) production as it is inhibited by anti-oxidant treatment. Pomolic acid also down-modulated the activity of the multidrug resistance associated protein 1 (MRP1) and inhibited migration of GBM cells. These results show that PA acts on several pathways of GBM drug resistance and therefore may be of potential interest for the treatment of this tumor.

Changes in O-Linked N-Acetylglucosamine (O-GlcNAc) Homeostasis Activate the p53 Pathway in Ovarian Cancer Cells.

O-GlcNAcylation is a dynamic post-translational modification consisting of the addition of a single N-acetylglucosamine sugar to serine and threonine residues in proteins by the enzyme O-linked ß-N-acetylglucosamine transferase (OGT), whereas the enzyme O-GlcNAcase (OGA) removes the modification. In cancer, tumor samples present with altered O-GlcNAcylation; however, changes in O-GlcNAcylation are not consistent between tumor types. Interestingly, the tumor suppressor p53 is modified by O-GlcNAc, and most solid tumors contain mutations in p53 leading to the loss of p53 function. Because ovarian cancer has a high frequency of p53 mutation rates, we decided to investigate the relationship between O-GlcNAcylation and p53 function in ovarian cancer. We measured a significant decrease in O-GlcNAcylation of tumor tissue in an ovarian tumor microarray. Furthermore, O-GlcNAcylation was increased, and OGA protein and mRNA levels were decreased in ovarian tumor cell lines not expressing the protein p53. Treatment with the OGA inhibitor Thiamet-G (TMG), silencing of OGA, or overexpression of OGA and OGT led to p53 stabilization, increased nuclear localization, and increased protein and mRNA levels of p53 target genes. These data suggest that changes in O-GlcNAc homeostasis activate the p53 pathway. Combination treatment of the chemotherapeutic cisplatin with TMG decreased tumor cell growth and enhanced cell cycle arrest without impairing cytotoxicity. The effects of TMG on tumor cell growth were partially dependent on wild type p53 activation. In conclusion, changes in O-GlcNAc homeostasis activate the wild type p53 pathway in ovarian cancer cells, and OGA inhibition has the potential as an adjuvant treatment for ovarian carcinoma.

O-GlcNAcylation: The Sweet Side of the Cancer.

O-GlcNAcylation is an O-linked ß-N-acetylglucosamine (O-GlcNAc) moiety linked to the serine or threonine residues in proteins. O-GlcNAcylation is a dynamic post-translational modification involved in a wide range of biological processes and diseases such as cancer. This modification can increase and decrease the activity of enzymes as well as interfere with protein stability and interaction. The modulatory capacity of O-GlcNAcylation, as well as protein phosphorylation, is of paramount importance in the regulation of metabolism and intracellular signaling of tumor cells. Thus, understanding the regulation of O-GlcNAcylation in tumor cells and their difference compared to non-tumor cells may elucidate new mechanisms related to tumor generation and development, could provide a new marker to diagnosis and prognosis in patients with cancer and indicate a new target to cancer chemotherapy.

Apoptosis-inducing effects of Melissa officinalis L. essential oil in glioblastoma multiforme cells.

Current therapies for glioblastoma multiforme (GBM) are not effective. This study investigated the activity of the M. officinalis essential oil (EO) and its major component (citral) in GBM cell lines. Both EO and citral decreased the viability and induced apoptosis of GBM cells as demonstrated by DNA fragmentation and caspase-3 activation. Antioxidant prevented citral-induced death, indicating its dependence on the production of reactive oxygen species. Citral downmodulated the activity and inhibited the expression of multidrug resistance associated protein 1 (MRP1). These results show that EO, through its major component, citral, may be of potential interest for the treatment of GBM.